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reversible protein phosphorylation is a major mechanism in the regulation of fundamental signaling events in all living organisms. in bacteria, four types of phosphorylation systems were described : (i) the two - component system (1), (ii) the phosphoenolpyruvate (pep) transferase system (pts) (2), (iii) the eukaryotic - like system (3) and (iv) the bacterial tyrosine kinase (by - kinase) system (4). those four types differ in the nature of the phosphorylated amino acids (s, t, y, h and d) and the phosphate donor (adenosine tri - phosphate (atp) or pep). by - kinases comprise two domains : a two - pass transmembrane activator domain (tad) that includes a large extra cellular part, and an intracellular catalytic domain (cd). the by - kinases cd encompasses three walker - like motifs (5), called a, a and b, and a c - terminal tyrosine - rich region named y cluster (yc). these two domains can either be linked in a single polypeptide (in proteobacteria and actinobacteria), or split into two different proteins, encoded by two adjacent genes (in firmicutes). in this case, it has been shown that by - kinases activity was effective only if the cd was interacting with the region following the second transmembrane segment of tad. a number of studies have demonstrated the importance of by - kinases in several facets of the physiology of the bacterial cell. more precisely, their best characterized function concerns the regulation of the export and the biosynthesis of bacterial extracellular polysaccharide that are recognized important virulence factors. since these enzymes are not homologs of their eukaryotic counterparts, by - kinases are particularly interesting in the search of new therapeutic targets to combat bacterial pathogens. we present here the bacterial tyrosine kinase database (bykdb), developed in order to collect, store and manage by - kinase sequences with standardized annotations. the sequences are identified by using an in silico workflow described in a previous work (6). diverse sequences identified by the workflow have been validated experimentally since then (dr mijakovic, personal communication). the sequences are automatically annotated and stored in the database by a second automated workflow. the database can be queried via a www interface and the query results can be further analyzed with the numerous integrated analysis tools. the bykdb is updated on a monthly basis from the uniprot knowledgebase (uniprotkb) (7), and stored in the postgresql relational database management system (rdbms). the programs for the sequence identification and annotation, as well as for the querying and the management of the database are implemented in java and sql programming languages. the first step of the database building procedure is the identification of by - kinase sequences, thanks to the it relies on the hmmer software package (version 3.0 of march 2010) (8). the by - kinases tad is matched by the pfam profile pf02706 (9), in a region including the first transmembrane segment. since this profile identifies proteins that are not by - kinases, and does not identify cd of two - parts by - kinases, it is not sufficient to unambiguously identify by - kinases. hmmsearch result sets are analyzed to lead to three clusters of sequences. the first cluster is constituted by sequences matching both hmm profiles (tadcd cluster). the second cluster contains sequences with the tad and cd encoded by two proteins (tad and the sequences of the third cluster only match the cd profile and have no identified tad (orphancd cluster). the sequences are further divided into two categories (confirmed and unsure) according to a filter (isbyk). this filter checks if a sequence contains all the necessary cd motifs (walker - like motifs and tyrosine cluster) with the right spacing between them (6). the annotatebyk process ensures the second step of the procedure, i.e. the sequence annotation. the annotation starts with the uniprotkb entries extracted according to the ac of the relevant sequences identified in the first step. indeed, the accession numbers, the creation date, the organism and the sequence of each entry are conserved. in the description line (de), the recommended name field is set to bacterial tyrosine kinase or to adaptator of bacterial tyrosine kinase depending on the type of sequences. in the keywords list (kw) are added the tadcd. the domains and motifs with their start and end positions in the sequence are also added to the feature (ft) lines, respectively as domain and site fields. besides, for each sequences of the second group, a cross - reference to the related tad entry is added and reciprocally for the tad entry. the user can navigate through the site, thanks to the menubar or the home page buttons. (a) the bykdb home page, with the resources accessible as clickable buttons (b) a pre - computed dataset of by - kinase and tad sequences and alignments, ordered by phylum and by cluster. (c) the multiple sequence alignment of firmicutes cd viewed in the editalignment applet. (a) the bykdb home page, with the resources accessible as clickable buttons (b) a pre - computed dataset of by - kinase and tad sequences and alignments, ordered by phylum and by cluster. (c) the multiple sequence alignment of firmicutes cd viewed in the editalignment applet. the annotate tool detailed results page for one input sequence. in the static part, the user can find general information about by - kinases with links to other external resources (byk link). the user can also access pre - computed datasets (dataset link) sorted according to the phylum and by - kinase cluster. the datasets include by - kinase sequences in pearson / fasta format and the corresponding multiple sequence alignment. furthermore, the alignments can be viewed and edited with the editalignment applet developed by our team. in the dynamic part (dynamic link), the user can extract his own datasets by combining multiple criteria (e.g. species and sequence length and sequence cluster). the datasets can be exported as fasta / pearson sequences, accession number lists and entry flat files for further analysis with the integrated analysis tools. the generic analysis tools (e.g. blast or clustal w) are available through the nps@ server (10), which is an integrated sequence analysis web server. the specialized analysis tool (annotate link) available on the site allows the annotation of one or several sequences, which can be useful to scan protein sequences from a newly sequenced bacterial genome in order to identify by - kinases and their adaptators. in the results page, the user can access the start / end positions and the sequences of the different domains and sites. furthermore, the domains and sites are mapped in color on the by - kinase sequence. the release 3.0 (october 2011) comprises 2,746 sequences, including 713 cd sequences with their corresponding tad sequences, 1,169 tadcd sequences and 151 orphancd sequences. bykdb collects by - kinase sequences, identified and annotated, thanks to a computer - automated system. the automatic annotation process used to generate bykdb entries guarantees harmonized annotations and allows efficient keyword searches. the bykdb website gives access to pre - computed static datasets and allows dynamic queries. the extracted data can be further analyzed with generic or specialized bioinformatics algorithms. in the future, the annotations will be enriched with new data (e.g. information about protein structure) and new analysis tools (e.g. phylogeny) will be added. bykdb is funded by the agence nationale de la recherche (anr-07-jcjc0125 - 01 bactyrkin) and the ple rhne - alpes de bioinformatique (prabi) platform by the groupement d'intrt scientifique infrastructures en biologie, sant et agronomie (gis ibisa). funding for open access charge : agence nationale de la recherche (anr-07-jcjc0125 - 01 bactyrkin). | bacterial tyrosine - kinases share no resemblance with their eukaryotic counterparts and they have been unified in a new protein family named by - kinases. these enzymes have been shown to control several biological functions in the bacterial cells. in recent years biochemical studies, sequence analyses and structure resolutions allowed the deciphering of a common signature. however, by - kinase sequence annotations in primary databases remain incomplete. this prompted us to develop a specialized database of computer - annotated by - kinase sequences : the bacterial protein tyrosine - kinase database (bykdb). by - kinase sequences are first identified, thanks to a workflow developed in a previous work. a second workflow annotates the uniprotkb entries in order to provide the bykdb entries. the database can be accessed through a web interface that allows static and dynamic queries and offers integrated sequence analysis tools. bykdb can be found at http://bykdb.ibcp.fr. |
the total removal of the larynx (total laryngectomy), performed in 1872 by the well - known viennese surgeon christian a. theodor billroth (18291894), was an epoch - making event in the history of surgery and laryngology. this paper presents the first surgeons who performed this operation. the first pole who performed a total laryngectomy (1877) was julian kosiski (18331914), head of the surgical clinic of imperial university warsaw. it was the 14th operation of this kind in the world. several laryngectomies were carried out by franciszek ksawery jawdyski (18511896), called the father of polish head and neck surgery, who was the first pole to excise a malignant neck cancer together with the lymph nodes. the next total laryngectomies were performed by the following polish surgeons : jan mikulicz - radecki (18501905), wadysaw h.s. krajewski (18551907), alfred obaliski (18431898). according to jan sdziak, by 1897 poland with 16 laryngectomies (per 188 carried out) had occupied the fourth position in the world. the failures of the first laryngectomies were due to such factors as the ignorance of shock pathophysiology, the inability to ensure the patency of the airways during and after the operation, which would result in lung and mediastinum infections, massive haemorrhages and so on. but the primary cause was the lack of constructive collaboration between the laryngologists and the surgeons. |
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the quest for newer materials is never ending in the field of restorative dentistry and endodontics. ideal characteristics lead to the introduction of mineral trioxide aggregate (mta) by torabinejad., in 1993. mta was recommended initially as a root - end filling material ; however, its use has been subsequently expanded to pulp capping, pulpotomy, apexogenesis, apical barrier formation in teeth with open apexes, repair of root perforations, and as root canal filling material. mta is composed of fine hydrophilic particles ; its main components are tricalcium silicate, tricalcium aluminate, tricalcium oxide, silicate oxide, and other mineral oxides. the original formulation, grey mta, was introduced first ; however, white mta was developed later as this version improved esthetics. mta powder sets in presence of water and results in formation of a colloidal gel, which solidifies to a hard structure in approximately 3 - 4h. mta is a biocompatible material, promotes cementum formation and apical root closure, it is an effective material in preventing leakage, as perforation repair and root - end filling material. the ph of mta is approximately 12.5 and is similar to that of calcium hydroxide, a material with proven antibacterial activity. despite its favorable properties a shorter setting time would be beneficial because it would allow less time for contaminants in the oral environment to adversely affect the material, allow safer placement of restorative material over it (pulp capping), and also shorten the period when washout of cement can occur. it is thus of interest to attempt to use chemicals to accelerate the setting time of mta. also mta has low compressive strength compared with most other materials, a reason for its limited application in restorative dentistry as this parameter is of major significance for indication of mta as coronal restorative material. the extended endodontic use and its potential of use in restorative dentistry necessitates the development of new formulation of the material, which optimizes both its strength and its setting time without compromising its ph and biocompatibility. mta and construction grade portland cement (pc) are very similar in chemical and physical characters. since setting time of pc can be accelerated by addition of certain accelerating admixtures ; addition of these construction grade pc setting accelerators to mta may induce a faster set. some of the commonly used cement accelerators are calcium chloride (cacl2), calcium formate (caf), disodium hydrogen orthophosphate (na2hpo4), and potassium chloride. however, it is important that these accelerants should not adversely affect the other properties of mta. therefore, this study was designed to evaluate the effect of 10% cacl2, 20% caf, and 15% na2hpo4 on the setting time, ph, and compressive strength of white mta. samples were prepared by mixing 0.8 g of white mta (pro root mta ; dentsply tulsa dental, ok) powder with various additives. each mix was prepared using a stainless steel cement spatula on a glass slab and was then immediately transferred into the molds. before mixing, the test materials, molds, pluggers, spatulas, and glass slabs were kept at room temperature for a period of 24 h. the samples were divided into four groups : group 1 (control) : 0.8 g of mta mixed with 0.3ml distilled water (n = 10). group 2 : 0.8 g mta with 0.08 g of cacl2 (lab chem., kanpur, india) mixed with 0.3 ml distilled water (n = 10). group 3 : 0.8 g mta with 0.16 g of caf (otto, mumbai, india) mixed with 0.3 ml of distilled water (n = 10). group 4 : 0.8 g mta mixed with 0.3 ml of 15% na2hpo4 (fisher scientific, mumbai, india ; n = 10). samples were prepared by mixing cement and placing them in circular acrylic mold (10.0 mm inner diameter and 5.0 mm height). for ph, 10specimens were prepared for each group by filling hydrated samples into a cylindrical teflon mold (6.0 mm in diameter and 12.0 mm high). specimens were individually immersed in holders containing 60 ml of deionized distilled water, sealed, and stored for 24 hours at 37c. each material was mixed and placed in a split stainless steel mold (4.0 mm inner diameter and 6.0 mm height). cement was then compacted into each mold using a spatula and further compacted using a dental plugger to ensure dense uniform sample with minimal porosity. once filled, the excess was scraped off with the edge of a glass microscopic slide to leave a flat uniform surface. no later than 120 s after mixing, the complete assembly was transferred to a cabinet maintained at 37c for 6 h, after which they were removed from the molds and checked visually for any air voids or chipped edges. all defective specimens were discarded, and 10 accepted samples were prepared for each time interval. the specimens were immersed in distilled water for 24 h, 3 days, and 7 days and maintained at 37c. setting time was determined using a vicat apparatus (aimil, new delhi, india). the vicat indenter is 400 5 g in weight containing a needle with a flat end and 1.0 0.1 mm in diameter. ninety seconds after mixing, the indenter needle was lowered vertically onto the surface of the test material. the process was repeated every 10 s until the needle failed to make a complete circular indentation in the test material. setting time was defined as the time elapsed between the end of mixing and the time when the needle failed to make a complete circular indentation on the test material. after 24 h, the immersed solution was mixed by vortexing for 30 s and ph was measured using a phmeter (si, mainz, germany), which was calibrated using standard solutions of ph 4.0, 7.0, and 12.0. compressive strength was measured by using a universal testing machine (instron 1195, norwood, ma, usa) at a crosshead speed of 1.0 mm / min. the maximum load needed to fracture each specimen was measured, and the compressive strength (c) was calculated in megapascals (mpa) according to formula : where p is the maximum load applied in newton and d is the mean diameter of the specimen in millimeters. comparison of mean values was done using analysis of variance (anova) with post - hoc games - howell test. samples were prepared by mixing cement and placing them in circular acrylic mold (10.0 mm inner diameter and 5.0 mm height). the assembly was placed in a cabinet at 37c and relative humidity of 95%. for ph, 10specimens were prepared for each group by filling hydrated samples into a cylindrical teflon mold (6.0 mm in diameter and 12.0 mm high). specimens were individually immersed in holders containing 60 ml of deionized distilled water, sealed, and stored for 24 hours at 37c. each material was mixed and placed in a split stainless steel mold (4.0 mm inner diameter and 6.0 mm height). cement was then compacted into each mold using a spatula and further compacted using a dental plugger to ensure dense uniform sample with minimal porosity. once filled, the excess was scraped off with the edge of a glass microscopic slide to leave a flat uniform surface. no later than 120 s after mixing, the complete assembly was transferred to a cabinet maintained at 37c for 6 h, after which they were removed from the molds and checked visually for any air voids or chipped edges. all defective specimens were discarded, and 10 accepted samples were prepared for each time interval. the specimens were immersed in distilled water for 24 h, 3 days, and 7 days and maintained at 37c. setting time was determined using a vicat apparatus (aimil, new delhi, india). the vicat indenter is 400 5 g in weight containing a needle with a flat end and 1.0 0.1 mm in diameter. ninety seconds after mixing, the indenter needle was lowered vertically onto the surface of the test material. the process was repeated every 10 s until the needle failed to make a complete circular indentation in the test material. setting time was defined as the time elapsed between the end of mixing and the time when the needle failed to make a complete circular indentation on the test material. after 24 h, the immersed solution was mixed by vortexing for 30 s and ph was measured using a phmeter (si, mainz, germany), which was calibrated using standard solutions of ph 4.0, 7.0, and 12.0. compressive strength was measured by using a universal testing machine (instron 1195, norwood, ma, usa) at a crosshead speed of 1.0 mm / min. the maximum load needed to fracture each specimen was measured, and the compressive strength (c) was calculated in megapascals (mpa) according to formula : where p is the maximum load applied in newton and d is the mean diameter of the specimen in millimeters. comparison of mean values was done using analysis of variance (anova) with post - hoc games - howell test. the setting time of mta mixed with the additives was significantly shorter than that of mta mixed with water (p < 0.001) [table 1 ]. setting time observed was shortest for group 3. setting time of the test materials in minutes (n = 10 for each group) the ph of mta mixed with cacl2 was lower than that of the control group (p < 0.001) ; however, when caf and na2hpo4 was added to mta, ph value obtained was higher than that of the control group (p < 0.001) [table 2 ]. ph of test materials (n = 10 for each group) the compressive strength of all the groups increased during a 7-day period. the compressive strength of mta mixed with the additives was lower than that of mta mixed with water (p < 0.001) [table 3 ]. for however, on day 3 the compressive strength of group 4 was found higher than other groups. compressive strengths of test materials in mpa (n = 10 for each group at each time interval) it was the intent of this research to incorporate additives used by the cement industry to mta to try to decrease its setting time and improve its compressive strength, without affecting its ph. the various additives used included 10% cacl2, 20% caf, and 15% na2hpo4. in this study, however, the setting time observed in this study was significantly shorter than the 2 h and 45 min as reported by torabinejad. this difference can be attributed to changes that may have been incorporated into the mta powder since it was first introduced. asgary., found that a significant change in composition has occurred since mta was first introduced. according to bortoluzzi., the penetration of cacl2 in the pores of cements could accelerate the reaction due to the hydration of silicates which reduces their crystallization time, thus hastening the final setting time of the material. cacl2 mixed with portland cement has already been confirmed to be nontoxic to human cells in vitro. addition of na2hpo4 reduced the setting time of mta to 31.60 2.91 min, a result similar to that obtained by ding., and na2hpo4 solution can be used as a cement liquid to accelerate the setting of the cement prepared from tricalcium phosphate as a cement powder. strong ionic interactions between phosphate and calcium ions lead to the formation of calcium silicate hydrate phase. caf resulted in markedly decreased setting time, that is, 12.40 2.07 min. caf is a highly soluble calcium compound that completely dissociates in solution to produce calcium ions. this might accelerate the dissociation step in hydration of cement, thus accelerating its setting time. this decrease may be because of the addition of calcium - based electrolytes like cacl2 which tends to suppress ionization of ca(oh)2, thus the percentage dissociation of ca(oh)2 decreases. however, such ph difference at an alkaline condition, might not affect the antimicrobial property of modified mta. mta mixed with na2hpo4 resulted in a ph value of 12.77 0.09, which is similar to result obtained by ding., and when mixed with caf the ph is 12.56 0.19, which is similar to result of wiltbank. the compressive strength for mta mixed with water after 7 days was 36.24 3.33 mp a. this result is contrary to those obtained originally by torabenajed., which maybe because of change in composition of mta since the time it was first introduced. with the addition of cacl2 to mta, compressive strength obtained was 33.37 3.18, this result was similar to that obtained by lee. the compressive strength of mta obtained with the addition of caf was 30.05 1.02 and that with addition of na2hpo4 was 29.32 1.13. huang., observed that 15% na2hpo4 used as liquid phase for mta resulted in a good diametral tensile strength value. although these additives reduced the setting time, they also reduced the compressive strength of mta ; which could be a disadvantage of these additives. compressive strength is an important parameter to consider when placing a filling material in a cavity that bears occlusal pressure. on the contrary, when used as root - end filling where minimal forces are applied, reduced compressive strength will not be a major drawback. one drawback of our study is that distilled water has been used as a storage medium for modified mta samples. however, previous studies have shown that different storage methods and media can influence dentine characteristics as well as physical properties of dental materials. parirokh., and and asgary., have shown that placing mta in tissue fluids such as phosphate buffer saline(pbs) storage medium results in formation of hydroxyapatite crystals over mta and causes a significant decrease in bacterial penetration. hence, future in vitro studies can use pbs as a storage medium to simulate environment in human body. within the limitation of this study, 10% cacl2, 20% caf, and 15% na2hpo4 significantly reduced the setting time of white mta and also maintained the ph at a high value. however, there was no improvement in the compressive strength of the material. based on the results, mta mixed with these additives can be recommended for single - visit procedures where compressive strength is not a major concern. however, further studies regarding their biocompatibility, antimicrobial properties, sealing abilities, dimensional stability, etc., are indicated before any recommendation for clinical use. | aims : this study examined the setting time, compressive strength, and ph of white mineral trioxide aggregate (mta) mixed with various additives : calcium chloride (cacl2), calcium formate (caf), disodium hydrogen orthophosphate (na2hpo4).materials and methods : group 1 (control) was obtained by mixing mta with distilled water. in groups 2 and 3, mta containing 10% cacl2 and 20% caf, respectively, was mixed with distilled water. in group 4, mta was mixed with 15% na2hpo4. setting time, compressive strength, and ph of each group were examined.statistical analysis used : analysis was done using statistical package for social sciences (spss) version 14. a p - value < 0.05 was considered statistically significant. comparison of mean values was done using analysis of variance (anova) with post - hoc games - howell test.results:the setting time of test groups were significantly shorter than that of control group (p < 0.001). the compressive strengths of test groups were lower than that of control group (p < 0.001). the ph value obtained for groups 3 and 4 were higher than that of the control group (p < 0.001).conclusions : study result showed that additives significantly reduced the setting time of mta and also maintained the ph at a high value. however, there was not much improvement in the compressive strength of the material. |
the open access publication charges for this paper has been waived by oxford university press. | pdbselect (http://bioinfo.tg.fh-giessen.de/pdbselect/) is a list of representative protein chains with low mutual sequence identity selected from the protein data bank (pdb) to enable unbiased statistics. the list increased from 155 chains in 1992 to more than 4500 chains in 2009. pdbfilter - select is an online service to generate user - defined selections. |
atrial fibrillation (af) represents the most common sustained supraventricular arrhythmia, which is also noteworthy for its growing incidence in the elderly population. it is associated with increased mortality, risk of stroke, and exacerbation of heart failure. since pharmacological treatment of arrhythmias have been found to be unsatisfactory (in terms of rhythm control), there have been a number of nonpharmacological treatment modalities introduced into routine clinical practice over the past 10 years. this is particularly true with regard to persistent forms of arrhythmia, which usually require repeated procedures to establish a stable sinus rhythm (sr). longstanding persistent af represents a significant therapeutic challenge due to left atrial (la) dilatation and fibrotic structural remodeling. ablation strategies include not only pulmonary vein (pv) isolation but also creation of transmural linear lines connecting anatomical barriers. various epicardial structures, such as the ligament of marshall, are often impossible to ablate from the endocardial surface. surgical treatment of af has shifted from open heart surgery and cutandsew techniques toward minimally invasive procedures using endoscopic instruments to isolate the pvs, create linear lesions, and occlude the left atrial appendage (laa)all on the beating heart. although this approach achieves higher arrhythmiafree success rates after a single procedure than catheter ablations, contiguous and transmural lesions can not always be guaranteed. the mitral isthmus and the tricuspid isthmus lines are particularly difficult to ablate from the epicardial surface of the heart. the recently introduced hybrid approach seems to be a very attractive alternative for overcoming the limitations of the epi or endocardial ablation approach alone. however, there is limited data regarding the staged approach on a welldefined cohort of patients with longstanding persistent af. published studies offer only a mix of different af types (paroxysmal, persistent, and longstanding persistent, with or without previous catheter ablation) and even employ heterogeneous ablation techniques. when patients with longstanding persistent af are included, they often represent only a small percentage of the sample. therefore, this study was designed to provide 1year results from a staged hybrid procedure in patients with longstanding persistent af using a combined endoscopic surgical radiofrequency (rf) ablation of the la with occlusion of laa, followed by an electrophysiological diagnostic study and a rf catheter ablation of the left and right atrium (ra) (primary endpoint). the catheter ablation took place 6 to 8 weeks after the surgical phase to allow healing and maturation of the surgical ablation sites. secondary end points included overall arrhythmiafree survival during the entire followup period, prevalence of transmural linear and circumferential lines after the surgical part of the hybrid procedure, and overall safety of both surgical and endocardial ablation procedures. patients with symptomatic drugrefractory, longstanding persistent af were included in an unblinded registry and followed prospectively. the definitions of paroxysmal, persistent, and longstanding persistent af were based on recent guidelines. all patients underwent the surgical part of the hybrid arrhythmia treatment, which followed a standardized protocol. six to 8 weeks later, patients were admitted for the transvenous catheter part of the procedure. to be recruited to the registry, patients had to have la dilation > 45 mm. exclusion criteria were the presence of severe coronary artery disease (> 60% stenosis of the main coronary arteries), moderate or severe heart valve disease, a history of stroke, previous catheter ablation, pulmonary surgery or cardiac surgery, inability to use anticoagulant drugs, or the presence of a thrombus in the laa. all patients underwent transesophageal echocardiography, cardiac computed tomography with contrast, and pulmonary function tests. patients older than 50 years or with symptoms of angina pectoris also underwent coronary angiography. transesophageal echocardiography and computed tomography scans were both repeated in all patients prior to rf catheter ablation ; of particular interest was any pv narrowing (defined as 20% to 50% reduction in the diameter of the vein with detected maximum pulsedwave doppler velocity at the orifice of the vein 90%. transmurality of ablation lines is considered to be of utmost importance for successful treatment of af according to the heart rhythm society (hrs)/european heart rhythm association (ehra)/european cardiac arrhythmia society (ecas) consensus. for the first time we were able to analyze data on the recurrence of electrical reconduction of previously epicardially ablated lines. despite direct visualization of the epicardial rf lesions, and despite the use of multiple burns during surgery (usually six to eight burns on each pair of pvs), acutely observed electrical isolation turned out to be only temporary in a significant proportion of patients, which was most likely due to tissue stunning and edema. endocardial procedures may be associated with a high number of falsenegative results (demonstration of entry or exit blocks that are only transient and do not endure longterm) and also falsepositive results in terms of early inducible arrhythmias, which usually fade away after maturation of the ablative lesions. furthermore, maturation of epicardial burns allows easier identification of boundaries of nonisolated substrate and target points during subsequent endocardial mapping. not surprisingly, linear ablation lines especially represented the weakest point of the epicardial approach. one possible explanation is the large amount of epicardial fat tissue in the areas of the ablation lines, which can dissipate rf energy. the use of different instruments for the creation of lines (linear pen for lines versus ablation clamps used for pvs) could also have contributed to the problems. there are no comparable data published in the literature on systematic delayed endocardial mapping in patients both with and without arrhythmia recurrence, with regard to completeness of linear lesions, after surgery. during the acute period, pison described the need for an endocardial touchup, to complete boxlesions during a simultaneous hybrid approach, in 36% of patients. completeness of lines and/or pv isolation is not directly related to the appearance of arrhythmias over the course of time. there is a lack of relevant data in the literature, especially postsurgery, and one can only speculate about the relationship between completeness of linear lesions and actual incidence of recurring arrhythmias. presence or absence of complete lines after surgery does not seem to affect acute arrhythmia recurrence in our study, since a similar proportion of patients with or without arrhythmia showed gaps in the surgical ablation lines (table 3). however, based on the knowledge gained from catheter ablation studies, any incomplete lines or pv reconduction could potentially hamper the longterm results of the procedure. it is also important to stress that the completion of both pv isolation and connecting linear lines was extremely easy in all patients thanks to the epicardial burns, which could be readily identified using bipolar voltage maps. apart from assessing chronic conduction properties of epicardial lesions, other potential advantages of a 2stage procedure may include (1) lower overall procedural time and consequently lower general anesthesia time, (2) fewer bleeding and infection complications, (3) the possibility of using a dedicated operating theater and an advanced threedimensional mapping system for targeting induced or ongoing ats, and (4) advantageous reimbursement in some countries, as well as other logistic benefits. although the benefits of a 2stage hybrid approach, as a treatment modality, are easy to imagine, its ultimate value and utility should be tested in randomized multicenter trials. serious complications from catheter ablations of af, in a highvolume center like ours, are extremely rare. in our series, we did not record any major or minor complications related to the endocardial procedure. however, there were 5 cases of phrenic nerve injury associated with the surgical part of the hybrid procedure. the first is a proximityrelated mechanical injury to the phrenic nerve during opening of the pericardium. after placing pericardial stitches, to prevent the pericardium from obstructing the surgeon 's view, manipulation with endoscopic tools while creating the inferior line can easily stretch or tear the pericardium, resulting in damage to the phrenic nerve. there is evidence that if the phrenic nerve is only partly damaged, the potential for healing is around 70% after 1 year and 90% after 2 years. in our series, onesided injury of the phrenic nerve with limited / no movement of the diaphragm does not necessarily lead to the sensation of dyspnea. therefore, such a complication is most likely underestimated in surgical literature since consistent followup examinations aimed at accessing diaphragm movement, using skiascopy, are usually missing. similarly, there is no data in the surgical literature that examines pv narrowing or stenosis after epicardial pv isolation. nonsignificant pv narrowings found in our study were most likely caused by overly distal clamping of the pvs during rf ablation. two conversions to openheart surgery, due to bleeding from the pulmonary artery, occurred during the early stages of our hybrid program, and 2 infectious complications were most likely related to extreme obesity and concomitant diabetes. one recorded pericardial bleed was directly caused by an overdose of anticoagulant drugs, and the complication was not deemed directly related to the surgery itself. the main limitation of the trial was that we did not include a control group of patients, in whom catheter ablation was not carried out after surgery. it might have happened that if catheter ablation was performed only in patients with arrhythmia recurrence, the overall clinical results would be similar and patients would not be exposed to an additional, albeit very low, risk of invasive procedure. therefore, it still remains to be elucidated whether endocardial ablation aimed at completing epicardial surgical ablation lines should be performed routinely in all patients. hybrid procedures have been recently proposed as a method to overcome the shortcomings of both surgical and catheterbased ablation procedures alone. currently, thoracoscopic minimally invasive surgery uses predominately bipolar rf energy. in a recent metaanalysis by gelsomino, bipolar rf energy was found to yield better results in terms of freedom from af. published results range from 85.7% to 92% in studies employing bipolar rf and from 36.8% to 88.9% in those utilizing monopolar rf. however, studies published to date, on the hybrid approach using bipolar rf energy, have included rather diverse patient groups, small samples, and extremely heterogeneous methodologies. a study by mahapatra included only 15 patients with persistent af, who were unsuccessfully treated, despite having a complex history, that is, after at least 1 catheter ablation and treatment with 1 aa drug. the authors performed an epicardial pv isolation, roof line, trigone line, superior vena cava isolation, laa amputation, and ablation of the ganglionated plexis. after 3 to 5 days, all patients were brought to the electrophysiological lab for cavotricuspid isthmus and coronary sinus ablation. in contrast to our study, which included all patients, be they in the sr or in any supraventricular arrhythmia, mahapatra checked pv isolation and completeness of the la linear lines only in patients with inducible arrhythmias, in whom they also tried to ablate regular ats or complex fractionated ecgs. the authors reported a success rate of 86.7% and 93.3%, off and on aa drugs, respectively, during a mean followup of 20.74.5 months. krul investigated 31 patients : 16 with paroxysmal, 13 with persistent, and 2 with longstanding persistent af. using a variation of the hybrid methodology, they performed epi and endocardial ablations, which were carried out simultaneously. pv isolation and ganglionated plexi ablation were performed in all patients, while roof and inferior lines were ablated in 13 and 8 patients, respectively. pison studied a cohort of 78 consecutive patients, again with a number of disease variations (29 with paroxysmal, 34 with persistent, and 15 with longstanding persistent af), who underwent a simultaneous epicardial and endocardial ablation. surgeons created lines around the pvs and a boxlesion on the posterior la, while electrophysiologists ascertained entry and exit blocks in the isolated regions and, if necessary, closed conduction gaps using cooltip rf ablation. in cases where af transitioned to la flutter, a mitral isthmus line (extending from left inferior pv toward the mitral annulus) was added from both the endo and epicardial sides. a cavotricuspid isthmus ablation was performed in patients with a history of typical ra flutter or whenever it was observed during the procedure. a superior vena cava ablation and a superior vena cava to inferior vena cava line were created, but only in patients with persistent and longstanding persistent af with a dilated ra. after an additional catheter ablation procedure in 10 patients, the success rate increased to 87% and 92% of patients, off and on aa drugs, respectively. in contrast to the abovementioned studies, our study followed a welldefined surgical protocol in a homogeneous group of patients with longstanding persistent af, who were naive to previous catheter ablations or surgery. the success rate of a staged hybrid approach using our protocol was astonishingly high. interestingly, the majority of recurrences occurred during the first 3 months after the procedure and midterm results seem to be promising and very stable, with no late recurrences (figure 4). finally, only 3 patients in our cohort were unable to maintain a stable sr : one patient remained in permanent af and repeated electrical cardioversions failed despite successful completion of the hybrid procedure and therapy with amiodarone. an extremely dilated la (la volume=210 ml !) with extensive fibrotic remodeling was considered to be the reason for failure. one at was confined to the laa ; however, ablating the arrhythmia would have probably led to complete electrical isolation of the unclipped laa and a consequent prothrombogenic state. therefore, an rf ablation was considered illadvised. the third patient, despite repeated ablations, suffered from multireentry ats, which were all confined to the ra. in 1 of our patients with successful catheter reablation, af was also maintained by a rotor in the laa, which was not excluded during the surgical part of the procedure. these data confirm the observation by di biase that, in up to 27% of redo cases, af is triggered or maintained by the laa and therefore excision or clipping of the laa (which causes its electrical isolation) may potentially eliminate this mechanism of arrhythmia recurrence. if the laa had been excluded in all patients, 2 of the failures would have most likely never occurred. we can only speculate that systematic exclusion of all laas would have also eliminated the paroxysms of af in 2 other patients in our study group, since 2 of 4 patients with a recurrence of paroxysmal af had no laa clip deployed. despite the fact that laa exclusion was an integral part of our surgical protocol wherever feasible and safe, the reason was that in the first 21 patients, we used firstgeneration atriclip devices, which did not include a distal device articulation as part of deployment, which led to implantation failures in a significant proportion of patients. in the remaining 30 patients, we utilized secondgeneration atriclips (atriclip pro), which raised our laa exclusion rate to > 90%. transmurality of ablation lines is considered to be of utmost importance for successful treatment of af according to the heart rhythm society (hrs)/european heart rhythm association (ehra)/european cardiac arrhythmia society (ecas) consensus. for the first time we were able to analyze data on the recurrence of electrical reconduction of previously epicardially ablated lines. despite direct visualization of the epicardial rf lesions, and despite the use of multiple burns during surgery (usually six to eight burns on each pair of pvs), acutely observed electrical isolation turned out to be only temporary in a significant proportion of patients, which was most likely due to tissue stunning and edema. endocardial procedures may be associated with a high number of falsenegative results (demonstration of entry or exit blocks that are only transient and do not endure longterm) and also falsepositive results in terms of early inducible arrhythmias, which usually fade away after maturation of the ablative lesions. furthermore, maturation of epicardial burns allows easier identification of boundaries of nonisolated substrate and target points during subsequent endocardial mapping. not surprisingly, linear ablation lines especially represented the weakest point of the epicardial approach. one possible explanation is the large amount of epicardial fat tissue in the areas of the ablation lines, which can dissipate rf energy. the use of different instruments for the creation of lines (linear pen for lines versus ablation clamps used for pvs) could also have contributed to the problems. there are no comparable data published in the literature on systematic delayed endocardial mapping in patients both with and without arrhythmia recurrence, with regard to completeness of linear lesions, after surgery. during the acute period, pison described the need for an endocardial touchup, to complete boxlesions during a simultaneous hybrid approach, in 36% of patients. completeness of lines and/or pv isolation is not directly related to the appearance of arrhythmias over the course of time. there is a lack of relevant data in the literature, especially postsurgery, and one can only speculate about the relationship between completeness of linear lesions and actual incidence of recurring arrhythmias. presence or absence of complete lines after surgery does not seem to affect acute arrhythmia recurrence in our study, since a similar proportion of patients with or without arrhythmia showed gaps in the surgical ablation lines (table 3). however, based on the knowledge gained from catheter ablation studies, any incomplete lines or pv reconduction could potentially hamper the longterm results of the procedure. it is also important to stress that the completion of both pv isolation and connecting linear lines was extremely easy in all patients thanks to the epicardial burns, which could be readily identified using bipolar voltage maps. apart from assessing chronic conduction properties of epicardial lesions, other potential advantages of a 2stage procedure may include (1) lower overall procedural time and consequently lower general anesthesia time, (2) fewer bleeding and infection complications, (3) the possibility of using a dedicated operating theater and an advanced threedimensional mapping system for targeting induced or ongoing ats, and (4) advantageous reimbursement in some countries, as well as other logistic benefits. although the benefits of a 2stage hybrid approach, as a treatment modality, are easy to imagine, its ultimate value and utility should be tested in randomized multicenter trials. serious complications from catheter ablations of af, in a highvolume center like ours, are extremely rare. in our series, we did not record any major or minor complications related to the endocardial procedure. however, there were 5 cases of phrenic nerve injury associated with the surgical part of the hybrid procedure. the first is a proximityrelated mechanical injury to the phrenic nerve during opening of the pericardium. after placing pericardial stitches, to prevent the pericardium from obstructing the surgeon 's view, manipulation with endoscopic tools while creating the inferior line can easily stretch or tear the pericardium, resulting in damage to the phrenic nerve. there is evidence that if the phrenic nerve is only partly damaged, the potential for healing is around 70% after 1 year and 90% after 2 years. in our series, onesided injury of the phrenic nerve with limited / no movement of the diaphragm does not necessarily lead to the sensation of dyspnea. therefore, such a complication is most likely underestimated in surgical literature since consistent followup examinations aimed at accessing diaphragm movement, using skiascopy, are usually missing. similarly, there is no data in the surgical literature that examines pv narrowing or stenosis after epicardial pv isolation. nonsignificant pv narrowings found in our study were most likely caused by overly distal clamping of the pvs during rf ablation. two conversions to openheart surgery, due to bleeding from the pulmonary artery, occurred during the early stages of our hybrid program, and 2 infectious complications were most likely related to extreme obesity and concomitant diabetes. one recorded pericardial bleed was directly caused by an overdose of anticoagulant drugs, and the complication was not deemed directly related to the surgery itself. the main limitation of the trial was that we did not include a control group of patients, in whom catheter ablation was not carried out after surgery. it might have happened that if catheter ablation was performed only in patients with arrhythmia recurrence, the overall clinical results would be similar and patients would not be exposed to an additional, albeit very low, risk of invasive procedure. therefore, it still remains to be elucidated whether endocardial ablation aimed at completing epicardial surgical ablation lines should be performed routinely in all patients. hybrid thoracoscopic and transvenous catheter ablation of longstanding persistent af shows extremely encouraging midterm results, which may represent an important and possibly more effective alternative to repeated rf catheter ablation therapy. complications during the surgical part of the procedure are mostly related to the learning curve and probably should be expected on introduction of the surgical component during development of, and transition to, a hybrid af treatment protocol. single thoracoscopic rf ablations fail to ensure permanent and durable transmural lesions in the la. of the two hybrid approaches, the staged hybrid approach offers several advantages over the simultaneous hybrid procedure. randomized multicenter clinical trials with longterm followup are needed to confirm the superiority of staged hybrid procedures over simultaneous procedures and over repeated catheter ablations using currently available novel technologies. | backgroundcatheter ablation of persistent atrial fibrillation yields an unsatisfactorily high number of failures. the hybrid approach has recently emerged as a technique that overcomes the limitations of both surgical and catheter procedures alone.methods and resultswe investigated the sequential (staged) hybrid method, which consists of a surgical thoracoscopic radiofrequency ablation procedure followed by radiofrequency catheter ablation 6 to 8 weeks later using the carto 3 mapping system. fifty consecutive patients (mean age 627 years, 32 males) with longstanding persistent atrial fibrillation (4134 months) and a dilated left atrium (> 45 mm) were included and prospectively followed in an unblinded registry. during the electrophysiological part of the study, all 4 pulmonary veins were found to be isolated in 36 (72%) patients and a complete boxlesion was confirmed in 14 (28%) patients. all gaps were successfully reablated. twelve months after the completed hybrid ablation, 47 patients (94%) were in normal sinus rhythm (4 patients with paroxysmal atrial fibrillation required propafenone and 1 patient underwent a redo catheter procedure). the majority of arrhythmias recurred during the first 3 months. beyond 12 months, there were no arrhythmia recurrences detected. the surgical part of the procedure was complicated by 7 (13.7%) major complications, while no serious adverse events were recorded during the radiofrequency catheter part of the procedure.conclusionsthe staged hybrid epicardial endocardial treatment of longstanding persistent atrial fibrillation seems to be extremely effective in maintenance of normal sinus rhythm compared to radiofrequency catheter or surgical ablation alone. epicardial ablation alone can not guarantee durable transmural lesions.clinical trial registrationurl : www.ablace.cz unique identifier : cz060520121617 |
olfaction contributes to the finding of food, hosts, reproductive partners, in the making of developmental decisions, and to the evasion from predators. studies into the olfactory system of the model nematode caenorhabditis elegans have yielded detailed descriptions of the molecular and cellular pathways that subserve the olfactory system [14 ]. these signaling pathways appear highly conserved across very divergent nematode species, and the sensory neurons have clear anatomical orthologs in distantly related nematodes. within the olfactory system of c. elegans, the ability to detect dilute volatile odors is mostly conferred by three pairs of neurons termed the amphid wing cells type a (awa), amphid wing cells type b (awb), and the amphid wing cells type c (awc) [3, 4 ]. these cells are primary sensory neurons located within the sensory amphid organ of the head that forms part of an anatomically distinct subclass of amphid neurons in that they do not extend processes through the amphid opening, but instead their distal ciliated endings terminate inside a glial sheath cell. here we describe a composite odor genetic network in c. elegans that encompasses all three pairs of volatile odor - detecting neurons. we used this network to identify orthologous genes of the odor network in the nematode pristionchus pacificus. we chose the nematode p. pacificus based upon three criteria : (1) it exhibits a similar lifestyle to that of c. elegans, in that they are both self - fertilizing hermaphrodites that will feed on bacteria, (2) conservation of the olfactory signaling pathway of p. pacificus to c. elegans has been validated experimentally [7, 8 ], and (3) because it is sufficiently divergent to generate meaningful divergence data (estimated divergence between caenorhabditis and pristionchus is between 280 and 430 million years ago [9, 10 ]). one of our goals in this study was to compare selective pressures across the nodes of the odor network by comparing substitutions at silent sites to that of nonsilent sites ; this analysis is best done using more divergent sequences. using this nematode odor network we searched for relationships between pathway position with divergence, diversity, and contribution across the network. then, we designed an interaction map of the network and investigated relationships between various metrics of interaction with molecular evolution, and contribution across the network. our data is not a definitive map of odor signaling in c. elegans but represents a snap shot of current data, and by uncovering robust associations between network topology, evolution, and function we may ultimately design a framework that facilitates a level of predictive power over novel nodes within the network. pristionchus pacificus orthologs were located by cross - referencing matches using the orthology databases : inparanoid_7, orthomcl database, and the oma orthology matrix browser. for each node in our network wormbase (version ws231) has defined a curated ortholog using wormbase - compara ; however, we corroborated these predictions using reciprocal blast searches, and, by inferring relationships by reconstructing phylogenetic trees (see figures s1s7 in supplementary materials available online at doi:10.1155/2012/548081), we outline all the evidence for orthology in table 1. orthologs were aligned using the multiple sequence alignment software muscle v3.6, and gaps were systematically stripped from all sequences after alignment, and phylogenies inferred using phyml. to determine orthologs, wormbase - compara uses the databases treefam [http://www.treefam.org/ ], inparanoid_7, kogs [http://www.ncbi.nlm.nih.gov/cog/ ], oma. the genes used in our network (table 1), and their corresponding ortholog in p. pacificus (wormbase identifier for p. pacificus is denoted ppaxxx), were goa-1 (go subunit protein : ppa - goa1) ; egl-30 (gq subunit protein : ppa - egl30) ; dgk-1 (dgk, diacylglycerol kinase theta : ppa - dgk1) ; eat-4 (vesicular glutamate transporter : ppa - eat4) ; egl-4/pkg-1 (protein kinase g : ppa - pkg1) ; tax-6 (calcineurin type a : ppa - tax6) ; odr-1 (receptor guanylyl cyclase : ppa17708) ; daf-11 (receptor guanylyl cyclase : ppa - daf11) ; tax-2 (cyclic nucleotide - gated channel subunit : ppa - tax2) ; tax-4 (cyclic nucleotide - gated channel subunit : ppa - tax4) ; odr-3 (g - protein subunit : ppa - odr3) ; gpa-3 (g - protein subunit : ppa - gpa3) ; gpa-5 (g protein subunit : ppa10789) ; gpa-13 (g - protein subunit : ppa - gpa13) ; arr-1 (arrestin : ppa - arr1) ; rgs-3 (regulator of g - protein signaling : ppa - rgs3). to detect c. elegans versus p. pacificus 1 : 1 candidate orthologs for the randomization study we selected orthologs from the orthology database, inparanoid_7. we only included orthologs that are represented by 100% bootstrap support, and from this approach we obtain 5,666 1 : 1 orthologs. synonymous (ds) and nonsynonymous (dn) substitution rates for orthologs were estimated using the methods of yang and nielsen as implemented in yn00 in the paml suite. to test the null hypothesis that there is no above average selective pressure on these genes, we performed a randomization test where we determined the average dn / ds value for 50,000 randomly assembled networks and compared with the average dn / ds value for our odor network. the network was treated as undirected and all network analyses available through cytoscape version 2.8.2 were examined ; these are average shortest path length, betweenness centrality, closeness centrality, clustering coefficient, degree, eccentricity, neighborhood connectivity, radiality, stress, and topological coefficient. the degree (k) of a node n is defined as the number of edges linked to n. the clustering coefficient (cn) reveals how connected the neighborhood of a node is by calculating the fraction of neighboring pairs, and for a node n it is defined as (1)cn=2enkn(kn1), where kn is the degree of n and en is the number of connected pairs between all neighbors of n. betweenness centrality (cb) is a measure of the fraction of shortest paths between node pairs (s, t) that pass through the node of interest, and for the node n it is calculated using the following formula : (2)cb(n)=sntst(n)st, where s and t are nodes different from n, st denotes the number of shortest paths from s to t, and st(n) is the number of shortest paths from s to t on which n lies. all chemotaxis indices (for both wildtype and mutant animals) were mined from previous publications (see references below). the chemotaxis index difference (c.i.diff) for each mutant within each neuron (awa, awb, and awc) was determined by calculating the difference between the wildtype chemotaxis index (c.i.wt) and mutant chemotaxis index (c.i.mut). (c.i.wt-awa) of 0.9, would have a c.i.diff-awa = 0.4 (i.e., subtract 0.5 from 0.9). in the case of long - term adaptation (lta) wildtype odortaxis : 0.9 (c.i.wt awa) ; 0.95 (c.i.wt awb) ; 0.85 (c.i.wt awc) [4, 23 ]. wildtype long - term adaptation : 0.5 (c.i.diff awa) ; 0.65 (c.i.diff awc) [24, 25 ]. odr-3 : 0.6 (c.i.diff awa) ; 0.7 (c.i.diff awb) ; 0.5 (c.i.diff awc) [23, 26 ]. egl-30 : 0.65 (c.i.diff awc).goa-1 : 0.6 (c.i.diff awa) ; 0.6 (c.i.diff awc). odr-1 : 0 (c.i.diff awa) ; 0.85 (c.i.diff awc) [31, 32 ]. tax-2 : 0.2 (c.i.diff awa) ; 0.8 (c.i.diff awb), 0.75 (c.i.diff awc) [23, 33 ]. tax-4 : 0.5 (c.i.diff awa) ; 0.75 (c.i.diff awc) [23, 34 ]. egl-4 : 0.8 (c.i.diff awa) ; 0.65 (c.i.diff awc) [25, 34 ]. the phenotype index (pi) was then calculated as follows : (3)p.i.=(c.i.diff - mutx - awaciwt - awa)2+(c.i.diff - mutx - awbciwt - awb)2+(c.i.diff - mutx - awcciwt - awc)2. the pi for lta mutants was measured by calculating the relative contribution of the lta mutant to the adaptation response. for example, if the lta response of wildtype animals normally decreases the c.i. value to 0.3 from 0.9, then the c.i.diff = 0.6 ; if the mutant is fully required (i.e., there is no lta response at all in the mutant) for the lta response, then the pi is calculated as follows : 0.6/0.6 = 1 ; which reflects the total contribution of the gene product to lta in wildtype animals but that which is defective in the lta mutant. the weighted phenotype index (wpi) was calculated for each group of mutants as follows : (4)i=1n(p.i.mut1)(wmut1)(wmut1), where p.i.mut is the phenotype index (p.i.) for the mutant x (mut), and wmut represents the number of odors for which mutant x (mut) exhibits a mutant phenotype. to compare divergence rate with pathway position in our network of dynamic odor transduction, we identified orthologous genes of the c. elegans odor network in p. pacificus, and divided the odor - signaling pathway members (figure 1(a)) within this network into 3 categories : (i) regulators class 1 (gpa-3 [35, 36 ], odr-3, gpa-5 [35, 36 ], gpa-13 [35, 36 ], arr-1 and rgs-3) ; (ii) regulators class 2 (egl-4, tax-6, odr-1, daf-11, tax-2, and tax-4) ; (iii) actuators (goa-1, egl-30, dgk-1, and eat-4). after binning pathway members into each category, we then calculated divergence within each group by comparing substitutions at silent sites to that of nonsilent sites and looked for patterns of correlation between topology and evolutionary rate. we did not observe a significant association between pathway position and divergence (pathway position : spearman rank - order correlation coefficient rs = 0.37, p = 0.15, kendall 's = 0.31, p = 0.13). we also designed an orthologous intragenus network using caenorhabditis japonica and compared substitutions at silent sites to that of non - silent sites and looked for a pattern of correlation with pathway position. using this intragenus network we also did not observe significant association between pathway position and divergence (pathway position : rs = 0.28, p = 0.3, kendall 's = 0.17, p = 0.4), suggesting that the pathway position alone may not shape constraint in our odor network. next, we designed an interaction map of the network and tested for correlations between divergence and various topological metrics (figure 1(b)). the interaction - based odor network is a composite map of the pathways within the three pairs of neurons that subserve volatile odor recognition in c. elegans (these cells are the awa, awb, and awc). edges may represent interactions from only one of the three odor sensing pairs of neurons, but placed in a composite framework of all three pairs of odor - sensing neurons to obtain an informative number of vertices. the network comprised 16 nodes with a network diameter of 5, a characteristic path length of 2.825, and an average number of neighbors equal to 2. for the interaction network we observed that a strong correlation between number of neighbors and average neighborhood connectivity (correlation = 0.857 ; coefficient of determination r = 0.648). plotting the number of neighbors against average clustering coefficient yields a strong pattern of correlation using a linear function (correlation = 0.868 ; coefficient of determination r = 0.753) ; we observed that a decrease in clustering coefficient with an increase in number of neighbors as is typical of many networks. network analysis was performed and the output of the analyses was tested for correlations with divergence by calculating the evolutionary rate for each node by comparing substitutions at silent sites to that of non - silent sites with the orthologous genes of the odor network in p. pacificus. we observed a significant association in two cases : (1) measures of betweenness centrality and (2) measures of degree. by comparing measures of betweenness or measures of degree with divergence we observed significant negative correlations (betweenness : rs = 0.48, p = 0.02, kendall 's = 0.42, p = 0.03 ; degree : rs = 0.57, p = 0.01, kendall 's = 0.44, p = 0.03). comparing measures of degree to measures of betweenness we observed a strong correlation (correlation = 0.936 ; r = 0.877) ; this is typical of networks containing nodes of high influence that increase their influence with increases in their number of edges and is not specific for our odor network. then, we calculated molecular diversity across the interaction network and searched for patterns of association with metrics from the following characteristics of the network : pathway position, betweenness centrality, and degree. in each case we did not observe a significant pattern of correlation (betweenness : rs = 0.35, p = 0.06, kendall 's = 0.3, p = 0.13 ; pathway position : rs = 0.4, p = 0.08, kendall 's = 0.37, p = 0.06 ; degree : rs = 0.47, p = 0.06, kendall 's = 0.38, p = 0.06). next, we developed a metric of contribution within the network we termed the weighted phenotype index (wpi), and compared the total contribution of each group with divergence when organized by pathway position, betweenness, or degree. in the case of pathway position we observed a weak correlation between divergence and contribution (r = 0.44), and in the case of betweenness or degree we observed strong negative correlations with contribution and divergence (figure 2(a) betweenness : r = 0.92 ; figure 2(b) degree : r = 0.76). we also examined the correlation between quantities of betweenness or degree for each node in our network with contribution, and again we observed robust correlations (figure 2(a) betweenness : r = 0.9 ; figure 2(b) degree : r = 0.79). from this analysis we uncovered patterns of association between measures of betweenness, and degree with evolutionary rate and contribution. the most highly connected nodes within our network are that of odr-3 and egl-4 (also called pkg-1). egl-4 is a protein kinase g that facilitates both primary signal transduction as well as desensitization responses within the network [25, 34 ]. to examine how molecular diversity varies across these hub vertices, we performed a sliding window examination of the coding region of each gene using 100-base - pair windows in increments of 20 base pairs. in the case of egl-4, we observed only a few polymorphic peaks, with each peak associating with areas between domains of functional importance (figure 3(a)) ; these are the n - terminal low - complexity domain (lc), the coiled - coil domain (cc), two cgmp - binding domains (cnmp), and the serine / threonine kinase domain (s / t k). in the case of odr-3, we observed more variability overall compared with that of egl-4, but again with most variable peaks associating with areas between domains of functional significance (figure 3(b)) ; in particular, the five alpha helices (g1g5) that comprise the gtpase domain, the receptor interaction c - terminal (ct) as well as the fatty acid modification site (m) (figure 3(b)). we observed similar scales for nucleotide diversity for both egl-4 (= 0.27) and odr-3 (= 0.25) with each gene exhibiting polymorphic peaks in areas between domains of functional importance. the similar pattern of diversity is in keeping with similar trends of divergence between egl-4 and odr-3 : egl-4 dn / ds = 0.0041, dn = 0.1183, ds = 29.1777 ; odr-3 dn / ds = 0.0054, dn = 0.1132, and ds = 21.1204. overall, nodes within the odor network are undergoing purifying selection, with an average dn / ds value across the network of 0.012 for p. pacificus versus c. elegans. to place this value in a context of global divergence rates between c. elegans and p. pacificus we examined divergence across 5,666 pairs of 1 : 1 orthologs, then generated randomized data sets (50,000 in total) comprising an equal number of nodes as our odor network (i.e., 16), and examined the frequency of mean dn / ds values in each case (figure 4, blue bars). from this analysis we found an average dn / ds value = 0.14 which is more than an order of magnitude higher (~11.6x) than what we observed for our dynamic odor network. to control for the number of g subunit proteins within our odor network we also conducted a randomization trial whereby dn / ds averages from 50,000 random data sets of 16 nodes that contain 6 g subunit genes in each case were generated (figure 4, red bars). in this control experiment we still observed a significantly higher average dn / ds value (dn / ds average = 0.106) than for our odor network. taken together, this suggests that the dynamic odor - signaling network is shaped by a more pronounced purifying selection than what guides global constraint across the genome. positional rate variation (prv) has been tested within numerous biosynthetic pathways where it has been demonstrated that upstream genes (perhaps on account of greater pleiotropy) undergo more selective constraint than downstream genes [4244 ]. however, it is unlikely that this effect holds true for all signaling pathways as input and network architecture will vary widely. in the case of sensory signaling pathways that subserve an environment versus perception arms race, the idea of upstream genes exhibiting greater diversity may be more applicable. this trend was observed in a study of the drosophila innate immune system where the authors demonstrated that recognition genes undergo higher levels of positive selection than immune effector genes. in our network we did not observe a significant correlation between pathway position and divergence, which was in keeping with previous observations from data on intragenus networks of odor signaling in caenorhabditis. however, by comparing measures of degree or centrality with divergence, we did observe significant negative correlations. this may suggest that pathway position is not the only factor in shaping constraint or change in our network, but rather network characteristics such as connectivity may play major roles in guiding molecular evolution. negative correlations between divergence with node relevance, and positive correlations between essentiality with node relevance have been reported previously and may represent a general trend of many biological networks [47, 48 ]. by comparing global levels of divergence between c. elegans and p. pacificus we found a pervasive theme of constraint across the genome ; however, through our randomization study (figure 4) we found that global levels of constraint are an order of magnitude lower than the level of constraint across the odor network. this suggests that the odor - signaling pathway represents a fixed circuit whose network properties are preserved by strong purifying selection. low levels of divergence have been reported within caenorhabditis for members of the olfactory pathway, and previously we have found that a large component in regulators class 1 and 2 has undergone extensive nematode - specific gene duplication events, namely, the g subunit proteins and guanylyl cyclase proteins [49, 50 ]. many of these genes are either exclusively or highly expressed in primary sensory neurons [26, 28, 31, 35, 36, 51 ]. this genetic expansion facilitates multiple capacities that shape developmental and survival strategies through intercellular and intracellular processing of polymodal sensory input. many of these duplicates are present in all four major clades of the phylum nematoda suggesting that the odor - signaling network arose early in nematode evolution and has undergone neofunctionalization events that are preserved by strong functional constraint. | the relationship between biological network architectures and evolution is unclear. within the phylum nematoda olfaction represents a critical survival tool. for nematodes, olfaction contributes to multiple processes including the finding of food, hosts, and reproductive partners, making developmental decisions, and evading predators. here we examine a dynamic nematode odor genetic network to investigate how divergence, diversity, and contribution are shaped by network topology. our findings describe connectivity frameworks and characteristics that correlate with molecular evolution and contribution across the olfactory network. our data helps guide the development of a robust evolutionary description of the nematode odor network that may eventually aid in the prediction of interactive and functional qualities of novel nodes. |
next - generation whole - genome and exome - sequenced cancer samples were previously sequenced by members of the international cancer genome consortium, the cancer genome atlas and other centres. high - confidence somatic substitutions were obtained from these consortia or other peer - reviewed publications not related to these consortia, filtered further for potential false positive calls using dbsnp, 1000 genomes, the nhlbi go exome sequencing project, the 69 complete genomics panel as well as a bespoke panel of bam files of unmatched normal tissues containing more than 120 normal genomes and 500 exomes. these data were then parsed through an algorithm previously developed to extract mutational signatures in human cancers called non - negative matrix factorisation (nnmf). six main substitution classes (c > a : g > t, c > g : g > c, c > t : g > a, t > a : a > t, t > c : a > g and t > g : a > c) were subdivided according to the 5 prime and 3 prime flanking sequence context. since there are six classes of base substitution and 16 possible sequence contexts for each mutated base (a, c, g or t at the 5 base and a, c, g or t at the 3 base), there are 96 possible mutated trinucleotides for each cancer. herewith, the convention for describing a mutated trinucleotide will be xpcpx, where x can be any base and the mutated base is underlined. the commonest signatures were signature 1a and signature 1b, both characterized by c > t mutations at a xpcpg trinucleotide (supplementary figure 1a), and signature 2 and signature 13, characterized by dominant c > t transitions at a tpcpx sequence context in signature 2 and c > g transversions at a tpcpx sequence context in signature 13 (supplementary figure 1a - b). signatures 1a/1b are likely to be caused by deamination at methylated cpgs whereas signatures 2/13 are thought to be due to the apobec family of cytidine deaminases. therefore, for the purposes of this analysis, signatures 2 and 13 are considered together. nnmf is able to estimate the number of mutations associated with extracted mutation signatures for individual cancers in a given set of samples (summarized in supplementary table 1b, figure 1c-1o). in this analysis, a total of 2,719 samples were previously characterized by nnmf and also had bam files available for inspection (supplementary table 1a - b). bam files were downloaded from cghub (https://cghub.ucsc.edu/) between 9 may 2013 and 26 june 2013. for the ease of tracking samples through this analysis, we have kept the naming convention attached to the cancer sample for tables and figures, even if the germline deletion polymorphism was sourced from a matched normal because the signatures of somatic mutagenesis will have been identified in the tumor samples in the first place. this is also for the purpose of continuity between publications. for samples originating from the sanger institute d denoting tumor samples and suffix b for the matched normal sample. in order to detect this deletion polymorphism from next - generation sequencing data, multiple loci within and flanking the coordinates of the deletion were sampled (supplementary figure 2a, supplementary table 2a) from bam files (overall workflow and directions to processing data in supplementary figure 2b). raw short read data had been aligned back to the reference genome (ncbi build 37) with duplicates and unmapped reads removed. externally sourced bam files were sourced from the tcga data hub https://browser.cghub.ucsc.edu/. matched normal bam files were sought for calling this deletion allele. however, a tumor bam file was used if a normal bam was not available (supplementary table 2b). samples that did not have bam files available for examination were excluded from analysis. in total, the apobec3a/3b polymorphism detection was sourced from 561 tumors (99 blca, 117 brca, 1 cesc, 19 hnsc, 2 kirp, 303 luad, 12 stad, 2 thca and 6 ucec) and 2158 normals. for samples with whole genome data, the expected sequencing depth in the absence of the deletion polymorphism, i.e. wild - type copy number (cn) of 2, d2, the expected depth in the presence of a heterozygous deletion allele, d1, is then given by d1 = d2 / 2 and the expected depth in the presence of a homozygous deletion allele, d0, was set to the expected number of misreads, estimated as d2/20. a maximum likelihood test was performed to identify the most likely cn from the set, with corresponding expected depths represented as poisson distributions { pois(d0), pois(d1), pois(d2) }, given the observed sequencing depths within the region of the deletion polymorphism (supplementary dataset 1). for exome - sequenced samples, an expectation - maximisation algorithm was utilized, with the copy number (cn) of each sample and the ratio of sequencing depth within and outside the deletion polymorphism used as latent variables. the cn of each sample was initialized as 2 and the depth ratio of loci within the deletion polymorphism region to those outside, r, was modelled non - parametrically by bootstrap resampling (n=1000). for samples with cn of 2, 1 and 0, respectively, the expected depths within the deletion polymorphism region are then given by din,2=rdoutdin,1=rdout2din,0=rdout20 at each maximisation step, the copy number of each sample was assigned as that whose distribution showed most overlap with din for that sample, after bootstrap resampling. at each expectation step, r was recalculated using bootstrap resampling of loci within just those samples classified as cn=2 in the previous maximisation step. the em algorithm was continued until no samples were reclassified from one iteration to the next, or for a maximum of 100 iterations (supplementary figure 2b, supplementary dataset 2). the results of the calling of the polymorphism status in all the samples is provided in supplementary table 2b. the reproducibility of the calling method was sought by examining the concordance between calling on the tumor and normal bam files from the same patient (supplementary note, supplementary table 2c, supplementary figure 2c) as well as concordance between genome- and exome - sequenced samples in the same patient. the dataset comprised genome - sequenced (123) as well as exome - sequenced (800) cancers. in order to perform the analyses, the rate of mutation was calculated for each cancer (rate of signature 2/13 per mb), correcting for whether the samples had been genome- or exome - sequenced. because the rates of signatures 2/13 were not normally distributed (supplementary 3a - d), a one - sided wilcoxon rank - sum test was performed to see whether carrying one copy of the deletion allele had an overall effect on the mutation rate of the signatures. we sought to include more cancer samples in order to increase the power of the analyses. there were no further available breast cancer samples with bam files ready for download, hence we sought inclusion of other cancer types that had previously been analyzed. however, it was noted that the distribution of rates of signatures 2/13 varied considerably between cancer - types (supplementary figure 3b - d) and clear outliers were present in all the cancer types skewing the distribution of mutation rates (supplementary figure 3a, d - e). some cancers were observed to have a strikingly high proportion of total mutations associated with signatures 2/13 and/or have higher rates of mutagenesis associated with this signature (supplementary figure 1c-1o, 5b). using the rate of signatures 2/13 mutagenesis, outliers were identified as patients with cancers that had a mutation rate exceeding 1.5 times the length of the interquartile range from the 75 percentile for each type of cancer. hypermutators although we do not suggest that there is an on - going biological process attached to this name. given the considerable variation of the mutation rates for different cancer tissue - types (supplementary figure 1a,1b), each cancer type was analyzed separately. a summary of the hypermutators versus non - hypermutators is provided in the supplementary table 3a. in theory, neighboring mutations could arise on either of two strands of a double - helix (supplementary figure 4a) particularly if they had arisen as independent events during different cycles of cell division. if more mutations are observed to occur on the same strand than expected by chance (supplementary figure 4b), this would imply one of two scenarios : either those neighboring mutations arose over different rounds of cell division with preferential targeting of one strand over another or they arose during a single round of cell division and potentially occurred in the same instance. we therefore sought to formally document that neighboring mutations are occurring on the same strand more often than expected or strand - coordinated mutagenesis. in order to demonstrate genome - wide strand coordination, analysis was carried out on all whole - genome sequence data for which bam files were available (supplementary table 4a). given a set of mutations, each occurring at a base of type (a, c, g, t) on the + strand, we identify all pairs of mutations and classify them as same if both mutations are of the same originating base and diff if not (i.e. first and second mutations of each pair respectively : a > x and a > x ; g > x and g > x ; c > x and c > x ; and t > x and t > x, with no prior selection for mutations at a tpc context and where x can be any base). the distance between successive pairs or intermutation distance is also calculated (supplementary figure 4c). the proportion of pairs of mutations that are expected to occur on the same base assuming randomly ordered mutations is given by pa2+pc2+pg2+pt2, where px is the fraction of mutations that occur at nucleotide x. to depict the deviation of the observed pairs of mutations found on the same strand from that of the expected pairs of mutations on the same strand, a standard forest plot was constructed (data of expected and observed same strand mutations for all 124 samples are provided in supplementary table 4a, columns b - e). for the reason of space, only a subset of samples were presented in figure 2a. because same - strand mutations were ascertained in an unbiased way from any mutation type (not restricted to just cytosine mutations at tpcs), to see whether strand - coordinated mutations were a particular feature of signatures 2/13, we sought a relationship between the degree of strand - coordination, given by the or of strand - coordination, and the fractional burden of signatures 2/13 in each cancer (supplementary figure 4d). we sought additional characteristics of the mutations in the whole - genome sequenced breast cancers that support the suggestion that mutations associated with signatures 2/13 have arisen due to the apobec family of enzymes (supplementary note, supplementary figure 4d, supplementary table 4). rna - seq derived expression data was obtained from the https://browser.cghub.ucsc.edu/ for relevant samples. in total, there were 1691 patients for whom comparable data were obtainable. expression levels for each apobec family member were standardized relative to the levels of tbp (tata - binding protein) and the relationship between the apobec3b expression levels and germline deletion allele status in these cancers (supplementary figure 5a - b, supplementary table 5a). the germline apobec3a/3b deletion polymorphism highlighted in this analysis was reported to display a strikingly differentiated worldwide distribution of allele frequencies5. the fst value (measuring population differentiation) was re - examined using the reported deletion allele frequencies and additional snp genotype data from the ceph - hgdp panel published after the cnp study. this value will depend on the way the populations are grouped, and needs to be compared with other variants of similar frequency to measure how unusual it is. we used two published grouping schemes, into five continental geographical / genetic groups (1. oceanians) or into 32 population groups, and matched snp frequencies measured as minor allele frequency to 0.1%. for the five continental groups, fst was 0.330 (97.4th percentile compared with 2,716 frequency - matched snps), and for the 32 population groups it was 0.285 (96.6th percentile compared to 2,059 frequency - matched snps). the level of population differentiation was thus higher than expected by chance, which can result from positive selection, and we therefore examined other statistics sensitive to positive selection. cross - population extended haplotype homozygosity (xp - ehh) and integrated haplotype score (ihs) values were obtained from the hgdp selection browser (http://hgdp.uchicago.edu/). these haplotype - based tests for positive selection utilise information 500 kb upstream and downstream of the deletion, and thus the two sides can be examined separately. neither side showed any significantly high xp - ehh (> 2.5) or ihs value (ihs>2.0) in any continental group or individual population. finally, we looked at allele frequency spectrum - based tests (tajima s d, fay & wu s h and nielsen.s composite likelihood ratio test) using the 1000 genomes phase 1 re - sequenced data in the east asian populations (chb, chs and jpt) (1000 g phase 1) in the regions surrounding the deletion, as described (1000 g pilot). there was no evidence for positive selection in these populations, although in this case, the power of these tests is limited because the frequency of the deletion in these populations is not high enough. however, there remains a lack of other evidence for positive selection and so we can not convincingly conclude that this deletion has been positively selected in human populations. next - generation whole - genome and exome - sequenced cancer samples were previously sequenced by members of the international cancer genome consortium, the cancer genome atlas and other centres. high - confidence somatic substitutions were obtained from these consortia or other peer - reviewed publications not related to these consortia, filtered further for potential false positive calls using dbsnp, 1000 genomes, the nhlbi go exome sequencing project, the 69 complete genomics panel as well as a bespoke panel of bam files of unmatched normal tissues containing more than 120 normal genomes and 500 exomes. these data were then parsed through an algorithm previously developed to extract mutational signatures in human cancers called non - negative matrix factorisation (nnmf). six main substitution classes (c > a : g > t, c > g : g > c, c > t : g > a, t > a : a > t, t > c : a > g and t > g : a > c) were subdivided according to the 5 prime and 3 prime flanking sequence context. since there are six classes of base substitution and 16 possible sequence contexts for each mutated base (a, c, g or t at the 5 base and a, c, g or t at the 3 base), there are 96 possible mutated trinucleotides for each cancer. herewith, the convention for describing a mutated trinucleotide will be xpcpx, where x can be any base and the mutated base is underlined. the commonest signatures were signature 1a and signature 1b, both characterized by c > t mutations at a xpcpg trinucleotide (supplementary figure 1a), and signature 2 and signature 13, characterized by dominant c > t transitions at a tpcpx sequence context in signature 2 and c > g transversions at a tpcpx sequence context in signature 13 (supplementary figure 1a - b). signatures 1a/1b are likely to be caused by deamination at methylated cpgs whereas signatures 2/13 are thought to be due to the apobec family of cytidine deaminases. therefore, for the purposes of this analysis, signatures 2 and 13 are considered together. nnmf is able to estimate the number of mutations associated with extracted mutation signatures for individual cancers in a given set of samples (summarized in supplementary table 1b, figure 1c-1o). in this analysis, a total of 2,719 samples were previously characterized by nnmf and also had bam files available for inspection (supplementary table 1a - b). bam files were downloaded from cghub (https://cghub.ucsc.edu/) between 9 may 2013 and 26 june 2013. for the ease of tracking samples through this analysis, we have kept the naming convention attached to the cancer sample for tables and figures, even if the germline deletion polymorphism was sourced from a matched normal because the signatures of somatic mutagenesis will have been identified in the tumor samples in the first place. this is also for the purpose of continuity between publications. for samples originating from the sanger institute d denoting tumor samples and suffix b for the matched normal sample. in order to detect this deletion polymorphism from next - generation sequencing data, multiple loci within and flanking the coordinates of the deletion were sampled (supplementary figure 2a, supplementary table 2a) from bam files (overall workflow and directions to processing data in supplementary figure 2b). raw short read data had been aligned back to the reference genome (ncbi build 37) with duplicates and unmapped reads removed. externally sourced bam files were sourced from the tcga data hub https://browser.cghub.ucsc.edu/. matched normal bam files were sought for calling this deletion allele. however, a tumor bam file was used if a normal bam was not available (supplementary table 2b). samples that did not have bam files available for examination were excluded from analysis. in total, the apobec3a/3b polymorphism detection was sourced from 561 tumors (99 blca, 117 brca, 1 cesc, 19 hnsc, 2 kirp, 303 luad, 12 stad, 2 thca and 6 ucec) and 2158 normals. for samples with whole genome data, the expected sequencing depth in the absence of the deletion polymorphism, i.e. wild - type copy number (cn) of 2, d2, the expected depth in the presence of a heterozygous deletion allele, d1, is then given by d1 = d2 / 2 and the expected depth in the presence of a homozygous deletion allele, d0, was set to the expected number of misreads, estimated as d2/20. a maximum likelihood test was performed to identify the most likely cn from the set, with corresponding expected depths represented as poisson distributions { pois(d0), pois(d1), pois(d2) }, given the observed sequencing depths within the region of the deletion polymorphism (supplementary dataset 1). for exome - sequenced samples, an expectation - maximisation algorithm was utilized, with the copy number (cn) of each sample and the ratio of sequencing depth within and outside the deletion polymorphism used as latent variables. the cn of each sample was initialized as 2 and the depth ratio of loci within the deletion polymorphism region to those outside, r, was modelled non - parametrically by bootstrap resampling (n=1000). for samples with cn of 2, 1 and 0, respectively, the expected depths within the deletion polymorphism region are then given by din,2=rdoutdin,1=rdout2din,0=rdout20 at each maximisation step, the copy number of each sample was assigned as that whose distribution showed most overlap with din for that sample, after bootstrap resampling. at each expectation step, r was recalculated using bootstrap resampling of loci within just those samples classified as cn=2 in the previous maximisation step. the em algorithm was continued until no samples were reclassified from one iteration to the next, or for a maximum of 100 iterations (supplementary figure 2b, supplementary dataset 2). the results of the calling of the polymorphism status in all the samples is provided in supplementary table 2b. the reproducibility of the calling method was sought by examining the concordance between calling on the tumor and normal bam files from the same patient (supplementary note, supplementary table 2c, supplementary figure 2c) as well as concordance between genome- and exome - sequenced samples in the same patient. the dataset comprised genome - sequenced (123) as well as exome - sequenced (800) cancers. in order to perform the analyses, the rate of mutation was calculated for each cancer (rate of signature 2/13 per mb), correcting for whether the samples had been genome- or exome - sequenced. because the rates of signatures 2/13 were not normally distributed (supplementary 3a - d), a one - sided wilcoxon rank - sum test was performed to see whether carrying one copy of the deletion allele had an overall effect on the mutation rate of the signatures. we sought to include more cancer samples in order to increase the power of the analyses. there were no further available breast cancer samples with bam files ready for download, hence we sought inclusion of other cancer types that had previously been analyzed. however, it was noted that the distribution of rates of signatures 2/13 varied considerably between cancer - types (supplementary figure 3b - d) and clear outliers were present in all the cancer types skewing the distribution of mutation rates (supplementary figure 3a, d - e). some cancers were observed to have a strikingly high proportion of total mutations associated with signatures 2/13 and/or have higher rates of mutagenesis associated with this signature (supplementary figure 1c-1o, 5b). using the rate of signatures 2/13 mutagenesis, outliers were identified as patients with cancers that had a mutation rate exceeding 1.5 times the length of the interquartile range from the 75 percentile for each type of cancer. hypermutators although we do not suggest that there is an on - going biological process attached to this name. given the considerable variation of the mutation rates for different cancer tissue - types (supplementary figure 1a,1b), each cancer type was analyzed separately. a summary of the hypermutators versus non - hypermutators is provided in the supplementary table 3a. in theory, neighboring mutations could arise on either of two strands of a double - helix (supplementary figure 4a) particularly if they had arisen as independent events during different cycles of cell division. if more mutations are observed to occur on the same strand than expected by chance (supplementary figure 4b), this would imply one of two scenarios : either those neighboring mutations arose over different rounds of cell division with preferential targeting of one strand over another or they arose during a single round of cell division and potentially occurred in the same instance. we therefore sought to formally document that neighboring mutations are occurring on the same strand more often than expected or strand - coordinated mutagenesis. in order to demonstrate genome - wide strand coordination, analysis was carried out on all whole - genome sequence data for which bam files were available (supplementary table 4a). given a set of mutations, each occurring at a base of type (a, c, g, t) on the + strand, we identify all pairs of mutations and classify them as same if both mutations are of the same originating base and diff if not (i.e. first and second mutations of each pair respectively : a > x and a > x ; g > x and g > x ; c > x and c > x ; and t > x and t > x, with no prior selection for mutations at a tpc context and where x can be any base). the distance between successive pairs or intermutation distance the proportion of pairs of mutations that are expected to occur on the same base assuming randomly ordered mutations is given by pa2+pc2+pg2+pt2, where px is the fraction of mutations that occur at nucleotide x. to depict the deviation of the observed pairs of mutations found on the same strand from that of the expected pairs of mutations on the same strand, a standard forest plot was constructed (data of expected and observed same strand mutations for all 124 samples are provided in supplementary table 4a, columns b - e). for the reason of space, only a subset of samples were presented in figure 2a. because same - strand mutations were ascertained in an unbiased way from any mutation type (not restricted to just cytosine mutations at tpcs), to see whether strand - coordinated mutations were a particular feature of signatures 2/13, we sought a relationship between the degree of strand - coordination, given by the or of strand - coordination, and the fractional burden of signatures 2/13 in each cancer (supplementary figure 4d). we sought additional characteristics of the mutations in the whole - genome sequenced breast cancers that support the suggestion that mutations associated with signatures 2/13 have arisen due to the apobec family of enzymes (supplementary note, supplementary figure 4d, supplementary table 4). rna - seq derived expression data was obtained from the https://browser.cghub.ucsc.edu/ for relevant samples. in total, there were 1691 patients for whom comparable data were obtainable. expression levels for each apobec family member were standardized relative to the levels of tbp (tata - binding protein) and the relationship between the apobec3b expression levels and germline deletion allele status in these cancers (supplementary figure 5a - b, supplementary table 5a). the germline apobec3a/3b deletion polymorphism highlighted in this analysis was reported to display a strikingly differentiated worldwide distribution of allele frequencies5. the fst value (measuring population differentiation) was re - examined using the reported deletion allele frequencies and additional snp genotype data from the ceph - hgdp panel published after the cnp study. this value will depend on the way the populations are grouped, and needs to be compared with other variants of similar frequency to measure how unusual it is. we used two published grouping schemes, into five continental geographical / genetic groups (1. oceanians) or into 32 population groups, and matched snp frequencies measured as minor allele frequency to 0.1%. for the five continental groups, fst was 0.330 (97.4th percentile compared with 2,716 frequency - matched snps), and for the 32 population groups it was 0.285 (96.6th percentile compared to 2,059 frequency - matched snps). the level of population differentiation was thus higher than expected by chance, which can result from positive selection, and we therefore examined other statistics sensitive to positive selection. cross - population extended haplotype homozygosity (xp - ehh) and integrated haplotype score (ihs) values were obtained from the hgdp selection browser (http://hgdp.uchicago.edu/). these haplotype - based tests for positive selection utilise information 500 kb upstream and downstream of the deletion, and neither side showed any significantly high xp - ehh (> 2.5) or ihs value (ihs>2.0) in any continental group or individual population. finally, we looked at allele frequency spectrum - based tests (tajima s d, fay & wu s h and nielsen.s composite likelihood ratio test) using the 1000 genomes phase 1 re - sequenced data in the east asian populations (chb, chs and jpt) (1000 g phase 1) in the regions surrounding the deletion, as described (1000 g pilot). there was no evidence for positive selection in these populations, although in this case, the power of these tests is limited because the frequency of the deletion in these populations is not high enough. however, there remains a lack of other evidence for positive selection and so we can not convincingly conclude that this deletion has been positively selected in human populations. | introductionthe somatic mutations in a cancer genome are the aggregate outcome of one or more mutational processes operative through the life of the cancer patient1 - 3. each mutational process leaves a characteristic mutational signature determined by the mechanisms of dna damage and repair that constitute it. a role was recently proposed for the apobec family of cytidine deaminases in generating particular genome - wide mutational signatures1,4 and a signature of localized hypermutation called kataegis1,4. a germline copy number polymorphism involving apobec3a and apobec3b, which effectively deletes apobec3b5, has been associated with a modest increased risk of breast cancer6 - 8. here, we show that breast cancers in carriers of the deletion show more mutations of the putative apobec - dependent genome - wide signatures than cancers in non - carriers. the results suggest that the apobec3a/3b germline deletion allele confers cancer susceptibility through increased activity of apobec - dependent mutational processes, although the mechanism by which this occurs remains unknown. |
brazil is a country whose population consists of several different nationalities, among them the japanese. japanese immigration occurred mainly from 1908 to 1935, and the large ethnic group living in brazil has prevented crossbreeding. as a result, japanese physical characteristics can be easily noted in japanese descendants residing in brazil. oriental eyelids have distinctive folds and contours that differentiate them from occidental eyelids due to inner anatomical relationships. the oriental eyelid shows a narrower tarsal plate, a higher level of subcutaneous fat, and a higher level of fat posterior to the orbital septum than is found in caucasian examples. the eyelid crease, when present, is located near the upper eyelid border due to the following anatomical features : the orbital septum fuses to the eyelid levator muscle aponeurosis at variable distances below superior tarsal border ; the preaponeurotic fat protrusion and its thick feature blocks the attachments of the aponeurosis to orbicular muscle and skin next to the superior tarsal border ; and the levator aponeurosis attaches to the orbicular muscle and skin next to the upper eyelid border (doxanas and anderson 1984 ; jeong 1999). in addition to this, there has been no comparison made between the eyelids measurement of native japanese and nikkeis living in other countries. there is no information available as to whether habits, climate condition, or the exposure to different environmental factors could induce variation in the eyelid and its contours. this study was constructed in order to analyze, in a quantitative form, the measurements of eyelid contours in japanese people living in japan and in nikkeis living in brazil by using digital images. a prospective observational study was performed between august 2004 and july 2005, evaluating 107 japanese descendents living in brazil (so paulo state), and 114 japanese living in japan (hamamatsu). the studied populations were required to have resided in their respective countries since birth or for more than 60 years. the protocol was approved by the ethics committee for human research at botucatu school of medicine (unesp). exclusion criteria included those with systemic diseases that could cause changes in palpebral position, those with ocular or eyelid diseases, those who had undergone palpebral surgery, those who would not authorize photographic records, and crossbreeding individuals. all images were taken by the same person using a digital camera (nikon coolpix 4100, china), with flash, positioned in the frontal plane parallel to and 30 cm from the facial plane, at pupil height with a metric scale stick on the face and looking at the camera lens. images were transferred to a computer running windows (microsoft, redmond, wa) and processed by scion image from the scion corporation (frederick, md). the following dimensions were analyzed : distance between medial canthi, distance between pupils (ipd), superior palpebral crease position (at the central part of the superior eyelid), the distance between superior palpebral margin and corneal reflex (mrd), horizontal width, height, area, and obliquity of the palpebral fissure (figure 1). data were analyzed by analysis of variance using a three factor model and respective multiple comparison tests. the distance between medial canthi tended to be larger in the japanese than in nikkeis, with men having significantly greater values than women in both groups. the ipd tended to be larger in the japanese, with men having larger ipd than women, and statistical difference for some age groups (table 2). horizontal fissure values were barely higher in the japanese, with statistical significance for japanese women from 20 to 29 years, and japanese men from 40 to 49 years. comparing individuals within age groups, japanese women over 50 years had much lower values than the rest (table 3). the smallest vertical fissure dimensions were found in the over 50 s (table 4). mrd dimensions were similar in both populations, but there was a tendency for higher values in japanese ; the only significant difference was in female nikkeis who had larger dimensions than their japanese counterparts. upper eyelid crease dimensions showed no statistical significance between group ; however, japanese women always had discretely higher values (table 6). measurements in the individuals over 50 years old were significantly smaller (table 7). comparison between age groups showed lower values as long as age advanced (table 8). the shape and characteristics of the palpebral fissure are influenced by race, as has been documented in several racial comparative studies (iosub 1985 ; leung 1990 ; kaimbo and kaimbo 1995 ; jeong 1999 ; hanada 2001). in the present study we evaluated members of the oriental race living in different localities, and who were therefore submitted to distinct exogenous factors. both populations consisted of similar numbers from both sexes and were divided into age groups to check for gender and age related differences (lam 1995 ; siqueira 2005). differences between the nikkeis and japanese were very subtle, and frequently without significantly statistical difference. distances between inner canthi and pupils tended to be higher in the japanese, and in men rather than women. for horizontal and vertical fissure, a reduction in palpebral fissure width was found mainly in women (in the group older than 50 years old) ; this has already been explored in literature (van den bosch 1999 ; siqueira 2005) and can be attributed to disinsertion and slackness of the palpebral structures which occur at individuals of advanced ages (nesi 1997). mean mrd values were higher in japanese than nikkeis in nearly all age groups, but differences were not statistically significant. as a consequence of lower vertical fissure values, mean mrd values were also lower in more elderly individuals. in relation to crease height, differences were small and variability high ; this meant that it was necessary to use medians and inter - quartile amplitude with this parameter. it is known that the oriental eyelid has a much lower superior palpebral crease position than a caucasian one, and it can even be absent due to particular anatomical differences. it should be noted that the japanese often use adhesive to artificially shape the upper eyelid crease, which can often remain even after discontinuing the treatment, and could explain their discreet larger values. discreet higher values in fissure width and height and mrd seen in japanese lead to their higher palpebral fissure areas. the oldest population had lower values for this parameter, which certainly link it with senility, when smaller vertical and horizontal palpebral fissures cause smaller palpebral fissure area. however, when analyzed apart this did not have the same tendencies as mrd or vertical and horizontal fissures. the palpebral fissure has a three - dimensional surface mainly shaped by the eyeball which pushes out and moulds both eyelid and all palpebral shape, including apexes and depressions, and the parabolic line of the superior lid, influencing the shape of the palpebral fissure (maulboisson 2000). evaluation of fissure obliquity in caucasians, japanese and descendents, and brazilian indians shows mean inclinations of 4.60, 9.39, and 9.34, respectively (hanada 2001). in this study, both groups had superior mean values (5.15 in nikkeis and 6.57 in japanese) to the caucasian population, but less than orientals from a previous study (hanada 2001). these results allow us to conclude that there are few differences in eyelids between orientals and the nikkeis who are living in brazil. environmental factors were not sufficiently strong to change the genetic load which has determined the shape of the oriental lids until the present. | objectivesquantitative evaluation of palpebral dimensions of japanese residents in japan and japanese descendant (nikkeis) who live in brazil, in order to define if environmental factors may influence these parameters.methodsa prospective study evaluating 107 nikkeis from brazil and 114 japanese residents in japan, aged 20 years or older. exclusion criteria were those with palpebral position alterations, prior palpebral surgery, and crossbreeding. images were obtained with a digital camera, 30 cm from the frontal plane at pupil height, with the individual in a primary position and the eye trained on the camera lens. images were transferred to computer and processed by the scion image program. measurements were made of distance between medial canthi, distance between pupils (ipd), superior eyelid crease position, distance between the superior lid margin and corneal reflexes (mrd), horizontal width, height, area, and obliquity of the palpebral fissure. data were analyzed using analysis of variance for a three factor model and respective multiple comparison tests.resultsjapanese residents and nikkeis living in brazil have similar measurements. statistical differences were found for some age groups concerning distance between pupils, horizontal, and vertical fissures, palpebral fissure area, and obliquity with native japanese presenting discretely higher measurements than nikkeis.conclusionenvironmental factors do not affect palpebral dimensions of nikkeis living in brazil. |
mosquitoes transmit some of the most deadly infectious diseases of humans. although malaria is the best known arthropod - transmitted viruses (arboviruses) include the causative agents of dengue, yellow fever, west nile virus, chikungunya, and many others. the life cycle of these viruses typically depends on transmission from a suitable vertebrate host via a mosquito vector to another suitable vertebrate, and so on for ever. for some of these viruses, such as dengue, humans are the only suitable vertebrate species across most or all of their range ; others, such as west nile virus, can infect a wide range of vertebrates. the mosquito is exposed to the pathogen when she (only female mosquitoes bite) takes a blood meal from an infectious vertebrate. the virus infects the mosquito, typically first in the midgut and then disseminating through the body. when the salivary glands become infected, the next time she takes a blood meal, her food source is exposed to the virus. if this individual becomes infected, for a period of time it will become infectious to other mosquitoes that bite it, and so the virus continues to propagate and spread. although insects lack the adaptive immune system of mammals, they are by no means merely passive hosts and vectors for these viruses ; rather, they have multiple innate immune defenses against the various microbial challenges they encounter. rna interference (rnai) is one of the mosquito 's major defenses against arboviruses, and suppression of this pathway has previously been shown to increase viral load in infected mosquitoes. two recent papers shed more light on the role of this system in insect antiviral innate immunity. writing in bmc microbiology, show that sindbis virus engineered to express a suppressor of rnai produces much more virus than normal in infected mosquitoes, and that this engineered virus is lethal to a range of mosquito species. previous studies used transient knockdown of components of the rnai pathway ; cirimotich. use a protein that binds to double - stranded rna (dsrna) and presumably protects it from processing in the rnai pathway. although either approach might have pleiotropic effects, both indicate a key role for the rnai pathway in reducing virus replication and titer. in this regard, in a recent paper in nature, saleh. show that drosophila can mount a systemic rnai - based response to viruses so that uninfected cells at distal locations can prepare a defense against infection. this response was shown to depend on a dsrna uptake pathway ; mutant flies defective in this pathway are hypersensitive to infection with drosophila c virus and sindbis virus. targeted rna interference against dengue virus infection. self - complementary rna with sequences from dengue virus is expressed from a promoter that expresses in the gut of the mosquito soon after a blood meal. these fragments are bound by the risc complex of proteins and one strand is removed. the risc complex is now primed to bind and cleave target sequences from an infecting dengue virus, preventing translation from the rna and replication of the virus. as well as their interest in terms of basic immunology, the mosquito 's antiviral defenses are significant from an applied perspective. if they could be artificially boosted to the point that infected mosquitoes do not themselves become infectious, mosquitoes that can not transmit a specific virus, or perhaps even a range of viruses, could be produced. antiviral rnai has already been used to confer resistance to dengue virus in transgenic mosquitoes, by expressing a hairpin rna corresponding to part of the virus. this long hairpin has the significant advantage of being relatively resistant to mutation of the virus target, as it presumably targets multiple viral sequences. constitutive expression of a large hairpin rna may be deleterious, but this potential problem was minimized by using a promoter that expresses only in the midgut the first cells to be infected and only following a blood meal. a virus - resistant strain of mosquitoes in the laboratory is, however, only a curiosity or a research tool. to have an impact on disease transmission, the virus - resistance gene(s) must spread within the vector population in the wild. for diseases such as dengue, where remarkably few competent vectors are required to sustain epidemic transmission, such a resistance gene would have to spread to a high allele frequency, so that practically all mosquitoes in the target population carried at least one copy. unfortunately, insertion and expression of a transgene imposes a fitness penalty ; this may be small, but will still tend to make the transgene decrease in frequency over time, even if a large number are initially introduced. if infection were itself highly deleterious, resistance might be a positive fitness trait, perhaps enough to cause the resistance gene to spread to fixation. but the viruses carried seem to have remarkably little negative impact on the mosquito vector. an infected mosquito does not clear so simply shortening the life expectancy of female mosquitoes is potentially an effective way to reduce transmission. a first step towards a genetic control strategy using this principle was recently achieved, using a pathogenic mutant version of the intracellular bacterium wolbachia pipiens, which reduces the lifespan of mosquitoes that carry it. if the resistance transgene will not spread through a population on its own, then further genetic tricks are needed to make it spread. natural self - spreading genetic systems include obligate bacterial endosymbionts such as wolbachia and selfish dna elements such as active transposons. however, artificial versions of self - spreading systems have proved remarkably difficult to construct, although a demonstration of spreading in drosophila of an artificial dna element based on the tribolium castaneum selfish dna system medea (maternal - effect dominant embryonic arrest) is a very promising development. can we get them to spread ? ' is important, but so is ' can we get them to stop ? ' both wolbachia and medea are extremely difficult to remove from a target population after release probably impossible in the case of wolbachia and also difficult or impossible to stop from spreading beyond the target population, perhaps even to all populations of the species worldwide. this is new territory for genetic engineering and such use or outcomes may well be controversial. however, it is not an entirely new concept analogies can be drawn with the introduction of exotic biocontrol agents, for which some of the same issues arise. (a) in population replacement strategies, the wild population is invaded by a heritable modification (e.g. transgene or pathogenic wolbachia) that reduces the vector competence of the mosquitoes that carry it. the number of competent vectors therefore declines, but the total number of (female) mosquitoes remains relatively constant, though possibly with some transient change during the invasion. (b) in contrast, a population suppression strategy aims to reduce the total number of vector mosquitoes. the two panels illustrate the changes in female population number and type over time for the two strategies. in both cases the situation will eventually reverse due to various pressures such as resistance, mutation, immigration, etc, unless some maintenance activities are undertaken. the strategy outlined above is commonly known as ' population replacement ' : a wild vector population is converted to a modified one in which the mosquitoes have reduced vectorial capacity. the other main strategy for genetic control of mosquitoes is ' population suppression '. here the objective is not to change the properties of the vector mosquitoes but to reduce their number, as in the case of the increased mortality induced by cirimotich.. this is a more familiar objective, in that it is also the aim of most source - reduction and chemical insecticide programs. the major current strategy in this area is based on the use of genetically sterile mosquitoes. in principle, large numbers of sterile male mosquitoes are released so that a wild female has a good chance of mating with a sterile male and so produces no or fewer progeny than usual. the population therefore tends to decline, and if enough sterile males can be released for long enough, the population collapses. this sterile insect technique (sit) has been used for decades to control some major agricultural pests, sterilizing the insects by irradiating them before release. applying conventional sit to mosquitoes has proved problematic, but genetic modifications should be able to overcome many of the key difficulties and limitations. the leading genetically modified sterile release system, known as ridl(release of insects carrying a dominant lethal), is ready to enter field trials for aedes aegypti. they tend to be extremely species - specific, as the modified insects will mate only with their own species. the self - spreading systems are hard to develop but may be relatively cheap to deploy, as the genetic system does much of the work. sterile - release methods such as ridlare relatively cheap to develop, but need regular releases of sterile insects to maintain sufficient sterile males in the field. this self - limiting nature stop releasing and the transgene will rapidly disappear from the field population may, however, be better accepted by the public and regulators, and these systems are likely to be the first ones used in the field. none of these systems should be seen as a ' magic bullet '. self - spreading systems will undoubtedly fail over time, due to mutation and pathogen evolution, and replacement versions will be required. sterile - release methods will be much more effective in the context of an integrated vector management program than on their own. all of these methods will have to be tested in the context of different health systems, cultures and ecosystems ; experience will determine where each is more or less valuable. nonetheless, these powerful genetics - based vector - control tools, about to emerge from the laboratory into the field, provide rare new hope for the control, and perhaps one day elimination, of some of the world 's major infectious diseases. this work is funded in part by a grant to the regents of the university of california from the foundation for the national institutes of health through the grand challenges in global health initiative. thanks to derric nimmo for creating the figures and to neil morrison for comments on the manuscript. | a recent paper in bmc microbiology shows how suppression of mosquito innate immunity against a virus that the mosquito can normally tolerate increases mosquito mortality. this is just one of several approaches that may soon bring genetics - based mosquito control methods from the laboratory into the field. |
pneumatosis intestinalis (pi) and portal venous gas (pvg) are individually worrying radiological signs. both are associated with severe mesenteric ischaemia and when present with symptoms of abdominal pain have a mortality rate in excess of 75%. however, both can exist either individually or concurrently without any underlying abdominal or systemic pathology. we describe a rare scenario where pi, pvg and pneumoperitoneum were found in an asymptomatic patient. a 78-year - old male presented to the surgical assessment unit, following an abnormal chest x - ray performed for a persistent productive cough with fever. the on - duty radiologist identified that this patient had significant free air under the diaphragm on his erect chest x - ray, and contacted the general practitioner directly who in turn organized admission to the surgical services. he reported a fever for 2 days prior to the time of presentation but denied further pyrexias in the previous 48 h. he still had an ongoing productive cough, but without chest pain or difficulty in breathing. he drank 10 units of alcohol per - week and he was an ex - smoker with a 15 pack - year history. on examination, the patient had non - tender and soft abdomen with no distension, organomegaly and bowel sounds were present. he had mild crepitations at the right lung base, but respiratory examination was otherwise unremarkable. a ct scan demonstrated significant free intra - peritoneal air (figs 13). circumferential and linear intra - mural gas within the small bowel was seen consistent with pi. small locules of air were seen in close proximity to the liver which was suggestive of pvg in the peripheral portal system. a faecal calprotectin level was also sent ; this was later found to be negative. figure 1:a coronal ct image of the abdomen taken at initial presentation, demonstrating free air under the diaphragm (pneumoperitoneum) and pvg, both marked on images. figure 2:an axial ct image of the abdomen taken at initial presentation, demonstrating pi marked on the image. figure 3:an axial ct image of the pelvis in lung view windows, taken at initial presentation clearly demonstrating intra - mural air within the bowel, marked on image. a coronal ct image of the abdomen taken at initial presentation, demonstrating free air under the diaphragm (pneumoperitoneum) and pvg, both marked on images. an axial ct image of the abdomen taken at initial presentation, demonstrating pi marked on the image. an axial ct image of the pelvis in lung view windows, taken at initial presentation clearly demonstrating intra - mural air within the bowel, marked on image. his observations remained stable, his abdominal examination remained unremarkable and an arterial blood gas was normal. he discharged home with open access but was to re - attend in 48 h for repeat ct scan and re - evaluation. this second scan demonstrated an increase within the intra - abdominal air, and the gas within the portal venous system became more prominent. the patient remained asymptomatic, observations remained normal and repeat blood tests were unremarkable. the patient was discharged with follow - up in clinic with a consultant general surgeon. upon review, he remained well and was discharged from clinic. this patient was reviewed again 2 months later when admitted for a separate admission under orthopaedics for the management of a fractured neck of femur following a mechanical fall. he was discharged home from orthopaedics following the placement of a dynamic hip screw with an uneventful recovery. pi is a rare radiographic finding with an incidence in the general population of 0.03%. the mechanical theory states an increase in pressure in the bowel lumen, lungs or mediastinum causes gas to dissect through the serosal / mucosal bowel wall. the bacterial theory states that gas - forming organisms enter the layers of the bowel wall. other associations of secondary pi include malignancy, chemotherapy, trauma, mechanical bowel disorders, copd, liver - failure and post - endoscopy. where pi is identified the mortality rate is 44%, and historically the presence of pi has necessitated a laparotomy. there is a rarer primary form of pi, referred to as pneumatosis cystoides intestinalis (pci) accounting for 15% of cases. the mortality for pvg ranges between 75 and 90%. the most common cause for pvg is acute mesenteric ischaemia accounting for 64% of cases. other associations include inflammatory bowel disease, acute intestinal obstruction, trauma, malignancy, chemotherapy, liver transplant and diverticulitis. idiopathic pvg has been described but makes up 2% of cases [2, 3 ]. both pi and pvg have been described concurrently, most commonly in symptomatic patients with severe intra - abdominal necrosis. a 2009 case series of 88 cases of pi / pvg established three distinct subgroups : mechanical, ischaemic and benign. of 88 patients, 19 were identified with benign pi including 6 patients with both pi and pvg. two had a history of malignancy, one with acute liver failure and one with copd. nine patients who were identified with benign pi and/or pvg were placed under observation, all making an uneventful recovery. thus a management algorithm for pi / pvg patients where patients are risk stratified into high-, medium- and low - risk groups was proposed, where low - risk patients are observed with medical treatment alone [4, 5 ]. pi can also be seen with pneumoperitoneum and it proposed that this is caused by the rupture of the intra - mural gas blebs. it is important to state that this rupture does not represent a true bowel perforation through all layers of bowel wall. most gas blebs exist within the submucosal and subserosal layers of the bowel but these blebs do not communicate with the lumen of the bowel. this case demonstrates that both the rare findings of pci and pvg can present together in the well - asymptomatic patient. this was made even more unusual as the patient had no risk factors for either of these structural abnormalities. whilst rare this has been described in literature before. however, this patient also had a significant pneumoperitoneum on ct imaging along with these other signs making this case particularly unusual. | pneumatosis intestinalis (pi) is defined as the presence of gas within the serosal or mucosal layer bowel wall. this sign is usually found upon radiographic imaging and is most commonly secondary to acute gastro - intestinal ischaemia. fifteen per cent of cases can present with a primary condition called pneumatosis cystoides intestinalis (pci). pci is usually a benign condition and patients are usually asymptomatic. portal venous gas (pvg) or the presence / accumulation of free gas within the hepatic portal vein. it is most commonly associated with acute bowel ischaemia, and when seen in the presence of ischaemia the mortality rate is between 75 and 90%. other associations include mechanical causes (e.g. obstruction), chemotherapy, liver transplant and diverticulitis. benign pi has previously been described with pvg, but usually in the presence of other associated conditions such as aids, malignancy or chemotherapy. some examples have been described without these associations, but not with free intra - peritoneal air. we describe a case of pci and pvg with pneumoperitoneum, investigations and ongoing management. |
although cerebral hyperperfusion syndrome (chs) is well documented after carotid endarterectomy and carotid artery stenting, only a few cases have been described after intracranial stenting. after the stenting versus aggressive medical therapy for intracranial arterial stenosis and vitesse stent ischemic therapy trials demonstrated the inferiority of intracranial stenting compared to aggressive medical treatment, this procedure has been largely abandoned. cerebral hyperperfusion is defined as a > 100% increase in cerebral blood flow (cbf) compared to the baseline and it is generally associated with postprocedural hypertension. chs has an estimated incidence of 0.4%2.7% after ce and usually presents with ipsilateral headache or migrainous phenomena, seizures, or intracerebral hemorrhage (ich). i would like to report an unusual case of chs following middle cerebral artery (mca) stenting. a 71-year - old man presented to us with fluctuating motor aphasia of 3 h duration. magnetic resonance imaging of the brain showed multiple acute infarcts in the left mca territory and he was started on antiplatelets and statins. by the next day morning, he had developed global aphasia and transient right - sided weakness. his blood pressure (bp) was 150/90 mm hg and he was taken up for a four - vessel digital subtraction angiography which showed a possible dissection with a thrombus in the distal left mca [figure 1 ]. after obtaining consent, a 4 mm 15 mm solitaire ab neurovascular modeling device (ev3, irvine, usa) was placed across the lesion into the superior mca division with good recanalization. postprocedure, the patient was sedated and ventilated, but had severe hypertension, exceeding 240/140 mm hg which was difficult to control even with multiple antihypertensives. the next morning, a routine computed tomography (ct) brain (12 h later) showed two discrete intracerebral hematomas (ich) in the left frontal and temporal areas [figure 1 ]. transcranial doppler (tcd) showed elevated mean flow velocities of > 130 cm / s in the left mca. coagulation parameters were normal. on examination, now he had a dense right hemiplegia and global aphasia. two weeks later, a repeat ct showed resolution of the ich and no fresh infarcts. the patient had a residual wernicke 's aphasia and right hemiplegia at follow - up even 6 months later. (a) top left panel shows an irregularity in the left middle cerebral artery m1 segment, suggestive of dissection. (c) bottom panels ; computed tomography scan images showing left temporal and frontal intracerebral hematomas risk factors for chs include age > 75 years, preexisting hypertension, high - grade stenosis with poor collateralization, decreased cerebrovascular reactivity, and increased peak flow velocities. reperfusion of ischemic territories can also lead to reperfusion injury, wherein oxidant production, complement activation, and increased microvascular permeability result in an impaired blood chs is a devastating complication because of the high morbidity and mortality of nearly 60%80% associated with this condition. after cerebral revascularization, the advent of severe headache, seizures, or focal neurological deficits after cerebral revascularization should be presumed to signify chs unless proved otherwise. tcd studies are helpful in monitoring elevated peak systolic velocities in the intracranial arteries as a marker of impending chs. in about 15% of patients, in such patients, near - infrared spectroscopy may be a useful option to monitor cbf. all critical care physicians should be aware of this entity for better monitoring and prevention of this postprocedural complication in the icu. | cerebral hyperperfusion syndrome (chs) is a rare complication following cerebral revascularization. it presents with ipsilateral headache, seizures, and intracerebral hemorrhage. it has mostly been described following extracranial carotid endarterectomy and stenting and it is very unusual after intracranial stenting. a 71-year - old man with a stuttering stroke was taken up for a cerebral angiogram (digital subtraction angiography), which showed a dissection of the distal left middle cerebral artery. this was recanalized with a solitaire ab stent. after 12 h, the patient developed a right hemiplegia and aphasia. computed tomography brain showed two discrete intracerebral hematomas in the left hemisphere. this is the first reported case of chs following intracranial stenting from india. |
moxifloxacin is a new 8-methoxy - quinolone with a broad spectrum of antibacterial activity against organisms gram - positive and gram - negative bacteria, anaerobes, and atypical organism such as mycoplasma and chlamydia spp. it has some characteristics such as a wide spectrum of bactericidal activity, a large volume of distribution, low plasma protein binding, and relatively low minimal inhibitory concentrations (mics) against susceptible target microorganisms [1, 2 ], the mic of moxifloxacin for mycobacterium ulcerans ranged from 0.0150.5 gml and for s. aureus from 0.030.12 gml. all these characteristics have made the moxifloxacin a drug of choice to treat many infectious diseases which could not be treated with older fluoroquinolones and other antimicrobials. the disposition study gives concentration versus time profile of drug which is prime step to generate pharmacokinetics data and subsequently to establish dosage regimens. the comparative disposition of long acting and conventional moxifloxacin helps to evaluate the difference in dosage regimens of both types of drugs and their pharmacokinetic profiles in animal body. no such kind of study has been reported to address such a comparative issue in live stock species ; hence, the present study was conducted to investigate the comparative profile of conventional and long acting moxifloxacin in sheep after single intravenous (iv) administration. five healthy adult male sheep weighing 3845 kg were used in the present study. the sheep were housed in well - ventilated appropriately spaced animal shed and fed with good quality fodder and concentration. all animals were acclimatized for the period of 15 days and observed clinically daily to confirm any illness or disease. the experimental protocol was approved by iaec and all the measures for the well being of experiment animal were taken as per cpcsea guideline. long acting moxifloxacin (10% moxifloxacin in solution with l - arginine, n - butyl alcohol, and benzyl alcohol) and conventional moxifloxacin injectable solution, moxifloxacin - base powder for standardization, were obtained from intas animal health, gujarat, india. water, acetonitrile, and tetrabutyl ammonium hydrogen sulfate of hplc grade were purchased from s. d. fine chem. m disodium hydrogen phosphate and hydrochloric acid of analytical grade were purchased from s. d. fine chem. five sheep were administered conventional and long acting moxifloxacin formulation at the dose rate of 5.5 and 7.5 mgkg through intravenous route via jugular vein in crossover design, respectively. washout period of 15 days was kept in between administration of conventional and long acting moxifloxacin. the blood samples were collected using needle (26 g, 0.45 mm 13 mm) disposable syringe (5 ml). blood samples (approximately 5 ml) were collected from each treated sheep in heparin containing test tubes with the help of an intravenous catheter (venflon) fixed into jugular vein at 0 time (before drug administration) and at 0.08 (5 min), 0.25 (15 min), 0.5 (30 min), 1, 2, 4, 8, 12, 24, 36, 48, 60, 72, and 96 h after drug administration. plasma was separated after centrifugation of blood samples at 1660 revolutions per minute (rpm) for 10 minutes. the plasma samples were transferred to cryovials (3 ml capacity) and stored at 40c until assayed for long acting moxifloxacin concentration using hplc procedure. plasma concentrations of moxifloxacin were measured using a modified hplc method previously reported by siefert. the hplc (agilent-1100) system was well equipped with a modal lc-9a (gradient solvent delivery pump), a modal rf-551 fluorescence detector, and a modal sil-6b automatic sampler and column heater (cto-6a). plasma samples were extracted in aliquots by adding 200 l of plasma to 200 l of acetonitrile. plasma proteins were precipitated by shaking in an ultrasonic bath followed by centrifugation for 10 min at 1660 rpm speed. supernatant was diluted four - fold with 0.067 m disodium hydrogen phosphate buffer with ph 7.5 and transferred to hplc autosampler vials. the hplc separation was performed with an injection a reserve phase c18 (supelco, 5, 4.6 150 mm) with using an injection volume of 50 l. the mobile phase consisted of acetonitrile (20%) and tetrabutyl ammonium hydrogen sulphate solution 10 g / l (80%). mobile phase was filtered by 0.22 m filter and degassed by sonicator and then pumped into column at a flow rate of 1.00 mlmin at ambient temperature. the fluorescence detection was performed at excitation wavelength of 296 nm excitation and an emission wavelength of 504 nm. the mean recovery of long acting moxifloxacin from plasma was 85.14% at 25 ng / ml. the sensitivity of long acting moxifloxacin assay was 25 ngml. the assay was sensitive, reproducible, and linearity was observed from 0.025 to 20 gml. the mean (se) plasma concentrations of conventional and long acting moxifloxacin following single - dose intravenous administration are tabulated in table 1. mean plasma concentration versus time profile plot of both formulations of moxifloxacin is illustrated in figure 1. the initial plasma drug concentration of long acting moxifloxacin was 7.212 0.107 gml achieved at 0.083 h (5 min). the lowest detectable plasma concentration of long acting moxifloxacin level as 0.016 0.001 ml was found at 72 h. the therapeutically effective concentration maintained from 0.083 to 72 h. the minimum inhibitory contraction of moxifloxacin is 0.10.5 gml. initially, the conventional moxifloxacin plasma level was 9.971 0.901 gml recorded at 0.083 h (5 min). the initial concentration of moxifloxacin rapidly declined to 5.596 0.901 gml at 0.167 h (15 min). thereafter, the concentration of the drug gradually diminished and after 24 h it was not detectable in plasma. the plasma concentrations of conventional moxifloxacin at 0.5, 1, 2, 4, 8, 12, and 24 h were 3.887 0.278, 2.496 0.007, 1.529 0.111, 0.544 0.012, 0.224 0.019, 0.067 0.008, and 0.015 0. gml, respectively. thereafter, plasma moxifloxacin level diminished gradually and was not detectable after 24 h of drug administration. the initial plasma drug concentration of long acting moxifloxacin was 7.227 0.13 gml achieved at 0.083 h (5 min), which rapidly declined to 5.646 0.044 gml at 0.166 h (15 min). the plasma concentrations of long acting moxifloxacin at 0.5, 1, 2, 4, 8, 12, 24, 36, 48, 60, and 72 h were 4.283 0.22, 2.853 0.136, 2.230 0.055, 1.712 0.077, 0.772 0.022, 0.368 0.019, 0.150 0.002, 0.076 0.002, 0.043 0.001, 0.026 0.001, and 0.016 0.002 gml, respectively. thereafter, plasma long acting moxifloxacin level diminished gradually and was not detectable at 96 h of drug administration. while conventional moxifloxacin administration resulted in rapid decline in plasma concentration and the drug was not detectable after 24 hours of administration. the long acting moxifloxacin administration provided effective therapeutic concentration for up to 72 hours after single - dose administration. this dose is advantageous with respect to cost of treatment and compliance to animal owner. conversely, while applying long acting moxifloxacin in sheep, initially the levels of drug remained low (7.227 gml at 0.083 h) as compared to its counter formulation (conventional) that reached initially to 9.917 gml (0.083 h). since moxifloxacin is a new generation fluoroquinolone antimicrobial drug, with some added quality of initial speedy high level with both types of formulations along with its low mic level, will definitely make this compound as the drug of choice for the treatment of infectious disorders in sheep. | this study describes disposition of long - acting moxifloxacin and conventional formulations of moxifloxacin in sheep after intravenous administration in five male sheep. long acting moxifloxacin solution (10% moxifloxacin in solution with l - arginine, n - butyl alcohol, and benzyl alcohol) and conventional moxifloxacin (10%) were injected in jugular vein. blood samples were collected from contralateral jugular vein in test tubes containing 3050 iu heparin (anticoagulant) periodically from 0.083 to 72 h of drug administration. drug concentrations in plasma were determined using high - performance liquid chromatography (hplc) with fluorescence detector. the mobile phase consisted of a mixture of buffer (10 gm of tetrabutyl ammonium hydrogen sulphate per liter - deionised water) and acetonitrile (80 : 20). the buffer was 0.067 m of disodium hydrogen phosphate with ph of 7.5. the flow rate was 1 mlmin1 at ambient temperature. the effluent was monitored at 296 nm excitation and 504 nm emissions wavelength. hplc with fluorescence detector method for plasma moxifloxacin assay was standardized with specific modification for plasma of sheep in the present study. after single - dose intravenous administration of long acting moxifloxacin the plasma concentration of 0.016 0.001 gml1 was maintained for up to 72 h. conventional formulation of moxifloxacin remained in body for up to 24 h of drug administration with the level of 0.015 0.005 gml1. |
the pediatric risk of mortality (prism) score is effective in predicting children 's mortality. information about the predictive value of prism score is very limited outside america and europe, especially in developing countries. especially in centers with limited pediatric intensive care unit (picu) beds, by helping physicians predict the mortality risk, prism score may help the medical group decide which patient benefits more from admission to picu. prism scoring system is a physiologic stability index that predicts mortality through normal physiologic disturbances during the period of disease. world health organization (who) estimated that 10 million children die every year of which 99% occurs in the developing countries. acute respiratory disease and malaria are the most common causes of death in under 5-year - old children in these countries. with good care in picu, physicians may save lives of one million children in developing countries such as pakistan. maysat introduced prism and pediatric index of mortality (pim) as two evaluating systems in picu worldwide. prism score is used in picu and needs observation during the first 24 hours of admission. but now prism iii scoring system is used to evaluate the severity of the disease after 12 and 24 hours of admission in picu. lacroix pointed out that prism score may be used in neonates, infants, children and adolescents with severe disease, but may not be used in preterm neonates and adults. tan aimed to evaluate risk factors that increase the mortality risk in picu, especially with the use of prism scoring system as an outcome predictor. a prospective study in india showed that a simple clinically scoring system will be useful in predicting severity of illness and outcome at admission in emergency. the purpose of present study was to evaluate the prism scoring system in predicting mortality rate in picu. this cohort study was performed on 1-month to 14-year - old patients admitted during an 18-month period in the picu of ali - ebne - abitaleb hospital of zahedan. each patient received a chart including vital signs, temperature, respiratory rate, heart rate and blood pressure. white blood cell count, platelets, liver enzymes (sgot, sgpt), bilirubin (total, direct), pt, ptt, glucose, blood urea, creatinine, sodium, potassium and calcium were measured by standard laboratory tests. arterial blood gas analysis including bicarbonate, paco2 and pao2 was carried out in each patient. the glasgow coma score (gcs) was calculated by the pediatric resident at admission. the patients were followed up during hospital stay by the resident doctor, and he reported the outcome as death or survival at the end of the hospital stay. prism score evaluation was done as recommendation of pollack this score is calculated from the most abnormal values of 14 physiological variables in the first 24 h and the patients age and operative status. the results were analyzed by kaplan - meier curve, roc curve, log rank (mantel - cox), cox and logistic regression model. spss 15 for windows (spss inc, chicago, illinois) was used for statistical analysis. of the 221 patients admitted to picu during the study period, 126 were males. table 1 one - hundred ten patients were less than 1 year old, 37 patients were 1 - 2 years old and 74 patients were more than 2 years old, of whom 22.7%, 27% and 16.2% respectively died. prism score 11 - 20 and 21 - 30 were observed in 45 and 19 cases, of whom 17 (37.8%) and 14 (73.8%) patients expired, respectively (table 1). survival of hospitalized patients in the picu according to age, gender and prism score picu : pediatric intensive care unit ; prism : pediatric risk of mortality outcome based on the type of disease according to time and rate of mortality, seven (87.5%) of the eight patients who were admitted at day 0 - 0.33 to picu died. five children (66.7%) died at day 0.34 - 0.5, six children (100%) died at day 0.51 - 1. however, probability of death significantly increased by higher prism scores (tables 3, 4, fig. the median age of survived children was 35 months in less than one year olds, 36 months in those between 1 - 2 years old and 45 months in more than 2 years old. the median age of survived children was 36 months for patients with prism score 0 - 10, 16 months for patients with prism score 11 - 20 and 4 months for patients with prism score 21 - 30. according to prism score, survival probability showed significant differences between groups, so with increase of the prism score, survival probability decreased (p0.001) (fig. 2). compared to children with prism score of 1 - 10, hazard of death increased 3.2 times and 8.3 times in children with a prism score of 11 - 20 and 21 - 30, respectively (table 1). according to disturbance of different systems, prediction of outcome by roc analysis according to roc curve analysis. under the curve area is 80.3% showing good outcome probability of survival by time in terms of prism score prediction of the probability of death according to prism score based on the logistic regression model prism : pediatric risk of mortality predictive model for the patient situation in hospitalized picu patients picu : pediatric intensive care unit ; hosmer - lemeshow chi - square=0.62 ; df=3 ; p. value=0.8 different care systems have been created to increase the quality of care for children who need special care. it is necessary to develop models which predict the mortality risk in picu in order to monitor the effectiveness of the cares carried out. they enable us to compare different units and evaluate the associations between the severity of diseases, hospitalization duration and the costs. it is important to know the accuracy of these scoring systems to estimate the mortality risk in icus of different groups and countries. prism is a physiologically based score that predicts the mortality risk according to the changes of normal values during disease period. qureshi indicate that by use of statistical analyses such as hosmer, odds ratio, spearman correlation test, roc, lemeshow goodness - of - fit, prism scoring system is a good predictor of mortality rate in picu. they found that prism and pim scoring systems had the same validity for estimating mortality. another study demonstrated that prism scoring system can predict mortality in children admitted to picu. martha vf observed that prism scoring system had good validity to predict mortality, but pim scoring system was not acceptable for this purpose. gemek stated that prism and prism iii scores are good scales to estimate the mortality rate in picu which is in harmony with our survey. a study by anu t showed that under the roc curve area in pim, pim2 and prism are higher than 0.8, but these models predict mortality less than that observed in picu. in our study, the area under the roc curve was more than 0.8. in a study, it was indicated that pim2 had the highest accuracy to differentiate risk groups ; therefore, it was thought to be the best model to predict mortality in picus in australia and new zealand. a prospective cohort study in iran revealed that pim2 is a good index for prediction of mortality in picu. in another study, it has been mentioned that pim2 shows significant differences between life and mortality in patients who were admitted in picu, but pim did not show significant relationship with homser lemeshow test. taylor showed that most patients admitted in picu had acceptable functional outcome and quality of life after discharge. shann stated that if the area under the curve of roc is equal to one, the model is perfect. an area between 0.9 and 0.99% is very good, between 0.8 and 0.89% is good and finally, between 0.7 and 0.79% is acceptable. the area under the curve was 80.3%, so it is good to predict mortality in picu admitted patients. choi concluded that pim and prism iii scoring systems are good predictors of mortality in picu and the validity of these models are high such as in our study. brady found that pim2 and prism iii are good scales to estimate picu mortality in the united kingdom. lacroix showed that pim and prism scores are able to estimate the severity of disease that was consistent with our study. another similar study showed that prism scoring system has high sensitivity to predict the outcome of children admitted in picu. our survey showed that prism scoring system is a good predicting value to evaluate death probability of children admitted to picu of our center. | objectivethe pediatric risk of mortality (prism) score is one of the scores used by many pediatricians for prediction of the mortality risk in the pediatric intensive care unit (picu). herein, we intend to evaluate the efficacy of prism score in prediction of mortality rate in picu.methodsin this cohort study, 221 children admitted during an 18-month period to picu, were enrolled. prism score and mortality risk were calculated. follow up was noted as death or discharge. results were analyzed by kaplan - meier curve, roc curve, log rank (mantel - cox), logistic regression model using spss 15.findingstotally, 57% of the patients were males. forty seven patients died during the study period. the prism score was 0 - 10 in 71%, 11 - 20 in 20.4% and 21 - 30 in 8.6%. prism score showed an increase of mortality from 10.2% in 0 - 10 score patients to 73.8% in 21 - 30 score ones. the survival time significantly decreased as prism score increased (p0.001). a 7.2 fold mortality risk was present in patients with score 21 - 30 compared with score 0 - 10. roc curve analysis for mortality according to prism score showed an under curve area of 80.3%.conclusionprism score is a good predictor for evaluation of mortality risk in picu. |
osteoid osteoma is an osteoblastic benign bone lesion that may be easily diagnosed when the typical clinical and radiological features are present. however, diagnosis may occasionally be difficult if the lesion is in an area not clearly seen on plain radiographs, or if it presents with atypical clinical findings.1, 2 a 34-year - old man with a subtrochanteric osteoid osteoma of the lateral cortex of the left femur is presented. this case is unusual because it was treated as a greater trochanteric pain syndrome for 9 months. the patient then sustained a transcervical fracture of the neck of the left femur that was fixed, but the osteoid osteoma remained undiagnosed for further 18 months. this report focuses to illustrate the clinical and radiological diagnostic problems in the presented patient and to examine any potential pathology that would be indicative of a pathological fracture. a 34-year - old white male presented with a history of mild spontaneous pain radiating to the lateral side of the left thigh and the knee. since he refused a radiographic examination, he was informed that a greater trochanter bursitis pain syndrome was the most likely diagnosis. were not significantly changed after 9 months. by that time he suffered a minimally displaced transcervical fracture of the neck of the left femur (fig. partial weight bearing was allowed only after the third month and full weight bearing after the sixth month postoperatively (fig. 2). the patient 's mild spontaneous pain over the lateral aspect of the thigh continued for 12 postoperative months. during his last consultation, 18 months postoperatively he reported that the pain had increased in intensity and awakened him nearly nightly during the last 6 months. he reported complete pain relief after taking the medication twice a day. on physical examination there was increased lateral temperature, pressure tenderness and a palpable bone overgrowth over the lateral side of the proximal thigh. there was an atrophy of his left thigh exceeding almost 3 cm compared with the right side. a new pelvic radiograph, that for the first time included the subtrochanteric region of the left femur, indicated a bone lesion consistent with osteoid osteoma (fig. tomograms of the left proximal femur indicated the subperiosteal location of the lesion (fig. en - block removal of a piece of reactive bone including the nidus was surgically performed. radiography in the operating theatre was needed to identify the nidus in the resected piece of bone (fig. the histologic examination confirmed the diagnosis of osteoid osteoma. the patient experienced immediate relief of symptoms from the first postoperative day. the patient was free of disease at the final follow - up 25 years after operation. diagnosis of osteoid osteoma is readily made by the history, the clinical presentation and the radiological features in most cases. the mean duration from the onset of symptoms to diagnosis of osteoid osteoma delayed diagnosis in most cases is due to atypical clinical features and from lack of awareness that plain radiographs in the early stages can be normal. misdiagnosis with prolonged impairment and sometimes overtreatment appears as a major problem concerning atypical cases.3, 4, 5 in addition, delayed diagnosis may be seen in unusual cases of osteoid osteoma presenting with slowly evolving neurological signs,5, 6, 7 as inflammatory joint disease and complex regional pain syndrome,8, 9, 10, 11, 12, 13 or mimicking sacroiliitis, femoroacetabular impingement syndrome and osteoarthritis. the misdiagnosis and treatment of an osteoid osteoma as greater trochanteric bursitis pain syndrome has not been previously reported. pain, tenderness, and sometimes swelling over the lateral aspect of the hip are usually attributed to a greater trochanter bursitis.17, 18 the differential diagnosis may include muscle strain, adipose dolorosa, local infections, stress fractures, osteonecrosis, neuropathy and soft tissue metastases.19, 20 although the initial evaluation of a patient may not require a radiological examination, cases with persisting or even deteriorating clinical symptoms may be sent for radiographs. the clinical suspicion of a bone tumor is the first step towards making a diagnosis ; and the awareness that osteoid osteoma may occur in young patients, should alert to the possibility of this uncommon entity. it should be emphasized that failure to carry out a proper radiographic survey in cases treated as greater trochanter bursitis pain syndrome may be associated with a considerable delay in the diagnosis of the underlying bone pathology. retrospective consideration of his pelvic radiographs following the hip fracture is indicative of a reactive bone formation at the lateral cortex of his proximal femur (fig. the question whether it was a pathological fracture or not can not be clearly answered. it has been previously reported that osteoid osteomas of the hip may present with atrophy of the thigh. this may be most likely related to fatty infiltration of the muscles due to the regional inflammation than to the limb disuse. in addition, regional osteoporosis due to osteoid osteoma has rarely been reported.24, 25 in the presented case, a 3 cm difference was determined between thigh measurements, but the atrophy was calculated 18 months following fixation of the hip fracture and this could be directly related to disuse muscle atrophy. retrospective evaluation of his pelvic radiographs at injury was suggestive of regional hip osteoporosis (fig. the diagnosis was missed for a further 18-month period following the fixation of the hip fracture. the severe deterioration of his symptoms necessitated a new radiographic investigation that for the first time included a sufficient portion of the subtrochanteric region of the femur. further diagnostic investigation of the bone lesion included only tomograms, since computed tomography and magnetic resonance imaging were not available by that time. the patient presented in this report is the first osteoid osteoma with a clinical presentation mimicking greater trochanteric bursitis pain syndrome. furthermore, it is the first subtrochanteric osteoid osteoma complicated by a hip fracture, although a clear involvement of the osteoid osteoma to bone pathology proximal to the border of cortical thickening could not be sufficiently proved. | a 34-year - old man with a subtrochanteric osteoid osteoma localized to the lateral cortex of the left femur is reported. the patient presented with mild spontaneous pain of the lateral thigh and knee. he refused a radiographic examination and was treated as a greater trochanteric pain syndrome for 9 months. he was then admitted with a transcervical fracture of the neck of the left femur after a fall from standing height. the fracture was fixed with 3 cannulated screws and healed uneventfully. his symptoms worsened after the first postoperative year. eighteen months postoperatively the pain was dull, worsening at night, and relieved only with anti - inflammatory drugs, and he had a limp. new radiographs and tomograms were indicative of a lateral subtrochanteric osteoid osteoma with a subperiosteal localization. the lesion was treated successfully with surgical excision of a piece of reactive bone including the nidus. |
influenza causes significant morbidity and mortality in the united states (us), with associated costs of 1077 billion dollars [13 ]. current us influenza surveillance activities include the reporting of the percentage of outpatient visits due to influenza - like illness (ili), reports of laboratory - confirmed influenza hospitalizations and pediatric deaths, pneumonia- and influenza - associated mortality, and viral culture for subtyping. the reporting lag associated with these surveillance measures is 14 weeks, which limits the implementation of prevention and control activities [57 ]. this has led to investigation of alternate data sources and analytic techniques that provide earlier notification of influenza activity. an article reviewing the timeliness of alternate data sources for influenza surveillance found that over - the - counter pharmaceutical sales, emergency visits, absenteeism, and health advice calls appeared to be more timely than ili reporting or virological confirmation, by 324 days. this lag results from both the timing of the surveillance event relative to the epidemic onset (affecting viral culture results, hospitalizations, and deaths) and the delays inherent in systems that report once weekly (affecting all systems). another study found that rapid influenza test positivity (the percentage of positive tests among all tests performed) increased 25 weeks prior to epidemic notification from ili surveillance. outpatient illness, in ambulatory clinics and urgent care settings, is generally the first recognized in the course of an influenza outbreak and is best suited as an early indicator of disease [5, 6, 10 ]. currently, outpatient influenza surveillance involves weekly reporting to the centers of disease control (cdc) of the percentage of visits that are due to ili from approximately 1300 outpatient care sites from all 50 states in the united states (us). since the first systematic reports of ili by outpatient providers for the 1982 - 83 season, little of the methodology has changed and several challenges exist. first, while some outpatient care sites now use electronic records for ili data collection, many still use paper - based data collection, which can be labor intensive. second, data transmission to cdc occurs in the week following data collection and is not publicly posted until the end of that week, creating a lag that diminishes the window of opportunity for implementing preventive measures. third, the ili case definition is not specific to influenza but can be associated with a variety of respiratory infections, potentially producing a false positive signal. the university of utah primary care research network (uupcrn) has 10 outpatient clinics and has participated in ili surveillance with the utah department of health (udoh) and cdc since 2004. the impetus for our study was the inherent lag in the current ili reporting system, and a variety of studies that suggested alternative data sources for surveillance could reduce this lag [9, 1215 ]. decreased lag could potentially create a window within which prevention and control activities might be conducted. using point - of - care (poc) test positivity as an early indicator of influenza activity, we retrospectively analyzed 4 seasons of influenza and created an alerting algorithm based on statistical process control (spc) charting. in the 5th season, we implemented the algorithm prospectively and evaluated the behavior of clinicians related to influenza identification and prevention in intervention clinics (received email notification of the early alert) compared with control clinics. finally, we comment on key lessons for implementing an effective and timely clinic - based surveillance and feedback system. the uupcrn is a population - based research network of 10 outpatient clinics and an associated urgent care located in the salt lake metropolitan area, where about 2 million of the state 's 2.5 million residents live [16, 17 ]. in fiscal year 2006 - 07, there were 320,000 uupcrn outpatient visits, with roughly one quarter of visits occurring among each of 4 age groups : 018, 1935, 3655, and 56 years and over. implementation of an electronic medical record (emr) designed by epic systems corporation (verona, wi) was begun in 2000. full operation with the emr was complete for 6 clinics in 2004 - 05, 7 in 2005 - 06, and all 10 for the remaining study years. the number of outpatient visits recorded in the emr and available for analysis during influenza season (october may) ranged from 139,303 in 2004 - 05 to 191,842 in 2007 - 08. in the us, influenza surveillance begins in early october and runs through mid - may of the following year, defined by morbidity and mortality weekly report (mmwr) weeks 40 through 20. all outpatient visits recorded in an emr for the uupcrn patient population were evaluated retrospectively for the seasons 2004 - 05 through 2007 - 08. prospective evaluation of the alerting system and associated clinician behaviors was completed for the 2008 - 09 season. the majority of us primary care providers report using influenza tests for ili patients, most of which are rapid tests. the median sensitivity of influenza rapid tests is 70%75%, and median specificity is 90%95%. by comparison, studies of the most predictive syndromic definition (fever plus cough) reported sensitivity of 64%78% and specificity of 55%67%, suggesting that rapid tests may have better predictive characteristics than a syndromic case definition [18, 19 ]. further, most rapid influenza tests can be conducted at the poc, eliminating the lag that accompanies off - site testing of patient samples. rapid influenza tests were introduced into the uupcrn clinics in december 2003 and were used in conjunction with viral culture for the remainder of the 2003 - 04 influenza season. in all subsequent seasons, uupcrn clinics used poc rapid tests almost exclusively. beginning with the 2003 - 04 influenza season, udoh began accepting rapid tests, in addition to viral culture, as laboratory confirmation for influenza surveillance. the uupcrn clinics contributed influenza surveillance data to udoh, as part of the sentinel providers network. the cdc case definition for ili isfever of 100f (37.8c) or higher, with cough and/or sore throat, in the absence of another known cause for these symptoms. fever of 100f (37.8c) or higher, with cough and/or sore throat, in the absence of another known cause for these symptoms. the implemented uupcrn case definition for ili required a measured temperature of 100f (37.8c) or higher and a chief complaint code of cough or sore throat or both as the reason for the visit. for comparison of uupcrn daily ili surveillance with traditional weekly ili surveillance, udoh provided weekly ili data collected for the cdc 's sentinel providers network for the seasons 2004 - 05 through 2007 - 08. test results were entered into the patient 's emr by the laboratory technician on the same day. an automated report extracted information from the emr and provided daily counts of the number of outpatient visits to uupcrn clinics, the number of patients who met the ili case definition, and the number who had a positive rapid influenza test. using the number of daily patient visits as the denominator, the proportion of patients with ili and the proportion of patients with a positive rapid influenza test were calculated for each day during the influenza season for the seasons 2004 - 05 through 2007 - 08. spc methodology was applied to the daily proportion of ili visits and of positive rapid influenza tests to build seasonal charts using statit custom qc (statware, inc.). process control charts generated a mean proportion (or center line) for each clinical indicator and confidence limits (or control limits) that defined the bounds for normal variation. we selected an upper control limit of 3 standard deviations (or 3 sigma) above the center line as the threshold for generating an alert of increased influenza activity. the upper control limit was calculated separately for weekends and weekdays, due to the lower number of patient visits on weekends when only the urgent care facility was open. timeliness was measured as the number of days between an early alert and the beginning of the influenza epidemic curve. using the western electric rules for identifying an action signal, the date on which 4 of 5 consecutive points exceeded the 1 sigma limit was defined as the epidemic onset. full - season charting (retrospective) used an entire season 's data to create the spc chart, and a full - season early alert was defined as the first signal exceeding the upper control limit prior to the epidemic onset. alternatively, we built the spc chart one day at a time, to simulate real - time surveillance (prospective). under this scenario, there were multiple single - day alerts. because these were potentially false alarms, we defined a real - time surveillance alert as a point that exceeded the upper control limit followed by a point that did not return to the center line. timeliness of spc in the uupcrn system was compared with other surveillance systems available to utah clinicians. these included influenza surveillance data posted to both the cdc and udoh websites [11, 22 ]. the data for each of these are reported once weekly for the mmwr weeks, defined as sunday through saturday. the utah sentinel providers send their data to udoh, where the data are posted on the wednesday following the end of the data collection week, resulting in a 4-day lag after the close of the data collection week. the udoh forwards these reports to cdc, where weekly data for all us sentinel providers are posted to cdc 's website on the friday following the end of the data collection week, resulting in a 6-day lag after the close of the data collection week. the web posting dates were defined as the notification date for the udoh and cdc systems. the uupcrn data were available the day following the clinic visit. assuming a 24-hour time frame to analyze the data and send an email to clinicians, the potential uupcrn notification date was defined as 2 days after generation of the real - time surveillance alert. this date was compared with the date udoh and cdc posted surveillance results on their websites. for the 2008 - 09 season, we implemented daily prospective charting of the proportion of patients with a positive rapid test. alerts were defined using the same methods described in the retrospective section (aforementioned). the early alert and the epidemic onset dates created the following time periods : prealert, early alert, and epidemic. the uupcrn clinics were categorized as academic clinics where residency training occurs (n = 2), and community clinics were stratified by annual patient volume (2 high volume, 4 intermediate volume, and 2 small volume). within each of these 4 strata, one clinic was randomized to receive early notification by email of the influenza alert and designated as intervention clinics. the remaining 6 control clinics did not receive the email, representing 56% of total patient visits. our plan was for the uupcrn medical director to send an email to clinicians in intervention clinics when the early alert was noted in the spc chart. the email would identify the date of the early alert and, based on historical data, estimate the date for the epidemic onset. the email would also include additional messages based on information from the centers for disease control and prevention (cdc) website [24, 25]:recommending vaccination because protective antibodies develop within 2 weeks after vaccination;providing guidance on antiviral therapy and prophylaxis. recommending vaccination because protective antibodies develop within 2 weeks after vaccination ; providing guidance on antiviral therapy and prophylaxis. the reason for visit, chief complaint(s), and vital signs were abstracted from the emr to classify patients visits as ili or non - ili. orders for rapid influenza testing, influenza immunization, and antiviral prescriptions were abstracted from the emr to assess the impact of the intervention on clinician behavior. the proportion of influenza - associated visits with a particular clinician behavior was calculated. to quantify the association between clinics receiving early notification and subsequent clinician behavior, an interaction term was created for intervention status and the time period prior to the epidemic onset (prealert and early alert). the 95% confidence intervals (cis) and p values for the interaction odds ratios (ors) were used to determine whether a statistically significant difference for clinician behavior was evident for intervention clinicians during the early alert period. analyses were conducted using stata, version 8.2 (statacorp, college station, tx). the spc charts for full - season charting of uupcrn data are provided for the 2007 - 08 season in figure 1. similar graphs were obtained for seasons 2004 - 05 through 2006 - 07 (data not shown). the smoothed curve generated with a 7-day moving average is shown for ili (upper graph) and rapid test positivity (lower graph) in figure 1(a). the horizontal lines define the center line (solid) and upper control limit (dashed), with data points exceeding the upper control limit labeled a. over the 4 seasons, the epidemic onset occurred once in december and january and twice in february (data not shown for the remaining 3 seasons). although the epidemic onset occurred in different months, several trends emerged from the 7-day moving average graphs. first, rapid test and ili peaks coincided in time, although rapid test peaks were of longer duration. second, the rapid test alert started earlier than the ili alert in all 4 seasons (range : 335 days). third, the rapid test data defined more than 1 peak in all 4 seasons while ili defined a single peak each season. in 2 seasons, the magnitude of these additional peaks was similar to the main peak. in 1 season, the additional peaks actually preceded the main peak. the spc charts for full - season charting of daily ili and positive rapid influenza tests in uupcrn are provided for the 2007 - 08 season in figures 1(b) and 1(c), respectively. using the daily positive rapid influenza tests data (figure 1(c)) the epidemic onset occurred on february 3rd, as defined by the western electric rules. these dates are identified with arrows in figures 1(b) and 1(c). by contrast, there was no clear epidemic peak evident on the ili graph and the percentage of visits due to ili - generated signals through the entire season, with 10 false alerts generated prior to january 19th. over the 4 seasons, the number of false alerts on the ili graph ranged from 5 to 10 (data not shown). the real - time surveillance graph of positive laboratory tests for the 2007 - 08 season is shown in figure 2. the modeled real - time surveillance alert occurred on january 10th (noted by an arrow), 9 days earlier than the alert generated in the full - season chart. alerts for modeled real - time surveillance occurred earlier than full - season charting in all 4 seasons. a comparison of the alert dates for full - season charting and real - time surveillance for each season is provided in table 1. the mean number of days that the early alert preceded the epidemic onset was 7.0 days for full - season charting and 15.5 days for modeled real - time surveillance (p = 0.039 for a one - sided t - test comparing these means). utah clinicians can access the udoh or cdc websites for updates on ili, with notification dates defined for this comparison as the day on which the new weekly surveillance summary was posted. the notification dates for cdc and udoh were compared with a theoretical notification date to uupcrn clinicians 2 days following the real - time surveillance alert. box plots for website posting in all 4 seasons for cdc, udoh, and the estimated uupcrn notification are provided in figure 3. two alerting thresholds for udoh were used for comparison, corresponding to the utah - specific ili baseline calculated during the years of the study (3.6% of visits due to ili) and the revised utah - specific baseline (1.7% of visits due to ili) that was used for the 2008 - 09 and later seasons. the latter baseline was closer to the regional baseline of 1.5%1.6% and is expected to be more accurate. the uupcrn theoretical notification date always occurred prior to the epidemic onset (median : 14.0 days). while the median notification date occurred prior to the epidemic onset for both the cdc and the revised udoh (1.7%) systems (18.0 and 5.0 days, resp.), each had a notification date after the uupcrn epidemic onset during 1 season. the udoh (3.6%) notification occurred after the uupcrn epidemic onset in 2 seasons (median : 16 days) and never exceeded the ili threshold in the other 2 seasons. the spc graph for the 2008 - 09 influenza season is provided in figure 4, with detail for the month preceding the alert shown in the inset. the time periods defined by the spc chart were prealert (oct 1, 2008jan 26, 2009), early alert (jan 27feb 13, 2009), and epidemic period (feb 14apr 12, 2009). for 2008 - 09, the early alert occurred 19 days prior to the epidemic onset. nationally and in utah, the 2008 - 09 season was mild, prior to the circulation of 2009 h1n1 in the late spring. this is reflected in the spc chart where the epidemic onset is followed by sporadic activity after the alert rather than the usual epidemic curve (as seen in figure 1(c)). the early signal was identified on january 27th, and an email was prepared on january 28th, which was consistent with our plan to notify clinicians within 2 days. however, due to delays in administrative processing, the email was not delivered to intervention clinicians until january 30th. even with the email delay, intervention clinicians were notified 15 days prior to the epidemic onset, representing a time during which they might have implemented prevention and control measures for their patients at risk for influenza infection. among patients with an influenza - associated visit during the prealert and early alert periods, 4.1% (range : 2.8%8.0%) had sufficient information documented in the emr to code them as ili cases for traditional surveillance ; 5.6% (range : 2.8%11.2%) had a rapid test performed ; 7.9% (range : 0.7%10.0%) received an influenza immunization ; 0.3% (range : 0.04%1.6%) received a prescription for an antiviral medication (see figure 5). as expected, influenza immunizations were higher in the prealert period (october january), corresponding with the timeframe when seasonal immunization for influenza is emphasized. other behaviors were higher in the early alert than in the prealert period, which was expected since they were responses to higher levels of respiratory illness including influenza. we used logistic regression to model the combined impact of notification status and time period for each clinician behavior. the interaction term compared clinician behavior in intervention clinics during the early alert period relative to the prealert period, as well as to clinicians in control clinics during both time periods. while not statistically significant at alpha = 0.05, the ors for clinician behaviors (figure 5) suggest that intervention clinicians were 40% more likely to perform rapid testing (or = 1.39, 95% ci = 0.932.08) and twice as likely to immunize against influenza (or = 2.11, 95% ci = 0.865.18) during the early alert period than their counterparts. we used spc charting to monitor the daily percentage of patient visits with a positive rapid influenza test identified from the emr of a population - based network of outpatient clinics. building the charts to simulate real - time surveillance, an early alert was identified for each of 4 seasons, occurring a median of 16 days prior to the epidemic onset. we estimated that notification of this early alert could be sent by email to uupcrn clinicians within 2 days of the alert. by comparison, the median notification date for the cdc website posting was similar to our early alert but had wider variability, including one season when cdc notification occurred after the uupcrn epidemic onset. in actual implementation during the 2008 - 09 season, the early alert occurred 19 days before the epidemic onset, but notification took 4 days rather than the hypothesized 2 days. there were increases in clinician performance of rapid testing and antiviral prescribing practicing at intervention clinics during the early alert period, but neither was statistically significant. other studies evaluating the timeliness of early signals in outpatient populations have been conducted and report a lead time of 724 days. three studies have used alternative modeling techniques with ili data and demonstrated earlier signals relative to traditional ili surveillance [2628 ]. a fourth study reported that the proportion of clinical visits with a positive rapid influenza test result began rising 25 weeks earlier than the proportion of ili. our finding of a 16-day lead time for the real - time surveillance is similar to these other studies in outpatient settings. further, our analytic method is relatively simple to implement and interpret visually, making it attractive to use in clinical settings that do not have access to the statistical expertise required to perform sophisticated modeling, as required by some of these studies. the use of rapid influenza test results stored in an emr has several potential advantages over the identification of ili cases for surveillance. first, rapid influenza tests are reported to have comparable sensitivity and higher specificity than a syndromic definition of fever and cough [14, 18, 19 ]. consequently, use of rapid influenza tests for surveillance could result in fewer false positive signals. second, the influenza test results are stored in the emr as part of usual patient care while identification of ili cases requires the use of a non - patient - related data collection form. thus, data captured from the emr is likely to be less labor intensive and more complete. health departments in hawaii and indiana that used automated electronic laboratory reporting have documented 2.3- and 4.4-fold increases in reporting, respectively [29, 30 ]. these same studies also demonstrated that reports were received 3.8 and 7.9 days earlier compared to paper - based methods. third, the emr data are available daily rather than weekly, enhancing the timeliness of analysis and notification and providing a window within which preventive activities could be initiated. it is possible that the uupcrn study population is not representative of the general population and that an early signal generated in our data would not reflect the timing of influenza activity in the broader population. we compared the uupcrn rapid influenza test results with ili reporting for both uupcrn and udoh, as well as with respiratory culture findings from the germwatch laboratory surveillance system in utah. the clinical and laboratory influenza indicators increased and decreased over the same time period for the seasons 2004 - 05 through 2007 - 08, suggesting that the uupcrn data are representative of the population of the salt lake metropolitan area. further, in 3 of the seasons the germwatch data demonstrated that the respiratory syncytial virus (rsv) epidemic started 37 weeks before the influenza epidemic, but our early alert signal coincided with the germwatch influenza epidemic and did not give a false signal associated with the earlier rsv epidemic. it is also possible that the use of influenza testing among uupcrn clinicians differs from their peers in utah. if uupcrn clinicians were more likely to use rapid testing, especially early in the season when the risk of false positive results is higher, our early signal based on test results might reflect false positives. differential use of influenza testing seems unlikely because udoh reported that 86% of 6,340 laboratory - confirmed influenza cases for the 2003 - 04 season were diagnosed by rapid test, suggesting that use of rapid tests is widespread. investigation of the impact of our rapid test analyses on practices outside uupcrn was beyond the scope of this study. because of their relative ease of acquisition, throat swabs are the most common samples available for rapid testing but generally have lower sensitivity than other clinical samples. it is possible that rapid tests in uupcrn were conducted predominantly using throat swab samples, which could further compound the problem of a high rate of false negative tests early in the season when the prevalence of influenza is low. we minimized the possibility of a false positive early signal by defining our real - time surveillance alert as a peak that contained a point exceeding the upper control limit followed by a point that did not return to the center line rather than using a single point for the alert. based on this conservative definition, the timeliness of our early alert was similar to that seen in other studies [2628 ]. however, further study using a confirmatory test such as viral culture or polymerase chain reaction would be required to determine whether these earlier peaks in our population are false positives or represent the earliest circulation of influenza in the population. our study demonstrated that poc influenza testing generated an early alert signal, averaging 16 days prior to the epidemic onset of influenza in the community. when this surveillance system was implemented prospectively, intervention clinicians received notification 15 days prior to the epidemic onset. in the outpatient setting, dissemination of relevant influenza surveillance information has the potential to aid clinicians in case management [14, 34 ]. we learned several lessons from our year of implementation that relate to the surveillance metrics, clinician notification, and the value of poc testing as a surveillance measure. in the mild 2008 - 09 season, the epidemic onset was difficult to recognize visually from the prospective graphic ; however, the surveillance metric (based on the western electric rules) we used provided an identifiable early alert, as well as start to the epidemic period. in hindsight, lack of a typical epidemic curve seen in the spc graph of the 2008 - 09 season may have been an early indication of the unusual season. in the future, curves deviating from the expected shape or timeframe should prompt investigation to determine potential reason(s) for the deviation. the goal of our study was to return surveillance information to clinicians, but we did not create an avenue for clinicians to return any clinical observations that were not part of the automatically extracted data. given the atypical 2008 - 09 season, clinicians may have had insights that would have been important in an investigation of the unusual shape of the epidemic curve. studies have reported that clinician perception of the care they provide, as well as actual diagnosis and use of resources, is improved when surveillance data are available to them [18, 19, 28, 34, 3638 ]. however, we did not find significant differences between intervention and control clinics with regard to the clinician behaviors we measured. it is possible that our nonsignificant results are a result of one or a combination of factors associated with our messaging practices. first, our email message was sent through an intranet limiting the message to a text summary. a study of utah clinicians documented a preference for time series graphs over textual summaries to represent trends for respiratory pathogens. clinician preference for frequency of such notifications has varied from weekly to quarterly when an outbreak occurs and likely reflects the fact that different circumstances require different notification frequencies [4042 ]. finally, we did not measure receipt of the email, whether it was opened, understood, agreed with, diffused to other clinical staff, or whether and when the prevention recommendations were implemented. additional evaluation of the format and frequency of data delivery, as well as clinician knowledge, opinions, and behaviors related to receipt of surveillance messages, appears warranted to determine the optimal circumstances for providing relevant information that improves clinical decision - making. the earlier notification from rapid test positivity, coupled with its ability to identify the epidemic period even in seasons when ili never exceeded the threshold, suggests that it is a more accurate surveillance measure than ili. the ili clinical case definition was first implemented for the 1982 - 83 season, prior to the availability of point - of - care influenza testing, and no substantive changes have been made in outpatient surveillance in the interim. one of the main concerns voiced about using rapid influenza tests is the low sensitivity (about 70%), resulting in poor predictive value when the prevalence is low, that is, early in the season [33, 37 ]. however, at a population level, rapid test positivity in uupcrn clinics was a consistently better indicator of the onset of the epidemic period than ili. our findings suggest two changes to improve surveillance for future influenza seasons : (1) a shift in the use of rapid influenza tests from diagnosis to surveillance and (2) a shift in influenza surveillance from ili to rapid test positivity. results from our study were based on rapid test positivity as the test was used in clinical practice ; further research is needed to determine the conditions (e.g., sampling strategies for clinicians and patient identification) under which this test would be used as a surveillance tool. | objectives. disease surveillance combines data collection and analysis with dissemination of findings to decision makers. the timeliness of these activities affects the ability to implement preventive measures. influenza surveillance has traditionally been hampered by delays in both data collection and dissemination. methods. we used statistical process control (spc) to evaluate the daily percentage of outpatient visits with a positive point - of - care (poc) influenza test in the university of utah primary care research network. results. retrospectively, poc testing generated an alert in each of 4 seasons (20042008, median 16 days before epidemic onset), suggesting that email notification of clinicians would be 9 days earlier than surveillance alerts posted to the utah department of health website. in the 2008 - 09 season, the algorithm generated a real - time alert 19 days before epidemic onset. clinicians in 4 intervention clinics received email notification of the alert within 4 days. compared with clinicians in 6 control clinics, intervention clinicians were 40% more likely to perform rapid testing (p = 0.105) and twice as likely to vaccinate for seasonal influenza (p = 0.104) after notification. conclusions. email notification of spc - generated alerts provided significantly earlier notification of the epidemic onset than traditional surveillance. clinician preventive behavior was not significantly different in intervention clinics. |
chiral gas chromatography (gc) analyses were performed by using a heptakis(2,3dioacetyl6otbutyldimethylsilyl)cyclodextrincolumn (machereynagel, dren, germany) on a gc14a gas chromatograph (shimadzu, tokyo, japan) and by using a heptakis(2,6odiomethyl3opentyl)cyclodextrincolumn (machereynagel, dren, germany) on a gc2010 gas chromatograph (shimadzu, tokyo, japan) equipped with an aoc20i autoinjector (shimadzu, tokyo, japan). chiral hplc analyses were performed by using the column odh (daicel chemical industries, osaka, japan) with nhexane and 2propanol as solvent on a hitachi elite lachrom device (hitachi high technologies america, san jose, usa). ag (zwingenberg, germany) and used as glycerolstabilized crude cell extracts or as lyophilisates (brsehaber., 2008). plasmids containing the genes for the esterases a3 (elend., 2006), ce (elend., 2006) and cl1 (schmeisser., 2003) were cloned as described. one equivalent (18 mmol of 1a or 3a, and 20 mmol of 2a) of the phenylalkyl carboxylic acid was stirred with 3 eq. of ethanol at 110c in toluene as solvent with some drops of concentrated sulfuric acid as catalyst. next, the solvent was evaporated and the product was purified by silica gel column chromatography (npentane / ethyl acetate, 10:1). using this procedure, 1b was obtained as light yellow oil (1.8 g, 9.3 mmol, 52% yield), 2b was obtained as colourless oil (2.6 g, 14.6 mmol, 73% yield) and 3b was obtained as colourless oil (2.5 g, 13 mmol, 72% yield). the structures were confirmed by nmr spectroscopy and matched literature data (yang., 1999). the test for hydrolytic activity towards the target substrates was based on a ph assay (baumann., 2000). wells were filled with 20 l of a substrate stock solution (200 mm in dmso), 5 l of a solution of the ph indicator bromothymol blue (btb, 16 mm in 16% ethanol and 84% water), 30 l of a glycerol stock solution of the different esterases and 145 l of sodium phosphate buffer (5 mm, ph 7.4) to get a final volume of 200 l. positive controls contained 5 l of btb solution, 175 l of sodium phosphate buffer (5 mm, ph 7.4) and 20 l of dmso containing the correspondent acids 1a3a at final concentrations of 120 mm. each mtp was then sealed with adhesive foil, incubated at 37c for 72 h and stirred at 400 r.p.m. in a thermoshaker (eppendorf, hamburg, germany). active esterases were then identified by visible colour changes and used in analytical scale experiments. to a stirred solution of the ethyl esters 1b3b (0.076 mmol of 1b or 2b, or 0.15 mmol of 3b) in 1.8 ml of sodium phosphate buffer (50 mm, ph 7.4) and 10% dmso if necessary, 200 l of esterase solution was added to a total volume of 2 ml. the reaction mixture was stirred at 1000 r.p.m. in a thermoshaker at 20c or 30c. samples (400 l) were taken after 2, 7 and 24 h. these samples were acidified with 20 l of 1 n hcl and subsequently extracted three times with 400 l diethyl ether. the combined organic layers were dried over anhydrous sodium sulfate and afterwards the organic solvent was evaporated in a speed vac (thermo fisher scientific, langenselbold, germany). the mixture was then treated with tms diazomethane (hashimoto., 1981) in order to convert the carboxylic acid (hydrolysis product) in its methyl ester to allow analysis of substrate and product concentration and optical purity in one run by gc. conversion and e values were then calculated from the enantiomeric excesses from substrate and product following the equations developed by chen and colleagues (1982). to a 40 mm solution of 1b3b (1 g) in sodium phosphate buffer (50 mm, ph 7.4) containing 10% (v / v) dmso, esterase (lyophilisate dissolved in buffer, units as given in table 2) was added and hydrolysis was performed at 20c or 30c. after tlc analysis indicated 50% conversion, the reaction was stopped by addition volume of 1 n hcl, and substrate and product were extracted three times with diethyl ether. after drying the organic phase over anhydrous na2so4, the solvent was removed by evaporation under reduced pressure and the product was purified by column chromatography (npentane / ethyl acetate). the purity of the biocatalysis products was confirmed by nmr spectroscopy and was in all cases > 99%. using this protocol, (r)()1b was obtained as a light yellow oil (411 mg, 21 mmol, 41.4% yield). (s)(+)1a was obtained as colourless oil (230 mg, 14 mmol, 27.7% yield, 72%ee) ; (s)(+)1b was obtained as light yellow oil (420 mg, 2.2 mmol, 42% yield). enantiomeric excess was determined after hydrolysis to 1a as 87%ee ; (r)()1a was obtained as light yellow oil (330 mg, 2 mmol, 38.5% yield, 98%ee) ; (r)()2b was obtained as colourless oil (164 mg, 9.2 mmol, 16.4% yield). enantiomeric excess was determined after hydrolysis to 2a as 82%ee ; (s)(+)2a was obtained as a white powder (181 mg, 12 mmol, 21.5% yield, 97%ee) ; (s)(+)2b was obtained as a colourless oil (218 mg, 12.2 mmol, 28.3% yield). enantiomeric excess was determined after hydrolysis to 2a as 92%ee ; (r)()2a was obtained as a white powder (180 mg, 12 mmol, 21.3%, 56%ee) ; (r)()3b was obtained as colourless oil (380 mg, 1.99 mmol, 38% yield). enantiomeric excess was determined after hydrolysis to 3a as 69%ee ; (s)(+)3a was obtained as yellow oil (360 mg, 2.18 mmol, 41.9% yield, > 99%ee) ; (s)(+)3b was obtained as colourless oil (150 mg, 0.78 mmol, 15% yield). enantiomeric excess was determined after hydrolysis to 3a as 97%ee ; (r)()3a was obtained after column chromatography (npentane / ethyl acetate) as yellow oil (330 mg, 2 mmol, 39% yield, 95%ee). chiral gas chromatography (gc) analyses were performed by using a heptakis(2,3dioacetyl6otbutyldimethylsilyl)cyclodextrincolumn (machereynagel, dren, germany) on a gc14a gas chromatograph (shimadzu, tokyo, japan) and by using a heptakis(2,6odiomethyl3opentyl)cyclodextrincolumn (machereynagel, dren, germany) on a gc2010 gas chromatograph (shimadzu, tokyo, japan) equipped with an aoc20i autoinjector (shimadzu, tokyo, japan). chiral hplc analyses were performed by using the column odh (daicel chemical industries, osaka, japan) with nhexane and 2propanol as solvent on a hitachi elite lachrom device (hitachi high technologies america, san jose, usa). ag (zwingenberg, germany) and used as glycerolstabilized crude cell extracts or as lyophilisates (brsehaber., 2008). plasmids containing the genes for the esterases a3 (elend., 2006), ce (elend., 2006) and cl1 (schmeisser., 2003) were cloned as described. one equivalent (18 mmol of 1a or 3a, and 20 mmol of 2a) of the phenylalkyl carboxylic acid was stirred with 3 eq. of ethanol at 110c in toluene as solvent with some drops of concentrated sulfuric acid as catalyst. next, the solvent was evaporated and the product was purified by silica gel column chromatography (npentane / ethyl acetate, 10:1). using this procedure, 1b was obtained as light yellow oil (1.8 g, 9.3 mmol, 52% yield), 2b was obtained as colourless oil (2.6 g, 14.6 mmol, 73% yield) and 3b was obtained as colourless oil (2.5 g, 13 mmol, 72% yield). the structures were confirmed by nmr spectroscopy and matched literature data (yang., 1999). the test for hydrolytic activity towards the target substrates was based on a ph assay (baumann., 2000). wells were filled with 20 l of a substrate stock solution (200 mm in dmso), 5 l of a solution of the ph indicator bromothymol blue (btb, 16 mm in 16% ethanol and 84% water), 30 l of a glycerol stock solution of the different esterases and 145 l of sodium phosphate buffer (5 mm, ph 7.4) to get a final volume of 200 l. positive controls contained 5 l of btb solution, 175 l of sodium phosphate buffer (5 mm, ph 7.4) and 20 l of dmso containing the correspondent acids 1a3a at final concentrations of 120 mm. each mtp was then sealed with adhesive foil, incubated at 37c for 72 h and stirred at 400 r.p.m. in a thermoshaker (eppendorf, hamburg, germany). active esterases were then identified by visible colour changes and used in analytical scale experiments. to a stirred solution of the ethyl esters 1b3b (0.076 mmol of 1b or 2b, or 0.15 mmol of 3b) in 1.8 ml of sodium phosphate buffer (50 mm, ph 7.4) and 10% dmso if necessary, 200 l of esterase solution was added to a total volume of 2 ml. the reaction mixture was stirred at 1000 r.p.m. in a thermoshaker at 20c or 30c. samples (400 l) were taken after 2, 7 and 24 h. these samples were acidified with 20 l of 1 n hcl and subsequently extracted three times with 400 l diethyl ether. the combined organic layers were dried over anhydrous sodium sulfate and afterwards the organic solvent was evaporated in a speed vac (thermo fisher scientific, langenselbold, germany). the mixture was then treated with tms diazomethane (hashimoto., 1981) in order to convert the carboxylic acid (hydrolysis product) in its methyl ester to allow analysis of substrate and product concentration and optical purity in one run by gc. conversion and e values were then calculated from the enantiomeric excesses from substrate and product following the equations developed by chen and colleagues (1982). to a 40 mm solution of 1b3b (1 g) in sodium phosphate buffer (50 mm, ph 7.4) containing 10% (v / v) dmso, esterase (lyophilisate dissolved in buffer, units as given in table 2) was added and hydrolysis was performed at 20c or 30c. after tlc analysis indicated 50% conversion, the reaction was stopped by addition volume of 1 n hcl, and substrate and product were extracted three times with diethyl ether. after drying the organic phase over anhydrous na2so4, the solvent was removed by evaporation under reduced pressure and the product was purified by column chromatography (npentane / ethyl acetate). the purity of the biocatalysis products was confirmed by nmr spectroscopy and was in all cases > 99%. using this protocol, (r)()1b was obtained as a light yellow oil (411 mg, 21 mmol, 41.4% yield). (s)(+)1a was obtained as colourless oil (230 mg, 14 mmol, 27.7% yield, 72%ee) ; (s)(+)1b was obtained as light yellow oil (420 mg, 2.2 mmol, 42% yield). enantiomeric excess was determined after hydrolysis to 1a as 87%ee ; (r)()1a was obtained as light yellow oil (330 mg, 2 mmol, 38.5% yield, 98%ee) ; (r)()2b was obtained as colourless oil (164 mg, 9.2 mmol, 16.4% yield). enantiomeric excess was determined after hydrolysis to 2a as 82%ee ; (s)(+)2a was obtained as a white powder (181 mg, 12 mmol, 21.5% yield, 97%ee) ; (s)(+)2b was obtained as a colourless oil (218 mg, 12.2 mmol, 28.3% yield). enantiomeric excess was determined after hydrolysis to 2a as 92%ee ; (r)()2a was obtained as a white powder (180 mg, 12 mmol, 21.3%, 56%ee) ; (r)()3b was obtained as colourless oil (380 mg, 1.99 mmol, 38% yield). enantiomeric excess was determined after hydrolysis to 3a as 69%ee ; (s)(+)3a was obtained as yellow oil (360 mg, 2.18 mmol, 41.9% yield, > 99%ee) ; (s)(+)3b was obtained as colourless oil (150 mg, 0.78 mmol, 15% yield). enantiomeric excess was determined after hydrolysis to 3a as 97%ee ; (r)()3a was obtained after column chromatography (npentane / ethyl acetate) as yellow oil (330 mg, 2 mmol, 39% yield, 95%ee). | summaryenantiomerically pure arylalkyl carboxylic acids are important synthetic intermediates for the preparation of a wide range of compounds with biological and pharmacological activities. a library of 83 enzymes isolated from the metagenome was searched for activity in the hydrolysis of ethyl esters of three racemic phenylalkyl carboxylic acids by a microtiter platebased screening using a phindicator assay. out of these, 20 enzymes were found to be active and were subjected to analytical scale biocatalysis in order to determine their enantioselectivity. the most enantioselective and also enantiocomplementary biocatalysts were then used for preparative scale reactions. thus, both enantiomers of each of the three phenylalkyl carboxylic acids studied could be obtained in excellent optical purity and high yields. |
menopause is associated with an increase in intra - abdominal fat, which is considered a major risk factor of atherosclerotic cardiovascular disease (cvd). however, whether an increased risk of cvd in postmenopausal women is due to altered body composition, changes in reproductive hormones, or some other physiological process associated with menopause has not been clearly established. it is also uncertain as to what extent androidal fat mass may influence cvd risk factors in healthy nonobese women. the risk of chronic disease is perceived to be considerably higher in obese compared to normal weight adults. however, gautier. recently reported that the effect of waist circumference, reflecting androidal fat mass, in increasing the risk for diabetes was more profound among men and women with a normal body mass index (bmi) of less than 25 kg / m than among those with above normal bmi. thus, it appears that efforts to reduce abdominal fat accumulation would be beneficial regardless of bmi category. this study was ancillary to the soy isoflavones for reducing bone loss (sirbl) study, a randomized, double - blind, placebo - controlled multicenter (iowa state university (isu) and university of california at davis (ucd)) clinical trial funded by the national institutes of health (nih). we examined contributors (androidal fat mass, duration of menopause, isoflavone treatment, family history of cvd, and dietary quality) to cardiometabolic risk in primarily normal and overweight healthy postmenopausal women. cardiometabolic outcomes included circulating low - density lipoprotein cholesterol (ldl - c), high - density lipoprotein cholesterol (hdl - c), triacylglycerol, glucose, homeostatic model assessment (homa) of insulin resistance, uric acid, c - reactive protein (crp), fibrinogen, and homocysteine (hcy). we hypothesized that consumption of soy isoflavones in tablet form would attenuate the anticipated increase in central fat (androidal) mass during the 36 months of treatment, which in turn may favorably modify circulating concentrations of cardiometabolic risk factors. we also hypothesized that androidal fat mass, other biologic factors, and dietary quality would influence these cardiometabolic risk factors, but that androidal fat mass would predominate. the parent study examined the effect of two doses (80 versus 120 mg / day) of soy protein - derived isoflavone versus placebo tablets for 36 months on bone loss in healthy postmenopausal women (45 to 65 years of age) who were at risk for osteoporosis. the parent study included power analysis on the primary outcome (lumbar spine bone mineral density) in the methodology and has been previously reported. many of these cardiometabolic risk factors were tested throughout the study (ldl - c, hdl - c, tag, glucose, and uric acid) whereas some were only tested through the 12 month time point (homa, crp, fibrinogen, and hcy). 200210884 - 2) approved our study protocol, consent form, and all participant - related materials. approvals for the dual - energy x - ray absorptiometry (dxa) procedures were obtained from each institution 's irb and state department of public health in iowa and california. at prebaseline, each participant received a detailed explanation of the study verbally and in writing before signing an informed consent form. we recruited (2003 to 2005) women throughout the state of iowa and in the greater sacramento and bay area regions in northern california primarily through direct mailing lists, stories in local newspapers, and local / regional radio advertisements. responders (n = 5,255) were initially screened via telephone to identify healthy women (without diseases or conditions, not taking hormones or medications) 65 years who had undergone natural menopause (cessation of menses 1 to 10 years), were not experiencing excessive vasomotor symptoms, nonsmokers (not currently smoking and who had not smoked in the past 6 months), and had a bmi between 18.5 through 29.9 (except for 9 women from ucd who did not meet the range of inclusion criterion : 8 women had bmi values that ranged from 30 to 32.7 and one woman had a bmi of 17.8, but were enrolled because they were deemed healthy). the parent sirbl project established the inclusion / exclusion criteria. we excluded vegans and high alcohol consumers (> 7 servings / week), as well as those who were diagnosed with chronic disease, had a first - degree relative with breast cancer, or who chronically used medication (current : cholesterol - lowering and/or antihypertensive ; past 3 months : antibiotics ; past 6 months : estrogen / progestogen creams, calcitonin ; past 12 months : oral hormones / estrogen or selective estrogen receptor modulators ; ever : bisphosphonates). women who met the initial screening criteria (n = 677) were invited to the clinic for further eligibility screening, including bmd assessment using dxa. the sirbl project focused on disease prevention rather than treatment ; thus, women with bmd lumbar spine (l1-l4) and/or proximal femur t - scores that were low (> 1.5 sd below young adult mean) or high (> 1.0 sd above mean) or with evidence of previous or existing spinal fractures were excluded. once each woman qualified based on bmd, fasting blood was drawn for a clinical chemistry profile. we excluded women with evidence of diabetes mellitus (fasted glucose 6.93 mmol / l (126 mg / dl)), abnormal renal, liver (elevated enzymes), and/or thyroid function, or elevated lipids (ldl - cholesterol > 4.10 mmol / l (160 mg / dl) ; triacylglycerol > 2.25 mmol / we excluded 13 women at ucd from this analysis because they did not meet the entry criteria (11 had thickened endometrium, 1 had breast cancer, 1 could not provide a baseline blood sample). we excluded an additional 19 women who did not have body composition data at either 12, 24, or 36 months because they dropped out of the study, resulting in a sample size of 224 women based upon androidal fat as our primary covariate of interest in this ancillary project. to meet the objectives of the parent project, participants at each location (isu, ucd) were stratified according to baseline proximal femur bmd (high, medium, and low) based upon nhanes iii database population values and randomly assigned to one of three treatment groups : placebo control, 80 mg isoflavones, or 120 mg isoflavones. tablets were provided by the archer daniels midland co. (decatur, il) ; tablet composition has been described previously. an independent researcher (patricia murphy) at isu confirmed that the actual isoflavone doses (mean sd, mg / d) were similar to those formulated and tested by archer daniels midland, respectively : control = 0 compared with 0.3 0.4 ; 80 mg = 89.5 5.0 compared with 84.3 4.5 ; 120 mg = 124.0 7.7 compared with 122.5 3.4. participants in each group were instructed to take three compressed tablets / d. to preserve the double - blind nature of the study, bottles did not indicate treatment assignment. for this study, we used anthropometric and body composition measurements that included weight, height, whole body lean and fat mass, and androidal fat mass, as well as fasting concentrations of lipids / lipoproteins, glucose, and uric acid. in addition, circulating insulin, homa (calculated as fasting glucose (mg / dl) fasting insulin (u / ml)/405), crp, fibrinogen, hcy, and red blood cell (rbc) folate concentrations were assessed at baseline and 12 months. standing height was taken twice (average value recorded) with a wall - mounted stadiometer (model s100 ; ayrton corp., prior lake, mn) and weight was measured at isu using a balance beam scale (abco health - o - meter ; bridgeview, il) and at ucd using an electronic scale (circuits and systems inc ; e. rockaway, ny). women wore hospital scrubs or shorts and a t - shirt, and removed their shoes, belts, watches, and jewelry for the duration of assessment. to ensure standardized data collection, body composition measurements were obtained by certified cross - trained dxa operators using matching dxa instruments (delphi w hologic inc ; bedford, ma) at each site that were calibrated daily. to further ensure quality control, one operator assessed overall composition from the whole body dxa scans for both sites. regional adiposity analysis was performed by one analyzer (lr) who sectioned each whole body dxa scan into waist, hip, and thigh regions based on bone landmarks using special software (discovery version 12.3:7). the hip region began below the fourth lumbar vertebrae and extended to the tip of the greater trochanter of the femur. androidal fat mass (kg) for each participant was the sum of waist and hip fat mass. phlebotomists collected fasted (9 h) blood samples between 7:00 and 8:00 am. we separated serum (allowed to clot for 30 min prior to centrifugation) and plasma from whole blood and centrifuged samples for 15 minutes (4c) at 1000 g, storing aliquots at 80c until analyses. certified clinical laboratories (labcorp ; kansas city, ks for isu and ucd medical center ; sacramento, ca for ucd) performed a chemistry panel (including serum glucose, lipid profile, and uric acid) on each participant at each time point. we measured the remaining analytes (serum insulin, serum crp, plasma fibrinogen, plasma hcy, and rbc folate) for each participant in batch from isu and ucd samples at baseline and 12 months in duplicate at isu. we used sufficient in - house sera / plasma as quality - control samples (frozen at 80c) to run with each kit to calculate interassay coefficient of variation (cv) ; we used duplicate samples to calculate intraassay cv. the low - to - normal and normal - to - high controls for each kit were well within the acceptable ranges. serum insulin (u / ml) concentration was determined with a radioimmunoassay kit (linco research, st charles, mo, usa) using a cobra ii series autogamma counting system (packard instrument company ; meriden, ct, usa). serum crp (mg / l) concentration was determined with a high - sensitivity sandwich enzyme - linked immunosorbent assay kit (alpco diagnostics ; salem, nh) and plasma (heparinized) fibrinogen (mg / ml) concentration was determined with a sandwich enzyme - linked immunosorbent assay kit (assaypro ; st. charles, mo) using a microtiter plate reader (elx808 ; bio - tek instruments, inc., the intra - assay cvs for crp and fibrinogen were 3.7% and 2.7%, respectively ; the interassay cvs for crp and fibrinogen were 6.0% and 2.3%, respectively. total hcy (mol / l) concentration was determined using a high - performance liquid chromatography (hplc) method adapted from araki and sako and ubbink.. the total hcy in plasma consists of free hcy (i.e., reduced plus oxidized hcy in the nonprotein fraction of plasma) and protein - bound hcy. n - acetylcysteine (1 mm) was added as an internal standard to the plasma samples prior to derivatization. the fluorescence intensities were measured with excitation at 385 nm and emission at 515 nm, using a jasco fp-1520 fluorescence detector. intracellular (rbc) folate (ng / ml) was measured using a radioactive immunoassay kit (mp biomedicals ; irvine, ca). because four hematocrit values were missing, rbc folate could not be calculated for these four samples. in addition, three baseline samples were incorrectly processed ; thus, rbc folate values have been presented for 217 women. the intra- and interassay cv for rbc folate were 3.2% and 11.8%, respectively. during the enrollment phase, trained interviewers administered three questionnaires to participants : a health and medical history [1012 ], a reproductive history, and a nutrition history [10, 11 ]. we also gathered data on prescription and over - the - counter medications at each time point, as well as previous and/or current use of herbal therapies or dietary supplements (which they were asked to discontinue prior to baseline testing). we assessed dietary intake at each time point using a semiquantitative food frequency questionnaire from block dietary data systems (berkeley, ca). a healthy eating index (hei) score, with higher scores representing greater adherence to federal dietary guidelines, was calculated for each participant based on this questionnaire and included in the statistical analyses as a covariate. we performed statistical analyses using version 2.10.1 of the r software, including version 3.1.96 of the nlme package software and considered results statistically significant (two sided) at p.05. our 3-year longitudinal analyses of ldl - c, hdl - c, triacylglycerol, and glucose included women with complete data at all time points, baseline through 36 months. our 1-year longitudinal analyses of fibrinogen, insulin, homa, hcy, and crp included women with complete data at baseline and 12 months. we constructed longitudinal models to identify significant contributors to each cardiometabolic risk factor (ldl - c, hdl - c, triacylglycerol, uric acid, glucose, insulin, homa, crp, fibrinogen, and hcy). each final longitudinal model included these obligatory variables : treatment (control versus combined treatment with 80 mg or 120 mg of isoflavones or, in other words, no treatment versus treatment), time point (baseline versus 12, 24, or 36 months), treatment by time point interaction, and site (isu versus ucd), as well as potential covariates that included androidal fat mass (kg) adjusted for height, time since last menstrual period (tlmp) (yr) (calculated for each woman : baseline test date date of her last menstrual period), family history of cvd coded as a categorical variable (none versus positive or none versus unknown), and hei score. additionally, the insulin model included glucose as a covariate, and the hcy model included rbc folate as a covariate. independent variables in modeling the outcomes of interest included those variables that were biologically plausible. preliminary models also included dietary fat intake (determined using the block food frequency questionnaire) and physical activity (determined using the paffenbarger physical activity recall), but those variables did not emerge as remotely significant contributors to any of the cardiometabolic risk factors and thus were not included in the final models. separate height adjustments of fat and fat - free mass have been suggested for children by wells and cole ; accordingly, we found a significant impact of height on androidal fat mass among the participants in our study based on a log - log regression analysis (parameter estimate 0.8286, p =.0058). this suggested the use of height - adjusted androidal fat mass (androidal fat mass (g)/height (cm)) (exponent of 0.8286 for height changed to 1 for ease of interpretation). in other words, we adjusted androidal fat mass for height because taller women typically had greater waist circumferences due to their larger frame size and thus appeared to be at higher risk for cvd compared with shorter women, whereas this is not necessarily the case. we performed log transformation of variables with skewed distributions : triacylglycerol, homa, crp, hcy, and rbc folate. restricted maximum likelihood (reml) estimation was used to obtain estimates of variances and correlations between repeated measures. model selection was guided by a stepwise backwards selection based on model diagnostics, such as akaike 's information criterion and bayes information criterion. an overall model fit was obtained based on a likelihood ratio test of the (maximum likelihood fitted) model at hand and the more parsimonious model of only obligatory covariates. significance indicates a failure to accept the null hypothesis of covariates contributing to the model in a random fashion. more specifically, significance indicates that the full model with all of the variables included explains a greater proportion of the variability in the outcome than the parsimonious model. body composition measurements, including androidal fat mass, did not change significantly during the course of the study. the isoflavone treatment did not have an effect on any of the body composition outcomes. the results of longitudinal analysis showed that each cardiometabolic outcome model was highly significant, with overall p - values.0001 for all but the ldl - c model (p =.002). androidal fat mass and site (isu versus ucd) were consistent predictors of all analytes (36 month data) assessed, including lipids / lipoproteins, glucose, and uric acid. in general, androidal fat mass was typically the strongest covariate (positive) in each model (except the model with hdl - c as the outcome, where it was significantly and negatively associated). time point also emerged as a significant covariate in the hdl - c and glucose models, indicating that the median concentrations of these analytes increased with time ; however, the analyte concentrations at subsequent time points were not significantly different compared to baseline. the median glucose concentration was 85 mg / dl at baseline and 88 mg / dl at 36 months. the median hdl - c concentration was 64 mg / dl at baseline and 65 mg / dl at 36 months. family history of cvd had a significant association with glucose : women who were unaware of their family history (do not know, n = 9), taking into account key factors in the model, had on average a 6.8 mg / dl higher glucose concentration compared with women who indicated that they had a positive family history. isoflavone treatment, tlmp, or hei did not influence any of the 36 month analytes. similar to the 36 month outcomes, each 12 month outcome model was highly significant (overall model p - values.0001), except for hcy (p =.23, data not shown). androidal fat mass was positively associated with each outcome (table 3) and was the strongest covariate (positive) in each model. the parameter estimate for site was negative, indicating that the women from ucd had lower values than the women at isu. in addition, tlmp (p =.0020) and tlmp - by - site interaction (p =.0021) contributed significantly, and a positive family history of cvd contributed marginally (p =.070) to fibrinogen concentration. the unadjusted mean insulin concentration was significantly greater (2.86 u / ml ; p.0001) at 12 months compared with baseline. isoflavone treatment or hei did not emerge as significant predictors in any of the 12 month models. the main objective of this study was to identify significant contributors to cardiometabolic risk in primarily normal and overweight (bmi 40% = obese). in conclusion, although cardiometabolic outcomes examined in this study were not affected by isoflavone treatment, each outcome (except for hcy) had a strong, significant association with androidal fat mass. thus, even small changes in androidal fat are likely to alter the cardiometabolic profile in healthy postmenopausal women. the sirbl clinical trial was supported mainly by a grant (ro1 ar046922) from the national institute of arthritis and musculoskeletal and skin diseases (niams). the project was also supported by the nutrition and wellness research center, iowa state university ; usda / ars, western human nutrition research center, clinical and translational science center, clinical research center at the university of california (1m01rr19975 - 01), and national center for medical research (ul1 rr024146). archer daniels midland company (decatur, il) donated soy isoflavone tablets (novasoy) and glaxosmithkline (moon township, pa) donated calcium and vitamin d supplements (os - cal). | we hypothesized that soy isoflavones would attenuate the anticipated increase in androidal fat mass in postmenopausal women during the 36-month treatment, and thereby favorably modify the circulating cardiometabolic risk factors : triacylglycerol, ldl - c, hdl - c, glucose, insulin, uric acid, c - reactive protein, fibrinogen, and homocysteine. we collected data on 224 healthy postmenopausal women at risk for osteoporosis (45.865 y, median bmi 24.5) who consumed placebo or soy isoflavones (80 or 120 mg / d) for 36 months and used longitudinal analysis to examine the contribution of isoflavone treatment, androidal fat mass, other biologic factors, and dietary quality to cardiometabolic outcomes. except for homocysteine, each cardiometabolic outcome model was significant (overall p - values from.0001 to.0028). androidal fat mass was typically the strongest covariate in each model. isoflavone treatment did not influence any of the outcomes. thus, androidal fat mass, but not isoflavonetreatment, is likely to alter the cardiometabolic profile in healthy postmenopausal women. |
cage : cap analysis of gene expression, a method based on selection of rnas containing the 5 cap modification and obtaining short sequences or tags near the 5 end of these rnas. encode : encyclopedia of dna elements, an nhgri - sponsored project aimed at empirically identifying functionally important element in the human genome sequence, http://www.genome.gov/10005107. genomic dark matter : usually refers to the portion of a genome that does not correspond to exons (coding or non - coding) of annotated mrnas. outward positioned primers to amplify toward the 5 and 3 end of an rna molecule from a point inside the molecule. rnaseq : a method to quantify and profile rna population in a cell based on massive sequencing of short (typically less than 100 bases) regions of a large number of rna molecules. typically, sequencing is conducted on cdna, rather than rna, thus cdnaseq would have been more appropriate. however, rnaseq is used for historical reasons. a note, since direct rna sequencing is now possible, a bona fide rnaseq analysis should somehow be distinguished from cdnaseq. single - molecule sequencing : a method where a single - molecule of a nucleic acid is sequenced directly as opposed methods that obtain sequence signal from a population of molecules. tiling array : a microarray platform designed to interrogate genomic sequence with a certain resolution set by the distance between the probes. opposite in concept to exon arrays where probes are designed only to the annotated regions of interest. vlinc : very long intergenic non - coding rna region, identified based on continuous rnaseq signal that spans genomic regions of 50 kb or longer (often much longer) in the area of genome where no annotated gene has been found. the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. | the mysteries surrounding the 9798% of the human genome that does not encode proteins have long captivated imagination of scientists. does the protein - coding, 23% of the genome carry the 9798% as a mere passenger and neutral cargo on the evolutionary path, or does the latter have biological function ? on one side of the debate, many commentaries have referred to the non - coding portion of the genome as selfish or junk dna (orgel and crick, 1980), while on the other side, authors have argued that it contains the real blueprint for organismal development (penman, 1995 ; mattick, 2003), and the mechanisms of developmental complexity. thus, this question could be referred to without much exaggeration as the most important issue in genetics today. |
fine needle aspiration cytology (fnac) has been applied routinely as a useful and indispensable method to diagnose thyroid lesions. however, it is still quite difficult to establish a precise diagnosis of follicular lesions of the thyroid preoperatively by cytology. the present study was undertaken to analyse the usefulness of quantitative estimations of various cytological nuclear features in specimens obtained by preoperative fnac that were confirmed by histopathological examination. the study was conducted on 36 patients who presented to the surgical outpatient department with thyroid lesions. all 36 cases were subjected to fine needle aspiration (fna) and tissue sections were obtained subsequently. a concise clinical history, examination, and details of relevant investigations were also obtained. these were helpful in reaching a probable clinical diagnosis as well as in cytohistological evaluation and formulations of the pathological diagnosis. the data included 10 cases each of adenomatoid goiter, follicular neoplasm, papillary carcinoma, 4 cases of medullary carcinoma and 2 cases of anaplastic carcinoma. their ages ranged from 28 to 50 years (average 45 years), and there were nine females and one male. the stained smears were examined with a standard microscope connected to a computerised video system and analyzed with image analyzer proplus v software. using 200 magnifications, three to five fields of vision were sampled randomly and a minimum of 10 nuclei per case were measured automatically and calculated. overlapping of the cells, oddly shaped nuclei and degenerative cells were excluded from the study. the mean nuclear area and the mean nuclear perimeter [figure 1 ] were measured by the calculations of pixels corresponding to the nuclei for the evaluation of the nuclear size in each case. to determine the variation in shape, the circular rate and the largest to the smallest dimension ratio of the nuclei (ls ratio) were calculated [figure 2 ]. in a round circle, if the object is elliptical, the circular rate becomes < 1 ; in contrast, the ls ratio is higher than 1. the coefficient of variation of the nuclear area (nacv) was calculated by expressing the variation in size in an individual case. statistical differences were determined by the analysis of variance (anova) test, both within the group and in between the groups. aspirate showing thyroid follicular epithelial cells highlighting nuclear perimeter (h and e, 200) aspirate showing thyroid follicular epithelial cells highlighting the largest to smallest (l / s) ratio (h and e, 200) the study examined 10 cases each of adenomatoid goiter, follicular neoplasm, papillary carcinoma, 4 cases of medullary carcinoma and 2 cases of anaplastic carcinoma. all clinical data, including the patient 's age, gender and tumor size, revealed no statistically significant difference among the five groups. among the parameters for the quantitative morphometrics of nuclei, the mean nuclear diameter and the perimeter were higher in anaplastic carcinomas and were the least for follicular neoplasms. the circular rate was significantly higher in the group with adenomatous goiter than with follicular neoplasm (p < 0.05). the adenomatous goiter patients had a higher ls ratio than the patients with follicular neoplasm (p < 0.05). the nacv value was 44.24 7.76% in adenomatous goiter, 24.02 4.11% in follicular neoplasm and 66.17 13.88% in papillary carcinoma which showed significant differences between all the groups (p < 0.0001). the data of the nuclear parameters are shown in tables 2 and 3 and figures 36. clinical characteristics of patients chart depicting nuclear area among variants (ag adenomatoid goiter ; fn follicular neoplasm ; pc papillary carcinoma ; mc medullary carcinoma ; ac anaplastic carcinoma) chart depicting perimeter among variants (ag adenomatoid goiter ; fn follicular neoplasm ; pc papillary carcinoma ; mc medullary carcinoma ; ac anaplastic carcinoma) chart depicting circular rate among variants (ag adenomatoid goiter ; fn follicular neoplasm ; pc papillary carcinoma) chart depicting (l / s) largest to smallest ratio among variants (ag adenomatoid goiter ; fn follicular neoplasm ; pc papillary carcinoma ; mc medullary carcinoma ; ac anaplastic carcinoma) however, it is difficult to establish a precise diagnosis of follicular lesions of thyroid preoperatively by cytology. the potential significance of this technique is to distinguish between benign, borderline and malignant lesions, for objective grading of invasive tumors, prediction of prognosis and therapeutic response. morphometry has been described for more than a century because the histological characteristics of normal and abnormal cells have been used as a measure of prognosis and as a way of predicting the cause of the disease. while adaptation of quantitative morphometric analysis as a clinical tool has been discussed in the literature for over 30 years, the usefulness of this approach has helped in cytological grading of breast lesions, but has not yet received widespread acceptance in thyroid lesions owing to limited references and subjectivity.[16 ] rajesh. studied histopathologically proven 19 cases of lobular carcinoma, 30 cases of ductal carcinoma, 10 cases of borderline lesions and 20 cases of benign breast lesions. anova showed that morphometric data may be useful in distinguishing invasive lobular carcinoma from infiltrating ductal carcinoma on cytological smears. however, data were not helpful in distinguishing benign and borderline lesions from invasive lobular carcinoma. nuclear area (na) and nacv were compared with prognostic factors and this analysis reflected the chemosensitivity and the risk of recurrence. there are studies where morphometry has been applied to other organs like nasopharynx, skin, urinary bladder and colon. in our study, we evaluated 10 cases each of adenomatoid goiter, follicular neoplasm, papillary carcinoma, 4 cases of medullary carcinoma and 2 cases of anaplastic carcinoma. the circular rate was significantly higher in the group with adenomatous goiter than with follicular neoplasm. also reported that nuclear features were not helpful in differentiation of follicular carcinomas from adenomas. nagashima. also evaluated variation in nuclear size by using nacv which showed significant differences among follicular carcinomas, adenomas and adenomatous goiters. in our study, the circular rate was higher in papillary carcinoma when compared to medullary carcinoma, which was not documented in any of the studies. fernandez - lopez. evaluated in their study, the prognostic value of the colorectal cancer in 90 patients. it was noticed that the patients with greater nuclear diameters also had a significant poor prognosis than the patients with cells of smaller diameter. this study asserted that the variations could be related to the survival of these patients. analyzed nuclear parameters like nuclear area, axis minor, diameter minor, radius minor, perimeter area in oligodendroglioma and found it to be statistically significant with regard to grading of oligodendrogliomas. studies including larger series of cases investigating detailed nuclear morphometric analysis of subtypes of thyroid carcinoma with longer periods of observation are required in order to demonstrate the association between clinical outcome and morphometric parameters and in order to standardize the morphometric method by giving a suitable cut - off value of nacv in thyroid lesions. when correctly applied with appropriate cutoffs, morphometry can help in preoperative assessment and may act as an adjunct to morphological features in thyroid lesions. | aim : to evaluate the cytomorphometric features in fine needle aspiration cytology (fnac) of thyroid lesions.materials and methods : fnac of 36 thyroid cases was reviewed. the study included 10 cases each of follicular lesion, adenomatous goiter, papillary carcinoma, 4 cases of medullary carcinoma and 2 cases of anaplastic carcinoma. their ages ranged from 28 to 50 years, and there were nine females and one male. morphometric analysis was done on aspiration smears from 36 thyroid lesions. hematoxylin and eosin stained smears were examined using image analyzer proplus v software. morphological parameters measured included mean nuclear diameter, mean nuclear perimeter, mean nuclear area, circular rate, largest to smallest dimension ratio (ls ratio) and coefficient of variation of nuclear area (nacv).statistical analysis : statistical evaluation was carried out using the analysis of variance (anova) test for the five variables, both within the group and in between the groups. the result was considered significant when p < 0.05.results:the follicular carcinomas had higher ls ratio than patients with adenomatous goiters. mean nuclear diameter and the mean nuclear perimeter were higher in anaplastic carcinomas when compared to other subtypes and were the least for follicular neoplasms.conclusion:when correctly applied, quantitative estimation of cytological nuclear features can play an important role in preoperative assessment and can complement morphological features in thyroid lesions. |
maculopathy associated with hypotony was first described by dellaporta in 1954,1 but the term hypotony maculopathy was coined by gass in 1977.2 chorioretinal folds can be identified with a number of conditions, but they are most frequently associated with hypotony.3 when folds extend to the macula, vision loss often results, leading to the term hypotony maculopathy. these changes are perhaps most common following glaucoma surgery and have been more frequently reported during the last 20 years since the initiation of antimetabolite adjuncts for trabeculectomy. conservative management may lead to spontaneous resolution, but some may require surgical intervention. if hypotony is the cause, normalizing the intraocular pressure (iop) will often lead to resolution of the folds. a subset of patients may require additional interventions including pressure patch, anterior chamber injection of viscoelastic, cryotherapy to a filtering bleb or to a cyclodialysis cleft, conjunctival compression sutures, or wound revision.3,4 furthermore, delayed normalization of the iop may result in permanent macular chorioretinal changes and poor vision.3 three cases of hypotony maculopathy following pars plana vitrectomy (ppv) documented by spectral domain ocular coherence tomography (sd - oct) are presented with differing underlying causes. the visual acuity improved after normalization of the iop and lessening of the folds was confirmed by sd - oct. a 47-year - old mexican - american man was treated for a macula - involving retinal detachment in the right eye. he was pseudophakic with a history of high myopia in both eyes and a prior manifest refraction of 22.00 d prior to cataract surgery. the best - corrected visual acuity (bcva) was 20/400 and a rhegmatogenous retinal detachment with several posterior breaks was noted. silicone oil was selected because of the patient s need to urgently return to mexico after surgery. at 5 months postoperatively, the bcva was 20/30, iop was 12 mmhg, the retina was attached, and there were no chorioretinal folds noted. at 1 year following surgery, he maintained a bcva 20/30 and iop 12 mmhg, and the silicone oil was removed. in the immediate postoperative period, the eye was noted to have a bcva of 2/200 and an iop of 3 mmhg. seven days later, an air bubble was injected to pressurize the eye with hypotony - induced chorioretinal folds. a repeat fluid the following day, the bcva was 20/400 with iop 4 mmhg and associated chorioretinal fold in the macula was documented by sd - oct (figure 1a). two weeks following surgery, the bcva was 20/200 with an improved iop of 10 mmhg and a persistent fold. two months after the silicone oil removal, the patient returned with a bcva of 20/25 and iop 9 mmhg. the chorioretinal fold was markedly improved but not completely absent (figure 1b). a 74-year - old hispanic woman presented after sustaining an accidental kick in the head from an infant resulting in a ruptured globe and extruded intraocular lens (iol) in the right eye. the bcva was light perception in the right eye and 20/25 in the left eye, and the iop was 5 mmhg and 20 mmhg in each eye, respectively. the patient underwent surgical repair for the 3-clock - hour superior scleral rupture from the site of prior cataract surgery and attained bcva of 20/200 with an iop of 17 mmhg at 2 weeks postoperatively. a 23-gauge ppv was performed for persistent vitreous hemorrhage, and the scleral wound was revised at that time. by postoperative month 1, her hypotony had resolved with an iop of 30 mmhg and she underwent secondary iol placement with scleral fixated iol and 25-gauge ppv. over the next 3 months, the patient had sustained hypotony and chorioretinal folds by sd - oct and a bcva of 20/50 (figure 2a and 2b). she again underwent revision of the initial scleral wound with resolution of the hypotony and by 1 month postoperative, her vision returned to 20/30, her iop normalized to 16 mmhg, and her chorioretinal folds by sd - oct resolved (figure 2c and 2d). an 86-year - old caucasian man was referred for management of a dislocated iol in the left eye 15 years after cataract surgery. the iol was in the posterior pole on initial examination with a bcva of 20/40 and an iop of 11 mmhg. the eye was found to be hypotonous, with pressures not exceeding 4 mmhg, during the first postoperative month. the patient also had a hyphema and vitreous hemorrhage with vision fluctuating from 20/400 to hand motions. he was found to have chorioretinal folds by sd - oct (figure 3a) and received an intravitreal injection of triamcinolone acetonide 4 mg/0.1 ml with minimal improvement in iop over the following 2 months. contact b - scan ultrasonography demonstrated a cyclodialysis cleft possibly exacerbated by a subluxed iol. he then underwent pupilloplasty and repositioning of the iol and sequential rounds of diode laser to the cleft over the following 2 months with slow increase in pressure from approximately 1 mmhg to 10 mmhg. the maculopathy persisted for several months after the cleft closure with a near visual acuity of jaeger 2. his pressure gradually decreased again, and he was treated with a 23-gauge ppv, membrane peeling, healon injection, and cryotherapy to the cyclodialysis cleft to induce elevation in iop. a vitreous chamber tap was performed but rebound increases in iop occurred, and the patient was taken back for a 23-gauge ppv and healon removal 3 days after initial injection. postoperatively, his iop remained in the low - teens and vision stabilized. his chorioretinal folds by sd - oct resolved (figure 3b) over the ensuing 2 months, and 17 months after his most recent surgery, the bcva had improved to 20/40 with a stable iop ranging from 9 to 12 mmhg. a 47-year - old mexican - american man was treated for a macula - involving retinal detachment in the right eye. he was pseudophakic with a history of high myopia in both eyes and a prior manifest refraction of 22.00 d prior to cataract surgery. the best - corrected visual acuity (bcva) was 20/400 and a rhegmatogenous retinal detachment with several posterior breaks was noted. silicone oil was selected because of the patient s need to urgently return to mexico after surgery. at 5 months postoperatively, the bcva was 20/30, iop was 12 mmhg, the retina was attached, and there were no chorioretinal folds noted. at 1 year following surgery, he maintained a bcva 20/30 and iop 12 mmhg, and the silicone oil was removed. in the immediate postoperative period, the eye was noted to have a bcva of 2/200 and an iop of 3 mmhg. seven days later, an air bubble was injected to pressurize the eye with hypotony - induced chorioretinal folds. a repeat fluid the following day, the bcva was 20/400 with iop 4 mmhg and associated chorioretinal fold in the macula was documented by sd - oct (figure 1a). two weeks following surgery, the bcva was 20/200 with an improved iop of 10 mmhg and a persistent fold. two months after the silicone oil removal, the patient returned with a bcva of 20/25 and iop 9 mmhg. a 74-year - old hispanic woman presented after sustaining an accidental kick in the head from an infant resulting in a ruptured globe and extruded intraocular lens (iol) in the right eye. the bcva was light perception in the right eye and 20/25 in the left eye, and the iop was 5 mmhg and 20 mmhg in each eye, respectively. the patient underwent surgical repair for the 3-clock - hour superior scleral rupture from the site of prior cataract surgery and attained bcva of 20/200 with an iop of 17 mmhg at 2 weeks postoperatively. a 23-gauge ppv was performed for persistent vitreous hemorrhage, and the scleral wound was revised at that time. by postoperative month 1, her hypotony had resolved with an iop of 30 mmhg and she underwent secondary iol placement with scleral fixated iol and 25-gauge ppv. over the next 3 months, the patient had sustained hypotony and chorioretinal folds by sd - oct and a bcva of 20/50 (figure 2a and 2b). she again underwent revision of the initial scleral wound with resolution of the hypotony and by 1 month postoperative, her vision returned to 20/30, her iop normalized to 16 mmhg, and her chorioretinal folds by sd - oct resolved (figure 2c and 2d). an 86-year - old caucasian man was referred for management of a dislocated iol in the left eye 15 years after cataract surgery. the iol was in the posterior pole on initial examination with a bcva of 20/40 and an iop of 11 mmhg. the eye was found to be hypotonous, with pressures not exceeding 4 mmhg, during the first postoperative month. the patient also had a hyphema and vitreous hemorrhage with vision fluctuating from 20/400 to hand motions. he was found to have chorioretinal folds by sd - oct (figure 3a) and received an intravitreal injection of triamcinolone acetonide 4 mg/0.1 ml with minimal improvement in iop over the following 2 months. contact b - scan ultrasonography demonstrated a cyclodialysis cleft possibly exacerbated by a subluxed iol. he then underwent pupilloplasty and repositioning of the iol and sequential rounds of diode laser to the cleft over the following 2 months with slow increase in pressure from approximately 1 mmhg to 10 mmhg. the maculopathy persisted for several months after the cleft closure with a near visual acuity of jaeger 2. his pressure gradually decreased again, and he was treated with a 23-gauge ppv, membrane peeling, healon injection, and cryotherapy to the cyclodialysis cleft to induce elevation in iop. a vitreous chamber tap was performed but rebound increases in iop occurred, and the patient was taken back for a 23-gauge ppv and healon removal 3 days after initial injection. postoperatively, his iop remained in the low - teens and vision stabilized. his chorioretinal folds by sd - oct resolved (figure 3b) over the ensuing 2 months, and 17 months after his most recent surgery, the bcva had improved to 20/40 with a stable iop ranging from 9 to 12 mmhg. chorioretinal folds were diagnosed clinically but confirmed by the use of fluorescein angiography prior to the routine use of sd - oct. a strictly retinal fold could be distinguished from a chorioretinal fold by the lack of involvement of the linear hyper and hypofluorescence of the choroid in the early stages. since the advent and documentation by sd - oct, chorioretinal folds that were previously unrecognized have become better documented, allowing improved correlation with visual acuity and iop.5 in addition to diagnostic assistance, sd - oct may aid in the follow - up of chorioretinal folds. clinically, patients with chorioretinal folds caused by hypotony are more likely to show swelling of the choroid around the optic nerve head and may produce concentric folding that can be confused for papilledema.2 management typically is directed to the source of hypotony in each individual case. with resolution of the hypotony, the macular chorioretinal folds may become less prominent or disappear. however, in prolonged hypotony, the folds may be evidenced by permanent pigmented lines corresponding to the troughs of the folds. in patients who underwent transconjunctival bleb resuturing for hypotony after trabeculectomy, there was no statistically significant difference in iop or bcva when comparing patients with short - term hypotony to those with long - term hypotony.6 long - term hypotony was defined as greater than 4 months. in that study, chorioretinal folds were only recorded in approximately half of the patients during the hypotonous period demonstrating that hypotony does not always lead to a maculopathy but also that these patients may have transiently had folds that spontaneously resolved between follow - up periods. there is the possibility that subtle chorioretinal folds were underdiagnosed in the absence of sd - oct. these findings also suggest that medical management can be successful while reserving surgical intervention for more severe hypotony and folds. in the current series, one of the three patients had documented chorioretinal folds for over a year prior to resolution and still regained baseline visual acuity and normalization of iop. furthermore, evidence has been presented in the form of case reports that delayed intervention, as much as 7 years, may still result in a good visual outcome.7 the chorioretinal folds that present in hypotony maculopathy are often oriented horizontally, radiating temporally from the optic nerve. in order to identify the folds on sd - oct, vertical line scans cases 2 and 3 in this series presented with horizontal folds that would not have been identified if only focusing on the horizontal line scans. this observation emphasizes the importance of two - dimensional sectioning and analysis when evaluating oct in these patients. the three cases of hypotony - induced chorioretinal folds following vitrectomy presented here highlight varied management approaches for resolution of the folds. chorioretinal folds resulting from hypotony maculopathy may resolve spontaneously as the iop normalizes as evidenced by case 1. in scenarios in which hypotony does not resolve with medical therapy, cases 2 and 3, surgical intervention may be considered and can result in good visual outcomes. despite different treatment regimens, all the three cases retained good visual acuity (20/2520/40). | chorioretinal folds may occur as a consequence of hypotony and can be a cause of vision loss when associated with macular involvement. in this report, the spectral domain ocular coherence tomography imaging of three patients with chorioretinal folds before and after management are presented. the cases had unique presentations and each underwent different management approaches, but the results included improved visual acuities and lessened chorioretinal folds. |
the median age of the sample was 60 years (range : 3587 years). all were in - patients in a coronary intensive care unit (getafe hospital, madrid) and were evaluated, in the first instance, within the first 4872 hours after the mi. a number of exclusion criteria were used : having suffered a similar episode on other occasions.being less than 18 years of age.not being fluent in spanish.having a mental condition (e.g. dementia or psychotic episode) impeding a coherent interview. not being fluent in spanish. having a mental condition (e.g. dementia or psychotic episode) impeding a coherent interview. after 45 months (mdn=4.63), follow - up was made with 48 of the patients (i.e., 63% of the original sample) and, after 13 months (mdn=13.21), 33 participants were evaluated (i.e., 43.4% of the original sample). in the analysis of the data from the follow - up, only the final group of 33 participants who completed the three evaluations of the study was included. the principal causes of attrition were : inability to contact the patient due to change of telephone or domicile (32.5%), lack of desire of patient to continue participation or being too busy (32.5%), death of the patient (18.6%), and difficulty in traveling to the hospital to perform the interview (16.3%). an analysis was made to ensure that the participants who continued in t2 and t3 had no socio - demographic or clinical difference to those who did not continue in the study. no significant difference was found with regard to any of the variables analyzed (age, gender, and clinical and psychological measures). the mi patients were interviewed for the first time in the coronary unit 4872 hours after their admission to the hospital (t1). after acceptance to participate in the study and the signing of consent forms, the evaluation protocol was applied for a period of approximately 60 min. five months after the mi (t2) and again after 13 months (t3), the patients were contacted by telephone and called to the hospital where the follow - up protocol was applied for duration of approximately 40 min. within a more extensive protocol, some of the instruments used in the three stages of the evaluation (t1, t2, and t3) were : the ptsd checklist (pcl - c ; weathers, litz, herman, huska, & keane, 1993). this questionnaire evaluates, on a scale of 1 to 5, the severity of the 17 symptoms covering the criteria b (re - experiencing), c (avoidance), and d (hyperarousal) of the dsm - iv (apa, 1994) for post traumatic stress disorder. the cronbach 's alpha values in our study were 0.80 (total score), 0.69 (re - experiencing), 0.68 (avoidance), and 0.62 (hyperarousal). the ptsd checklist (pcl - c ; weathers, litz, herman, huska, & keane, 1993). this questionnaire evaluates, on a scale of 1 to 5, the severity of the 17 symptoms covering the criteria b (re - experiencing), c (avoidance), and d (hyperarousal) of the dsm - iv (apa, 1994) for post traumatic stress disorder. the cronbach 's alpha values in our study were 0.80 (total score), 0.69 (re - experiencing), 0.68 (avoidance), and 0.62 (hyperarousal). first, in order to determine the probable prevalence of ptsd, we implemented the criterion most used in the literature (i.e., total score>44 ; blanchard. second, in order to verify the percentage of participants likely to have separate symptom clusters (i.e., criteria b, c, and d of the dsm - iv - tr (apa, 2000) for ptsd), the percentage of participants meeting each criterion of the pcl - c was analyzed. we considered the criteria met (see vzquez, prez - sales, & matt, 2006) if the participant had a severity rating of 4 or more on the 5-point likert scale in the minimum number of symptoms required for each criterion of the dsm - iv - tr (i.e., one of the five for re - experiencing, three of the seven for avoidance, and two of the five for hyperarousal). three items on a 110 likert - type scale were used, referring to a subjective reaction to the mi : perception of danger (did you feel that your life was in danger ?), if the situation was traumatic for the patient (would you describe the event as traumatic for you ?) and perception of the severity of the situation (to what point would you describe what has happened to you as severe ?). goldberg health questionnaire (ghq-12) (goldberg & williams, 1996). the objective of this questionnaire, widely used in epidemiological studies in the general population, is to evaluate general distress. it consists of 12 items with 4 request options on a likert - type scale. as some authors have proposed recently (hu, steward - brown, twigg, & weich, 2007), the scoring can be divided by separately taking into account the six items that evaluate a state of positive mental health (e.g., have you been able to concentrate on things ?) and the six items that evaluate a state of negative mental health or general distress (e.g., the cronbach 's alpha values were 0.65 (ghq12), 0.51 (ghq6 : positive mental health), and 0.74 (ghq6 : general distress). life orientation test revised (lot - r, scheier, carver, & bridges, 1994). the test measures dispositional optimism or the generalized predisposition towards the expectation of positive results. it consists of six specific items of which three evaluate optimism and three evaluate pessimism. an additional item was added, in the same answer format, which specifically asked about the patient 's expectations with regard to a complete recovery from the infarction (i believe i 'm going to come out of this heart problem i have completely fine). positive and negative affect schedules (watson, clark, & tellegen, 1988). the panas is a widely used instrument for measuring affect by means of a listing of 20 emotions (10 positive and 10 negative). the cronbach 's alpha values were 0.84 for the positive subscale and 0.82 for the negative subscale. perceived importance of the heart failure. three items on a 110 likert - type scale were used, referring to a subjective reaction to the mi : perception of danger (did you feel that your life was in danger ?), if the situation was traumatic for the patient (would you describe the event as traumatic for you ?) and perception of the severity of the situation (to what point would you describe what has happened to you as severe ?). the objective of this questionnaire, widely used in epidemiological studies in the general population, is to evaluate general distress. it consists of 12 items with 4 request options on a likert - type scale. as some authors have proposed recently (hu, steward - brown, twigg, & weich, 2007), the scoring can be divided by separately taking into account the six items that evaluate a state of positive mental health (e.g., have you been able to concentrate on things ?) and the six items that evaluate a state of negative mental health or general distress (e.g., the cronbach 's alpha values were 0.65 (ghq12), 0.51 (ghq6 : positive mental health), and 0.74 (ghq6 : general distress). life orientation test revised (lot - r, scheier, carver, & bridges, 1994). the test measures dispositional optimism or the generalized predisposition towards the expectation of positive results. it consists of six specific items of which three evaluate optimism and three evaluate pessimism. an additional item was added, in the same answer format, which specifically asked about the patient 's expectations with regard to a complete recovery from the infarction (i believe i 'm going to come out of this heart problem i have completely fine). positive and negative affect schedules (watson, clark, & tellegen, 1988). the panas is a widely used instrument for measuring affect by means of a listing of 20 emotions (10 positive and 10 negative). the cronbach 's alpha values were 0.84 for the positive subscale and 0.82 for the negative subscale. the median age of the sample was 60 years (range : 3587 years). all were in - patients in a coronary intensive care unit (getafe hospital, madrid) and were evaluated, in the first instance, within the first 4872 hours after the mi. a number of exclusion criteria were used : having suffered a similar episode on other occasions.being less than 18 years of age.not being fluent in spanish.having a mental condition (e.g. dementia or psychotic episode) impeding a coherent interview. not being fluent in spanish. having a mental condition (e.g. dementia or psychotic episode) impeding a coherent interview. after 45 months (mdn=4.63), follow - up was made with 48 of the patients (i.e., 63% of the original sample) and, after 13 months (mdn=13.21), 33 participants were evaluated (i.e., 43.4% of the original sample). in the analysis of the data from the follow - up, only the final group of 33 participants who completed the three evaluations of the study was included. the principal causes of attrition were : inability to contact the patient due to change of telephone or domicile (32.5%), lack of desire of patient to continue participation or being too busy (32.5%), death of the patient (18.6%), and difficulty in traveling to the hospital to perform the interview (16.3%). an analysis was made to ensure that the participants who continued in t2 and t3 had no socio - demographic or clinical difference to those who did not continue in the study. no significant difference was found with regard to any of the variables analyzed (age, gender, and clinical and psychological measures). the mi patients were interviewed for the first time in the coronary unit 4872 hours after their admission to the hospital (t1). after acceptance to participate in the study and the signing of consent forms, the evaluation protocol was applied for a period of approximately 60 min. five months after the mi (t2) and again after 13 months (t3), the patients were contacted by telephone and called to the hospital where the follow - up protocol was applied for duration of approximately 40 min. within a more extensive protocol, some of the instruments used in the three stages of the evaluation (t1, t2, and t3) were : the ptsd checklist (pcl - c ; weathers, litz, herman, huska, & keane, 1993). this questionnaire evaluates, on a scale of 1 to 5, the severity of the 17 symptoms covering the criteria b (re - experiencing), c (avoidance), and d (hyperarousal) of the dsm - iv (apa, 1994) for post traumatic stress disorder. the cronbach 's alpha values in our study were 0.80 (total score), 0.69 (re - experiencing), 0.68 (avoidance), and 0.62 (hyperarousal). the ptsd checklist (pcl - c ; weathers, litz, herman, huska, & keane, 1993). this questionnaire evaluates, on a scale of 1 to 5, the severity of the 17 symptoms covering the criteria b (re - experiencing), c (avoidance), and d (hyperarousal) of the dsm - iv (apa, 1994) for post traumatic stress disorder. the cronbach 's alpha values in our study were 0.80 (total score), 0.69 (re - experiencing), 0.68 (avoidance), and 0.62 (hyperarousal). first, in order to determine the probable prevalence of ptsd, we implemented the criterion most used in the literature (i.e., total score>44 ; blanchard., 2004). second, in order to verify the percentage of participants likely to have separate symptom clusters (i.e., criteria b, c, and d of the dsm - iv - tr (apa, 2000) for ptsd), the percentage of participants meeting each criterion of the pcl - c was analyzed. we considered the criteria met (see vzquez, prez - sales, & matt, 2006) if the participant had a severity rating of 4 or more on the 5-point likert scale in the minimum number of symptoms required for each criterion of the dsm - iv - tr (i.e., one of the five for re - experiencing, three of the seven for avoidance, and two of the five for hyperarousal). three items on a 110 likert - type scale were used, referring to a subjective reaction to the mi : perception of danger (did you feel that your life was in danger ?), if the situation was traumatic for the patient (would you describe the event as traumatic for you ?) and perception of the severity of the situation (to what point would you describe what has happened to you as severe ?). the objective of this questionnaire, widely used in epidemiological studies in the general population, is to evaluate general distress. it consists of 12 items with 4 request options on a likert - type scale. as some authors have proposed recently (hu, steward - brown, twigg, & weich, 2007), the scoring can be divided by separately taking into account the six items that evaluate a state of positive mental health (e.g., have you been able to concentrate on things ?) and the six items that evaluate a state of negative mental health or general distress (e.g., the cronbach 's alpha values were 0.65 (ghq12), 0.51 (ghq6 : positive mental health), and 0.74 (ghq6 : general distress). life orientation test revised (lot - r, scheier, carver, & bridges, 1994). the test measures dispositional optimism or the generalized predisposition towards the expectation of positive results. it consists of six specific items of which three evaluate optimism and three evaluate pessimism. an additional item was added, in the same answer format, which specifically asked about the patient 's expectations with regard to a complete recovery from the infarction (i believe i 'm going to come out of this heart problem i have completely fine). positive and negative affect schedules (watson, clark, & tellegen, 1988). the panas is a widely used instrument for measuring affect by means of a listing of 20 emotions (10 positive and 10 negative). the cronbach 's alpha values were 0.84 for the positive subscale and 0.82 for the negative subscale. perceived importance of the heart failure. three items on a 110 likert - type scale were used, referring to a subjective reaction to the mi : perception of danger (did you feel that your life was in danger ?), if the situation was traumatic for the patient (would you describe the event as traumatic for you ?) and perception of the severity of the situation (to what point would you describe what has happened to you as severe ?). the objective of this questionnaire, widely used in epidemiological studies in the general population, is to evaluate general distress. it consists of 12 items with 4 request options on a likert - type scale. as some authors have proposed recently (hu, steward - brown, twigg, & weich, 2007), the scoring can be divided by separately taking into account the six items that evaluate a state of positive mental health (e.g., have you been able to concentrate on things ?) and the six items that evaluate a state of negative mental health or general distress (e.g., did you feel constantly under strain ?). the cronbach 's alpha values were 0.65 (ghq12), 0.51 (ghq6 : positive mental health), and 0.74 (ghq6 : general distress). life orientation test revised (lot - r, scheier, carver, & bridges, 1994). the test measures dispositional optimism or the generalized predisposition towards the expectation of positive results. it consists of six specific items of which three evaluate optimism and three evaluate pessimism. an additional item was added, in the same answer format, which specifically asked about the patient 's expectations with regard to a complete recovery from the infarction (i believe i 'm going to come out of this heart problem i have completely fine). positive and negative affect schedules (watson, clark, & tellegen, 1988). the panas is a widely used instrument for measuring affect by means of a listing of 20 emotions (10 positive and 10 negative). the cronbach 's alpha values were 0.84 for the positive subscale and 0.82 for the negative subscale. table 2 presents the data obtained in each of the questionnaires of the evaluation protocol. characteristics, in the three times of assessment, of the sample who completed all the follow - ups (n=33) notes : pcl, the ptsd checklist ; ghq, goldberg health questionnaire (12- and six - item versions) ; lot - r, life orientation testrevised ; panas, positive and negative affective schedules. in the first place, a series of repeated measures anovas revealed no significant time differences on participants scores on pcl total score [f (2, 29)=2.29, ns ] and on two subscales : pcl - re - experiencing [f (2, 31)=2.69, ns ] and pcl - avoidance [f (2,29)=0.45, ns ]. nevertheless, significant differences were encountered in the scale of hyperarousal [f (2, 31)=3.66, p=0.038 ]. a post - hoc bonferroni analysis showed that hyperarousal was significantly greater in t2 than in t1 (p44, the probable prevalence of significant stress reaction in t1 is very low, rising to a prevalence of 11.1% after 5 months. finally, the number of cases with probable diagnoses of ptsd declines 13 months after the event (3.1%). probable prevalence of ptsd based on a selected cutoff score for the ptsd checklist (pcl - c) and percentage of patients fulfilling criteria for each of the three symptom clusters in regard to the prevalence of specific syndromes (see measures section), it was found that, at each point of measurement, re - experiencing was the criterion that appeared in the greatest number of participants while the criterion of avoidance was that of least prevalence in the patients evaluated (table 3). the progression of the three steps over time was similar : the moment at which most subjects met the criteria was at five months after the mi, while mitigating later. table 2 presents the data obtained in each of the questionnaires of the evaluation protocol. characteristics, in the three times of assessment, of the sample who completed all the follow - ups (n=33) notes : pcl, the ptsd checklist ; ghq, goldberg health questionnaire (12- and six - item versions) ; lot - r, life orientation testrevised ; panas, positive and negative affective schedules. in the first place, a series of repeated measures anovas revealed no significant time differences on participants scores on pcl total score [f (2, 29)=2.29, ns ] and on two subscales : pcl - re - experiencing [f (2, 31)=2.69, ns ] and pcl - avoidance [f (2,29)=0.45, ns ]. nevertheless, significant differences were encountered in the scale of hyperarousal [f (2, 31)=3.66, p=0.038 ]. a post - hoc bonferroni analysis showed that hyperarousal was significantly greater in t2 than in t1 (p44, the probable prevalence of significant stress reaction in t1 is very low, rising to a prevalence of 11.1% after 5 months. finally, the number of cases with probable diagnoses of ptsd declines 13 months after the event (3.1%). probable prevalence of ptsd based on a selected cutoff score for the ptsd checklist (pcl - c) and percentage of patients fulfilling criteria for each of the three symptom clusters in regard to the prevalence of specific syndromes (see measures section), it was found that, at each point of measurement, re - experiencing was the criterion that appeared in the greatest number of participants while the criterion of avoidance was that of least prevalence in the patients evaluated (table 3). the progression of the three steps over time was similar : the moment at which most subjects met the criteria was at five months after the mi, while mitigating later. the sudden and brusque occurrence of a mi is a potentially serious and life - threatening condition. it is interesting that, during the first few days, the heart attack was not considered to be a highly traumatic event by the participants but, 5 months and 13 months after the mi, it was considered so. nevertheless, this perceived severity and the perception of the event as traumatic did not correspond to a grand estimation that one 's life was in danger. probably, the fact that the patient was treated rapidly and clinically stabilized in a safe, hospital environment, leads to a weakening of the perception of risk, which in turn can explain the relatively low prevalence of symptoms related to stress encountered in this study and in studies of similar design (bennett., it is likely that rapid medical intervention and hospitalization may prevent people from developing intense sensations of horror or despair, which can be considered a necessary prerequisite for the diagnosis of ptsd and a strong predictor of the severity of this condition (brewin, andrews, & rose, 2000)., it is more likely that these differences reveal that at t1 (i.e., only a few days after the event), mi patients may not have enough perspective to judge the medical and psychosocial consequences of the event. interestingly, contrary to other studies (ginzburg., 2003), the high perception of the event as severe and traumatic did not correspond to a high level of ptsd in our sample. it is possible that this elevated perception of seriousness may be the result of the impact of the heart attack on other areas of functioning (e.g., work performance, physical and sexual activity, perception of disability, and limitations) rather than the direct effects of the mi itself. in any case, the study reveals that the perception of the event can change significantly with the passing of time and it is necessary that clinicians be aware of this factor. one of the advantages of the design of our study was the ability to be able to make an immediate evaluation of the patient 's affective state during the first hours following the heart attack. in general we have found that there are no significant changes either in total symptomatology in the pcl - c or in patterns of symptoms related to the stress (i.e., re - experiencing and avoidance) over time (i.e., 5 months and 13 months later). the only exception was a significant increase of hyperarousal from t1 to t2, returning in t3 to its basal level. as changes in arousal are not a common clinical characteristic of mi (thygesen, alpert, & white, 2007), it is likely that the increases in hyperarousal detected in our sample are instead part of a psychological reaction in response to this serious medical condition. there were neither significant changes with regard to positive emotions nor optimism present from the onset. these positive emotions were significantly greater than the negative ones at all times of measurement, so it is not probable that the affective well - being that was observed from the first days after the event results from an effect of illusory optimism that has been described elsewhere (zoellner & maercker, 2006). it is possible that these positive emotions not only have an important role as buffer factors for negative emotions (fredrickson, 2001), but that they are also important for a better recuperation from the medical problem (howell, kern, & lyubomirsky, 2007 ; vzquez, hervs, rahona, & gomez, 2009). in the case of trauma - related disorders, recent investigations have made clear that positive emotions and cognitions are not necessarily absent (fredrickson, tugade, waugh, & larkin, 2003 ; vzquez & hervs, 2010). although there are few references to positive emotions in patients having suffered an mi (affleck, tennen, & croog, 1987 ; brummett, morey, boyle, & mark, 2009), the finding that these patients are capable of optimism and of feeling relatively intense positive emotions is important. in fact, there is growing evidence showing that resilience is mediated by a complex network of mediational cognitive and motivational processes (lyubomirsky, 2001) in which positive emotions experienced during and after the trauma may have an important role in the attenuation of psychopathological problems (vzquez & hervs, 2010). the pattern of results of our study reveals that, in general, a myocardial infarct, with adequate medical treatment, has a relatively minor effect on the appearance of psychopathological symptoms related to post - traumatic stress. this corresponds also with the low prevalence of psychopathology found in previous studies (ginzburg., 2003 ; hari., 2010) and even in medical problems such as cancer (shelby, golden - kreutz, & andersen, 2008). the results obtained with regard to the prevalence of probable cases of ptsd in our sample are consistent with those found in the literature. we found 11.1% of probable cases at 5 months from the mi and 3.1% at one year following the event. in similar studies, with first - time mi patients and with a comparable measurement period, for example, levels of ptsd of 9.8% are found at 6 months, (shemesh., 2001). although it is difficult to compare our results with other longitudinal studies, due to the fact that none have three measurement periods, and none coincide with regard to the time between measurements, published studies usually show this decrease in the prevalence of ptsd over a period of time (ginzburg., 2003 in a more general manner, the findings of our study corroborate that most people show resilience in adverse situations (wessely, 2004). studies of the general population indicate that whereas traumatic events, as defined in dsm - iv, may affect more than 50% of the general population in the course of their lives (breslau, davis, & andreski, 1995 ; darves - bornoz., 2008), only 13% (515%, if the less severe forms are included) will show ptsd (alonso., 2004 ; kessler., 1995). in the case of physical illnesses, the european study of the epidemiology of mental health showed, in a sample of 8,796 subjects from the general population, that while 10% of the sample had suffered a serious illness, only 2% of these patients had developed ptsd as a result of that illness (darves - bornoz., 2008). the study, nevertheless, has some limitations. in the first place, although the initial number of participants was relatively high, various conditions (including a high death rate) decreased the resulting final sample. however, as has been explained above, the analyses revealed that the final samples of the study in t2 and t3 did not differ significantly from the initial sample either in socio - demographic (age, sex, socio - economical level) or psychopathological variables. secondly, it is possible that the evaluation of the symptoms related to post - traumatic stress could have been better dealt with using structured diagnostic interviews. we chose the pcl - c as it is a relatively efficient instrument to carry out a diagnosis of probable ptsd although it takes into account only the symptom criteria of dsm - iv - tr (i.e., symptom clusters b, c, and d). nevertheless, our results are similar to those obtained in other studies that have employed this style of questionnaires (van driel & op den velde, 1995 ; rocha., 2008). we wish to stress that, in t1, the results of the plc - c inform only of symptoms related to stress, as less than a month had passed since the appearance of the cause of stress. the sole study that has evaluated patients so soon after the mi was that of ginzburg. (2003) who, 4 days after the mi, evaluated the the presence of probable acute stress disorder by using a questionnaire (cardena, 1996). in any case, our investigation shows that one must be cautious with regard to the pyschopathological implications of important physical conditions. the sufferance of a severe and unexpected physical condition does not guarantee the appearance of symptoms related to the trauma. therefore, it is necessary to be very cautious in the use and abuse of the term trauma to characterize common medical problems even when they may be potentially life - threatening. there is no conflict of interest in the present study for any of the authors. | background there is a controversy as to whether the diagnostic umbrella of post - traumatic stress reactions is directly applicable to serious health conditions like myocardial infarction (mi).objective the principal objective of this study was to examine longitudinally the prevalence of posttraumatic stress - related symptoms, throughout three measurement periods, for patients who had suffered a first mi. in addition to the analysis of symptoms related to stress and general distress, the presence of and temporary evolution of positive emotions and optimism in these patients was also evaluated.design a longitudinal study with three periods of evaluation after the mi (time 1 (t1) : 4872 hours, time 2 (t2) : 5 months, and time 3 (t3) : 13 months). results in t1 few symptoms related to the stressful event were found. the probable prevalence of ptsd was 811% at 5 months after the mi and 03% 13 months after the event. with regard to subjective severity of the infarction, although in the first instance patients did not regard the event as excessively traumatic, in the periods t2 and t3 this perception increased significantly [f (2, 32)=20.00 ; p=0.0001 ]. at all times during the measurement period the mean positive affect was significantly greater than the negative affect. conclusions as the results show, the probable prevalence of ptsd, as well as the severity of different symptom clusters, is low at all times of the evaluation. the diagnostic implications of these findings are discussed as well as the uses and abuses of diagnostic labels to characterize the psychological experiences lived through after a potentially life - threatening health problem. |
. interactions of connective tissue cells chiefly fibroblasts with numerous inflammatory mediators and cytokines present at the site of injury initiate an inflammatory response that destroys the collagenous and non - collagenous proteins in the extracellular matrix of the periodontium. cementum of the root becomes exposed to the environment of periodontal pocket or the oral cavity and alterations may occur within exposed hard tissue. structural and compositional changes in the matrix components of cementum have been reported in patients with periodontitis. a number of systemic conditions may affect the host 's immune response to oral pathogens and may exacerbate the periodontal disease. diabetes mellitus, a chronic metabolic disorder that constitutes a major worldwide health problem, is one such condition. many systemic complications such as micro- and macro - angiopathies, neuropathy, and nephropathy are recognized as common complications of diabetes. periodontal disease has been recognized as the sixth complication of diabetes. in large populations, changes in gingiva, alveolar bone, and periodontal ligament have been reported in diabetics. tendency toward enlarged gingivae, sessile or pedunculated gingival polyps, polypoid gingival proliferations, abscess formation, periodontitis, and loosened tooth are the most frequent findings. high levels of glucose present in the crevicular spaces of diabetics for extended period of time can adversely affect the periodontal ligament (pdl) cell function. changes in the density and mineralization of sharpeys fibers on the depository surfaces of alveolar bone have been reported in streptozotocin - induced diabetic swiss mice. inhibition of collagen matrix formation, increased time for mineralization of osteoid, and reduced bone turnover have also been reported in diabetes. however, changes in cementum have received little attention, probably because cementum of root was considered an inactive or unresponsive tissue. histologic sections of human teeth provide evidence that cementum of the root surface is not inactive. it is affected by changes in immediate environment as evidenced by the root surface alterations that occur in periodontitis. defect in defense mechanism may facilitate entry of bacteria and bacterial substances into cementum producing altered root surfaces. formation of advanced glycated end products in response to elevated glucose concentration may modulate the function of cell residing in periodontal ligament altering its ability to repair or regenerate the mineralized tissue. therefore, the aim of the present study was to evaluate and compare the thickness of cementum in diseased and healthy areas of roots of teeth with advanced chronic periodontitis from non - diabetic and type 2 diabetic patients. a comparative observational study was carried out to evaluate and compare the thickness of cementum in diseased and healthy areas of roots of teeth with chronic periodontitis from non - diabetic and type 2 diabetic patients. an informed consent of all the subjects who participated in this investigation was obtained after the possible discomforts, risks, and procedures were fully explained. thirty root specimens, 15 from non - diabetic (group i), and 15 from diabetic patients (group ii) with chronic periodontitis indicated for extraction were selected. out of the 30 specimens, 19 were central incisors (14 maxillary and 5 mandibular) and 11 lateral incisors (8 maxillary and 3 mandibular incisors). diagnosed as chronic periodontitisno previous history of root planning and periodontal flap surgery received by the patientvital teeth with no caries or filling or restorationgrade iii mobility and presence of available bone support, according to the radiography, not exceeding one third of root length. diagnosed as chronic periodontitis no previous history of root planning and periodontal flap surgery received by the patient vital teeth with no caries or filling or restoration grade iii mobility and presence of available bone support, according to the radiography, not exceeding one third of root length. diagnosed as chronic advanced periodontitispatients with type 2 diabetes with a history of diabetes of ranging from 4 to 6 yearsno previous history of root planning and periodontal flap surgery received by the patientvital teeth with no caries or filling or restorationgrade iii mobility and presence of available bone support, according to the radiography, not exceeding one third of root length. diagnosed as chronic advanced periodontitis patients with type 2 diabetes with a history of diabetes of ranging from 4 to 6 years no previous history of root planning and periodontal flap surgery received by the patient vital teeth with no caries or filling or restoration grade iii mobility and presence of available bone support, according to the radiography, not exceeding one third of root length. patients with any systemic diseases or prejudicial habits (tabagism)teeth with caries and subgingival restorationsfractured toothroot canal treated toothteeth in hypofunction missing antagonist, submerged teeth. patients with any systemic diseases or prejudicial habits (tabagism) teeth with caries and subgingival restorations root canal treated tooth teeth in hypofunction missing antagonist, submerged teeth. diabetic patients with any other systemic diseases or prejudicial habits (tabagism)teeth with caries and subgingival restorationsfractured toothroot canal treated toothteeth in hypofunction - missing antagonist, submerged teeth. diabetic patients with any other systemic diseases or prejudicial habits (tabagism) teeth with caries and subgingival restorations root canal treated tooth teeth in hypofunction - missing antagonist, submerged teeth. following local infiltrative anesthesia, with 2% lignocaine with 1:80,000 adrenaline, the level of the gingival margin was marked using a diamond bur. clinical attachment level (cal) and probing depth (pd) measurements were done. all clinical measurements were taken to nearest 0.5 mm using unc-15 probe following which the tooth was extracted. after the extraction, cal was marked by the notch on the extracted tooth using a diamond bur. area of the root surface above the cal (notch) was designated as diseased while the area below the cal (notch) was designated as healthy. the teeth were then fixed with buffered 10% formaldehyde for 48 hours to preserve biofilm coating. the tooth samples were then sectioned corono apically in mesiodistal plane using a thin diamond disc mounted on a slow speed hand piece with distilled water as a coolant solution [figure 1 ]. one half of each specimen was dehydrated in ascending strengths of ethanol to 100% and mounted on to brass metal stub. schematic drawing of the sectioning procedure examination was performed above and below the notch (clinical attachment level) [figure 2 ]. schematic drawing of the evaluated root section by using these micrographs, the thickness of the cementum layers for the healthy and diseased part was measured at three different sites and the average value was calculated [figures 36 ]. the same examiner repeated the readings 3 weeks later in order to evaluate the intraexaminer agreement. measurement of thickness of cementum in non - diabetics (group i) at healthy area (200) measurement of thickness of cementum in non - diabetics (group i) at diseased area (200) measurement of thickness of cementum in diabetics (group ii) at healthy area (200) measurement of thickness of cementum in diabetics (group ii) at diseased area (200) descriptive statistical analysis was carried out in the present study. results of continuous measurements were presented as mean sd (min - max) and results of categorical measurements were presented in number (%). the student t test (two tailed, independent) was used to find the significance of study parameters on continuous scale between two groups (group i and group ii) and the student t test (two tailed, dependent) was used to find the significance of study parameters on continuous scale within each group (above the notch and below the notch). diagnosed as chronic periodontitisno previous history of root planning and periodontal flap surgery received by the patientvital teeth with no caries or filling or restorationgrade iii mobility and presence of available bone support, according to the radiography, not exceeding one third of root length. diagnosed as chronic periodontitis no previous history of root planning and periodontal flap surgery received by the patient vital teeth with no caries or filling or restoration grade iii mobility and presence of available bone support, according to the radiography, not exceeding one third of root length. diagnosed as chronic advanced periodontitispatients with type 2 diabetes with a history of diabetes of ranging from 4 to 6 yearsno previous history of root planning and periodontal flap surgery received by the patientvital teeth with no caries or filling or restorationgrade iii mobility and presence of available bone support, according to the radiography, not exceeding one third of root length. diagnosed as chronic advanced periodontitis patients with type 2 diabetes with a history of diabetes of ranging from 4 to 6 years no previous history of root planning and periodontal flap surgery received by the patient vital teeth with no caries or filling or restoration grade iii mobility and presence of available bone support, according to the radiography, not exceeding one third of root length. patients with any systemic diseases or prejudicial habits (tabagism)teeth with caries and subgingival restorationsfractured toothroot canal treated toothteeth in hypofunction missing antagonist, submerged teeth. patients with any systemic diseases or prejudicial habits (tabagism) teeth with caries and subgingival restorations root canal treated tooth teeth in hypofunction missing antagonist, submerged teeth. diabetic patients with any other systemic diseases or prejudicial habits (tabagism)teeth with caries and subgingival restorationsfractured toothroot canal treated toothteeth in hypofunction - missing antagonist, submerged teeth. diabetic patients with any other systemic diseases or prejudicial habits (tabagism) teeth with caries and subgingival restorations root canal treated tooth teeth in hypofunction - missing antagonist, submerged teeth. following local infiltrative anesthesia, with 2% lignocaine with 1:80,000 adrenaline, the level of the gingival margin was marked using a diamond bur. clinical attachment level (cal) and probing depth (pd) measurements were done. all clinical measurements were taken to nearest 0.5 mm using unc-15 probe following which the tooth was extracted. after the extraction, cal was marked by the notch on the extracted tooth using a diamond bur. area of the root surface above the cal (notch) was designated as diseased while the area below the cal (notch) was designated as healthy. the teeth were then fixed with buffered 10% formaldehyde for 48 hours to preserve biofilm coating. the tooth samples were then sectioned corono apically in mesiodistal plane using a thin diamond disc mounted on a slow speed hand piece with distilled water as a coolant solution [figure 1 ]. one half of each specimen was dehydrated in ascending strengths of ethanol to 100% and mounted on to brass metal stub. schematic drawing of the sectioning procedure examination was performed above and below the notch (clinical attachment level) [figure 2 ]. schematic drawing of the evaluated root section by using these micrographs, the thickness of the cementum layers for the healthy and diseased part was measured at three different sites and the average value was calculated [figures 36 ]. the same examiner repeated the readings 3 weeks later in order to evaluate the intraexaminer agreement. measurement of thickness of cementum in non - diabetics (group i) at healthy area (200) measurement of thickness of cementum in non - diabetics (group i) at diseased area (200) measurement of thickness of cementum in diabetics (group ii) at healthy area (200) measurement of thickness of cementum in diabetics (group ii) at diseased area (200) descriptive statistical analysis was carried out in the present study. results of continuous measurements were presented as mean sd (min - max) and results of categorical measurements were presented in number (%). the student t test (two tailed, independent) was used to find the significance of study parameters on continuous scale between two groups (group i and group ii) and the student t test (two tailed, dependent) was used to find the significance of study parameters on continuous scale within each group (above the notch and below the notch). fifteen root specimen from non - diabetic (group i) and 15 root specimen from diabetic patients (group ii) were evaluated with sem. the mean age of patients was 48.4 years, (non - diabetic patients : 43 years and diabetic patients : 53.8 years) of which 73.3% were males and 26.7% females (22 males, eight females). the mean values of pd and cal for group i was 2.33 1.59 mm and 4.80 1.14 mm, respectively. the mean values of pd and cal for group ii was 3.07 1.53 mm and 5.53 1.59 mm, respectively. although the mean values for pd and cal were higher in group ii compared to group i, the differences were not statistically significant. the mean thickness of cementum in group i in healthy areas was 93.04 25.44 m (53.95 - 133.72 m) while in diseased areas was 89.77 22.47 m (45.34 - 132.95 m). the mean thickness of cementum for group ii in healthy areas was 105.38 41.34 m (55.95 - 133.72 m) and in diseased areas was 104.11 38.18 m (50.58 - 168.50 m). the value of thickness of cementum in both group i and ii was higher in healthy areas compared to diseased areas although the differences were not statistically significant. thickness of cementum in both healthy and diseased areas was higher in group ii as compared to group i, but was not statistically significant. thus, irrespective of the type of specimen, the thickness of cementum was found to be higher in healthy areas compared to diseased areas, but was not statistically significant. cementum is a thin layer of mesenchymal tissue covering the dentine of the root and is one of supporting tissues of the periodontium. the other tissues that support the tooth are the alveolar bone and the periodontal ligament. it is characterized by an inflammatory process that gradually spreads in an apical direction, causing resorption of alveolar bone as well as loss of soft tissue attached to the tooth. it is believed that 5% to 10% of the adult population is affected by severe periodontitis. the disease usually begins at around the age of 40 and progresses slowly with periods of exacerbation and remission. a number of systemic factors modify the course of disease and diabetes mellitus is one of them. cementum is a biologically responsive tissue affected by environmental stimuli and may undergo alterations as a result of pathologic changes in the immediate environment. therefore, the aim of the study was to evaluate and compare the changes in the thickness of root surfaces of teeth with advanced chronic periodontitis from non - diabetic and diabetic patients. thirty root specimens, 15 from non - diabetic (group i) and 15 from diabetic patients (group ii) with advanced periodontitis were included in the study. the two groups did not differ with respect to pd and cal, allowing the assumption of homogenecity of patient population. diabetic patients with a history of diabetes of ranging from 4 to 6 years were included in the study. degree of metabolic control and duration of diabetes have been closely associated with periodontal disease severity. patients in whom diabetes was uncontrolled as determined by fasting blood glucose 70 to 110 mg / dl were referred to physician. a number of tools have been used for assessing the thickness of diseased root surfaces. decalcification of the root specimens for analysis under light microscopy is the most commonly used methodology. sem is a tool which gives three - dimensional pictures at ultra - structural level. the thickness of cementum is affected by various factors such as tooth type, history of eruption, function, and age of individual. to eliminate the effect of these factors, areas above the clinical attachment level as identified by a notch were designated as diseased while that below the notch were designated as healthy. thus, each tooth served as its own control for studying the diseased and healthy cementum. following careful extraction of teeth, the root specimens were sectioned longitudinally, so that the labial and lingual surfaces of the teeth were available for study. mesial or distal sites were not used for evaluating the thickness of cementum as it has been found that the mean cementum width on the mesial side of the root is slightly less than on the distal side which seems to represent a functional effect related to mesioocclusal migration of the teeth. rate of development of extrinsic cementum at distal sites has been shown to be three times than at mesial sites. thus, the thickness of cementum was assessed only on the labial aspect of tooth. cementum thickness is higher at cervicopalatal areas and apicolabial surfaces of maxillary single rooted teeth and at cervicolabial and apicolingual surface of mandibular single rooted teeth. in this study, measurement of thickness was done at three different areas in healthy and diseased portions of the roots and average of each was taken. in this study, the mean values of thickness of cementum in diseased areas were lesser than in healthy areas, both in non - diabetic and diabetic patients. the results of this study are in agreement with an earlier study which revealed a significant decrease in the thickness of cementum on the diseased root surfaces compared to the healthy surfaces. the decrease in thickness of cementum in diseased areas of root surfaces is attributed to decrease or loss of collagen cross - banding at or near the cemental surfaces. the possible mechanisms for the collagen decrease or loss are action of collagenase and other proteolytic enzymes responsible for the breakdown of collagen in surrounding connective tissue before cementum is exposed and to the action of bacteria and salivary enzymes producing the changes after cementum is exposed to the oral environment. interruption of continuous cementum deposition during inflammatory process by altering the environment from the healthy to diseased sites affects the physiologic deposition of cementum. reduction of periodontium by marginal periodontitis may result in destruction of progenitor cells which have the potential to form essential structural components of the periodontium. these factors could have been responsible for the decreased thickness of cementum in diseased areas of the root. thus, within the limits of the present study it can be concluded that the mean thickness of cementum in diseased area was less in comparison to mean thickness of cementum in healthy areas in both group i and group ii although the differences were not statistically significant. the mean thickness of cementum was higher in group ii compared to group i in both healthy and diseased areas although the differences were not statistically significant. | background : it is suggested that diabetes plays an important role in tooth loss. the periodontal structure and alveolar bone architecture in diabetics have revealed alterations. however, changes in cementum of diabetics with periodontal disease have not been evaluated adequately. thus, the aim of the study is to evaluate and compare the thickness of cementum in diseased and healthy areas of roots of teeth with chronic periodontitis from non - diabetic (group i) and type 2 diabetic patients (group ii).materials and methods : thirty incisors indicated for extraction as a result of chronic periodontitis from group i and group ii were selected. measurements of probing depth (pd) and clinical attachment loss (cal) were taken prior to extractions. area of the root surface above the cal (notch) was designated as diseased, while the area below the cal (notch) was designated as healthy. the extracted tooth samples were sectioned longitudinally and were evaluated by scanning electron microscopy (sem). descriptive statistical analysis was performed. results of continuous measurements were presented as mean sd.results:the results showed that in both groups the thickness of cementum in diseased areas was lower in comparison to the thickness in healthy areas. the mean thickness of cementum in both healthy and diseased areas was higher in group ii compared to group i, though the differences in both were not statistically significant.conclusion:within the limits of the present study, group ii showed greater mean thickness of cementum in both healthy and diseased areas compared to group i, though the differences were not statistically significant. |
today, many middle - aged women suffer from urinary incontinence, because of the decrease in the strength of their pelvic floor and urethral sphincter muscles due to pregnancy, delivery and aging1,2,3,4. urologic diseases, particularly urinary incontinence, tend to decrease people s self - esteem and cause them to feel anxiety and humiliation1, 2. the number of urinary incontinence patients has gradually increased every year and a medical industry has grown up around this problem1, 2. in other words, more and more people are likely to be diagnosed with urinary incontinence and incur a heavy financial burden attempting to treat it1, 2. urinary incontinence can be mitigated by several types of treatment, including surgery, medication, and behavior therapy3, 4. particularly, behavior therapy, such as pelvic muscle strength training, is actively applied as a primary treatment, since it tends to have a lower incidence of side effects compared to surgical treatment or medication3, 4. although physical exercise is recommended for patients, muscle strength training through dumbbells or weight machines would increase the possibility of injury among middle - aged females with diminished physical ability2. in addition, physical exercise programs need to be more interesting and recreational if they are to succeed in engaging the continuous participation of the patients. thus, this study investigated the effect of pelvic muscle strength training through belly dancing, one of well - known recreational dances, on the urinary incontinence - related muscles and vaginal pressure in middle - aged women, in order to provide practical data for establishing an effective training program focusing on mitigating and preventing urinary incontinence. a total of 24 middle - aged women, who were diagnosed with urinary incontinence by doctor, from d city, south korea participated in this study. the subjects were randomly divided into two groups : the experimental group (n=12 ; age=52.3 4.1 yrs ; height=157.3 2.2 cm ; weight 59.2 3.4 kg) and the control group (n=12 ; age=51.4 5.1 yrs ; height=158.4 3.2 cm ; weight 59.8 2.9 kg). the experimental group was requested to participate in a belly dancing program (90 min, 2 times a week, for 12 weeks), while the control group did not participate in such a program. the belly dancing program, focusing on pelvic muscle strength training (table 1table 1.belly dance exercise programtypeexercisetime (min)warm - upleg shimmy, body shimmy, pelvic tilt, hip drop / lift, hip slide, hip thrust, hip circle, hip bump20belly dance movesleg shimmy, body shimmy, pelvic tilt, hip drop / lift, hip slide, hip thrust hip circle, hip bump, hip twist, belly roll, undulation, figure eight rib cage, hip shimmy, choo choo - shimmy60cool - downsame as warm - up10intensity = rpe 810 (week 1 to 2), rpe 1112 (week 3 to 8), rpe 1314 (week 9 to 12)), was established with two sports training and conditioning experts and three current belly dancing instructors. the intensity of the training was decided by rating the perceived exertion (rating of perceive exertion : rpe)5. the rpe was gradually increased from 8 to 14 (week 1 to 2 : rpe 810, week 3 to 8 : rpe 1112, week 9 to 12 : rpe 1314). all of the subjects underwent the measurement of their vaginal pressure and urinary incontinence - related muscles pre- and post - experiment. their vaginal pressure was measured by vaginal pressure measurement equipment (hmt-2000, hmt, korea). particularly, the maximum pressure and duration of their pelvic floor muscle contraction were recorded by one well - trained and experienced nurse. the strength of their urinary incontinence - related muscles, specifically their adductor muscle, was measured by a one repetition maximum strength test (80% of 1 repetition maximum) by using a hip adductor weight machine (t-8003, technogym, italy) after sufficient warm - up exercises. the data obtained were processed using spss 18.0. and standard deviations of the two groups were calculated and the paired t - test was conducted to examine the difference between the pre- and post - experimental values. all of the subjects were educated about the content and purpose of the experiment and provided written informed consent before beginning the study, in accordance with the ethical principles of the declaration of helsinki. intensity = rpe 810 (week 1 to 2), rpe 1112 (week 3 to 8), rpe 1314 (week 9 to 12) the results of the paired t - test by group are presented in table 2table 2.result of pared t - testtypebaseline12 weeksexercise group (n=12)adductor muscle (repeat)8.35 0.6512.54 0.70maximum vaginal contraction pressure (mmhg)18.70 1.5032.50 3.59maximum vaginal contraction duration (sec)3.88 0.506.55 0.35control group (n=12)adductor muscle (repeat)8.55 0.508.53 0.65maximum vaginal contraction pressure (mmhg)19.50 1.7519.35 2.30maximum vaginal contraction duration (sec)3.95 0.203.91 0.27values are mean sd. significantly different (p<0.001) between pre and post in the experimental group.. after 12 weeks of the belly dancing intervention, the experimental group showed a significant difference between their pre- and post - experiment values, while the control group did not demonstrate any significant change. firstly, the maximum pressure of the pelvic floor muscle contraction was significantly increased from 18.7 1.5 mmhg to 32.5 3.6 mmhg in the experimental group (p<0.05). secondly, the experimental group showed a significant increase in the duration of their maximum pelvic floor muscle contraction from 3.9 0.5 sec to 6.6 0.4 sec. thirdly, the one repetition maximum of the adductor muscle was significantly increased from 8.4 0.7 to 12.5 0.7. values are mean sd. significantly different (p<0.001) between pre and post in the experimental group. about 55.7% of korean middle - aged women experience symptoms of urinary incontinence, which is considered as one of the most important health issues around the world by the world health organization (who) and needs to be controlled consistently6, 7. for instance, over 30%, 50% and 53.7% of middle - aged women in the u.s., canada and taiwan, respectively, are considered as urinary incontinence patients8,9,10. thus, this study tried to examine the effect of a belly dancing program on the urinary incontinence - related muscles and vaginal pressure in middle - aged women. the result of this study indicated that 12 weeks of the belly dancing intervention significantly increased the maximum pressure of the pelvic floor muscle contraction and its duration and the adductor muscle strength. s study11, 10 weeks of pelvic exercise increased the maximum pressure of the pelvic floor muscle contraction among middle - aged women. mun.12 also found that the degree of urinary incontinence was improved by a pelvic muscle exercise intervention, due to the resulting increased maximum pressure of the pelvic floor muscle contraction and its duration. shin & park13 indicated that 12 weeks of pelvic exercise significantly improved many symptoms of urinary incontinence, such as urinary frequency and nocturia. na.2 found that the weakened muscles around the pelvis caused by aging and delivery could be strengthened through regular physical activity, thereby, in turn, relieving or preventing urinary incontinence. ko.14 suggested that belly dancing, in particular, increases the bone density in middle - aged women. yoon.15 found that 6 weeks of adductor exercise increased the adductor muscle strength and pressure of the pelvic floor muscle contraction, showing similar effects to the kegel and other sphincter exercises. in conclusion, a regular pelvic exercise program could be actively applied as an effective non - surgical treatment for urinary incontinence. this study included a pelvic exercise program, in the form of recreational dancing, that females could follow more easily and participate in for longer by engaging their interest. although the results of this study indicated the effectiveness of pelvic exercise in reducing urinary incontinence, more specific and diverse research is necessary to identify how such exercises can be tuned to the patient s individual characteristics (e.g., age, gender, previous exercise experience, degree of urinary incontinence, etc.). in addition, future study would identify the effect of exercise on urinary incontinence by types such as stress, urge, overflow, and functional incontinence. | [purpose ] this study examined the effect of belly dancing on the urinary incontinence - related muscles and vaginal pressure in middle - aged women to provide fundamental data for establishing an effective training program focusing on mitigating and preventing urinary incontinence. [subjects and methods ] the subjects included 24 middle - aged women, who have been diagnosed with urinary incontinence. the subjects were randomly divided into two groups, viz. the experimental group (n=12) and control group (n=12). the experimental group underwent a belly dancing program focusing on pelvis moves. [results ] in the experimental group, the urinary incontinence - related muscle strength and vaginal pressure were increased, while the control group showed no significant change. [conclusion ] belly dancing focusing on pelvis moves had a positive effect on the urinary incontinence - related muscle strength and vaginal pressure, suggesting that a recreational dance program focusing on pelvic exercise can be used to prevent and relieve the symptoms of urinary incontinence as a non - surgical treatment. |
serous cystic neoplasm (scn) of the pancreas is a very rare benign lesion, accounting for only 1% to 2% of all pancreatic tumors. in 2010, several new types of scn were added to the world health organization criteria for pancreatic tumor classification ; this includes the mixed scns and pancreatic neuroendocrine tumors (pannets). these are termed a mixed serous neuroendocrine neoplasm (msnn), and defined as a tumor containing 2 components with different pathologies. several cases of msnn have been reported, including distinctly separated and intimately admixed tumors. here, a 73-year - old chinese woman was admitted to our hospital in august 2010, having suffered from an incidental mild epigastric pain for 10 months. the patient had no complaints involving jaundice, vomiting, or weight loss ; however, she had hypertension, and underwent an oophorotomy in 2006 because of an ovarian physiologic cyst. examination on admission revealed impaired liver function with increased levels of alt (133 u / l) and ast (116 u / l). the level of amylase was normal and all of the tumor markers, including ca199 (17.9 u / ml), ca242 (6.1 u / ml), cea (2.46 ng / ml), afp (15.7 ng / ml), and ca125 (10.2 u / ml), were within normal limits. a cystic tumor with a thin capsule at the pancreatic tail, which measured 3.4 2.5 cm in diameter, was detected using ultrasonography (fig. 1). enhanced computed tomography (ct) revealed an ill - defined hypovascular tumor, approximately 3.3 2.6 cm in diameter, located in the pancreatic tail and adhering to the splenic artery. preoperative ct and ultrasonography scans of our patient, with an ill - defined cystic lesion located in the pancreatic tail, and the arrow points the mural nodules. the tumor was 3.3 2.6 cm in ct scans and 3.4 2.5 cm in ultrasonography. laparoscopic distal pancreatectomy with splenectomy was performed, during which a 3-cm tumor was found encroaching on the pancreatic tail. the resected pancreatic tissue measured 9.0 6.0 2.5 cm, together with a unilocular cystic tumor measuring 2.5 2.0 1.0 cm. the cyst was filled with serous fluid while the inner surface was white and smooth. a focal gray, solid ill - defined mass (approximately 1.2 1.0 0.5 cm in diameter) hematoxylin and eosin staining of the surgical specimen demonstrated that the cyst was formed by a thin fibrous wall, which was lined with a single layer of cuboidal or flattened epithelial cells exhibiting partial denudation (fig. the cell cytoplasm was pale to clear while the nucleus was round to oval and centrally located. cytological atypia was minimal and there were no invasive or metastatic features present. according to the definition of lee and kimura, serous oligocystic adenoma was identified. (b) lining cuboidal epithelial cells (arrow) with a pale or clear cytoplasm and a centrally located round nucleus. (c) the collisional growth of 2 components with the pancreatic neuroendocrine tumor (pannet) growing into the wall of serous oligocystic adenoma. (f) immunohistochemistry staining for chromogranin a. neuroendocrine tumor cells were positive (300). the adherent solid mass was composed of cells arranged in a trabecular or banding pattern. the size and shape of these cells were uniform with a low karyoplasmic ratio, and there was no mucin inside. in addition, the nucleolus was not obvious, and small nucleoli could occasionally be seen. nuclear fission was scarce and appeared in less than 1 in every 10 high power fields of view. perineural invasion and fat tissue permeation were noted, while the lymph nodes examined were negative. in accordance with these findings, a pannet (g1) with uncertain behavior was considered ; it was positive for the expression of chromogranin, synaptophysin, and ki67 (index < 1%) (fig. intriguingly, we found these 2 components exhibited a collision type of growth, with the pannet component growing into the wall of the scn (fig. 2c and d). the patient started to suffer from diabetes shortly after surgery. during a 54-month follow - up, there were no signs of tumor recurrence or metastasis, and the complaints of epigastric pain and abnormal liver function evident before surgery had disappeared. the patient gave her informed consent for treatment and inclusion in this study, after having been provided with all the necessary information. the world health organization classified the scns into 5 types in 2010 : serous microcystic adenoma, serous macrocystic / oligocystic adenoma, von hippel lindau (vhl)-associated scn, solid serous adenoma / neoplasm, and msnn. to investigate msnn, the pubmed, embase, and science citation index databases were systematically searched until february 2015. endocrine tumor / neoplasm, islet cell tumor / neoplasm, and pancreas. the citation lists associated with all of the studies retrieved were used to identify other potentially relevant publications. the search results were then screened according to the following inclusion criteria : any form of publications focused on msnn, without a third pancreatic tumor ; and presentation of sufficient data, including clinicopathological characteristics and histopathological features. studies were excluded if they met the following criteria : were not published in english ; and duplicated data from the same sample of patients. hough reported a case of diffuse cystic changes in the pancreas associated with pannets, but detailed data was not available. baek and jung reported the same patient in korea, while heresbach reported a case of cystadenocarcinoma with msnn in the pancreatic head. thus, 14 patients remained for analysis in the current study after excluding these 3 studies. the baseline characteristics of the included studies are summarized in table 1. as for statistical analysis, we used fisher test, the t - test, and the t-test when comparing different groups. clinical features of msnn (15 cases). to the best of our knowledge, after reviewing the other cases, we found that our case was the first where the mixed lesion arose in the pancreatic tail ; it was also the first case with an intimately, partially overlapped but mostly separated mixed scn and pannet. among these patients, 13 were female and 2 were male. the chief complaints were abdominal pain, nausea / vomiting, weight loss, and jaundice, in 46.7%, 26.7%, 20.0%, and 13.3% of patients respectively. however, no endocrine syndrome was detected in any of the patients (table 1). the results of laboratory tests in these cases were mostly within the reference range, except for the case reported by hsieh that involved an increased level of ca199. all patients underwent preoperative ultrasonography and/or enhanced thin - sliced spiral ct and/or magnetic resonance imaging scans. the unifocal cystic lesions were often located in the head of the pancreas (50% [10 cases ]) with varying sizes, from 1.3 cm to 13 cm in greatest dimension. most tumors were hypoechoic in ultrasonic images and exhibited low density in ct scans, with or without enhancement after contrast injection. the medical history in all 15 patients was investigated ; gallbladder stones and diabetes mellitus were most frequently seen with an incidence of 5/15. all 15 patients accepted intentional radical surgery ; there were 7 pancreaticoduodenectomies, 6 distal pancreatectomies with splenectomy and 2 total pancreatectomies. the longest follow - up time was 2 years (not including our patient). none of these patients had died of tumor recurrence or distant metastasis by the time they were reported. we analyzed all 15 cases with regard to different clinical, morphological, and histological appearances, and found that msnn could be subdivided into several types (fig. the diffuse type : the entire pancreas was involved with numerous serous cysts and pannets, which could be located in any region of the pancreas. the 2 components grew together and jointly constituted the tumor tissue ; these components could not be distinctly divided. the solitary type : there were isolated scns and pannets arising in the pancreas without any intermingling area, and the transitional zone was normal, resembling 2 different tumors coinciding in one organ. the collision type : as in the case reported by us, most parts of these 2 components were separated with a partially intermixed or overlapping zone. there were 5 patients with diffuse scn associated with pannet, 5 with mixed ones, and 4 with solitary ones. we noted that the diffuse type was mostly associated with vhl disease (3 confirmed, 1 suspected, and 1 uncertain), and that all of the pannet components were well - differentiated endocrine carcinomas. all the mixed type of msnn presented as serous microcystic adenoma with larger cysts as compared with those of the solitary type (mean 9.1:1.9 cm ; p < 0.05). serous microcystic adenoma or serous oligocystic adenoma could both be discovered in the solitary type, in which all of the pannets were benign. there were no significant differences between the mixed and solitary types regarding patient age or the location of the mass. our case was an unequivocal type of coexistent scn and pannet which was intimately adjacent to a partially intermingling area, and was termed a collision type combination (fig. only one case of the collision type has been reported, and it seemed to have the same clinical and histological features as mixed msnn. because of the coexistence of 2 types of tumor, we should consider them together when discussing the surgical indication and prognosis. nevertheless, there is a challenge in accurately distinguishing scn from mucinous cystic neoplasms, which are highly suspected of being malignant. pannets are usually regarded as being potentially malignant because their behavior is difficult to predict from their histopathological features and cellular products. surgery is considered to be the first - line therapy for patients with pannets when the lesion is resectable. as suggested by slukvin, msnn may have a higher malignant potential than scn alone. among these patients, including our case, malignant biological behavior such as perineural, lymphatic, and duodenal invasion appeared in 33.3% (5/15) of all the cases, which is much higher than that of scn or. as a result, once a diagnosis of msnn is made, we are more inclined to surgical treatment ; in the cases that have been followed up, this lesion seemed to have a better prognosis after curative surgery. however, the robustness of our treatment recommendation is diminished by the small number of cases. evaluation of the malignant potential and prognostic outcomes of mixed tumors requires a long - term follow - up and data from additional reports of such cases. high potential for malignancy has been reported in vhl - associated pannets. among the patients, including our case, 3 vhl - scns were described ; all these cases had diffuse lesions and invasion. despite the fact that no metastasis was seen, these 3 cases should be attributed to neuroendocrine carcinoma. therefore, close attention should be paid to the presentation of diffuse msnn regarding vhl disease. the world health organization classified the scns into 5 types in 2010 : serous microcystic adenoma, serous macrocystic / oligocystic adenoma, von hippel lindau (vhl)-associated scn, solid serous adenoma / neoplasm, and msnn. to investigate msnn, the pubmed, embase, and science citation index databases were systematically searched until february 2015. endocrine tumor / neoplasm, islet cell tumor / neoplasm, and pancreas. the citation lists associated with all of the studies retrieved were used to identify other potentially relevant publications. the search results were then screened according to the following inclusion criteria : any form of publications focused on msnn, without a third pancreatic tumor ; and presentation of sufficient data, including clinicopathological characteristics and histopathological features. studies were excluded if they met the following criteria : were not published in english ; and duplicated data from the same sample of patients. hough reported a case of diffuse cystic changes in the pancreas associated with pannets, but detailed data was not available. baek and jung reported the same patient in korea, while heresbach reported a case of cystadenocarcinoma with msnn in the pancreatic head. thus, 14 patients remained for analysis in the current study after excluding these 3 studies. the baseline characteristics of the included studies are summarized in table 1. as for statistical analysis, we used fisher test, the t - test, and the t-test when comparing different groups. clinical features of msnn (15 cases). to the best of our knowledge, after reviewing the other cases, we found that our case was the first where the mixed lesion arose in the pancreatic tail ; it was also the first case with an intimately, partially overlapped but mostly separated mixed scn and pannet. among these patients, 13 were female and 2 were male. the chief complaints were abdominal pain, nausea / vomiting, weight loss, and jaundice, in 46.7%, 26.7%, 20.0%, and 13.3% of patients respectively. however, no endocrine syndrome was detected in any of the patients (table 1). the results of laboratory tests in these cases were mostly within the reference range, except for the case reported by hsieh that involved an increased level of ca199. all patients underwent preoperative ultrasonography and/or enhanced thin - sliced spiral ct and/or magnetic resonance imaging scans. the unifocal cystic lesions were often located in the head of the pancreas (50% [10 cases ]) with varying sizes, from 1.3 cm to 13 cm in greatest dimension. most tumors were hypoechoic in ultrasonic images and exhibited low density in ct scans, with or without enhancement after contrast injection. the medical history in all 15 patients was investigated ; gallbladder stones and diabetes mellitus were most frequently seen with an incidence of 5/15. all 15 patients accepted intentional radical surgery ; there were 7 pancreaticoduodenectomies, 6 distal pancreatectomies with splenectomy and 2 total pancreatectomies. the longest follow - up time was 2 years (not including our patient). none of these patients had died of tumor recurrence or distant metastasis by the time they were reported. we analyzed all 15 cases with regard to different clinical, morphological, and histological appearances, and found that msnn could be subdivided into several types (fig. the diffuse type : the entire pancreas was involved with numerous serous cysts and pannets, which could be located in any region of the pancreas. the 2 components grew together and jointly constituted the tumor tissue ; these components could not be distinctly divided. the solitary type : there were isolated scns and pannets arising in the pancreas without any intermingling area, and the transitional zone was normal, resembling 2 different tumors coinciding in one organ. the collision type : as in the case reported by us, most parts of these 2 components were separated with a partially intermixed or overlapping zone. there were 5 patients with diffuse scn associated with pannet, 5 with mixed ones, and 4 with solitary ones. we noted that the diffuse type was mostly associated with vhl disease (3 confirmed, 1 suspected, and 1 uncertain), and that all of the pannet components were well - differentiated endocrine carcinomas. all the mixed type of msnn presented as serous microcystic adenoma with larger cysts as compared with those of the solitary type (mean 9.1:1.9 cm ; p < 0.05). serous microcystic adenoma or serous oligocystic adenoma could both be discovered in the solitary type, in which all of the pannets were benign. there were no significant differences between the mixed and solitary types regarding patient age or the location of the mass. our case was an unequivocal type of coexistent scn and pannet which was intimately adjacent to a partially intermingling area, and was termed a collision type combination (fig. only one case of the collision type has been reported, and it seemed to have the same clinical and histological features as mixed msnn. because of the coexistence of 2 types of tumor, we should consider them together when discussing the surgical indication and prognosis. nevertheless, there is a challenge in accurately distinguishing scn from mucinous cystic neoplasms, which are highly suspected of being malignant. pannets are usually regarded as being potentially malignant because their behavior is difficult to predict from their histopathological features and cellular products. surgery is considered to be the first - line therapy for patients with pannets when the lesion is resectable. generally speaking, the prognosis of msnn depends on the malignant component. as suggested by slukvin, msnn may have a higher malignant potential than scn alone. among these patients, including our case, malignant biological behavior such as perineural, lymphatic, and duodenal invasion appeared in 33.3% (5/15) of all the cases, which is much higher than that of scn or. as a result, once a diagnosis of msnn is made, we are more inclined to surgical treatment ; in the cases that have been followed up, this lesion seemed to have a better prognosis after curative surgery. however, the robustness of our treatment recommendation is diminished by the small number of cases. evaluation of the malignant potential and prognostic outcomes of mixed tumors requires a long - term follow - up and data from additional reports of such cases. high potential for malignancy has been reported in vhl - associated pannets. among the patients, including our case, 3 vhl - scns were described ; all these cases had diffuse lesions and invasion. despite the fact that no metastasis was seen, these 3 cases should be attributed to neuroendocrine carcinoma. therefore, close attention should be paid to the presentation of diffuse msnn regarding vhl disease. msnn is a distinct clinicopathological entity rather than an incidental concurrence of 2 separate tumors. to the best of our knowledge, we are the first to report a collisional case of msnn, followed by a literature review and analysis based on its morphological and clinicopathological appearance. different types of msnn exhibit various features that could be helpful in indicating the prognosis. | abstractintroduction : the aim of this study was to report a new case of mixed serous neuroendocrine neoplasm (msnn) and review the literature concerning this type of lesion, which was added to the world health organization classification of pancreatic tumors in 2010.results:a 73-year - old woman presented with a pancreatic mass. the lesion was an intriguing combination of serous cystic neoplasm (scn) and pancreatic neuroendocrine tumor (pannet), in which the pannet component grew into the wall of the serous oligocystic adenoma. we searched different databases for studies that had investigated msnn. a total of 15 patients (age, 2878), including the patient in the present study, were evaluated. we discuss these cases in detail especially regarding morphology and pathology ; our case was the only one involving a collision type combination.conclusion:although msnn is recognized as a variant of scn, it is quite different from scn or pannet. a new morphological analysis of msnn may help in elucidating its histogenesis and prognosis. |
the childbearing years pose potential obesity risk for women because of excessive gestational weight gain (gwg) and postpartum weight retention [24 ]. excess gwg and postpartum weight retention result in suboptimal pre- and perinatal health and predispose both mothers and their infants to long - term chronic diseases. in response to the mounting evidence, the institute of medicine (iom) revised its 1990 recommendations and released new gwg recommendations in 2009 [5, 6 ]. these guidelines are specific to a woman 's prepregnancy body mass index (bmi) ; for example, underweight women (bmi of 75%) were not aware that their gestational weight gain goals differed by prepregnancy weight status. these results are confirmed by anderson and colleagues, who also found that women received inadequate or conflicting information about pregnancy nutrition and gwg from health care providers. this suggests an important opportunity and avenue for education in all settings where pregnant women are routinely seen. one of the complications of determining appropriate gwg is that guidelines are based on four categories of prepregnancy weight : underweight, normal weight, overweight, or obese. in the focus group discussions of how to differentiate the pregnancy weight tracker tool based on prepregnancy weight status, participants offered different ideas for how to identify the woman 's weight category. some participants wanted the weight designation (underweight, normal weight, overweight, and obese) listed on the pregnancy weight tracker because it was an accurate reflection of their weight status ; others expressed concern that the labels posed a negative stigma. i agree, some people might not be able to accept that [they are overweight ]. i do n't know, but i agree you should n't get mad, because it 's the truth if you 're overweight. for a majority of caucasian and hispanic participants, the designation of overweight or obese on the tracker was a sensitive issue, and they preferred not to have these labels on the pregnancy weight tracker ; instead, the participants suggested color coding the four weight ranges and including the weight range goals on the pregnancy weight tracker. one participant commented, you do n't know how sensitive a person may be when it comes to words like obese ' or overweight ', and like she said, what does it mean ? how do you define that for a person ? another participant suggested distinguishing the different weight categories by color : no, i do n't think [overweight or obese ] should be on the pregnancy weight tracker. having different colors for different weights is better. when asked if participants were interested in having a way to track their weight gain during pregnancy, all the participants indicated a strong interest in having a tool to track their weight during pregnancy. most women reported a willingness to use a pregnancy weight tracker if wic provided one ; also, the women suggested that the pregnancy weight tracker would be helpful to facilitate discussion of their weight gain goals at regular prenatal visits with their healthcare provider, as well as wic visits. sized pregnancy weight tracker for their purse and half sheet to be used at home, to be placed either on the refrigerator or in the bathroom. participants shared affirmatively that they were interested in having accountability with using the pregnancy weight tracker, along with educational handouts. participants shared that having a pregnancy weight tracker (gwg tracker) on hand to discuss with wic staff and their doctors would encourage them to monitor their own weight and stay on track with weight gain goals. one participant suggested that the pregnancy weight tracker should be a routine part of wic services, they ask about our address and income when we come, and they should ask about that [pregnancy weight tracker ] too when they give it out. findings from this study suggest that participants face significant barriers to being able to gain the appropriate amount of weight and are very interested in tools and education to help stay on target with weight gain. results of focus groups analyses were utilized to design a gwg tracker and accompanying educational handout for use with pregnant moms enrolling in wic. a gwg tracker was developed based on their feedback (compact size so participants can carry it with them and color coded rather than identifying their weight status). the first barrier discussed by the participants was the theme of eating for two. this perception of having to eat for two contributed to participants feeling the need to eat more, which then encouraged higher gestational weight gain rather than discouraging it. these findings are confirmed in other qualitative research studies with african - american and puerto rican mothers [20, 21, 28 ]. this suggestion from family members to eat for two is derived from motivation and desire to optimize pregnancy outcomes and fetal growth. if mothers do not perceive excess weight gain to be problematic, there will be little buy - in to follow the iom guidelines. these perceptions of threat and susceptibility are at the heart of the health belief model ; as such, these findings suggested the need to direct nutrition education to target debunking this myth of eating for two. the educational handout developed for the intervention phase served to debunk the myth in the following ways : (1) providing clarification for weight gain distribution during pregnancy ; (2) explaining how excess weight gain leads to complications with pregnancy and delivery ; (3) providing iom weight gain ranges so participants are aware and can track their weights during pregnancy. we found that a majority of the focus group participants were concerned about gaining too much weight but had many misperceptions and barriers that needed to be clarified and corrected ; these findings are corroborated by another study. figure 2 provides a sample gwg tracker tool that was developed for use during the focus group discussions ; focus group participant feedback and suggestions led to the final gwg tracker. this gwg tracker tool allows participants to be accountable about their weight gain goals and facilitates discussions with their doctors about weight gain during prenatal visits and with wic nutritionists. finally, placement of the gwg goals on the pregnancy weight tracker builds awareness among participants of appropriate weight gain during their pregnancy. building this awareness among wic participants for appropriate gwg provides important perceived benefits which, based on the health belief model [29, 30 ], may go a long way in raising consciousness among wic participants. while this qualitative study provided important grounding for the development of an intervention study to prevent excessive gwg among wic participants, it is not without its limitations. because the sample is specific to the wic population, these findings may have limited generalizability to the entire population of pregnant women who have varying socioeconomic and cultural backgrounds. second, the hispanic population of wic participants is primarily from mexico, and this may not be generalizable to wic programs in other parts of the us. finally, while it is possible that our findings may have been impacted by the presence of women with different bmi classifications within a focus group, our moderators used qualitative methodology skills to encourage all participants to share. it would be beneficial in future studies to also further stratify focus groups by bmi classifications to ensure a more homogenous grouping of women based on weight groupings. despite these limitations, the benefit of this qualitative study was to allow for an unhurried and open discussion of the iom gestational weight gain guidelines and challenges to appropriate weight gain during pregnancy in the wic program ; the findings from these discussions were applied directly to inform the development of the intervention. findings from this study have important implications for how perinatal health is conceptualized and assessed. these findings illustrate the complex nature of weight gain during pregnancy, particularly among low - income ethnic minority groups. this work extends the discussion beyond a simplistic and unidimensional conceptualization to one which considers the multilevel and complex socioecological landscape in which immigrant communities navigate their health decisions. employing qualitative research methods in maternal and child health research with immigrant groups allow for findings and interpretations to be grounded in the cultural context of daily lived experiences. second, narratives give voice to and illuminate our understanding of how individual experiences collectively and simultaneously affect pregnancy outcomes, thus providing a more holistic perspective on the impact of culture on perinatal health. at a practical level, this holistic approach allows for a better understanding of the contextual environment in which many of these low - income families live ; as a result, we will be able to design wic - based programming and health - related interventions that will address these barriers and move us forward in bridging the gap in health disparities. | women of reproductive age are particularly at risk of obesity because of excessive gestational weight gain (gwg) and postpartum weight retention, resulting in poor health outcomes for both mothers and infants. the purpose of this qualitative study was to examine perceptions and barriers to gwg among low - income women in the wic program to inform the development of an intervention study. eleven focus groups were conducted and stratified by ethnicity, and each group included women of varying age, parity, and prepregnancy bmi ranges. participants reported receiving pressure from spouse and family members to eat for two among multiple barriers to appropriate weight gain during pregnancy. participants were concerned about gaining too much weight but had minimal knowledge of weight gain goals during pregnancy. receiving regular weight monitoring was reported, but participants had inconsistent discussions about weight gain with healthcare providers. most were not aware of the iom guidelines nor the fact that gestational weight gain goals differed by prepregnancy weight status. results of these focus groups analyses informed the design of a pregnancy weight tracker and accompanying educational handout for use in an intervention study. these findings suggest an important opportunity for gwg education in all settings where pregnant women are seen. |
a cross - legged position is that in which the lower legs are folded towards the body, crossing each other at the ankle or calf, with both ankles on the floor. this posture has various names originating from different cultural backgrounds : in english, it is referred to as indian style ; in many european languages, as turkish style ; and in korea, as yangban style. sitting on the floor in a cross - legged position is common in the oriental culture but not in the western culture. low back pain (lbp) associated with sitting on the chair has been widely studied. some agree that prolonged static sitting and reduced lumbar lordosis are risk factors for lbp4,5,8,15). however, to the best of our knowledge, the effect of sitting on the floor on the sagittal lumbar curve has not been studied. it is just presumed that sitting on the floor is worse for lbp than standing or sitting on a chair. this study is designed for radiographic comparison of lumbar lordosis when standing, sitting on a chair, and sitting on the floor. the aim of the present study is to determine the clinical significance of sitting on the floor on lumbar spinal alignment. thirty asymptomatic volunteers among the hospital staff without a history of spinal pathology were recruited. the study population comprised 11 women and 19 men with a mean age of 29.8 (sd, 6.2) years (range, 23 - 52 years). lateral radiographs of the lumbar spine were obtained when standing, sitting on a chair, and sitting on the floor (fig. each person was asked to stand erect comfortably. the individual 's arms were flexed ; hands, placed on the clavicle ; and the knees, held in extension. for chair sitting, the height of the chair was adjusted to allow each participant 's hips and knees to flex approximately to 90 and for their feet to rest comfortably on the floor. for floor - sitting, each person was asked to sit crossed - legged on the floor in a comfortable position. the distance from the radiographic source to the film was maintained at 230 cm for exposure. an independent observer measured all radiographic assessments by using a measuring program with a built - in picture - archiving communication system (piview ; infinitt co. ltd., segmental lumbar lordosis (sl) between l1 to s1 and whole lumbar lordosis (wl) were assessed using the cobb 's angle in each position. sl was defined as the cobb 's angle between the superior endplate line of the upper vertebra and the inferior endplate line of the lower vertebra. sl at l5-s1 was defined as the cobb 's angle between the superior endplate line of l5 and the endplate line of s1. wl was defined as the cobb 's angle between the superior endplate line of l1 and the superior endplate line of s1. chicago, il, usa). a probability value of less than 0.05 was considered significant. analysis of variance or the kruskal - wallis test was used for statistical analysis to compare the variables among the 3 groups. if a significant difference was found, the bonferroni post hoc test was used to determine which groups were significantly different. thirty asymptomatic volunteers among the hospital staff without a history of spinal pathology were recruited. the study population comprised 11 women and 19 men with a mean age of 29.8 (sd, 6.2) years (range, 23 - 52 years). lateral radiographs of the lumbar spine were obtained when standing, sitting on a chair, and sitting on the floor (fig. each person was asked to stand erect comfortably. the individual 's arms were flexed ; hands, placed on the clavicle ; and the knees, held in extension. for chair sitting, the height of the chair was adjusted to allow each participant 's hips and knees to flex approximately to 90 and for their feet to rest comfortably on the floor. for floor - sitting, each person was asked to sit crossed - legged on the floor in a comfortable position. the distance from the radiographic source to the film was maintained at 230 cm for exposure. an independent observer measured all radiographic assessments by using a measuring program with a built - in picture - archiving communication system (piview ; infinitt co. ltd., segmental lumbar lordosis (sl) between l1 to s1 and whole lumbar lordosis (wl) were assessed using the cobb 's angle in each position. sl was defined as the cobb 's angle between the superior endplate line of the upper vertebra and the inferior endplate line of the lower vertebra. sl at l5-s1 was defined as the cobb 's angle between the superior endplate line of l5 and the endplate line of s1. wl was defined as the cobb 's angle between the superior endplate line of l1 and the superior endplate line of s1. chicago, il, usa). a probability value of less than 0.05 was considered significant. analysis of variance or the kruskal - wallis test was used for statistical analysis to compare the variables among the 3 groups. if a significant difference was found, the bonferroni post hoc test was used to determine which groups were significantly different. there were significant decreases in sl at all lumbar spine levels, except at the l1 - 2 level, when sitting on the floor as compared to when standing or sitting on a chair. wl when sitting on the floor was decreased to -13.9 (sd, 13.6) degrees from -50.0 (sd, 9.2) degrees observed in the standing position (72.9% reduction). table 3 shows the contribution of sl change to wl change during a change in the position. l5-s1 and l4 - 5 contributed the most to the change in wl during a change in position. a change in position from standing to sitting on a chair resulted in a change in wl of a mean value of 47% and 32.6% at the l5-s1 and l4 - 5 levels, respectively. a change in position from standing to sitting on the floor resulted in a change in wl of a mean value of 40.2% and 37.9% at the l5-s1 and l4 - 5 levels, respectively. a change in position from sitting on the chair to sitting on the floor resulted in a change of 62.2% in wl at the l4 - 5 level. there was more loss in wl when sitting on the floor than when sitting on the chair. each sl below the l2 - 3 level also significantly decreased when sitting on the floor. most of the wl is determined by the l4-s1 level, and most of the change in wl observed during a change in position is also attributed to the l4-s1 level. when sitting comfortably on the floor, wl below the l1 - 2 level became more kyphotic and a decrease in sl was prominent at the l4 - 5 and l5-s1 levels. this implies relaxation of the thoracic erector spinae and lumbar multifidus muscle14). in comparison with the chair - sitting position, sl at the l4 - 5 level was greatly decreased than other levels during the floor - sitting position ; this reveals high compressive loading at this level. in the case of low lumbar fusion, especially at the l4 - 5 level, adjacent segment hypermobility is expected when sitting on the floor. because loss of segmental motion at the l4 - 5 level is shown to be redistributed among multiple cranial segments, in most cases at the first cranial adjacent level2), individuals used to sitting on the floor are more prone to adjacent segment hypermobility after low lumbar fusion. akamaru.1) demonstrated that hypolordotic fusion at the l4 - 5 level caused the greatest amount of flexion - extension motion at the l3 - 4 level. bae.3) reported that postoperative hypolordotic sl is a risk factor for adjacent segment degeneration. we speculate that hypolordotic fusion might aggravate adjacent segment motion and degeneration in the sitting position. surgeons should remember to avoid hypolordotic low lumbar fusion in individuals who are used to sitting on the floor. low lumbar fusion, especially in the hypolordotic state, is highly associated with hyperflexion at the adjacent segment when sitting on the floor ; this brings about deactivation of the local lumbar stabilizing muscle and increases connective tissue strain12). deactivation of the local spinal stabilizing muscle that is known to be fatigue - resistant exerts high compressive loads on the spine7,10). hence, patients may feel uncomfortable and face difficulty when sitting on the floor after low lumbar fusion surgery. loss of sl is associated with an increased incidence of low back pain and increased rate of degenerative changes observed at the adjacent levels. to decrease segmental motion at the adjacent segment after low lumbar fusion in the sitting position, it may be beneficial to fuse the lumbar spine in normal or hyperlordotic alignment1). o'sullivan.14) suggested a lumbopelvic sitting position in which participants were instructed to rotate their pelvis anteriorly to obtain a neutral lordosis in the lumbar spine and relax their thorax. this resulted in increased coactivation of the superficial lumbar multifidus and internal oblique and is capable of providing a local stabilizing effect on the lumbopelvic region without high compressive loading. however, this position may not be easily achieved when sitting on the floor because of ischial tuberosities. when sitting, the spinal load is carried mainly by the ischial tuberosities15), which make it difficult to rotate the pelvis anteriorly when sitting on the floor. makhsous.13) proposed that sitting on a chair with reduced ischial support and enhanced lumbar support could decrease sitting - related low back pain. it is presumed that lumbar support provided by a cushion for sitting on the floor helps reduce sitting load and enhances neutral lumbar lordosis by rotating the pelvis anteriorly. contribution to the loss of lordosis when sitting on a chair and on the floor was the greatest at levels l5-s1, and it showed relatively less influence on the change in lordosis when switching positions from sitting on a chair to sitting on the floor. this implies hypermobility at the l5-s1 level in any sitting position, which explains the remarkable incidence of nonunion at this level6). using the clinical significance of this data, we should 1) explain to patients before they undergo surgery that they would experience difficulty when sitting on the floor after fusion surgery, 2) create greater lordotic fusion for the low lumbar spine, 3) recommend patients a change in lifestyle, and 4) perform more rigid fixation for the l5-s1 fusion. because this study included only healthy volunteers, there exists a limitation to explain the changes of sl and wl after low lumbar fusion. we are planning to perform similar study in fusion group and expect to report this result in near future. the results of this study suggest sitting on the floor affects the sagittal lumbar curve by decreasing lordosis at each level, especially at the l4 - 5 and l5-s1 levels. in the case of low lumbar fusion, hyperflexion is expected at the adjacent segment when sitting on the floor. in this respect, sitting on the floor can easily aggravate lbp. to avoid this, sitting with a lordotic lumbar curve is important. surgeons should remember to create sufficient lordosis when performing lower lumbar fusion surgery in patients with an oriental life style. | objectiveto compare radiographic analysis on the sagittal lumbar curve when standing, sitting on a chair, and sitting on the floor.methodsthirty asymptomatic volunteers without a history of spinal pathology were recruited. the study population comprised 11 women and 19 men with a mean age of 29.8 years. an independent observer assessed whole lumbar lordosis (wl) and segmental lordosis (sl) between l1 and s1 using the cobb 's angle on lateral radiographs of the lumbar spine obtained from normal individuals when standing, sitting on a chair, and sitting on the floor. wl and sl at each segment were compared for each position.resultswl when sitting on the floor was reduced by 72.9% than the average of that in the standing position. of the total decrease in wl, 78% occurred between l4 to s1. there were significant decreases in sl at all lumbar spinal levels, except l1 - 2, when sitting on the floor as compared to when standing and sitting on a chair. changes in wl between the positions when sitting on a chair and when sitting on the floor were mostly contributed by the loss of sl at the l4 - 5 and l5-s1 levels.conclusionwhen sitting on the floor, wl is relatively low ; this is mostly because of decreasing lordosis at the l4 - 5 and l5-s1 levels. in the case of lower lumbar fusion, hyperflexion is expected at the adjacent segment when sitting on the floor. to avoid this, sitting with a lordotic lumbar curve is important. surgeons should remember to create sufficient lordosis when performing lower lumbar fusion surgery in patients with an oriental life style. |
hospitals, as the most important healthcare organizations, have a leading role in health promotion in society, and their coordinated action with a set of political, social, and cultural factors leads to people s health. since this sector is the main consumer of a large part of the healthcare budget in a country, full attention to its performance and costs is of particular importance. annually, immense sums of money are expended on buildings, maintenance, and reconstruction of hospitals around the world, but evidence is rare regarding achievement of the expected benefits from such spending. in 1989, the world health organization (who) estimated that about 40% of the resources available to the health sector in the u.s. were wasted, and it showed that the resources that can be saved by increased efficiency were very appreciable (1). financing by the public sector can not be responsive to the ever - increasing expenses in the health system. obligations to finance by public sector as well as increasing costs have created the need for public - sector hospitals to reduce their expenses (2). these factors have led governments pursuing various approaches to deal with these problems as well as ways to curb costs, raise capital, and ultimately achieve better health outcomes through more active participation of the private sector in providing services and also financing this sector (3). to benefit from the capabilities of public and private sectors in the form of a hybrid model, the public - private partnership (ppp) model was introduced in 1990, and it has been used constructively in recent years to reform healthcare sectors in many countries. in fact, ppp can be a powerful political tool for improving and promoting the survival and quality of services in public hospitals (4). ppp is trying to implement some of the principles of the private sector, including economic balance and revenues through a rigorous financial management to solve some major problems in the public sector (5). one of the fairly comprehensive definitions is as follows : contractual agreement between public sector and private sector in which the private sector executes some government activities on behalf of the private sector within a specified time, in a framework of negotiations, with a risk - adjusted extent, and determined executive rewards. in fact, ppp includes a range of traditional methods of procurement of services by the public sector and full privatization (6). there is a growing belief that partnership between public and private sectors can lead to increased efficiency, equality, accountability, quality, and accessibility in the health system. in ppp, the sectors can make use of mutually competitive advantages of each other in the fields of technology, knowledge, resources, skills, and management capabilities (7). examination of different experiences of various countries on the use of different options of ppp indicates the impact of these options on the cost of providing services, amount of operation, access of the population covered, and other performance indicators of public hospitals (8). the results of several studies have suggested that several factors have been identified for the implementation and success of executing ppp projects. for example, some researchers have considered various factors to be important factors and requirements in the implementation of ppp (911). these factors include proper understanding of the project, stable economic and political conditions, adequate economic justification, sufficient initial capital, strong governance and management, appropriate allocation of risks, enough experience, use of reputable and competent contractors, and innovation in funding. therefore, in order to achieve success in the implementation of ppp and its development and promotion, these factors should be considered to avoid any failure in the implementation and operation of these ppp projects. in iran, more than half of the funds of the health sector are allotted to hospitals ; however, the low hospital bed occupancy rate compared with that of developed countries reflects the fact that the available resources are not exploited properly, and it would necessitate the use of appropriate management methods. therefore, policy - makers and senior officials are seeking ways to get out of this situation and implement appropriate policies and measures to improve the situation of hospitals in the country, especially in the public sector. taking advantage of the potential capacity of the private sector in the hospital system is one of these policies (12). considering the obligations in upstream documents, such as the overall policies of article 44 of the constitution of the islamic republic of iran, on better and more interactions with the private sector (provision 136), and given that the foundations of this model have been defined in the budget laws in 2015 and in the laws of the fifth socio - economic plan, it seems necessary to conduct a study to identify strategies to develop and promote ppps in the provision of hospital services in iran in order to raise awareness among policy - makers and managers in the field of health planning and decision - making. it seems that implementing and promoting the ppp model can help develop some areas of health services, as it has done in different fields of the economy earlier (13). overall, we can conclude that a managed ppp can even aid seemingly unlikely aspects of the public health sector, such as education and personnel management very much and help public hospitals overcome many of their age - long shortcomings and more recent constraints. this was an exploratory qualitative research study that sued content analysis and was conducted for a specific purpose in 2015. the statistical population consisted of experts and professionals of the health system and individuals familiar with ppp models and roles in the development of the model in hospital services in iran. this method is one of the most common methods of sampling in qualitative studies in which the group of participants is selected based on pre - specified criteria related to specific research questions (14). in this approach, participants are handpicked by the researcher because they have the features or the phenomena considered or they have rich information about specific cases (15). likewise, in order to increase the diversity of attitudes, the purposive sampling method with maximum diversity was used in such a way that, after the first sample selection, the sample was asked to introduce other experts (16). the criterion for sample size in this study was similar to qualitative studies in terms of reaching the saturation level of data, and, when new codes were not obtained, the sampling was finished. accordingly, 18 professionals and experts in iran s health system were interviewed. of these individuals, nine representatives of the public sector (ministry of health, university of medical sciences, and public hospitals) and nine representatives of the private sector (moheb institution, moheb hospital) were enrolled in the study. at first, the interview guide was prepared according to the theoretical background of the study and review of the related literature in a semi - structured form with seven questions. in order to determine the validity and reliability of the significance of the questions from the participants perspectives, three pilot interviews were conducted with individuals who were not among the selected participants ; according to their attitudes and in necessity, the required amendments were made to remove the defects. ultimately, 10 final questions were decided upon to conduct the main interviews of this study. accordingly, first, general questions were asked to start the interviews ; then, the process of interviews was directed based on the participant s answers to questions. interviews were conducted by appointment, and the locations of the interviews were chosen by the participants. thus, after coordination, all interviews were conducted in the participants workplaces, and the duration of each interview ranged from 30 to 90 minutes, depending on the conditions and the willingness of the participants. all interviews were recorded, and, immediately after the end of each session, they were written down with the consent of the participants to determine the data saturation time and increase the accuracy and preciseness of transcribing the texts. in order to consider the ethical issues of this study, the informed consent of the participants was obtained, and they were assured that their participation and recording the interviews were voluntary. they also were assured that their information would remain confidential, available only to the researcher, and that they had the right to withdraw at any stage of the study. this method is a specialized technique for processing scientific data, and it is used to determine the presence of certain words and concepts in the text to summarize, describe, and interpret the data (17, 18). to this end, the interviewer transcribed the contents after each interview. the transcriptions of the interviews were studied several times to obtain a general understanding of them. in addition, the transcribed texts for each interview were given to the relevant interviewees so they could provide comments or revisions. the interview texts were fed into maxqda software, version 10, and the initial codes were extracted. then, with the cooperation and the use of the opinions of the participants and the members of the research team, the final codes and themes of the study were extracted. to consolidate the results, different strategies were used, such as member checks, submission of the transcribed and interpreted texts to the participants, and reception of their comments and suggestions, peer checks, and peer debriefing (19). approval of the transcribed texts and the extracted codes and themes was conducted by two members of the research team with no conflict of interest regarding the subject of the study. of the 18 interviewees present, all were male with the average age and working experience of 48 8.94 and 23 7.81 years, respectively. according to the results of the study, four main themes and 20 sub - themes related to the strategies to develop and promote ppp in the provision of hospital services in iran one of the factors that can provide the context for the development and promotion of ppp in the provision of medical services in iran is changes in current policies and laws. in this regard, stable government policies could provide for the development of ppp., they can not say everything as they like, [for instance ] a president might come and say that i will reduce the bank interest to 5%, one might come and abandon it ; then, another one might come and say that i will reduce the bank interest to zero. (p5). reform and restructuring of insurance in iran was one of the other issues that, according to many interviewees, needed attention for the development of ppp. now, we have just one contract. it can cover a limited part of the services [as a result ] insurance is not perfect. in some situations, people need more than that and it should have a better coverage of insurance. (p11). some participants believed that the ministry of health should only have the task of trusteeship and policy - making and that it should not be involved in executive affairs. the ministry of health is required to do as a trustee, as a supervisor, as a policy - maker and legislator. the private sector or ppps, charities, and other organizations come and provide services. the ministry of health should only supervise and have the role of a trustee and policy - maker. (p8, p12). some of the interviewees considered the formation of a board of trustees in hospitals as a starting point for the development of ppp and, consequently, chain hospitals. in this regard, one of the senior managers of the health system stated that : formation of chain hospitals needs that a hospital becomes an independent economic unit called the board of trustees. after that, this independent economic unit can invest to establish other branches in other provinces and even it is likely to do it with an external or internal partner. (p17). another issue that was identified to develop and promote ppp in iran s hospitals was socio - cultural changes. change in the mindset and belief of individuals regarding the use of the capacities of the private sector is one of the important things noted by the participants. they believed that the development and promotion of ppp requires changes in mentality and culture. in my opinion, most of the problems are related to cultural ones ; i mean the cultural mindset that is prevailing in the parliament, in government, in the ministry of health is in a way that they need no help, but when you analyze it ; you find that the need is very serious and you should help them as soon as possible. if the cultural mindset shifts and they feel that they should make use of the capacities of the private sector, regard, one of the interviewees believed that : in the healthcare sector, we should consider the issue of utilitarian in the private sector. we think people involved in the government are very good and all people in the private sector are bad. improvement in current mechanisms and processes is another important issue necessary to develop and promote ppp. one of these mechanisms highlighted by the majority of interviewees was creating a strong and single entity for managing relationships with private sector. there should be a committee to issue the qualifications of private partners. in all countries, there is a very strong consortium that sets up the contracts, approves the qualifications of private sector, and monitors everything. we need such a framework which is supported and does not change with any changes in the government. clarification of existing patterns and their management were among the important issues taken into account by many participants for the development and promotion of ppp in iran. in this regard, one of the interviewees said that : if ppp wants to promote, the example cases that have already started need at first a good management to have a good outcome. now, our status and the activities we started (moheb institution) lead the non - governmental organizations have no tendency to this issue due to the problems that we were involved in. they say that you got into this work, did your best, got forward, and got involved in all the problems and challenges ; so why the private sector should start the same thing. the government should start, come forward, and help and facilitate everything and ask the private sector to get forward. if these problems are not solved in this model, it can not be expanded. we have to start from pilot cases. because it helps us do it better and create a good motivation for the rest of private investors. another strategy suggested to develop and promote ppp was the formulation of proper and understandable contracts. my suggestion is to have someone outside of the health system in iran, perhaps i should say an international consultant who studies our laws and then develops a strong legal agreement based on the laws in iran in order to assure the investors that their capital is provided. the government is also confident that its rights are met according to the contract, and the investor and the government start to debate. we have problems with the agreements which are irrational and ambiguous. (p2). providing conditions and the background for attracting investment is another strategy that was mentioned by some individuals. in this respect, one of the participants believed that : to make such partnerships happen in iran, we need to do something that would help the investor prefer to invest in hospitals rather than in other places. there must be government guarantees for return on capital and earnings to the investor because no one tends to invest somewhere without any return on investment. one of the things that sets the stage for attracting investment in the health sector is considering incentives and motives awarded by the government. the most important thing in the economy of health is individuals participation in providing services through investment. to attract investors in the provision of healthcare services, we need to define different incentives from the government. the government has a number of facilities which can be given to the private sector to keep the total cost low ; in other words, to increase the earnings. for example, the government can delete the price of land from this equation. (p4). government guarantees for investors were one of the other issues that was highlighted by participants in order to develop and promote ppp. for example, one of the participants believed that : the government needs to think ahead to develop its services and to play a prominent role in this regard. for example, the government needs to ensure that there is a market, there is a guarantee about the existing risks, and take these risks from the investors such as domestic risks and exchange rate risks. (p10). one of the factors that can provide the context for the development and promotion of ppp in the provision of medical services in iran is changes in current policies and laws. in this regard, stable government policies could provide for the development of ppp., they can not say everything as they like, [for instance ] a president might come and say that i will reduce the bank interest to 5%, one might come and abandon it ; then, another one might come and say that i will reduce the bank interest to zero. reform and restructuring of insurance in iran was one of the other issues that, according to many interviewees, needed attention for the development of ppp. in this it can cover a limited part of the services [as a result ] insurance is not perfect. in some situations, people need more than that and it should have a better coverage of insurance. (p11). some participants believed that the ministry of health should only have the task of trusteeship and policy - making and that it should not be involved in executive affairs. the ministry of health is required to do as a trustee, as a supervisor, as a policy - maker and legislator. the private sector or ppps, charities, and other organizations come and provide services. the ministry of health should only supervise and have the role of a trustee and policy - maker. (p8, p12). some of the interviewees considered the formation of a board of trustees in hospitals as a starting point for the development of ppp and, consequently, chain hospitals. in this regard, one of the senior managers of the health system stated that : formation of chain hospitals needs that a hospital becomes an independent economic unit called the board of trustees. after that, this independent economic unit can invest to establish other branches in other provinces and even it is likely to do it with an external or internal partner. (p17). another issue that was identified to develop and promote ppp in iran s hospitals was socio - cultural changes. change in the mindset and belief of individuals regarding the use of the capacities of the private sector is one of the important things noted by the participants. they believed that the development and promotion of ppp requires changes in mentality and culture. in my opinion, most of the problems are related to cultural ones ; i mean the cultural mindset that is prevailing in the parliament, in government, in the ministry of health is in a way that they need no help, but when you analyze it ; you find that the need is very serious and you should help them as soon as possible. if the cultural mindset shifts and they feel that they should make use of the capacities of the private sector, (p2, p6). in this regard, one of the interviewees believed that : in the healthcare sector, we should consider the issue of utilitarian in the private sector. we think people involved in the government are very good and all people in the private sector are bad. improvement in current mechanisms and processes is another important issue necessary to develop and promote ppp. one of these mechanisms highlighted by the majority of interviewees was creating a strong and single entity for managing relationships with private sector. there should be a committee to issue the qualifications of private partners. in all countries, there is a very strong consortium that sets up the contracts, approves the qualifications of private sector, and monitors everything. we need such a framework which is supported and does not change with any changes in the government. (p14). clarification of existing patterns and their management were among the important issues taken into account by many participants for the development and promotion of ppp in iran. in this regard, one of the interviewees said that : if ppp wants to promote, the example cases that have already started need at first a good management to have a good outcome. now, our status and the activities we started (moheb institution) lead the non - governmental organizations have no tendency to this issue due to the problems that we were involved in. they say that you got into this work, did your best, got forward, and got involved in all the problems and challenges ; so why the private sector should start the same thing. the government should start, come forward, and help and facilitate everything and ask the private sector to get forward. if these problems are not solved in this model, it can not be expanded. we have to start from pilot cases. because it helps us do it better and create a good motivation for the rest of private investors. another strategy suggested to develop and promote ppp was the formulation of proper and understandable contracts. my suggestion is to have someone outside of the health system in iran, perhaps i should say an international consultant who studies our laws and then develops a strong legal agreement based on the laws in iran in order to assure the investors that their capital is provided. the government is also confident that its rights are met according to the contract, and the investor and the government start to debate. we have problems with the agreements which are irrational and ambiguous. (p2). providing conditions and the background for attracting investment is another strategy that was mentioned by some individuals. in this respect, one of the participants believed that : to make such partnerships happen in iran, we need to do something that would help the investor prefer to invest in hospitals rather than in other places. there must be government guarantees for return on capital and earnings to the investor because no one tends to invest somewhere without any return on investment. one of the things that sets the stage for attracting investment in the health sector is considering incentives and motives awarded by the government. the most important thing in the economy of health is individuals participation in providing services through investment. to attract investors in the provision of healthcare services, we need to define different incentives from the government. the government has a number of facilities which can be given to the private sector to keep the total cost low ; in other words, to increase the earnings. for example, the government can delete the price of land from this equation. (p4). government guarantees for investors were one of the other issues that was highlighted by participants in order to develop and promote ppp. for example, one of the participants believed that : the government needs to think ahead to develop its services and to play a prominent role in this regard. for example, the government needs to ensure that there is a market, there is a guarantee about the existing risks, and take these risks from the investors such as domestic risks and exchange rate risks. (p10). most of the individuals who believed in the provision of healthcare services by a centralized public system in the past have argued that the private sector is an indispensable part of a national program of providing healthcare services and that the use of various methods of private - sector partnership can effectively contribute to improved quality, reduced costs, and better management of resources and efficiency (20). there should be prerequisites and arrangements for the development of these partnerships so that they can be effective and bring valuable results and outcomes. four main strategies that were identified as the main factors in the development and promotion of ppps in the provision of hospital services in iran were changes in policies and laws, socio - cultural changes, improvement of current mechanisms and processes, and capital and financial capacity building. in terms of political and legal strategies, different countries have taken various measures in order to implement, develop, and promote ppp. for example, a separate chapter on ppp was considered in the constitution in thailand. in the uk, as one of the pioneers in ppp, units of ppp were established at the level of ministries, and incentives to the private sector were recommended. in germany, policies to support small firms to join ppps were adopted (21). although partnership with the private sector and the use of its capacities in iran s 20-year prospective plan and the provisions of the fourth and the fifth development plans were considered, the development and promotion of such partnerships in the healthcare sector require changes and amendments to existing laws and policies. it is better to approve legal provisions and regulations to strengthen ppp contracts by the government and the judicial system. in this regard, grimsy and lewis concluded that conflicts are inevitable in ppp without an efficient and optimal legal framework, and they stated that revisions in existing laws and policies must be taken into account for ppp projects to succeed (22). another strategy identified to develop and promote ppp in the provision of hospital services in iran was socio - cultural changes. in a study conducted in sri lanka, various strategies, such as changes in the mindset of health workforce, provision of education to the political leaders in the country, and culture - building in terms of accepting ppp in order to provide health services, were suggested for the development of ppp in the healthcare industry (23), which partially confirmed the results in our study. government support is vital in the early years of the development of ppp projects or in a new market. without adequate support by the government improvement of current mechanisms and processes was one of the other strategies that was suggested for the development and promotion of ppp. in this respect, the results of a systematic study revealed the effectiveness of ppp projects on several factors, including writing a contract, role of partners, legal framework, and stakeholders partnership (24). in fact, the temporary and pilot implementation of the program has advantages that examine the feasibility of the program and can identify the strengths and weaknesses for the implementation of future programs (25). the ppp models of hasheminejad hospital and moheb institution in iran were relatively successful examples among public hospitals, which provided capital entry into the health sector and could be promoted and expanded according to the existing laws and capacities. the experiences in other countries may not be exactly applicable in iran, but it seems that the localization of this model and its adaptation with the conditions of the country could well change the administration of educational medical centers in iran. financial and capital capacity building was considered as one of the other important factors for the development of ppp in hospital services in iran. in fact, governments should provide the groundwork for attracting investment in health sector. to do so, incentives and motives for the entry of investors in this area should be taken into account. such incentives and motives for private investors can provide an appropriate context for investment in the health sector. stimuli and incentives, such as loans with reasonable interest rates and guarantees for reasonable returns on earnings, can be used in the regard. if governments provide these for the private sector, investors will be more motivated to finance in underprivileged areas, and this leads to a reduction in the financial burden on the governments to provide public services (26). in this regard, evidence suggests that the allocation of low - interest loans for ppp projects in thailand, providing loans and facilities from the development fund in india, and allocation of funds and loans from the banks to the private partners in the uk have been some of the strategies and incentives for ppp development in the health sector (21). in the unique experience of ppp in iran s health sector (moheb hospital), bank facilities were awarded to the private sector partner as an incentive to join the ppp projects ; therefore, this kind of facilities and other financial incentives should continue for further development of ppp projects and motivate investors and the government to take steps to buy back the services from the private sector, consider reasonable profits for investors, and create confidence among them. this study encountered several limitations as follows : first, triangulation could not occur because the limited number of participants did not allow the researchers to use a mix - method design. identification of the factors that affect the development and promotion of ppp models before their implementation and execution can be very effective. in this study, various factors, such as changes in policies and laws, socio - cultural changes, correction of current mechanisms and processes, and financial and capital capacity building, were identified as the most effective strategies in developing and promoting ppp in the provision of hospital services in iran. accordingly, given that this study was conducted using the content analysis method by means of interviews to collect the data, it is recommended that more precise investigations and case studies be conducted to extract and explore the influential components, the implementation process, and the achievements. furthermore, given that the present results and their comparison with other international findings showed the importance of moving towards the implementation of ppp, it was recommended that appropriate studies be conducted before implementing this model in qualified hospitals in iran. | introductionpublic - private partnerships (ppps) have been constructively considered in recent years to reform health sectors in many countries. this study aimed at explaining the strategies to develop and promote ppps in the provision of hospital services in iran.methodsthis qualitative study was conducted in 2015. the study population consisted of experts and professionals of the health system and individuals familiar with ppps and roles in the development of such models in hospital services in iran that were selected through purposeful sampling, and semi - structured interviews were used for data collection. to this end, 18 experts and professionals of the health system and individuals familiar with ppps and roles in the development of such models in hospital services in iran were selected. the data were analyzed and classified using maxqda10 software and content analysis, respectively.resultsaccording to the results of this study, four main themes and 20 subthemes in terms of strategies to develop and promote ppps in the provision of hospital services in iran were identified, including changes in policies and laws, socio - cultural changes, improvement of mechanisms and current processes, and financial and capital capacity building.conclusionto develop and promote ppps in the provision of hospital services, there is a need to consider prerequisites and measures to help such partnerships to be effective and produce valuable results. |
echinococcus multilocularis is one of the rare types of parasitic infestations in the northern hemisphere which can involve several organs, especially the liver. however, there is not enough information about liver involvement related to this parasite in human beings (hepatic alveolar echinococcosis) (1). liver involvement of this parasite is a life - threatening infestation, especially when diagnosis is delayed, which itself can be life - threatening (2). in this review, we discuss all of the published cases of hepatic ae from iran in regard to epidemiology, clinical presentation, diagnosis, and protocols of treatment. the adult form of this tapeworm lives in the intestines of natural definitive hosts, which can be wild carnivores such as red fox or domestic dogs and cats. the eggs released by the adult tapeworm are passed in the stool of the definitive hosts, which then is ingested by the intermediate hosts, such as small rodents or red foxes. however, there are other accidental intermediate hosts, such as humans. ingestion of the eggs by humans usually occurs because of the contamination of food or water by the fecal material of definitive hosts. after the ingestion of the eggs by humans, the larvae (metacestodes) travel to the liver, where they create alveolar small vesicles and alveolar cysts (3 - 5). as mentioned, hepatic ae is caused by the infestation of humans, as an accidental intermediate host, by the larval stage of e. multilocularis. the echinococcal metacestodes grow in the liver and create an alveolar - like structure, which consists of several vesicles surrounded by large granuloma. human ae is a severe and emerging disease which, with delayed diagnosis, can be life - threatening (2). ae is considered one of the most dangerous parasitic infestations of human beings in the northern hemisphere. new ae cases in western and central europe have been estimated in the range of 170 - 200 per year. the highest reported numbers of ae from europe were from france, germany, switzerland, lithuania, and poland, and it is extremely rare in the eastern and central parts of europe (6). high parasite prevalence is also reported in the middle east and arabic north africa. in the middle east, iran, iraq, and turkey are known as areas where ae is endemic (7). however, from these regions, where the parasite is known to be present, there is a scarcity of information about recent distribution and frequency (8). the adult form of this tapeworm lives in the intestines of natural definitive hosts, which can be wild carnivores such as red fox or domestic dogs and cats. the eggs released by the adult tapeworm are passed in the stool of the definitive hosts, which then is ingested by the intermediate hosts, such as small rodents or red foxes. however, there are other accidental intermediate hosts, such as humans. ingestion of the eggs by humans usually occurs because of the contamination of food or water by the fecal material of definitive hosts. after the ingestion of the eggs by humans, the larvae (metacestodes) travel to the liver, where they create alveolar small vesicles and alveolar cysts (3 - 5). as mentioned, hepatic ae is caused by the infestation of humans, as an accidental intermediate host, by the larval stage of e. multilocularis. the echinococcal metacestodes grow in the liver and create an alveolar - like structure, which consists of several vesicles surrounded by large granuloma. human ae is a severe and emerging disease which, with delayed diagnosis, can be life - threatening (2). ae is considered one of the most dangerous parasitic infestations of human beings in the northern hemisphere. new ae cases in western and central europe have been estimated in the range of 170 - 200 per year. the highest reported numbers of ae from europe were from france, germany, switzerland, lithuania, and poland, and it is extremely rare in the eastern and central parts of europe (6). high parasite prevalence is also reported in the middle east and arabic north africa. in the middle east, iran, iraq, and turkey are known as areas where ae is endemic (7). however, from these regions, where the parasite is known to be present, there is a scarcity of information about recent distribution and frequency (8). in this brief review, the published cases of hepatic ae from iran were retrieved for review via a search in pubmed, scopus, google scholar, iranmedex, scientific information database (sid), magiran, and irandoc (1995 - 2015), using the keywords echinococcus multilocularis and iran, alveolar hydatid cyst and iran, liver and alveolar hydatid cyst and iran, and hepatic alveolar hydatid cyst and iran. the following inclusion criteria were employed : 1, articles must be written in english or farsi ; 2, articles must have been published between 1995 and 2015 ; 3, cases must have been confirmed by pathologic examination. during the 20 years between 1995 and 2015, there were 24 published cases of hepatic ae from iran (9 - 14). there were 20 female patients and 4 male (f / m = 5/1). the age range of these patients was 21 - 74 years (42.6 12.2). twenty - two patients were from the northeast part of the country, i.e., khorasan razavi province, one patient was from ardebil province, and one case was reported from the south, i.e., khuzestan province. clinical presentation of the patients was mostly right upper quadrant (ruq) pain in 20 (83%) patients, anorexia and weight loss in 2 (8.3%) patients, jaundice in 5 (20.8%) patients, and fever in 4 (16.6%) patients. the most common sign in physical examination was hepatomegaly and liver mass in all 24 patients, which was tender in two of them (8.3%). one patient presented with cough, bloody sputum, and fatigue, secondary to lung metastasis (13). duration of the disease at the time of diagnosis was from 20 days to 5 years (35.6 27.5 months). in all of the cases, sonographic findings were described as large (from 10 - 30 cm) solid or cystic liver masses with heterogeneous echogenicity and irregular margins. magnetic resonance image (mri) and ct scan, used in a few cases, revealed large multiloculated liver masses with heterogeneous density and irregular and infiltrative margin. fifteen cases had a mass in the right lobe of the liver, seven cases in the left lobe, and in two patients, there were multiple liver masses in both liver lobes. in most of the published cases, however, in four reported cases, wbc was increased in two of the patients (5,200 - 15,500/ml). liver enzymes, including alanine aminotransferase (alt), aspartate aminotransferase (ast), and alkaline phosphatase (alp) were mostly abnormal. alt was 10 - 268 iu / l (74.9 78.6) (normal < 28 iu / l), ast was 18 - 262 iu / l (79.83 80.4) (normal < 28 iu / l), and alp was 90 - 1919 iu / l (578.7 595) (normal < 150 bilirubin level was reported in 21 patients as between 0.3 to 29.3 mg / dl (6.5 8.7) with a normal of < 1.2 mg / dl. table 1 shows the primary characteristics of the 24 patients with hepatic ae published from iran. iu / l) ; alp, alkaline phosphatase (normal < 150 the gold standard for final diagnosis in all of the cases of hepatic ae from iran was tissue biopsy and pathologic diagnosis, either with a wedge biopsy or needle biopsy. most of the cases were treated by a combination of surgery and antiparasitic medical therapy, i.e., albendazole, for six months to two years. nine cases had large, unresectable liver masses, one of which was scheduled for liver transplantation, but the patient did not accept it. however, she responded to medical therapy and, after six months follow - up, she remained alive (9, 10). postoperative complications for patients who underwent resection included postsurgical abscess, cholangitis, and biliopulmonary fistula (13, 14). death was reported in three patients, secondary to lung metastasis, infection, and fistula (13). the larval form rapidly proliferates and produces an alveolar - like pattern of microvesicles in different tissues of the body, especially the liver (15). therefore, the characteristic feature of ae is its tumor - like pattern of growth. the liver is the first and most common organ to be affected by larval infestation. hepatic ae is a rare and life - threatening parasitic disease, which develops as a result of intrahepatic growth of the echinococcus multilocularis (4). liver involvement related to echinococcus multilocularis was first observed by virchow in 1855, who published an article entitled echinococcus - tumor of the liver (16). the who - iwge pnm classification system was established in 2001, based on imaging findings, as an international standardization for evaluation of diagnostic and therapeutic modalities. p is the degree of extension of the parasite, n indicates the involvement of nearby organs, and m is considered metastasis. this classification was developed to evaluate the extension of the disease and therapeutic approaches (15). ae is characterized by primary lesions in the liver, producing tumor - like, multi - vesicular, infiltrating structures. early diagnosis of ae by characteristic signs and symptoms, imaging modalities, immunologic tests (such as elisa), histopathological examination, or molecular analysis of tissue materials can be life - saving and will significantly improve the prognosis of ae patients. recently developed and improved serological and molecular tests for ae have been shown to be reliable (17). however, in most patients, characteristic symptoms are not present, and in most geographic areas of world, as in many parts of iran, these tests are not available. ultrasonography, ct scan, and mri are the most common imaging modalities for the diagnosis of ae in the liver. large liver masses with mixed echogenicity and irregular margin are the most common sonographic findings (18). it is recommended to perform ct scans in cases where liver - involved ae is suspected, but in most of the published cases from iran, no ct report was mentioned. ae in ct presents with a hypodense liver mass, very similar to a malignant infiltrative process with irregular borders. ct scan has the capacity to determine the extension of the disease, as well as the number, size, and localization (12). magnetic resonance imaging (mri) is also a good modality to diagnose the extension of the disease. in mri, the liver mass of ae presents as an irregular, solid - cystic mass, with honeycomb appearance in t2-weighted images (19). fluorodeoxyglucose positron emission tomography (fdg - pet) is a noninvasive modality for the detection of metabolic activity in ae. delayed images would increase its sensitivity for the assessment of the metabolism of the lesion and its reliability for the continuation or withdrawal of antiparasitic treatment (20). pet - ct can be used to monitor suppression of periparasitic inflammatory activity and has an important role for excluding the metastatic spread. however, expensive and sophisticated equipment is not widely available, which limits its use for a routine procedure (19). the most common presentation is extensive necrosis, with small vesicles and delicate membranes scattered in the necrosis. in our experience, the most common pitfall for the pathologist is missing the above - mentioned structures and calling the biopsy unsatisfactory, because of extensive necrosis (9, 10). this places emphasis on the role of the pathologist in the diagnosis of ae in the liver, because delayed diagnosis of hepatic ae leads to advanced disease with unresectable masses and poor prognosis (21). in these circumstances, for very large, unresectable masses, the only surgical therapeutic modality can be liver transplantation (22). in addition to surgical approaches, considering long - term (six months to two years) medical antiparasitic therapy (e.g., albendazole) is very important to decrease the risk of recurrence and postsurgical complications (23). according to the previous studies in the english literature, although liver hydatid cyst, secondary to echinococcus granulosus, is a very common disease in iran, very few cases of hepatic alveolar hydatid cyst have been published from iran (12). despite the rarity of this disease, thus, it is very important to consider this disease in the differential diagnosis of patients with liver mass, especially those from endemic areas (e.g., northeast iran) (9, 10). | contextechinococcus multilocularis is the cause of alveolar hydatid disease, which most commonly involves the liver in more than 90% of cases. this disease is endemic in northern iran. however, there are very few published cases from iran. in this article, we will review all of the published cases of hepatic alveolar echinococcosis from iran regarding all aspects, including clinical, paraclinical, and treatment protocols.evidence acquisitionin this brief review, the published cases of hepatic alveolar echinococcosis (ae) from iran were retrieved for review via a search in pubmed, scopus, google scholar, iranmedex, scientific information database (sid), magiran, and irandoc (1995 - 2015) using the keywords echinococcus multilocularis and iran, alveolar hydatid cyst and iran, liver and alveolar hydatid cyst and iran, and hepatic alveolar hydatid cyst and iran. the following inclusion criteria were employed : 1, articles must be written in english or farsi ; 2, articles must have been published between 1995 and 2015 ; 3) cases must have been confirmed by pathological diagnosis.resultsthere were 24 published cases of liver - involved alveolar hydatid cyst from iran. the disease was more common in young- to middle - aged women in northeast iran. the most common presenting signs and symptoms were abdominal pain with hepatomegaly and liver mass. most of the patients were treated by surgery and albendazole. the few unresectable liver masses were treated by medical therapy. no liver transplantation for this disease was reported from iran.conclusionshepatic alveolar hydatid cyst should be considered one of the important differential diagnoses of liver masses, especially in endemic areas of the world. |
appendectomy for acute appendicitis is one of the most common emergency general surgery procedures performed in the united states. while novel nonoperative treatments for acute appendicitis are emerging, the mainstay of treatment for nonperforated appendicitis remains laparoscopic removal of the appendix. in the current healthcare environment, methods to maximize the value of care delivered are being explored. identifying methods to achieve the choice of instruments used for any given surgical procedure has traditionally been left to the discretion of each individual surgeon, within the confines of what instruments are available at that particular hospital. the ability to reduce costs in laparoscopic introducing less expensive instruments for achieving the same outcomes is another method of reducing costs. the current literature most commonly describes using either a suture ligature device or an endoscopic stapler to transect the appendix. our institution was interested in the use of polymeric clips for securing the appendiceal base, which has been reported in the literature outside of the united states. polymeric clips are currently in use for numerous procedures, but have not been reported in the united states for use during appendectomy. the purpose of this study was to examine the outcomes of appendectomy performed with polymeric clips. we hypothesized that the use of polymeric clips for ligation of the appendiceal stump would be feasible and would not lead to significant perioperative complications. in this prospective study, we examined a cohort of patients who underwent laparoscopic appendectomy with polymeric clips (hem - o - lok, weck polymer locking ligation system, teleflex, research triangle park, north carolina, usa). the trial included patients 18 years old or older who were scheduled for laparoscopic appendectomy for a diagnosis of appendicitis. the diagnosis of appendicitis was made by the consulting surgeon after review of the patient 's history and imaging. all patients in this study had undergone computer - assisted tomography (ct) before surgical consultation. patients with a diagnosis of appendicitis and who were scheduled for a laparoscopic appendectomy were approached by a member of the research team and were prospectively asked to consent to participate in the study. patients were excluded if they were less than 18 years of age, had signs of appendiceal rupture or abscess on preoperative ct, or were unable to provide informed consent. three surgeons experienced in minimally invasive techniques who routinely perform a variety of laparoscopic general surgery procedures performed all of the appendectomy procedures and were occasionally assisted by surgical trainees with various experience levels (residents and fellows). standard 3-port laparoscopic appendectomy techniques were used, and all appendiceal bases were ligated with polymeric clips. a clip applier placed through a 10-mm port was used to apply the x - large clips. two clips were left on the appendiceal stump, and a third clip was applied toward the specimen side of the appendix before sharp scissor division of the appendix. tissue dissection and mesoappendix ligation techniques were left up to the discretion of the surgeon ; bipolar vessel - sealing technology and monopolar electrocautery dissection were the methods most commonly used. the exposed appendiceal stump mucosa was fulgurated with electrocautery (figure 1). a, placement of polymeric clip on appendiceal base. our primary outcome of interest was the incidence of major surgical complications : appendiceal stump dehiscence, abscess, and hemorrhage. our secondary outcomes of interest were other complications (pneumonia, urinary tract infection, myocardial infarction, pulmonary embolism, deep vein thrombosis, and death) and length of stay (los). data on patient, operative, and hospitalization characteristics and on postoperative follow - up were gathered from review of medical records. los was determined by chart review and was calculated from the date of surgical consult or date of admission until the date of discharge. patient characteristics included sex, age, white blood cell (wbc) count before the operation, preoperative imaging, smoking history, body mass index (bmi, kg / m), american society of anesthesiologists (asa) physical status classification, and final pathology. operative characteristics included stump closure technique, intraoperative complications, estimated blood loss (ebl), and other concurrent procedures. follow - up characteristics included time to follow - up in surgical clinic, total length of follow - up time in our healthcare system (in any clinic), and all postoperative complications (presenting either during hospitalization or after discharge). all analyses were performed using stata (statacorp, college station, texas, usa). a total of 26 patients were enrolled in our study, and 25 underwent laparoscopic appendectomy with polymeric clips. one patient was unable to have the clips placed because of inflammation of the appendiceal base. the maximum diameter of tissue that can be ligated by the xl (largest size) clip is 16 mm, and acute tissue inflammation resulting in appendiceal base diameter beyond 16 mm precluded use of the clip in that patient. the patients were 56% female with a mean age of 41 years and average bmi of 29 kg / m. one patient underwent ct on postoperative day 2 for abdominal pain and ileus, which revealed a small amount of persistent fluid in the pelvis. however, the patient did not require drainage or other invasive interventions, and symptoms resolved spontaneously. one patient presented to the emergency department for evaluation of continued abdominal pain several days after surgery, but no physiological abnormalities were identified. one patient had placement of an intraoperative drain due to radiologic concerns over possible concurrent diverticulitis in the descending colon. this patient 's clinical condition improved after appendectomy, and the drain was removed without incident. mean length of stay was 1 d and mean length of follow - up within our health system was 81 d. twelve percent of the patients did not come for a follow - up appointment in our health system. additional patient - related and perioperative characteristics can be found in tables 1, 2, and 3. postoperative characteristics of patients undergoing laparoscopic appendectomy with a polymeric clip used to secure the appendiceal stump n = 25 patients. other incidental ct findings included nephrolithiasis / large staghorn calculus, ovarian cyst / dominant follicle, cholelithiasis, focal diverticulitis of the descending colon, constipation, atherosclerosis, medical duodenal diverticulum, suspected hepatic cyst, small hiatal hernia, colonic diverticulosis, hepatic steatosis, bilateral renal cysts, intrauterine device, borderline retroperitoneal and mesenteric adenopathy, small right inguinal hernia containing fat only, mild elevation right hemidiaphragm, mild biliary dilatation. e, emergent. postoperative characteristics of patients undergoing laparoscopic appendectomy using polymeric clip to secure the appendiceal stump postoperative characteristics of patients undergoing laparoscopic appendectomy with polymeric clip to secure the appendiceal stump laparoscopic appendectomy for acute appendicitis has been shown to be a safe alternative to the standard open procedure. initial reports suggested that the laparoscopic approach offers shorter los and decreased incidence of wound infection in exchange for higher operative cost and a potential for increased incidence of intra - abdominal infection. however, more recent studies suggest that the complication rates are now similar, and efforts are ongoing to reduce costs. furthermore, if operative cost differences remain, this may be compensated for by shorter los and quicker return to work for laparoscopic procedures. in our institution, laparoscopic appendectomy is the most common treatment for acute appendicitis, but a variety of techniques have been used for transecting the appendix. the use of endoscopic linear staplers to control the appendix base is the most common technique and has been shown to be safe and effective. however, there is a significant cost associated with this application ; stapler handles and loads usually cost several hundred dollars. institution - specific pricing schedules prevent detailed comparison of stapler / load costs, but there is no question that polymeric clips are much less expensive than endoscopic staplers. use of an endoloop (ethicon, llc, cincinnati, ohio, usa) or similar suture ligature technique has been advocated as a less expensive option that can be placed through a 5-mm port, which is another proposed benefit. however, they can be more difficult to apply, and this may be why their use has been limited at our institution. furthermore, endoloops are associated with increased operative time, in comparison to polymeric clips and endostaplers. proficient use of endoloop is part of the fundamentals of laparoscopic surgery (fls) curriculum, and surgeons who have completed this training should therefore feel comfortable using this technique but may still take more time to do so. we believe that the application of polymeric clips represents a more basic skill and is a readily applicable and cost - effective option for surgeons who may not have completed fls training but continue to perform basic laparoscopic procedures. the polymeric clip offers significant cost savings when compared to endoscopic staplers, and provides a simpler application when compared to suture ligature techniques. recently, several international studies have demonstrated the effective use of a polymeric clip for transecting the appendix. polymeric clips are made of nonconductive, inert, nonabsorbable material and are capable of securing vessels and tissue bundles up to 16 mm in thickness. the clips have a hinged locking mechanism, as well as integrated teeth, which prevent slippage. because it is nonmetallic, it is safe to use electrocautery near the clip without risk of transmitting current. the only known contraindications to use of polymeric clips are severe inflammation with associated poor tissue quality, and tissue diameter larger than can be encompassed by the clip. sound surgical judgment should determine applicability of polymeric clip ligation of acutely inflamed tissue, and in one of our cases, an intraoperative decision was made to avoid clip ligation of an acutely inflamed appendiceal base with a diameter approaching the maximum size of 16 mm. of note, all other published studies to date examining the use of polymeric clips to secure the appendiceal stump have demonstrated it to be safe and cost - effective (table 4). prior studies examining use of polymeric clips in laparoscopic appendectomy pc, polymeric clip ; ot, operative time ; los, length of stay ; usd, u.s. dollars. polymeric clips are routinely used during laparoscopic cholecystectomy for clipping the cystic duct and artery, during laparoscopic urologic procedures for control of vessels and ureters, and in numerous other gynecologic and general surgery procedures. based on our clinical experience and the positive international reports in the literature, we conducted the first prospective clinical series using polymeric clips in the united states. we were particularly interested in expanding their use to appendectomies while maintaining our established, safe surgical outcomes profile and capturing potential cost savings. the single minor complication we encountered was associated with a superficial suture abscess, which developed at the umbilical incision site. one patient (4%) experienced postoperative pain and prolonged ileus caused by persistent pelvic fluid observed on ct scan. however, given the prompt and spontaneous resolution of this complication without any invasive intervention, we did not classify this event as significant. of note, the reported rate of postoperative intra - abdominal abscess in the literature ranges from 1 to 24%. clip migration is the most commonly reported complication of polymeric clips in the literature, although it has not been reported after laparoscopic appendectomy. it has been reported to happen as soon as 5 weeks after radical prostatectomy and as long as 8 months after a laparoscopic cholecystectomy. other theoretical complications related specifically to this technique include chronic pain, granulomas, or foreign body reaction due to the nonabsorbable nature of the clips. given that the mean length of follow - up in our health system was almost 3 months, we feel that we would have been aware of any occurrences of early migration. although it is possible that later migrations may yet occur, this complication remains extremely rare overall. we believe that the polymeric clips provided a significant cost savings without compromising patient safety outcomes. at our institution and in other studies, in contrast, a stapler plus just one staple load would contribute several hundred dollars to the operative cost. of note, we used the polymeric clips, not only for control of the appendix, but also for ligation and control of the mesoappendix in most cases. this application resulted in greater cost savings since a vessel sealing bipolar device or ultrasonic dissection device would contribute even greater additional costs to the operation. our average operative time was 45 min with only 1 case where the clip could not be applied because of significant appendiceal tissue inflammation. in fact, we had initially estimated a sample size of 40 patients to be recruited prospectively over a 1-year period. however, we did not anticipate the difficulty in obtaining investigational research consent in acutely ill patients presenting with abdominal pain and appendicitis. given their acute pain as well as their stress and anxiety in anticipation of unexpected impending surgery, we found them often unwilling to engage in a discussion regarding participation in a research study. we extended the initial study period beyond 12 months, and approached a larger number of patients than expected. however, we were able to obtain consent from only 26 patients and closed our recruitment at 18 months. another limitation is that 3 patients did not have follow - up in our system after discharge. in addition, the mean follow - up was 81 d, meaning that we could have missed some longer term complications. however long - term complications are rare after an appendectomy, and prolonged follow - up would have placed an undue burden on the patients. the single minor complication we encountered was associated with a superficial suture abscess, which developed at the umbilical incision site. one patient (4%) experienced postoperative pain and prolonged ileus caused by persistent pelvic fluid observed on ct scan. however, given the prompt and spontaneous resolution of this complication without any invasive intervention, we did not classify this event as significant. of note, the reported rate of postoperative intra - abdominal abscess in the literature ranges from 1 to 24%. clip migration is the most commonly reported complication of polymeric clips in the literature, although it has not been reported after laparoscopic appendectomy. it has been reported to happen as soon as 5 weeks after radical prostatectomy and as long as 8 months after a laparoscopic cholecystectomy. other theoretical complications related specifically to this technique include chronic pain, granulomas, or foreign body reaction due to the nonabsorbable nature of the clips. given that the mean length of follow - up in our health system was almost 3 months, we feel that we would have been aware of any occurrences of early migration. although it is possible that later migrations may yet occur, this complication remains extremely rare overall. we believe that the polymeric clips provided a significant cost savings without compromising patient safety outcomes. at our institution and in other studies, in contrast, a stapler plus just one staple load would contribute several hundred dollars to the operative cost. of note, we used the polymeric clips, not only for control of the appendix, but also for ligation and control of the mesoappendix in most cases. this application resulted in greater cost savings since a vessel sealing bipolar device or ultrasonic dissection device would contribute even greater additional costs to the operation. our average operative time was 45 min with only 1 case where the clip could not be applied because of significant appendiceal tissue inflammation. in fact, we had initially estimated a sample size of 40 patients to be recruited prospectively over a 1-year period. however, we did not anticipate the difficulty in obtaining investigational research consent in acutely ill patients presenting with abdominal pain and appendicitis. given their acute pain as well as their stress and anxiety in anticipation of unexpected impending surgery, we found them often unwilling to engage in a discussion regarding participation in a research study. we extended the initial study period beyond 12 months, and approached a larger number of patients than expected. however, we were able to obtain consent from only 26 patients and closed our recruitment at 18 months. another limitation is that 3 patients did not have follow - up in our system after discharge. in addition, the mean follow - up was 81 d, meaning that we could have missed some longer term complications. however long - term complications are rare after an appendectomy, and prolonged follow - up would have placed an undue burden on the patients. in this prospective study of the use of polymeric clips in laparoscopic appendectomy, our findings support previous international conclusions that polymeric clips are safe, efficient, and effective for securing the appendiceal stump. although the absolute value of cost savings varies by specific health system and individual pricing contracts, we confirmed that polymeric clips offer the ability to secure the appendiceal stump at a reduced cost, without increasing operative time or overall length of stay. thus, we believe polymeric clips can provide increased value of healthcare to patients undergoing laparoscopic appendectomy. | background and objectives : laparoscopic appendectomy remains one of the most common emergency general surgical procedures in the united states. in an era of increasing focus on costs in medical care, we sought to evaluate the use of polymeric clips to secure the appendiceal base during laparoscopic appendectomy.methods:we performed a prospective cohort study of patients undergoing laparoscopic appendectomy from april 2013 through september 2014 at a single academic teaching institution. polymeric clips were used to secure the appendiceal stump. tissue dissection techniques and control of the mesoappendix were the operating surgeon 's choice. clinical outcomes are reported.results:a total of 25 patients (56% women ; mean age, 41 y ; body mass index of 29 kg / m2) were enrolled in our study and underwent laparoscopic appendectomy with polymeric clips. one patient was unable to have polymeric clips placed due to inflammation of the appendiceal base. there were no major perioperative complications. one patient developed a suture abscess in the umbilical incision, and another had prolonged ileus with computed tomography demonstrating persistent pelvic fluid that did not require intervention. median length of stay was 1 d and mean length of follow - up was 81 d. the use of polymeric clips contributed $ 32 to the overall operative cost.conclusions:polymeric clips are a safe alternative for securing the appendiceal base in laparoscopic appendectomy. they offer significant cost savings without any evidence of increased complications. |
a 57-year - old man was referred to samsung medical center with abrupt confusion, fever, and loss of vision in both eyes. the patient was in his usual state of health until two weeks prior, when he began to develop sudden loss of vision in his right eye. in another hospital, he was diagnosed with retinal vasculitis in his right eye and was prescribed oral prednisolone, 60 mg / day for three days, followed by 120 mg / day for three days. the retinal vasculitis did not respond to oral prednisolone, so he was treated with intravenous methylprednisolone, 1 g / day for five days. two days after initiation of intravenous methylprednisolone, he complained of decreased vision in his left eye. one week after initiation of intravenous methylprednisolone, he became drowsy and high fever occurred, and he was then referred to samsung medical center. on neurological examination, he was drowsy and confused to time and place orientation. his brain magnetic resonance imaging (mri) showed a hyper - intense lesion in the left medial temporal and bilateral medial occipital areas on t2-weight images, suggestive of hse (fig. cerebrospinal fluid (csf) study revealed 11 cells / ml (lymphocytes 90%), low protein (19.3 mg / dl), and normal range glucose concentration (63 mg / dl compared to 110 mg / dl in serum). an empirical antiviral agent one week later pcr of the csf was revealed to be positive for hsv-1 dna. on the night of admission, his electroencephalography showed sharp waves in left or right anterior temporal areas (f7t7, f8t8 maximum) with a frequency of 1/min and diffuse slow waves. an antiepileptic drug (phenytoin 300 mg / d) was administered and no more epileptic seizure recurred. on the day of admission, his visual acuity was perception of light in both eyes. fundus examination showed retinal hemorrhage, confluent retinal necrosis affecting almost the whole retina, vascular sheathing involving both arterioles and venules, optic disc swelling, and exudative retinal detachment in both eyes (fig. infectious retinitis including acute retinal necrosis was suspected, but in terms of the location of necrosis, his clinical features were not typical of arn diagnostic vitrectomy confirmed the presence of hsv-1. after administration of intravenous acyclovir for 14 days, his systemic symptoms consistent with encephalitis slowly resolved, but his vision was not restored. on follow - up examination, retinal necrosis did not progress, but he still suffered from severe bilateral ocular pain with partial improvement of the exudative retinal detachment (fig. 2c, d). thus, intravitreal ganciclovir injection (5,000/0.05 g / ml) causes pain relief and decreased intraocular inflammation. atrophy was found six weeks later, thus vitrectomy with silicone oil tamponade was done in the right eye. cases with arn following hse have been uncommonly reported,59 and to our knowledge, this is the first report of the reverse situation, hse following arn, in even an immune - competent patient. arn associated with hse is known to be caused by axonal transmission of a reactivated latent virus from the brain to the retina, and retinitis occasionally occurs after the encephalitis. as shown by our patient, this is consistent with the finding that herpes virus uses bidirectional fast - axonal transport in neurons.11 arn usually presents as retinal necrosis with discrete borders located in the peripheral retina, rapid progression, circumferential spread, occlusive vasculopathy with arterial involvement, and a prominent inflammatory reaction in the vitreous and anterior chamber.3 our patient was initially diagnosed with retinal vasculitis and treated with a high - dose systemic corticosteroid, which was not effective. corticosteroids are known to affect cellular immunity and may promote viral replication. intraocular or systemic corticosteroids may induce retinal necrosis in patients with viral infection. thus, it is recommended that corticosteroids should be used in combination with antiviral agents or after beginning antiviral therapy.12 in this patient, the contralateral eye became infected, and hse occurred during initial corticosteroid therapy without antiviral medication. in cases with an unusual clinical course, especially in retinal vasculitis with arterial involvement,, arn may be a risk factor for hse, even in an immune - competent patient. the virus may reach the brain from the eye by a trans - axonal route. systemic steroid therapy may have exacerbated herpes viral infection in the retina, with subsequent spread to the contralateral eye and brain. | acute retinal necrosis (arn), a viral retinal disease with poor visual prognosis, following herpes simplex encephalitis (hse) are uncommonly seen, and there has been no case yet reported of the reverse situation. we herein present the reverse situation, an immune - competent patient with hse following arn. a 57-year - old man who had been under steroid therapy for retinal vasculitis the prior two weeks, presented with abrupt confusion and high fever. his cerebrospinal fluid study and brain magnetic resonance imaging revealed typical hse. ophthalmic examination and polymerase chain reaction of the vitreous specimen revealed arn by herpes simplex virus type 2. intravenous acyclovir treatment improved his encephalitis symptoms and retinal necrosis. this case implies that arn may be a risk factor for hse and the virus may reach the brain from the eye. inappropriate administration of a systemic steroid may exacerbate herpes viral infection in the retina, with subsequent spread to the brain. |
anterior choroidal artery (achoa) aneurysms (achoaa) are rare and count for less than 5% of all intracranial aneurysms. most of the time, achoaa arise from the posterior wall of the internal carotid artery (ica) and are located at the proximal segment near the origin10). distal achoaas (daa) are even more exceptional with only 46 cases reported in the literature2). it is important to recognize such a peculiar location regarding specific problems raised for the treatment. in this report we describe for the first time the usefulness of motor - evoked potentials (mep) to monitor and prevent post - operative motor impairment in distal anterior choroidal artery (achoa) aneurysm surgery. a 35 years old woman, non - smoker, with no past medical history was admitted in emergency for acute headache and vomiting compatible with an acute meningeal syndrome. clinical examination showed drowsiness, mild neck stiffness but no focal neurological sign (wfns ii). however, her level of consciousness worsened until coma for which she was subsequently intubated. a computed tomography (ct) demonstrated a pure intra ventricular haemorrhage (fisher iv), with no associated subarachnoid haemorrhage and no visible vascular malformation on the ct angiography (cta). an external ventricular drain (evd) was inserted in emergency to treat the obstructive hydrocephalus. a digital subtraction angiography (dsa) failed to highlight the source of the bleeding. eight days after her admission, a second vascular imagery workup spotted a left saccular daa (fig. reviewing the first vascular images (cta and dsa), the aneurysm was already present but barely visible as mostly thrombosed. treatment options were discussed with the neuroradiologists and a neurosurgical approach was chosen regarding the small diameter of the achoa and the distal location of the aneurysm, making an endovascular option not suitable. a standard temporo - pterional bone flap was cut and, under neuronavigation guidance, a trans - sulcal approach between the superior and the middle temporal gyrus (t12) was performed to access the left temporal horn. inside the ventricle, a partially thrombosed, 6 mm in diameter aneurysm was found (fig. regarding the high ratio aneurysm size / achoa diameter, a standard neck clipping was not achievable (fig. 2c, therefore it was decided to excise the aneurysm en bloc with sacrifice of the distal part of the achoa. before, a temporary clipping of the achoa was realized just upstream the aneurysm under monitoring of mep (fig. the left distal portion of achoa was definitively occluded with 2 weck clips (horizon, tfx medical, athlone, ireland) and the aneurysm removed (fig. the procedure went uneventful and the patient recovered gradually with neither post - operative focal neurology nor hydrocephalus. we found no cause of increased hemodynamic stress within the left achoa which can contribute to the formation and rupture of this aneurysm type. one year after the surgery, she remains well [glasgow outcome scale (gos) v ]. the achoa arises from the infero - lateral wall of the internal carotid artery, mostly as a single vessel, and runs posteriorly in the crural cistern in close association with the optic tract. passing through the ambient cistern it enters the choroidal fissure to reach the choroidal plexus of the temporal horn. paradoxically to its tiny mean outer diameter of only about 0.70 mm, it has an important functional role. even though the achoa vascular territory shows large variations among individuals, the invariable terminal fields of supply of this artery are the posterior two thirds of the posterior limb of the internal capsule, most of the globus pallidus, the beginning of the optic radiation, the lateral aspect of the lateral geniculate body, and the middle third of the crus cerebri. therefore, occlusion of these branches generally results in various neurological problems, which include contralateral hemiplegia, hemianesthesia and, hemianopia. however, the blood supply from the plexal segment feeds mainly the choroid plexus extending from the temporal horn to the atrium and usually has anastomosis with the lateral posterior choroidal artery on the surface of the choroid plexus. the number of reported cases of moyamoya disease associated with peripheral aneurysms has been increasing, and some hypotheses as to the mechanism of peripheral aneurysm formation have been offered. among 46 daa cases published, 21 were operated with 2 failed surgeries, 9 were treated by an endovascular procedure, 13 were managed conservatively and for 3 the treatment is not given. clinical presentation of daa rupture is often typical of an acute meningeal syndromefocal neurological signs. the first reported case presented with symptoms of an obstructive hydrocephalus and a paediatric case was revealed by a hemiplegia following an ischemic stroke of the globus pallidus. however ct scan images may be misleading as the bleeding is more likely to be intra - ventricular or intra - parenchymal rather than in the subarachnoid spaces. vascular imagery diagnosis of daa can also be difficult regarding their unusual location, their small size or thrombosed sac. treatment options of daa are mainly surgical regarding the achoa gauge making difficult its endovascular catheterisation. the clinical features including clinical presentation according the wfns sah grading scale, appearance of subarachnoid haemorrhage on ct according the fisher grade, location of aneurysm, cause, surgical pathway treatment and, outcome according the gos, including ours are summarized in table 1. the location of the daas was in the temporal horn of the lateral ventricle, the so - called plexal segment, in the largest number (66.7%). trapping of the parent artery and excision of the aneurysm was the most common surgical procedure applied as, like in our case the ratio aneurysm size on achoa diameter was not suitable for clipping. only in four cases, the authors report a successful clipping of the aneurysm with preservation of the feeding artery17192122). regarding the localization of the daa, different surgical pathways have been described. via a trans - temporal / trans - parietal way for daa located inside the ventricles or through a trans - sylvian / trans - choroidal fissure approach which allows proximal control of the parent vessel before aneurysm dissection. image guided surgery with vascular sequences fused with parenchymal (mri) images can be useful for a more accurate daa targeting, especially for those located inside the ventricle. an important aspect of daa surgery, in contrast to that for proximal aneurysm, is considered to be approaching the aneurysm with minimal brain damage, as most of them are reached through a trans - cerebral way. endoscopic procedure can be achieved in case of intra - ventricular preferentially unruptured aneurysms11). in two reported cases, in the first one the aneurysm could not be identify during the surgery. in the second case reported by kasamo.8) the clip could not be applied on the aneurysm due to many moyamoya vessels surrounding the aneurysm, on contrary to what wrote some previous authors which did n't most likely read the original article and copy mistake of previous authors2). intra - operative ischaemic events during intracranial aneurysms clipping are the leading cause of permanent post - operative morbidity and mortality1016. mep, recorded from muscles following motor cortex trans - cranial electrical stimulation, are commonly used for intra operative monitoring of pyramidal tract functional integrity. therefore, they are useful for aneurysms which are supplied by arteries involved in pyramidal tract vascularization101216). among them, achoa has a particular significance as its first branches which rise from the cisternal segment, generally supply the posterior limb of the internal capsule that contains cortico - spinal fibers. surgical treatment of proximal achoa aneurysms is known to involve a significant risk of vascularization insufficiency. achoa branches have a variable origin that need better to be investigate before a surgical procedure. but due to their tiny diameters, dsa is often not enough accurate to pinpoint them, especially those reaching the internal capsule. obliteration of a normal achoa beyond the plexal point, where the artery enters the choroid plexus, is usually considered relatively safe. however this is not the case of patients with abnormal (moyamoya) achoa providing collateral vessels to a large territory of the surrounding brain tissue. thus, it is important to assess the functional effect of artery occlusion in order to adapt the surgical procedure. intra operatively, this can be easily done by mep monitoring. the absence of branches to cerebral tissue after the anterior choroidal artery 's ventricular penetration should allow trapping of the very peripheral aneurysm. however, this may be difficult to verify per operatively due to the limited surgical exposure. sacrifice of achoa 's distal portion, even if constant anastomosis exist with the vertebro - basilar system, especially the postero - lateral choroidal and posterior cerebral artery, may lead to a stroke. this risk can be minimized by careful effort to preserve the achoa patency based on achoa - avoiding clip placement and intra operative monitoring. mep modifications precede the occurrence of an irreversible ischaemic lesion and may provide the basis for immediate corrective procedures. significant alteration of the mep after temporary clamping is likely due to ischaemia and should thereby prevent a definitive vessel occlusion. in this last situation where the blood flow must be preserve, a non - deleterious treatment must be achieved. if a proper neck clipping ca n't be done, wrapping of the aneurysm may be a solution. risking a post - operative hemiplegia, especially in a pre - operative non disabled a patient, is not acceptable. regarding the diameter of the achoa and the neck shape, an endovascular procedure may be attempt subsequently if the neurosurgical procedure failed to prevent safely a risk of re bleeding. on contrary other intra operative neurophysiological monitoring like somato - sensory evoked potentials can also be studied in the same goal. even if mep is a well known tool, none of the 19 previous surgical cases mentioned the use of them per operatively. although many authors have trapped the plexal segment and resected these aneurysms without any neurologic damage, we think that, whenever possible, this artery should be preserved. due to techniques improvements, more and more daa are likely to be treated by endovascular procedure during which mep study has also shown its usefulness5). patients with severe clinical presentation due to an important intracerebral bleeding were more likely to be dead or highly disabled after neurosurgical treatment. nowadays, surgical aneurysms are usually the most complicated to treat. mep monitoring is useful to prevent ischaemic stroke and severe post surgical outcome, at a time where the vascular neurosurgeons ought to keep post operative complications as low as possible relatively to the embolization. daa are exceptional and may be difficult to diagnosed, as they often present with intra ventricularintra cerebral haemorrhage. surgical approaches of daa are different from proximal achoa aneurysms and their complex management rerequired careful surgical planning for which per operative motor - evoked potentials monitoring is of great value in order to prevent catastrophic outcome and safely assume that the distal part of the achoa can be safely occluded if necessary. | a 35 years old woman presented with an acute meningeal syndrome following an intra ventricular haemorrhage without subarachnoid haemorrhage. the angiography demonstrated a 6 mm partially thrombosed saccular aneurysm at the plexal point of the right anterior choroidal artery (achoa). it was surgically approached inside the ventricle through a trans - temporal corticotomy. the aneurysm was excised after distal exclusion of the feeding artery under motor - evoked potentials monitoring. of the 19 cases of distal achoa aneurysm neurosurgical treatment, this is the only one performed under electrophysiology monitoring, a simple and safe method to detect and prevent motor tract ischemia. we discuss this rare case, along with a comprehensible review of the literature of the previous surgical cases of distal achoa aneurysms. |
cyanotic congenital heart disease is still a common occurrence and many patients with uncorrected cardiac lesions present for various non - cardiac surgery. this case report is aimed at presenting a case of labor epidural analgesia and subsequently epidural anaesthesia in a patient with uncorrected pentalogy of fallot. a 22-year - old primigravida was admitted to the labour ward with history of 8-months amenorrhoea. she was known to have cyanotic congenital heart disease since childhood, and was not on any regular medications or follow - up. she also gave a history of palpitations, giddiness and blurring of vision, which improved over the course of pregnancy. echocardiogram [figure 1 ] at the 4 month revealed pentology of fallot (pof) and hence she was advised therapeutic termination of pregnancy, for which she had refused. echocardiography image of the patient with pentology of fallot showing atrial and ventricular septal defect on pre - anesthetic check up, grade ii clubbing and peripheral cyanosis were present. electrocardiogram (ecg) showed normal sinus rhythm, right axis deviation and right bundle branch block. echocardiography revealed malpositioned great vessels, double - outlet right ventricle with severe pulmonary stenosis, large subaortic ventricular septal defect with significant overriding of aorta, moderate - sized atrial septal defect with left arterial blood gas showed ph 7.38, pao2 48 mmhg, paco2 34 mmhg and sao2 of 84%. as the patient was getting frequent painful contractions that were associated with tachycardia, the obstetricians requested for labour analgesia. with a strict aseptic technique, in sitting posture at l3/4 interspace, 25 g fentanyl was injected intrathecally using a 25 g bevel cut spinal needle. this was followed by placement of an 18 g epidural catheter at the l2/3 interspace. the patient had excellent pain relief immediately, with a visual analogue score of 0. continuous electrocardiography (ecg), pulse oximetry and non - invasive blood pressure monitoring were performed during the labour. labour progressed normally with an outlet forceps - assisted vaginal delivery of a healthy male baby within 2 h. apgar at birth was 8/10 and 10/10 after 5 min. five hours following delivery, the patient started complaining of severe pain at the episiotomy site. examination revealed a huge vulval haematoma that required immediate drainage. in the operating theatre, under full cardiac monitoring, epidural analgesia was provided with 10 ml of 0.125% bupivcaine and 50 g of fentanyl. the patient was haemodynamically stable, with the oxygen saturation between 80 and 85% throughout the surgical procedure. the rest of the post - partum period was uneventful and the patient was discharged on the 7 day of admission to the hospital. tetralogy of fallot (tof) is the most common cyanotic congenital heart defect seen in around 56% of congenital heart malformations. it is characterized by anterior and superior infundibular septal displacement, ventricular septal defect, aortic override, infundibular obstruction and right ventricular hypertrophy. when the tof is associated with atrial septal defect, it is commonly referred to as pof. there is an association between tof and chromosomal defects seen in 15% of the population, with the possibility of genetic transmission to the offsprings. there is fall in systemic vascular resistance (svr), increase in blood volume and increase in cardiac output secondary to increased heart rate and stroke volume, and a physiological left ventricular hypertrophy. the pathophysiology of tof depends on the level of right ventricular outflow obstruction. when the obstruction is mild, the patients present with increased pulmonary blood flow (acyanotic fallot or rose tetrology). acyanotic fallot is more commonly seen in adults, while children present with significant right ventricular outflow obstruction, right left shunt and cyanosis. maternal cyanotic cardiac lesions lead to an impaired uteroplacental perfusion and diminished foetal oxygenation, leading to a higher incidence of miscarriages, premature births and low birth weight babies. anaesthetic management for patients with cyanotic heart disease requires a thorough understanding of the pathophysiology, events and medications, which alters the magnitude of the right - to - left intracardiac shunt. decreased svr, increased pulmonary vascular resistance and increased myocardial contractility increase the magnitude of the right - to - left shunt. the choice of anaeshetic technique is crucial in the management of patients with cyanotic heart disease. sympathetic blockade seen in regional techniques may increase shunt by decreasing svr, leading to decreased ventricular filling and cardiac output. pharmacological agents that decrease svr increase the magnitude of the right - to - left shunt and accentuate arterial hypoxemia. also, intermittent positive pressure ventilation and positive end expiratory pressure cause increases in pulmonary vascular resistance and decrease pulmonary blood flow. providing adequate analgesia, maintenance of uterine placental perfusion, preventing aortocaval compression and minimization of sympathetic block by maintaining intravascular volume are the goals of anaesthetic management. in cyanotic congenital heart diseases (tof, pof), administration of labour analgesia during early labour is advisable to limit increases in pulmonary vascular resistance and consequent right - to - left shunting. small incremental doses of local anaesthetic drugs with opioids are preferred over large bolus doses to avoid abrupt reduction in svr, which may cause reversal of shunt flow and hypoxemia. intravenous fluids should be carefully monitored to prevent deficits or excesses, leading to shunt worsening and patient 's decompensation. adequate pain relief with minor haemodynamic changes should be aimed when regional anaesthesia is considered. shrivastava. reported administration of epidural block to a patient with uncorrected tof for caesarean section. mendes. described a case where labour analgesia was administered in uncorrected tof that was diagnosed during gestation. as an institutional practice, we employ combined spinal epidural labour analgesia, which is an effective and safe technique. it allows the use of subarachnoid opioids, such as fentanyl or sufentanil, leading to faster analgesic effect, lower sympathetic and motor block. placing an epidural catheter allows administration of low concentration of local anaesthetic with minimal haemodynamic changes. the pathophysiology and the haemodynamic derangements of pof are more or less similar to tof, eventually depending on the magnitude of shunt. the anaesthetic management of patients with pof is not commonly reported in the literature owing to the rarity of occurrence. recent advances in cardiac interventions and surgery have enabled more number of women with congenital heart disease to thrive well into their childbearing years. although pregnant women with congenital heart disease are rarely reported around the world, a thorough understanding of the pathophysiology and anaesthetic management of these patients is essential. the choice of anaesthesia in these patients requires thorough understanding of problems, careful titration of drugs and maintaining haemodynamic stability. our patient with pof was managed with labour analgesia and subsequently epidural anaesthesia for drainage of vulval haematoma with successful outcome. | tetraology of fallot is the most common congenital heart disease causing intracardiac right - left shunts. it is characterized by presence of ventricular septal defect, aortic overriding, pulmonary artery outflow obstruction and right ventricular hypertrophy. when these features are associated with atrial septal defect, it is often referred to as pentology of fallot (pof). if the lesions remain uncorrected, they can cause significant morbidity and mortality to the patient. pregnancy and labour in such a patient present with significant haemodynamic changes, which can be challenging to the anaesthesiologist. our patient with pof was managed with labour analgesia and subsequently epidural anaesthesia for drainage of vulval haematoma with successful outcome. |
the second - order evolution equation (1)l[u]ut+uux+uxx=0 was proposed by burgers as a one - dimensional turbulence model. (1) is also applied to model other phenomena in physics, chemistry, mathematical biology, etc. a fairly complete review of properties of the burgers equation can be found in [3, chapter 4 ]. (1) is linearized to the heat equation vt+vxx=0 using the so - called hopf cole transformation u=2vx / v [4, p. 102 ]. at the same time, due to the importance of the burgers equation for various applications, the exhaustive study of its properties in the framework of symmetry analysis is still topical. lie symmetries of the burgers equation and some of its generalizations were studied since the 1960s. the maximal lie invariance algebra gb of eq. (1) was first computed by katkov in the course of group classification of differential equations of the general form ut+uux=(f(u)ux)x. the algebra gb is spanned by the vector fields pt=t, d=2tt+xxuu, k = t2t+txx+(xut)u, px=x, g = tx+u. the complete point symmetry group gb of eq. (1) consists of the transformations t=t+t+,x=x+1t+0t+,u=(t+)ux+10, where (,,,,,0,1) is an arbitrary set of constants defined up to a nonzero multiplier, and =2>0. up to composition with continuous point symmetries, the group gb contains the single discrete symmetry (t, x, u)(t,x,u). generally, reductions of partial differential equations using their lie symmetries do not provide sufficiently large families of exact solutions of these equations. the nonclassical method of reduction was proposed in (see also) in order to utilize a wider class of vector fields than lie symmetries. later such vector fields were called nonclassical symmetries or conditional symmetries [911 ] or reduction operators. the notion of nonclassical symmetries can be extended in several directions, e.g., to the concept of weak symmetry introduced in, which is also closely related to compatibility theory of differential equations and the general method of differential constraints [1418 ]. generalized notions of ansatzes and reductions associated with weak symmetries were intensively discussed, e.g., in [13,1921 ], see also references therein. a reduction operator of eq. (1) is a vector field of the general form (2)q=(t, x, u)t+(t, x, u)x+(t, x, u)u, where the coefficients and do not simultaneously vanish, which allows one to construct an ansatz reducing the initial eq. see, e.g., for the general definition of involutive families of reduction operators. the multiplication by nonvanishing functions of (t, x, u) generates an equivalence relation on the set of reduction operators. the determining equations on coefficients of a reduction operator q are derived from the conditional invariance criterion (3)q(2)l[u]lq(2)=0. here q(2) is the second prolongation of the vector field q, l is the manifold in the second - order jet space j(2) that corresponds to the burgers equation l[u]=0, and q(2) is the manifold in the same jet space determined by the invariant surface condition q[u]=0 jointly with its differential consequences dtq[u]=0 and dxq[u]=0, q[u]=utux is the characteristic of the vector field q, dt and dx are the operators of total differentiation with respect to t and x, respectively. in view of the evolution kind of the burgers equation it is natural to partition the set of its reduction operators into two subsets, singular and regular, depending on whether or not the coefficient vanishes. up to the above equivalence relation, one can assume (,)=(0,1) and =1 for singular and regular reduction operators of the burgers equation, respectively. it is the burgers equation that was first considered from the nonclassical symmetry point of view after the prominent paper. namely, in the determining equations for regular reduction operators of (1) were derived under the gauge =1 and a few of their particular solutions satisfying the additional constraint u=0 were constructed. the determining equations for both regular and singular nonclassical symmetries of (1) were presented in. therein the regular case was studied in detail under the gauge =1, for which the consideration was shown to be partitioned into three cases, u=0, u=1 and u=12. the case u=0 proved to result merely in nonclassical symmetries which are equivalent to lie symmetries. (within the framework of the direct method, the same result was earlier obtained in in terms of the corresponding ansatzes and reductions.) the unique reduction operator t+ux satisfying the constraint u=1 was also found and used for reducing the burgers equation. for the case u=12 some particular solutions of the determining equations jointly with the corresponding ansatzes and invariant solutions of the burgers equation were constructed. the above consideration of regular nonclassical symmetries from was extended in with more particular solutions satisfying the constraint u=12. still wider families of particular solutions of the determining equations in this case were given in. in an algorithmic procedure to derive determining equations for nonclassical symmetries was proposed, and the burgers equation was one of the illustrative examples for application of this procedure. the system sb of determining equations for the case u=12 was not well investigated for a surprisingly long time although the study of the analogous system sh for regular reduction operators of the linear heat equation vt+vxx=0, whose form is very similar to sb, had already been completed in. see also for preliminary results on sh and for further generalizations to (1 + 1)-dimensional second - order linear evolution equations. namely, this system was reduced by a differential substitution to the uncoupled system of three copies of the linear heat equation. as shown in, the systems sb and sh as well as the substitutions linearizing them can be interpreted in terms of the matrix burgers equation and the matrix hopf cole transformation. the above in fact means that the case u=12 can be referred to as no - go. in general, looking for reduction operators in a family of vector fields is said to result in a no - go case if the corresponding system of determining equations for coefficients of reduction operators is reduced to a well - determined system whose general solution can not be represented in a closed form, and, moreover, solving this system is equivalent, in a certain sense, to solving the initial equation. singular reduction operators of the burgers equation were in fact not studied until, where no - go results of on reduction operators with =0 for the linear heat equation were extended to general evolution equations of order greater than one. these results were treated in within the framework of singular reduction operators. in this paper we intend to enhance and complete the above results on nonclassical symmetries and reductions of the burgers equation. in particular, extending methods from we present a new proof on the linearization of the system sb and show that solutions of this system are expressed via triples of solutions of the burgers equation. we first exhaustively describe all possible nonclassical reductions of the burgers equation to single ordinary differential equations including reductions associated with the no - go case =1 and u=12. a part of the description is the assertion stating that any lie reduction of the burgers equation is equivalent via the hopf cole transformation to a parameterized family of lie reductions of the linear heat equation. as the burgers equation (1) is a (1 + 1)-dimensional second - order evolution equation, every reduction operator of the form (2) with =0 is singular since the corresponding reduced equation is of a lower (namely, the first) order. all basic results on such reduction operators of (1) follow from the general results on singular reduction operators of co - order singularity one. after setting =1 in q due to the equivalence of reduction operators, the conditional invariance criterion (3) implies a single determining equation on the single coefficient =(t, x, u), (4)t+ux+2+xx+2xu+2uu=0. namely, the composition of the differential substitution =x/u with u0, where is a smooth function of (t, x, u), and the hodograph transformation, where the new independent variables are t=t, x=x and = and the new dependent variable is u=u, reduces eq. (1) on the function u=u(t,x,) with playing the role of a parameter. moreover, up to equivalences of reduction operators and solution families, there exists a bijection between one - parameter families of solutions of eq. namely, each operator of the above kind corresponds to the family of solutions which are invariant with respect to this operator. the problems of construction of all one - parameter solution families of (1) and the exhaustive description of its reduction operators with zero coefficients of t are completely equivalent. given a family f={u = f(t, x,) } of solutions of (1) parameterized by a single essential parameter, the corresponding singular reduction operator is q=x(x/u)u, where the function is obtained by solving the equality u = f(t, x,) with respect to, =(t, x, u). the ansatz u = f(t, x,()), where =t, associated with q, reduces eq. the simplicity of the reduced equation is explained by the specific choice of the ansatz based on knowing the one - parameter family f of solutions. now we look for regular reduction operators of the burgers equation (1), which are of the form (2) with nonvanishing values of the coefficient. up to equivalence of reduction operators, for any regular operator q we can set =1. in view of this gauge we do not need to use the differential consequences in order to derive the determining equations, i.e. it suffices to take into account only the equations ut+uux+uxx=0 and utux=0 in the course of confining to the manifold lq(2) in the conditional invariance criterion (3). substituting the expressions for ut and uxx obtained from these equations into the differential function q(2)l[u ] and splitting the result with respect to ux, we get (5)uu=0,2xu2u+2uu+uu=0,2xu+2u+t+uxxx2x=0,t+ux+xx+2x=0. integrating the first two equations, we represent the functions and as polynomials of u with the coefficients depending on t and x, (6)=1u+0,=131(11)u3+(x1+10)u2+1u+0. then we split the third equation of the system (5) with respect to u and get a system of differential equations on the functions 1, 0, 1 and 0, (7)1(11)(21 + 1)=0,10(21 + 1)+41x1=0,3xx1 + 2(x10+1x0)+(21 + 1)1t1+x0=0,t0 + 20x0+xx0(21 + 1)02x1=0. the further consideration depends on the choice among the three possible solutions of the first equation of the system (7). we rewrite the last equation of (5) in terms of 1, 0, 1 and 0 and split it with respect to u severally for each value of 1. the corresponding vector field q1=t+ux is a unique reduction operator (up to the equivalence relation) for the burgers equation in this case. the set of q1-invariant solutions consists of two families, one of which is two - parameter and the other is one - parameter. the two - parameter family is formed by the functions u=(x+c1)/(t+c0), where c1 and c0 are arbitrary constants, and all these solutions are lie - invariant and equivalent to the scale - invariant solution u = x / t. they are invariant with respect to shifts of both t and x. the optimal way for the construction of q1-invariant solutions of (1) is to integrate at first the equation l[u]+q1[u]=uxx=0, which gives the representation u=(t)x+(t) with some smooth functions and of t. the generalized vector field q=(l[u]+q1[u])u = uxxu, is equivalent to the evolutionary representative q1[u]u of q1 on the set of solutions of (1) and hence it is a generalized conditional symmetry of (1), cf. this is why the associated ansatz u=(t)x+(t) reduces the burgers equation (1) to a system of two ordinary differential equations with respect to the functions and, t+2=0,t+=0. a complete set of functionally independent integrals of the equation q1[u]=0 consists of xut and u. therefore, directly with q1 we construct the implicit ansatz u=(), where =xut (resp. the case 1=0 is discussed in in detail (see also), where it is noted that all corresponding solutions of the burgers equation are lie - invariant. the system (7) with 1=0 implies 1=x0 and 0=t0 + 20x0 + 3xx0, and splitting the last eq. (5) with respect to u we derive xx0=0,tt0 + 20tx0 + 4t0x0 + 40(x0)2=0. then we have 0=01(t)x+00(t), where the coefficients 01 and 00 satisfy the system tt01 + 601t01 + 4(01)3=0,tt00 + 401t00 + 2t0100 + 4(01)200=0. the transformation 01=t/2, 00=/ maps this system to the system of two simple uncoupled equations ttt=0 and tt=0 for the functions =(t) and =(t). hence 01=c2t+c1c2t2 + 2c1t+c0,00=c4t+c3c2t2 + 2c1t+c0, where c0, the substitution of the expressions obtained for 1, 0 and 0=01x+00 into (6) gives q=t+(c2t+c1)x+c4t+c3c2t2 + 2c1t+c0x+(c2t+c1)u+c2x+c4c2t2 + 2c1t+c0u. it is easy to see that the operator q differs from a lie symmetry operator of the burgers equation by the multiplier (c2t2 + 2c1t+c0)1. proposition 1there is a bijection between reduction operators of the burgers equation of the general formq=t+(t, x)x+(1(t, x)u+0(t, x))uand one - dimensional algebras spanned by lie symmetry operators of this equation with nonzero coefficients of t. there is a bijection between reduction operators of the burgers equation of the general formq=t+(t, x)x+(1(t, x)u+0(t, x))uand one - dimensional algebras spanned by lie symmetry operators of this equation with nonzero coefficients of t. the bijection is established by the equivalence relation of reduction operators. in other words, any nonclassical reduction of the burgers equation with respect to a vector field with =1 and u=0 is in fact a lie reduction. at the same time, any lie solution of the burgers equation is obtained from a lie solution of the linear heat equation via the hopf cole transformation. before presenting the corresponding rigorous assertion, we recall that the maximal lie invariance algebra gh of the linear heat equation vt+vxx=0 is spanned by the vector fields pt=t, d=2tt+xx, k=t2t+txx+(14x212t)vv, px=x, g=tx+12xvv, i=vv, h(t, x)v, where h(t, x) runs through the set of solutions of this equation. we associate any vector field q = c0pt+c1d+c2k+c3px+c4 g from the maximal lie invariance algebra gb of the eq. proposition 2a solution u of the burgers equation (1) is invariant with respect to a vector field q from gb if and only if u=2vx / v for some q-invariant solution v of the linear heat equation vt+vxx=0, where is a constant and q=qigh. a solution u of the burgers equation (1) is invariant with respect to a vector field q from gb if and only if u=2vx / v for some q-invariant solution v of the linear heat equation vt+vxx=0, where is a constant and q=qigh. proofif smooth functions u and v depending on t and x are related via the hopf cole transformation, u=2vx / v, then q[u]=q[2vx / v]=2(q[v]/v)x.suppose that u is a q - invariant solution of (1). then the function v can be assumed to be a solution of the linear heat equation vt+vxx=0, and (q[v]/v)x = q[u]/2=0, i.e. q[v]=v for some smooth function of t. acting by the operator t = dt+dxx on both sides of the last equation, we derive tq[v]=q[tv]2(c2t+c1)tv=tv+tv. in view of tv=0 and tq[v]=0 as then q[v]v = q[v]=0, the function v is a q-invariant.conversely, if for some constant the function v is a q-invariant solution of the linear heat equation vt+vxx=0 then the function u=2vx / v is a solution of the burgers equation (1) and q[u]=q[2vx / v]=2(q[v]/v)x=2(q[v]/v)x=0, i.e., the function u is q - invariant. if smooth functions u and v depending on t and x are related via the hopf cole transformation, u=2vx / v, then q[u]=q[2vx / v]=2(q[v]/v)x. then the function v can be assumed to be a solution of the linear heat equation vt+vxx=0, and (q[v]/v)x = q[u]/2=0, i.e. q[v]=v for some smooth function of t. acting by the operator t = dt+dxx on both sides of the last equation, we derive tq[v]=q[tv]2(c2t+c1)tv=tv+tv. in view of tv=0 and tq[v]=0 conversely, if for some constant the function v is a q-invariant solution of the linear heat equation vt+vxx=0 then the function u=2vx / v is a solution of the burgers equation (1) and q[u]=q[2vx / v]=2(q[v]/v)x=2(q[v]/v)x=0, i.e., the function u is q - invariant. the case 1=12 leads to reduction operators of the general form (8)q=t+(12u+0)x+(14u302u2+1u+0)u, where the coefficients 0, 1 and 0 are smooth functions of t and x satisfying the system of differential equations (9)t0 + 20x0+xx02x1=0,t1 + 2x01+xx1+x0=0,t0 + 2x00+xx0=0 derived from (7). as a differential substitution reduces the system (9) to an uncoupled system of three copies of the linear heat equation, the general solution of (9) can not be represented in a closed form. this result appears to directly follow from the fact that q is a reduction operator of eq. moreover, we show that solutions of the system (9) can be represented via solutions of the uncoupled system of three copies of the burgers equation. theorem 1any solution of the determining system (9) on the coefficients of reduction operators of the form (8) is represented as(10)0=(w(v))xw(v),1=|v,vxx, vxxx|w(v),0=2w(vx)w(v),where v=(v1,v2,v3) is a triple of linear independent solutions of the heat equation vt+vxx=0, w(v)=|v,vx, vxx| and w(vx)=|vx, vxx, vxxx| are the wronskians of this triple and the triple of the corresponding derivatives with respect to x, respectively, and |p,q,r| denotes the determinant of the matrix constructed from ternary columns p, q and r. conversely, any triple (0,1,0) admitting the representation (10) satisfies the system (9). any solution of the determining system (9) on the coefficients of reduction operators of the form (8) is represented as(10)0=(w(v))xw(v),1=|v,vxx, vxxx|w(v),0=2w(vx)w(v),where v=(v1,v2,v3) is a triple of linear independent solutions of the heat equation vt+vxx=0, w(v)=|v,vx, vxx| and w(vx)=|vx, vxx, vxxx| are the wronskians of this triple and the triple of the corresponding derivatives with respect to x, respectively, and |p,q,r| denotes the determinant of the matrix constructed from ternary columns p, q and r. conversely, any triple (0,1,0) admitting the representation (10) satisfies the system (9). the set of q - invariant solutions of the burgers equation l[u]=0 coincides with the set of solutions of the system l[u]=0 and q[u]=0, and it is parameterized by two arbitrary constants as q is a regular reduction operator of the burgers equation. for convenience we recombine the equations of the above system in the following way : l[u]=0, l[u]+q[u]=0. the hopf cole transformation u=2vx / v maps this system to the linear system (11)vt+vxx=0,vxxx0vxx+1vx+120v=0.,vn of t and x be linear independent solutions of the system (11). then the equation (12)u=2c1vx1++cnvxnc1v1++cnvn where c1,,cn are arbitrary constants which are not simultaneously zero, defines a family of q - invariant solutions of the burgers equation parameterized by n1 essential constants, and hence n3 as the number of such parameters can not be greater than two. in other words, the dimension of the space v of solutions of the system (11) does not exceed three.,vn of t and x form a basis of the space v, where n = dimv. then the expression (12) represents the general solution of the system l[u]=0 and q[u]=0, which contains n1 essential constant parameters. therefore, n1=2, i.e. n=3.consider a basis { v1,v2,v3 } of the space v. by definition, the elements of v are solutions of the heat equation vt+vxx=0, which is linear and evolutionary. hence the usual linear independence of them implies the linear independence of them over the ring of smooth functions of t, i.e. the wronskian w(v) of the functions v1, v2 and v3 with respect to x does not vanish. see, e.g., note 5 in. substituting the elements of the basis into the second equation of (11), we obtain a well - defined system of linear algebraic equations, vxxxi0vxxi+1vxi+120vi=0,i=1,2,3, for the coefficients 0, 1, 0, or, in the matrix form, mq=vxxx, where v=(v1v2v3),m=(v1vx1vxx1v2vx2vxx2v3vx3vxx3),q=(12010). solving this system with respect to 0, 1 and 0, we derive the representation (10).as the proof can be turned back, the inverse statement is also true. the set of q - invariant solutions of the burgers equation l[u]=0 coincides with the set of solutions of the system l[u]=0 and q[u]=0, and it is parameterized by two arbitrary constants as q is a regular reduction operator of the burgers equation. for convenience we recombine the equations of the above system in the following way : l[u]=0, l[u]+q[u]=0. the hopf cole transformation u=2vx,vn of t and x be linear independent solutions of the system (11). then the equation (12)u=2c1vx1++cnvxnc1v1++cnvn where c1,,cn are arbitrary constants which are not simultaneously zero, defines a family of q - invariant solutions of the burgers equation parameterized by n1 essential constants, and hence n3 as the number of such parameters can not be greater than two. in other words, the dimension of the space v of solutions of the system (11) does not exceed three.,vn of t and x form a basis of the space v, where n = dimv. then the expression (12) represents the general solution of the system l[u]=0 and q[u]=0, which contains n1 essential constant parameters. consider a basis { v1,v2,v3 } of the space v. by definition, the elements of v are solutions of the heat equation vt+vxx=0, which is linear and evolutionary. hence the usual linear independence of them implies the linear independence of them over the ring of smooth functions of t, i.e. the wronskian w(v) of the functions v1, v2 and v3 with respect to x does not vanish. see, e.g., note 5 in. substituting the elements of the basis into the second equation of (11), we obtain a well - defined system of linear algebraic equations, vxxxi0vxxi+1vxi+120vi=0,i=1,2,3, for the coefficients 0, 1, 0, or, in the matrix form, mq=vxxx, where v=(v1v2v3),m=(v1vx1vxx1v2vx2vxx2v3vx3vxx3),q=(12010). solving this system with respect to 0, 1 and 0, we derive the representation (10). as the proof can be turned back, the inverse statement is also true. corollary 1the coefficients of the reduction operator (8) of the burgers equation can be represented in the form(13)0=12|e,u,z||e,u,y|,1=14|e,y,z||e,u,y|,0=14|u,y,z||e,u,y|,where the columns e, y and z consist of three units, the expressions yi=2uxi+(ui)2 and zi=4uxxi+6uiuxi+(ui)3, respectively, i=1,2,3, and u is a column of three solutions of the burgers equation with |e,u,y|0. the coefficients of the reduction operator (8) of the burgers equation can be represented in the form(13)0=12|e,u,z||e,u,y|,1=14|e,y,z||e,u,y|,0=14|u,y,z||e,u,y|,where the columns e, y and z consist of three units, the expressions yi=2uxi+(ui)2 and zi=4uxxi+6uiuxi+(ui)3, respectively, i=1,2,3, and u is a column of three solutions of the burgers equation with |e,u,y|0. proofthe connection between solutions of the heat equation and the burgers equation via the hopf cole transformation 2vxi / vi = ui gives the expressions vxxivi=12uxi+14(ui)2,vxxxivi=34uiuxi+18(ui)3 note that the determinant |e,u,y| is nonvanishing as the wronskian w(v) is the same. the connection between solutions of the heat equation and the burgers equation via the hopf cole transformation 2vxi / vi = ui gives the expressions vxxivi=12uxi+14(ui)2,vxxxivi=34uiuxi+18(ui)3 note that the determinant |e,u,y| is nonvanishing as the wronskian w(v) is the same. corollary 2the representations (10) and (12), where n=3, explicitly define the one - to - one correspondence between reduction operators of the form (8) and families of solutions of the burgers equation that are invariant with respect to these operators. the representations (10) and (12), where n=3, explicitly define the one - to - one correspondence between reduction operators of the form (8) and families of solutions of the burgers equation that are invariant with respect to these operators. it looks very difficult to explicitly construct an ansatz for u by the direct integration of the invariant surface condition that corresponds to an operator of the form (8) in the case of an arbitrary solution of system (9). even for a simple solution of system (9), carrying out the corresponding reduction of the burgers equation (1) see, e.g.,, where a few such reductions were implemented. a better way for using an operator of the form (8) for reducing the burgers equation is to consider, instead of q, the generalized vector field q=(l[u]+q[u])u which is equivalent to the evolutionary representative q[u]u of q and is a generalized conditional symmetry of (1), cf. (1) with respect to the generalized vector field q is somehow presented in the proof of theorem 1, and corollary 2 exhaustively describes the family of q - invariant solutions of (1). at the same time, knowing the representation (12), where n=3, for q - invariant solutions of (1) allows us to easily construct an ansatz for u associated with the operator q and then to reduce the burgers equation (1) using this ansatz. setting c1=1 and c2=0 (resp. c1=0 and c2=1) and assuming c3 to be an arbitrary constant, we derive two integrals of the equation q[u]=0, =v1u2vx1v3u2vx3,=v2u2vx2v3u2vx3, which are correctly defined for u2vx3/v3 (we can always assume this condition to be satisfied for a specific solution u up to renumbering the functions v1, v2 and v3). therefore, the general solution of the equation q[u]=0 is implicitly represented in the form f(,)=0, where f is an arbitrary nonconstant function of its arguments. up to permutation of and then the equality f(,)=0 implies the implicit ansatz =() for the unknown function u = u(t, x), where and are the new dependent and independent variables, respectively. the standard way for deriving the corresponding reduced equation via the computation of the expressions for derivatives ut, ux and uxx implied by the ansatz and the subsequent substitution of the expressions into (1) is too cumbersome. instead of this, we act on the ansatz =() by the operator dt+dxx and then make the substitutions ut+uxx=uux and vti+vxxi=0, i=1,2,3. as a result, we derive the equation dx=0. the differential function dx does not vanish for solutions of (1) which are implicitly represented in the form =() for some smooth function of. indeed, suppose that dx=0 (i.e., =0(t)) for such a solution u = u0(t, x). then we have u0=2(vx20vx3)/(v20v3), which implies that for some nonvanishing smooth function of t the expression (v2+0v3) gives a solution of the linear heat equation vt+vxx=0. as the functions v2 and v3 are linearly independent solutions of the same equation, this is possible only if 0 is a constant. therefore, the substitution u = u0(t, x) into results in the constant value 0=(0) and hence u0=2(vx10vx3)/(v10v3). comparing the two expressions for the solution u = u0(t, x), we conclude that the functions v1, v2 and v3 are linearly dependent and thus arrive at a contradiction. as dx0, the reduced equation takes the form =0. its general solution is =c1+c2, which completely agrees with the representation (12) and the ansatz =(). the above reduced equation is obtained for an arbitrary operator q of the form (8) and it is much simpler than particular reduced equations derived in. this fact is explained by that the ansatz =(), whose construction is based on the representation (12) for q - invariant solutions of the burgers equation (1), is in better agreement with the structure of this equation than particular ansatzes given in. the corresponding vector field q1=t+ux is a unique reduction operator (up to the equivalence relation) for the burgers equation in this case. the set of q1-invariant solutions consists of two families, one of which is two - parameter and the other is one - parameter. the two - parameter family is formed by the functions u=(x+c1)/(t+c0), where c1 and c0 are arbitrary constants, and all these solutions are lie - invariant and equivalent to the scale - invariant solution u = x / t. they are invariant with respect to shifts of both t and x. the optimal way for the construction of q1-invariant solutions of (1) is to integrate at first the equation l[u]+q1[u]=uxx=0, which gives the representation u=(t)x+(t) with some smooth functions and of t. the generalized vector field q=(l[u]+q1[u])u = uxxu, is equivalent to the evolutionary representative q1[u]u of q1 on the set of solutions of (1) and hence it is a generalized conditional symmetry of (1), cf. this is why the associated ansatz u=(t)x+(t) reduces the burgers equation (1) to a system of two ordinary differential equations with respect to the functions and, t+2=0,t+=0. a complete set of functionally independent integrals of the equation q1[u]=0 consists of xut and u. therefore, directly with q1 we construct the implicit ansatz u=(), where =xut (resp. the case 1=0 is discussed in in detail (see also), where it is noted that all corresponding solutions of the burgers equation are lie - invariant. the system (7) with 1=0 implies 1=x0 and 0=t0 + 20x0 + 3xx0, and splitting the last eq. (5) with respect to u we derive xx0=0,tt0 + 20tx0 + 4t0x0 + 40(x0)2=0. then we have 0=01(t)x+00(t), where the coefficients 01 and 00 satisfy the system tt01 + 601t01 + 4(01)3=0,tt00 + 401t00 + 2t0100 + 4(01)200=0. the transformation 01=t/2, 00=/ maps this system to the system of two simple uncoupled equations ttt=0 and tt=0 for the functions =(t) and =(t). hence the substitution of the expressions obtained for 1, 0 and 0=01x+00 into (6) gives q=t+(c2t+c1)x+c4t+c3c2t2 + 2c1t+c0x+(c2t+c1)u+c2x+c4c2t2 + 2c1t+c0u. it is easy to see that the operator q differs from a lie symmetry operator of the burgers equation by the multiplier (c2t2 + 2c1t+c0)1. proposition 1there is a bijection between reduction operators of the burgers equation of the general formq=t+(t, x)x+(1(t, x)u+0(t, x))uand one - dimensional algebras spanned by lie symmetry operators of this equation with nonzero coefficients of t. there is a bijection between reduction operators of the burgers equation of the general formq=t+(t, x)x+(1(t, x)u+0(t, x))uand one - dimensional algebras spanned by lie symmetry operators of this equation with nonzero coefficients of t. the bijection is established by the equivalence relation of reduction operators. in other words, any nonclassical reduction of the burgers equation with respect to a vector field with =1 and u=0 is in fact a lie reduction. at the same time, any lie solution of the burgers equation is obtained from a lie solution of the linear heat equation via the hopf cole transformation. before presenting the corresponding rigorous assertion, we recall that the maximal lie invariance algebra gh of the linear heat equation vt+vxx=0 is spanned by the vector fields pt=t, d=2tt+xx, k=t2t+txx+(14x212t)vv, px=x, g=tx+12xvv, i=vv, h(t, x)v, where h(t, x) runs through the set of solutions of this equation. we associate any vector field q = c0pt+c1d+c2k+c3px+c4 g from the maximal lie invariance algebra gb of the eq. proposition 2a solution u of the burgers equation (1) is invariant with respect to a vector field q from gb if and only if u=2vx / v for some q-invariant solution v of the linear heat equation vt+vxx=0, where is a constant and q=qigh. a solution u of the burgers equation (1) is invariant with respect to a vector field q from gb if and only if u=2vx / v for some q-invariant solution v of the linear heat equation vt+vxx=0, where is a constant and q=qigh. proofif smooth functions u and v depending on t and x are related via the hopf cole transformation, u=2vx / v, then q[u]=q[2vx / v]=2(q[v]/v)x.suppose that u is a q - invariant solution of (1). then the function v can be assumed to be a solution of the linear heat equation vt+vxx=0, and (q[v]/v)x = q[u]/2=0, i.e. q[v]=v for some smooth function of t. acting by the operator t = dt+dxx on both sides of the last equation, we derive tq[v]=q[tv]2(c2t+c1)tv=tv+tv. in view of tv=0 and tq[v]=0, this implies that t=0, i.e., is a constant. as then q[v]v = q[v]=0, the function v is a q-invariant.conversely, if for some constant the function v is a q-invariant solution of the linear heat equation vt+vxx=0 then the function u=2vx / v is a solution of the burgers equation (1) and q[u]=q[2vx / v]=2(q[v]/v)x=2(q[v]/v)x=0, i.e., the function u is q - invariant. if smooth functions u and v depending on t and x are related via the hopf cole transformation, u=2vx / v, then q[u]=q[2vx then the function v can be assumed to be a solution of the linear heat equation vt+vxx=0, and (q[v]/v)x = q[u]/2=0, i.e. q[v]=v for some smooth function of t. acting by the operator t = dt+dxx on both sides of the last equation, we derive tq[v]=q[tv]2(c2t+c1)tv=tv+tv. in view of tv=0 and tq[v]=0, this implies that t=0, i.e., is a constant. conversely, if for some constant the function v is a q-invariant solution of the linear heat equation vt+vxx=0 then the function u=2vx / v is a solution of the burgers equation (1) and q[u]=q[2vx / v]=2(q[v]/v)x=2(q[v]/v)x=0, i.e., the function u is q - invariant. the case 1=12 leads to reduction operators of the general form (8)q=t+(12u+0)x+(14u302u2+1u+0)u, where the coefficients 0, 1 and 0 are smooth functions of t and x satisfying the system of differential equations (9)t0 + 20x0+xx02x1=0,t1 + 2x01+xx1+x0=0,t0 + 2x00+xx0=0 derived from (7). as a differential substitution reduces the system (9) to an uncoupled system of three copies of the linear heat equation, the general solution of (9) can not be represented in a closed form. this result appears to directly follow from the fact that q is a reduction operator of eq. moreover, we show that solutions of the system (9) can be represented via solutions of the uncoupled system of three copies of the burgers equation. theorem 1any solution of the determining system (9) on the coefficients of reduction operators of the form (8) is represented as(10)0=(w(v))xw(v),1=|v,vxx, vxxx|w(v),0=2w(vx)w(v),where v=(v1,v2,v3) is a triple of linear independent solutions of the heat equation vt+vxx=0, w(v)=|v,vx, vxx| and w(vx)=|vx, vxx, vxxx| are the wronskians of this triple and the triple of the corresponding derivatives with respect to x, respectively, and |p,q,r| denotes the determinant of the matrix constructed from ternary columns p, q and r. conversely, any triple (0,1,0) admitting the representation (10) satisfies the system (9). any solution of the determining system (9) on the coefficients of reduction operators of the form (8) is represented as(10)0=(w(v))xw(v),1=|v,vxx, vxxx|w(v),0=2w(vx)w(v),where v=(v1,v2,v3) is a triple of linear independent solutions of the heat equation vt+vxx=0, w(v)=|v,vx, vxx| and w(vx)=|vx, vxx, vxxx| are the wronskians of this triple and the triple of the corresponding derivatives with respect to x, respectively, and |p,q,r| denotes the determinant of the matrix constructed from ternary columns p, q and r. conversely, any triple (0,1,0) admitting the representation (10) satisfies the system (9). the set of q - invariant solutions of the burgers equation l[u]=0 coincides with the set of solutions of the system l[u]=0 and q[u]=0, and it is parameterized by two arbitrary constants as q is a regular reduction operator of the burgers equation. for convenience we recombine the equations of the above system in the following way : l[u]=0, l[u]+q[u]=0. the hopf cole transformation u=2vx,vn of t and x be linear independent solutions of the system (11). then the equation (12)u=2c1vx1++cnvxnc1v1++cnvn where c1,,cn are arbitrary constants which are not simultaneously zero, defines a family of q - invariant solutions of the burgers equation parameterized by n1 essential constants, and hence n3 as the number of such parameters can not be greater than two. in other words, the dimension of the space v of solutions of the system (11) does not exceed three.,vn of t and x form a basis of the space v, where n = dimv. then the expression (12) represents the general solution of the system l[u]=0 and q[u]=0, which contains n1 essential constant parameters. therefore, n1=2, i.e. n=3.consider a basis { v1,v2,v3 } of the space v. by definition, the elements of v are solutions of the heat equation vt+vxx=0, which is linear and evolutionary. hence the usual linear independence of them implies the linear independence of them over the ring of smooth functions of t, i.e. the wronskian w(v) of the functions v1, v2 and v3 with respect to x does not vanish. see, e.g., note 5 in. substituting the elements of the basis into the second equation of (11), we obtain a well - defined system of linear algebraic equations, vxxxi0vxxi+1vxi+120vi=0,i=1,2,3, for the coefficients 0, 1, 0, or, in the matrix form, mq=vxxx, where v=(v1v2v3),m=(v1vx1vxx1v2vx2vxx2v3vx3vxx3),q=(12010). solving this system with respect to 0, 1 and 0, we derive the representation (10).as the proof can be turned back, the inverse statement is also true. the set of q - invariant solutions of the burgers equation l[u]=0 coincides with the set of solutions of the system l[u]=0 and q[u]=0, and it is parameterized by two arbitrary constants as q is a regular reduction operator of the burgers equation. for convenience we recombine the equations of the above system in the following way : l[u]=0, l[u]+q[u]=0. the hopf cole transformation u=2vx let for some integer n functions v1,,vn of t and x be linear independent solutions of the system (11).,cn are arbitrary constants which are not simultaneously zero, defines a family of q - invariant solutions of the burgers equation parameterized by n1 essential constants, and hence n3 as the number of such parameters can not be greater than two. in other words, the dimension of the space v of solutions of the system (11) does not exceed three.,vn of t and x form a basis of the space v, where n = dimv. then the expression (12) represents the general solution of the system l[u]=0 and q[u]=0, which contains n1 essential constant parameters. consider a basis { v1,v2,v3 } of the space v. by definition, the elements of v are solutions of the heat equation vt+vxx=0, which is linear and evolutionary. hence the usual linear independence of them implies the linear independence of them over the ring of smooth functions of t, i.e. the wronskian w(v) of the functions v1, v2 and v3 with respect to x does not vanish. see, e.g., note 5 in. substituting the elements of the basis into the second equation of (11), we obtain a well - defined system of linear algebraic equations, vxxxi0vxxi+1vxi+120vi=0,i=1,2,3, for the coefficients 0, 1, 0, or, in the matrix form, mq=vxxx, where v=(v1v2v3),m=(v1vx1vxx1v2vx2vxx2v3vx3vxx3),q=(12010). solving this system with respect to 0, 1 and 0 corollary 1the coefficients of the reduction operator (8) of the burgers equation can be represented in the form(13)0=12|e,u,z||e,u,y|,1=14|e,y,z||e,u,y|,0=14|u,y,z||e,u,y|,where the columns e, y and z consist of three units, the expressions yi=2uxi+(ui)2 and zi=4uxxi+6uiuxi+(ui)3, respectively, i=1,2,3, and u is a column of three solutions of the burgers equation with |e,u,y|0. the coefficients of the reduction operator (8) of the burgers equation can be represented in the form(13)0=12|e,u,z||e,u,y|,1=14|e,y,z||e,u,y|,0=14|u,y,z||e,u,y|,where the columns e, y and z consist of three units, the expressions yi=2uxi+(ui)2 and zi=4uxxi+6uiuxi+(ui)3, respectively, i=1,2,3, and u is a column of three solutions of the burgers equation with |e,u,y|0. proofthe connection between solutions of the heat equation and the burgers equation via the hopf cole transformation 2vxi / vi = ui gives the expressions vxxivi=12uxi+14(ui)2,vxxxivi=34uiuxi+18(ui)3 note that the determinant |e,u,y| is nonvanishing as the wronskian w(v) is the same. the connection between solutions of the heat equation and the burgers equation via the hopf cole transformation 2vxi / vi = ui gives the expressions vxxivi=12uxi+14(ui)2,vxxxivi=34uiuxi+18(ui)3 note that the determinant |e,u,y| is nonvanishing as the wronskian w(v) is the same. corollary 2the representations (10) and (12), where n=3, explicitly define the one - to - one correspondence between reduction operators of the form (8) and families of solutions of the burgers equation that are invariant with respect to these operators. the representations (10) and (12), where n=3, explicitly define the one - to - one correspondence between reduction operators of the form (8) and families of solutions of the burgers equation that are invariant with respect to these operators. it looks very difficult to explicitly construct an ansatz for u by the direct integration of the invariant surface condition that corresponds to an operator of the form (8) in the case of an arbitrary solution of system (9). even for a simple solution of system (9), carrying out the corresponding reduction of the burgers equation (1) see, e.g.,, where a few such reductions were implemented. a better way for using an operator of the form (8) for reducing the burgers equation is to consider, instead of q, the generalized vector field q=(l[u]+q[u])u which is equivalent to the evolutionary representative q[u]u of q and is a generalized conditional symmetry of (1), cf. (1) with respect to the generalized vector field q is somehow presented in the proof of theorem 1, and corollary 2 exhaustively describes the family of q - invariant solutions of (1). at the same time, knowing the representation (12), where n=3, for q - invariant solutions of (1) allows us to easily construct an ansatz for u associated with the operator q and then to reduce the burgers equation (1) using this ansatz. setting c1=1 and c2=0 (resp. c1=0 and c2=1) and assuming c3 to be an arbitrary constant, we derive two integrals of the equation q[u]=0, =v1u2vx1v3u2vx3,=v2u2vx2v3u2vx3, which are correctly defined for u2vx3/v3 (we can always assume this condition to be satisfied for a specific solution u up to renumbering the functions v1, v2 and v3). therefore, the general solution of the equation q[u]=0 is implicitly represented in the form f(,)=0, where f is an arbitrary nonconstant function of its arguments. up to permutation of and then the equality f(,)=0 implies the implicit ansatz =() for the unknown function u = u(t, x), where and are the new dependent and independent variables, respectively. the standard way for deriving the corresponding reduced equation via the computation of the expressions for derivatives ut, ux and uxx implied by the ansatz and the subsequent substitution of the expressions into (1) is too cumbersome. instead of this, we act on the ansatz =() by the operator dt+dxx and then make the substitutions ut+uxx=uux and vti+vxxi=0, i=1,2,3. as a result the differential function dx does not vanish for solutions of (1) which are implicitly represented in the form =() for some smooth function of. indeed, suppose that dx=0 (i.e., =0(t)) for such a solution u = u0(t, x). then we have u0=2(vx20vx3)/(v20v3), which implies that for some nonvanishing smooth function of t the expression (v2+0v3) gives a solution of the linear heat equation vt+vxx=0. as the functions v2 and v3 are linearly independent solutions of the same equation, this is possible only if 0 is a constant. therefore, the substitution u = u0(t, x) into results in the constant value 0=(0) and hence u0=2(vx10vx3)/(v10v3). comparing the two expressions for the solution u = u0(t, x), we conclude that the functions v1, v2 and v3 are linearly dependent and thus arrive at a contradiction. its general solution is =c1+c2, which completely agrees with the representation (12) and the ansatz =(). the above reduced equation is obtained for an arbitrary operator q of the form (8) and it is much simpler than particular reduced equations derived in. this fact is explained by that the ansatz =(), whose construction is based on the representation (12) for q - invariant solutions of the burgers equation (1), is in better agreement with the structure of this equation than particular ansatzes given in. the aim of this paper is to arrange, enhance and complete the description of the nonclassical reductions of the burgers equation, including lie reductions. although this problem had been considered in a number of papers, certain of its aspects needed additional investigation. the set of reduction operators of the burgers equation is naturally partitioned into two subsets, which consist of singular and regular reduction operators, respectively. basic properties of singular reduction operators, whose coefficients of t vanish, are quite common for (1 + 1)-dimensional evolution equations and properly formulated in no - go terms, and the main property is the existence of a bijection between equivalence classes of singular reduction operators and one - parameter families of solutions which are different up to re - parameterization. at the same time, the subset of regular reduction operators, whose coefficients of t do not vanish, is of specific structure. the representatives of equivalence classes of such operators, whose coefficients of t equal one, are partitioned into three sets, namely, the unique operator t+ux with u=1, the family of operators with u=0, each of which is equivalent to a lie symmetry operator, and the family of operators with u=12, which have the form (8) with the coefficients 0, 1 and 0 satisfying the system (9). we present a new optimized proof of the no - go theorem on the system (9) of determining equations for reduction operators with =1 and u=12, which is directly based on properties of such operators. as a consequence of the theorem, it is detected that the coefficients of reduction operators in this case admit the representation in terms of solutions of the uncoupled system formed by three copies of the burgers equation. any lie reduction of the burgers equation proves to be equivalent via the hopf cole transformation to a parameterized family of lie reductions of the linear heat equation. we also carry out all possible nonclassical reductions of the burgers equation to single ordinary differential equations. regular reduction operators with the coefficient gauged to one give purely nonclassical reductions of the burgers equation only if u{1,12}. it is obvious that all corresponding ansatzes are necessarily implicit and hence they can not be constructed using the direct method by clarkson and kruskal. see related discussions on the connection between nonclassical symmetries and the direct method, e.g., in. finding reduction operators for the burgers equation, one faces two kinds of no - go cases. the first kind is given by singular reduction operators and is in fact related to lowering the equation order to one in the course of reduction. this is why similar no - go results are true for any (1 + 1)-dimensional partial differential equation possessing a family of reduction operators of singularity co - order one which is parameterized by an arbitrary smooth function of all independent and dependent variables. for each such family, the system of determining equations consists of a single partial differential equation on the function parameterizing the family, and this equation is equivalent, in a certain sense, to the original equation, where the variable tuple is implicitly augmented with an additional parametric variable. the second kind given by regular reduction operators with =1 and u=12 is more specific and definitely related to the fact that the burgers equation is linearized by the hopf cole transformation to the linear heat equation. the corresponding system of determining equations is a system of three (1 + 1)-dimensional evolution equations, which is reduced by differential substitutions to the uncoupled system of three copies of the linear heat equation as well as to the uncoupled system of three copies of the burgers equation. similar no - go results are known only for linear (1 + 1)-dimensional evolution equations of the second order. it looks possible to extend these results to the entire class of generalized burgers equations which are linearized by the hopf cole transformation, and the optimized proof of theorem 1 creates a significant prerequisite for this. the question whether there exist no - go cases of other kinds related to regular reduction operators of single evolution equations is still open. | the solution of the problem on reduction operators and nonclassical reductions of the burgers equation is systematically treated and completed. a new proof of the theorem on the special no - go case of regular reduction operators is presented, and the representation of the coefficients of operators in terms of solutions of the initial equation is constructed for this case. all possible nonclassical reductions of the burgers equation to single ordinary differential equations are exhaustively described. any lie reduction of the burgers equation proves to be equivalent via the hopf cole transformation to a parameterized family of lie reductions of the linear heat equation. |
the threats of terrorism and criminal bombing are becoming ever more serious problems for all countries. to find hidden explosives, various types of detectors for explosives bulk detection includes x - ray imaging, nuclear quadrupole resonance (nqr), and neutron techniques that are used to determine the shape and size of suspicious objects in luggage, letters, packages, etc. however, bulk detection is not capable of easily identifying what the suspicious objects are. trace detection, on the other hand, including chemical - analysis methods such as chemical luminescence, ion mobility spectroscopy, and mass spectrometry, has been applied to the detection of trace contaminants that are present on a passenger s body, clothes, and luggage. trace detection permits specific chemicals to be identified, and its selectivity is higher than that of bulk detection. however, it can not determine the actual amount of chemicals in a suspicious item such as luggage. as mentioned above, the characteristics of bulk detection and trace detection are different. to improve security at important facilities, therefore, the combined use of bulk detection and trace detection is recommended. at current security checkpoints at most airports, not all carry - on luggage is tested by trace detector, although all luggage is examined by x - ray imaging. one of the reasons for this is that the use of a trace detector is not so popular, because its throughput is not sufficiently rapid to permit the method to be applied for all passengers and luggage. typical test procedures for a trace detector are as follows : a security guard at a security checkpoint wipes the surface of the luggage with a wiping - sheet to collect any contaminants that are adhered to the luggage, inserts the wiping - sheet into the heating unit of the trace detector, and waits to see if the alarm of the trace detector is activated or not. the total analysis time from wiping to detection is typically 30 s per test, when applied to human screening. to shorten this inspection time, we evaluated the use of mass spectrometry because its high separation and high sensitivity might possibility lead to much higher throughput. in the mass spectrometry studies, both vapor or particle detection was used for volatile explosives or less - volatile explosives, respectively. the use of a mass spectrometer for the detection of vapor derived from explosives using various ambient ionization sources such as atmospheric pressure chemical ionization (apci) and secondary electrospray ionization (sesi) ionized the vapor molecules derived from an explosive has been reported in previous studies. reported on the detection of very low concentrations of vapor (below 25 ppq) produced by an explosive vapor using a laboratory based triple - quadrupole mass spectrometer, but a much higher sensitivity would be needed to achieve the real - time detection of less volatile explosives such as like rdx, and petn. many researchers who are involved in the detection of explosive are still seeking a method that is faster and more accurate in this field. before starting the development of systems for detecting explosives, we focussed on the development of a highly robust, miniaturized version of a mass spectrometer. a technology for on - site applications would only be acceptable if they met the user s requirements for usability, size, cost, and related issues. although it was not specifically designed for detecting explosives but was originally designed for an environmental applications, i.e., monitoring dioxin precursors, we developed the counter - flow introduction apci depicted in fig. 1. the direction of the high - speed air opposite the electric field can avoid overlapping the secondary ionization (ion molecular reaction) area and the nox abundant area. because ions are first generated by corona discharge they do not perturb the nox abundant area, ions that are generated first have more chances to react with target molecules like explosives. this less - overlapping configuration improved the ionization efficiency for negative ions by about 3 times. as another advantage of this configuration is that it extended the maintenance frequency to typically every 6 months ; our conventional apci ion source required irregular maintenance between a few hours and a week under this condition. we conclude that the high speed air flow prevents the deposition of dust around the tip of the needle electrode, which makes the corona discharge much more stable. the robust ionization source has been a key technology and is deployed in our mass spectrometers for on - site applications. we aimed to develop a miniature mass spectrometer that meets, not only size requirements but also has sufficient sensitivity and selectivity for detecting explosives for purposes of trace detection. since an ion trap has intrinsic characteristics such as small dimension, and less - strict vacuum conditions, we developed several types of linear ion trap mass spectrometers, such as an axially resonant excitation (arex) linear ion trap mass spectrometer, and a wire type linear ion trap. 2, inserted in a set of vane lenses between each quadrupole rod, produces ab electrostatic potential that is approximately harmonic along the central axis of the quadrupole field. after ions with a specific m / z are resonantly oscillated in the axial direction, they are mass selectively ejected in the axial direction. at a high scan rate of 11 th / ms, the arex achieved a high ejection efficiency of more than 60%, which is more than three times higher than a conventional mass selective axial ejection method from a linear trap using a fringing field. in contrast, cid excitation in a conventional linear ion trap is in a pseudo - harmonic radio frequency potential that destabilizes fragment ions whose m / z is less than 1/4 that of the precursor ions. ecd was achieved in an axial magnetic field of about 0.2 tesla superimposed along the arex linear ion trap mass spectrometer. we observed that sequential ecd / cid inside the trap improved the sequence coverage for a peptide. in another linear ion trap, the wire type linear ion trap shown in fig. 3, a set consisting of a trap wire lens and an extraction wire lens positioned orthogonally to each other was placed between the quadrupole rods. the trap wire lens confines the ions inside the trap, and the extraction wire axially extracts ions from the trap. the ions are resonantly excited in the direction perpendicular to the trap wire lens by applying a supplemental alternating current to the excitation lenses. excited ions with a large motion pass over the trap wire lens, while unexcited ions remain trapped inside. ions that have passed over the trap wire lens are then extracted by the extraction wire lens. the characteristics of mass - selective ejection with a direct current extraction field were investigated by both simulations and experiments. a mass resolving power of m/m=1300 was achieved at a scan rate of 500 th / s. the dependence of ejection efficiency on trap wire lens bias was measured, and an ejection efficiency of 20% at a scan rate of 500 th / s was achieved by optimizing the dc bias on the trap wire lens. for producing portable mass spectrometers used in the illicit drug detection applications, we developed a mass spectrometer with discontinuous sample gas introduction. the study was intended to produce a mass spectrometer that was sufficiently compact that it would be portable and be used in an onsite analysis. the development of a small mass spectrometer required the use of a compact pump whose displacement was small, thus decreasing the sensitivity of that spectrometer. to obtain a high sensitivity with a small mass spectrometer, we integrated some novel techniques : a highly sensitive ionization source and the efficient extraction of sample vapor. the low - pressure dielectric barrier discharge ionization (lp - dbdi) source made it possible to increase the conductance between the source and the mass analyzer, compared with ambient ionization sources, thus enhancing the efficiency of ion transfer from the ionization source to the mass analyzer. we also developed a vacuumed headspace and probe heating methods for efficiently transporting the sample vapor to the ionization source. a prototype portable mass spectrometer using those novel techniques was found to be sufficiently sensitive to permit the detection of 0.1 ppm methamphetamine, 1 ppm amphetamine, 1 ppm 3,4-methylenedioxymethamphetamine, and 10 ppm cocaine in a liquid. we started the development of a high - throughput explosive detection system using these component devices in 2007. we first developed a high - throughput portal system for detecting triacetone triperoxide (tatp) vapor emitted from passengers and luggage, as shown in fig. the portal system consists of a push - pull air sampler, a counter flow introduction apci ion source, and a wire - type linear ion trap mass spectrometer. tatp signals were clearly obtained within 2 s after the subject passed through the portal system. by optimizing the shape of the nozzle that controls the air flow of the vapor sampler, tatp vapor could be detected at a high throughput, i.e., 1200 persons / hour. the false - positive rate of the detection portal system for tatp was evaluated by a field test performed at a train station. a multimarker logic to determine was adopted, and no false - positive alarms were obtained for over 3000 passengers during the field test. moreover, acetone, which is an inflammable liquid, could be detected from the passengers during the field test. we therefore concluded from these results that this detection portal system would be useful for detecting dangerous chemicals that have high vapor pressure (such as tatp and inflammable liquids). the detection limits of the vapor portal were between the sub - ppb and ppb level, which is not sufficient to permit explosive vapors such as tnt, and rdx to be detected. in 2010, we started another development of a high - throughput explosive detection system for detecting more comprehensive explosives including less volatile explosives such as tnt, and rdx. this detector consists of an automated particle sampler for collecting contaminants in explosives and an ion - trap mass spectrometer for analyzing them. the automated sampler, which uses a cyclone concentrator and mass spectrometer in this manner enables the rapid detection of explosives particles that had adhered to the surface of a synthetic leather sheet within a few seconds. figure 5 shows the experimental setup of the prototype sampler, which consists of an air nozzle (inner diameter : 1.5 mm), evacuation port, cyclone particle concentrator, vaporizer, and a homemade mass spectrometer. a wire type linear ion trap mass spectrometer with counter - flow introduction atmospheric pressure chemical ionization (apci) explosive particles are wiped off with a sampling probe, which is an aluminium rod with a diameter of 2 mm. this wiping operation was made to cover only the tip of probe with particles from the sample reservoir. first, the explosive particles on a sheet are detached by an air jet pulse with a duration of 100 ms. particles detached by the air jet pulse are collected from the evacuation port and then introduced into the cyclone particle concentrator. the mechanism and structure of the concentrator has been described in the literature, and our dimensions are given in the caption for fig. the pumping speed from the evacuation port into the concentrator is about 80 l / min. particles accumulate near the bottom of the concentrator as the result of centrifugal wind and gravity forces. the vaporizer, which is placed at the bottom of the concentrator, has a transfer line between the concentrator and the mass spectrometer. because the vaporizer is constantly heated at about 200c, explosive vapor is then introduced into the mass spectrometer at a flow rate of about 1 l / min. to prevent particles from reaching the spectrometer, a metal filter with a filtration accuracy of 1 m (nippon seisen co., ltd., explosive particles wiped off with the sampling probe were deposited onto and directly injected into the vaporizer after removing the cyclone particle concentrator (number of trials, n=20). by comparing the calibration curve and the signal intensity at the direct injections, the amount of tnt contained in a single deposit of particles was estimated to be about 47 ng (c.v. of 24%). the weight of deposited silica (c18) particles with the sample probe was calculated to be about 47 g by calculating the amount of tnt of 47 ng and mass fraction of 0.1%. in the following, we calculate the amounts of explosive particles from the calculated deposit particle weight of 47 g and each mass fraction. we investigated the dependencies versus several parameters such as the nozzle gas pressure, angles of gas injections onto the object, distance between the nozzle and objects, pumping speed into the cyclone using the experimental setup. an example for determining the optimum pressure inside the jet nozzle, and the pumping speed are explained in the following. figures 6(a) and (b) show surface images on the synthetic leather sheet before and after an air jet pulse at a nozzle pressure of 0.05 mpa is introduced. these images were taken with a fluorescence microscope after silica (c18) particles were prepared by doping with the fluorescent dye fluorescein. from the images, we calculated a detachment efficiency of 86.4%. figure 7 shows changes in detachment efficiency against nozzle pressure. the detachment efficiency increased with the pressure increment and was saturated at more than 80% over 0.05 mpa. since a higher nozzle pressure might spread the detached particles with a strong air flow and degrade the subsequent collection efficiency into the evacuation port, a nozzle pressure of 0.05 mpa was found to be optimum for this configuration. the detection efficiency reached a maximum value of about 20% when optimal conditions were employed. the cyclone particle concentrator enriches the explosive particles by about 80 times compared to the absence of a concentration process. figures 8(a) and (b) show mass spectra when explosive particles were directly deposited into the metal filter inside the vaporizer using the setup shown in fig. molecular ions of m with a peak of m / z 227 were dominantly observed in the tnt mass spectrum of fig. unlike tnt, adduct ions of [m+no2 ] with a peak of m / z 268 were dominant in the rdx mass spectrum shown in fig. these spectra show that tnt and rdx particles can be vaporized with minimal fragmentation at a temperature of 200c. we used the intensities of the dominant peaks at m / z 227 and m / z 268 for the tnt and rdx concentration index. figures 9(a) and (b) show the signal time response after the explosive particles are exposed to an air jet pulse. an air jet pulse was injected at a timing of 1.0 s, depicted as arrows in the figures. the signal for tnt rose in about 1 s and returned to the former level in less than 10 s after the air jet pulse. the time necessary for the transportation of the particles from the sample sheet to the mesh, and the transportation time of the vapor from the mesh to the mass spectrometer are estimated to be about 50, and 100 ms, respectively. in comparison, the signal of rdx increased in 2 s and then returned to the former level in about 15 s. the time response for rdx was slower than that for tnt, which can be explained by the difference of their volatility. the vapor pressure of rdx is much lower than that of tnt, which means rdx is vaporized more slowly than tnt with heating. if we take 1 s of data after signal - rising as its explosive s concentration, detection is accomplished in 2 s for tnt and in 3 s for rdx after the air jet pulse. as the next step towards practical security applications of the high - throughput explosive detection system, we installed the automated particle sampler in a commercial boarding gate and baggage screener to check all passengers and baggage boarding an aircraft for traces explosives. we designed these prototypes using several parameters such as the nozzle gas pressure, angles of gas injection onto the object, and distance between the nozzle and objects, which we obtained in the sample testing described above. the prototype is based on a commercial boarding gate used at airports (nippon signal, co., ltd., 10(a), the boarding gate consists of a main unit, a sub unit, and a footboard. to install the particle sampler and the mass spectrometer in the main unit, the width of the side cover of the main unit is 15 cm wider than that of the original cover. the controllers of the particle sampler and the mass spectrometer are installed in the sub unit. an air compressor (not shown in the figure) is needed to supply compressed air to the boarding gate. however, the air compressor does not always need to be located nearby the boarding gate because compressed gas may be supplied via a long tube. an enlargement of the electronic - ticket (e - ticket) reader is shown in fig. when a passenger places his / her ic - card or e - ticket on the reader, an air jet is emitted from the nozzle. particles that had adhered to the e - ticket and/or the passenger s hand are blown by the jet and collected by the inlet. the main challenge to installing the detectors into a gate system is the simultaneous detection of various explosives. we carried out the simultaneous detection of tnt and tatp as an example where the switching of the ionization polarity to detect nitro - compounds (which have strong electron affinity) and peroxides (which have a strong proton affinity) was confirmed. to change the polarity of the ion source and the mass spectrometer, the interval between the negative and positive mass analysis was set to 0.3 s. the simulants of tnt and tatp particles that had adhered to the surface of the plastic card, and the plastic card was tested by the boarding gate set up. the results for detecting tnt and tatp the data acquisition frequency in the case of the polarity switching analysis was expanded from every 0.25 s in the case of only the negative mode to every 0.9 s, but the signals for tnt and tatp were clearly obtained, as shown in fig. the findings reported herein show that the high - throughput detection for both positive and negative ionized explosives could be performed within 3 s after a passenger places his / her ic - card or e - ticket on the reader. for baggage screening, on the other hand, the trace - explosives detector was combined with a conventional x - ray baggage screener shown in fig. when a bag is put on the conveyor belt of the automated particle sampler, compressed air jets are emitted from nozzles. the trace analysis is finished, while an x - ray image of the bag is being obtained by the x - ray baggage screener. in both security equipment cases, the simulated tnt, rdx, and tatp particles that adhered to the detection targets were successfully detected. accordingly, the developed security equipment will be useful tools for improving airport security in the near future. we started the development of a high - throughput explosive detection system using these component devices in 2007. we first developed a high - throughput portal system for detecting triacetone triperoxide (tatp) vapor emitted from passengers and luggage, as shown in fig. the portal system consists of a push - pull air sampler, a counter flow introduction apci ion source, and a wire - type linear ion trap mass spectrometer. tatp signals were clearly obtained within 2 s after the subject passed through the portal system. by optimizing the shape of the nozzle that controls the air flow of the vapor sampler, tatp vapor could be detected at a high throughput, i.e., 1200 persons / hour. the false - positive rate of the detection portal system for tatp was evaluated by a field test performed at a train station. a multimarker logic to determine was adopted, and no false - positive alarms were obtained for over 3000 passengers during the field test. moreover, acetone, which is an inflammable liquid, could be detected from the passengers during the field test. we therefore concluded from these results that this detection portal system would be useful for detecting dangerous chemicals that have high vapor pressure (such as tatp and inflammable liquids). the detection limits of the vapor portal were between the sub - ppb and ppb level, which is not sufficient to permit explosive vapors such as tnt, and rdx to be detected. in 2010, we started another development of a high - throughput explosive detection system for detecting more comprehensive explosives including less volatile explosives such as tnt, and rdx. this detector consists of an automated particle sampler for collecting contaminants in explosives and an ion - trap mass spectrometer for analyzing them. the automated sampler, which uses a cyclone concentrator and mass spectrometer in this manner enables the rapid detection of explosives particles that had adhered to the surface of a synthetic leather sheet within a few seconds. figure 5 shows the experimental setup of the prototype sampler, which consists of an air nozzle (inner diameter : 1.5 mm), evacuation port, cyclone particle concentrator, vaporizer, and a homemade mass spectrometer. a wire type linear ion trap mass spectrometer with counter - flow introduction atmospheric pressure chemical ionization (apci) records the mass spectrum at a rate of about 4 spectra / s. explosive particles are wiped off with a sampling probe, which is an aluminium rod with a diameter of 2 mm. this wiping operation was made to cover only the tip of probe with particles from the sample reservoir. first, the explosive particles on a sheet are detached by an air jet pulse with a duration of 100 ms. particles detached by the air jet pulse are collected from the evacuation port and then introduced into the cyclone particle concentrator. the mechanism and structure of the concentrator has been described in the literature, and our dimensions are given in the caption for fig. the pumping speed from the evacuation port into the concentrator is about 80 l / min. particles accumulate near the bottom of the concentrator as the result of centrifugal wind and gravity forces. the vaporizer, which is placed at the bottom of the concentrator, has a transfer line between the concentrator and the mass spectrometer. because the vaporizer is constantly heated at about 200c, the explosives particles that arrive here are converted into vapor. explosive vapor is then introduced into the mass spectrometer at a flow rate of about 1 l / min. to prevent particles from reaching the spectrometer, a metal filter with a filtration accuracy of 1 m (nippon seisen co., ltd., explosive particles wiped off with the sampling probe were deposited onto and directly injected into the vaporizer after removing the cyclone particle concentrator (number of trials, n=20). by comparing the calibration curve and the signal intensity at the direct injections, the amount of tnt contained in a single deposit of particles the weight of deposited silica (c18) particles with the sample probe was calculated to be about 47 g by calculating the amount of tnt of 47 ng and mass fraction of 0.1%. in the following, we calculate the amounts of explosive particles from the calculated deposit particle weight of 47 g and each mass fraction. we investigated the dependencies versus several parameters such as the nozzle gas pressure, angles of gas injections onto the object, distance between the nozzle and objects, pumping speed into the cyclone using the experimental setup. an example for determining the optimum pressure inside the jet nozzle, and the pumping speed are explained in the following. figures 6(a) and (b) show surface images on the synthetic leather sheet before and after an air jet pulse at a nozzle pressure of 0.05 mpa is introduced. these images were taken with a fluorescence microscope after silica (c18) particles were prepared by doping with the fluorescent dye fluorescein. from the images, we calculated a detachment efficiency of 86.4%. figure 7 shows changes in detachment efficiency against nozzle pressure. the detachment efficiency increased with the pressure increment and was saturated at more than 80% over 0.05 mpa. since a higher nozzle pressure might spread the detached particles with a strong air flow and degrade the subsequent collection efficiency into the evacuation port, a nozzle pressure of 0.05 mpa was found to be optimum for this configuration. the detection efficiency reached a maximum value of about 20% when optimal conditions were employed. the cyclone particle concentrator enriches the explosive particles by about 80 times compared to the absence of a concentration process. figures 8(a) and (b) show mass spectra when explosive particles were directly deposited into the metal filter inside the vaporizer using the setup shown in fig. molecular ions of m with a peak of m / z 227 were dominantly observed in the tnt mass spectrum of fig. unlike tnt, adduct ions of [m+no2 ] with a peak of m / z 268 were dominant in the rdx mass spectrum shown in fig. these spectra show that tnt and rdx particles can be vaporized with minimal fragmentation at a temperature of 200c. we used the intensities of the dominant peaks at m / z 227 and m / z 268 for the tnt and rdx concentration index. figures 9(a) and (b) show the signal time response after the explosive particles are exposed to an air jet pulse. an air jet pulse was injected at a timing of 1.0 s, depicted as arrows in the figures. the signal for tnt rose in about 1 s and returned to the former level in less than 10 s after the air jet pulse. the time necessary for the transportation of the particles from the sample sheet to the mesh, and the transportation time of the vapor from the mesh to the mass spectrometer are estimated to be about 50, and 100 ms, respectively. in comparison, the signal of rdx increased in 2 s and then returned to the former level in about 15 s. the time response for rdx was slower than that for tnt, which can be explained by the difference of their volatility. the vapor pressure of rdx is much lower than that of tnt, which means rdx is vaporized more slowly than tnt with heating. if we take 1 s of data after signal - rising as its explosive s concentration, detection is accomplished in 2 s for tnt and in 3 s for rdx after the air jet pulse. as the next step towards practical security applications of the high - throughput explosive detection system, we installed the automated particle sampler in a commercial boarding gate and baggage screener to check all passengers and baggage boarding an aircraft for traces explosives. we designed these prototypes using several parameters such as the nozzle gas pressure, angles of gas injection onto the object, and distance between the nozzle and objects, which we obtained in the sample testing described above. the prototype is based on a commercial boarding gate used at airports (nippon signal, co., ltd., 10(a), the boarding gate consists of a main unit, a sub unit, and a footboard. to install the particle sampler and the mass spectrometer in the main unit, the width of the side cover of the main unit is 15 cm wider than that of the original cover. the controllers of the particle sampler and the mass spectrometer are installed in the sub unit. an air compressor (not shown in the figure) is needed to supply compressed air to the boarding gate. however, the air compressor does not always need to be located nearby the boarding gate because compressed gas may be supplied via a long tube. an enlargement of the electronic - ticket (e - ticket) reader is shown in fig. when a passenger places his / her ic - card or e - ticket on the reader, an air jet is emitted from the nozzle. particles that had adhered to the e - ticket and/or the passenger s hand are blown by the jet and collected by the inlet. the main challenge to installing the detectors into a gate system is the simultaneous detection of various explosives. we carried out the simultaneous detection of tnt and tatp as an example where the switching of the ionization polarity to detect nitro - compounds (which have strong electron affinity) and peroxides (which have a strong proton affinity) was confirmed. to change the polarity of the ion source and the mass spectrometer, the interval between the negative and positive mass analysis was set to 0.3 s. the simulants of tnt and tatp particles that had adhered to the surface of the plastic card, and the data acquisition frequency in the case of the polarity switching analysis was expanded from every 0.25 s in the case of only the negative mode to every 0.9 s, but the signals for tnt and tatp were clearly obtained, as shown in fig. the findings reported herein show that the high - throughput detection for both positive and negative ionized explosives could be performed within 3 s after a passenger places his / her ic - card or e - ticket on the reader. for baggage screening, on the other hand, the trace - explosives detector was combined with a conventional x - ray baggage screener shown in fig. when a bag is put on the conveyor belt of the automated particle sampler, compressed air jets are emitted from nozzles. the trace analysis is finished, while an x - ray image of the bag is being obtained by the x - ray baggage screener. in both security equipment cases, the simulated tnt, rdx, and tatp particles that adhered to the detection targets were successfully detected. accordingly, the developed security equipment will be useful tools for improving airport security in the near future. the instrument includes a counter - flow introduction apci, a miniature mass spectrometer, and an automated sampling explosive detector system combined with these component devices. these developments have been ongoing for nearly 20 years in order to realize on - site mass spectrometry. counter flow introduction apci enhances robustness, ion trap development reduces the size of the mass spectrometry, permitting it to meet the requirements needed for on - site application. the detection of explosives at the level of the boarding gate and baggage screener were demonstrated, where the automated particle sampling using cyclone concentrator successfully reduced the inspection time and labour - intensive sampling process. | the development of a robust ionization source using the counter - flow apci, miniature mass spectrometer, and an automated sampling system for detecting explosives are described. these development efforts using mass spectrometry were made in order to improve the efficiencies of on - site detection in areas such as security, environmental, and industrial applications. a development team, including the author, has struggled for nearly 20 years to enhance the robustness and reduce the size of mass spectrometers to meet the requirements needed for on - site applications. this article focuses on the recent results related to the detection of explosive materials where automated particle sampling using a cyclone concentrator permitted the inspection time to be successfully reduced to 3 s. |
animals : all experiments were performed using female wistar imamichi rats, 910 weeks of age, 218.45 2.09 g (mean se) in weight, and obtained from the institute of animal reproduction (kasumigaura, japan). the animals were reared in a room with the temperature controlled at 22 2c, humidity at 50 5%, with ventilation 11 times per hr, lighting at 12:12-hr light / dark cycle (light cycle, 7:0019:00) and given standard chow (ce-2 ; nihon clea, tokyo, japan). the present experiments were performed following the provisions approved by the animal research committee of tottori university. experimental design : a total of 40 animals were divided into 2 groups as follows : (1) saline - treated control rats (n=20), (2) mtx - treated rats (n=20). day 0 of gestation (gd 0) was designated as the day when the presence of a vaginal plug was identified. the rats received intraperitoneal injections (i.p.) with mtx (30 mg / kg body weight) or saline (the control) on gd 13. the specific timing of mtx administration was selected, because the injection of dna damaging chemicals, such as ethylnitrosourea (enu), 6-mercaptopurine (6-mp), busulfan, t-2 toxin, 1--d - arabinofuranosylcytosine (ara - c), 5-azacytidine (5azc) [39, 40 ] and 5-fluorouracil (5-fu), on gd13 induced apoptosis in neuroepithelial cells of telencephalon in rat fetal brain. the dose level in the present study was decided, because this dose was equivalent to 10% of the ld50 in intraperitoneal injection in rats or 1530% of dosage used in high - dose cancer therapy in human [12, 15, 23 ]. fetus samples were collected under pentobarbital anesthesia (100 mg / kg, i.p.) 6, 12, 24, 36 and 48 hr, respectively, after mtx administration. they were removed from the uterus and weighed, and their body lengths were measured. histopathological examination : for histopathological examination, all fetuses were fixed in 10% neutral buffered formalin and then embedded in paraffin. the tissues of telencephalon, diencephalon, mesencephalon and metencephalon in 12 fetal brain samples per group consisted of 3 fetuses ramdomly selected per dam in each group were sectioned at 2 m thickness, stained with hematoxylin and eosin and examined with light microscopy. the number of pyknotic cells or mitotic cells was counted from over 1,000 neuroepithelial cells in the telencephalon, diencephalon, mesencephalon and metencephalon for each fetus by light microscopy, and the pyknotic index and mitotic index were calculated as the percentage of pyknotic cells or mitotic cells from out of the total number of neuroepithelial cells counted. tunel method : dna - fragmented neuroepithelial cells in telencephalon were detected by terminal deoxynucleotidyl - transferase (tdt)-mediated deoxyuridine triphosphate - digoxigenin (dutp) nick - end labeling (tunel), which was performed using an in situ apoptosis detection kit (trevigen, inc., the number of tunel - positive cells was obtained from over 1,000 neuroepithelial cells in the telencephalon for each fetus by light microscopy, and the tunel - index was calculated as the percentage of tunel - positive cells out of the total number of neuroepithelial cells counted. immunohistochemical examinations of rat fetuses : immunohistochemical staining was performed by a labeled - polymer method using histofine simple stain max - po (r) (nichirei, tokyo, japan). to retrieve the antigen, tissue sections for the detection of cleaved caspase-3 antigen were immersed in citrate buffer, ph 6.0 (dako, glostrup, denmark) and autoclaved for 15 min at 121c ; and tissue sections for the detection of phospho - histone h3 antigen were immersed in citrate buffer, ph 6.0 (dako) and microwaved for 15 min. endogenous peroxidase activity was quenched by immersing the sections in 3% hydrogen peroxide in methanol for 15 min. the sections were incubated with the cleaved caspase-3 rabbit polyclonal antibody (1:300 dilution ; cell signaling technology, inc., danvers, ma, u.s.a.) at 4c overnight ; and the sections were incubated with the phospho - histone h3 rabbit monoclonal antibody (1:1,500 dilution ; abcam, tokyo, japan) for 30 min at room temperature. then, these sections were treated with histofine simple stain max - po (r) (nichirei) for 30 min at room temperature. they were exposed to a 3,3-diaminobenzidine solution containing hydrogen peroxide (nichirei) to facilitate a peroxidase color reaction and then counterstained with mayer s hematoxylin. the number of cleaved caspase-3- or phospho - histone h3-positive cells was counted from over 1,000 neuroepithelial cells in the telencephalon for each fetus by light microscopy, and the cleaved caspase-3- or phospho - histone h3-index was calculated as the percentage of cleaved caspase-3- or phospho - histone h3-positive cells out of the total number of neuroepithelial cells counted. statistical analysis : means standard error (se) of the individual litter value was calculated. effects of mtx on rat fetuses : six, 12 and 24 hr after mtx treatment, there were no significant differences in the number of living fetuses per litter and the fetal mortality rates between the control group and mtx - treated group (table 1table 1. of damstotal no. of live fetusesliving fetuses per litterdead fetus ratio (%) 6 hrcontrol45814.59 0.509.03 3.03mtx45714.25 0.633.14 1.8212 hrcontrol45614.00 0.417.93 3.00mtx46315.75 2.5015.92 10.6724 hrcontrol45814.50 1.447.63 4.81mtx45714.25 0.956.48 4.4436 hrcontrol46215.50 1.193.58 3.58mtx44411.00 0.41 30.30 5.4148 hrcontrol45614.00 1.004.73 3.15mtx410.25 0.2598.53 1.48values are expressed as means se.,, : significantly different from control at p<0.05, p<0.01, p<0.001, respectively (student s t - test). : significantly different from control at p<0.001 (welch s t - test).) the number of living fetuses significantly declined, and fetal mortality rates significantly increased at 36 hr in the mtx - treated group, compared to those of the control group (table 1). almost all fetuses died by 48 hr after mtx treatment (table 1).,, : significantly different from control at p<0.05, p<0.01, p<0.001, respectively (student s t - test). : significantly different from control at p<0.001 (welch s t - test). histopathological findings of rat fetal brain : in the control group, pyknotic changes in neuroepithelial cells were rarely observed in any layers of the telencephalic wall throughout the experimental period (figs. 1 and 2fig. 1.histopathological findings of telencephalic wall of rat fetal brain in the control group 6 hr after treatment (a) and the mtx - treated group at 6 (b), 12 (c), 24 (d) and 36 hr (e), respectively. 2.changes in the pyknotic index (%) in telencephalic wall of rat fetal brain., : significantly different from control at p<0.05, p<0.001, respectively (welch s t - test).) histopathological findings of telencephalic wall of rat fetal brain in the control group 6 hr after treatment (a) and the mtx - treated group at 6 (b), 12 (c), 24 (d) and 36 hr (e), respectively. bar=30 m. changes in the pyknotic index (%) in telencephalic wall of rat fetal brain., : significantly different from control at p<0.05, p<0.001, respectively (welch s t - test). six hr after treatment in the mtx - treated group, pyknotic neuroepithelial cells appeared in the telencephalic wall and scattered throughout that wall (fig. then, 1236 hr after mtx treatment, pyknotic neuroepithelial cells drastically increased and were diffusely distributed throughout the telencephalic wall (figs., neuroepithelial cells were eliminated and showed low cell density in the telencephalon of mtx - treated group (fig. although mitotic neuroepithelial cells were located along the ventricular layer of the telencephalic wall in the control group, they were rarely observed in the same region at 636 hr in the mtx - treated group (fig. 1b1e). in the telencephalon of the control group, tunel - positive neuroepithelial cells were rarely observed in any layers of the telencephalic wall throughout the experimental period (figs 3.tunel-positive cells in telencephalic wall of rat fetal brain in the control group 6 hr after treatment (a) and the mtx - treated group at 6 (b), 12 (c), 24 (d) and 36 hr (e). most of the pyknotic neuroepithelial cells were positively stained by the tunel method at 12, 24 and 36 hr in the mtx - treated group (fig. 3c3e), while there were few pyknotic neuroepithelial cells positively stained by the tunel method at 6 hr (fig. the index of tunel - positive neuroepithelial cells in the telencephalic wall significantly increased at 12 and 24 hr and peaked at 36 hr (fig. 4.changes in the tunel index (%) in telencephalic wall of rat fetal brain., : significantly different from control at p<0.01, p<0.001, respectively (welch s t - test).). tunel - positive cells in telencephalic wall of rat fetal brain in the control group 6 hr after treatment (a) and the mtx - treated group at 6 (b), 12 (c), 24 (d) and 36 hr (e). bar=30 m. changes in the tunel index (%) in telencephalic wall of rat fetal brain., : significantly different from control at p<0.01, p<0.001, respectively (welch s t - test). cleaved caspase-3-positive neuroepithelial cells were rarely observed in the telencephalon of control group throughout the experimental period (figs. 5a and 6fig. 5.immunohistochemical expression of cleaved caspase-3 in telencephalic wall of rat fetal brain in the control group 6 hr after treatment (a) and the mtx - treated group at 6 (b), 12 (c), 24 (d) and 36 hr (e). bar=30 m), although in the mtx - treated group, almost all pyknotic neuroepithelial cells were immunohistochemically positive for cleaved caspase-3 throughout the experimental period (fig. 5b5e). in the mtx - treated group, the index of cleaved caspase-3-positive neuroepithelial cells in the telencephalon significantly increased at 6, 12 and 24 hr and peaked at 36 hr (fig., : significantly different from control at p<0.05, p<0.001, respectively (welch s t - test).) immunohistochemical expression of cleaved caspase-3 in telencephalic wall of rat fetal brain in the control group 6 hr after treatment (a) and the mtx - treated group at 6 (b), 12 (c), 24 (d) and 36 hr (e). bar=30 m cleaved caspase-3 index., : significantly different from control at p<0.05, p<0.001, respectively (welch s t - test). phospho - histone h3-positive neuroepithelial cells were located along the ventricular layer of the telencephalic wall in the control group, while there were fewer phospho - histone h3-positive cells at 636 hr in the same region of the mtx - treated group than in the control group (fig. 7.immunohistochemical expression of phospho - histone h3 in telencephalic wall of rat fetal brain in the control group 6 hr after treatment (a) and the mtx - treated group at 6 (b), 12 (c), 24 (d) and 36 hr (e), respectively. the index of phospho - histone h3-positive neuroepithelial cells in telencephalon decreased significantly at 6 hr compared with the control group and maintained that low level throughout the experimental period (fig. values are expressed as means se. : significantly different from control at p<0.001 (student s t - test). : significantly different from control at p<0.01 (welch s t - test).). immunohistochemical expression of phospho - histone h3 in telencephalic wall of rat fetal brain in the control group 6 hr after treatment (a) and the mtx - treated group at 6 (b), 12 (c), 24 (d) and 36 hr (e), respectively. values are expressed as means se. : significantly different from control at p<0.001 (student s t - test). : significantly different from control at p<0.01 (welch s t - test). in the mtx - treated group, pyknotic indices of telencephalon and diencephalon significantly increased at 1236 hr, and those of the mesencephalon significantly increased at 24 and 36 hr, compared to the control group (table 2table 2.pyknotic index (%) in rat fetal brain treated by mtx6 hr12 hr24 hr36 hrtelencephaloncontrol0.03 0.030.05 0.050.10 0.070.28 0.14mtx2.68 0.8519.98 2.4534.82 2.1743.33 3.07diencephaloncontrol0.61 0.280.79 0.300.66 0.360.81 0.30mtx1.61 0.3814.31 1.5320.88 2.4420.65 1.67mesencephaloncontrol0.39 0.040.41 0.110.29 0.110.47 0.12mtx0.37 0.187.78 2.8826.90 5.4536.56 3.99metencephaloncontrol0.00 0.000.00 0.000.00 0.000.07 0.05mtx0.04 0.020.00 0.002.34 1.523.88 0.72values are expressed as means se (%).,, : significantly different from control at p<0.05, p<0.01, p<0.001 respectively (welch s t - test).) the pyknotic index of metencephalon significantly increased at 36 hr compared to control group, and it showed a clearly lower value, compared to those of the telencephalon, diencephalon and mesencephalon (table 2). in the mtx - treated group, there were fewer pyknotic neuroepithelial cells in the metencephalon than in the telencephalon, diencephalon and mesencephalon. in the mtx - treated group, while mitotic indices of telencephalon, diencephalon and mesencephalon decreased significantly at 636 hr, that of metencephalon decreased significantly at 6 and 12 hr compared to the control group (table 3table 3. mitotic index (%) in rat fetal brain treated by mtx6 hr12 hr24 hr36 hrtelencephaloncontrol4.56 0.204.48 0.254.82 0.244.50 0.09mtx0.53 0.050.24 0.030.16 0.080.22 0.13diencephaloncontrol3.01 0.283.30 0.332.56 0.363.66 0.28mtx0.37 0.080.57 0.090.37 0.090.40 0.20mesencephaloncontrol3.64 0.433.76 0.353.51 0.444.53 0.34mtx0.64 0.170.96 0.100.22 0.071.13 0.27metencephaloncontrol3.12 0.643.25 0.251.37 0.251.25 0.10mtx0.67 0.081.40 0.351.53 0.471.60 0.26values are expressed as means se (%)., : significantly different from control at p<0.01, p<0.001 respectively (student s t - test).,, : significantly different from control at p<0.05, p<0.01, p<0.001 respectively (welch s t - test).).,, : significantly different from control at p<0.05, p<0.01, p<0.001 respectively (welch s t - test). values are expressed as means se (%)., : significantly different from control at p<0.01, p<0.001 respectively (student s t - test).,, : significantly different from control at p<0.05, p<0.01, p<0.001 respectively (welch s t - test). it has been reported that microcephaly can occur as one of the central nervous system anomalies of mtx embryopathy in humans [1, 8 ]. the pathogenesis of microcephaly was closely associated with excessive neuronal death [7, 31, 38 ]. the cause of mtx - induced microcephaly may be also associated with excessive neuronal death. in the present study, the majority of pyknotic neuroepithelial cells in the telencephalic wall were positive for tunel staining and cleaved caspase-3. cleavage of caspase-3 is known to be involved in cell apoptosis and is recognized as an apoptosis marker. these results indicate that pyknotic changes induced by mtx in the present study are caused by apoptosis. in the present study, pyknotic neuroepithelial cells in the telencephalon at 6 hr were positive for cleaved caspase-3, but negative for tunel staining. this result may reflect that the cleavage of caspase-3 precedes dna fragmentation in the process of the neuroepithelial cell apoptosis induced by mtx. the previous studies showed that dna - damaging chemical administration at gd 12 or 13 induces apoptosis in neuroepithelial cells of the telencephalic wall of fetal brain [19,20,21, 28, 30, 39,40,41,42 ]. these apoptotic neuroepithelial cells are mainly observed in the dorsal layer after treatment with enu [6, 19,20,21 ] in the medial to dorsal layer after treatment with hydroxyurea, 5-fu [6, 42 ], busulfan and etoposide and in the ventral to medial layers after treatment with 5azc [6, 39, 40 ]. in the present study, apoptotic neuroepithelial cells localized throughout all layers of the telencephalic wall, but their distribution was different from those of apoptotic neuroepithelial cells induced by the above - mentioned other dna - damaging chemicals (table 4table 4.location of apoptotic cells in the ventricular zone of telencephalon after treatment with dna - damaging chemicalslocation of apoptotic cells in the ventricular zone of telencephalonethylnitrosoureadorsal layerhydroxyurea, busulfan, etoposide, 5-fluorouracilmedial to dorsal layers5-azacytidineventral to medial layersmethotrexateall layers). it is known that in the ventricular zone of fetal brain, the positions of the nuclei of neuroepithelial cells are correlated with their cell cycle phase. s - phase nuclei are located in the dorsal layer of the ventricular zone. they migrate inward during the g2 phase, and mitosis occurs at the ventricular surface. then, they migrate outward during the g1 phase and enter the s phase again. in brief, the nuclei during the g1 or g2 phase are in the middle layer of the ventricular zone. the distribution of apoptotic neuroepithelial cells throughout all layers of the telencephalic wall in the present study may reflect that mtx induces apoptosis of neuroepithelial cells in all phases of the cell cycle or that apoptosis occurs independently of cell cycle arrest. whereas a previous study showed that apoptosis of the human hepatoma hepg2 cells induced by folate deficiency was specific for s- and/or g2-phase arrest, the present result suggests that mtx induces apoptosis of neuroepithelial cells through a mechanism other than s- and/or g2-phase arrest. in the present study, the pyknotic, tunel and cleaved caspase-3 indices of neuroepithelial cells in the telencephalon peaked 36 hr after treatment with mtx at gd 13 (table 5table 5. timing of the maximal level of cell death indices and minimal level of cell proliferation indices in the telencephalon after dna - damaging chemicals treatmentmaximal level of cell death indicesminimal level of cell proliferation indicespyknosistunelcleaved caspase-3mitosisphospho - histone h31--d - arabinofuranosylcytosine9 hrethylnitrosourea12 hrt-2 toxin12 hr5-azacytide12 hr24 hr busulfan48 hr48 hr6-mercaptopurine36 hr36 hrmethotrexate36 hr36 hr36 hr636 hr636 hr). the index of pyknotic cells in the telencephalon peaked 912 hr after treatment with ara - c and enu. the index of tunel - positive cells peaked 12 hr after treatment with t-2 toxin and 5azc [34, 39 ], and the index of cleaved caspse-3-positive cells peaked 48 hr after treatment with busulfan. on the other hand, in the present study, mitotic and phospho - histone h3 indices in telencephalon treated with mtx decreased significantly at 6 hr and maintained that low level throughout the experimental period (table 5). the mitotic cell index in the telencephalon significantly decreased at 30 hr and reached its lowest level 36 hr after treatment with 6-mp, while the mitotic cell index peaked 6 hr, decreased thereafter and reached the minimal level 24 hr after treatment with 5azc. the index of phospho - histone h3-positive cells significantly decreased at 24 hr and reached its minimal level 48 hr after treatment with busulfan, while it reached the lowest level 36 hr after treatment with 6-mp. the distribution of pyknotic neuroepithelial cells and the time - course changes of the indices of pyknotic and mitotic neuroepithelial cells in the telencephalic wall were different from those of the previously - mentioned other dna - damaging chemicals. these differences may reflect the disparity in mechanisms of apoptosis and the inhibition of cell proliferation in neuroepithelial cells among dna - damaging chemicals. further investigations should be conducted to clarify the true cause of these differences. in the present study, mtx induced fewer pyknotic changes of neuroepithelial cells in the metencephalon than in other brain regions. the result of a previous study suggested that cell - proliferative activity was associated with the sensitivity to mtx. the different pyknotic indices of the mtx - treated group among the brain regions in the present study may reflect the disparity of cell - proliferative activity. in a previous study, mtx inhibited the development of individual brain parts to varying degrees in chick embryos. the same study suggested that the different effects of mtx on various brain parts may be due to different schedules of cell proliferation among individual brain parts. other previous studies demonstrated that busulfan or 5-fu treatment at gd13 in rat fetus induced pyknotic changes of neuroepithelial cells in metencephalon to the same degree as those in diencephalon and mesencephalon [29, 42 ]. these results suggest that there are differences in sensitivities to mtx, busulfan and 5-fu of neuroepithelial cells in the metencephalon. there are several studies describing brain malformation induced by mtx in human and rabbits [2, 16, 35 ]. a report of seidahmed. described that 2.5 mg of mtx 3 times a day for 7 days, for a total of 52.5 mg at 6 weeks of gestation, induced alobar holoprosencephaly, cerebellar hypoplasia and agenesis of the corpus callosum in human. reported that mtx 5 mg / day treatment for 14 days at the 5th week post - conception induced brain anomalies including semilobar holoprosencephaly and hydrocephalus in human. in rabbits, mtx 19.2 mg / kg administration during gd 1015 induced various anomalies including hydrocephalus. in the present study, single dose - administration of mtx 30 mg / kg on gd 13 induced fetal death 48 hr after treatment, and the cause of fetal death in the present study may be relevant to dose, number and timing of mtx administration. in conclusion, mtx administration of 30 mg / kg on gd 13 induced apoptosis of neuroepithial cells and inhibited mitosis of these cells in telencephalon 636 hr after treatment. the distribution of apoptotic neuroepithelial cells in the telencephalic wall and the whole brain and the time - course changes of the indices of apoptotic and mitotic neuroepithelial cells were different from other dna - damaging chemicals reported previously. while the detailed mechanisms of mtx - induced neuroepithelial cell damage in fetal brain remain unclear, the present results provide fundamental information about the fetal brain damage induced by mtx. the present results serve to elucidate the pathogenesis of nervous system anomalies resulting from failed pregnancy termination with mtx or when mothers who are taking mtx for medical reasons become pregnant inadvertently. to our knowledge, this is the first report demonstrating histopathological findings of fetal brain damage induced by mtx. | abstractpregnant rats were treated with 30 mg / kg of methotrexate (mtx) on gestation day 13, and fetal brains were examined histopathologically from 6 to 48 hr after the treatment. in the telencephalon of the control group, there were few pyknotic neuroepithelial cells throughout the experimental period. six hr after mtx treatment, several pyknotic neuroepithelial cells scattered throughout the telencephalic wall. at 1236 hr, pyknotic neuroepithelial cells increased significantly and were diffusely distributed throughout the telencephalic wall. neuroepithelial cells were eliminated and showed sparse cell density at 36 hr in the telencephalon. almost all fetuses died at 48 hr. most of the pyknotic neuroepithelial cells were positively stained by the tunel method and positive for cleaved caspase-3. while mitotic and phospho - histone h3-positive neuroepithelial cells were located along the ventricular layer of telencephalon in the control group, they were rarely observed in the same region at 636 hr in the mtx - treated group. mtx induced few pyknotic changes to neuroepithelial cells in the metencephalon, compared to other parts of brain. the distribution of apoptotic neuroepithelial cells and the time - course changes of the indices of apoptotic and mitotic neuroepithelial cells were different from those of other dna - damaging chemicals reported previously. the difference may reflect the disparity in mechanisms of apoptosis and the inhibition of cell proliferation in neuroepithelial cells induced by mtx. to our knowledge, this is the first report demonstrating histopathological findings of fetal brain damage induced by mtx. |
the dpp design and methods have been described (1). in brief, eligible participants had impaired glucose tolerance and elevated fasting glucose levels, bmi 24 kg / m (> 22 kg / m for asian americans), and were at least age 25 years (1). the lifestyle intervention (12) had two specific goals : to lose at least 7% of body weight and increase moderate intensity activity to at least 150 min / week. lifestyle coaches met with participants individually over the first 24 weeks to review a 16-session core curriculum that focused on diet, physical activity, and behavioral modification strategies. after the first 6 months, lifestyle coaches tailored individual sessions to the needs of each participant and also offered group classes / campaigns three times per year to help improve activity levels and weight loss. dpp participants in the lifestyle intervention arm were recruited for this substudy between 1998 and 2001 ; 18 of 27 dpp centers agreed to participate and obtained approval from their institutional review boards. of the nine centers that did not participate, two were not invited because they had already randomized almost all of their center cohorts ; the other seven (four of which were american indian centers) declined to participate to minimize participant burden because they had elected to participate in other ancillary studies. of the final 293 lifestyle participants who were randomized, written informed consent was obtained from 274 (94%), representing almost 25% of the entire lifestyle intervention cohort. at baseline, the entire lifestyle intervention cohort was aged 50.6 11.3 years, 32% male, and weighed 94.1 20.8 kg. by race ethnicity, the cohort was 54% white, 18.9% black, 16.5% hispanic, 5.6% american indian, and 5.3% asian. as reported previously (4), the subgroup in the current study was representative of the total lifestyle group with regard to age, sex, initial weight, and race - ethnicity, except that american indian centers did not participate (table 1). the mean weight loss and percentage of participants achieving the 7% weight loss goal at 6 months and end of study were also similar in this subgroup as in the entire dpp lifestyle cohort, who lost 6.9 4.5% at 6 months and 4.9 7.4% at study - end (1,3). pretreatment demographic, weight history, psychological and behavioral characteristics participants were asked to complete self - administered questionnaires assessing weight loss history and psychological and behavioral variables at baseline after randomization to the lifestyle intervention group and again at 6 months. ninety - eight percent of the study subjects completed the questionnaires at 6 months, which was the data used in the current analyses. participants reported their weight history, including age of onset of obesity, frequency of weight cycling (change of 20 pounds or more), and number of previous attempts with self - implemented and formal weight loss programs. the dpp lifestyle intervention was based on social cognitive theory, which was the theoretical framework for selection of the psychological and behavioral measures included in this study (12). the psychological and behavioral variables selected have been frequently cited as important predictors of weight loss and have the potential to be modified (411). the validated psychological and behavioral measures used have been previously described (4) and are briefly summarized. the weight efficacy lifestyle questionnaire assesses confidence in resisting overeating in 20 tempting situations (5). the 16-item low - fat diet self - efficacy scale measures confidence in performing healthy dietary behaviors (13). the 5-item exercise self - efficacy scale (6) measures self - confidence to persist with exercise in various situations (14). the 30-item perceived stress questionnaire (15) measures frequency of feeling pressured by life events. participants also answered one question regarding how often they felt deprived while dieting and one question regarding how often they fantasized a lot about favorite foods while dieting (craving) (4). all dpp participants completed beck depression and anxiety inventories (16,17) at baseline and annually. the 5-item emotional eating questionnaire measures frequency of eating due to negative emotions (6). a 5-item version of the binge eating scale measures binge eating severity (18,19). binge eating frequency was assessed using the first three questions from the questionnaire on eating and weight patterns (20). the restraint subscale of dutch eating behavior questionnaire (21) rates frequency of using 10 different restraint behaviors. the 22-item fat - related diet questionnaire (22) measures dietary patterns related to selecting low - fat diets. all dpp participants completed the modifiable activity questionnaire (maq) at baseline and annually and low level physical activity recall (lopar) at baseline, 6 months, and annually. the maq assesses occupational and leisure activity during the past year, whereas lopar provides a general measure of physical activity (occupational, leisure, and home) during the preceding week (23). to minimize the issue of multiple comparisons, weight outcome at end of study short - term weight outcomes at 6 months are presented separately to permit comparison of predictors at the end of the initial intervention period with predictors of long - term weight outcomes at end of study. this comparison distinguishes between predictors of the intervention effect across time to observe which effects are persistent. multivariate models were constructed using hierarchical logistic regression to assess the association of weight outcomes (the dichotomous measure of whether the 7% weight loss goal was met) with each variable block (demographics, weight loss history, cognitive / psychological factors, and behaviors). in the first block of variables, race - ethnicity, age, sex, and baseline weight were forced into the models. stepwise regression was performed using variables from remaining blocks, one block at a time. a value of p = 0.05 was required to enter the model, although the p value could become nonsignificant after adding subsequent variables or after adjusting for cluster effect. stepwise regression was used to select variables from each successive block for entry into the model, retaining any variables from previous blocks. once this final model was constructed, we used generalized estimating equations to adjust models for study site and center cluster effect ; the results of this analysis represent the key findings of the study. for cognitive / psychological and behavioral factors, baseline levels and change scores because baseline levels and change scores are confounded with each other ; the worse the baseline score, the greater the improvement in that factor. each pair of variables was entered into the model, and the significance level of each variable was assessed. if one or both variables of a pair were significantly related to the outcome, those pairs of variables were evaluated in terms of their contribution to the model r ; the pair of variables that made the largest contribution to r was entered into the model. then, remaining variable pairs in that block were evaluated for entry into the model, following the same procedure. results for multivariate analyses of weight outcomes are presented for two models : 1) after all demographic and statistically significant weight history and cognitive / psychological variables had entered the model ; and 2) after all statistically significant behavioral variables had been added to the first model. this analytic strategy allows us to determine whether behavioral variables explained the association of other factors with weight outcomes. we also present the r at the end of each block to assess the contribution of each set of factors to weight outcomes. because results other than those for the multivariate analysis of the end - of - study outcome are supplementary analyses designed to provide a context for interpreting the primary results, we do not adjust for multiple comparisons. all analyses were performed using sas 9.1 software (sas institute, inc., cary, nc). dpp participants in the lifestyle intervention arm were recruited for this substudy between 1998 and 2001 ; 18 of 27 dpp centers agreed to participate and obtained approval from their institutional review boards. of the nine centers that did not participate, two were not invited because they had already randomized almost all of their center cohorts ; the other seven (four of which were american indian centers) declined to participate to minimize participant burden because they had elected to participate in other ancillary studies. of the final 293 lifestyle participants who were randomized, written informed consent was obtained from 274 (94%), representing almost 25% of the entire lifestyle intervention cohort. at baseline, the entire lifestyle intervention cohort was aged 50.6 11.3 years, 32% male, and weighed 94.1 20.8 kg. by race ethnicity, the cohort was 54% white, 18.9% black, 16.5% hispanic, 5.6% american indian, and 5.3% asian. as reported previously (4), the subgroup in the current study was representative of the total lifestyle group with regard to age, sex, initial weight, and race - ethnicity, except that american indian centers did not participate (table 1). the mean weight loss and percentage of participants achieving the 7% weight loss goal at 6 months and end of study were also similar in this subgroup as in the entire dpp lifestyle cohort, who lost 6.9 4.5% at 6 months and 4.9 7.4% at study - end (1,3). participants were asked to complete self - administered questionnaires assessing weight loss history and psychological and behavioral variables at baseline after randomization to the lifestyle intervention group and again at 6 months. ninety - eight percent of the study subjects completed the questionnaires at 6 months, which was the data used in the current analyses. participants reported their weight history, including age of onset of obesity, frequency of weight cycling (change of 20 pounds or more), and number of previous attempts with self - implemented and formal weight loss programs. the dpp lifestyle intervention was based on social cognitive theory, which was the theoretical framework for selection of the psychological and behavioral measures included in this study (12). the psychological and behavioral variables selected have been frequently cited as important predictors of weight loss and have the potential to be modified (411). the validated psychological and behavioral measures used have been previously described (4) and are briefly summarized. the weight efficacy lifestyle questionnaire assesses confidence in resisting overeating in 20 tempting situations (5). the 16-item low - fat diet self - efficacy scale measures confidence in performing healthy dietary behaviors (13). the 5-item exercise self - efficacy scale (6) measures self - confidence to persist with exercise in various situations (14). the 30-item perceived stress questionnaire (15) measures frequency of feeling pressured by life events. participants also answered one question regarding how often they felt deprived while dieting and one question regarding how often they fantasized a lot about favorite foods while dieting (craving) (4). all dpp participants completed beck depression and anxiety inventories (16,17) at baseline and annually. the 5-item emotional eating questionnaire measures frequency of eating due to negative emotions (6). a 5-item version of the binge eating scale measures binge eating severity (18,19). binge eating frequency was assessed using the first three questions from the questionnaire on eating and weight patterns (20). the restraint subscale of dutch eating behavior questionnaire (21) rates frequency of using 10 different restraint behaviors. the 22-item fat - related diet questionnaire (22) measures dietary patterns related to selecting low - fat diets. all dpp participants completed the modifiable activity questionnaire (maq) at baseline and annually and low level physical activity recall (lopar) at baseline, 6 months, and annually. the maq assesses occupational and leisure activity during the past year, whereas lopar provides a general measure of physical activity (occupational, leisure, and home) during the preceding week (23). the weight efficacy lifestyle questionnaire assesses confidence in resisting overeating in 20 tempting situations (5). the 16-item low - fat diet self - efficacy scale measures confidence in performing healthy dietary behaviors (13). the 5-item exercise self - efficacy scale (6) measures self - confidence to persist with exercise in various situations (14). the 30-item perceived stress questionnaire (15) measures frequency of feeling pressured by life events. participants also answered one question regarding how often they felt deprived while dieting and one question regarding how often they fantasized a lot about favorite foods while dieting (craving) (4). all dpp participants completed beck depression and anxiety inventories (16,17) at baseline and annually. the 5-item emotional eating questionnaire measures frequency of eating due to negative emotions (6). a 5-item version of the binge eating scale measures binge eating severity (18,19). binge eating frequency was assessed using the first three questions from the questionnaire on eating and weight patterns (20). the restraint subscale of dutch eating behavior questionnaire (21) rates frequency of using 10 different restraint behaviors. the 22-item fat - related diet questionnaire (22) measures dietary patterns related to selecting low - fat diets. all dpp participants completed the modifiable activity questionnaire (maq) at baseline and annually and low level physical activity recall (lopar) at baseline, 6 months, and annually. the maq assesses occupational and leisure activity during the past year, whereas lopar provides a general measure of physical activity (occupational, leisure, and home) during the preceding week (23). to minimize the issue of multiple comparisons, weight outcome at end of study was defined as the primary outcome. short - term weight outcomes at 6 months are presented separately to permit comparison of predictors at the end of the initial intervention period with predictors of long - term weight outcomes at end of study. this comparison distinguishes between predictors of the intervention effect across time to observe which effects are persistent. multivariate models were constructed using hierarchical logistic regression to assess the association of weight outcomes (the dichotomous measure of whether the 7% weight loss goal was met) with each variable block (demographics, weight loss history, cognitive / psychological factors, and behaviors). in the first block of variables, race - ethnicity, age, sex, and baseline weight were forced into the models. stepwise regression was performed using variables from remaining blocks, one block at a time. a value of p = 0.05 was required to enter the model, although the p value could become nonsignificant after adding subsequent variables or after adjusting for cluster effect. stepwise regression was used to select variables from each successive block for entry into the model, retaining any variables from previous blocks. once this final model was constructed, we used generalized estimating equations to adjust models for study site and center cluster effect ; the results of this analysis represent the key findings of the study. for cognitive / psychological and behavioral factors, variables were assessed as pairs baseline levels and change scores because baseline levels and change scores are confounded with each other ; the worse the baseline score, the greater the improvement in that factor. each pair of variables was entered into the model, and the significance level of each variable was assessed. if one or both variables of a pair were significantly related to the outcome, those pairs of variables were evaluated in terms of their contribution to the model r ; the pair of variables that made the largest contribution to r was entered into the model. then, remaining variable pairs in that block were evaluated for entry into the model, following the same procedure. results for multivariate analyses of weight outcomes are presented for two models : 1) after all demographic and statistically significant weight history and cognitive / psychological variables had entered the model ; and 2) after all statistically significant behavioral variables had been added to the first model. this analytic strategy allows us to determine whether behavioral variables explained the association of other factors with weight outcomes. we also present the r at the end of each block to assess the contribution of each set of factors to weight outcomes. because results other than those for the multivariate analysis of the end - of - study outcome are supplementary analyses designed to provide a context for interpreting the primary results, we do not adjust for multiple comparisons. all analyses were performed using sas 9.1 software (sas institute, inc., cary, nc). baseline characteristics related to weight history, previous weight loss experiences, and scores on psychological and behavioral variables that were potential predictors of weight outcomes are reported in table 1. the 6-month changes in psychological and behavioral variables selected to represent putative predictors for end - of - study long - term weight outcomes are reported in table 2. statistically significant improvements were documented in all measured psychological and behavioral factors, except for perceived stress, after completion of the 6-month core curriculum. changes in psychological and behavioral factors and weight outcomes race (being white compared with black), older age, older age when first overweight, and fewer past self - implemented weight loss attempts were the independent, nonmodifiable, pretreatment predictors of achieving 7% weight loss at end of study. greater exercise self - efficacy, greater dietary restraint, fewer high - fat dietary behaviors, and a more sedentary activity level were independent, modifiable, pretreatment predictors of achieving the 7% weight loss goal at end of study (table 3). at baseline, lower activity levels were correlated with larger increases in activity (pearson correlation coefficient : r = 0.49 ; p < 0.0001). fewer previous formal weight loss programs (nonmodifiable) and less frequent emotional eating (modifiable) were positive predictors of achieving 7% weight loss at 6 months but not at end of study. at baseline, more previous weight loss attempts were associated with younger age when first overweight (r = 0.35, p < 0.0001), more frequent weight cycling (r = 0.52, p < 0.0001), more cravings (r = 0.36, p < 0.0001) and feeling deprived while dieting (r = 0.43, p < 0.0001), and less self - efficacy for weight loss (r = 0.22, p < 0.0001). once behavioral variables were entered into the regression (model 2), they explained the effect of race and age when first overweight on end - of - study weight outcomes. multivariate predictors (odds ratios and ci) of categorical weight loss outcomes (adjusted for cluster) only 6-month improvements in low - fat diet self - efficacy and dietary restraint skills remained important as predictors of achieving 7% weight loss at end of study. once behavioral variables were entered into the regression (model 2), they explained the effect of changes in low - fat diet self - efficacy on weight outcomes. although baseline and early changes in perceived stress, feelings of being deprived while dieting, binge eating severity, weight loss self - efficacy, and exercise self - efficacy were significantly (p < 0.05) associated with ability to achieve weight loss outcomes at 6 months and end of study (data not shown), these variables were not independent predictors of weight outcomes when confounders were eliminated. depression, anxiety, binge eating frequency, and craving were not significantly associated with weight outcomes (data not shown). race (being white compared with black), older age, older age when first overweight, and fewer past self - implemented weight loss attempts were the independent, nonmodifiable, pretreatment predictors of achieving 7% weight loss at end of study. greater exercise self - efficacy, greater dietary restraint, fewer high - fat dietary behaviors, and a more sedentary activity level were independent, modifiable, pretreatment predictors of achieving the 7% weight loss goal at end of study (table 3). at baseline, lower activity levels were correlated with larger increases in activity (pearson correlation coefficient : r = 0.49 ; p < 0.0001). fewer previous formal weight loss programs (nonmodifiable) and less frequent emotional eating (modifiable) were positive predictors of achieving 7% weight loss at 6 months but not at end of study. at baseline, more previous weight loss attempts were associated with younger age when first overweight (r = 0.35, p < 0.0001), more frequent weight cycling (r = 0.52, p < 0.0001), more cravings (r = 0.36, p < 0.0001) and feeling deprived while dieting (r = 0.43, p < 0.0001), and less self - efficacy for weight loss (r = 0.22, p < 0.0001). once behavioral variables were entered into the regression (model 2), they explained the effect of race and age when first overweight on end - of - study weight outcomes. multivariate predictors (odds ratios and ci) of categorical weight loss outcomes (adjusted for cluster) only 6-month improvements in low - fat diet self - efficacy and dietary restraint skills remained important as predictors of achieving 7% weight loss at end of study. once behavioral variables were entered into the regression (model 2), they explained the effect of changes in low - fat diet self - efficacy on weight outcomes. although baseline and early changes in perceived stress, feelings of being deprived while dieting, binge eating severity, weight loss self - efficacy, and exercise self - efficacy were significantly (p < 0.05) associated with ability to achieve weight loss outcomes at 6 months and end of study (data not shown), these variables were not independent predictors of weight outcomes when confounders were eliminated. depression, anxiety, binge eating frequency, and craving were not significantly associated with weight outcomes (data not shown). this study expands on previous research by examining the relative importance of pretreatment demographic, weight history, psychological and behavioral characteristics, and early changes in cognitive and behavioral factors targeted by the intervention in predicting short- and long - term weight outcomes in an ethnically diverse group of men and women. moreover, the multivariate models identify the nonmodifiable demographic and weight history factors and the modifiable psychological and behavioral factors that independently influence success in achieving weight loss. understanding mediators of success (and failure) in lifestyle intervention programs is critical to maximize success. moving beyond previous analyses showing that race, older age, and higher initial body weight are highly significant predictors of weight loss (24), we have now found that several additional baseline patient characteristics were independent and durable predictors of successful weight loss with the dpp lifestyle program at end of study in multivariate models : older age when first overweight, fewer previous self - implemented weight loss attempts, greater exercise self - efficacy, fewer fat - related dietary behaviors, greater dietary restraint, and a more sedentary activity level. in addition, improvements in low - fat diet self - efficacy and dietary restraint skills were independent and durable predictors of end - of - study long - term weight outcomes. consistent with previous findings (9,11), more previous weight loss attempts, whether formal or self - implemented programs, was an important independent predictor of inability to achieve 7% weight loss. those with previous weight loss attempts also reported a profile of being younger when first overweight, more frequent weight cycling, more cravings and feeling deprived while dieting, and less self - efficacy for weight loss. these patterns of repeated negative experiences with previous programs and associated weight cycling may indicate the degree of experience with repeated failure and potentially affect long - term motivation and self - efficacy. it is noteworthy that lower overall activity level at baseline was an independent predictor of greater likelihood of achieving 7% weight loss at end of study. those with lower activity levels at baseline had larger increases in activity, which has been previously reported to predict greater weight loss (2,3). our findings that less frequent emotional eating at baseline was an independent predictor of achieving a 7% weight loss at 6 months and that greater dietary restraint and exercise self - efficacy at baseline are important factors in predicting 2- to 3-year weight outcomes are consistent with those of teixeira. the persistent interference of emotional eating in achieving weight loss goals (6) at the 6- and 12-month follow - up is important to consider, as others have reported that emotional eaters lose less weight with standard behavioral treatment programs and seem to lose more weight with acceptance - based behavioral treatment (2628). this approach uses acceptance and mindfulness processes, along with commitment and behavior change processes, to produce psychological flexibility ; the ability to defuse from difficult thoughts and accept difficult feelings while persisting in value - based action (29). notably, emotional eating at baseline was not an independent predictor of weight loss at end of study, implying that other factors became more important in determining longer - term weight outcomes. the significant improvements in low - fat diet self - efficacy, fat - related dietary behaviors, and dietary restraint after participation in the dpp core curriculum may have helped participants to manage the effect of any emotional eating better by end of study. we expand on previous research by confirming that low - fat diet self - efficacy and dietary restraint skills are important modifiable, persistent predictors of long - term weight outcomes durably after 23 years of follow - up. improvement in low - fat diet self - efficacy during the core curriculum was the most important independent psychological predictor of long - term weight outcomes. for each unit improvement in low - fat diet self - efficacy score, there was almost a threefold greater likelihood of achieving 7% weight loss at end of study. behavioral variables explained the relationship between low - fat diet self - efficacy and end - of - study long - term weight outcomes, which may explain why there have been conflicting reports on the relationship of diet self - efficacy with weight loss outcomes (9,11). improvement in dietary restraint skills was the most important independent behavioral predictor of achieving long - term weight outcomes. for each unit increase in dietary restraint change score, there was a 4.3-fold greater likelihood of achieving 7% weight loss at end of study. the dpp core curriculum resulted in significant improvements in low - fat diet self - efficacy and dietary restraint skills, each of which predicted better weight loss outcomes. the core curriculum focused on learning skills, such as setting small achievable goals, options for reducing fat intake, self - monitoring and balancing of fat gram intake, stimulus control, managing stress / high - risk situations, and problem solving, which could explain these improvements. improvements in dietary restraint skills appear to be key to sustainable lifestyle change and weight loss. when participants learn to respond to lapses in meeting fat gram or activity goals by compensating with improved behaviors at the next opportunity, they are likely to feel more self - efficacious and less likely to engage in dichotomous thinking that is related to weight regain (10). clinically, these results point to the importance of a flexible approach to modifying fat- related dietary behaviors and to flexible dietary restraint as potential determinants of sustainable lifestyle change and weight loss (25). in the dpp, participants learned that there were three ways that they could choose to eat less fat in a given situation : eat smaller portions of the high - fat food, eat the high - fat food less often, or substitute a lower - fat alternative. this flexible approach to self - managing fat intake goals allowed each participant to tailor his or her own means to long - term success with lifestyle change and did not rely on adhering to a specific or rigid diet plan. they practiced the skill of responding to an increase in weight or an overeating episode by eating less than usual on the following day(s) to compensate for and balance out fat and calorie intake so that on average they still met their goals over a given week. the clinical implications of these results may begin to address the issues raised by the study to help improve early evaluation and management of risk factors leading to diabetes (shield) study, which underscored the need for programs that facilitate the development of lifestyle change skills to fill the critical gap that exists between knowing what to do and knowing how to do it for people with type 2 diabetes or at risk to develop it (30). strengths of this study are inclusion of an ethnically diverse group of men and women, very high retention rate, weight loss success levels that were defined a priori, sufficient follow - up time to study weight loss, use of a large number and broad scope of baseline and follow - up assessments, and examination of associations between psychological and behavioral factors targeted by the 6-month core curriculum and subsequent end - of - study weight outcomes. the findings here must be interpreted with several limitations in mind. although we conducted longitudinal analyses, the design is observational because we did not have a control group. we studied only one - quarter of the dpp lifestyle cohort ; however, they resembled the entire cohort with regard to baseline characteristics and weight outcomes achieved, including univariate associations among age, race, and weight loss (3,24). another limitation is that the clinical trial participants we studied may not be representative of all individuals with prediabetes seeking to lose weight. we acknowledge that nonsignificant associations with weight outcomes may be due to the narrow range of some of the factors (depression and anxiety scores were low, with little variance in this subgroup likely due to screening procedures for study entry). we have expanded on previous research by identifying which pretreatment factors and psychological and behavioral factors targeted by the intervention are independent predictors of weight loss, by examining how long these associations persist, and by determining which factors explain the associations of other factors with weight loss. our findings offer important insights into which modifiable and nonmodifiable pretreatment characteristics and which cognitive - behavioral factors targeted by the intervention were associated with successful long - term weight loss in dpp. health care providers who are translating the dpp lifestyle intervention should be aware of participant characteristics that may hamper or enhance success with weight loss and consider prioritizing strategies that improve self - efficacy and dietary restraint skills to maximize sustainable weight outcomes. the findings presented here on the most important independent predictors of short - term and long - term weight outcomes may also help researchers in community settings to streamline and prioritize the number and type of survey measures used as they translate these results to real world practice settings. future research is needed to examine whether taking action on the basis of these results is effective in improving weight loss outcomes, a key component of diabetes prevention and treatment. | objectiveto identify the most important pretreatment characteristics and changes in psychological and behavioral factors that predict weight outcomes in the diabetes prevention program (dpp).research design and methodsapproximately 25% of dpp lifestyle intervention participants (n = 274) completed questionnaires to assess weight history and psychological and behavioral factors at baseline and 6 months after completion of the 16-session core curriculum. the change in variables from baseline to 6 months was assessed with t tests. multivariate models using hierarchical logistic regression assessed the association of weight outcomes at end of study with each demographic, weight loss history, psychological, and behavioral factor.resultsat end of study, 40.5% had achieved the dpp 7% weight loss goal. several baseline measures (older age, race, older age when first overweight, fewer self - implemented weight loss attempts, greater exercise self - efficacy, greater dietary restraint, fewer fat - related dietary behaviors, more sedentary activity level) were independent predictors of successful end - of - study weight loss with the dpp lifestyle program. the dpp core curriculum resulted in significant improvements in many psychological and behavioral targets. changes in low - fat diet self - efficacy and dietary restraint skills predicted better long - term weight loss, and the association of low - fat diet self - efficacy with weight outcomes was explained by dietary behaviors.conclusionshealth care providers who translate the dpp lifestyle intervention should be aware of pretreatment characteristics that may hamper or enhance weight loss, consider prioritizing strategies to improve low - fat diet self - efficacy and dietary restraint skills, and examine whether taking these actions improves weight loss outcomes. |
kidney epithelium in collecting duct contacts with hypotonic fluid which osmolality varies significantly and the cells continually expose to osmotic stress. alterations in luminal osmolality would influence cell volume. to avoid damage and perform their functions, these processes, termed regulatory volume decrease (rvd), occur through activation of specific transporters in the plasma membrane that mediate net fluxes of osmotically active molecules. despite its importance, very little is known about cell - volume regulation inkidney outer medulla collecting duct (omcd) cells. cells recruit special mechanisms to correct acute swelling in hypotonic medium [1, 2 ]. cell swelling has the effect of increasing k efflux, this occurs through channels distinct from those responsible for basal k conductance. obviously, both k and cl concentrations play key roles in governing these volume - regulatory responses. the predominant pathway for rvd is the opening of k channels which are usually large conductance [3, 4 ]. these transporters can be quite nonselective and can include more than one type of channels. in cells containing na channels like collecting duct principal cells, na fluxes can influence the volume - regulatory response but the role of intracellular sodium in rvd is almost unstudied. from another hand, the mechanism by which cells sense changes in cell volume and activates it is important to study the changes of cell volume and [na]i during the process of rvd for better understanding of the mechanisms involved in this reaction. the current study was undertaken to investigate the time course of changes in cell volume and sodium concentration during the volume - regulatory response after acute swelling in hypotonic medium. wistar rats weighting 150200 g (breeding laboratory of experimental animals, institute of cytology and genetics, novosibirsk, russia) were kept in individual cages and received standard diet. before the experiments, the rats were anaesthetized with pentobarbital (50 mg / kg i.p.), the kidneys were extracted and placed in ice - cold pbs, and a suspension of collecting duct fragments was prepared. a superfusion chamber was constructed as an acrylic block mounted on the objective of the upright microscope (water immersion 65 magnification, numerical aperture 1.1). the perfusate flow rate was generally 20 ml / min, which gave complete solution exchange in under 200 milliseconds. fluorescence measurements of cell volume were performed as it was described [6, 7 ]. fragments of outer medullary collecting duct (omcd) on glass plate were loaded with calcein by incubation with calcein - am (molecular probes inc., ore, usa) (5.0 m) for 25 minutes at 23c. the glass plate bearing the fragments of the omcd was positioned on the stage of a microscope (lomo - r8, st. calcein fluorescence was measured continuously with halogen light source, calcein filter set (480 nm excitation, 490 nm dichroic mirror, 535 nm emission), photomultiplier detector has pinhole diaphragm to select the cells of interest at the end of fragment where cells expose apical surface to bath solution, and 14-bit analog - to - digital converter. loading conditions and filter set for fluorescence measurements were the same as for calcein. tissue from the outer medulla zone was cut from the isolated kidneys and squeezed through a needle (0.45 mm i.d.) in the ice - cold calcium - free pbs. the resulting suspension was filtered through nylon mesh, diluted 10 times with eagle mem culture medium and centrifuged (100 g, 10 minutes, 4c). sediment containing the tubules was diluted with culture medium to an appropriate concentration of about 10 fragments per microliter. the solutions used were based on pbs (137 mm nacl, 4.7 mm na2hpo4, 2.7 mm kcl, 1.5 mm kh2hpo4, 0.5 mm mgcl2, 0.05 mm cacl2, 280 mosm / l, ph = 7, 27, 1) and contained 1.0 mg / ml glucose. this solution was chosen so as to be able to degas it without affecting its ph. to create osmotic challenges, bath solutions were changed from normal pbs to pbs diluted with distilled water (1:1). isotonic solutions with decreased [na]o were prepared by substitution part of sodium in pbs by nmdg (n - methyl - d - glucamine, sigma - aldrich, moscow, russia). for measurements of sodium efflux in isotonic medium fluorescent measurements were calibrated plotting the relative values of fluorescence as an x value, against synchronized data of microscopic image analysis, obtained by the method published before. a perfusion chamber, described above, was mounted on the objective of the microscope (water immersion 65 magnification, numerical aperture 1.1, 1.6 magnification photo adapter). the glass plate bearing the fragments of the omcd was positioned on the stage of a microscope for acquisition of transmitted light images by a ccd camera. twelve - bit monochrome images were captured at a rate of 15 frames per second, stored on a personal computer. cell volume was calculated from measurements of calcein fluorescence according this calibration. in vivocalibration of sodium green was accomplished by exposing the fragments to various extracellular [na ] ([na]o) in the presence of 0.1 u/l nystatin. the solutions with various [na]o were prepared by substitution part of sodium in pbs by nmdg. for calibration were used solutions with 138, 14, and 5 mm [na]o. a calibration was performed at the end of each experiment (see figure 2). ion fluxes that appeared during cell swelling seem to be critically involved in the regulation of cell volume. however, the nature of the channels and the mechanism of their activation are poorly understood. it is also not clear to what extent this activation can explain the regulatory volume decrease. the goal of this study is to examine the time course of cell volume changes and concomitant variations in [na]i after hypotonic shock. in hypotonic medium, principal cells in omcd fragments swell very fast, and the volume increases more than 60% (see figures 2 and 3). the evaluation of characteristic time of swelling (1) gives the value 0.65 0.05 seconds (n = 12), which is in a good agreement with our previous results. in our experiments, basolateral and apical surfaces of the cells on the end of microdissected fragment are in contact with medium. fast swelling of the principal cells is the consequence of the high water permeability determined by the water channels that are expressed in basolateral and apical membranes [8, 9 ]. it was shown that abundance of water channels correlates with faster rvd [10, 11 ]. probably the water channels are part of the mechanism, which is activated in cell volume decrease response. after cell volume reaches the peak of swelling, the rvd begins without lag period. the kinetics of this process is slower than swelling, and the characteristic time of volume decreasing to steady - state level (2) is 8.9 1.1 seconds (n = 12) (see figure 4). the published data show that cells in cell lines derived from kidney epithelia, rccd, cos-7, a6 have a significantly slower rate of rvd, and 2 is at least one order more [1113 ]. there is a discrepancy of results obtained on microdissected fragments of omcd and published data obtained on cell lines. most probably the difference is due to the loss of some ion transporters, which could not be activated by hypoosmotic shock in cultured cells. after restoration of the medium osmolality to normotonic, the cells shrink and the volume stabilizes on significantly lower level in comparison with control level (see figure 4). this decrease probably is due to the loss of cellular osmolites during rvd. salt withdrawal procedure could result in responses that depend on the type of cation removed. in this study, we reduced the osmolality by diluting pbs with water or removing nacl from the bath. the procedure causes drastic changes in ionic strength that could interfere with the mechanisms involved in volume recovery. hypotonic medium causes decrease of [na]i and stabilization on the low level (see figures 2 and 5). the established level of [na]i during the hypotonic shock is the result of interaction of three main processes : dilution of osmolites with water that enters the cell, activity of na, k - atpase, and ion flux into the cell through channels activated by swelling, which could be permeable for sodium. comparing the rates of normalized cell volume and [na]i shows that cell volume grows about two times faster than [na]i decreases (0.72 0.05 and 0.30 0.02 s correspondingly, p <.01). this could be explained if during swelling water enters the cell accompanied by sodium. to block cell swelling and water entry, we created the transmembrane sodium gradient with normotonic pbs where 69 mm of na was substituted by nmdg. in the absence of cell swelling with virtually zero water entry, the of [na]i decrease is significantly faster in comparison with the same parameter in hypoosmotic shock (2.6 0.26 and 8.9 0.98 seconds, n = 8 and n = 12, correspondently, p <.01). these results could be accepted as evaluation of sodium efflux through na, k - atpase. the evaluation of sodium content per cell, made by multiplication of synchronized measurements of calcein and sodium green fluorescence, shows that peak of cell volume correlates with the temporary increase of this calculated parameter (see figures 2 and 5). these results indicate that the water comes into cell during the swelling together with sodium. this could happen if swelling activated several types of channels which could pass ions with high conductivity and probably there are some channels which could pass water. probably, this sodium flux aggravates the cell swelling in the hypotonic medium if [na]o is higher than [na]i but during the phase of cell volume decreasing these channels being active could promote the outcome of potassium from the cell. the outcome of the ions, mainly potassium, should be facilitated by partial depolarization which could happened in these cells during swelling. one of the most probable transporters active in normal conditions and which probably takes a part in volume regulating response is epithelial sodium channel (enac). this channel is expressed in apical membranes of principal cells, and there are data that indicate that enac could be involved in rvd [15, 16 ]. to evaluate the putative influence of enac on [na]i during hypoosmotic challenge, we blocked it with amiloride. the addition of amiloride to the concentration of 10 m in bathing solutions caused decrease of control steady - state level of [na]i (see figure 6), but did not influence this parameter in hypotonic medium and after returning the normotonic pbs. amiloride did not influence significantly the relative swelling during hypoosmotic challenge (0.65 0.01 control, 0.77 0.05 amiloride, ns). the ineffectiveness of amiloride means that contribution of enac in sodium entry in our model of hypotonic shock is negligible and can not be detected in our experiments. the results of these experiments indicate that in principal cells hypoosmotic shock causes activation of the transporters, which have high permeability for water and probably provide sodium and potassium transport across cell membrane in dependence of the ion electrochemical gradient which is forming during swelling. | the purpose of this study was to investigate the time course of the volume - regulatory response and intracellular sodium concentration ([na+]i) in the principal cells of rat kidney outer medulla collecting duct (omcd) epithelia during acute swelling in hypotonic medium. hypotonic shock was created by pbs diluted with 50% of water. changes in cell volume were measured with calcein quenching method. intracellular sodium concentration was studied with fluorescence dye sodium green. principal cells of microdissected omcd fragments swelled very fast. the characteristic time of swelling (1) was 0.65 0.05 seconds, and the volume increased more than 60% (92.9 5.6 and 151.3 9.8 m3 control and peak volumes correspondently, p <.01). after cell volume reached the peak of swelling, the rvd began without lag period. the characteristic time of volume decreasing to new steady - state level (2) was 8.9 1.1 seconds. in hypoosmotic medium, cell volume stabilized on higher level in comparison with control (110.3 8.3 m3, p <.01). after restoration of the medium osmolality to normotonic, cell volume stabilized on significantly low level in comparison with control level (71.4 6.1 m3, p <.01). during the hypoosmotic shock, [na+]i decreased from control level in isotonic pbs to the low level in hypoosmotic solution (27.7 1.4 and 5.8 0.23 mm, p <.01). calculation of sodium content per cell has shown the significant sodium entry into the cells, which caused a temporary increase correlated with the peak of cell volume caused by swelling. the conclusion is made that in our model of hypoosmotic shock, swelling activates transporters with high permeability for na+ that provides sodium flux into the cells. |
extraosseous soft - tissue uptake of technetium-99 m methylene diphosphonate (tc-99 m mdp) can occur in number of occasions. cancer patients those undergoing bone scintigraphy will have abnormal uptake, which should be differentiated from benign and metastasis in a planar bone scan. single - photon emission tomography - computed tomography (spect / ct) help in localizing uptake to the muscle hematoma is not reported yet. a 79-year - old man with prostate cancer and coagulation disorder was referred for a bone scan because of left shoulder pain. a bone scan was performed to assess for any skeletal involvement, especially in the shoulder. a total of 20 mci (740 mbq) mdp was injected intravenously and imaging was performed 3 h later using a large field of view, dual headed gamma camera fitted with a low - energy, high - resolution collimator. anterior whole - body images showed intense uptake in the left axilla [figure 1 ]. spect ct of the chest [figures 2 and 3 ] was performed to rule out whether the uptake is in the humerus, but it showed a hematoma in the left brachialis muscle, which he developed due to his coagulation disorder. a hematoma was confirmed on fine - needle aspiration and resolved spontaneously over the next 4 - 5 weeks. follow - up bone scan carried out after 6 months showed resolution of uptake in the left axilla [figure 4 ]. technetium-99 m methylene diphosphonate whole body bone scan showing increased uptake noted in the left axilla (arrow) and no other site of metastasis coronal computed tomography images showing hematoma in the left brachialis muscle fused coronal single - photon emission tomography - computed tomography images localises tracer uptake to the hematoma in the left brachialis muscle follow - up technetium-99 m methylene diphosphonate whole body scan shows resolution of uptake in the left axilla after 6 months localization of bone imaging agents like tc-99 m mdp and tc-99 m hydroxymethylene diphosphonate has previously been described in abdominal and pelvic hematomas. extraosseous soft - tissue concentration of these radiotracers in the chest has also been reported in soft - tissue hematoma, a benign intrapulmonary calcified focus, squamous cell carcinoma of the pleura and pulmonary alveolar microlithiasis. proposed mechanism leading to increased extraosseous tc-99 m mdp uptake include extracellular fluid expansion, enhanced regional vascularity and permeability, diminished lymphatic or venous removal from the affected area, and elevated tissue calcium concentration. tissue damage from inflammation, infection or physical trauma results in localized hyperemia, edema or calcium (and hemosiderin) deposition based on their pathophysiologic characteristics. because of advent of spect ct, it is possible to localize exact site of tracer uptake and characterize the lesion to guide further management. spect / ct localizing the uptake to the hematoma in the left brachialis muscle help in ruling out metastasis. | we report a case of 79-year - old male with prostate cancer and coagulation disorder presented with left shoulder pain. he underwent bone scintigraphy to rule out metastasis, which showed intense foci of tracer activity in the left axilla. hybrid single - photon emission tomography - computed tomography (spect / ct) of the shoulder region localized tracer uptake to the left brachialis muscle hematoma. |
congenital pouch colon (cpc) is a relatively rare form of ano - rectal malformation (arm) in which part or whole of colon is converted into a pouch - like dilatation that ends blindly and communicates with the urogenital tract by means of a fistula[1, 2 ]. pathogenesis and embryology of cpc are not well understood, but dietary, environmental and familial factors have been hypothesized to contribute to the pathology. high number of cases with increased fetal death rate (10 - 33%) of type iv cpc has been reported. renal agenesis, i.e., absence of one or both of the kidneys, is often incidentally diagnosed by abdominal ultrasound or computed tomography (ct) scans secondary to another condition or during routine investigation for other diseases like arm. absence of one or both testes in a phenotypic male with normal karyotype is defined as congenital anorchism. the present study was aimed at reporting a rare case of type iv congenital pouch colon with associated anomaly of unilateral anorchism along with unilateral renal agenesis. a 1-day old male baby, born of non - consanguineous marriage and from a low economic family, was admitted with history of gradual distention of abdomen, bilious vomiting and failure of passage of meconium with meconuria and pneumaturia since birth. the baby was delivered normally at the 39 week of gestation (bw 2.25 kg ; apgar score 5 - 7) by a young mother (20 yrs ; primigravida, primipara) in a peripheral health center. the baby had normal body temperature, high respiratory rates and racing pulse with depressed neonatal reflexes. physical examination revealed a flat bottom with no anal opening and normal genitalia with empty right scrotal sac. abdominal examination revealed distended flanks with bulged umbilicus, tympanic on percussion, and sluggish peristaltic sound. routine blood count revealed normal hemogram and total cell counts with normal renal function test. the invertogram in ap and lateral view showed a large globular loop of bowel with single air fluid level occupying more than 50% of the total abdominal width. abdominal and perineal sonogram showed absence of left kidney and right testis with air in urinary bladder. analysis of peripheral blood leukocytes cultured for 72 hrs in presence of mitogen, revealed normal male child with 46,xy karyotype (fig. karyotype of the subject studied by gtg - banding analysis anorectal malformation (arm) with recto - vesical or recto - prostatic urethral fistula. exploratory laparotomy, done on the 2-day old baby, confirmed type iv cpc communicating with bladder neck. associated malrotation of the midgut without volvulus was detected and left renal fossa was empty. pouch was excised and low descending colostomy performed, malrotation was corrected (ladd 's procedure) and appendectomy done. neither testicular tissue nor structures of spermatic cord was detected in the right posterior abdominal wall. anorectal malformation (arm) with recto - vesical or recto - prostatic urethral fistula. exploratory laparotomy, done on the 2-day old baby, confirmed type iv cpc communicating with bladder neck. associated malrotation of the midgut without volvulus was detected and left renal fossa was empty. pouch was excised and low descending colostomy performed, malrotation was corrected (ladd 's procedure) and appendectomy done. neither testicular tissue nor structures of spermatic cord was detected in the right posterior abdominal wall. congenital pouch colon is an uncommon type of arm which has been studied in detail in recent times. higher numbers of cpc cases were reported from india as compared to other countries[2, 57 ]. types i, ii and iii cpc are usually diagnosed during preoperative evaluation period in arms and there are typically large air fluid levels in plain abdominal x - ray[2, 810 ]. however, type iv cpc does not have a characteristic appearance on plain x - ray and was diagnosed during surgery for colostomy[3, 10 ]. a few earlier reports have shown the presence of abnormal histology in the excised colon. however, in the present case, excised pouch histopathology revealed presence of normal muscle tissue as well as few ganglion cells. incidence of associated cardiac, vertebral and genitourinary anomalies is very high in cpc and therefore, evaluation of these systems is mandatory in all cases of arm, more so in suspected cases of cpc[2, 12 ]. increased risk of fetal renal agenesis or dysgenesis was observed in mothers addicted to alcohol, smoking etc[14, 15 ]. lower maternal weight gain during pregnancy has also been reported. in the present case, while no association with addiction was observed, a history of suboptimal maternal weight gain during antenatal period was noticed. diagnosis of congenital anorchia is suspected in a patient exhibiting male external genitalia, 46,xy karyotype together with absence of testis in scrotal sac. surgical exploration performed to repair the cpc in the present case failed to find any testicular elements or mullerian structures confirming diagnosis of unilateral congenital anorchia on the right side. though the incidence of cpc is increasing, to the best of our knowledge, presence of type iv cpc with renal agenesis and monorchism in a male boy with normal karyotype is an extremely rare presentation and is described here to bring up to date of the medical fraternity. | backgroundcongenital pouch colon, also known as congenital short colon or pouch colon syndrome, is a rare condition that occurs in association with anorectal malformations ; colon is either partially or completely replaced by pouch - like dilatation and communicates with the urogenital tract by means of a fistula. this anomaly is exclusively seen in northern parts of india with only a few cases reported from elsewhere.case presentationa 1-day old neonate was presented with abdominal distension due to lack of passage of meconium. clinical and radiological investigations revealed ano - rectal malformation. incidental findings were left sided renal agenesis and right sided anorchia. laparotomy revealed congenital pouch colon which was dealt accordingly. the baby is now healthy and awaiting further reconstructive surgery.conclusionalthough urogenital anomalies are not uncommon with congenital pouch colon, the finding of renal agenesis with unilateral anorchia is quite rare. |
as a service to our authors and readers, this journal provides supporting information supplied by the authors. such materials are peer reviewed and may be reorganized for online delivery, but are not copyedited or typeset. technical support issues arising from supporting information (other than missing files) should be addressed to the authors. | abstractthree types of chlorideconducting membranes based on polyvinyl chloride, commercial gelatin, and polyvinyldifluoridehexafluoropolymer are introduced in this report. the polymers are mixed with chloridecontaining salts, such as tetrabutylammonium chloride, and cast to form membranes. we studied the structural properties, thermal stability, and electrochemical response of the membranes to understand chloride migration and transport. finally, the membranes are tested in a prototype solidstate chlorideion battery setup. the feasibility of the membranes for their potential use in anion batteries is discussed. |
type 2 diabetes (t2d) is a chronic disease with an increasing incidence worldwide. it remains the leading cause of nontraumatic lower - extremity amputation [1, 2 ]. the annual incidence of diabetic foot ulcers varies between 1.9% and 2.2%, with a prevalence of 7.5% to 12%. the risk of amputation is associated with the presence of sensory peripheral neuropathy, peripheral vascular disease, charcot joint, ulceration, and the presence of infection. fifty - six percent of diabetic foot ulcers will develop infection and 20% of them will end up in a lower - extremity amputation. a number of chemical mediators are involved in the tissue repair process such as cytokines and growth factors. within this complex process, the transforming growth factor beta (tgf-) plays a key role regulating the development, differentiation, growth, and apoptosis of most cells. tgf- in turn activates monocytes by increasing gene expression of proinflammatory cytokines such as interleukin 1 (il-1) and tumor necrosis factor alpha (tnf-).. the natural substrates of mmps include major proteins of the extracellular matrix (ecm) such as collagen, elastin, and proteoglycans. therapies including growth factors, bioengineered skin, tissue grafts, hyperbaric oxygen, negative pressure wound therapy, and other novel approaches to stimulating wound healing have demonstrated healing rates of around 40% in noninfected diabetic foot ulcers. 1-phenyl-5-methyl-2-[1h]-pyridone (pirfenidone) is a synthetic chemical molecule that acts as a selective cytokine regulator, providing its action by anti - inflammatory and specific antifibrotic properties. pirfenidone acts as a modulator of tnf-, tgf-, fibroblast growth factor (fgf), platelet derived growth factor (pdgf), and vascular endothelial growth factor (vegf), which are cytokines involved in the inflammatory - fibrotic process. this results in a reduced expression of tgf- by a direct inhibition of furin, a proprotein convertase. pirfenidone has shown utility in the treatment of patients with wounds, burns, and scars without serious adverse events. topical pirfenidone may improve healing of diabetic foot ulcers and could be an option as an adjuvant therapy. therefore, the aim of this study is to evaluate the effect of pirfenidone added to conventional treatment on noninfected diabetic foot ulcers assessing the rate of complete wound closure and the change in ulcer size. the protocol was approved by the comite de etica en investigacion del instituto nacional de ciencias medicas y nutricion. subjects who attended the diabetic foot clinic at the instituto nacional de ciencias medicas y nutricion salvador zubiran in mexico city were recruited. grade 0 is a patient at risk or with a postulcerative diabetic foot without ulcer. grade 1 is a full - thickness ulcer not involving tendon, capsule, or bone. grade 2 is an ulcer that involves tendon or capsule, without abscess or osteomyelitis. the inclusion criteria were the following : men and women with t2d, being older than 18 years of age, being with a diabetic foot ulcer grade wagner 1 or 2, ulcer size 1 cm, and being with duration of at least 8 weeks. the exclusion criteria included infected ulcers, presence of an ankle brachial index (abi) < 0.4, ulcers due to a different cause such as venous insufficiency, inability to attend to the weekly evaluations, use of systemic or topical diabetic foot ulcer treatments, immunosuppressant treatment, connective tissue diseases, pregnancy, and lactation. the elimination criteria were an attendance to < 75% of the evaluations, allergy to pirfenidone, development of ulcer infection, and occurrence of other serious diseases requiring hospitalization. conventional treatment consisted of weekly ulcer cleansing with saline, debridement using a surgical blade, maintenance of a moist environment, and covering with sterile gauzes. in addition, patients were instructed to perform daily cleansing with saline - moistened gauzes and offloading the affected extremity. group 1 received conventional treatment in combination with topical pirfenidone for the first eight weeks and at the end of this period they were switched to conventional treatment only for the remaining 8 weeks. group 2 received conventional treatment only for the first 8 weeks and at the end of this period they were switched to conventional treatment in combination with topical pirfenidone for another 8 weeks. the body mass index was calculated as the weight in kilograms divided by the squared height in meters. in addition, all ulcers were categorized using the classification of the international working group on the diabetic foot (iwgdf), abbreviated with the acronym pedis, which stands for perfusion, extent (size), depth (tissue loss), infection, and sensation (neuropathy). to evaluate the presence of lower - extremity vascular disease, the abi was calculated and classified as follows : normal from 0.9 to 1.3, peripheral artery disease (pad) 0.9, and severe pad < 0.4. after debridement, the length and width of the ulcer were measured with a standard ruler, the maximal size was calculated in cm, and a photograph was taken. biochemical parameters included complete blood cell count, erythrocyte sedimentation rate, c - reactive protein, glucose, creatinine, uric acid, total cholesterol, high density lipoprotein- (hdl-) cholesterol, low density lipoprotein- (ldl-) cholesterol, triglycerides, glycosylated hemoglobin, aspartate aminotransferase, alanine aminotransferase, and 24 h albuminuria. patients were reinforced about offloading their affected extremities, and adherence to treatment was assessed by requesting the empty tubes. finally, possible harm including burning, redness, itching, and hypergranulation was monitored by physical examination and questioning. complete wound closure was defined as full epithelialization of the ulcer with absence of drainage. percentage of closure was estimated by calculating the ulcer size in cm at visit 1 and at visit 8. the size at the end of each period was subtracted to the initial ulcer size (week 1 and week 8) and the change was estimated and expressed as a percentage. a sample size of 50 ulcers was calculated estimating a 25% change in ulcer size with pirfenidone treatment, with an alpha error of 5% (z = 1.96) and a beta error of 10% (power of 90%). participants were randomly assigned in blocks to one of the two groups with the use of sealed envelopes using a random sequence numbers generator. normal distributed variables were described as means and standard deviation. in the case of nonnormal distributed variables, differences between groups were evaluated with independent student 's t - tests or u mann - whitney tests, as appropriate. forty patients with 55 ulcers were randomized to pirfenidone (n = 20) or conventional (n = 20) treatment. participants ' characteristics were not different between the groups except for the body mass index, which was slightly higher in the group assigned to receive conventional treatment first (29 vs 25.3 kg / m, p = 0.048). we do not consider that this slight difference in the body mass index may have affected the results. however, according to the abi classification, in group 1 there were more patients with pad and in group 2 there were more patients with noncompressible arteries. we excluded patients with severe pad and do not consider that these differences affected the results., 52.4% of the ulcers assigned to group 1 healed compared with 14.3% of group 2 (p = 0.025). after the crossover, the remaining 22 ulcers switched treatments and 30.8% of ulcers that received pirfenidone (during 8 to 16 weeks) healed compared with none in the conventional only group (p = 0.081)., the median percentage reduction in ulcer size was 100% [73100 ] in group 1 compared with a 57.5% [28.974 ] reduction in group 2, p = 0.011 (figure 2(a)). at 16 weeks, the median percentage reduction was 93% [42.7100 ] in the pirfenidone group compared with a 21.8% [1677.5 ] size reduction in the conventional group, p = 0.050 (figure 2(b)). figure 3(a) shows an ulcer assigned to group 1 and figure 3(b) an ulcer assigned to group 2. no serious adverse events were observed during the course of the study in any treatment group. one patient in group 2 developed hypergranulation during pirfenidone treatment which resolved spontaneously. in group 2, a patient was diagnosed with thyroid carcinoma during conventional treatment and was eliminated from the study (figure 1 and table 4). during the first 8 weeks, 10 ulcers (8 patients) developed osteomyelitis. four ulcers (4 patients) were in group 1 and 6 ulcers (4 patients) in group 2 ; one of them had a supracondylar amputation. this study demonstrates that pirfenidone added to conventional treatment is superior to conventional treatment alone. treatment with pirfenidone decreased the size and increased the rate of complete healing of chronic noninfected diabetic foot ulcers. a greater number of ulcers achieved complete wound closure when receiving pirfenidone treatment. pirfenidone enhanced successful healing in addition to the standard ulcer care. a key component of the intervention was the weekly ulcer debridement and offloading the affected foot. debridement enables removal of devitalized and necrotic tissue and promotes the beginning of the healing process. chronic diabetic foot ulcers represent a therapeutic challenge and their treatment involves debridement, frequent assessment, identification and treatment of infection, revascularization if indicated, and satisfactory offloading the foot. analysis of the evidence regarding the effectiveness of interventions to enhance healing of chronic diabetic foot ulcers is difficult. there is not strong evidence to choose a specific dressing or topical medication in preference to another. products designed to improve wound biochemistry and cell biology to promote wound healing demonstrate an ulcer healing rate between 40% and 80%. however, the evidence to support their use is not robust and further rigorously designed blinded trials are needed. a crossover design was chosen because otherwise it would not have been possible to control variables that could influence significantly the healing process such as ulcer size, ulcer depth, glycemic control, physical activity, and weight. in summary, this study demonstrates that the addition of topical pirfenidone to conventional treatment significantly improves the healing of chronic diabetic noninfected foot ulcers. | only 30 percent of chronic diabetic foot ulcers heal after 20 weeks of standard treatment. pirfenidone is a drug with biological, anti - inflammatory, and antifibrotic effects. the aim of this study was to evaluate the effect of topical pirfenidone added to conventional treatment in noninfected chronic diabetic foot ulcers. this was a randomized crossover study. group 1 received topical pirfenidone plus conventional treatment for 8 weeks ; after this period, they were switched to receive conventional treatment only for 8 more weeks. in group 2, the order of the treatments was the opposite. the end points were complete ulcer healing and size reduction. final data were obtained from 35 ulcers in 24 patients. fifty - two percent of ulcers treated with pirfenidone healed before 8 weeks versus 14.3% treated with conventional treatment only (p = 0.025). between 8 and 16 weeks, 30.8% ulcers that received pirfenidone healed versus 0% with conventional treatment (p = 0.081). by week 8, the reduction in ulcer size was 100% [73100 ] with pirfenidone versus 57.5% with conventional treatment [28.974 ] (p = 0.011). by week 16, the reduction was 93% [42.7100 ] with pirfenidone and 21.8% [877.5 ] with conventional treatment (p = 0.050). the addition of topical pirfenidone to conventional treatment significantly improves the healing of chronic diabetic noninfected foot ulcers. |
when robert merton wrote about the sociology of science in the 1970s, the central task at hand was explaining how a set of social norms and practices yielded knowledge what was different about science compared to the humanities and the professions (merton, 1973). this paper addresses a related, but somewhat different aspect of science how reliable knowledge can be turned into social benefit, using genomics as a case in point. the value of a science commons a pool of knowledge that is widely available at little or no cost is the central focus. the zone of intersection between reliable knowledge and useful knowledge falls squarely into what the late donald stokes described as pasteur s quadrant, (1997) where research results both contribute insight into the workings of nature and at the same time find practical application. the value of having knowledge widely and freely (or almost freely) available is particularly salient in pasteur s quadrant. knowledge is more likely to advance, and to be applied, if it is available at little or no expense to a broad array of scientists and innovators. these features of network efficiency are well known in software and other fields characterized by widely distributed cumulative innovation under mantras such as to many eyes, every bug is shallow, and theoretically described by benckler (2002). i shamelessly steal the term science commons from the new organization of that name that has spun out of the creative commons movement. science commons is dedicated to making it easier for scientists, universities, and industries to use literature, data, and other scientific intellectual property and to share their knowledge with others. science commons works within current copyright and patent law to promote legal and technical mechanisms that remove barriers to sharing (while i endorse their mission, they may not endorse my agenda or analysis. i have no direct connection to the organization, and do not speak for it.) the main approach in what follows is historical, using background on how the science commons functioned in genomics to illustrate the role of a commons in general. genomics will be the main topic, occasionally straying into collateral fields of biomedical research such as bioinformatics or molecular and cellular biology when they provide better examples. there is some fuzziness around the edges of what constitutes a science commons, and how it relates to the public domain. there can be variants of many terms marching under the banner of open science or public research. open access, for example, can mean free access to view information, but not necessarily freedom to use it in all ways without restriction. the information in patents, for example, is openly available, but users may need to get permission or pay fees to use a patented invention (including some basic methods used in science). to some, open science means no one can fence it in. access to information, say through viral licensing or copyleft, may be conditioned on agreeing not to restrict subsequent users. information may also simply be put into the public domain for any and all subsequent uses, by deposit at a freely available public database, for example. i focus on this last meaning, with information available to all at low or no cost. but again, this may not mean completely unfettered use, as sequence information in genbank may be covered by patent claims. jensen and murray, for example, noted that more than 4,000 human dna sequences are subject to claims in us patents (jensen & murray, 2005). sometimes there are restrictions on use of information and materials in the science commons, but those restrictions must also impose low or no costs to subsequent users, or else that information has left the science commons (e.g., through subsequent patenting, copyright, or database protection). there is no bright line dividing the science commons from proprietary r&d, and indeed in the case of some sequences, materials, and methods in molecular biology, the expense associated with use of genomic information may depend more on licensing terms and practices than what is or is not patented and one person s reasonable terms may be out of reach for some users. some of the data shown below, for example, are drawn from a free public database, the dna patent database at georgetown university (dna patent database, 2005). that database is, in turn, drawn from the freely available us patent and trademark database (uspto database, 2005). but the search engine and database used to generate the dna patent database are derived from an intermediate subscription database that is not widely or freely available, but available to subscribers at several thousand dollars a year, through the delphion patent database (delphion database, 2005). this is a major tool for our research, and for us the subscription cost is balanced by ease of use and reliability. we pay for delphion for its special features (such as corporate trees that track ownership of patents) and because its search results have proven more reliable than several alternatives, including the uspto s own computers and software available in northern virginia. we are happy to pay for a proprietary database because it helps us do our work and the price is reasonable, within reach of our nonprofit institution. delphion does not restrict our use, and does not prevent our creating a free public database. i raise the example of a pay - for - use database sandwiched between two public resource databases to hint that the story will get complicated, and to signal early that this is not a diatribe against for - profit intrusions into research. this is important because many of the points that come out later will seem unfriendly to purveyors of databases. those objections are not deep - seated rejections of capitalism in science, but rather pragmatic judgments about adverse effects of particular policies. innovation depends on how much information is produced as well as how widely and easily it is shared. funding of r&d is a major determinant of how much research is conducted, and thereby how much information is created. some reasons are obvious, but some are not so obvious, and some are even counterintuitive.one final conceptual point will be helpful to flag before proceeding into the narrative. there is extensive overlap between academic health research and the science commons in molecular biology. academic science is important in many fields, not just the life sciences. in all lines of scientific and technical work, universities and nonprofit research institutions and government laboratories (academic research institutions) play key roles. everyone is trained in academe, not just academic scientists, but also those working in industry. and within industry, academic training is not just for those doing r&d, but also managers and professionals. academe is also one place where the norms of mertonian science have real traction, where the norms of openness, community, mutual criticism, and fair allocation of credit are supposed to be respected, at least as an ideal. in some circumstances, however, academic science is done under strictures of secrecy, or results are made available only at great cost or encumbered by restrictions on use. great science goes on in industry, including or even particularly in the life sciences, but no one expects the norms of openness to prevail in industrial r&d, even if in some circumstances at some times scientists in companies publish in the open literature, present their findings at open scientific conferences, make materials freely available, and contribute data to public databases. when industrial r&d is widely shared openly, results flowing from industrial r&d can become part of the science commons, and there are several instances of this in the stories to follow. in sum, most academic research contributes to the science commons, and some industrial r&d also does so. it remains true nonetheless, that most of the science commons at least in the life sciences is based on academic research funded by government and nonprofit organizations, and most academic research probably enlarges the science commons, although to my knowledge no one has quantitatively assessed what fraction of the research funded by government and nonprofit organizations remains in the science commons. policies put in place over the past three decades have raised concerns about how big the science commons will be, and in particular, whether and to what degree government and nonprofit funders and academic research institutions will maintain it. richard nelson of columbia university, in particular, has expressed concerns about intrusions on open science, based on his decades of studying the innovation process as an economist (nelson, 2006). genomics became the grounds for a vigorous, sometimes even vicious, fight over what should or should not be in the public domain, and under what conditions. how much genomic data should be in the science commons has been a matter of explicit policy - making in government, nonprofits, academic institutions, and private firms since 1992 or 1993, when the commercial promise of genomics became apparent, and private funding for genomics in for - profit companies began to accelerate. several features of genomics make it an interesting field to study as an instance of the science commons. it is clearly derived from a scientific project that was initially conceived as a public works project to construct maps and derive a reference sequence of the human genome and other genomes. the original intent of the human genome project was to produce information and tools to make that information useful and valuable. some commercial uses were foreseen from the beginning, but the main focus was on producing public data of permanent scientific value. it caught a wave of enthusiasm for the new biotechnology that had become both scientifically hot and also a darling on wall street. cetus was founded in 1971 and turned to recombinant dna techniques soon after they were discovered (stanley cohen, co - inventor of recombinant dna, joined the cetus board in 1975). those companies went public with high - profile stock offerings in 1980 and 1981, raising sums that startled the markets (smith hughes, 2001). the origins of the human genome project were not in commercial biotechnology, however, but in publicly funded science. the ideas behind the human genome project began to appear in 1985, while the embers of biotechnology were still warm but too distant from this particular part of molecular biology to catch fire. walter gilbert tried to start genome corp. in 1987, for example, and had to resign from a national research council study as a consequence.) scientists conceived a grand idea and focused on the scientific value of having a reference human genomic sequence (cook - deegan, 1994).1 commercial interest lagged for several years, until in 1991 a conflict over patenting short sequence tags derived from human genes blew up into a major controversy, and created commercial interest in human genomic sequencing. j. craig venter, a scientist in nih s intramural (government laboratory) research program, started using automated dna sequencing machines rapidly to identify sequences unique to human genes. a genentech lawyer, max hensley, contacted the nih technology licensing lawyer, reid adler, who in turn contacted venter about filing a patent application on his method and the resulting dna sequences. the method was eventually given over to the public domain through a statutory registration of invention, but the patent application for the sequences themselves continued through the patent examination process. that 1991 patent application generated tremendous controversy until 1994, when nih director harold varmus decided to abandon the patents, following the advice of patent scholars rebecca eisenberg and robert merges (1995). the controversy at nih paradoxically induced interest in commercial biotechnology circles. in 1991 and 1992, noise over darth venter s turn to the dark side (by patenting dna sequences from gene fragments) attracted the attention of scientist randall scott at incyte in california, and incyte began to focus on dna sequencing of human genes. through 1994, several other companies including human genome sciences, mercator genetics, genset, myriad genetics, millennium pharmaceuticals, genome therapeutics (renamed from collaborate research), hyseq, and sequenom were formed around the idea of mapping and/or the sequencing the human genome, or turned from other pursuits to those ends. one company illustrates the public - science origins of private genomics in particular : human genome sciences. wallace steinberg, a former johnson & johnson executive who had started several biotech companies after leaving j&j, decided to meet venter, having read about him amidst the patenting controversy. he talked venter into leaving nih to form a nonprofit research unit, eventually named the institute for genomic research (tigr), by promising venter $ 70 million ($ 85 million by the time the deal was done) (cook - deegan, 1994). that was enough to build a larger sequencing and sequence - analysis facility than existed anywhere else at the time. (hgsi), was formed as a for - profit corporation that would own the patent rights to tigr s results as well as pursuing its own research leads. there were also plans to form additional companies, industrial genome sciences and plant genome sciences, to exploit different opportunities deriving from high - throughput sequencing and other genomic technologies. haseltine also had his roots in academic science, most notably from his work on hiv / aids at harvard. the first boomlet in genomics startups in the early 1990s paralleled a significant increase in pharmaceutical r&d among established pharma and biotech firms that started in the early 1980s. a pharmaceutical r&d arms race of sorts began in the early 1980s, and during that decade, firms delved ever more deeply into molecular and cellular biology to bolster their absorptive capacity for drug discovery (cockburn & henderson, 1998 ; fabrizio, 2005, unpublished data), recognizing the importance of rapid and effective use of public domain science to their business plans. indeed, their ability to tap public science was one of the indicators of firms success in pharmaceuticals (fabrizio, 2004). by historical happenstance, this birth of genomics out of publicly funded science took place as patent rights were being expanded and strengthened, by a combination of changes in legislation, in court decisions, and in patent offices. in academia, the major change was the bayh - dole act of 1980, which gave grantees and contractors rights and indeed a mandate to seek patents on federally funded research results. mowery and his coauthors review this history and some of its consequences in their book, ivory tower and industrial innovation, which combines economic empiricism, historical research, and policy analysis (mowery., 2004). foreseeable to some immediately, to others a few years after its launch took root as a field in american academe under the new bayh - dole regime. academic institutions began to patent much more frequently after 1980, and genomics is one of the areas where this effect was pronounced. moreover, patent rights were being expanded and strengthened in many areas of american law, including biotechnology. the court of appeals for the federal circuit (cafc) was formed in 1982. it was designed to handle appeals of certain cases, including appeals of federal district court decisions about patent litigation. it and the patent office expanded the kinds of inventions that could be patented (including software and business methods, for example) and tended to strengthen the hand of patent - holders relative to those contesting patent rights (jaffe & lerner, 2005). in the hands of the cafc, more territory could be enclosed and patent fences generally got higher. these developments had a particularly strong impact on areas of rapid innovation, including both wet lab biotechnology and bioinformatics, fields directly relevant to genomics. three other factors are not related to changes in policy, but nonetheless make genomics a useful field to study for this policy history : (1) the story was compressed into a decade, so its narrative is shorter and crisper, (2) there was intense media coverage, producing an ample public record of events, and (3) the patentable inventions arising from genomics can be tracked because it is possible to identify relevant patents. patents resulting from genomics r&d almost always make patent claims that use terms distinctive to dna and rna, which can be used to create a searchable patent database mapping to genomics research.2 a science commons can supply information needed to achieve social benefit that for - profit markets in goods and services may fail to achieve. moreover, even in markets well served by the profit motive, a science commons can in some circumstances improve efficiency, when many disparate firms can draw on a common pool of knowledge and data, rather than having to construct the same information firm - by - firm at substantial cost because of duplication. one theoretical rationale for this effect has been set forth by benkler (2002). the cases arising in genomics suggest that network theory may have some practical applications in the real world of science and its application. i will illustrate three social goals that can benefit from a robust scientific commons in genomics : advancing science, improving public health, and creating a shared foundation for productively diverse forms of industrial r&d and commercialization. but first, some historical background. the beginning of the human genome project was marked by conflict between scientists who thought it was a poor use of resources versus those who thought it was a useful and efficient way to spend public research dollars. by broadening the project to include maps, tools, and organisms in addition to the human a 1988 report of the national research council reported that consensus (national research council, 1998). that did not eliminate all conflict, however, because the question of which federal agency should play the larger role remained unresolved, and both the national institutes of health and the department of energy assumed active roles, in a roughly 21 ratio of funding. and even as the rival agencies in the us settled into a generally amicable cooperative framework, other nations began to engage in genomics r&d. the 1991 controversy over gene - tagging sequences erupted while the genome project was getting underway. as that controversy died down, an even more public conflict over sequencing the entire genome exploded in 1998, pitting a private company against the public sector genome project. the heads of the two private organizations, venter (tigr) and haseltine (hgsi), never sang close harmony, despite their supposed corporate matrimony. as tigr moved away from human gene sequencing and into microbial sequencing, including proof of principle that whole - genome shotgun sequencing could work, the noise from the tigr - hgsi conflict got downright cacophonous. tigr s scientific interests hardly coincided with what hgsi would want from its r&d partner, and two alpha - males confined in close corporate space found themselves in frequent conflict. in 1997, tigr and hgsi severed their ties, with tigr foregoing rights to future payments and hgsi foregoing rights to future tigr discoveries (tigr, 1997). venter became a free agent, heading up a free - standing nonprofit research institute, until michael hunkapiller of applied biosystems approached him with another big idea (shreeve, 2004). in discussions with its parent company (then perkin - elmer cetus, which became applera), applied biosystems had begun to think seriously about sequencing the human genome with private funds. it would be a high - profile use of a promising new dna sequencing instrument that was much faster and more scalable than existing sequencers. the question was whether the methods tigr had used on smaller genomes, such as bacteria, could work on the human genome, and produce a final sequence faster than the public genome project. if so, a company could charge both for access to the data, and for access to informatic tools to mine the data. in order to charge users, the company would need a truly impressive bioinformatic capacity, and great tools for analyzing sequence data. if a private company decided to sequence the genome, it might even kick up a market for sequencing instruments, including applied biosystems machines, among the publicly funded laboratories doing dna sequencing, who would buy the same machines to compete with the new genomic sequencing company. in may 1998, craig venter became the head of a company, later named celera genomics, which would carry out the sequencing and pull together the computing infrastructure to assemble it into a reference sequence, and then begin to interpret the sequence information. celera s 1998 establishment inaugurated another boom in genomics startups, this one entailing many more companies and much more money than the 19921994 boomlet. the initial kernel of the media snowball was an exclusive to nicholas wade of the new york times in may 1998 (wade, 1998). thus began a privately financed scientific effort at celera running into the hundreds of millions of dollars that competed head to head with the publicly financed human genome project. the drama played out over 3 years and became the biggest story in science, and one of the most visible general interest stories of its period. the story is often told as a race, competition between venter at celera and the public human genome project whose most conspicuous spokesmen were francis collins in the united states and sir john sulston in the united kingdom. collins was director of the national human genome research institute at nih, and sulston directed the sanger institute affiliated with the university of cambridge and funded mainly by the wellcome trust of london (with additional funding from the uk medical research council). the usual narrative strategy was to use the metaphor of a race, but in fact there were not just two human genome projects running in parallel, there were many. a consortium of laboratories funded by government agencies and nonprofit organizations in north america, europe, and japan constituted the sulston emerged as the champion of that faction, emphasizing open science, rapid sharing of data and materials, and a passionate appeal to refrain from patenting bits of the human genome except when they could foreseeably induce investment in developing end - products such as therapeutic proteins. sulston s model for the human genome project was the biology of the worm (ankeny, 2001)a close - knit community of scientists who studied nematodes, and had made immense scientific progress in a hub - and - spoke model of biology. one at the university of cambridge and another at washington university in saint louis did high - tech, whiz - bang, expensive mapping and sequencing projects on the worm genome. those hubs shared data quickly and widely with the spokes a vibrant network of smaller laboratories throughout the world. sulston wrote the common thread with georgina ferry to tell the genome story from his point of view (sulston & ferry, 2002). his was the public works model of genomics, with public funding producing a valuable scientific resource. the sanger institute was the trust s foremost research institution, and john sulston its most visible scientist. nih and francis collins, as a government organization and employee, respectively, had to be more cautious in their rhetoric and were, most of the time. the wellcome trust sponsored a bermuda meeting of the major sequencing centers throughout the world in 1996 (despite the exotic sound of it, the weather was miserable, it was off - season, and the site was chosen deliberately to be neutral, not in the usa or europe). one theme of the meeting was how to make sequence data widely available, modeled on the worm science world. a set of bermuda rules emerged from the meeting, mandating daily disclosure of dna sequence data. the pledge to rapidly share data was linked to a plea not to patent dna, unless a gene or dna sequence had been studied further to show its function or practical utility. that kind of functional biology was not the business of the publicly funded dna sequencing centers, so it was in effect a no patents venter was present at the beginning of the bermuda meeting, in his pre - celera days as head of tigr, but fittingly he left early and was gone by the time the bermuda rules were agreed. the wellcome trust played another important role in 1998, soon after venter announced his intention to sequence the genome at a new startup company. wellcome reacted to the announcement of the new company by proposing to do a faster, better public genome sequence by increasing its commitment to fund genomic sequencing through the public project. wellcome s move, in turn, bolstered funding from the us government, uk government, and other government and nonprofit funders of the public genome project. in addition to the upstart startup celera and the public genome project, the private firms hgsi and incyte were in effect conducting a different kind of genome project in parallel call it a human genes project. both companies had been sequencing genes for 56 years before celera was even formed, and had been sending in patent applications the whole time. hgsi had one main client smithkline beecham (which later merged with glaxo wellcome to become glaxo smithkline). the business strategies of incyte and hgsi were both initially based on sequencing human genes. randall scott at incyte was part of a scientific network with many links to the public genome project. indeed, at times incyte was contemplated as a partner in the public project (shreeve, 2004). hgsi had some academic and industrial collaborations beyond smithkline beecham, but far fewer than incyte. and scott of incyte was in the public genome family, or at least ate some meals with them ; haseltine was never welcome at the table. haseltine reinforced his role as troublemaker for the public genome project when he wrote a 1998 editorial to the new york times arguing congress should pull the plug on the public project because it was already being done without tax money by his company and others (haseltine, 1998). haseltine argued government funds for dna sequencing would be better spent on smaller projects in individual laboratories to understand gene sequences. first, it assumed almost all the value of sequencing came from gene sequences, whereas molecular genetics has become focused on many regulatory processes that happen at the rna and dna level and are never translated into protein. it seems a safe bet that a lot of biology would never be approachable if we only got protein - coding sequences. haseltine is an excellent scientist and knew this full well, although for commercial purposes, he could certainly make a good case that the most rapid returns were likely to come from coding sequences. this argument conflated commercial value with scientific value, but as an argument about public support for science, it is simply wrong, as the complexity of gene regulation is becoming obvious, and the importance of dna sequences in addition to protein - coding regions is becoming apparent. gene - based strategies made eminently good sense when hunting for drug targets, because drugs are designed to interact with proteins that are secreted outside of cells, that bind dna, or that extend outward from the surface of cells. but as a tool to understand biology, the entire sequence was a much more powerful tool than just protein - coding regions. the fact that genes were being sequenced by companies did no one but those companies any good if the sequences were not public. academic scientists could, of course, approach hgsi or incyte or other companies to collaborate, getting access to their data, but relatively few did so. the reason was that such collaboration came with strings or ropes, or even cables. the constraints were patent rights that is, exclusive property rights that were routinely being granted for full - length genes by the us patent and trademark office. collaboration with hgsi or incyte meant nondisclosure agreements, publication review, and rights on resulting intellectual property. sometimes this made sense, but it was not terribly attractive for those mainly interested in advancing science. a central condition of collaboration was control of information and constraints on open sharing of data. it made sense in a business context, but as a public works project in science, it made none. and to argue that proprietary gene sequencing was a substitute for public funding of genomic sequencing was ridiculous. scientists could of course wait for patents to issue from privately sequenced genes, but that was not really a practical option because of the many - year delay. perhaps scientists could hope hgsi and incyte would publish the sequences voluntarily someday, but the companies would do that only when patents issued, or if it suited their business needs. the companies did publish, but only very selectively. to academic scientists in the field, waiting for companies to do the work would be surrendering to the competition in any event. pharmaceutical companies working with the incyte and hgsi played from power money and the ability to generate the data themselves if need be with their huge r&d war chests. small academic laboratories were on the other end of the power curve, with relatively little leverage. academic laboratories had a much better alternative, to scan the public genbank for genes of interest, at no cost and with no strings attached. genbank and other databases received sequences from thousands of laboratories throughout the world, as well as (eventually) the output of major dna sequencing centers. incyte and hgsi drew regularly on genbank data, but company gene sequence data made their way back to public sequence databases only when a patent issued, or if the company chose to publish an article in the scientific literature.3 in effect, company projects built on the foundation laid by the public genome project and drew regularly upon its data, but only occasionally contributed data back to public databases. this was sensible business practice, but it was misleading for haseltine to imply that leaving the genome projects to companies and small laboratories would produce a genome project with the desired features of the concerted public project. one forthright way to make haseltine s case would have been to indeed allow the private gene sequencing firms to proceed, but for the government and nonprofit funders to pay to make the data public. there are two reasons haseltine may have chosen not to take his arguments to this logical conclusion. first, the offer would likely have been refused by the companies, because their business plan was precisely to keep sequence data proprietary until they could be patented. government procurement of the data would have vitiated this business plan, and turned the companies into contractors. it would have been embarrassingly high, and certainly would have undercut the argument the work could be done with no tax dollars. but pushed to its conclusion, haseltine s line of argument could have made a clean case it might have made sense for the government to buy this particular genomic real estate and dedicate it to the science commons, if the private sector could produce the data faster and cheaper. haseltine started from the premise that human gene sequence data were valuable and who could argue with that ? from 1998 until february 2001, when nature and science published rival articles containing draft reference sequences of the human genome prepared by the public genome project (lander., 2001) and by celera (venter., 2001), there were in effect two competing projects focused on sequencing the entire human genome, and in parallel also several other genome projects focused on expressed sequences and bits and pieces of the genome of interest to research communities in both public and private sectors. in addition to the two companies sequencing human genes, many other companies were mapping and sequencing parts of the human genome. and thousands of laboratories were contributing sequencing and mapping information to databases and to scientific publications. by the time the initial genomic sequence publications came out, the ratio of private to public funding appeared to be roughly two private dollars for every one government or nonprofit dollar (see fig. 1genomics research funding, 2000, source : world survey of funding for genomics research, stanford university, 2001 (unpublished data from robert cook deegan, amber johnson, and carmie chan, stanford - in - washington program, based on a survey of over 200 funders) genomics research funding, 2000, source : world survey of funding for genomics research, stanford university, 2001 (unpublished data from robert cook deegan, amber johnson, and carmie chan, stanford - in - washington program, based on a survey of over 200 funders) in 2001, the financial genome bubble burst. at the end of 2000, 74 publicly traded firms were valued at $ 94 billion, of which the largest 15 accounted for approximately $ 50 billion. by the end of 2002, those 15 firms market value had dropped to $ 10 billion, but their reported r&d expenditures nonetheless climbed from $ 1 to $ 1.7 billion (kaufman, johnson & cook - deegan, 2004, unpublished data). these data make three simple points : first, the private sector has invested heavily in genomics, but those investments are made in expectation of financial return. that is quite different from the public and nonprofit funding of genomics, which is mainly intended to produce public goods knowledge and materials that are widely available to advance knowledge and combat disease. second, private r&d investment is a powerful complement to the public and nonprofit funding. private r&d follows public r&d in time, it draws on the science commons but does not necessarily contribute back to it. if successful, private r&d investment can create wealth and jobs as well as the social benefit from developing goods and services that would otherwise not be produced. this benefit is real, but it is distinct from the social value of the science commons. genomics also provides several examples of private funding to augment the science commons, such as the snp consortium, and the merck funding to washington university to fund gene sequencing (cook - deegan & mccormack, 2001). and third, and most to the point for policy purposes, it would be foolhardy to generalize from the happy circumstances when private r&d expands the science commons to expect private r&d to substitute for the science commons except in unusual circumstances, usually related to the grounds of competition among firms in a particular industrial sector. private industrial r&d will sometimes find it useful to contribute to the science commons, but expecting industry to do so always and consistently would be foolhardy. to see why having a healthy science commons matters, we move away from genomics to make a general point about health research. murphy and topel estimated that the gains in life expectancy from medical research 1970 to 1990 were staggering in the range of $ 2.8 trillion per year ($ 1.5 trillion of this from cardiovascular disease reduction alone) (murphy & topel, 1999). many of the health benefits of discovering new information about health and disease come not from drugs or vaccines or medical services, but from individuals acting on information. cutler and kadiyala attributed 2/3 of the health gains in cardiovascular disease reduction to effects of public information, such as stopping or reducing tobacco use, changing diet, getting more exercise, or monitoring one s blood pressure. the second largest determinant was technological change, such as introduction of new drugs and services, followed by increasing cigarette taxes to reduce tobacco use (cutler & kadiyala, 2001). the estimated return on investment in medical treatment was 41, but on the public information it was 301. cutler and kadiyala s result can not be generalized, because smoking is a very large risk factor that is sui generis, and cardiovascular disease has proven far more malleable to many kinds of interventions than nonlung cancer and other chronic diseases. the path from scientific understanding of cause to prevention of cancer, diabetes, arthritis, and alzheimer s disease, among others, appears far less linear, and so the value of public information about risk is correspondingly less powerful and has less impact on health outcomes. few if any risk factors will ever be found to rival tobacco use as predictors of poor health. but the finding that information can have value irrespective of being translated into products and services in a paying market is nonetheless important. even if public information will not be quite as powerful in reducing other chronic diseases as it has been for cardiovascular disease, the vector is likely to point in the same direction. we can not say that public information will always prove more powerful than information channeled into new drugs, vaccines, biologics, devices, and medical services sold for profit in the health care system. where there are public health benefits from public research results, however and the probability there will be no such public health effects of genomics seems vanishingly small the health science commons is essential, because it alone can supply the public information benefits. both words in public information we need new information that arises from science, but to capture social benefits based on that knowledge itself, we also need it to be public. the 2002 report from the world health organization, genomics and world health gave the example of fosmidomycin (advisory committee on health research, 2002). this drug is currently being testing to treat malaria in africa (missinou., 2002). that use came to light as a consequence of sequencing the genome of the malaria parasite, and noticing a metabolic pathway not previously known to exist. the compound fosmidomycin was known to inhibit the pathway, and had been developed as a treatment for urinary tract infections. when the new possible use to treat malaria was revealed, fosmidomycin was pulled off the shelf and moved into clinical trials against malaria. this is a treatment that may never turn a profit for any company, but the social returns could be enormous if fosmidomycin works, because so many millions of people are infected with malaria. if not fosmidomycin, then perhaps other findings will lead to prevention or treatment of malaria, enabled by now having the full genomic sequence available for host, pathogen, and mosquito vector (gardner. the information about these organisms available worldwide is essential to accruing the benefits of research. there is only a weak world market for drugs to treat malaria because it is largely an affliction in resource - poor populations. the usual profit motives of the intellectual property system can not create incentives where there is no prospect of profit to pull products through an expensive discovery and testing process. but networks of nonprofit organizations, such as the malaria vaccine initiative, the global fund, the who essential medicines program, and other sources of public capital might nonetheless be capable of discovering and developing new treatments despite the unlikely prospect of commercial profit.5 in theory, public funds might induce a sufficient incentive to motivate profit - driven investments for diseases of poor people living in poor countries, but it is not true now, and betting that money will be found could prove wrong. having a scientific commons with information relevant to vaccines and treatments many of the scientists most motivated to study such diseases work in resource - poor countries ; they do not have rich resources, but they do have strong motivation, as well as computers and access to public databases. strains of the coronavirus that causes sars were identified and sequenced within a month by at least three laboratories in asia, canada, and the united states. that sequence information was shared widely, and a chip to detect the virus was available for research and possible clinical use just a few months later. making progress with such alacrity requires strong norms of open science, with obvious social benefit.6 many of the infectious diseases that plague mankind have long eluded measures to combat them. in many cases, this is because they are difficult to grow in tissue culture, and therefore research progress is slow. with new technology, the genomes of hundreds of nasty bugs have been fully sequenced, giving scientists an entirely new tool to develop drugs, vaccines, and control measures. it is far from clear that this will tilt the battle decisively in favor of humans over schistosomes, trypanosomes, plasmodia, bacteria, viruses, and other organisms that maim and kill humans by the billions, but it is a new line of attack. in the case of organisms on the select agent list of bioterror bugs, there is now extensive research underway to develop preventive and treatment measures. for most infectious agents that afflict those in the poorest parts of the world, however, the prospect of profit will not create a demand - pull for innovation that could improve billions of human lives, unless indirect incentives such as prizes or guaranteed payments for effective remedies by third parties serve as surrogates for paying markets. unlike the public information case described above, however, here the market failure has a different cause. it is not due to the fact that the research results are public goods, but because the potential users are deeply impoverished, and the economic incentives for drug development in advanced economies do not prevail. nongovernment organizations around the globe, including major funders such as the gates foundation, the tb alliance, the global fund, and others, are attempting to use philanthropy, government funding, and creative networking to address this form of market failure. their efforts depend critically on access to scientific and technical information at low or no cost. another likely use of genomic information will be newborn screening, as more diseases are characterized, linked to possible intervention, and incorporated into routine testing. this must be done with care to avoid harms and false positives, but as knowledge accumulates, the list of conditions that can be treated will lengthen, and costs of testing should drop. any benefits from newborn screening are unlikely to arise from strong profit motives, however, as most testing is done by state - funded laboratories in the united states and government public health programs in most other countries. [two - thirds of us states spent between $ 20 and $ 40 per infant for all screening in 2002, and no state spent more than $ 61 (us general accounting office, 2003) ]. this is far less than most single genetic tests, or even routine medical laboratory tests. newborn screening is now, and will likely continue to be, a public health service (newborn screening steering committee, 2005). any shift to dna - based testing, or addition of tests beyond the current testing regimes, will face very serious cost constraints, and advances are unlikely to result from prospect of ample profits in this market. even if we were to stipulate that the public information impact of health research might be less important in the future than it has been in the past, does it diminish the role and importance of the science commons ? in this section, the focus is not on social benefits foregone for lack of a robust commons. instead, the argument shifts to efficiency gains to private r&d that follow from being able to draw upon the commons. several lines of research corroborate the intuition that a pool of public information and materials must surely raise all ships to the benefit of each. the case is likely to be stronger in health research than in other lines of research, just because of the well known deep mutualism between public and private r&d in health research. the late edwin mansfield s surveys of industrial leaders clearly showed that executives in firms believed their lines of business - related r&d depended on academic research, and pharmaceuticals to a greater degree than any other sector he characterized (mansfield, 1995). narin and colleagues have repeatedly shown how industrial publications cite academic research, and patents related to pharmaceuticals and biotechnology cite academic research far more heavily than most other kinds of inventions (narin & olivastro, 1992). when steve mccormack and i read through the more than 1,000 dna - based us patents issued 19801993, we found that 42%were assigned to universities (14% to private ; 9% to public), nonprofit institutions (13%), or government (six percent), compared to less than three percent academic ownership of patents overall (mccormack & cook - deegan, 19961997, unpublished data). this is a tenfold enrichment of academic involvement in life sciences compared to most other kinds of invention. the 1997 survey of the association of university technology managers was the last year for which the questions made it possible to analyze life sciences separately from physical sciences. that year, life sciences accounted for 70 percent of licenses and 87% of income (massing, 1998).7 industries closest to health research depend on academe, and academic institutions are more heavily involved in technology transfer activities related to the life sciences. if we were looking for a place where public science matters to industry, life sciences would be a good place to start. beyond the special role of academic institutions as the training grounds for both technical and nontechnical workers in the knowledge economy, academic institutions also play a unique role in creating and sustaining the science commons. it is worth noting that the studies above generally focus on academic r&d, not specifically on the science commons, or only open science. recall that universities and nonprofit research centers do not always practice open science, and some elements in the commons come from private industry r&d. while we can not be completely sure, it is quite likely that the main explanation for the importance of academic research is that it is open, producing data and materials available to all. the most direct line of evidence for this comes from the carnegie - mellon survey of industrial r&d managers. cohen, nelson, and walsh conclude that public research has a substantial impact on industrial r&d in a few industries, particularly pharmaceuticals, andthe most important channels for accessing public research appear to be the public and personal channels (such as publications, conferences, and informal interactions), rather than, say, licenses or cooperative ventures. finally, we find that large firms are more likely to use public research than small firms, with the exception that start - up firms also make particular use of public research, especially in pharmaceuticals (cohen., 2002). the most important channels for accessing public research appear to be the public and personal channels (such as publications, conferences, and informal interactions), rather than, say, licenses or cooperative ventures. finally, we find that large firms are more likely to use public research than small firms, with the exception that start - up firms also make particular use of public research, especially in pharmaceuticals (cohen. this certainly corroborates the stories of genomics startup companies, including companies like celera, depending heavily on their recent past in academic research, and their ongoing collaborations with (and sometimes customers and markets in) academic research. and it confirms the role of large firms in preferring to draw inputs from a science commons, rather than having to collect atomized, individually expensive fragments of proprietary technologies and data. the history of genomics provides many examples of this, but two are particularly famous. one salient example is the decision in the period 19881991 by the national institutes of health not to sequence human genes (i.e., protein - coding regions), but instead to focus on systematically mapping and sequencing the entire genome (cook - deegan, 2003). that decision opened the way for private firms human genome sciences and incyte to fill the void, attracting private capital to do what the public sector had chosen not to do. because it fell victim to the law of unintended effects, nih s decision not to pursue cdna sequencing, however well - intended and understandable, was a mistake in retrospect. the story behind that decision is mainly about the sociology of science, not a theory of the science commons, but it is instructive nonetheless. the decision not to sequence protein - coding regions was initially about fairness between big labs and small ones, not about commercial prospects. as the genome project took shape, the importance of maps of humans and various model organisms was apparent. what kinds of maps deserved substantial funding and concerted effort remained, however, a matter of ongoing dispute. gene map based on cdna technology that is, making dna copies of the messenger rna translated into protein within cells. construction of cdna libraries was standard fare, and remains a seminal technology in efforts to study expression of many genes through microarray technologies. one question left open during the early debates about the human genome project, 19871991, was whether the genome project would include gene sequencing to start sequencing efforts with dna known to code for protein, and therefore certain to provide codes for most of the important building blocks of cells, while also providing targets for drug development. a technical means to isolate the rna that is translated into proteins was readily available. this was called cdna technology, complementary to the messenger rna that is exported from the nucleus of the cell to its cytoplasm to be translated into protein. in fact, one could take it a step further and look for genes coding proteins likely to be of particular biological significance and focus on just those cdnas coding for secreted proteins and peptides (such as hormones or neurotransmitters), for receptor or transporter molecules extending outside the cell (with many trans - membrane domains), or proteins that bind dna (with zinc fingers), etc. these functional motifs could be predicted, if imperfectly, from dna sequence data. one logical strategy to start the dna sequencing program was to sequence cdnas of particular interest first, then other cdnas, and then turn to genomic dna between genes. (dna between genes would still be of interest because such sequences were likely to house regulatory signals for turning genes on and off, and affecting the timing of gene expression, as well as structures involved in cell division and the 3-dimensional shape of dna in cells.) at one of the first public discussions of the human genome project, at cold spring harbor in june 1986, walter gilbert responded to one attack on the idea of sequencing the genome by noting, of course you would start by sequencing the cdnas when the congressional office of technology assessment presented a plausible budget for funding the genome project, it included a cdna sequencing component (us congress, office of technology assessment, 1988). the department of energy did pursue some cdna sequencing, but nih s genome program did not. it was a matter of some discussion, but in the end it was largely james watson s call, as director of the relevant nih center. first, it was already going to happen, since incentives to find genes were strong with funding from other nih institutes, but incentives for individual labs to produce whole genomic sequence data were entirely dependent on genome project funding. if big sequencing centers did cdna sequencing, they would inevitably also be at least tempted to pause to characterize particularly interesting genes, and turn to the fascinating biology sure to follow. there were two problems with this : (1) it would distract them from the major task at hand of deriving a complete reference sequence of the entire genome, and (2) it would give them an unfair advantage over the thousands of smaller laboratories lacking the dna sequencing firepower. it was the nih decision not to fund cdna sequencing that left the door open to incyte and hgsi to follow human cdna sequencing with private funding, because in the absence of a big public effort, the low - hanging fruit of the genome was there to be plucked, sequenced, and shipped off with claims to the patent office. when incyte and hgsi began to go down this path, those who saw genes as increasingly important inputs to their r&d efforts particularly large pharmaceutical companies got concerned, for two reasons. one was that the us patent and trademark office was obviously patent - friendly, industry - oriented, and seemingly tone - deaf to the concerns of scientists about enclosing the public domain. if patents were granted, then any firm making, using or selling a gene or gene fragment could be hit up for a piece of the action by the company that first sequenced it. incyte and hgsi were clearly capable of filing patent applications on hundreds of thousands of gene tags, and thousands of full - length genes. moreover, the small genomic startups had a running start on large pharmaceutical firms, the plodding apatosaurus s of the biotech jurassic. merck decided to take action (williamson, 1999).8 it stepped forward to fund a public domain sequencing effort, starting with gene fragments and moving on to full - length cdnas. the work was to be done at washington university in saint louis, home of one of the largest public genome sequencing facilities, and the data were to be moved quickly into the public domain. merck funded the work through a nonprofit arm and had no privileged access to the data. here was a large company funding data to flow into the science commons where it would be freely available to all. four reasons suggest themselves : (1) it poisoned the well for incyte, hgsi, and other startup firms, creating an open, academic competitor (albeit funded by industry) to shut the window on securing exclusive property rights on genes, and thus limiting the number of genes that would be have to be licensed ; (2) it built good will with scientists, vital collaborators in merck s drug discovery efforts ; (3) it was great pr ; and (4) it took advantage of nonprofit funding. if merck paid for it as corporate r&d, it could deduct the r&d as an expense, but would also have to justify public domain science at stockholder expense. through a nonprofit arm, merck funded great science, burnished merck s image, and enhanced merck s future freedom to operate cleanly, without having to appropriate any returns on an investment. the snp consortium story started 5 years later, but followed the same general outline, with an added level of sophistication. during the late 1990s, it became apparent that there were many single - base - pair differences in dna sequence among individuals. these were dubbed single nucleotide polymorphisms, or snps, because of molecular biologists penchant for impenetrable polysyllabic neologism (ipn) and three - letter acronyms (tlas). single nucleotide polymorphisms could be used as dna markers, to trace inheritance, to look for associations with diseases or traits, and to study population differences. many genomic firms, including celera, began to signal they were finding snps and filing patent applications. given the uncertainty about what the patent office would allow to be claimed in patents, it seemed possible patents on snps would be granted, meaning anyone using patented snps would need to get a license. this raised the prospect of needing to get licenses on hundreds or even thousands of snp sequences from some unknown (but potentially large) number of patent owners. the court of appeals for the federal circuit had instructed the patent office that the nonobvious criterion for dna sequence was met by any new dna sequence, so obvious did not mean obvious how to find it but sequence determined and in hand. the patent office was signaling it might permit patents for any plausible utility, demonstrated or not, and related to biological function or not (doll, 1998). this was just the kind of nightmare that michael heller and rebecca eisenberg had speculated might arise in their classic 1998 article on the anticommonssituations when too many exclusive rights upstream needed to be assembled, thus thwarting the development of final products, such as drugs, vaccines, biologics, or instruments (heller & eisenberg, 1998). this threat awakened some companies and scientific institutions to forge an alliance to defeat patent rights in snps (the snp consortium, 2005 ; holden, 2002 ; thorisson & stein, 2003). the snp consortium was founded in 1999 to first discover snps, file patent applications, map and characterize the snps, and then finally abandon the patent applications. the expense and paperwork of this elaborate dance were intended to ensure snps landed in the public domain unfettered by patent rights. it was deemed necessary as a defensive strategy to ensure that consortium members would have standing as inventors should disputes arise about priority for related inventions (in patent parlance, interference proceedings, the administrative procedure to determine the real first inventor). here a group of private firms of various sizes found common cause in defeating patents on research tools. they valued their freedom to operate highly and the threat of patenting sufficiently to pay for a complicated, expensive procedure to enlarge the public domain. private firms that dearly loved patents for their own products were working together with academic institutions to defeat patents ? one interpretation might be that the public sector failed to support lines of research with a strong need for a science commons sufficiently. but members of the public genome project were well aware of the need for unfettered access to snps and were as worried about the problem as the private firms that wanted to use snps in their research. the issue here was the presence of many different kinds of genomics firms, some of which saw an opportunity to create and sell access to snp research tools. it was no accident that this episode played out during the genomics bubble years, 19982001, when seemingly any startup with omics in its name could raise millions in private placements and months later (before any products hit the market) tens of millions through initial public offerings of stock. it was conceivable that a company could raise private capital to find snps based on a possible paying market to use them in research. the public sector was simply not going to be able to mount a systematic snp initiative fast enough and large enough to compete, and other companies wanted to avoid having to deal with the snp upstart firms (yes, celera was one of the firms with an interest in snps). one interpretation of this story is that the market, some market somewhere, solved the problem. the wonder of capitalism worked its magic by creating public domain resources at private expense to forestall the undue private appropriation of rents from research tools. can we learn to relax, and assume that excesses of the patent system will be compensated by enlightened capitalists guarding their long - term best interests and future freedom to operate ? the merck gene index and snp consortium show the answer is sometimes yes. the nagging worry is that sometimes the answer may be no. a final historical pastiche before closing out the arguments. consider again the prospect of an alternative universe in which free access to data about the medical literature and scientific data we take for granted in health research might instead be constrained by exclusive proprietary rights. if the history and geography had been different and database firms had turned their attention to genomics just a bit sooner, the story might have been quite different. as it was, the early algorithms for interpreting dna sequence such as the blast and smith waterman algorithms were developed by individuals committed to open science. in more recent years, patents have begun to issue on bioinformatic methods relevant to genomics. in some cases, these patents confer incentives to support products marketed by firms, with service teams and development teams to improve their quality.9 how this story will play out remains to be seen, but the ideas of open genomics are being tested in the real world along - side more proprietary models. the early years of the human genome project were marked by many decisions about the disposition of crucial databases. human genetic disease and variation was lovingly cataloged by a team surrounding its founder, victor mckusick of johns hopkins university, in online mendelian inheritance in man (omim). many databases were established to retain data on human genetic maps of various types, and similar databases for other organisms. dna sequence data were collected primarily by a trio of databases in the united states, europe, and japan, and these shared data among themselves. there was, in effect, just one major, central dna sequence database beginning in the early 1980s. creating and coordinating these databases, including the sequence databases, was its own titanic struggle (smith, 1990), but the battle was waged with only glancing concern for commercial potential. the databases contain many errors (pennissi, 1999), and creating financial incentives sufficient to encourage careful curation and maintenance is one reason to support proprietary rights in making databases. but that step should not be taken lightly, and now we have a decade - long experiment in the real world to inform such decisions, with strong protection in europe and only copyright and contractual protections for databases in the united states. how different it might have been had the genome project begun in europe, just a decade later, when the european community saw fit to create a new exclusive right in databases as an incentive for companies to create and maintain valuable data. the impacts of this new form of intellectual property have received particular attention from the scientific community. the landmark report on the topic was the bits of power report from the national research council (1997), which has led to a line of further work. much of the most advanced work has focused on weather, remote imaging and other huge and complex data sets. there may be cause for worry, and not just for scientists, but for the innovation system as a whole. it may be that free access to data generated at government and nonprofit expense is far more efficient, and a more powerful prime for the economic engine, than allowing every incremental advance to form the basis for rent - seeking. in the patent - happy united states that moves toward ever - longer copyright and data generated at government expense and published by the government can not be copyrighted, and are thus freely available to anyone who wants to use them. it turns out that when it comes to data about the weather, it is the europeans who are scrooge, charging for access. and yet us businesses that provide weather information to various kinds of users have flourished, and the us market for such information is vastly larger than in europe, despite the nearly equal size of the economies of the european union and the united states. an analysis by peter weiss of the national weather service concludes, the primary reason for the european weather risk management and commercial meteorology markets lag so far behind the us is the restrictive data policies of a number of european national meteorological services (weiss, 2002). given that genomic databases and most health research databases are publicly administered and protect strong norms of open sharing, concern over database protections could prove a sideshow. perhaps it is silly to think that dna sequence might have been housed in a proprietary database owned by reed elsevier, springer, or thomson. but some databases do straddle nonprofit and for - profit worlds, and if a strong us database right were created, the rules of the game could change. swissprot, a database with information about proteins of interest in molecular biology, has been the subject of dispute, both about how to fund it, and about its pricing and access policies driven by trying to ensure its long - term financial survival. the analogies between weather and dna sequence data are not exact, but careful thinking about policies bearing on health research data, including genomic data, is crucial, because the creation of a us database right similar to the european counterpart remains a distinct possibility. the various genome projects, both public and private, pursued quite disparate policies about sharing of data and materials. proprietary technologies and data were created, mainly by private startup firms, and they contributed to the pace and success of the human genome project. deliberate policies of funding organizations, especially the wellcome trust and national human genome research institute and other funders of the public genome project created and preserved a large and important science commons of genomic data and technologies for analyzing dna structure and function. agreements such as the bermuda rules, privately funded initiatives such as the merck gene index, and public - private hybrids such as the snp consortium were deliberately designed to promote broad access to data and materials. genome projects spanned a full range of openness, from rapid open access under the bermuda rules, to subscription - based access to genomic data and analytical tools at moderate cost (e.g., celera), to highly proprietary gene - sequencing with public disclosure mainly limited to patents as they were granted and published (human genome sciences and incyte). the practical public information benefits from having information widely and inexpensively available, such as public health advances from new knowledge about health risk, reinforce the benefits for science, where a broad network of investigators can draw on masses of information. it is, however, a powerful argument for the need to support open science and a healthy science commons upon which both public and private science can draw. without explicit policies to foster the science commons, this valuable pool of knowledge would have been shallower, and a less productive fountain of social benefits. science is not just about creating knowledge, it is also about making it widely available and making it useful. deliberate policies to promote open access and low - cost use enable some social benefits that profit - driven r&d can not. public genomics is, however, a creature of deliberate policies, not just to fund the science but also to ensure that the results are shared. it is not a system that can be left to mindless self - assembly or politics as usual. without an expansive science commons, many benefits would be lost and private genomics would be vastly less productive and valuable. | the science commons, knowledge that is widely accessible at low or no cost, is a uniquely important input to scientific advance and cumulative technological innovation. it is primarily, although not exclusively, funded by government and nonprofit sources. much of it is produced at academic research centers, although some academic science is proprietary and some privately funded r&d enters the science commons. science in general aspires to mertonian norms of openness, universality, objectivity, and critical inquiry. the science commons diverges from proprietary science primarily in being open and being very broadly available. these features make the science commons particularly valuable for advancing knowledge, for training innovators who will ultimately work in both public and private sectors, and in providing a common stock of knowledge upon which all players both public and private can draw readily. open science plays two important roles that proprietary r&d can not : it enables practical benefits even in the absence of profitable markets for goods and services, and its lays a shared foundation for subsequent private r&d. the history of genomics in the period 19922004, covering two periods when genomic startup firms attracted significant private r&d investment, illustrates these features of how a science commons contributes value. commercial interest in genomics was intense during this period. fierce competition between private sector and public sector genomics programs was highly visible. seemingly anomalous behavior, such as private firms funding open science, can be explained by unusual business dynamics between established firms wanting to preserve a robust science commons to prevent startup firms from limiting established firms freedom to operate. deliberate policies to create and protect a large science commons were pursued by nonprofit and government funders of genomics research, such as the wellcome trust and national institutes of health. these policies were crucial to keeping genomic data and research tools widely available at low cost. |
a 67-year - old post - menopausal woman visited the clinic due to vaginal bleeding. levels of serum tumor markers, including carbohydrate antigen 125 and carbohydrate antigen 19 - 9, were normal. curettage of the endometrial lesion showed endometrioid adenocarcinoma, the international federation of gynecology and obstetrics (figo) g1. there was an ill - demarcated grayish mass, measuring 43.73.5 cm, on the anterior wall of the endometrium considered to be endometrial origin. there was another well - demarcated heterogeneous white to yellowish partially hemorrhagic and fibrotic mass, measuring 654.4 cm, on the posterior wall of the myometrium. the cut surface of the mass showed an ill - demarcated yellowish necrotic lesion, enclosed by a whitish fibrotic area. 1). the endometrial lesion had the histologic appearance of endometrioid carcinoma, the same as the previous curettage (fig. the histologic findings of the myometrial mass were characterized by infiltration of spindle to ovoid cells into the surrounding leiomyoma. frequent mitotic activity was encountered in up to 14/50 high - power fields (hpf). immunohistochemical staining was performed on the myometrial lesion for cd10, caldesmon, desmin, -smooth muscle actin (sma), human melanoma black 45 (hmb-45), melan - a, transcription factor e3 (tfe3), and ki-67. the infiltrating tumor cells were positive for sma, hmb-45, and tfe3 with a low ki-67 index of about 5%, but they were negative for cd10, caldesmon, and desmin (fig. pecomas, which have no known normal tissue counterpart, are unique in that they react immunohistochemically for both melanocytic and myoid markers. reviewed 26 cases of pecomas of soft tissue and gynecologic origin. according to that report, all cases demonstrated expression of at least one of the melanocytic markers (hmb-45, melan - a, and microphthalmia transcription factor), and hmb-45 showed the highest rate of positivity at 92%. they also showed positivity for myoid markers with sma (80%) and desmin (36%). our case demonstrated positivity for both hmb-45 and melan - a, and melanin pigments were observed in some cells. among the myoid markers, our case was positive for sma and negative for desmin and caldesmon. the existing leiomyoma was positive for desmin and caldesmon, so the pecoma was clearly defined from the large preexisting leiomyoma at the peripheral portion of the bulky mass. we reviewed all case series published in english available in pubmed that included more than three cases of pecoma in the female genital tract, for a total of 63 cases. the results (table 1) [6 - 8 ] show that all (100%) of the tested cases showed positivity for hmb-45, and 52.8% were positive for melan - a. myoid markers including sma, caldesmon, and desmin were positive in 74.5%, 66.0%, and 94.4% of tested cases, respectively. cd10 immunostaining was performed in 23 cases and showed positivity in six cases (26.1%). these results confirm the diagnostic value of immunohistochemistry profiling in uterine pecoma, especially the use of melanocytic and myoid markers, due to their high rates of positivity. the differential diagnosis of uterine pecoma includes uterine smooth muscle tumor, endometrial stromal tumor, gastrointestinal stromal tumor (gist) with secondary involvement of the uterus, and other sarcomas such as alveolar soft part sarcoma (asps). paraganglioma, metastatic melanoma / clear cell sarcoma of soft part, and rarely, carcinoma should also be considered in the differential diagnosis due to their epithelioid cytomorphology. positivity enables a differential diagnosis of pecoma from uterine smooth muscle tumor, endometrial stromal sarcoma, and asps. there was a report of late pulmonary and renal metastatic pecomas with initial misdiagnosis as uterine leiomyosarcomas. cd10, which usually shows diffuse and strong immunoreactivity in endometrial stromal tumors, may be helpful in the differential diagnosis because 25% of uterine pecomas are reported to be positive for cd10. the exclusion of gist from pecoma may be possible on the basis of cd34 staining, as well as c - kit positivity and melanocytic marker negativity in gists. metastatic melanoma and/or clear cell sarcoma can be distinguished from pecoma by the strong s-100 protein immunoreactivity of the former and their muscle marker negativity. tfe3 is a member of the mit family of transcription factors, and tfe3 gene fusions have been found in some neoplasms such as asps and a subset group of renal cell carcinoma. the group harboring tfe3 gene fusion is reported to be more common in young patients, to be unrelated to tuberous sclerosis, to show alveolar architecture, and to have more epithelioid than spindle cell cytology and minimal immunoreactivity for myocytic markers. because of the small number of cases, however, it is difficult to define these cases as a single distinct group showing specific clinical characteristics compared with conventional pecomas. there was a report of a concomitant pecoma and an endometrioid carcinoma with synchronous / metastatic bilateral ovary carcinomas and uterine leiomyoma. the case had a finding common to ours in that there was a well differentiated endometrial carcinoma involving a depth of more than half of the myometrium with a synchronous separate pecoma. in that case, however, the pathologic examination revealed benign features of pecoma and it presented as a subserosal mass, unlike our case, which presented as a protruding endometrial mass and showed malignant features. criteria for malignancy of pecoma of the female genital tract are currently not clearly defined due to insufficient case studies. according to criteria that are currently accepted, pecomas are classified into three categories : benign, uncertain malignant potential, and malignant. benign is defined to exhibit the following gross or histologic features : gross size 5 cm regardless of cellular features. satisfying the malignant category refers to cases which show two or more of the following features : gross size > 5 cm, infiltrative growth, high - grade nuclear features, necrosis, vascular invasion, or a mitotic index 1/50 hpf. according to the largest single series of pecomas of gynecologic origin, the current classification system was very specific and sensitive for the diagnosis of malignant pecoma in the gynecologic tract. | perivascular epithelioid cell tumors (pecomas) refers to a family of mesenchymal neoplasms composed of angiomyolipomas, clear cell sugar tumors of the lung, and lymphangioleiomyomatoses. these tumors have a distinctive and common component of perivascular epithelioid cells that show an association with blood vessel walls and immunohistochemically display myomelanocytic differentiation. the unique neoplasms have been shown to have an expanded range through a variety of case reports, including visceral, intra - abdominal, soft tissue, and bone tumors. the retroperitoneum, abdominopelvic region, and uterus have been reported to be the most common sites. most pecomas follow a benign course. however, reports of malignant pecomas are increasing. many papers have described uterine pecomas, but to our knowledge, there have not yet been any reports of a malignant pecoma arising concomitant with another epithelial tumor and mesenchymal tumor. we report herein the case of a 67-year - old woman who experienced a malignant uterine pecoma infiltrating a preexisting intramural leiomyoma with synchronous well differentiated endometrial carcinoma and multiple liver and lung metastases. |
crouzon syndrome is a genetic disorder showing autosomal dominant trait. originating from the mutation of fibroblast growth factor receptor-2 (fgfr2), this syndrome affects the first branchial arch, which is the precursor of the maxilla and mandible. the most notable characteristic of crouzon syndrome is cranial synostosis (a union between adjacent bones or parts of a single bone), but it is usually presented as brachycephaly (fusion of the coronal suture), which results in the appearance of a short and broad head. the craniofacial skeletal findings are believed to result from the cranial base, possibly resulting in premature suture fusion of both the cranial base and cranial sutures. premature synostosis of the coronal, sagittal, and lambdoid sutures begins in intrauterine life and growth restriction in the neighboring bones lead to abnormal bone growth and produce facial deformities. multiple sutural synostosis initiate the premature fusion of the skull base sutures causing midfacial hypoplasia, shallow orbits, a foreshortened nasal dorsum, maxillary hypoplasia, and, in severe cases, upper airway obstruction. although usually presented at birth, craniofacial deformities are prominently presented with time, leading to mental retardation in 12% of the cases. physical features include;[57 ] craniosynostosis, which mostly affects the coronal and sagittal sutures, starting from intrauterine life up to 23 years of age.due to the lateral and anteroposterior flattening of the acrocranium, anteroposterior diameter of the head is smaller than transverse diameter.vertical growth pattern of the cranium leads to high and widened forehead.hypoplastic maxilla and wide face produce pseudoprognathism and malocclusions.oral peculiarities include narrowed and high v - shaped maxillary dental arch, overcrowding or widely spacing of the upper teeth, cleft palate and bifid uvula.deviation of the nasal septum, narrowed or obliterated anterior nares, nasopharyngeal narrowing and wide beaked nose are present that cause upper airway obstruction. the nose is curved in shape resembling a parrot nose.ocular proptosis is presented, that is caused by very shallow orbits.hypertelorism, divergent squint, and downward slanting of the upper eyelids cause frog face craniosynostosis, which mostly affects the coronal and sagittal sutures, starting from intrauterine life up to 23 years of age. due to the lateral and anteroposterior flattening of the acrocranium, oral peculiarities include narrowed and high v - shaped maxillary dental arch, overcrowding or widely spacing of the upper teeth, cleft palate and bifid uvula. deviation of the nasal septum, narrowed or obliterated anterior nares, nasopharyngeal narrowing and wide beaked nose are present that cause upper airway obstruction. hypertelorism, divergent squint, and downward slanting of the upper eyelids cause frog face appearance. this case report presents a choice of prosthetic treatment for a partially edentulous patient with crouzon syndrome. a 25-year - old male patient was referred to istanbul university faculty of dentistry by his dentist as a complicated partial edentulous case. the patient was free of systemic diseases and was not receiving any medication. the patient apparently represented physical means of crouzon syndrome like craniosynostosis, involving high and widened forehead, parrot like nose, deep v shaped maxillary arch, proptosis of the eyes, abundant, and thick dark hair, and as the most distinct feature anteroposterior flattening of the cranium [figures 1 and 2 ]. facial view before prosthetic treatment saggital view before treatment dental history revealed that both primary and permanent dentitions were complete initially but a removable partial denture was fabricated at the age of 10 due to early tooth loss caused by severe periodontal destruction. unfortunately, the patient had not used the dentures as he could not have adopted himself to it. the patient 's main expectation was to improve the reduced chewing ability, which was caused by excessive number of tooth loss. there were a total of 14 remaining teeth, 8 in the maxilla and 6 in the mandible, most of which were malpositioned and periodontally involved [figures 3 and 4 ]. the probing depths of most teeth were 2 to 3 mm, which was 7 mm in the mesial sight of the maxillary right first molar, 5 mm in the mesial sight of the second left incisor, 4 mm in the maxillary left canine and the mandibular left first molar. right maxillary first molar, left maxillary first incisor, both mandibular first incisors, and right mandibular first molar teeth exhibited a mobility of mod 1 according to miller 's index. the intermaxillary relation of the jaws was class iii resulting nearly in a circular non - occlusion [figure 5 ]. maxillary arch before treatment mandibular arch before treatment intermaxillary relationship before treatment diagnostic casts were prepared via alginate impressions. casts were mounted on a parallelometer and were analyzed on the basis of present undercuts, potential guiding surfaces and retentive areas of the remaining teeth for a possible removable partial denture. a telescopic overdenture for the maxilla and a single piece fixed prosthesis for the mandible were planned for restoring the dentition. among the malpositioned teeth, both mandibular first molars were severely tipped to the mesial sides but only left molar had to receive endodontic treatment for proper tooth preparation. the maxillary right first molar, which had a severe soft tissue recession on the buccal side, was prepared at the enamel - cement border to ease the oral care. final impressions for ceramic veneers and telescopic copings were made with an addition type silicone (panasil ; kettenbach, eschenburg, germany) three days after tooth preparations. the copings of the telescopic crowns were cast with retention pearls on the sides and mandibular framework for ceramic veneers was cast as one piece. the try in for the copings and mandibular single piece cast framework were done as in routine. before making a secondary impression over the copings from the maxillary arch, the retention pearls on the copings alsip, u.s.a.) to make sure that the primers do not change position during impression making. the final impression for the telescopic crowns was also made with the same addition type silicone. the telescopic crowns were cast over the copings with retention pearls all over to be embedded in the acrylic resin during polymerization. the framework of the removable partial denture was cast with a non - precious alloy and the telescopic crowns were soldered to the framework. the height of the rims, lip support, and inclination of the occlusion plane were adjusted in the mouth before determining occlusal vertical dimension. the occlusal vertical dimension was then determined with both niswonger 's and silverman 's methods. the centric relation was recorded with inter - occlusal registration method. in the try - in session, the consents of the patient and his family were taken on the basis of aesthetics and comfort. the maxillary removable prosthesis was finished with conventional procedures and the mandibular fixed prosthesis was glazed [figure 6 ]. mandibular prosthesis was cemented with resin - type cement (panavia f, kuraray dental, okayama, japan) and the copings of the telescopic overdenture were cemented with zinc polycarboxylate cement (adhesor carbofine, spofa dental, jicin, czech republic) via closed mouth technique. verbal and written instructions about the care of the prosthesis and the abutment teeth were given to the patient and his family. early recalls were made on the first day, first week, and two weeks after the prostheses were delivered to the patient. as the cooperation of the patient was poor about the care of prostheses and the abutment teeth, monthly recalls were made during the following year to consolidate the oral care. later recalls revealed positive feedback, the remaining abutment teeth and the periodontal structures were healthy and the prostheses were in good condition, still serving satisfactory function and aesthetics [figures 710 ]. after prosthetic rehabilitation, as stated by his family, the patient was further socialized and at the age of 25 he was employed for the first time in his life. maxillary telescopic overdenture intra - oral view without the overdenture after one year intra - oral view after one year facial view after one year saggital view after one year multiple staged surgery is the general treatment plan in infancy, in order to stage reconstruction to coincide with facial growth patterns, visceral function, and psychosocial development. to maximize surgical outcomes and patient satisfaction, an interdisciplinary approach, including the pediatric dentist orthodontist, within a developmental context is needed at early ages. however, in adult patients surgical management may not be preferred for various reasons by the patient, as in our case. a variety of treatment plans ranging from a conventional removable partial denture to fixed partial dentures over remaining teeth or with additional support from implants were evaluated for reconstructing the mandibular arch. as the patients former experience of a conventional clasp retained removable partial denture was a failure, this choice of treatment was ignored. the cost of implant therapy was the reason to eliminate this choice. for restoring the maxillary dentition fixed partial dentures or an overdenture type removable prosthesis were the choices of treatment. extreme class iii relation of the jaws restrained the possibility of constructing the maxillary arch with fixed partial dentures, even in a cross bite occlusion. besides these factors, to achieve maximum support, retention, stability and to benefit from the splinting effect, a telescopic overdenture was the final choice. there were certainly more complicated treatment options, which mostly included quiet invasive orthognatic surgical procedures. as the patient refused surgical treatment, these options were initially eliminated. usually, multiple surgeries to prevent maxillary hypoplasia and cranial synostosis are planned starting from the early ages. most available papers on crouzon syndrome present either information about the syndrome itself or multidisciplinary treatment procedures to overcome the craniofacial deformities disturbing the growth pattern and the physical appearance. the dental treatment of a patient is usually neglected due to the invasive procedures during early ages when permanent dental treatment is not possible. therefore, such patients receive dental care mostly after the treatment for the vital features of the syndrome. this situation has led to a lack of literature about the dental care and treatment of adult patients with crouzon syndrome. prosthodontic treatment seems to be a good choice to restore not only the dental arches but also facial contours as well for selected patients with crouzon syndrome. the type of prostheses, the supporting structures or implants should be evaluated for each patient. prosthetic reconstruction of function and aesthetics for crouzon patients avoiding further surgical applications appears out to be a powerful alternative to improve the quality of life and patients satisfaction. | crouzon syndrome is a rare genetic disorder, which can be defined as a variation of craniofacial dysostosis caused by the premature obliteration and ossification of two or more sutures. the growth pattern results in pseudoprognathism and malocclusions including an overcrowded or a widely spaced dentition. specifically maxillary arch is narrow, high, and v - shaped. cleft palate and bifid uvula are other possible features in the oral cavity. this report describes a non - surgical treatment model to overcome the remaining significant class iii intermaxillary relation and excessive tooth loss to recover function and aesthetics for a 25-year - old crouzon patient. |
data were obtained from a sample of children residing in a ukrainian region where mn mining operations were active (near the cities ordzhonikidze and marhanets of dnipropetrovsk [dnk ] oblast). these data were compared with records of children living in the territory of novotroitskoye in the dnk region, an environmentally neutral region that served as a control, where the medical and health facilities are located. they spent at least 8 hours each day, 5 days per week at daycare facilities, and during this time had at least two similar meals a day. soil samples collected near a mn ore agglomeration plant and a waste depository were analyzed at the ecotoxicology laboratory of dnk state agrarian university. soil was collected at depths of 010 cm and at distances of 0.10.2, 0.5, and 1.52 km from the ore agglomeration plant and the waste depository in the direction of the predominant winds in the area. each soil specimen was formed by combining at least five samples of soil collected from different spots at the designated plots. one kilogram specimens were prepared for chemical analyses using heavy metal extraction in ammonium acetate buffer (ph=4.8, adjusted with 1n hcl). atomic absorption spectrophotometry was used to detect metal and metalloid concentrations in the soil samples (16, 17). the same atomic absorption technique was used to measure the content of heavy metals in hair samples of 166 randomly selected children from the mn area and 56 control children from the reference region. hair was cut from the back of the head (occipital area), using stainless steel scissors, as close to the scalp as possible. when possible, 2 cm of hair closest to the scalp was used for testing. children with short hair supplied less than a 2 cm length, but still a sufficient sample for analysis. case data were collected from 683 preschool children (57 years old) living in the mn mining region. the control data were collected from 56 children of the same age from the reference area. the children 's previous medical histories were reviewed from the medical charts kept by the daycare facilities. respiratory function was tested by a peak flow meter in the 166 children randomly selected for hair analysis (18). healthy, had lived in the area at least 3 years, and attended one of the randomly selected daycare facilities. saliva (1.52.0 ml) was collected from the anterior floor of the mouth with a sterilized syringe, placed in an airtight test tube, and transferred to the laboratory on ice. the supernatant liquid was diluted 10 times with normal saline and analyzed for siga using an enzyme immunoassay system (19). the test was performed on 37 children from one randomly selected daycare center from the mn area and all 56 control children. the cost of analyses and limited budget determined the number of tests that could be performed. serum samples (finger - pricks) were analyzed for a1pi using a semiquantitative procedure (20). a micronucleus (mnuc) analysis in buccal cells for cytogenetic monitoring was employed to estimate the general mutagen background in a randomly selected group of 56 children from the mining region and 23 children from the control region. the rehabilitation program tested in the mining region included the combined oral administration of humic substances (humics), carotene oil (pro - vitamin a), enterosorbents (pectin), and probiotics (acidophilus). all were officially approved and previously tested for food supplements (21, 22). a humic food additive in the form of a 0.05% solution of humic acid was used for 21 days according to instructions from the pharmaceutical committee of the ukraine ministry of health. treatment was provided for 2 months to the 37 children in the mn mining area who were previously included in the hair, siga, and a1pi testing. the children 's legal guardians all gave verbal informed consent to allow their children to participate in the study. means and standard error of the mean were determined for each study variable. results with p - values less than or equal to 0.05 were considered statistically significant. when the means of two independent samples (exposed vs. reference groups) were compared, unpaired t - tests were used. paired t - tests were used to analyze values for the exposed group before and after rehabilitation measures were implemented, and to determine whether there were differences associated with the treatment. the simultaneous analysis of two variables was performed ; correlations between hair concentration of mn and various physical measures such as weight, height, bmi, and hyperplasia of the thyroid gland were also investigated. soil samples collected near a mn ore agglomeration plant and a waste depository were analyzed at the ecotoxicology laboratory of dnk state agrarian university. soil was collected at depths of 010 cm and at distances of 0.10.2, 0.5, and 1.52 km from the ore agglomeration plant and the waste depository in the direction of the predominant winds in the area. each soil specimen was formed by combining at least five samples of soil collected from different spots at the designated plots. one kilogram specimens were prepared for chemical analyses using heavy metal extraction in ammonium acetate buffer (ph=4.8, adjusted with 1n hcl). atomic absorption spectrophotometry was used to detect metal and metalloid concentrations in the soil samples (16, 17). the same atomic absorption technique was used to measure the content of heavy metals in hair samples of 166 randomly selected children from the mn area and 56 control children from the reference region. hair was cut from the back of the head (occipital area), using stainless steel scissors, as close to the scalp as possible. when possible, 2 cm of hair closest to the scalp was used for testing. children with short hair supplied less than a 2 cm length, but still a sufficient sample for analysis. case data were collected from 683 preschool children (57 years old) living in the mn mining region. the control data were collected from 56 children of the same age from the reference area. the children 's previous medical histories were reviewed from the medical charts kept by the daycare facilities. respiratory function was tested by a peak flow meter in the 166 children randomly selected for hair analysis (18). healthy, had lived in the area at least 3 years, and attended one of the randomly selected daycare facilities. saliva (1.52.0 ml) was collected from the anterior floor of the mouth with a sterilized syringe, placed in an airtight test tube, and transferred to the laboratory on ice. the supernatant liquid was diluted 10 times with normal saline and analyzed for siga using an enzyme immunoassay system (19). the test was performed on 37 children from one randomly selected daycare center from the mn area and all 56 control children. the cost of analyses and limited budget determined the number of tests that could be performed. serum samples (finger - pricks) were analyzed for a1pi using a semiquantitative procedure (20). a micronucleus (mnuc) analysis in buccal cells for cytogenetic monitoring was employed to estimate the general mutagen background in a randomly selected group of 56 children from the mining region and 23 children from the control region. the rehabilitation program tested in the mining region included the combined oral administration of humic substances (humics), carotene oil (pro - vitamin a), enterosorbents (pectin), and probiotics (acidophilus). all were officially approved and previously tested for food supplements (21, 22). a humic food additive in the form of a 0.05% solution of humic acid was used for 21 days according to instructions from the pharmaceutical committee of the ukraine ministry of health. treatment was provided for 2 months to the 37 children in the mn mining area who were previously included in the hair, siga, and a1pi testing. the children 's legal guardians all gave verbal informed consent to allow their children to participate in the study. results with p - values less than or equal to 0.05 were considered statistically significant. when the means of two independent samples (exposed vs. reference groups) were compared, unpaired t - tests were used. paired t - tests were used to analyze values for the exposed group before and after rehabilitation measures were implemented, and to determine whether there were differences associated with the treatment. the simultaneous analysis of two variables was performed ; correlations between hair concentration of mn and various physical measures such as weight, height, bmi, and hyperplasia of the thyroid gland were also investigated. the distribution of mn, zn, and cu in soil samples taken near the mn ore agglomeration plant and mn ore waste depository is shown in table 1 and table 2. the black soil of the steppe was used as a measure of the mn control level and averaged 220 mg mn / kg soil.results mn, zn, and cu distribution in soil samples taken near manganese ore agglomeration plant, (msem), mg / kg mn, zn, and cu spreading in upper layer (010 cm) of arable lands neighboring manganese ore slime depository, (msem), mg / kg among the 683 children in the mn region, 53% had disproportional growth (deviation from normal height : weight charts), and 33% had rickets - like skeletal deformities versus 15% in the control area. bowed legs, abnormal curvature of the spine, pelvic deformities, and breastbone projections were the most frequently observed skeletal abnormalities. growth and developmental problems were significantly more common in the mn mining area compared with the control region (p<0.05). these findings correlated (r=0.41) with mn levels in the hair of the affected children. the degree of detectable hyperplasia of the thyroid gland, as determined by physical examination, also correlated with the level of mn detected in hair (r=0.57). the average amount of mn in hair of children living in the contaminated region was 3.660.33 mcg / g versus 1.840.52 mcg / g for children in the control region (p<0.01). the chart review revealed that 53% of the mothers of the children enrolled in the mn - affected area had chronic diseases, 46.5% of which involved a complicated pregnancy and/or delivery. these percentages were significantly higher than those observed in the control region (p<0.01). the children from the mn mining region had a mean siga of 104.414.2 mcg / ml versus 49.76.1 mcg / ml among the control children (p<0.05). serum a1pi was also significantly higher in the mn - mining region, at 4.930.21 g / l compared with 2.910.22 although the average level of a1pi was higher among the group living in the mn - affected area, 11% of the children in this group displayed a1pi levels below the average for the control children. children with immunologic disturbances had peak flow meter readings 20% lower than the group average. the mean level of mnuc in the cells of children residing in the mn - affected region was 5.8 times higher than that of the controls (table 3). an estimation of total mutagen background of the environment in marhanets on mnuc test in cells of oral cavity mucosa of preschool - aged children an antimutagen effect was observed in 87.5% of cases. following 2 months of rehabilitation treatment, the concentration of mn decreased significantly in the hair of children living in the contaminated region from 3.660.32 the siga rose further, to 187.328.3 mcg / ml (p<0.05), following the rehabilitation. at the end of the 2-month treatment period we did not observe any noteworthy changes in growth and/or skeletal deformities the distribution of mn, zn, and cu in soil samples taken near the mn ore agglomeration plant and mn ore waste depository is shown in table 1 and table 2. the black soil of the steppe was used as a measure of the mn control level and averaged 220 mg mn / kg soil.results mn, zn, and cu distribution in soil samples taken near manganese ore agglomeration plant, (msem), mg / kg mn, zn, and cu spreading in upper layer (010 cm) of arable lands neighboring manganese ore slime depository, (msem), mg / kg among the 683 children in the mn region, 53% had disproportional growth (deviation from normal height : weight charts), and 33% had rickets - like skeletal deformities versus 15% in the control area. bowed legs, abnormal curvature of the spine, pelvic deformities, and breastbone projections were the most frequently observed skeletal abnormalities. growth and developmental problems were significantly more common in the mn mining area compared with the control region (p<0.05). these findings correlated (r=0.41) with mn levels in the hair of the affected children. the degree of detectable hyperplasia of the thyroid gland, as determined by physical examination, also correlated with the level of mn detected in hair (r=0.57). the average amount of mn in hair of children living in the contaminated region was 3.660.33 mcg / g versus 1.840.52 mcg / g for children in the control region (p<0.01). the chart review revealed that 53% of the mothers of the children enrolled in the mn - affected area had chronic diseases, 46.5% of which involved a complicated pregnancy and/or delivery. these percentages were significantly higher than those observed in the control region (p<0.01). the children from the mn mining region had a mean siga of 104.414.2 mcg / ml versus 49.76.1 mcg / ml among the control children (p<0.05). serum a1pi was also significantly higher in the mn - mining region, at 4.930.21 g / l compared with 2.910.22 although the average level of a1pi was higher among the group living in the mn - affected area, 11% of the children in this group displayed a1pi levels below the average for the control children. children with immunologic disturbances had peak flow meter readings 20% lower than the group average. the mean level of mnuc in the cells of children residing in the mn - affected region was 5.8 times higher than that of the controls (table 3). an estimation of total mutagen background of the environment in marhanets on mnuc test in cells of oral cavity mucosa of preschool - aged children following 2 months of rehabilitation treatment, the concentration of mn decreased significantly in the hair of children living in the contaminated region from 3.660.32 the siga rose further, to 187.328.3 mcg / ml (p<0.05), following the rehabilitation. at the end of the 2-month treatment period we did not observe any noteworthy changes in growth and/or skeletal deformities occupational exposure to mn is characterized by contact with high concentrations of mn in air for short periods of time. chronic environmental exposure to mn may consist of low mn concentrations in air over long periods, but food grown in mn - rich soil can also be a major source of mn for human populations (1). the population evaluated in this study has a low migration rate, which led us to hypothesise that long - term, low - dose mn exposure might produce observable effects on the growth and development of children living in the area. we suspected that children might be exposed to mn through different routes such as food, water, and air. the level of mn contamination in the study area was significantly higher than the control area. reclamation of abandoned mined land in the region is critically important in order to reduce exposure levels for the local population (23, 24). the abandoned mined areas lower the water table and reduce crop yields in the areas surrounding the mines. approximately 9,000 ha of mined land have been covered with soil, but most of these areas were inadequately reclaimed. another 10,000 ha were left abandoned, with no reclamation, more than 30 years ago. an estimated additional 150,000 ha show obvious signs of adverse effects of being non - restored, mined land. through clinical evaluation of the children, we unexpectedly discovered that the major adverse effect of mn exposure was on bone formation. although we did not measure levels of vitamin d, calcium, or phosphorus, our control region, which had the same sunlight exposure, had fewer than half of the number of cases of rickets as in the area of mn contamination. a literature search revealed the same phenomenon had been reported in experimental animals (25). mn administered orally to young rats resulted in morphologic changes in the growth plate that histologically resembled rickets (25). we observed increased levels of siga and serum a1pi, which could represent compensatory protective mechanisms to counter mn toxicity. the antibody siga plays a critical role in mucosal immunity (26), and the secretory component is directly involved in the siga function in vivo (27). elevation of siga concentrations, as was found in our study, might reflect the intensity of the immune response needed to protect the body from external antigens. the a1pi is an anti - protease that can protect tissues from enzymes secreted by inflammatory cells (28). the concentration of a1pi can rise substantially in cases of acute inflammation (28). the 11% of children from the study region who displayed a1pi levels below the average for controls might have exhausted their protective mechanisms. it is a cost - effective, non - invasive method of detecting cytogenetic damage in an exposed pediatric population. micronuclei in exfoliated buccal cells emerge during mitosis of the basal layer of the epithelium as extra - chromosomal dna particles when chromosome fragments or whole chromosomes lag behind and fail to be included in the main nuclei of the daughter cells (29). the formation of micronuclei is induced by substances that cause breakage of chromosomes (clastogens), as well as by agents that affect the spindle apparatus (aneugens) (30). an increase in the number of cells with mnuc might be a characteristic of human cell genetic damage and reflect a mutagenic impact of the environment (31, 32). it shows that exposure to mn is associated with an increase in dna damage compared with the reference population. the results of the rehabilitation studies demonstrated that natural adaptogens can promote a positive effect by decreasing the frequency of occurrence of genetic pathologies in somatic cells. the rehabilitation measures could not prevent the inhalation of mn particles, but by giving exposed children enterosorbents, we may have prevented some enteric absorption of the metal, and facilitated excretion of mn. humics and carotene oil possess immuno - modulating effects that positively influenced the immunologic status of the children. eubiotics support the stability of the enteric flora and have been found to be beneficial in a previous study in children with disturbed immunity (33, 34). a limitation in our research was the absence of neurocognitive and neurodevelopmental measurements in the pediatric population. future work in this area should evaluate the effects of mn on the nervous system. our study demonstrates the observable influence of residing near open mn mines on the health of a pediatric population. major effects identified included deficits in bone growth and immune function and somatic cell mutation. these data contribute to a better understanding of mn - related toxicity, and can be used by health authorities to make evidence - based decisions to improve the health of children living near mn mines. the implementation of social and medical rehabilitation measures may protect the next generation living in unfavorable environmental conditions from developing the symptoms of chronic mn toxicity. the authors have not received any funding or benefits from industry to conduct this study. | introduction : chronic manganese (mn) exposure is a health hazard associated with the mining and processing of mn ores. children living in an area with increased environmental exposure to mn may have symptoms of chronic toxicity that are different from adults who experience occupational exposure. the aim of the study was to compare health outcomes in a pediatric population living near open mn mines with a group of children from a reference area and then to develop and implement preventive / rehabilitation measures to protect the children in the mining region.methods:after environmental assessment, a group of 683 children living in a mn - rich region of ukraine were screened by clinical evaluation, detection of siga (37 children), micronucleus analysis (56 children), and hair mn content (166 children).results : impaired growth and rickets - like skeletal deformities were observed in 33% of the children. this was a significantly higher percentage than in children in the reference region (15%). the children from the mn - mining region also had increased salivary levels of immunoglobulin a (104.414.2 mcg / ml vs. 49.76.1 mcg / ml among the controls (p<0.05), increased serum alpha 1 proteinase inhibitor levels (4.930.21 g / l compared with 2.910.22 g / l for controls ; p<0.001) and greater numbers of micronuclei in the mucous cells of the oral cavity (0.0700.008 vs. 0.0120.009, p<0.001).conclusions : these findings indicate the deleterious health consequences of living in a mn - mining area. medical rehabilitation programs were conducted and produced positive results, but further validation of their effectiveness is required. the study provided background information to formulate evidence - based decisions about public health in a region of high mn exposure. |
(r. tomentosa), also named as rose myrtle, is an evergreen shrub in the family myrtaceae and mainly grown in southeast asian countries, especially southern china, japan, and thailand. the edible berries of r. tomentosa are dark violet and bell - shaped and have been historically used as a folk medicine to treat diarrhea, dysentery, and traumatic hemorrhage. our previous study has reported the total content of flavonoids of r. tomentosa berries and identified six of flavonoids (myricetin, quercetin, dihydromyricetin, kaempferol, quercetin 7, 4-diglucoside, and vitexin) by uplc - ms / ms. however, flavonoids are sensitive to environmental factors such as light, heat, ph, and o2 and of low water solubility. after oral administration, the flavonoids undergo degradation in the drastic acidic stomach environment, resulting in reduction of their health and therapeutical benefits. therefore, microencapsulation is a technology that envelops sensitive ingredients in a protective coating material in order to prevent these ingredients from adverse reaction, volatile loss, or nutritional deterioration. spray drying is the most prevalent technology for microencapsulation due to its higher equipment availability and cheaper operational cost. the choice of coating materials in spray drying microencapsulation critically influences every stage of production (emulsification before drying and retention of bioactive and volatile compounds during drying) and product stability. gum arabic of excellent emulsification property and maltodextrin of low viscosity and good solubility are used frequently for spray drying microencapsulation process [9, 10 ]. different ratios of maltodextrin to gum arabic and different dextrose equivalents have been widely used to encapsulate various compounds such as unsaturated fatty acids, essential oils, plant extracts, and fruit and vegetable juices [1115 ]. glycerol monostearate is one of the most important emulsifiers which provide interfacial interactions thus enhancing emulsification. therefore, in order to maintain the stability and bioactivity of flavonoids from r. tomentosa berries, the optimal microencapsulation condition and the antioxidant capacities of flavonoid microcapsules produced at the optimal condition were studied in this paper. r. tomentosa berries were purchased from guangzhou medicine market (guangzhou, guangdong, china). rutin was purchased from the national institute for the control of pharmaceutical and biological products (beijing, china). ab-8 macroporous resin was purchased from the chemical plant of nankai university (tianjin, china). 2, 2-diphenyl-1-picrylhydrazyl (dpph) was purchased from sigma chemical co. (st. louis, mo, usa). all other chemicals used for analysis were of analytical reagent grade, obtained from guangzhou chemical reagent corporation (guangzhou, guangdong, china). the air - dried fruits were powdered (40 mesh) and extracted for 4 hr twice with 95% ethanol under reflux (70c). the extract redissolved in water was then extracted with petroleum ether for 2 times and the water - soluble fraction was purified by ab-8 macroporous resin eluted with 40% ethanol. the collected solution was concentrated and dried to powder format. then the pure fe was obtained and stored at 5c for the further usage. the emulsion solutions were prepared according to the conditions designed in box - behnken design and response surface method (rsm) (tables 1 and 2). the gum arabic and maltodextrin were previously dissolved in distilled water (5060c) separately for 1 hr and then mixed together for 5 min. gsm and fe (from 2.2) were then slowly added to the coating solutions with constant stirring. the emulsified solution was further homogenized at 40 mpa for 5 min by gyb60 - 6s high pressure homogenizer (donghua high pressure homogenizer factory, shanghai, china), followed by feeding into a mini spray dryer sy-6000 (shiyuan bio. co., shanghai, china) with 0.7 mm diameter nozzle. according to the preliminary study, the inlet temperature was set at 150 2c and the outlet temperature was 100 5c. the pressure of compressed air for the flow of the spray was 4.0 mpa and the feeding rate was 30%. the end product fems were kept in self - sealing bags which were stored in a desiccated container at 5c before analysis. 100 mg of fems was dispersed in 1 ml 50% ethanol aqueous solution and subsequently mixed using a sonicator (kq-200kdb, shumei, china) for 15 min. tfcwhole was expressed as milligrams of rutin equivalents per gram of microcapsule weight. to determine the tfcsurface, 100 mg of fems was dispersed in 1 ml of a mix of ethanol and methanol (1 : 1, v / v) by vortexing at room temperature for 1 min, followed by filtration. the tfcsurface was measured with the same method described in tfcwhole section. encapsulation efficiency (ee) was calculated from the following equation : (1)ee(%)=tfcwholetfcsurfacetfcwhole100, where tfcwhole was the total amount of flavonoids in fems and tfcsurface was the amount of flavonoids presented in the surface of fems. response surface method (rsm) was employed to investigate the effect of independent parameters including md to ga ratio, (md : ga, x1), total solid content (x2), gsm content (x3), and core to coating ratio (core : coating, x4) on the process of microencapsulation. a box - behnken design (bbd) with four variables and three levels consisting of 29 experimental runs the range and levels of the independent parameters based on the preliminary experiment were presented in table 1. the experimental points contain 24 factorial points and 5 center points (table 2). the experimental values were fitted under a second - order model in the form of quadratic polynomial equation (2)y=0+i=1kixi+i=1kiixi2+i=1 j=1 + 1jixixj+, where y was response (ee, %). 0, i, ii, and ji were constant coefficients of intercept, linear, quadratic, and interaction terms, respectively. 2 g of the sample was dried in an oven (sfg-02b, huangshi, china) at 105c until a constant weight, and the moisture content was calculated in terms of weight loss. 2 g of the sample was added into 50 ml of distilled water, and the mixture was agitated in a 100 ml glass beaker with a magnetic stirrer (c - mag ms4, ika, germany) at 1000 rpm for 20 min the volume of weighed sample was determined using a graduated cylinder, and the bulk density was calculated by the quotient of weight and volume. prior to scanning electron microscopy (sem) analysis, fems were placed on a stub using double - side adhesive tape and then coated with gold. the analysis was carried out by using a scanning electron microscope philips xl-30 esem (netherlands) of low vacuum operated at 10 kv. a 2.0 ml of sample at various concentrations (dissolved in 50% ethanol aqueous solution) was mixed with 2.0 ml of 200 m dpph solution. the mixture was kept at room temperature for 30 min before measuring its absorbance at 517 nm. equation (3) shows the radical scavenging activity (rsa) formula : (3)rsa(%)=a0a1a0100, where a0 was the absorbance of pure dpph and a1 was the absorbance of dpph in the presence of various extracts. the lipid system was thoroughly homogenized (70 0.5c) for 30 min and stored at 65 0.5c in a water bath with stirring every 24 hr. pov was determined using na2s2o3 titrimetric method : (4)pov(meq / kg)=sn1000w, where s was the volume of na2s2o3, n was the normality of na2s2o3, and w was the weight of sample. statistical analyses of the data were performed with one - way analysis of variance (anova) or student 's t - test (spss 16.0). significant differences (p < 0.05) between the means were determined using tukey 's multiple range test. as shown in table 1, md : ga (w / w), solid content (sc, %), gms content (%), and core : coating (w / w) were investigated in the ranges of 1 : 1.51.5 : 1 (w / w), 2030%, 0.20.4%, and 1 : 93 : 7, respectively. the response values (ee, %) ranged from 86 to 92% (table 2) which were comparable to those reported in other literatures using gum arabic or / and maltodextrins (table 5). after regression fitting, the quadratic equation expressing the relationship between ee (y) and influence factors (xi) is modeled as follows : (5)y=90.61 + 0.90x1 + 0.30x2 + 0.55x3 2.16x4 + 0.038x1x20.11x1x3 0.52x1x4 + 0.11x2x3 + 0.047x2x4 + 0.16x3x41.15x12 0.38x220.54x321.18x42. in table 3, the results demonstrated that the regression model could predict 97.43% of ee measured values (p < 0.0001, r = 0.9743). the adeq precision of 21.046 (higher than 4) indicated that the model with an adequate noise ratio could be applied to this experimental design. x2, x2, x1x4 had significant effects on ee (p < 0.05) and x1, x3, x4, x1, x3, x4 were highly significant (p < 0.01) and thus other forms of variables had negligible effects. based on the regression coefficients and the p value, md : ga (x1) and core : coating (x4) were the most critical factors to yield high ee, followed by total solid content (x2) and gsm content (x3). in addition, x1 and x4 were both extremely significant at first level (p < 0.0001) and second level (p < 0.001), indicating that minor changes of md : ga and/or core : coating could affect the ee significantly. adversely, total solid content (x2) and gms content (x3) impacted ee more significantly at first level (p = 0.0214 for x2 and p = 0.003 for x3). the low p value of x1x4 (p < 0.05) (table 3) indicated the interactive effect of x1 and x4 (figure 1(c)). at any given value of total solid (2030%) or gms content (0.20.4%), a decrease of md : ga resulted in an increase of the ee (figures 1(a), 1(b), and 1(c)) with the highest ee at md : ga at 1 : 1.3 (w / w). overall, the condition with md : ga at 1 : 1.3 (w / w), solid content at 27.4%, gms content at 0.25%, and core : coating at 3 : 7 resulted in the maximum value of ee (91.75%). in this study, md : ga was identified to be critical factor to microencapsulate r. tomentosa flavonoids extract (p < 0.0001) with optimized ratio at 1 : 1.3 (w / w). described that md to ga ratios from 10 : 0 to 3 : 2 increased the microencapsulation efficiency of phenolic compounds from sour cherry pomace after freeze drying process. similarly, idham. also used 3 : 2 as md : ga ratio to microencapsulate purified anthocyanin from hibiscus resulting in optimal efficiency at 99.87% and high retention and stability of anthocyanin - rich microcapsules. however, previous research also showed that a high concentration of ga in the emulsion solution reduced encapsulation efficiency. described the decrease of encapsulation of maqui leaf extracts with more than 15% gum arabic in emulsions (water - oil base) owing to the less solubility of plant extracts in the higher viscosity of coating material solutions. in addition, tonon. also reported that 6% of ga and 6% of md (20 de) demonstrated similar efficiencies on microencapsulating aai pulp (table 5) which might be attributed by the fact that pectin and other polysaccharides in the aai pulp also acted as coating materials. in contrast to md : ga ratio, an increase of core : coating increased the ee (figures 1(c), 1(e), and 1(f)) within our experimental limits (1 : 9 to 3 : 7), demonstrating a controversial result to that reported by cilek. who revealed that better encapsulation efficiencies were obtained when core : coating was 1 : 20 instead of 1 : 10. in the current study, the purified flavonoids extract was composed of myricetin (c15h10o8), quercetin (c27h30o17), dihydromyricetin (c15h12o8), kaempferol (c15h10o6), quercetin 7, 4-diglucoside (c27h30o17), and vitexin (c21h20o10) according to our previous study. the large amount of hydroxyl groups from flavonoids could rapidly form hydrogen bonds when presented in solution resulting in the formation of nonstarch polysaccharide - flavonoid complex via hydrogen bonding. among all the nonstarch polysaccharides, gum arabic has been used mostly to form flavonoids - polysaccharide complex in the wine industry due to its high proportion of anion fraction contributed by glucuronic arabinogalactan. in the light, gum arabic could actively link to r. tomentosa flavonoids extract when they came in contact in the aqueous emulsion and was able to retain flavonoids extract throughout the spray drying process. the maltodextrin used in this study had 510 dextrose equivalents, which showed better retentions of flavors and polyphenols and higher yields while having a very low viscosity at high concentration [32, 33 ]. therefore, the highly active interaction between r. tomentosa flavonoids extract and coating solution especially gum arabic along with the functionality of maltodextrin (510 de) contributed to the superior microencapsulating properties of the coating solution, which might explain that the higher the core : coating ratio the better the yield obtained in this study. the effect of solid content demonstrated weakest impacts among all the variables on the final encapsulation efficiency in this study (p = 0.0214) (table 3). 27.4% of solid content in the emulsion was the optimal solid content to yield maximum encapsulation efficiency, agreeing with the results published by robert. in which in which 20.1% and 24.2% of maltodextrin yielded the optimal encapsulation of pomegranate juice (15.97brix) and pomegranate extract (13.80brix). in this study, the solid content higher than 27.4% generating the microcapsules with less encapsulated flavonoids extract might be explained by the reduction of carrier solubility resulting in reduction of encapsulated extract. gms is a type of hydrophobic surfactant and foam stabilizer which were added to mango pulp (15 kg/1000 kg mango solid) and edible film formula (0.6%) before the drying processes [35, 36 ]. an appropriate percentage of gms increased the interaction between flavonoids extract and coating solution as well as solubility and dispersibility of final microcapsules. however, because of the hydrophobic and foam inducing properties of gms, higher concentration of gms in the coating solution might adversely reduce the hydrogen bonding between flavonoids extract and coating material thus exposing more noncapsulated flavonoids extract on the surface of final powder which was indicated by the lower encapsulation efficiencies with the gms percentage higher than 0.25%. as shown in table 4, bulk density was 0.35 g / cm comparable to the bulk densities reported in previous studies using maltodextrin and/or gum arabic as coating materials and spray drying process [15, 20 ] (table 5). moisture content was 3.27% which was within the range of powder ingredients used in the food industry (3 - 4%)., high solid content (27.4%) in the feeding solution contributed to the low moisture content in the final fems. the high solubility (92.35%) of fems was contributed by hydrophilic properties of coating materials, mainly gum arabic and maltodextrin, and also the exposure of hydrophilic groups on the fems surface after spraying drying. in this study, fems possessed both high solubility and relatively high bulk density making it an ideal powder for food product application. most of the microcapsules were observed as irregularly spherical with some porous and dented spots on the surface. the formation of the dented surfaces was attributed to the shrinkage of the particles induced by the high temperature during spray drying process. the morphology of fems was similar to other microcapsules manufactured through spray drying using gum arabic and maltodextrin as coating materials [18, 29 ]. the irregularly sphere particle observed under sem reflected on the high solubility and good bulk density of fems. after microencapsulation and spray drying, antioxidants were retained 95% in fems. as shown in figure 3(a), the dpph - rsa of fems increased with the increase of flavonoid concentrations, and the scavenging activity reached a maximum value (70.96%) at 20 g / ml. the dpph - rsa of fems was also compared to other antioxidants including rutin, nonencapsulated flavonoids extract (fe), citric acid, and vitamin c (vc) at the same concentration of 20 g / ml (figure 3(b)). rsa of fems was close to fe (73.27%), higher than rutin (35.15%) and citric acid (12.21%), and weaker than vc (87.08%). results indicated that, after microencapsulation process and high temperature spray drying, fems demonstrated similar dpph - rsa as fe. unlike the reduction of antioxidant activity observed in black carrot anthocyanins microcapsules, the process exerted little effect on the dpph - rsa of fe from r. tomentosa which might be explained by the better thermal stability of flavonoids than that of anthocyanin. the inhibiting ability of fems to lard peroxidation was investigated and compared with other antioxidants (figure 4). as shown in control (figure 4), under high temperature induction, the lipid peroxidation of lard was generated, and the pov value increased over time. the addition of antioxidants retarded the lipid peroxidation, thus slowing down the increment of pov value. increase of fems significantly prevented lipid oxidation after 10 days of incubation (p < 0.05). initially, pov values were higher under the fems treatments than those under the same concentrations of other antioxidants. however, after six days, lower pov values were tested in fems. at 0.1% level the coating of fems helped to prevent the oxidation of flavonoids caused by environmental factors (i.e., temperature, ph, and light) and slowly release lipid antioxidants to the system so that fems surpassed fe in inhibiting peroxidation. the slow release of antioxidant activities could associate with the high stability and long half - life time of bioactive compounds after being coated by gum arabic or / and maltodextrin [18, 19, 21 ] (table 5). in this study, the microencapsulation conditions of flavonoids from the berries of r. tomentosa were optimized. among maltodextrin to gum arabic ratio, solid content, glycerol monostearate, and core to coating ratio, maltodextrin to gum arabic ratio and core to coating ratio were identified as two critical factors and had interaction (p < 0.05). with 91.75% of encapsulation efficiency under the optimal condition, the fems were of antioxidant activities with good powder qualities in terms of bulk density, moisture content, and solubility. this study successfully accomplished the production of flavonoid rich microcapsules from r. tomentosa berries by spray drying at bench top scale. future studies, however, need to investigate the stabilities of fems during storage and different food applications and also in vivo bioactivities as well. | the optimal condition of spray drying purified flavonoids extract from r. tomentosa berries was studied by response surface methodology. the optimized condition for microencapsulation was of maltodextrin to gum arabic ratio 1 : 1.3, total solid content 27.4%, glycerol monostearate content 0.25%, and core to coating material ratio 3 : 7, resulting in ee 91.75%. prepared at the optimized condition, the flavonoids extract microcapsules (fems) were irregularly spherical particles with low moisture content (3.27%), high solubility (92.35%), and high bulk density (0.346 g / cm3). dpph radical scavenging activity of fems was not decreased after spray drying (p > 0.05) and higher than those in citric acid and rutin at the same concentration. moreover, fems effectively retarded the oxidation of fresh lard during the 10-day storage period compared with vitamin c, nonencapsulated flavonoids extract, and rutin. therefore, fems produced at the optimized condition could be used as powder ingredients with antioxidant capacities. |
during a 16-year period (1995 - 2010), a total of 21 patients with primary thymic neuroendocrine tumors had been surgically treated at the department of thoracic and cardiovascular surgery of samsung medical center in seoul, korea. note that these 21 cases included no cases of pulmonary neuroendocrine tumors and tumors involving both lung and thymus. clinical data and follow - up data were obtained through a retrospective analysis of the medical records, and these data include sex, age, initial symptoms and signs, a notable past history, gross findings, treatment and follow - ups. the postoperative staging was done based on the classification system of masaoka.10 for thymomas. all the patients were followed up until january of 2012 during a median follow - up period of 34.95 months. for histological classification, the histopathologic features were assessed and these include tumor differentiation, mitotic rate, presence or absence of necrosis, presence of local invasion and lymph node metastasis. the tumors were classified into four types : tc, ac, lcnec, and scnec according to the who criteria based on the histopathologic differentiation as shown below : tc : a well - differentiated tumor with mitotic figures, fewer than 2 mitoses per 10 high power fields (hpfs) and no necrosisac : a well - differentiated tumor with mitotic figures, 2 - 10 mitoses per 10 hpfs and/or presence of necrosisscnec : a poorly - differentiated tumor with small cell cytology, mitotic figures with more than 10 mitoses per 10 hpfs and extensive areas of necrosislcnec : a poorly - differentiated tumor with non - small cell nec, mitotic figures with more than 10 mitoses per 10 hpfs and extensive areas of necrosis.6 tc : a well - differentiated tumor with mitotic figures, fewer than 2 mitoses per 10 high power fields (hpfs) and no necrosis ac : a well - differentiated tumor with mitotic figures, 2 - 10 mitoses per 10 hpfs and/or presence of necrosis scnec : a poorly - differentiated tumor with small cell cytology, mitotic figures with more than 10 mitoses per 10 hpfs and extensive areas of necrosis lcnec : a poorly - differentiated tumor with non - small cell nec, mitotic figures with more than 10 mitoses per 10 hpfs and extensive areas of necrosis.6 in the current study, we used representative formalin - fixed, paraffin - embedded tissue sections for the immunohistochemical staining in our series of 21 cases of primary thymic neuroendocrine tumors. to evaluate neuroendocrine differentiation of the thymic neoplasm, synaptophysin, chromogranin or cd56 immunohistochemical staining was performed on 3-m thick sections from each case using a biotin - avidin - peroxidase method on a bond - max autostainer (leica, wetzlar, germany) after retrieval with t / e buffer (cd56) or citrate buffer (chromogranin and synaptophysin). we also used primary antibodies to synaptophysin (1:100, dako, glostrup, denmark), chromogranin (1:400, dako), cd56 (1 : 200, novocastra, newcastle upon tyne, uk) and adrenocorticotrophic hormone (acth ; 1:100, dako). during a 16-year period (1995 - 2010), a total of 21 patients with primary thymic neuroendocrine tumors had been surgically treated at the department of thoracic and cardiovascular surgery of samsung medical center in seoul, korea. note that these 21 cases included no cases of pulmonary neuroendocrine tumors and tumors involving both lung and thymus. clinical data and follow - up data were obtained through a retrospective analysis of the medical records, and these data include sex, age, initial symptoms and signs, a notable past history, gross findings, treatment and follow - ups. the postoperative staging was done based on the classification system of masaoka.10 for thymomas. all the patients were followed up until january of 2012 during a median follow - up period of 34.95 months. for histological classification, the histopathologic features were assessed and these include tumor differentiation, mitotic rate, presence or absence of necrosis, presence of local invasion and lymph node metastasis. the tumors were classified into four types : tc, ac, lcnec, and scnec according to the who criteria based on the histopathologic differentiation as shown below : tc : a well - differentiated tumor with mitotic figures, fewer than 2 mitoses per 10 high power fields (hpfs) and no necrosisac : a well - differentiated tumor with mitotic figures, 2 - 10 mitoses per 10 hpfs and/or presence of necrosisscnec : a poorly - differentiated tumor with small cell cytology, mitotic figures with more than 10 mitoses per 10 hpfs and extensive areas of necrosislcnec : a poorly - differentiated tumor with non - small cell nec, mitotic figures with more than 10 mitoses per 10 hpfs and extensive areas of necrosis.6 tc : a well - differentiated tumor with mitotic figures, fewer than 2 mitoses per 10 high power fields (hpfs) and no necrosis ac : a well - differentiated tumor with mitotic figures, 2 - 10 mitoses per 10 hpfs and/or presence of necrosis scnec : a poorly - differentiated tumor with small cell cytology, mitotic figures with more than 10 mitoses per 10 hpfs and extensive areas of necrosis lcnec : a poorly - differentiated tumor with non - small cell nec, mitotic figures with more than 10 mitoses per 10 hpfs and extensive areas of necrosis.6 in the current study, we used representative formalin - fixed, paraffin - embedded tissue sections for the immunohistochemical staining in our series of 21 cases of primary thymic neuroendocrine tumors. to evaluate neuroendocrine differentiation of the thymic neoplasm, synaptophysin, chromogranin or cd56 immunohistochemical staining was performed on 3-m thick sections from each case using a biotin - avidin - peroxidase method on a bond - max autostainer (leica, wetzlar, germany) after retrieval with t / e buffer (cd56) or citrate buffer (chromogranin and synaptophysin). we also used primary antibodies to synaptophysin (1:100, dako, glostrup, denmark), chromogranin (1:400, dako), cd56 (1 : 200, novocastra, newcastle upon tyne, uk) and adrenocorticotrophic hormone (acth ; 1:100, dako). in our series, there was a male predilection with a male - to - female ratio of 15:6. in addition, the mean age of patients was 49 years (range, 20 to 72 years). clinical and radiologic impressions were thymoma or lymphoma preoperatively in most cases of our series. postoperatively, the concurrent chemoradiation therapy (ccrt) and the radiotherapy (rt) alone were preferentially performed for cases of lcnec and ac, respectively. during the follow up period, a local recurrence or a distant metastasis were observed in 100% (3/3) of patients with lcnec and 27.8% (5/18) of those with ac. common sites of metastasis included lung, lymph node, brain, lumbar spine, mediastinum, bone, and liver. the cause of death was tumor progression in 100% (3/3) of patients with lcnec and 16.7% (3/18) of those with ac. all 21 cases were divided into two types according to the who criteria : ac (n=18) and large cell nec (n=3). the tumors were described as a large, soft and whitish - tan to brown mass with the greatest diameter of 1.8 - 21 cm (fig. the mean size of the mass was 6.81 cm and 8.27 cm in cases of ac and lcnec, respectively. some of the tumors showed an infiltrative growth pattern to the adjacent organs such as the pericardium. on cut sections, the tumors revealed focal areas of hemorrhage and necrosis. in particular, lcnec showed extensive areas of necrosis, which is the hallmark of poorly - differentiated neuroendocrine thymic carcinomas (fig. ac showed a punctate necrosis with a maximum of 13 (0 - 10) mitoses or it was devoid of necrosis but showed a proliferation with a mitotic rate of up to 9 mitoses per 10 hpf (fig. thymic lcnec was defined as a poorly - differentiated non - small cell nec with a mitotic rate of > 10 mitoses per 10 hpf. 11 frequently revealed lymphatic emboli and six, undergoing a lymph node dissection, demonstrated a nodal metastasis. finally, tumors associated with ectopic hormone secretion were also found to share the characteristic features of cytoplasm. neuroendocrine differentiation was revealed by positive immunohistochemistry for neuroendocrine markers including synaptophysin, chromogranin and cd56. in all the cases, at least two of these markers were expressed. there was no definite difference in the degree of immunohistochemical staining between cases of lcnec and those of ac (fig. but the degree of immunohistochemical staining was lower in cases of lcnec as compared with those of ac (fig. in addition, we also performed an immunohistochemical staining of acth for tumors with ectopic acth secretion. in our series, there was a male predilection with a male - to - female ratio of 15:6. in addition, the mean age of patients was 49 years (range, 20 to 72 years). clinical and radiologic impressions were thymoma or lymphoma preoperatively in most cases of our series. postoperatively, the concurrent chemoradiation therapy (ccrt) and the radiotherapy (rt) alone were preferentially performed for cases of lcnec and ac, respectively. during the follow up period, a local recurrence or a distant metastasis were observed in 100% (3/3) of patients with lcnec and 27.8% (5/18) of those with ac. common sites of metastasis included lung, lymph node, brain, lumbar spine, mediastinum, bone, and liver. the cause of death was tumor progression in 100% (3/3) of patients with lcnec and 16.7% (3/18) of those with ac. all 21 cases were divided into two types according to the who criteria : ac (n=18) and large cell nec (n=3). in addition the tumors were described as a large, soft and whitish - tan to brown mass with the greatest diameter of 1.8 - 21 cm (fig. the mean size of the mass was 6.81 cm and 8.27 cm in cases of ac and lcnec, respectively. some of the tumors showed an infiltrative growth pattern to the adjacent organs such as the pericardium. on cut sections, the tumors revealed focal areas of hemorrhage and necrosis. in particular, lcnec showed extensive areas of necrosis, which is the hallmark of poorly - differentiated neuroendocrine thymic carcinomas (fig. ac showed a punctate necrosis with a maximum of 13 (0 - 10) mitoses or it was devoid of necrosis but showed a proliferation with a mitotic rate of up to 9 mitoses per 10 hpf (fig. thymic lcnec was defined as a poorly - differentiated non - small cell nec with a mitotic rate of > 10 mitoses per 10 hpf. 11 frequently revealed lymphatic emboli and six, undergoing a lymph node dissection, demonstrated a nodal metastasis. finally, tumors associated with ectopic hormone secretion were also found to share the characteristic features of cytoplasm. neuroendocrine differentiation was revealed by positive immunohistochemistry for neuroendocrine markers including synaptophysin, chromogranin and cd56. in all the cases, there was no definite difference in the degree of immunohistochemical staining between cases of lcnec and those of ac (fig. but the degree of immunohistochemical staining was lower in cases of lcnec as compared with those of ac (fig. in addition, we also performed an immunohistochemical staining of acth for tumors with ectopic acth secretion. thymic epithelial tumors, predominantly or exclusively composed of neuroendocrine cells, are classified as necs of the thymus. thymic necs are rare, constituting 2 - 5% of thymic epithelial tumors.6 there is a sufficient amount of statistical data for pulmonary necs. but there is a paucity of data about the clinicopathologic correlations among the thymic necs. considering this, the first edition of the who classification of tumors of the thymus suggests that pathologists classify thymic neuroendocrine tumors based on the same criteria as necs of the lung.6 it is known that a vast majority of carcinoids in the thymus would correspond to acs if the same criteria are applied as the lung.6 in addition, several studies have reported that a substantial number of cases of ac are associated with ectopic hormone secretion.5,11,12 in the current study, our clinical series of patients (n=21) comprised 18 cases of ac and 3 cases of lcnec. furthermore, these three cases of ac were accompanied by cushing 's syndrome. it is well known that ac shows a better clinical behavior than lcnec. however there were two patients with ac, associated with ectopic hormone secretion, both of whom died of tumor progression and metastasis although they had a small - sized tumor and low mitotic counts. furthermore, there was another patient with ectopic hormone secretion who developed a pulmonary metastasis. in conclusion, our results indicate that acs associated with ectopic hormone secretion show a poorer prognosis as compared with conventional ones. in our series this implies that tc is a very rare entity unlike the pulmonary necs. over the past few decades, a three - tiered pathologic classification has been used and necs are classified into low grade, intermediate - grade, and high - grade ones.13,14 based on this classification system, some previously diagnosed cases of low- or intermediate - grade necs actually correspond to ac. those who were diagnosed with low or intermediate grade neuroendocrine carcinoma underwent post - operative ccrt while only post - operative rt was added to ac patients according to the current treatment trend. with regard to the clinical behavior of necs, our results are in agreement with the previous reports that thymic neuroendocrine tumors show a poor prognosis. during the follow - up period, 100% (3/3) of patients with lcnec and 16.7% (3/18) of those with however no statistical analysis has been attempted to determine whether the classification of tumors is not dependent on a prognosis of patients because of a limited number of cases enrolled in the current study. thymic neuroendocrine tumors are histologically classified according to tumor differentiation, presence or absence of necrosis and mitotic counts.6 acs are therefore classified as a carcinoid tumor having architectural features of the classic type but exhibiting 2 - 10 mitoses per 10 hpf and/or foci of necrosis. ac is actually classified as a group of well - differentiated nec. in our series, cases 12, 15, and 16 were classified as an ac despite a high mitotic index of > 10 mitoses per 10 hpf. in these cases, high mitotic counts may pose a diagnostic challenge to pathologists. however, tumor differentiation is one of the most important factors by which pathologists can make a differential diagnosis of carcinoid tumor from scnec and lcnec. according to the who criteria, both tc and ac are classified a well - differentiated tumor and both scnec and lcnec are classified a poorly differentiated tumor. cases 12, 15, and 16 showed the similar characteristics of carcinoid tumor and they had a lack of the typical histologic characteristics of lcnec such as extensive necrosis or poorly - differentiated pattern. not including high mitotic counts, the histologic findings did not seem to correlate with a poorly - differentiated carcinoma which usually reveals aggressive clinical behavior. in addition, some tumors exhibited large tumor size and mitotic counts varied depending on the part of the tumor. considering all lcnec patients died of the tumor, the fact those patients were alive without evidence of relapse might support out diagnosis. it can also be presumed that the current who classification needs a more detailed explanation because it might pose a diagnostic challenge to pathologists. it has been reported that even " innocent " looking and encapsulated carcinoids bear a significant risk for recurrence, metastasis and tumor - associated death.6 on the other hand, one of the recent studies has shown a better prognosis of atypical thymic carcinoids as compared to pulmonary carcinoids.6 in our series, 16.7% (3/18) of patients with ac patients died of tumor progression, two of whom had a concurrent presence of ectopic hormone secretion. further, regarding the view that thymic neuroendcrine tumors are clinically more aggressive than morphologically identical neuroendocrine tumors of the lung, we also observed that primary thymic lcnecs tended to exhibit less neuroendocrine differentiation, such as trabeculae, nesting, rossettes and perilobular palisading patterns, which is commonly seen in pulmonary lcnecs. this may suggest that thymic lcnecs are higher - grade tumors than pulmonary lcnecs. in conclusion, regarding the tumor classification, our results showed that a vast majority of carcinoids in the thymus correspond to acs. some cases of ac exhibited a large size, solid pattern and they showed aggressive clinical behavior, which highlights the spectrum of histologic appearances of thymic necs. | backgroundthymic neuroendocrine carcinomas (necs) are uncommon, for which there is no established information available because of a limited number of epidemiological study in asia.methodswe reviewed 21 cases of surgically resected thymic necs, and evaluated their pathological and clinical features.resultsit showed male predominance (male / female ratio, 15/6) with wide age range from 20 to 72 years (mean age, 49 years). all 21 cases were divided into two types according to the world health organization criteria : atypical carcinoid (n=18) and large cell nec (n=3). three cases of atypical carcinoid (ac) were associated with ectopic cushing 's syndrome. all the patients (3/3) with large cell nec (3/3) and 16.7% (3/18) of those with ac died of tumor progression. common sites of metastasis included lung, lymph node, brain, lumbar spine, mediastinum, bone, and liver.conclusionsin conclusion, thymic neuroendocrine tumors carry a poor prognosis. regarding the tumor classification, our results showed that a vast majority of carcinoids in the thymus correspond to acs. in addition, our results also indicate that typical carcinoid is a very rare entity. some cases of ac exhibited a large size, solid pattern and they showed aggressive clinical behavior, which highlights the spectrum of histologic appearances of thymic necs. |
intake data from adults 19 + years of age (y) participating in the nhanes 20032006 who self - identified as nhw (n=4,811), nhb (n=2,062), or ma (1,950) (26, 27) were obtained from a single in - person 24-h dietary recall interview using an automated multiple - pass method (28, 29). data from pregnant or lactating women (n=632) or data judged incomplete or unreliable (n=71) by the food surveys research group were excluded from these analyses. this research did not require institutional review board review, as it does not constitute original research on human subjects (31). the usda dietary sources of nutrients (dsn) database (32) was used to define food groups. the dsn database was originally developed for use with the continuing survey of food intake by individuals 19941996. for this study, the dsn database was updated for application to more recent food consumption survey releases. the food grouping and disaggregation rules used to update the dsn database were similar to those methods reported by others who have described dsn in the american diet (3335). the more than 130 dsn food groups were collapsed into 51 categories, as described previously and with a previously published table showing the food categories (8). this is an aggregation level consistent with that used by the usda food surveys research group when defining food groups (36). disaggregation of foods allows for a more accurate assessment of nutrients in food groups (35). categories of food include survey foods or ingredients of recipes for home- or restaurant - prepared foods that were disaggregated. none of the baked goods (neither home - baked nor commercially prepared baked goods) and none of the manufactured foods were disaggregated. if foods were not disaggregated in the dsn database, the food and nutrient database for dietary studies (37) (fndds) codes were assigned to dsn food groups (fndds versions 2.0 and 3.0 were used in 20032004 and 20052006 nhanes, respectively). the ingredients of disaggregated survey food recipes (coded using the usda nutrient database for standard reference [sr ] food codes) were linked to the appropriate food composition databases using the sr - link file of the fndds [versions 2.0 and 3.0 link sr releases 18 and 20, respectively (38) ]. recipe calculations were performed to determine proportions of the disaggregated survey foods assigned to the 51 dsn food groups. ingredients of a recipe for a grain - based mixture such as spaghetti and meat sauce, for example, included spaghetti, tomato sauce / paste, carrots, onions, garlic, olive oil, ground meat, and cheese, which were each classed to respective dsn groups. ingredients added in food preparation, such as salt added to the sauce, were disaggregated to separate the sodium that was added to the sauce from that occurs naturally in foods. analyses were conducted using sudaan 9.0.3 (2007 ; research triangle institute, research triangle park, nc). appropriate weighting factors were used in analyses to adjust for oversampling of selected groups, survey non - response of some individuals, and day of the week the interview was conducted (39). mean and standard errors of energy and nutrient intakes from the total diet and from each food group were determined using proc descript of sudaan. using the mean intake data, the average percentage of total dietary intake of energy and nutrients contributed from each food grouping tests for comparisons of means of energy and nutrients among three ethnic groups were performed using bonferroni 's correction (p<0.05/3 = p<0.0167) to adjust the significance level for multiple comparisons. intake data from adults 19 + years of age (y) participating in the nhanes 20032006 who self - identified as nhw (n=4,811), nhb (n=2,062), or ma (1,950) (26, 27) were obtained from a single in - person 24-h dietary recall interview using an automated multiple - pass method (28, 29). data from pregnant or lactating women (n=632) or data judged incomplete or unreliable (n=71) by the food surveys research group were excluded from these analyses. this research did not require institutional review board review, as it does not constitute original research on human subjects (31). the usda dietary sources of nutrients (dsn) database (32) was used to define food groups. the dsn database was originally developed for use with the continuing survey of food intake by individuals 19941996. for this study, the dsn database was updated for application to more recent food consumption survey releases. the food grouping and disaggregation rules used to update the dsn database were similar to those methods reported by others who have described dsn in the american diet (3335). the more than 130 dsn food groups were collapsed into 51 categories, as described previously and with a previously published table showing the food categories (8). this is an aggregation level consistent with that used by the usda food surveys research group when defining food groups (36). disaggregation of foods allows for a more accurate assessment of nutrients in food groups (35). categories of food include survey foods or ingredients of recipes for home- or restaurant - prepared foods that were disaggregated. none of the baked goods (neither home - baked nor commercially prepared baked goods) and none of the manufactured foods were disaggregated. if foods were not disaggregated in the dsn database, the food and nutrient database for dietary studies (37) (fndds) codes were assigned to dsn food groups (fndds versions 2.0 and 3.0 were used in 20032004 and 20052006 nhanes, respectively). the ingredients of disaggregated survey food recipes (coded using the usda nutrient database for standard reference [sr ] food codes) were linked to the appropriate food composition databases using the sr - link file of the fndds [versions 2.0 and 3.0 link sr releases 18 and 20, respectively (38) ]. recipe calculations were performed to determine proportions of the disaggregated survey foods assigned to the 51 dsn food groups. ingredients of a recipe for a grain - based mixture such as spaghetti and meat sauce, for example, included spaghetti, tomato sauce / paste, carrots, onions, garlic, olive oil, ground meat, and cheese, which were each classed to respective dsn groups. ingredients added in food preparation, such as salt added to the sauce, were disaggregated to separate the sodium that was added to the sauce from that occurs naturally in foods. analyses were conducted using sudaan 9.0.3 (2007 ; research triangle institute, research triangle park, nc). appropriate weighting factors were used in analyses to adjust for oversampling of selected groups, survey non - response of some individuals, and day of the week the interview was conducted (39). mean and standard errors of energy and nutrient intakes from the total diet and from each food group were determined using proc descript of sudaan. using the mean intake data, the average percentage of total dietary intake of energy and nutrients contributed from each food grouping tests for comparisons of means of energy and nutrients among three ethnic groups were performed using bonferroni 's correction (p<0.05/3 = p<0.0167) to adjust the significance level for multiple comparisons. nhb consumed less energy than ma ; however, energy intake for nhw was not different from the other two ethnic groups (table 1). mas consumed more energy from grain products than nhw and nhb. in all ethnic groups, meat / poultry / fish (mpf) contributed the next highest percentage of energy : nhw, nhb, and ma. nhw had lower energy intake from mpf than nhb ; however, there were no other differences. for nhw, energy intake from desserts and sweets ranked third and was significantly higher than that seen in nhb and ma. for nhb and ma, non - dairy beverages were the third ranking energy source and intake was significantly higher than that seen in nhw. dairy products were the fifth highest source of energy for nhw and ma and the sixth highest for nhb ; energy intake from dairy products was lowest in nhb. the only difference was that nhw had a higher energy intake from vegetables than nhb. mean energy (kj) intake from disaggregated food groups, and ranked percentage contribution to daily energy intake : adults 19 + years by ethnic group source : nhanes, 20032006, day 1, all aged 19 years and older (n=8,823). mean sfa intake was higher in nhw than nhb ; however, neither was different from intake by ma (table 2). for nhw, dairy products provided the highest number of grams of sfa in the diet ; for nhb and ma, dairy products provided the second highest number of grams of sfa. for nhw, mpf provided the second highest number of grams of sfa in the diet ; mpf was the highest source of grams of sfa in the diets of nhb and ma. nhw consumed significantly fewer grams of sfa from the mpf group than nhb, but not than ma. in all three ethnic groups, fats and oils contributed the third highest number of grams of sfa : nhw, nhb, and ma ; nhw had the highest intake of sfa from this food group. desserts and grain products were also major contributors of sfa in the diet in all three ethnic groups. mean saturated fatty acid (g) intake from disaggregated food groups, and ranked percentage contribution to daily saturated fatty acid intake : adults aged 19 + years by ethnic group source : nhanes, 20032006, day 1, all aged 19 years and older (n=8,823). defined as 100% fruit juice. the only significant difference in added sugars intake was that intake from nhw was lower than that of nhb (table 3). for nhw, desserts and sweets contributed the highest number of grams of added sugars to the diet ; for nhb and ma this food group contributed the second highest number of grams of added sugars. ma consumed the fewest number of grams from this food group when compared with nhw and nhb. for nhb and ma, beverages contributed the highest number of grams of added sugars ; for nhw, beverages contributed the second highest number of grams. both nhb and ma consumed significantly more grams of added sugars from beverages than nhw. mean added sugars (g) intake from disaggregated food groups, and ranked percentage contribution to daily added sugars intake : adults aged 19 + years by ethnic group source : nhanes, 20032006, day 1, all aged 19 years and older (n=8,823). nhw consumed significantly more sodium than nhb (table 4). in all three ethnic groups, other foods contributed the highest amounts of sodium to the diet, with ma consuming the highest amount from this food group compared with nhw or nhb. in all three ethnic groups, grain products were the next highest contributor of sodium to the diet, although no differences were observed among the groups. mean sodium (mg) intake from disaggregated food groups, and ranked percentage contribution to daily sodium intake : adults 19 + years by ethnic group source : nhanes, 20032006, day 1, all aged 19 years and older (n=8,823). defined as 100% fruit juice. nhb had the lowest mean dietary fiber intake of all three ethnic groups, and ma had the highest of all groups (table 5). grain products contributed the most fiber to the diet of all three groups ; ma had the highest dietary fiber intake from grain and nhb had the lowest. nhw consumed more dietary fiber from vegetables than nhb, but not more than ma ; ma consumed more fiber from vegetables than nhb. for nhw and nhb, fruit provided the third highest source of fiber. nhw had higher dietary fiber intake from fruit than nhb ; however, ma had the highest dietary fiber intake from fruit. ma consumed higher amounts of dietary fiber from eggs, legumes, nuts, and seeds than either nhw or nhb, which was the lowest. mean dietary fiber (g) intake from disaggregated food groups, and ranked percentage contribution to daily dietary fiber intake : adults 19 + years by ethnic group source : nhanes, 20032006, day 1, all aged 19 years and older (n=8,823). vitamin d intake (table 6) was highest in nhw when compared with nhb or ma ; nhb intake was not significantly different from ma intake. dairy products were the principal source of vitamin d in the diet for all three ethnic groups, with nhb having the lowest intake ; nhw intake was not different from ma intake. intake was lowest in ma ; intake in nhw was not different from that of nhb. mean vitamin d (g) intake from disaggregated food groups, and ranked percentage contribution to daily vitamin d : adults 19 + years by ethnic group source : nhanes, 20032006, day 1, all aged 19 years and older (n=8,823). n / a values result from a non - definable number due to division by zero. calcium intake (table 7) was lowest in nhb ; calcium intake was not different in nhw when compared with intake from ma. in all three ethnic groups, nhw had the highest intake of calcium from dairy products and nhb had the lowest. mean calcium (mg) intake from disaggregated food groups, and ranked percentage contribution to daily calcium : adults 19 + years by ethnic group source : nhanes, 20032006, day 1, all aged 19 years and older (n=8,823). nhw intake of potassium was higher than nhb ; no other significant differences were observed (table 8). vegetables were the highest ranking source of dietary potassium in the diet of nhw and ma ; nhw consumed significantly more dietary potassium from vegetables than nhb. for nhb, mpf was the highest ranking source of dietary potassium ; however, intake from mpf was not higher than nhw or ma. for ma, eggs, legumes, nuts, and seeds were a major source of potassium ; intake of potassium in this group was higher than nhw and nhb. grain products, fruit, dairy, and beverages also contributed relatively higher amounts of potassium to the diet in all three ethnic groups. mean potassium (mg) intake from disaggregated food groups, and ranked percentage contribution to daily potassium intake : adults aged 19 + years by ethnic group source : nhanes, 20032006, day 1, all aged 19 years and older (n=8,823). nhb consumed less energy than ma ; however, energy intake for nhw was not different from the other two ethnic groups (table 1). mas consumed more energy from grain products than nhw and nhb. in all ethnic groups, meat / poultry / fish (mpf) contributed the next highest percentage of energy : nhw, nhb, and ma. nhw had lower energy intake from mpf than nhb ; however, there were no other differences. for nhw, energy intake from desserts and sweets ranked third and was significantly higher than that seen in nhb and ma. for nhb and ma, non - dairy beverages were the third ranking energy source and intake was significantly higher than that seen in nhw. dairy products were the fifth highest source of energy for nhw and ma and the sixth highest for nhb ; energy intake from dairy products was lowest in nhb. the only difference was that nhw had a higher energy intake from vegetables than nhb. mean energy (kj) intake from disaggregated food groups, and ranked percentage contribution to daily energy intake : adults 19 + years by ethnic group source : nhanes, 20032006, day 1, all aged 19 years and older (n=8,823). mean sfa intake was higher in nhw than nhb ; however, neither was different from intake by ma (table 2). for nhw, dairy products provided the highest number of grams of sfa in the diet ; for nhb and ma, dairy products provided the second highest number of grams of sfa. for nhw, mpf provided the second highest number of grams of sfa in the diet ; mpf was the highest source of grams of sfa in the diets of nhb and ma. nhw consumed significantly fewer grams of sfa from the mpf group than nhb, but not than ma. in all three ethnic groups, fats and oils contributed the third highest number of grams of sfa : nhw, nhb, and ma ; nhw had the highest intake of sfa from this food group. desserts and grain products were also major contributors of sfa in the diet in all three ethnic groups. mean saturated fatty acid (g) intake from disaggregated food groups, and ranked percentage contribution to daily saturated fatty acid intake : adults aged 19 + years by ethnic group source : nhanes, 20032006, day 1, all aged 19 years and older (n=8,823). defined as 100% fruit juice. the only significant difference in added sugars intake was that intake from nhw was lower than that of nhb (table 3). for nhw, desserts and sweets contributed the highest number of grams of added sugars to the diet ; for nhb and ma this food group contributed the second highest number of grams of added sugars. ma consumed the fewest number of grams from this food group when compared with nhw and nhb. for nhb and ma, beverages contributed the highest number of grams of added sugars ; for nhw, beverages contributed the second highest number of grams. both nhb and ma consumed significantly more grams of added sugars from beverages than nhw. mean added sugars (g) intake from disaggregated food groups, and ranked percentage contribution to daily added sugars intake : adults aged 19 + years by ethnic group source : nhanes, 20032006, day 1, all aged 19 years and older (n=8,823). nhw consumed significantly more sodium than nhb (table 4). in all three ethnic groups, other foods contributed the highest amounts of sodium to the diet, with ma consuming the highest amount from this food group compared with nhw or nhb. in all three ethnic groups, grain products were the next highest contributor of sodium to the diet, although no differences were observed among the groups. mean sodium (mg) intake from disaggregated food groups, and ranked percentage contribution to daily sodium intake : adults 19 + years by ethnic group source : nhanes, 20032006, day 1, all aged 19 years and older (n=8,823). defined as 100% fruit juice. nhb had the lowest mean dietary fiber intake of all three ethnic groups, and ma had the highest of all groups (table 5). grain products contributed the most fiber to the diet of all three groups ; ma had the highest dietary fiber intake from grain and nhb had the lowest. nhw consumed more dietary fiber from vegetables than nhb, but not more than ma ; ma consumed more fiber from vegetables than nhb. for nhw and nhb, nhw had higher dietary fiber intake from fruit than nhb ; however, ma had the highest dietary fiber intake from fruit. ma consumed higher amounts of dietary fiber from eggs, legumes, nuts, and seeds than either nhw or nhb, which was the lowest. mean dietary fiber (g) intake from disaggregated food groups, and ranked percentage contribution to daily dietary fiber intake : adults 19 + years by ethnic group source : nhanes, 20032006, day 1, all aged 19 years and older (n=8,823). vitamin d intake (table 6) was highest in nhw when compared with nhb or ma ; nhb intake was not significantly different from ma intake. dairy products were the principal source of vitamin d in the diet for all three ethnic groups, with nhb having the lowest intake ; nhw intake was not different from ma intake. intake was lowest in ma ; intake in nhw was not different from that of nhb. mean vitamin d (g) intake from disaggregated food groups, and ranked percentage contribution to daily vitamin d : adults 19 + years by ethnic group source : nhanes, 20032006, day 1, all aged 19 years and older (n=8,823). n / a values result from a non - definable number due to division by zero. values in bold face are significantly different. calcium intake (table 7) was lowest in nhb ; calcium intake was not different in nhw when compared with intake from ma. in all three ethnic groups, nhw had the highest intake of calcium from dairy products and nhb had the lowest. mean calcium (mg) intake from disaggregated food groups, and ranked percentage contribution to daily calcium : adults 19 + years by ethnic group source : nhanes, 20032006, day 1, all aged 19 years and older (n=8,823). nhw intake of potassium was higher than nhb ; no other significant differences were observed (table 8). vegetables were the highest ranking source of dietary potassium in the diet of nhw and ma ; nhw consumed significantly more dietary potassium from vegetables than nhb. for nhb, mpf was the highest ranking source of dietary potassium ; however, intake from mpf was not higher than nhw or ma. for ma, eggs, legumes, nuts, and seeds were a major source of potassium ; intake of potassium in this group was higher than nhw and nhb. grain products, fruit, dairy, and beverages also contributed relatively higher amounts of potassium to the diet in all three ethnic groups. mean potassium (mg) intake from disaggregated food groups, and ranked percentage contribution to daily potassium intake : adults aged 19 + years by ethnic group source : nhanes, 20032006, day 1, all aged 19 years and older (n=8,823). faced with a high prevalence of overweight and obesity, and the difficulty balancing dietary recommendations with energy intake, it was important to identify principal food sources of energy and examine their potential nutrient contributions to the diet in different ethnic groups. this is the first study to examine specific foods and their contribution to energy, nutrients of public health concern, and nutrients to limit in three ethnic groups in the united states. this study, along with previous studies (8, 34, 35), showed that energy - dense, nutrient - poor foods were major contributors to dietary energy intake, making it difficult for individuals to achieve nutrient recommendations without exceeding energy limits. the study also showed significant differences in energy intake and in the food sources contributing to energy and nutrient intakes among nhb, nhw, and ma. in all three ethnic groups, dairy products and mpf were the two major sources of sfa in the diet, likely reflecting the choice of full - fat dairy products and fatty meats. in nhb, sfa from dairy products was lower than in nhw and ma ; however, this is likely due to ma and nhb not consuming the recommended intake of dairy foods (41), rather than selection of low - fat options. although the role of sfa in heart disease (42) and type 2 diabetes (43) has recently been questioned, it is still premature to change recommendations. however, the higher prevalence of these diseases in ma and nhb merits further study of dietary and other potential causes (44) so that ethnic - specific interventions can be devised to reduce the risk of chronic disease (45). ma had the highest intake of fiber ; however, mean intake of dietary fiber in each ethnic group was well below the recommended intakes for adults (46), although the results were not inconsistent with other studies (47). although grain products were the highest contributor of dietary fiber to the diet, it is unlikely that the grains consumed by those participating in this study were whole grains since other studies have shown that intake of whole grains is very low (48). fruit, vegetables, nut, and legumes were the other sources of fiber in the diet. fruit contributed very little dietary fiber to any ethnic group, especially to the diets of nhb. the total fiber intake from fruit was less than that found in one medium apple (3.4 g) (36), suggesting that the fruit recommendation was not being met (6). vegetables contributed more fiber than fruit to the diet ; however, amounts were small, especially among nhb. fiber intake from legumes was highest in ma, confirming results from a previous study that showed ma and other hispanics (49) were more likely to consume legumes, a rich source of dietary fiber, than not. part of the failure to meet the dietary fiber recommendations may be a lack of awareness of fiber - rich foods and of the dietary recommendations (5, 50). all three ethnic groups studied consumed less than half of the recommended intake of 10 g of vitamin d / day (52) and less than the recommended intakes of 1,0001,200 mg calcium / day (52). the nutrient contribution of milk and dairy products plays an important role in helping americans meet recommendations for these nutrients and for potassium (53). although all ethnic groups had average intakes below recommendations, nhb had the lowest intake of these nutrients. this confirms results from previous studies (54) and is likely the result of the low intake of milk and other dairy foods by nhb (10). a major barrier to dairy consumption among nhb and ma is actual or perceived lactose intolerance (41). many adults who are lactose intolerant can consume up to one cup of milk at a time without symptoms (55) ; however, those that are lactose intolerant need to understand this and to understand the importance of dairy foods in the diet. strategies are available to help those that are lactose intolerant or perceive themselves to be, to consume more dairy products (55). it is important to recognize that in addition to lactose intolerance that cultural food and dietary practices also contribute to low dairy intake (55) ; however, it is very difficult to meet the recommendations for vitamin d, calcium, and potassium without dairy foods in the diet. excess dietary sodium, especially when coupled with low potassium intake, has been linked with hypertension, cardiovascular disease, and osteoporosis (22, 56, 57). mean sodium intakes were high in all ethnic groups, but were more than double the recommendation of 1,500 mg for nhb promulgated by the 2010 dga (3). control of sodium intake is critical since nhb adults have one of the highest prevalence rates for hypertension in the world, with more than 40% of nhb adults having hypertension (19). nhb adults also have a higher prevalence of stroke and stroke mortality than nhw (19). it is critical that individuals make good food choices to help reduce their risk of hypertension, and ultimately stroke and chronic kidney disease ; the latter are also found disproportionally among nhb (19, 58). the fact that grain products were the leading source of dietary sodium in all three ethnic groups was not surprising, since bread has been shown to be a major contributor of sodium to the diet (8). nhb also consumed less than half the recommended intake of potassium ; although the nhw and ma consumed slightly more potassium, mean intake fell far short of the recommended level of 4,700 mg / day for adults (53). food choices contributing to potassium included vegetables, meat, dairy, grains, and fruit ; however, not enough of these foods was consumed and it was clear that recommendations for these food groups were not met. reducing dietary sodium and increasing fruit, vegetables, and dairy products to increase potassium intake are major public health objectives (3). a dietary approaches to stop hypertension diet, high in potassium, calcium, and magnesium and low in sodium has been shown to be more effective in lowering blood pressure in nhb than in nhw (59). overall, nutrient intake of nutrients to limit and those of public health concern is poor, but intake of minorities is generally poorer than that of nhw. questions not answered in this paper revolve the intake of hispanics other than ma, the influence of acculturation (60, 61) on dietary intake, and barriers to intake for all ethnic groups. but what is clear is that ethnic interventions need to be designed to improve overall intake of all groups. understanding of barriers and enabling factors to consumption of specific foods need further study to design these interventions. food grouping can have a major influence on the ranked order of food groups ; thus, caution is advised when comparing these data to previous reports if there were differences in the level of aggregation (i.e. the number of food groups) or disaggregation procedures used to include ingredients in food groups. foods were grouped and the groups were collapsed, making it difficult to study the effect of specific foods by the different ethnic groups. study outcomes are based on self - reported data that tend to underestimate energy intake (62). another limitation is the possible under - reporting of food intake by some groups, including overweight and obese individuals which have been previously documented in other studies (63). however, the mean of the intake distribution drawn from a large, representative sample of a group is not affected by day - to - day variation, and since the contribution of food sources is based on mean intake data from nhanes, the use of a single 24-h recall was appropriate (64). further, because the food grouping in the usda dsn database does not include ingredients of manufactured foods, disaggregated foods represent mixtures that are prepared from recipes. the usda reduces the sodium content of mixtures if the respondent never, rarely, or occasionally uses salt in cooking, and the food was prepared at home ; therefore, a large portion of the salt was added to recipes for foods prepared by restaurants, schools, and other establishments (data not shown). finally, the updated vitamin d database that usda released (65) was appropriate for use with the 20052006 nhanes dietary intake data ; however, since vitamin d intake from foods consumed in 20032004 was determined using the updated food composition data, the 20032004 intake data may not have been representative of that time period. food grouping can have a major influence on the ranked order of food groups ; thus, caution is advised when comparing these data to previous reports if there were differences in the level of aggregation (i.e. the number of food groups) or disaggregation procedures used to include ingredients in food groups. foods were grouped and the groups were collapsed, making it difficult to study the effect of specific foods by the different ethnic groups. study outcomes are based on self - reported data that tend to underestimate energy intake (62). another limitation is the possible under - reporting of food intake by some groups, including overweight and obese individuals which have been previously documented in other studies (63). however, the mean of the intake distribution drawn from a large, representative sample of a group is not affected by day - to - day variation, and since the contribution of food sources is based on mean intake data from nhanes, the use of a single 24-h recall was appropriate (64). further, because the food grouping in the usda dsn database does not include ingredients of manufactured foods, disaggregated foods represent mixtures that are prepared from recipes. the usda reduces the sodium content of mixtures if the respondent never, rarely, or occasionally uses salt in cooking, and the food was prepared at home ; therefore, a large portion of the salt was added to recipes for foods prepared by restaurants, schools, and other establishments (data not shown). finally, the updated vitamin d database that usda released (65) was appropriate for use with the 20052006 nhanes dietary intake data ; however, since vitamin d intake from foods consumed in 20032004 was determined using the updated food composition data, the 20032004 intake data may not have been representative of that time period. any variation in food composition data meeting nutrient intake recommendations while staying within energy needs has proven to be challenging for many americans, and as a result, individuals may be overweight or obese while being undernourished. this study suggested that nutrient intake by nhw, nhb, and ma could be improved by improving food choices. ideally, foods containing a constellation of the nutrients of public health concern (3) should be selected without unduly increasing energy intake or contributing to intakes of added sugars, sfa, or sodium. thus, consumption of whole grains, fruit, vegetables, and low - fat dairy should be increased, calling for profound changes in the diet of all three ethnic groups. programs tailored to each ethnic group should be developed and provided to these groups. in these programs, specific barriers and enablers support was provided by the national dairy council and administered by the dairy research institute and the usda agricultural research service through specific cooperative agreement 58 - 6250 - 6 - 003. the funding sources had no input into the study design, the interpretation of the results, or drafting the manuscript. | backgroundidentification of current food sources of energy and nutrients among us non - hispanic whites (nhw), non - hispanic blacks (nhb), and mexican american (ma) adults is needed to help with public health efforts in implementing culturally sensitive and feasible dietary recommendations.objectivethe objective of this study was to determine the food sources of energy and nutrients to limit [saturated fatty acids (sfa), added sugars, and sodium ] and nutrients of public health concern (dietary fiber, vitamin d, calcium, and potassium) by nhw, nhb, and ma adults.designthis was a cross - sectional analysis of a nationally representative sample of nwh (n=4,811), nhb (2,062), and ma (n=1,950) adults 19 + years. the 20032006 nhanes 24-h recall (day 1) dietary intake data were analyzed. an updated usda dietary source nutrient database was developed using current food composition databases. food grouping included ingredients from disaggregated mixtures. mean energy and nutrient intakes from food sources were sample - weighted. percentages of total dietary intake contributed from food sources were ranked.resultsmultiple differences in intake among ethnic groups were seen for energy and all nutrients examined. for example, energy intake was higher in ma as compared to nhb ; sfa, added sugars, and sodium intakes were higher in nhw than nhb ; dietary fiber was highest in ma and lowest in nhb ; vitamin d was highest in nhw ; calcium was lowest in nhb ; and potassium was higher in nhw as compared to nhb. food sources of these nutrients also varied.conclusionidentification of intake of nutrients to limit and of public health concern can help health professionals implement appropriate dietary recommendations and plan interventions that are ethnically appropriate. |
mycosis fungoides (mf) is the commonest variant of primary cutaneous t cell lymphoma, accounting for almost 50% of all primary cutaneous lymphomas [1, 2 ]. typically, neoplastic t cells localize to the skin and produce patches, plaques, tumors or erythroderma. it is characterized by a relatively consistent constellation of clinical, histologic, immunophenotypic and molecular aberrations. mf typically manifests as an indolent cutaneous eruption with erythematous scaly patches or plaques and may progress to generalized erythroderma, cutaneous tumors, or extracutaneous involvement. tumors, however, develop only in a minority of patients from patches, plaques, or de novo. leonine facies results from malignant t cell infiltration leading to extensive thickening and skin fold accentuation. classically, mf shows a cd2 +, cd3 +, cd4 +, cd8, cd30, cd45ro+ immunophenotype. rare cases with a t suppressor cd8+/cd4 phenotype have been reported [8, 9 ]. this report describes the clinical, histopathologic and immunohistochemical features of a patient with mf mimicking psoriasis vulgaris. a 52-year - old white man was referred to the department of dermatology, university of palermo (italy) with a six - month history of psoriasiform plaques with superficial erosion and fissuring on the palms (fig. red violet and infiltrated plaques with moderate hyperkeratosis and scaling developed on the scalp and upper limps accompanied by itching. four months prior to referral he developed red to violaceous nodules which coalesced to form large plaques on the face. skin examination at the time of hospital admission, approximately six months from the onset of his illness, revealed reddish brown ulcerated tumors and indurated erythematous plaques with superficial erosion on the forehead, glabella, cheeks (fig. 1 a), scalp and neck. in addition, numerous plaques, accompanied by itching, were found on trunk and extremities in a generalized distribution. plaques appeared as sharply delineated, scaly, elevated lesions that were dusky red to violaceous and variably indurated (fig the blood count showed normal red cell count and a slight leukocytosis (11.42 10/l) comprised of 74.6% neutrophils, 7.2% eosinophils, 0.7% basophils, 11.1% lymphocytes and 6.4% monocytes. further laboratory investigations of the blood showed alpha 1-globulin 5.25% and an erythrocyte sedimentation rate of 44 mm / h. a biopsy specimen was obtained from affected areas of the face, fixed in 10% buffered formalin and embedded in paraffin. for routine histology chest x - ray, total body computed tomography scanning and excisional biopsy of the inguinal lymph node were obtained. review of the face biopsy on hematoxylin and eosin showed a dense, partly subepidermal and band - like, partly perivascular and periadnexal dermal infiltrate (fig. 2 a) containing a high number of atypical lymphocytes, eosinophils, plasma cells and macrophages. many of the lymphocytes were pleomorphic, hyperchromatic, hyperconvoluted and had an increased cytoplasmic to nuclear ratio. mild acanthosis, hyperkeratosis, basal layer damage, edema and fibrosis of the papillary dermis and moderate proliferation of postcapillary venules were also seen. immunohistologically the malignant cells expressed a mature peripheral t cell phenotype (cd3 +, cd4 +, cd45ro+, cd8, cd20) (fig. total body computed tomography scanning was obtained documenting retroauricular, occipital, deep cervical lymphatic chain, submandibular, submental, axillary, subclavian and inguinal enlarged lymph nodes without visceral involvement. findings were consistent with tumor stage mf (stage iib, t3 n1 m0). mf is generally an indolent malignancy, with slow progression over years or even decades. the onset of the disease is often insidious and initial cutaneous symptoms may be difficult to distinguish from various inflammatory dermatoses such as chronic eczema, psoriasis, atopic dermatitis, etc. [8, 11 ]. as the disease progresses, patches may evolve over a variable period of time into infiltrated plaques with a more generalized distribution. tumors develop only in a minority of patients, although it is common to have patch, plaque and tumor lesions simultaneously on different parts of the body. visceral dissemination (lungs, spleen, liver, gastrointestinal tract) may develop subsequently. extracutaneous dissemination is directly correlated to the extent of cutaneous disease [7, 10 ]. we report a patient who first showed hyperkeratotic psoriasiform plaques on the palms and soles. involvement of the palms and/or soles occurs at some time during the course of the disease in 11.5% of patients. mycosis fungoides palmaris et plantaris is a rare variant of mf - type cutaneous t cell lymphoma predominantly affecting or initially presenting on the palms and/or soles. the prevalence of mycosis fungoides palmaris et plantaris among mf patients is 0.6%. in most cases the disease shows a benign course ; the lesions remain confined to the initial areas without dissemination and extracutaneous spread [12, 13 ]. in our patient tumors in the skin and lymphadenopathy developed rapidly over a two - month time span without lengthy preceding erythematous or plaque stages. the time interval between onset of skin changes and definite pathologic diagnosis of mf is 46 years [10, 14 ]. in the present case the lesions developed into a leonine clinical appearance of the face accompanied by lymphadenopathy, which reminded us the tumor stage mf. based on the early involvement of extracutaneous sites, the course of this patient may be more aggressive than typical patch - stage disease, in which mortality is not significantly different from matched controls. the presence of tumor stage disease is associated with a worse prognosis (stage iib : median survival of 2.9 years). mf is an uncommon lymphoma with a wide spectrum of clinicopathological manifestations and unpredictable course. diagnosis can be difficult in its early stages given the considerable overlap with more common benign dermatoses. | mycosis fungoides (mf) is an uncommon primary cutaneous lymphoma with a wide spectrum of clinicopathological manifestations. diagnosis can be difficult in its early stages given the considerable overlap with more common benign dermatoses. we report an unusual case of mf in a 52-year - old male presenting with psoriasiform plaques on the palms and the soles who rapidly developed additional lesions on the scalp, limps and trunk. punch biopsy of the face was obtained for routine histology and immunohistochemical stains. chest x - ray, total body computed tomography scanning and excisional biopsy of the inguinal lymph node were performed. review of the face biopsy revealed a diffuse dermal infiltrate containing a high number of atypical lymphocytes showing a cd3 +, cd4 +, cd45ro+, cd8, cd20 immunophenotype and epidermotropism. findings were consistent with tumor stage mf (stage iib, t3 n1 m0). we report a rare presentation of mf mimicking psoriasis vulgaris. |
the masectomized tissue of an 11-year - old female yorkshire terrier with large intestinal and abdominal tissues were obtained from a hwanggum animal medical center (daegu, korea) for evaluation of tumors. a laparatomy revealed masses that were black to reddish colored and 2 - 3 mm in diameter ; they were multifocally located on the serosal membrane of the large intestine and visceral peritoneum of the sublumbar region. the subcutaneous lesion of the right mammary gland showed a black to reddish mass with black to reddish petechia and ecchymosis. tissues samples for light microscopy were fixed in 10% neutral buffered formalin, paraffin embedded, and stained with hematoxylin and eosin (h&e). for immunohistochemistry, tissue sections were deparaffinized in xylene, rehydrated in graded alcohol series, incubated in a solution of 0.3% hydrogen peroxide in methanol for 30 minutes and microwaved at 750w for 10 min in 10 mmol / l citrate buffer, ph6.0. tissue sections were washed with phosphate - buffered saline (pbs) and then immunostained with primary antibody. the primary antibodies used recognized s100 protein (diluted in 1:200, dakocytomation, carpinteria, ca, usa), vimentin (diluted in 1:100, dakocytomation), protein kinase c- (pkc- ; diluted in 1:100, santa cruz biotechnogy, santa cruz, ca, usa). the avidin - biotin - peroxidase complex (abc) solution of the abc kit (vector laboratories, burlingame, ca, usa) with 3,3-diaminobenzidine (zymed laboratories, san francisco, ca, usa) was used for detection. the most extensively invaded lesions, skin and mammary glands showed abnormal hyperplasia of melanocytes in the dermal layers with hyperactivated epidermis melanocytes (figure 1a). melanocytic tumor cells had invaded into the dermal lymphatic channels (figure 1b) and micro vessels and were hyperpigmented in the dermal reticular layer and deeper layers. normal mammary glands were invaded and destroyed by tumor cells (figures 1c and 1d). pleomorphic round cells were arranged in sheets or clusters. there were also myoepithelial cells exhibiting hyperplasia. histologic evaluation of the mass in the sublumbar region revealed melanocytes intermingled with abdominal connective tissue and invasiveness of the micro - vessels (figures 1e and 1f). the neoplastic cells were fusiform and epithelioid in the peritoneum. in a section of the large intestinal mass, melanocytes had invaded the muscular layer and the metastasis of melanocytic tumor cells through the blood vessels (figures 1 g and 1h). immunohistochemically, mononuclear amelanotic cells were positive for cytoplasmic vimentin staining (figure 2a) and multinucleated cells containing melanin granules were positive for intranuclear and cytoplasmic s100 staining (figure 2b). the tumor cells are closely associated with the external surfaces of microvessels which expressed pkc- (figure 2c) and weakly positive for pkc-. our interpretation of these observations was that the multifocally invaded tumor, classified as a malignant melanoma, had at least three distinct lesions with very similar hyperplasia types and metastasis into blood vessels. melanoma may be one of the few neoplasms in animals whose location is an important prognostic indicator in its own right. there is no obvious relationship between histologic characteristics, including mitotic figures and pigmentation, and survival rate. the initial definitive diagnosis of melanoma is usually done by histologic evaluation, with cytopathology used as a screen before biopsy or as an adjunct to biopsy. morphology of the melanoma in the metastatic site is frequently similar to the primary tumor. vascular and/or lymphatic invasion has been thought to be a direct manifestation of metastasis for progress and prognostic factors. angiotropism in malignant melanoma has been observed in previous studies by conventional microscopy in routine tissue sections in human [13,16 - 19 ]. to our knowledge, there have been no reports of the origin of the potential mechanism of melanoma. we evaluated the expression of vimentin, s100 protein and pkc-. s100 staining characteristics have also been useful in the diagnosis of canine amelanotic melanomas. the -isozyme of pkc is widely expressed in tissues and abnormal levels are found in tissues such as proliferating pulmonary arteriolar smooth muscle cells and adventitial fibroblasts after chronic exposure to hypoxia in mammals [22 - 26 ]. in our study, we carefully had to distinguish angiotropism from entrapment or engulfment of vessels by the melanoma. pkc- activation appeared in the epithelium of microvascular channels and some tumor cells at the periphery of the vessels. there are a number of known activation signals for pkc- such as platelet - derived growth factor (pdgf), vascular endothelial growth factor (vegf), epidermal growth factor (egf) and fibroblast growth factor (fgf). although the latter feature may in fact constitute angiotropism and metastasis, we have no means at present to verify if and when such entrapment is biologically significant. from our results, there was also vascular / lymphatic invasion, which has been thought to be a direct manifestation of metastasis progress therefore, we consider the significance of recording vascular / lymphatic invasion until further studies are better able to sort out the problems of angiotropism versus a true intraluminal tumor. although our study involved only a single case of melanoma, it is of interest that the angiotropic melanoma presented had detailed, interesting findings of histopathology and immunohistochemistry. these results showed that black masses of the serosal membrane of the large intestine and peritoneum originated from the skin. the metastasis of most tumors usually occurs in the lung and other solid organs in clinical cases. this report describes the occurrence of an angiotropic metastatic melanoma in the serosal membranes of the large intestine rather than the internal regions of the solid organ. | an eleven - year - old spayed female yorkshire terrier presented with a sublumbar mass and upon ultrasonographic examination, was revealed to have a mammary gland tumor. black to reddish colored masses, located in the visceral peritoneum of the sublumbar region was observed on laparotomy with masectomy of the right side. in the laparotomy, we observed reddish masses multifocally located in the serosal membrane of the large intestine. histopathologic examination of the intestinal and abdominal mass showed highly invasiveness into the muscle and metastasis of melanocytic tumor cells through the blood vessels. the mammary glands showed abnormal hyperplasia of melanocytes, destruction of the normal glands by tumor cells and infiltration of some lymphocytes in the pool of melanocytic cells. we have identified a malignant melanoma containing an angiotumoral complex in which tumor cells occupied a pericytic location along the microvessels with intravasation determined by immunohistochemistry for s100 protein and protein kinase c-. histologic findings in this dog lead to a diagnosis of an angiotropic metastatic malignant melanoma. |
we provide some background on the main task at hand and relevant motivation, before the control methodology is elaborated. this procedure is successfully performed in human oocytes as well as other species such as mouse and bovine. first, a holding pipette is used to immobilize an individual oocyte with a slight suction. then an injection pipette (with outer diameter of about 8 m), which contains the sperm head to be injected, is forced into the cell. the piercing through the zona layer and the membrane needs to be achieved with minimal biological damage to facilitate rapid healing. a significant amount of research effort has been devoted towards developing microscopic instruments for icsi from this perspective alone. however, its piercing performance is successful only by using a small mercury droplet in the pipette tip [2, 3 ]. without this addition, undesirable lateral oscillations occur at the tip and severely hamper the performance of piercing. due to high toxicity of mercury, on the other hand, the piezo - assisted icsi procedure is forbidden in many biological laboratories. in recent years, an improved remedial technology, called rotationally oscillating drill (ros - drill), this technique shows comparable results to those obtained by the piezo - assisted icsi process, with one major difference, that ros - drill does not have the mercury problem. the injection pipette is connected to a small - precision micromotor which is controlled to track a desired sinusoidal trajectory : (1)d = adsin(2fdt), where ad is the oscillation amplitude (typically around 0.2 deg), and fd is the frequency (in the range of 500700 hz). these selections are based on a simple bandwidth analysis ; at this frequency range of external stimuli the cell membrane is not expected to follow the pipette tip motion. the ensuing relative rotational motion between the pipette and the cell membrane creates a clean piercing action, which facilitates the rapid healing of membrane after piercing. considerable amount of experimental effort has been invested to demonstrate the validity of ros - drill technology. it is shown that the success rate in icsi by ros - drill is comparable to that of piezo - assisted technology, provided that the pipette oscillations are maintained as close as possible to the desired trajectory in (1). many healthy mouse offspring are produced using the ros - drill methodology, as shown in figure 1(a). these biological tests are conducted by a group of experimentalists from the university of connecticut and the university of california, davis, usa. two outstanding requirements are noted in these experiments towards an acceptable ros - drill performance : (i) rotational oscillation to track the harmonic trajectory very closely ; (ii) the flexible pipette to be concentric to the rotational axis of ros - drill. the later condition is shown to satisfy because of the extreme bending compliance and whirling effects [2, 3 ]. the smooth variation of the rotational motion in accordance with a harmonic function is the most natural desired trajectory. the contribution of this paper is a proper control law which can serve the objective under severe sensing constraints. overarching restriction in this study comes from the pricing aspect. in order to make the ros - drill accepted by a wide range of ivf clinics, the cost of this automated device must remain within $ 1500 per copy ; otherwise, it would be difficult to compete against the commonly - used but inefficient method adopted by trained icsi specialists. let us take a closer look at the physics of the ros - drill microinjection procedure. the rotational stiffness of the pipette holder and the pipette including the extremely fine tip are assumed to be high. consequently, the angular displacement,, of the injection pipette is transmitted from the shaft of the micromotor to the pipette tip without a loss. the lateral (bending) vibrations are the major concern at the tip, and the present operating scheme is intended to suppress them to ignorable levels especially when compared with those oscillations caused by the piezo - assisted icsi [2, 3 ]. with these assumptions, the major objective of this study is directed to insure desirably precise harmonic motion tracking capabilities at the pipette tip [68 ] despite the very coarse sensory capabilities. for position servoing, in general, the sensors are expected to have high resolution vis - - vis the range of the intended motion, which can yield a desirable tracking capability. for instance, in representing a harmonic trajectory, one expects to have a minimum of 10 discrete data points per cycle. however, resolution of digital encoders is limited by the number of slots on a rotating disk, through which the encoder 's light beam travels. although advances in encoder technology have wonderfully progressed to increase the number of slots in order to improve the resolution of the encoders, the trade - off between resolution and cost is unavoidable. in such applications as ros - drill where the cost limitations are very stringent, sensor resolution is often compromised. recent model - based speed observers make the velocity estimate robust using an interesting disturbance observer. in another effort, kwon. bautista - quintero and pont propose an h - infinity control algorithm for sensor - constrained mechatronic systems using the position sensors with relatively low resolutions. they demonstrate how this procedure allows a faithful reproduction of observed motion starting from a limited sensing ability using relatively common (and inexpensive) microcontrollers. furthermore, the methods which deal with control applications with low - resolution sensors usually have high computational demands to compensate the sensory shortfall. this aspect quickly makes the method prohibitive in a cost - sensitive design such as ros - drill. we wish to familiarize the reader with the components of the first - generation ros - drill setup and the current improvements. the first - generation design having a 512 lines / revolution - encoder with quadrature signature is the finest selection we could find within the cost and spatial confines. it allows a maximum of 2-step reading over the desired peak - to - peak stroke (note that the desired amplitude is 0.2 deg) [4, 12 ]. this sensor makes our best observation of a ros - drill harmonic cycle with a 2-step representation, which is a colossal handicap to perform the control. the spirit of the proposed control scheme and the focus of this paper are primarily on this crucial feature. moving on to another component, the first trial generation of ros - drill employs a plc (programmable logic controller) as its digital controller, which has a maximum sampling speed of 1 khz. this constraint clearly limits the maximum frequency of the controlled trajectory to 500 hz. most recently reported icsi tests use injection pipette oscillations up to 0.3 deg amplitude and maximum frequency of 500 hz. these oscillations last a certain length of time which we name the duration of oscillation, d. typically, d varies within 250500 msec. the preliminary reports claim that ros - drill - assisted icsi results in embryo survival, embryo development, birth, and weaning rates comparable to those of piezo - assisted icsi using mercury. although these biological results are very promising, the trajectory tracking performance of the existing prototype is not satisfactory mainly because of the low resolution of position sensor and low control sampling rate of plc. this work represents an effort to further improve this performance. in the second - generation (current) ros - drill microinjector prototypes, the low - sampling - frequency matter is considerably improved by replacing the plc with a microcontroller (which brings the sampling speed from 1000 hz to 10000 hz) and the encoder resolution issue by selecting one - level higher - capacity sensor (1000 lines and 0.09 degree resolution after quadrature). however, despite this upgrade, the position sensor with the resolution of 0.09 degrees still presents the biggest hurdle in this control system design because the required harmonic motion of 0.2 degree amplitude displays only 4-step peak - to - peak encoder recording, which is still low. in, a look - up - table - based adaptively the most appropriate pid (proportional, integral, and derivative) control gains are a priori selected corresponding to the given operating frequency using dynamic simulations. for a given desired trajectory, d, with ad and fd attributes, an ad hoc 3d search routine is performed using the representative simulink simulation program. this yields a set of feedback gains, which are then utilized through a look - up adaptation structure. they are regarded as nominal control gains as they are tuned based on the nominal model of ros - drill, and the control design is considered as the first adaptation stage towards the second - generation ros - drill. an important point to discuss for this first adaptation stage is that the look - up - table - based control law can handle the potential parameter uncertainties, disturbances, and the unmodeled mechanical properties of the system only via the supervisory interference from the user. for instance, the external load originating from the membrane resistance torque may vary with the type of species and it may affect the control performance of the system. still worse is the extremely stringent requirement of concentricity on the shaft of motor and pipette holder via a coupling and ensuing resistance to the servosystem. in order to achieve better updates of the control gains, furthermore, the low - resolution encoder is a range indicator (as discussed later) rather than a measurement device. the text is organized as follows. in the first part of the paper, a dedicated novel adaptive feedback control method, the second - stage adaptive law is developed to update the control gains in situ, which is validated by experiments. in the second part of the paper, a stochastic perspective is presented which correlates the encoder readings with the actual angular motion. the highlighted feature in this study is the unusually low - resolution sensor (encoder). figure 2 gives a description of the operating principle of an optical encoder which is our main sensor. for simplicity, we overlook the quadrature effect and depict an encoder disk as the combination of transparent and dark lines. the small circle indicates the position of light beam. the angular motion denotes the rotational motion of the encoder. at the start, the encoder is declared at its zero position and = 0. we now present a critical argument on the position detection ability of the optical encoder. it is about an encoder offset angle, which we will denote by a, 0 a2 > 0) and define the complete actual peak - to - peak stroke as a12 = a1 + a2. the blue line represents the rotational motion being sensed by the encoder and the encoder reading is denoted as enc. the peak - to - peak angular stroke which is recorded by the encoder is named aenc, which is an integer multiple of (e.g., 4 in figure 3). we denote the average of a12 and aenc over a certain number of oscillations by a12 and aenc, respectively. the bias of actual angular motion is defined as the distance of center of a12 from zero and it is expressed as (2)a12-b = a1a22. for a certain frequency, the hypothetical actual oscillations of the pipette can be described by the bias and the amplitude of actual angular motion. let us express as (3)=a12-b+asint, where = 2f and a and f are amplitude and frequency of harmonic wave, respectively, and we have a12 = 2a. to summarize these definitions, we list them again as follows:(i)resolution of position sensor = ;(ii)encoder offset angle = a (deg);(iii)upper stroke of actual angular position from rest = a1 > 0 (deg);(iv)lower stroke of actual angular position from rest = a2 > 0 (deg);(v)actual peak - to - peak angular stroke = a12 = a1 + a2 (deg);(vi)bias of actual angular stroke = a12-b;(vii)peak - to - peak angular stroke detected by the encoder = aenc (deg);(viii)average actual angular stroke over a certain number of oscillations = a12 (deg);(ix)average encoder stroke over a certain number of oscillations = aenc (deg). resolution of position sensor = ; encoder offset angle = a (deg) ; upper stroke of actual angular position from rest = a1 > 0 (deg) ; lower stroke of actual angular position from rest = a2 > 0 (deg) ; actual peak - to - peak angular stroke = a12 = a1 + a2 (deg) ; bias of actual angular stroke = a12-b ; peak - to - peak angular stroke detected by the encoder = aenc (deg) ; average actual angular stroke over a certain number of oscillations = a12 (deg) ; average encoder stroke over a certain number of oscillations = aenc (deg). this section presents a brief review of the earlier work, which establishes the departure points of the present effort. in ros - drill application, the desired angular trajectory requires high control sampling frequency, fs, in the microcontroller (such as 10 khz) in order to perform a meaningful tracking. this imposes some further constraints on the limited computational capabilities of selected microcontroller. keeping these restrictions in mind, the ros - drill microinjector can be considered as a simple rotational mass, which is attached to a torque - generating dc servomotor (figure 1). this yields a transfer function of ros - drill system and the corresponding frequency response creates the magnification factor as a function of, kp, ki, and kd : (4)m=|g(s = j)|=m(fd, kp, ki, kd), where = 2 fd and kp, ki, and kd are the control gains. the objective of the control is to achieve a flat response over a given range of operating frequencies. we wish to emphasize that the phase angle between the desired harmonic input and the resulting output is not considered as a part of the performance description. another important point to stress is that since we have limited access to the actual rotationally oscillating motion via the coarse measurements of the encoder, this flat response characteristic can be achieved, at best, by enforcing the peak - to - peak strokes of the encoder readings to be equal to aenc = floor (2ad/) deg. it is shown in that m is much more sensitive to the kd variations rather than kp and ki. we compare the effects of variation of these three control gains around their nominal values (k / k). this results in some percentage variation of magnification factor (m / m). among the three gains (kd, kp, and ki), the most effective one is kd, from this perspective. thus, the tuning of the magnification factor can be achieved more directly by utilizing only kd variations. this tuning methodology needs to be further adapted to the actual sample data system when using a low - resolution encoders. the logic steps of this tuning methodology, or the adaptive update laws of kd, form the main contributions of this study, and they will be detailed in the following sections. in this section, we present a realistic dynamic model of the system using matlab - simulink platform. the encoder block is created to reflect the limitations of the position sensor as closely as possible to the reality. two pieces of information are used from the quad signature encoder outputs : one is the angular step counter, and the other is the velocity of the angular motion. these data divide the angular movement space into four sections : (5)(i) >0, >0(ii) >0, 0. notice that only one of these four cases will be active at any sampling period ; thus, the summation of the four outputs will actually declare the one - step angular accumulation. the angular measurements, ad, in zero - order - hold (z - o - h) mode is created with sampling time ts just to simulate the sample data procedures of the microcontroller, see figures 5(a) and 5(b). the integration step size for the simulation routine is chosen to be one - tenth of the sampling period of microcontroller (10 s), which is sufficient to represent the transient behavior within a sampling period with a desirable fidelity. the amplifier gain, k, in figure 4 is chosen by the user based on experimental knowledge such that the saturation in the d / a converter is prevented. the section enclosed by dotted lines in figure 4 is the dynamic model for ros - drill microinjector itself. using this simulink tool, we first determine some starting values of the control gains kp, ki, and kd using a pole - placement - based method as described in. then, we systematically vary kd. the objective in this tuning procedure is to obtain m = 1 as close as possible for the particular interval of frequencies. for a desired motion of d = adsin(2 fd t), m = 1 implies a peak - to - peak stroke of 2ad. this is the continuous rotational angle and the corresponding peak - to - peak encoder recording should be aenc = floor (2ad/). we continue the systematic variations of kd until the frequency response amplitude condition, m = 1, is achieved for the given operating frequency. we then repeat the same operation for other fd values on a list of potential (i.e., biologically required) operating frequencies. this set of kd gains will form a look - up table which can be used by the real - time control program on the microcontroller. once the user identifies a preferred harmonic frequency, the program selects a kd feedback gain from a given list and adaptively sets the new control logic. this completes the first adaptation scheme, which follows the steps below:(a)declare the desired amplitude and frequency via gui (ad, fd) ; (b)select the corresponding control gain (kd) using the look - up table;(c)observe the encoder registrations of peak - to - peak strokes, aenc;(d)evaluate the average of aenc over a predetermined duration d, aenc;(e)signal the operator the direction of the error e = floor (2ad/)-aenc when it is outside a tolerance range, |e| > tolerance. notice that ad is typically not an integer multiple of. therefore, we deploy 2ad/ to create a comparable basis with aenc;(f)manually adjust the gain, kd, as supervisory fine tuning and repeat the steps (c)(f) until no violation of tolerance in (e) remains. declare the desired amplitude and frequency via gui (ad, fd) ; select the corresponding control gain (kd) using the look - up table ; observe the encoder registrations of peak - to - peak strokes, aenc ; evaluate the average of aenc over a predetermined duration d, aenc ; signal the operator the direction of the error e = floor (2ad/)-aenc when it is outside a tolerance range, |e| > tolerance. notice that ad is typically not an integer multiple of. therefore, we deploy 2ad/ to create a comparable basis with aenc ; manually adjust the gain, kd, as supervisory fine tuning and repeat the steps (c)(f) until no violation of tolerance in (e) remains. this procedure is shown to perform the task so that the peak - to - peak strokes of ros - drill are guaranteed to stay desirably close to aenc = floor (2ad/). figure 6 shows that experimental setup, the mechanical device along with the controller box. a faulhaber an optical encoder with 1000 lines is utilized as position sensor, enabling the angular resolution at 0.09 deg (with quad - signature characteristics). it is important to clarify that this is the best option for an encoder in terms of size, resolution, and costs demanded by the application. a control box contains the components that handle the logical operations between the sensor (encoder) and the actuator (micromotor). its main unit (the cpu and i / o device) is a silicon lab 's c8051f121 controller. this box also contains the necessary peripheral circuits such as the converter of encoder 's quadrature - signatures into the rotational pulse counter and direction determination of the rotation, optoisolators, and power output chip which feeds directly into the dc micromotor. we wish to give an idea to the reader about the microcontroller 's time management structure. for the aimed ros - drill application, 10 khz siso (single input - single output) control sampling rate is selected, and the c8051f121 micro - controller can easily accommodate this speed. out of this 100 s total loop time, 71.8 s is used for sensing, control logic evaluation, and d / a conversion ; the remaining 28.9 s is the idle time which can be devoted to other applications (such as filtering) to be added later. in order to crossvalidate the capabilities of the tracking control, we use an independent angular motion monitoring tool. the motion validations provided in the entire text are from this sensing channel instead of the unit which handled the same task in the loop. the look - up - table - based control law offered in, as we summarized above, can handle the potential parameter uncertainties, disturbances, and the unmodeled mechanical properties of the system only via the supervisory interference from the user, see step (f) above. for a typical application such as ros - drill, these uncertainties originate mainly from friction - related sources, which are impossible to model accurately. we include them in our simulink model (figure 4) as some arbitrary combinations of viscous and coulomb friction effects, just to get a feel. the control objective is again to achieve a sustained peak - to - peak stroke of aenc = floor (2ad/) as the encoder registers it (as an integer multiple of). the average deviation of the aenc (over a predetermined number of cycles) from this desired aenc = floor (2ad/) is used as the error for tuning of kd. one must pay attention, however, to another angle of these encoder readings. during the experiments, it is commonly observed that aenc fluctuates typically between two successive steps (say 3 and 4). this feature occurs due to the random offset angle a as mentioned above, but also stems from the transient regimes where the oscillations are not yet set into periodically repeated format. to discriminate the two causes of the same effect is an impossible task. throughout this study, we consider the motion to have reached a steady regime for simplicity, therefore disqualifying the latter cause. the effects of the transient behavior are left for a future document. in this paper, we confine the execution of supervisory gain adjustments : (a) through a windowing and averaging procedure ; (b) with sufficient update - and - wait period. in short, therefore, kd can change only after a certain number of oscillations. in the icsi experiment, typically entire duration of oscillations, the tuning of kd is expected to be completed within a small fraction of d and in the very early stages of the period d. after extensive tests using simulink with additional disturbances imposed, it is observed that the angular position of the servo system () reaches the steady state no later than 5 oscillations for all the operating frequencies (from 400 to 1000 hz). therefore, we use the average of peak - to - peak strokes within 15 cycles of oscillations, which we denote by aenc, to assess the performance of current control gains and to update them. obviously, if aenc is equal to aenc = floor (2ad/) that is considered to be satisfactory for the objectives of the control. in summary, this adaptation scheme follows the steps listed below:(a, b, c) are identical to the first adaptation scheme above;(d) evaluate the average of aenc over 15 cycles, aenc ; (e) utilize an update law for kd adjustments if the error, e, is outside a tolerance range, that is, |e| > tolerance. if the error is within the tolerance, no kd update is needed.(f) repeat step (d). are identical to the first adaptation scheme above ; evaluate the average of aenc over 15 cycles, aenc ; utilize an update law for kd adjustments if the error, e, is outside a tolerance range, that is, |e| > tolerance. these steps will again guarantee the execution of the desired aenc using an adaptive control gain update law. for the sake of simplicity, from this point onwards we will take ad = 0.2 degrees and the corresponding aenc = 4 = 0.36 degrees. let us define kd(n) as the nth update of the derivative gain and e(n) as ensuing amplitude error which is obtained over 15 oscillatory cycles (an informed selection based on observation of experimental data), after kd(n) is applied. that is, (6)e(n)=4aenc. for the next 15-cycle period, we use a new feedback gain with the following update law:(7a)kd(n)=kd(n1)+kd(n), where (7b)kd(n)=ce(n1),and c is an update constant. this update process is performed in the following sequences:(i) start with kd(0) which is taken from a look - up table as explained in section 3. (ii) determine the resulting e(0) at the end of the following 15 cycles. (iii) for n = 1, use kd(1) = c e(0) and evaluate kd(1) from (7a).(iv) again after 15 cycles of using this control gain, determine e(1). (v)(a) if |e(1)| |e(0)|, evaluate kd(2) = c e(1), kd(2) = kd(1) + kd(2), assign kd - ref = kd(1) and eref = e(1), and go to (vi).(b) if |e(1)| > |e(0)|, evaluate kd(2) = 0.5 c e(0), kd(2) = kd(0) + kd(2) and determine e(2). if |e(2)| |e(0)|, assign kd - ref = kd(2) and eref = e(2), go to (vi). if |e(2)| > |e(0)|, repeat (vb) but this time with kd(3) = 0.5 0.5c e(0) and continue until the absolute error falls below e(0). assign the current kd(n) value to kd - ref, current e(n) value to eref and move to (vi).(vi) after 15 cycles, determine the new e(n) and go to (va). start with kd(0) which is taken from a look - up table as explained in section 3. determine the resulting e(0) at the end of the following 15 cycles. for n = 1, use kd(1) = c e(0) and evaluate kd(1) from (7a). again after 15 cycles of using this control gain, determine e(1). (a) if |e(1)| |e(0)|, evaluate kd(2) = c e(1), kd(2) = kd(1) + kd(2), assign kd - ref = kd(1) and eref = e(1), and go to (vi).(b) if |e(1)| > |e(0)|, evaluate kd(2) = 0.5 c e(0), kd(2) = kd(0) + kd(2) and determine e(2). if |e(2)| |e(0)|, assign kd - ref = kd(2) and eref = e(2), go to (vi). if |e(2)| > |e(0)|, repeat (vb) but this time with kd(3) = 0.5 0.5c e(0) and continue until the absolute error falls below e(0). assign the current kd(n) value to kd - ref, current e(n) value to eref and move to (vi). if |e(1)| |e(0)|, evaluate kd(2) = c e(1), kd(2) = kd(1) + kd(2), assign kd - ref = kd(1) and eref = e(1), and go to (vi). if |e(1)| > |e(0)|, evaluate kd(2) = 0.5 c e(0), kd(2) = kd(0) + kd(2) and determine e(2). if |e(2)| |e(0)|, assign kd - ref = kd(2) and eref = e(2), go to (vi). if |e(2)| > |e(0)|, repeat (vb) but this time with kd(3) = 0.5 0.5c e(0) and continue until the absolute error falls below e(0). assign the current kd(n) value to kd - ref, current e(n) value to eref and move to (vi). after 15 cycles, determine the new e(n) and go to (va). some nuances on this gain adaptation process are discussed later in this section over an example data with the role of kd - ref and eref. the following portion is devoted to the selection of the update constant c. uncertain friction term is modeled as some combination (in simulink model of figure 4) of viscous and coulomb frictions : (8)tf(t)=tcsgn()+b, where tc is the coulomb friction torque and b is the viscous friction coefficient. note that different friction scenarios refer to the model in figure 4 with the same system parameters but with combination of different tc and b. scenarios with various combinations of frictions are artificially created by varying tc and b components in (8). the selection of c is performed offline via the following steps:(a)estimate the bounds of tc and b based on experimental studies. (b)deploy kd(0) in simulink and find the corresponding e(0) over a certain number of cycles (say 15). e(0), using systematically increasing c values (starting from zero), so that the resulting error e(1) becomes desirably small (e.g., 75% as we used in our tests) compared with e(0) under most adverse friction conditions. at the same time, we note that kd should be upper - bounded as it multiplies the encoder - based angular speed and tends to saturate the actuator input (u in figure 4). note that the determination of c becomes critical from the concern of minimizing the number of gain updates before the error converges to zero. using the proposed method, determination of constant c deploy kd(0) in simulink and find the corresponding e(0) over a certain number of cycles (say 15). determine kd(1) = c e(0), using systematically increasing c values (starting from zero), so that the resulting error e(1) becomes desirably small (e.g., 75% as we used in our tests) compared with e(0) under most adverse friction conditions. at the same time, we note that kd should be upper - bounded as it multiplies the encoder - based angular speed and tends to saturate the actuator input (u in figure 4). note that the determination of c becomes critical from the concern of minimizing the number of gain updates before the error converges to zero. using the proposed method, determination of constant c the following portion is devoted to the convergence of e to zero and the determination of kd over some experimental data. reference kd, kd - ref is the updated derivative gain which leads to smaller amplitude error |eref| than that which evolves under the previous kd - ref. and the corresponding new error is denoted by eref. to ensure that e converges to zero, the update law of kd always moves the gain in the direction of smaller |eref|. the update of kd - ref is based on eref (i.e., kd = c eref). if the resultant |e| |eref|, kd(1) is bisected for the third round of tuning (k = 2 in table 1 and figure 7), kd(2) = kd - ref + kd(1)/2. because |e(2)| < |eref|, kd(2) and e(2) are taken as new kd - ref and eref, respectively. the controlled oscillation of pipette lasts for a predetermined duration and then the pipette is returned to starting zero position in order to prevent the wrap - around effect (on the tubing attachments). in figure 7, at 0.98 sec, oscillation of pipette ends and begins to return to zero position. figure 8 shows the flowchart of this monitoring and on - line tuning mechanism. the first part of the paper, which is related to the new, second - stage adaptation scheme, ends here. in what follows, we will demonstrate the influence mechanism for the actual angular motion, if the encoder reading of the stroke is aenc = 4 just to provide a better insight to the reader. the above control scheme is designed to perform an adaptively updating logic to assure a given aenc peak - to - peak stroke. let us take aenc = 4 for the sake of simplified arguments, without loss of generality. we query which operational conditions are satisfied if aenc = 4 reading is guaranteed, next. note that the derivations pertaining to aenc = 4 in what follows can be generalized with ease to cases when desired control performance is aenc = 2m, for m = 1,2,. to clarify some definitions about encoder reading, we list them as follows:(i)a duration of oscillations = d;(ii)absolute deviation of a12 from its nearest odd integer multiple of =. a duration of oscillations = d ; absolute deviation of a12 from its nearest odd integer multiple of =. a very important observation is stated next. one can see from figure 3 that for the encoder to register 4-step stroke (i.e., aenc = 4), the following are the necessary and sufficient conditions:(9a)a+2<a1<a+3,(9b)a+<a2<a+2.by summing (9a) and (9b), we obtain (10)3<a12<5. here, we define as the absolute deviation of a12 from the nearest odd integer multiple of and 0 < <. for 3 < a12 < 5, we have(11a) either 3<a12<4, =a123(11b) or 4a12<5, =5a12.for the simplicity of expressions, let us denote (12)a12-b=2a2. note that the offset angle is an uniformly distributed random variable between zero and. hence, a12-b is also an uniformly distributed random variable but between /2 and /2. proposition 1. for any harmonic motion with (2 m 1) < a12 < (2 m + 1), peak - to - peak encoder reading will always be aenc = 2m if and only if the bias of stroke (a12-b) satisfies (13)2<a12-ba12-b<2. furthermore if a12-b = a12-b, for such a harmonic motion the encoder reading will always be 2m. for any harmonic motion with (2 m 1) < a12 < (2 m + 1), peak - to - peak encoder reading will always be aenc = 2m if and only if the bias of stroke (a12-b) satisfies (13)2<a12-ba12-b<2. furthermore if a12-b = a12-b, for such a harmonic motion the encoder reading will always be 2m. proofwithout loss of generality, let us take m = 2 and prove the sufficiency of (13) for aenc = 4. by substituting (2) and (12) into (13), we obtain(14a)2a<a1a2<2a+. (a) first consider the interval 3 < a12 < 4. note from (11a) : (14b)a12=a1+a2=3+.using (14a) and (14b) once for a1 and again for a2, we obtain(15a)a+2<a1<a+2+,(15b)a+<a2<a++.keeping in mind that 0 < <, one can see that satisfying (15a) and (15b) automatically satisfies (9a) and (9b), which are the necessary and sufficient conditions for aenc = 4 measurement (as mentioned at the beginning of section 5).(b) one should next consider the interval 4 < a12 < 5. note from (11b) : (16)a12=a1+a2=5. using (14a) and (16) once for a1 and again for a2, we obtain(17a)a+3<a1<a+3,(17b)a+2<a2<a+2.with the condition 0 < <, (17a) and (17b) guarantee the fulfillment of (9a) and (9b), respectively. again this completes the proof of sufficiency of proposition 1. without loss of generality, let us take m = 2 and prove the sufficiency of (13) for aenc = 4. by substituting (2) and (12) into (13), we obtain(14a)2a<a1a2<2a+. (a) first consider the interval 3 < a12 < 4. note from (11a) : (14b)a12=a1+a2=3+.using (14a) and (14b) once for a1 and again for a2, we obtain(15a)a+2<a1<a+2+,(15b)a+<a2<a++.keeping in mind that 0 < <, one can see that satisfying (15a) and (15b) automatically satisfies (9a) and (9b), which are the necessary and sufficient conditions for aenc = 4 measurement (as mentioned at the beginning of section 5). (b) one should next consider the interval 4 < a12 < 5. note from (11b) : (16)a12=a1+a2=5. using (14a) and (16) once for a1 and again for a2, we obtain(17a)a+3<a1<a+3,(17b)a+2<a2<a+2.with the condition 0 < <, (17a) and (17b) guarantee the fulfillment of (9a) and (9b), respectively. again we take into account that aenc = 4 and, therefore, ((9a) and (9b)) conditions hold. 4 first. under this assumption, the right inequalities of ((9a) and (9b)) are satisfied automatically. the left inequality of (9a) is a + 2 < a1 and it can be expressed as(18a)(a1+a23)<a1a2(2a). similarly, the left inequality of (9b) is a + < a2 and it can be rewritten as (18b)a1a2(2a)<a1+a23.using the definitions in (2) and (12) and the constraint (14b), the combined inequalities of ((18a) and (18b)) render exactly the conditions given in (13). < 5. we also claim that this condition, this time, together with the right inequalities of ((9a) and (9b)), will satisfy the left inequalities of ((9a) and (9b)). following the similar procedure above, with the right inequality of (9a), a1 < a + 3, we arrive at(19a)a1a2(2a)<5a1a2. the right inequality of (9b), a2 < a + 2, creates (19b)(5a1a2)<a1a2(2a).once again, using the definitions in (2) and (12) and the constraint (16), the combined inequalities of ((19a) and (19b)) render exactly the conditions given in (13). furthermore, if a12-b = a12-b, (13) automatically holds for any a12 within 3 < a12 < 5 ; therefore, the causality follows. this proof for m = 2 can be extended for cases when aenc = 2m, for m = 1,2,. the important implications of proposition 1 can be summarized in the following logical sequences:(i)a12-b is a random number (due to the term a). however, a remains unchanged during the oscillatory period d ; thus, a12-b remains constant in the same period.(ii)bounds expressed by (13) suggest that the bias, a12-b, of each cycle during d stays within a bounded distance from a12-b (i.e., /2) if and only if we maintain the encoder reading of aenc = 2m.(iii)in essence, if a control mechanism which is described earlier assures the aenc = 2m, this also brings a guarantee for the boundedness of |a12-b - a12-b|</2, where is defined as (11a) and (11b). this implies that the bias a12-b has an attractive bonding to a12-b. it can fluctuate around this value but can not run away from it. a remains unchanged during the oscillatory period d ; thus, a12-b remains constant in the same period. bounds expressed by (13) suggest that the bias, a12-b, of each cycle during d stays within a bounded distance from a12-b (i.e., /2) if and only if we maintain the encoder reading of aenc = 2m. in essence, if a control mechanism which is described earlier assures the aenc = 2m, this also brings a guarantee for the boundedness of |a12-b - a12-b|</2, where is defined as (11a) and (11b). this implies that the bias a12-b has an attractive bonding to a12-b. it can fluctuate around this value but can not run away from it. a novel control system with low - resolution encoder for the desired harmonic trajectory is studied on a cellular microinjection technology called the ros - drill (rotationally oscillating drill). in the first part of paper, a novel adaptive control logic is developed to facilitate the tracking of the harmonic rotational motion under uncertainties, especially frictions. we demonstrate via dynamics simulations first, followed by experiments that the tracking of the harmonic rotational motion is achieved with desirable fidelity. in the second part, a stochastic analysis connecting the actual motions and their low - resolution sensory recordings is presented. it is observed that when the control structure guarantees a fixed peak - to - peak stroke, the bias of actual angular motion is bounded in a range and it is attracted to a certain predefined value. | a novel control methodology which uses a low - resolution encoder is presented for a cellular microinjection technology called the ros - drill (rotationally oscillating drill). it is developed primarily for icsi (intracytoplasmic sperm injection) operations, with the objective of generating a desired oscillatory motion at the tip of a micro glass pipette. it is an inexpensive setup, which creates high - frequency (higher than 500 hz) and small - amplitude (around 0.2 deg) rotational oscillations at the tip of an injection pipette. these rotational oscillations enable the pipette to drill into cell membranes with minimum biological damage. such a motion control procedure presents no particular difficulty when it uses sufficiently precise motion sensors. however, size, costs, and accessibility of technology to the hardware components severely constrain the sensory capabilities. consequently, the control mission and the trajectory tracking are adversely affected. this paper presents two contributions : (a) a dedicated novel adaptive feedback control method to achieve a satisfactory trajectory tracking capability. we demonstrate via experiments that the tracking of the harmonic rotational motion is achieved with desirable fidelity ; (b) some important analytical features and related observations associated with the controlled harmonic motion which is created by the low - resolution feedback control structure. |
according to the world health organization (who), diabetes is likely to be one of the most substantial threats to human health in the 21 century ; 346 million people worldwide have diabetes. type 2 diabetes typically develops in older people. among people aged 65 years and older, type 2 diabetes is diagnosed 10 times more frequently than in people younger than age 45 years. however, it has been noticed that the average age of patients with type 2 diabetes mellitus is decreasing. depression is associated with a variety of diabetes complications (diabetic retinopathy, nephropathy, neuropathy, and macrovascular complications). after the diagnosis of diabetes mellitus, people must change their everyday lifestyle and adapt to the new conditions of life ; also, they experience many negative emotions that cause lower social adaptation, and they experience decreased efficiency and disability. the prevalence of the symptoms of anxiety (32%) and depression (22.4%) in patients with diabetes is considerably higher than in general population samples (10%). diabetes doubles the likelihood of comorbid depression, which is present in approximately 30% of patients with type 1 or type 2 diabetes. a study by khuwaja. found that the prevalence of depression and anxiety among adults with type 2 diabetes was 44% and 58%, respectively. both depression and anxiety different studies have been performed and are currently being carried out in a number of countries all over the world to analyze the influence of various factors, including disease, its duration, social factors, complications, and treatment methods, on the emotional state of diabetic patients. evidence from several studies suggests that detection and effective treatment of depression can improve diabetic control. the presence of symptoms of depression significantly impairs the health - related quality of life among individuals with type 2 diabetes. anxiety and depression symptoms often remain unrecognized and, thus, untreated. screening for and treatment of anxiety and depression may lead to a better medical prognosis and better quality of life. it is important to estimate the prevalence of anxiety and depression symptoms and factors associated with these symptoms in patients with diabetes. there is limited or controversial information regarding factors associated with anxiety and depression in patients with type 2 diabetes. the aim of this study was to evaluate prevalence and determinants of anxiety and depression symptoms in patients with type 2 diabetes (t2 dm). patients were selected for the survey by applying the following inclusion criteria : men and women, 35 years of age or older, with type 2 diabetes mellitus, and without any acute diseases diagnosed during the survey. the hospital anxiety and depression scale (hads) was used to measure depression and anxiety for the evaluation of depressive state and anxiety. it is well - validated instrument and has been used in other chronic disease patient populations. hads consists of 14 statements : 7 questions of the questionnaire are designed for anxiety symptoms, and the other 7 statements evaluate depression symptoms. four possible answers (03) are given for the each statement, and the subjects must choose the one that most precisely reflects their condition last week. the results for the depression and anxiety are summed and evaluated separately : a score of 07 means there is no depression or anxiety and a score of 8 or over means there is depression or anxiety. a brief patient health questionnaire was used to collect demographic data, duration of the disease (in years), complications, and arterial hypertension. the study was approved by lithuanian bioethics committee and the survey was conducted in compliance with the requirements of the professional ethics of sociological - medical research. in total, 1500 patients were invited to participate in study. before presenting the survey form, after agreeing to participate in the survey, the subjects filled out the provided forms independently. according to the duration of the disease, the patients were distributed into 4 groups : patients who had diabetes for up to 5 years, patients who had diabetes for 610 years, those who had the disease for 1115 years, and those who had diabetes for 16 years or more. we evaluated the distribution of the respondents according to the following complications of diabetes : nephropathy, retinopathy, angiopathy, and polyneuropathy of legs. also, they were asked if they were diagnosed with arterial hypertension, and whether they used medicines for the treatment of hypertension. the prevalence of anxiety and depression symptoms were assessed using hads cut - off points : 07 indicating there is no depression or anxiety and 8 and over indicating there is depression or anxiety. pearson s test was used for categorical data analysis and the extended mantel - haenszel test was used for trend. the anxiety and depression scores were dichotomized prior to regression analysis : 07 was defined as a normal score, 821 was defined as a mild to severe anxiety or depression score. the odds ratios (or) for anxiety and depression socio - demographic variables were specified in block 1 and health status and behavior variables were specified in block 2. the most patients were in age groups of 5564 years (38.1%) and 6574 years (32.2%) (mean age 59.3 years (median 59.0). more than 70% of all respondents who participated in the study had diabetes mellitus complications (72.2%). retinopathy (57.5%) and angiopathy (54.6%) were the most common complications of t2 dm. arterial hypertension (ah) was self - reported by 74.4% of patients with t2 dm. the majority of patients (71.5%) had no depression symptoms (normal score 7). the prevalence mild to severe depression score was 28.5% (95% ci 25.731.4) (table 2). depression symptoms were more prevalent in females (32.3%) than in males (21.8%) (p<0.001). anxiety was more frequent among females (46.8%) than among males (34.7%) (p<0.001). a significant negative trend was observed between prevalence of anxiety and depression, and age and education (p for trend < 0.001). the prevalence of anxiety and depression symptoms among unmarried people was 55.6% (95%ci 35.374.5), while among married people it was 36.9% (95%ci 33.240.7) and 25% (95% ci21.828.5), respectively. anxiety and depression were more prevalent among retired (49.5% [95% ci 44.854.3 ] and 33.3% [95% ci 28.937.9 ], respectively) and disabled persons (41.1% [95% ci31.751.1 ] and 40.2% [95% ci 30.850.1 ], respectively) compared with blue - collar and white - collar workers. therefore, the prevalence of anxiety and depression symptoms were dependent from gender, age, marital status, education, and social status. the prevalence of depression symptoms was higher (p=0.007) among obese (31.0%, 95% ci 27.1834.97) than among overweight respondents (23.3% [95%ci 19.4527.59 ]). however, the prevalence of anxiety in these groups of patients was similar (40.3% and 43.0%, p=0.384). prevalence of anxiety and depression symptoms was associated with duration of diabetes, diabetes complications, and self - reported arterial hypertension. the prevalence of anxiety and depression symptoms increases with increasing duration of the disease (p<0.001). our study results confirmed that chronic complications of diabetes mellitus affected the emotional state of patients. the prevalence of anxiety and depression symptoms was higher among patients with complications (45.7% [95% ci 42.0349.34 ] and 33.5% [95% ci 30.137.0 ]) and self - reported arterial hypertension (45.7% [95% ci 42.149.3 ] and 31.7% [95% ci 28.435.2 ]). anxiety and depression symptoms were more prevalent among insulin users (47.9% [95% ci43.252.6 ] and 39.1% [95% ci 34.643.8 ], respectively). the prevalence of anxiety and depression symptoms was lower among respondents who had changed their diet (37.2% [95% ci 34.140.4 ] and 24.7% [95% ci 21.927.6 ], respectively). increased physical activity after t2 dm diagnosis was statistically significantly (p=0.004) associated with lower prevalence of anxiety symptoms (36.1% [95% ci 31.141.3 ]). we found no statistically significant association between smoking and prevalence of anxiety and depression symptoms by bivariate analysis (table 3). results of the multivariable analysis for factors associated with anxiety and depression symptoms are given in table 4 and table 5. age (older age), education (incomplete secondary, secondary vs. elementary), social status (white - collar workers vs. blue - collar workers), duration of diabetes, and complications were risk factors significantly associated with higher anxiety scores. self - reported arterial hypertension, increased physical activity after t2 dm diagnosis, and smoking were not significantly associated with anxiety. protective factors significantly associated with lower depression scores were : marital status, education, retiree (vs. blue - collar workers), and changed nutrition habits after t2 dm diagnosis. age (older age) and duration of diabetes were associated with higher depression symptoms. gender, complications, insulin using, self - reported arterial hypertension, increased physical activity after dm diagnosis, and smoking were not significantly associated with higher depression scores. diabetes particularly its complications not only causes impairment of the physical health, but at the same time promotes occurrence of psycho - emotional and social problems. after the diagnosis of diabetes mellitus, people must change their everyday lifestyle and adapt to the new conditions the life. they experience many negative emotions that cause lower social adaptation, and they face disability and decreased efficiency. co - morbid major depression is a significant problem among patients with t2 dm and/or coronary heart disease and this negatively impacts quality of life. most epidemiological studies indicate that diabetes is associated with an increased risk of depression. in our study, depression was detected in 28.5% of the subjects : 32.3% for females, and 21.8% of males. anxiety was found in 42.4% of the subjects. anxiety like depression was more common (p<0.001) in females (46.8%) than in males (34.7%). tellez - zenteno and cardiel reported a 39% prevalence of depression, and the risk for depression in females was more than twice as high (or 2.95, ci 1.505.82, p=0.006) as in males. our study shows that insulin use did not affect the states of depression and anxiety, but it could lead to a longer duration of the disease and increase complications. in addition, insulin - treated patients had more advanced disease and poor patient performance status. according to the literature, complications of t2 dm negatively affect patient health, and health impairment due to t2 dm complications increases the risk of depression. depressive states were significantly related to the complications of diabetes, the presence of other chronic diseases, anxiety, and poorer quality of life. in our study anxiety was more frequent among females (46.8%) than among males (34.7%) (p<0.001). previous studies showed that different chronic complications influence the depressive state : neuropathy, cardiovascular diseases, retinopathy, and nephropathy have a direct impact on the depressive state of diabetic patients. studies showed that depression and anxiety rate depended on the duration of the disease. according to a study performed in norway (the had scale was used), the probability that patients with type 2 diabetes would develop depression was the same as in the general population. the diabetes itself as a disease does not have any impact on the depressive state (oro 1.08 ; 95%, ci 0.701.65 ; p=0.740) the strongest influence is exerted by complications and concomitant chronic diseases (oro 1.38 ; 95%, ci 1.101.74 ; p<0.005). our study shows a significant negative trend between prevalence of anxiety and depression, and age and education (p for trend < 0.001). a significant association between depression and diabetic complications has been identified. according to a meta - analysis by de groot., the effect sizes for each complication were : 0.17 for retinopathy, 0.20 for macrovascular complications, 0.25 for nephropathy, 0.28 for neuropathy, and 0.32 for sexual dysfunction. the overall effect size was small to moderate (r=0.25), and was comparable between the 2 types of diabetes. the data of our study confirmed that chronic complications of diabetes mellitus affected the emotional state of patients. the prevalence of anxiety and depression symptoms was higher among patients with complications (45.7% [95% ci42.0349.34 ] and 33.5% [95% ci 30.137.0 ]) and self - reported arterial hypertension (45.7% [95% ci 42.149.3 ] and 31.7% [95% ci 28.435.2 ]). a canadian study found that smoking and depression are strongly associated in patients with type 2 diabetes, and this association appears to be strongest for moderate / heavy smokers. however, we did not find a statistically significant association between smoking and prevalence of anxiety and depression symptoms by bivariate analysis. duration of the disease was relevant to lower rating of all fields of quality of the life except for evaluation of role limitations due to emotional problems, emotional state, and mental health. in our study the results of the multivariable analysis for factors associated with anxiety and depression symptoms shows that diabetes duration was a risk factor significantly associated with higher scores of anxiety among patients with t2 dm. our results confirmed the study results of malaysian research findings that individuals with diabetes of less than 2-year duration were 1.6 times more likely to have depressive symptoms than individuals with diabetes of longer duration. females and asian indians were 1.4 times more likely to have depressive symptoms compared to males and malay diabetic individuals. the prevalence of depression and anxiety among type 2 diabetes mellitus patients was statistically significantly higher in females than in males (p<0.001). a significant association between depression and diabetic complications was identified (p<0.05). diabetes duration was a risk factor significantly associated with higher scores of anxiety among the patients with t2 dm. | backgrounddepression is associated with a variety of diabetes complications, including diabetic retinopathy, nephropathy, neuropathy, and macrovascular complications. the prevalence of the symptoms of anxiety (32%) and depression (22.4%) in patients with diabetes is considerably higher than in general population samples (10%). the aim of this study was to evaluate the prevalence and determinants of anxiety and depression symptoms in patients with type 2 diabetes (t2dm).material / methodsthis survey was conducted during 20072010. in total, 1500 patients were invited to participate in the study. the hospital anxiety and depression scale (hads) was used to measure depression and anxiety for the evaluation of the depressive state and anxiety. statistical analysis was carried out using spss 17.0.resultsmore than 70% of all respondents who participated in the study had diabetes mellitus complications (72.2%). the prevalence of mild to severe depression score was 28.5% (95% ci 25.731.4). the prevalence of anxiety was 42.4% (95% ci 39.345.5). anxiety was more frequent among females (46.8%) than among males (34.7%) (p<0.001). a significant negative trend was observed between prevalence of anxiety and depression, and age and education (p for trend < 0.001).conclusionsa significant association between depression and diabetic complications was identified (p<0.05). duration of diabetes was a risk factor significantly associated with higher scores of anxiety among the patients with t2 dm. |
many clinical cases of genital self - mutilation (gsm) were reported in the literature. however, the voluntary cutting of the genital organ remains extremely rare. in this paper, we report a case of genital self - mutilation committed in a jail while associating a depression episode., we will discuss the epidemiological and psychopathological aspects of the self - mutilation of the borderline patients with a focus on the genital self - mutilation. our male patient was 25-years - old, he was hospitalized in march 2003 in the department of psychiatry of the university hospital of fez (morocco) for genital self - mutilation while attempting to suicide. this act was carried out during the incarceration stage in the civil prison of fez. the patient belongs to large family composed of 11 brothers and sisters ; the family belongs to a low - income class of the rural area of fez (morocco). the patient received a severe family education from his father demonstrating a full use of authority, power and violence. the patient has spent seven years to learn the holly book of koran. then he practiced several jobs such agriculture and shoemaker at early age without achieving any professional stability. he believed that people do not try to understand him ; they do not identify his concerns, issues and needs ; and he believed that he might be rejected any time. the relationship of the patient with women was restricted and limited since he feared to be abandoned. concerning the sexuality however, the patient expressed that his not confident about his sexual identity and virility. in addition, neither familial psychiatric history disorders or mood disorders nor suicidal behaviors or addiction behavior were recorded. through his 15 years old, a succession of various psychiatric symptoms occurred in the patient with collection of clinical aspects. evermore, the patient reported episodically fluctuating anxiety in abandoned or accompanied contexts and sometimes - associating periods of depersonalization and derealization. the patient revealed also episodes of a permanent feeling of emptiness and lack of goals in life, with a frequent feeling of uselessness and underestimation. an excessive and abusive consumption of cannabis and alcohol was reported during episodes of anxiety and distress. during his 19-years old, the patient attempted to commit suicide using organo - phosphorics chemicals, this required hospitalization in the intensive care department along ten days. a year later, the patient demonstrated a brief psychotic disorder associating persecuting delusions, hypochondria and a diabolic possession. thus, while he was walking with his girlfriend, two young men from his village tried to humiliate him and captivate the girl away with them. the patient could not control his aggressive behavior and he wounded one of his aggressors that died in the emergency department of the hospital. he was condemned guilty and sentenced for ten years of jail. since his incarceration, the anxiety disorder, emptiness and abandon feelings were worsening. the patient was self - isolating in the prison cell and established very few relationships with the prison colleagues. the feeling of abandon has increased by the incarceration circumstances and the rare family visits. the patient started the self - mutilation in the prison iteratively commencing with the left arm and the thorax [figures 1 and 2 ] ; these occurred in a context of infringement of impulsion, abandon and anxiety. he reported that these self - destructive acts allowed him to discard fear and tension feelings without being determined to suicide. two weeks earlier the gsm, the patient has acquired a massive anxiety, a feeling of emptiness and uselessness. accordingly, a strong desire to die was subsequent. the patient decided to cut his genital organ since he always believes that this organ is the center of his existence. he started to organize the means to accomplish his act with a complete dissimulation of his suicidal intention. by the day of the gsm act, our patient borrowed a razor cutter from another detainee in the prison pretending that he wanted to shave. the patient took the opportunity of the absence of other detainees and prison guards who were in their morning break, he has hided himself in a corner of the prison cell and proceeded to completely cut his genital organ using the razor cutter around 10 am [figure 3 ]. the image clearly demonstrates the self - mutilation at the thoracic level the image clearly demonstrates the self - mutilation at the neck level the image clearly demonstrates the self - mutilation at the neck level the patient was transferred to the surgical unit of the emergency department, where he immediately followed a re - implantation of the genital organ with a realignment of the urethra. however, necrosis was been noticed and treated during the control of the implant. unfortunately, the necrosis caused tissues shrinking, and the urological surgery team removed the necrotized graft and proceeded to a plastic surgery of the remaining base of the genital organ [figure 4 ]. however, the patient completely refused any surgery and therapeutical approach despite all explanations provided about the consequences including the vital prognosis. the image clearly demonstrates the complete cut of the genital organ afterward, the patient was hospitalized in the psychiatric department where he showed a clear depressive syndrome, a self - culpability regarding the execution and declared that he has lost his virility. our case adopted a desperate vision of his future but he did not reveal any intention to recommit suicide. in addition, none of symptoms such disorganized speech, delusional, or perceptive disorders were noticed. the somatic examination was without particularity besides a routine biological assessment that was conducted and was negative. the psychometric test dibr (diagnostic interview for borderline - revised) revealed a borderline personality. during the admission in the psychiatry department, the patient demonstrated a progressive improvement of the symptomatology while using anxiolytic and antidepressant treatments including clomipramine at 150 mg / day and diazepam 20 mg / day. additionally the desperate, hopeless and self - culpability feelings have decreased within 6 weeks. during the hospitalization stage, the patient left the hospital after the stabilization of his clinical state lasting 64 days of hospitalization. in addition, the patient refused to talk about his self - destructive act and declared that he does not want to remember it again. one year later, the patient was examined in the jail and neither self - mutilation nor suicidal behavioral signs were recorded. since he left hospital, he declared that his act was committed without his determination and god would forgive him. he superficially mentioned the self - mutilation with avoiding to talk about the circumstances again. indeed, the gsm of a borderline personality diagnosis was evoked first considering the omnipresent fear of being abandoned, the permanent dysphoria, the instability characterizing the interpersonal relationship, the life sentimental, the recurrent suicidal behaviors, the psychiatric disorders alternation of varied categories and the significant values recorded in psychometric tests. various facts allowed suggesting recurrent major depressive disorders including the re - occurrence of episodic suicidal attempt during depressive state and the suicidal goal behind the genital self - mutilation however, a combination of depressive episodes with fear of abandon and the occurrence of psychiatric disorders such psychotic, obsession, substance abuse and substance dependence disorders were not supporting this hypothesis. the atypical gsm of the borderline personality might suggest a schizophrenia diagnosis, which was discarded considering the absence of any element of disorganization and the preservation of a good lucidity toward reality. an antisocial personality diagnosis was also envisaged considering the relational and professional instability, the impulsivity of repetitive self - mutilations. however, the biographical data, the interaction of the patient with events and the prevalence of fear of separation allowed withdrawing this diagnosis. the self - mutilation is an intentional injury committed by a patient on his / her body without an apparent intention to pass away. this definition opposes the durkeim definition that considers these acts such a slight figure of suicide. several denominations were used to describe the self - mutilation acts. the first scientific description of the self - mutilation act was given by strch in 1901, and reported a case of a 27-year - old male that self - mutilated in because of success lack with women. according to joyce., the rate of self - mutilation in the general population is comprised between 0.75% and 4%, while it varies between 5.5% and 77% in hospitalized psychiatric population and between 50% to 80% in borderline personality patients. patients used various means for self - mutilation and might select any part of their bodies. zanarini., studied 290 borderline patients during 10 years ; they reported high prevalence of self - destructive acts in this category of patients as well as large varieties of methods and means used for this purpose. hence, more than 70% of patients revealed a history of multiple sequences of self - mutilation and 60% have previously undergone a suicide attempt. in the same longitudinal study, a prospective periodical follow - up of two years revealed a significant decrease of self - destructive behavior in 75% of patients ; the improvement was mainly due to medical and psychotherapeutical treatments and to the maturation cumulated by the age increase of patients during treatment stage. the gsm achievement circumstances correlated with long surgical treatment mostly combined to coordination lack between the surgical and psychiatric care departments. the most reported gsm cases in the literature are not associated to another mutilation form of the body. generally, the masculine gsm concerns bourses including contents and the penis. lesions vary in intensity from the simple superficial cutaneous laceration to almost complete ablation of testicles and cutting of the penis. compared to males, the gsm of females is exceptional and the most frequent lesions are cuts or scarifications. the literature review of greisheiner and groves achieved on 52 gsm cases, demonstrated that 87% of gsm patients are caused by psychotic context including 28.5% schizophrenic. literature review of 110 cases showed that most of patients have suffered of psychoses associating sexual conflicts and delirious disorder about mystic - religious topics ; while non - psychotic cases have presented sexual identity disorders. the psychiatric disorders consisted of psychosis, erotomania, dysmorphophobia disorder, histrionic personality, munchausen 's syndrome, alcoholic dementia, temporal lobe epilepsy, and sexual identity disorders. lennon., reported a case of mood disorder during a manic episode. however, tharoor., reported a 72 years old patient that committed gsm secondary to familial conflicts without any remarkable psychiatric disorder history. to our knowledge, fayed., reported a 17-year - old girl who was admitted in the gynecological emergency department for an acute hemorrhage resulting from a gsm using an electric knife. the self - destructive act was accomplished during an episode of dissociation of a borderline personality. three years earlier, this young girl was a victim of sexual abuse without penetration by three people ; and she kept the incident secrete. the investigation of the risk factors of the self - mutilation behavior in borderline patients found factors counting loss of physical integrity, confusion, the feeling of guiltiness, self - underestimation, the dependence or the abusive consumption of a substance. the presence of maltreatment during childhood including physical or effective negligence, sexual abuse is mostly mentioned as a predictive factor of the self - mutilation in borderline personality patients. in - contrast, functions were attributed to these acts such coping with dissociative states, stress communication to others, symbolic expression of emotions especially guiltiness, regulation of dysphoric element such an anti - suicide role. authors insisted on the addictive nature through deliverance of endorphin and dopamine in blood, and on the distractive function consisting of shifting patient attention from moral pain to physical pain. subsequently, 60% of borderline patients did not report any pain feeling during the self - mutilation acts. bohus has achieved two perception tests of cold and pain in 12 borderline patients and 19 witness women (the cold pressure test, the tourniquet pain test). results showed a significant decrease of pain perception in borderline patients compared to the witness group regardless of their stress state. they noted that there was neither a modification of sensory discriminative component of the pain nor a deficit of attention. this approach would involve either a serotoninergic dysfunction yielding impulsive and aggressive aspects or a low limit of excitability in the secondary limbic structure with repeated emotional trauma during a fast evolution. indeed our clinical case illustrates the issue that is often raised in the literature such : what is the suicidal component in the self -mutilation behaviors ? these included the patient suicidal intention, the physical damage resulting of the self - destructive act, the frequency or the chronicity of the act supporting the self - mutilation and used methods. they found that these varied for the self mutilation acts and are identical for the suicide attempts. fulwiter.,studied 31 self - mutilated prisoner patients and noticed that 15 cases reported their act targeting an attempt to commit suicide, while 16 patients simply targeted hurting themselves. the suicide attempt was associated to affective disorders, while the mutilation injuries were related to a history of hyperactivity and dysthymia or to an anxiety disorder during childhood. in addition, greilsheimer and groves reported a series of 53 patients of gsm including five cases with suicidal profile. jason., presented three chinese patients followed for psychotic disorder while they committed gsm for suicidal purposes. recently, tharoor., reported a 72 years old married patient without major medical or psychiatric history who tried to suicide using gsm. in fact, it is believed in chinese and indian cultures that the penis is the source of the life. hence destroying this organ might lead to death. in addition, it is to highlight that our patient does not have any associated delirious religious believes. indeed the original aspects of the genital self - mutilation in a borderline personality of the psychiatric profile discussed have demonstrated almost unique case. the suicidal aspect of this self - destructive act constituted another originality of our case study. therefore, our paper might contribute to better understanding and clarification of unknown aspects of the profile of the borderline personality behaviors. | self - mutilation acts are known to characterize the borderline personality disorders. however, voluntary cutting of the male genital organ remains extremely rare. the present paper reports a case of a 25-years - old young male with a borderline personality. the patient committed a genital self - mutilation (gsm) targeting suicide during incarceration stage in jail. in addition, a discussion of the epidemiological and psychopathological aspects of the self - mutilation of borderline patients was been conducted. a particular interest is attributed to the genital self - mutilation and a review of the literature is presented. |
chickenpox (varicella) is a highly contagious disease recognized by a characteristic and often pathognomonic maculopapular vesicular rash. varicella may lead to serious complications, including secondary bacterial skin and soft - tissue infections, cerebellitis, encephalitis, pneumonia, and coagulopathy. more than 95% of the non - vaccinated populations were infected with varicella, and most of them are younger than 20 years. children younger than 10 years of age, particularly those between 3 and 6 years, exhibit a high incidence of infection when they do not receive varicella vaccine as part of a universal vaccination program. in 1998, the world health organization (who) recommended routine childhood varicella vaccination in countries where chickenpox significantly affects public health. since the introduction of universal vaccination, a number of countries have witnessed declines in varicella incidence, hospitalization, and/or mortality, including the united states of america, australia, italy, germany, and taiwan. varicella vaccination provides long - term effectiveness in preventing varicella and may reduce the risk of herpes zoster. furthermore, varicella vaccination is cost - effective in the asian regions. in singapore, the benefit cost ratio of vaccination is 2.25, which indicates that every us$1.00 invested in vaccination would save us$2.25. similar findings were reported in 2 studies in taiwan, which reported a benefit cost ratio of 2.06 and 0.9, respectively. although varicella is a global health issue, disease burden varies by geographical region, climate, population density, and socioeconomic development. for example, the onset of varicella infection may be delayed in tropical countries because of virus inactivation by high ambient temperature and humidity. in hong kong, a city with subtropical climate, the average annual varicella notification and hospital attendance rates in children younger than 18 years are 981 and 285 per 100,000 individuals, respectively. moreover, complication rate is 47% among hospitalized cases. even though the varicella complication rate in hong kong is comparable with international data, the varicella vaccine was only introduced into hong kong 's universal childhood immunization program (cip) as a free but optional vaccine in july 2014 for children born on or after january 1, 2013. before the implementation of universal varicella vaccination, children in hong kong a survey conducted by hong kong 's department of health in 2009 reported that only 32.4% of local - born children had received varicella vaccine at the time. a 2012 survey of kindergartens revealed the varicella infection rates was 19.5% and the uptake of varicella vaccination rate was 57.6%. although varicella vaccine coverage has increased in hong kong, it remains below that of the united states (79.9%92.0%). previously noted barriers to vaccination in hong kong include uncertainties about the effectiveness and side effects of the vaccine and the lack of recommendations from doctors or the government. cross - sectional surveys conducted in the united states and turkey showed that positive parental health beliefs on the effect of varicella vaccine may affect parental decisions to vaccinate their children. however, both studies did not correlate parental health beliefs and vaccination status. in the present study, we investigate the varicella vaccination rates among preschool (kindergarten) and primary schoolchildren before the vaccine 's incorporation into hong kong 's universal cip ; the association between the rates and health beliefs and demographic characteristics of the parents of these children ; and the motivators and barriers of having the vaccination. a complete list of kindergarten and primary schools was downloaded from hong kong 's education bureau (www.edb.gov.hk), which comprise 948 kindergartens and 615 local primary schools. fourteen kindergarten (1.5%) and 5 primary schools (0.8%) were randomly selected and their school principals were invited to participate. although no data are available on the age - specific varicella cumulative incidence in hong kong, several overseas studies reported varicella seroprevalence rates in children. various countries such as england, italy, australia, and korea have varicella seroprevalence rates ranging from 58% to 75% by the age of 5 years, and 83% to 93% by the age of 12 years. from these figures, the sample size was estimated to be 1400 kindergarten students (350 cases for each age group within 25 years of age), and 1610 primary school students (230 cases for each age group within 612 years of age), which sum up to a total of 3010 students. this estimation gave a precision of 5% at a confidence interval of 95% for age - specific varicella cumulative incidence in our schoolchildren population. according to the education statistics (www.edb.gov.hk) and by assuming that the number of students in each kindergarten and primary school in hong kong are around 100 and 400, respectively, we determined that 14 kindergartens and 5 primary schools can satisfy the sample size. after obtaining consent from the principals, questionnaire packages that contain information, consent forms, and self - administered questionnaires were distributed to the students. the students were then asked to bring home the packages for filling out by their respective parent or guardian. the questionnaire consisted of 4 parts : demographic information of the children and the respondents, children 's history of varicella infection and vaccination, 10 parental health belief statements regarding the varicella vaccine, knowledge on varicella transmission and therapy, and reasons / barriers for their having / not having varicella vaccination. most questions in the survey were in multiple - choice format, except for personal health beliefs and motivators / barriers for vaccination, in which participants were requested to answer with numbers in pre - specified ranges. each of the 10 parental health belief statements were scored from 1 (completely disagree) to 6 (completely agree). the motivators / barriers of vaccination were graded using a 6-point likert scale from 0 (completely disagree) to 5 (completely agree). only the parents of vaccinated children were asked to answer on the motivators of vaccination, whereas only the parents of unvaccinated children were asked to answer on the barriers. perceived knowledge and chance of infection were assessed by categorical responses ranging from very inadequate / very unlikely to very adequate / very likely. completed questionnaires were collected, and data checking and entry into a pre - developed spss database were performed by student assistants. median and interquartile ranges were calculated by vaccination status for the variables that used the likert scale and subsequently compared by employing the mann crude odds ratios (ors) and their 95% confidence intervals were computed for each variable by vaccination status. ors with p values 50% of respondents strongly agreed with these statements (table 2). for respondents with unvaccinated children, only 3 statements obtained median scores of 5. all but one statement showed significant differences between the vaccinated and nonvaccinated groups (table 2). parental health beliefs on having their children vaccinated against varicella infection adjusted ors and their 95% confidence intervals from the logistic regression were calculated based on the final model selected using the stepwise procedure (table 3). children who were studying in senior primary school, respondents with educational attainments higher than the secondary level, family income of $40,000, very adequate perceived knowledge on varicella, very high perceived chance of children being infected by varicella, discussion with a family doctor, and willingness to pay > $ 300 for the vaccine were associated with higher vaccination rates. except for my child may not need the chickenpox vaccine because chickenpox is only a minor illness, these health beliefs are as follows : the chickenpox vaccine is very effective in preventing chickenpox, my child is very likely to contract chickenpox if he / she does not receive the vaccine, the chickenpox vaccine is worthwhile even if it does not provide lifelong immunity to chickenpox for my child, and i am uncomfortable with the number of shots my child is receiving. the results from the application of the generalized linear mixed model with school as random effect are also presented in table 3. multivariate analysis of the associations between demographic characteristics and the rate of varicella vaccination reasons for varicella vaccination were scored from 0 (not important) to 5 (very important). the median scores for recommendation by family doctors and recommendation by specialists were both 5, which implies that > 50% of respondents thought that these recommendations were very important (table 4). recommendation on tv / newspaper / magazine obtained the lowest median score (3) among the 6 recommendations. of the parents with vaccinated children, 71.8%, 69.0%, and 45.7% rated family doctors, specialists, and the government, respectively, as very important motivators of vaccination. reported motivators for vaccination among parents of vaccinated students four statements on the barriers to varicella vaccination (worry about adverse side effects, not sure about the effectiveness of the vaccine, no recommendation from doctors, and no recommendation from the government / department of health) obtained the highest median scores (3). by contrast, 4 other statements (do nt know where to vaccinate, not aware of the existence of the vaccine, child fear of injection, and child is too young) obtained the lowest median scores (0), which shows that > 50% respondents thought that these factors were unimportant barriers (table 5). of the parents of unvaccinated children, 21.3%, 22.3%, 21.7%, and 26.0% rated adverse effects, uncertain effectiveness, no recommendation from doctors, and no recommendation from the government, respectively, as very important barriers to vaccination. varicella can pose serious threats of disease to fetuses, neonates, and non - immune or immunocompromised children and adults. the present study found that 69.0% of hong kong preschool and school children were reported to have received vaccination against varicella, although the vaccine was not yet included in hong kong 's cip at the time. this rate is higher than that in a similar study in hong kong reported a few years ago. the rate increase may be due to the educational campaigns conducted by the government and the discussions on including the vaccine in the cip over the recent years. the vaccine has been included as a free optional vaccine in the cip program since 2014. hong kong 's rate of varicella vaccination appears slightly higher than those of several other countries that also do not include the vaccine in their routine immunization programs. the corresponding vaccination rates for these countries were 61.1% in athens, greece, 50% in japan, and 62.0% in shandong province, china. the higher rate of vaccination in hong kong may indicate the greater awareness or value placed on varicella prevention by hong kong parents. this study showed that parents with higher educational attainments, greater family income, greater perceived knowledge of varicella, and higher perceived chance of infection were significantly associated with positive vaccination. in the united states, higher maternal educational levels positively correlated with varicella vaccination. similarly, families with better parental educational attainments and higher family income showed greater vaccination rates in turkey and taiwan. promoting parental knowledge on vaccination is a key factor in improving immunization rates, especially in families with lower educational backgrounds and from cities with low socioeconomic ranking, as observed in israel. discussion with a family doctor is significantly associated with a greater propensity for vaccination, which indicates that an expert 's opinion importantly influences the perspective of the parents. recommendation from family doctors and recommendation from specialists were the major reasons that the parents allowed their children to receive the vaccine. by contrast, other parents did not seek vaccination for their children mainly because of their lack of knowledge about the vaccine and lack of recommendation from the experts or government officials. a previous study conducted in sweden also showed the great importance of the role of well - trained healthcare staff in providing advice and discussing measles, mumps, and rubella vaccination with concerned parents. the results may be explained by the relationship between health beliefs and other parental attitudes. the chickenpox vaccine is very effective in preventing chickenpox and my child may not need the chickenpox vaccine because chickenpox is only a minor illness may be related to the perceived knowledge of parents on vaccination. parents are more willing to vaccinate their children when with adequate awareness on the effectiveness of vaccine and potential complications of varicella. my child is very likely to contract chickenpox if he / she does not receive the vaccine may be associated with the perceived chance of children being infected. parents were more likely to allow vaccination of their children if they perceive that their children are otherwise at risk. the chickenpox vaccine is worthwhile even if it does not provide lifelong immunity to chickenpox for my child was also associated with parental knowledge. when promoting varicella vaccination, clarifying the period of vaccine effectiveness is important. although children can not be immunized throughout their lives, long - term health benefits still encourage vaccination. surprisingly, i am uncomfortable with the number of shots my child is receiving positively correlated with vaccination. greater awareness on the risk of infection and more knowledge on vaccination may have outweighed the discomfort parents feel while their children are being vaccinated. this explanation, however, should be justified in future studies. despite its effectiveness in preventing varicella in the united states, the varicella vaccine has not been included in the immunization program of many countries, including japan and mainland china. furthermore, our survey was completed only before varicella vaccination was added to hong kong 's cip. this immunization program is funded by the hong kong government and includes vaccines that are free of cost. the government announced in december 2012 the possible addition of the varicella vaccine to the cip, which was then launched to fruition in july 2014. the present study indicated that 94.5% of the respondents would allow their children to be vaccinated if the vaccine was included in the cip. furthermore, high vaccination rates (95%) have been reported for all other vaccines included in cip. the strength of this study lies in the large sample of > 2700 participants recruited from 14 kindergartens and 5 primary schools randomly selected across hong kong. however, an important limitation of this study is the potential for recall bias ; the vaccination status of the children reported by parents was not validated by official records. another limitation is that the number of children without varicella infection is likely less than the figure reported because of possible previous unrecognized infection. furthermore, the total of 19 schools recruited only accounted for a small proportion of all the schools in hong kong, which might limit the generalizability despite the large sample size. this suggestion could explain the greater likelihood of varicella vaccination among parents who felt uncomfortable for their children receiving more shots of vaccines, as revealed in present study. this survey has provided valuable information on the association between parental attitudes and varicella vaccination in the period before the inclusion of the vaccine to hong kong 's cip. our study suggested that to increase vaccination rates, government officials should organize more promotional activities to raise parents awareness on the inclusion of the vaccine in the cip as a free optional vaccine. once varicella vaccine is implemented as part of hong kong 's cip for a period of time and anticipated high vaccination rates are achieved, conducting a follow - up study would be valuable to demonstrate the effect of the policy change on parental attitudes and practices regarding varicella vaccination. if possible, evidence in terms of morbidity and complications could be gathered by reviewing hospital admission data to shed greater light on the general impact of the cip inclusion. in countries with universal immunization programs that still exclude the varicella vaccine, these countries do not include united states, australia, italy, germany, and taiwan, which have already been providing universal varicella vaccination. we hope to witness a future increase in the number of countries that would follow the who recommendation on including the vaccine in their routine immunization programs. among parents of hong kong preschool and schoolchildren, parental attitudes and demographic characteristics were associated with rates of varicella vaccination. as emphasized in the study, health professionals and the government play important roles in promoting varicella vaccination to the public. | abstractthis study investigates parental attitudes and factors associated with varicella vaccination among preschool and schoolchildren prior to introduction of the vaccine into hong kong 's universal childhood immunization program.fourteen kindergartens and 5 primary schools in hong kong were randomly selected in 2013. parents of the students were invited to answer the self - administered questionnaires. acquired information included demographic characteristics and socioeconomic statuses of families, children 's history of chickenpox infection and vaccination, and reasons for getting children vaccinated. logistic regression was applied to examine the factors associated with vaccination.from the 3484 completed questionnaires, the calculated rates of varicella infection and vaccination were 20.7% and 69.0%, respectively. barriers to vaccination included parental uncertainties about vaccine effectiveness, lack of recommendation from the government, and concerns on adverse effects. overall, 71.8%, 69.0%, and 45.7% of the parents rated family doctors, specialists, and the government, respectively, as very important motivators of vaccination. higher parental educational level and family income, better perceived knowledge of varicella and chance of infection, discussion with a family doctor, and positive health belief towards vaccination were associated with vaccination (all p < 0.05).the rate of vaccination in hong kong was higher than that of some other countries that also did not include the vaccine in their routine immunization programs. more positive parental attitudes, higher socioeconomic status, and discussion with a family doctor are associated with greater vaccination rates. the important roles that health professionals and the government play in promoting varicella vaccination were emphasized. |
deep vein thrombosis (dvt) and pulmonary embolism (pe) are expressions of venous thromboembolism (vte). cancer patients with active disease are at higher than average risk compared to a non - cancer population.1 in one study, cancer patients with solid tumors had a vte prevalence of 7.8% (95% confidence interval [ci ] : 6.2%9.4%) or 1 event per 12.8 patients.2 following the occurrence of an initial event, the risk of recurrence remains high.1,3,4 as a result, extended thromboprophylaxis in cancer patients is recommended by the american college of chest physicians and other groups in order to prevent recurrent vte.35 for many years, vitamin k antagonists (vka), such as warfarin, were used for extended thromboprophylaxis in cancer patients. however, current international guidelines recommend low - molecular - weight heparin (lmwh) monotherapy over vka for the secondary prevention of cancer - associated vte.4,6,7 the advantages of lmwh over vka are a more favorable safety profile, fewer interactions with food and other drugs and a more predictable anticoagulation response, which eliminates the need for continuous blood monitoring.4,6,7 the disadvantages associated with lmwh are the need for daily subcutaneous (sc) administration for up to 6 months and the higher drug acquisition cost compared to vka. the three lmwh that have been investigated in randomized comparative trials against vka in the setting of secondary vte prophylaxis in cancer patients are enoxaparin, tinzaparin, and dalteparin. there were two small trials comparing enoxaparin to vka and both failed to demonstrate significant improvements over vka.8,9 similarly, there were also two tinzaparin trials that failed to reach statistical significance, one being a large well - powered multinational study that enrolled 900 cancer patients.10,11 the only lmwh to have demonstrated a statistically and clinically meaningful benefit in terms of vte risk reduction has been dalteparin. in the multinational clot (comparison of low - molecular - weight heparin versus oral anticoagulant therapy for the prevention of recurrent venous thromboembolism in patients with cancer) trial, patients were to receive dalteparin (200 iu / kg sc once daily in the first month, then 150 iu / kg sc once daily from months 2 to 6) or 57 days of dalteparin (200 iu / kg sc once daily) overlapped with and followed by an oral vka for up to 6 months.12 the main inclusion criteria were prior acute symptomatic vte, and the primary end point was the first objectively documented, symptomatic recurrent dvt, or pe during the 6-month study period. during the course of the study, 676 cancer patients with a newly diagnosed vte were enrolled. by the end of the study, 27 of 336 (8.0%) patients in the dalteparin group developed a recurrent vte compared to 53 of 336 (15.8%) in the vka control group (hazard ratio [hr]=0.48 ; p=0.002). safety was comparable between groups with no significant differences in major bleeding events (dalteparin=6% vs. vka=4% ; p=0.27).12 dalteparin also provides benefits across important patient subgroups. in a recent post hoc analysis of the clot trial conducted by woodruff, dalteparin patients with moderate (crcl 3059 ml / min) to severe (crcl 1529 ml / min) renal impairment at randomization had lower absolute rates of vte than those in the vka group (3% vs. 17% ; p=0.011), and the frequency of major bleeding was similar between subgroups (2.0% vs. 2.4% ; p=0.46).13 the original clot trial collected health care resource data, and this was initially used to perform a cost utility analysis from the canadian health care system perspective.14 in that study, the main conclusion was that dalteparin was economically attractive to vka with the cost per quality - adjusted life year (qaly) being $ can13,800.14 however in that analysis, health state utilities were estimated from a sample of oncology health care professionals (i.e., patient surrogates), which is not recommended by guidelines for the economic evaluation of drugs.15,16 in addition, the economic value of dalteparin as an alternative to vka was not assessed in patient subgroups of special clinical interest, such as those with renal impairment. in this study, an updated pharmacoeconomic analysis was conducted to determine the economic value of dalteparin from the perspective of the publicly funded canadian health care system. therefore, the two main questions addressed in the current analysis were as follows : does the use of dalteparin for secondary vte prophylaxis in cancer patients provide good economic value using 2015 costs of care and more appropriate sources of clinical utility ? and what is the economic value of dalteparin when used as secondary prophylaxis in cancer patients with moderate - to - severe renal impairment ? patients groups within the clot trial have been previously described,12 but they were well balanced in terms of important prognostic factors, such as age, performance status, presence of metastatic disease, history of prior vte, and baseline renal function. health care resource use data that were collected from the clot trial were extracted and used for a patient - level economic analysis. the extracted data included information on dose and duration of therapy for dalteparin and the vka, biochemistry and hematology tests, the international normalization ratio (inr) in the case of vka, patient telephone contact, unscheduled clinical visits, diagnostic tests associated with vte recurrence, hospital length of stay, blood transfusions, and the occurrence of major bleeding events that were possibly or probably related to the study drugs. not all aspects of health care resource use were captured in the clot trial. the actual type of intervention for the treatment of patients who developed recurrent vte or for the management of related side effects was not collected. as a result, canadian and international literature was reviewed to identify costs for the management of dvt, pe, fatal pe, and for adverse events consisting of heparin - induced thrombocytopenia, hematochezia, hematoma, hematuria, hematemesis, melena, retroperitoneal bleeding, intracranial bleeding, and intraperitoneal bleeding.1722 in addition, only events of grade iii (national cancer institute s common terminology criteria for adverse events, nci ctcae v4.0) were included in the economic analysis. unless it was otherwise specified within the clot trial database, an assumption was made that all patients who developed dvts were treated in the outpatient setting. the unit costs for the individual health care resource items were obtained from the ontario ministry of health and the university health network in toronto. all costs were reported in 2015 canadian dollars, and cost estimates from previous years were converted into 2015 dollars using the consumer price index as reported by the bank of canada. the daily cost of supplies for dalteparin administration and the monthly pharmacy dispensing fee (i.e., $ can11.99) were also included in the analysis. the end product of this process was a comprehensive database consisting of patient demographic, clinical outcomes, and 2015 total cost of care for each patient enrolled into the clot trial. the final database was then used to conduct a patient - level univariate economic analysis between patients randomized to receive dalteparin or vka within the clot trial. cancer patients with renal impairment and acute vte are a clinically high - risk subgroup. this consisted of a univariate economic analysis on the renally impaired subgroup to determine the magnitude of the cost difference between patients treated with dalteparin relative to those who received vka within the clot trial. this cost difference was then used in the subsequent cost analysis to estimate the incremental cost per vte avoided and qaly gained with dalteparin within the renally impaired subgroup. for the intention to treat (itt) population and the renally impaired subgroup, the total cost of therapy between patients randomized into the dalteparin and vka group was compared using the unpaired t - test and the nonparametric wilcoxon rank - sum test. the cutoff significance for all of the statistical tests was set at the p<0.05 level and there was no adjustment for multiplicity. all of the statistical analyses were performed using stata, release 11.0 (stata corp., college station, tx, usa). a health state utility is a value between 0 and 1 where the former represents death and the latter a state of optimal quality of life.23 in this study, there were four relevant health states associated with secondary vte prophylaxis in cancer patients. they consisted of treatment with a vka or dalteparin for up to 6 months to prevent a recurrent vte within the general clot trial population and then in the patient subgroup with renal impairment. using the time - trade - off technique, healthy months equivalence for the time spent in each health state.24,25 gains in healthy months equivalence were then converted into qalys, dividing by 12 months. to illustrate the process therefore, the utility would be 0.5, on a scale between 0 and 1 (6 months of healthy months equivalence/12 months in the given health state). it has been recommended by the task force from the international society of pharmacoeconomics and outcomes research and the guidelines developed by the canadian agency for drugs and technology in health that health state utilities be measured from a randomly selected sample from the general public.16,25 therefore, within this study, subjects consisted of 24 volunteers from the general canadian tax paying public that were selected through a multistage, random cluster sampling technique with postal code as the unit of selection. to be eligible for the survey, individuals had to be 18 years of age, have permanent resident status in canada, be indirectly supporting the health care system through tax contributions, and give informed consent to participate in the interview. respondents were interviewed face - to - face by a trained field investigator via a door - to - door contact strategy. the only personal information recorded was age, marital status, education, household income, number of children, history of vte, familiarity with the cost of cancer drugs, and whether they had drug insurance. participants were assured that participation was voluntary and they were free to withdraw at any time. permission and approval to conduct the study was received by the quorum institutional review board, which is a central ethics review board that is fully compliant with relevant us and canadian regulations. after informed consent was obtained, respondents were presented with information about the natural history of vte in patients with cancer, followed by a description of the vka and dalteparin administration protocol. this included information on the method of administration, monitoring requirements and the associated risks and benefits. during the final part of the interview, this consisted of the risk for recurrent dvt, fatal and nonfatal pe, and major bleeding events associated with each therapy. they considered being equivalent to the time spent in each of the less than optimal health states described. these measures were used to weigh the duration within each health state by the quality of life experienced by a patient living through that time period. the interview process was then repeated with the presentation of data for patients with moderate - to - severe renal impairment. the two components that comprised the pharmacoeconomic analysis consisted of the incremental cost per vte avoided and the incremental cost per qaly gained when dalteparin was used as an alternative to vka to prevent recurrent vte in cancer patients. these estimates were determined for both the full itt clot population and the renally impaired subgroup. the two economic outcomes were calculated by dividing the difference in cost between dalteparin and vka therapy (numerator) by the difference in vte avoided or qalys gained (denominator). to test for uncertainly in the point estimates, this consisted of reanalyzing the primary outcomes using the upper and lower 95% cis of the differences in total treatment costs and utility estimates. cancer patients with renal impairment and acute vte are a clinically high - risk subgroup. this consisted of a univariate economic analysis on the renally impaired subgroup to determine the magnitude of the cost difference between patients treated with dalteparin relative to those who received vka within the clot trial. this cost difference was then used in the subsequent cost analysis to estimate the incremental cost per vte avoided and qaly gained with dalteparin within the renally impaired subgroup. for the intention to treat (itt) population and the renally impaired subgroup, the total cost of therapy between patients randomized into the dalteparin and vka group was compared using the unpaired t - test and the nonparametric wilcoxon rank - sum test. the cutoff significance for all of the statistical tests was set at the p<0.05 level and there was no adjustment for multiplicity. all of the statistical analyses were performed using stata, release 11.0 (stata corp., college station, tx, usa). a health state utility is a value between 0 and 1 where the former represents death and the latter a state of optimal quality of life.23 in this study, there were four relevant health states associated with secondary vte prophylaxis in cancer patients. they consisted of treatment with a vka or dalteparin for up to 6 months to prevent a recurrent vte within the general clot trial population and then in the patient subgroup with renal impairment. using the time - trade - off technique, healthy months equivalence for the time spent in each health state.24,25 gains in healthy months equivalence were then converted into qalys, dividing by 12 months. to illustrate the process therefore, the utility would be 0.5, on a scale between 0 and 1 (6 months of healthy months equivalence/12 months in the given health state). it has been recommended by the task force from the international society of pharmacoeconomics and outcomes research and the guidelines developed by the canadian agency for drugs and technology in health that health state utilities be measured from a randomly selected sample from the general public.16,25 therefore, within this study, subjects consisted of 24 volunteers from the general canadian tax paying public that were selected through a multistage, random cluster sampling technique with postal code as the unit of selection. to be eligible for the survey, individuals had to be 18 years of age, have permanent resident status in canada, be indirectly supporting the health care system through tax contributions, and give informed consent to participate in the interview. respondents were interviewed face - to - face by a trained field investigator via a door - to - door contact strategy. the only personal information recorded was age, marital status, education, household income, number of children, history of vte, familiarity with the cost of cancer drugs, and whether they had drug insurance. participants were assured that participation was voluntary and they were free to withdraw at any time. permission and approval to conduct the study was received by the quorum institutional review board, which is a central ethics review board that is fully compliant with relevant us and canadian regulations. after informed consent was obtained, respondents were presented with information about the natural history of vte in patients with cancer, followed by a description of the vka and dalteparin administration protocol. this included information on the method of administration, monitoring requirements and the associated risks and benefits. during the final part of the interview, this consisted of the risk for recurrent dvt, fatal and nonfatal pe, and major bleeding events associated with each therapy. they considered being equivalent to the time spent in each of the less than optimal health states described. these measures were used to weigh the duration within each health state by the quality of life experienced by a patient living through that time period. the interview process was then repeated with the presentation of data for patients with moderate - to - severe renal impairment. the two components that comprised the pharmacoeconomic analysis consisted of the incremental cost per vte avoided and the incremental cost per qaly gained when dalteparin was used as an alternative to vka to prevent recurrent vte in cancer patients. these estimates were determined for both the full itt clot population and the renally impaired subgroup. the two economic outcomes were calculated by dividing the difference in cost between dalteparin and vka therapy (numerator) by the difference in vte avoided or qalys gained (denominator). to test for uncertainly in the point estimates, this consisted of reanalyzing the primary outcomes using the upper and lower 95% cis of the differences in total treatment costs and utility estimates. for the original clot trial report, a comparison of baseline patient and clinical variables suggested that patients randomized into the dalteparin and vka groups were well balanced.12 however in the recent subgroup analysis, there were slightly more patients in the vka group who had moderate - to - severe renal impairment at baseline (26.0% vs. 21.9%), but the difference failed to reach statistical significance (p=0.21).13 a comparison of health care resource use data collected during the clot trial indicated that patients in the experimental group received dalteparin for a mean of 126.3 days compared to 8.0 days in the vka group (table 1). after the initial 8 days of therapy with dalteparin, patients in the control group received oral vka therapy only, which was given for a mean of 116.9 days. a comparison of laboratory and diagnostic test use indicated similar levels of utilization, with the exception of inr measurements, which are required for oral anticoagulation therapy with vkas. the number of blood transfusions, units given and inr measurements associated with the transfusions were also higher in the vka group (table 1). the comparative analysis on health care resource use increased use of some resources was also noted in this patient subgroup (table 2). the clot trial was powered to detect statistically significant differences in the number of recurrent vte between treatment groups over the 6-month trial horizon. a statistically significant reduction in the occurrence of new vte in patients randomized to the dalteparin group was reported (table 3). in patients who received therapy in the outpatient setting, there was a hospital admission rate (regardless of cause) of 25.1% in the dalteparin group compared to 28.5% in the control (p=0.33 ; not statistically significant). when the cause for admission was limited to a recurrent vte, bleeding or heparin - induced thrombocytopenia, the rates of hospital admission were 3.2% for dalteparin and 3.8% in the oral therapy group (p=0.68). overall, these rates translated to 32 additional hospital days for dalteparin patients and 40 days in the vka group (table 3). similar benefits in favor of dalteparin were also reported in the renally impaired subgroup (table 3). the analysis was continued with the application of canadian unit costs to all health care resources used by patients from the itt clot population. dalteparin patients had higher costs for drug therapy and for the treatment of major bleeding events relative to the vka control group (table 4). in contrast, the vka group had increased costs for laboratory monitoring, blood transfusions, and for the management of vte recurrences. when all of the costs were combined, the total mean cost for all clot patients randomized into the dalteparin group was $ 5,771 (95% ci : $ 5,324$6,219) compared to $ 2,569 (95% ci : $ 2,328$2,810) in the vka group (p<0.001). in the subgroup analysis consisting of renally impaired patients, the differences in cost between the dalteparin and vka groups remained statistically significant ($ 5,000 [95% ci : $ 4,248$5,569 ] vs. $ 2,671 [95% ci : $ 2,079$3,264 ] ; p<0.001) but treatment preferences and health state utilities for each of the four outcomes were measured from a sample of 24 members of the tax paying general public that were selected through a random multistage sampling strategy. the mean age of respondents was 52 years (range : 3273) with 11 of 24 (45.8%) being female. overall, 23 of 24 respondents were married, 15 (62.5%) had postsecondary school education, 11 (45.8%) were working full time, and 12 (57.1%) had annual incomes that exceeded $ 100,000. the data revealed that only 1 of 24 subjects (4.2%) had previously experienced a vte, which was treated with warfarin. when the background information was presented on both drugs, respondents were asked to select their preferred secondary prophylactic intervention from an overall clot trial perspective. overall, 21 of 24 (87.5%) respondents selected dalteparin over a vka as their drug of choice. healthy month equivalence scores and health state utilities for each alternative were then estimated from the sample. the higher preferences for dalteparin translated into higher health state utility scores (measured as healthy month equivalence). the utility of the dalteparin health state was almost twofold higher than that seen with treatment with a vka suggesting improved quality of life (table 5). the higher utility scores for dalteparin were due to a combination of factors such as improved efficacy, a demonstrated safety profile with extended use and the ability to eliminate continuous inr monitoring. the main positive attribute that respondents indicated about vka was the oral route of administration. overall, the gain in healthy month equivalence was ~1.67 months with dalteparin for the full clot population, which corresponded to an additional gain of ~0.14 qalys. the utility assessment was then extended to patients with moderate - to - severe renal impairment. under this treatment setting where dalteparin demonstrated an even greater effect size (i.e., number needed to treat=7 vs. 13 for the overall population), 23 of 24 respondents (96%) selected dalteparin as their preferred treatment. this higher preference translated into a healthy month equivalence gain of 2.0 months, which corresponded to an additional gain of ~0.17 qalys (table 5). from the results in the itt clot population, dalteparin was associated with an incremental cost of ~$41,200 (95% ci : $ 34,300$46,200) per vte avoided (table 5). one of the limitations of only reporting the cost per vte avoided is that it does not incorporate the patient quality of life and utility gain associated with the avoidance of dvt or pe. therefore, the incremental cost of dalteparin (i.e., $ 3,202) was combined with the 0.14 qalys gained (a gain of 1.67 health months equivalence corresponds to a gain of 0.14 qaly). the findings revealed an incremental cost of ~$23,100 per qaly gained (95% ci : $ 19,200$25,800). the analysis was also done for the renally impaired patient subgroup. when used in patients with moderate - to - severe renal impairment, dalteparin was associated with an incremental cost per vte avoided and qaly gained of $ 16,400 and $ 14,000, respectively (table 5). a series of one - way sensitivity analyses were conducted using the 95% ci of the differences in healthy months equivalent scores (1.182.15) and cost ($ 2,668$3,588) in the full itt clot population. under the worst - case scenario for dalteparin where the highest cost difference between the two treatments was used (i.e., $ 3,588) along with the lowest gain in healthy months equivalence (i.e., 1.18), the cost per qaly gained with dalteparin increased to ~$36,500. under the best - case scenario for dalteparin where the lowest cost difference (i.e., $ 2,668) was used along with the highest gain in healthy months equivalence (i.e., 2.15), the cost per qaly gained with dalteparin was reduced to $ 14,900 in the itt clot population. repeating the analysis in patients with renal impairment, the cost per vte avoided and qaly gained changed to $ 23,500 and $ 6,800 under the two worst- and best - case scenarios, respectively. these findings imply that the base case cost per vte avoided and qaly gained estimates were stable. treatment preferences and health state utilities for each of the four outcomes were measured from a sample of 24 members of the tax paying general public that were selected through a random multistage sampling strategy. the mean age of respondents was 52 years (range : 3273) with 11 of 24 (45.8%) being female. overall, 23 of 24 respondents were married, 15 (62.5%) had postsecondary school education, 11 (45.8%) were working full time, and 12 (57.1%) had annual incomes that exceeded $ 100,000. the data revealed that only 1 of 24 subjects (4.2%) had previously experienced a vte, which was treated with warfarin. when the background information was presented on both drugs, respondents were asked to select their preferred secondary prophylactic intervention from an overall clot trial perspective. overall, 21 of 24 (87.5%) respondents selected dalteparin over a vka as their drug of choice. healthy month equivalence scores and health state utilities for each alternative were then estimated from the sample. the higher preferences for dalteparin translated into higher health state utility scores (measured as healthy month equivalence). the utility of the dalteparin health state was almost twofold higher than that seen with treatment with a vka suggesting improved quality of life (table 5). the higher utility scores for dalteparin were due to a combination of factors such as improved efficacy, a demonstrated safety profile with extended use and the ability to eliminate continuous inr monitoring. the main positive attribute that respondents indicated about vka was the oral route of administration. overall, the gain in healthy month equivalence was ~1.67 months with dalteparin for the full clot population, which corresponded to an additional gain of ~0.14 qalys. the utility assessment was then extended to patients with moderate - to - severe renal impairment. under this treatment setting where dalteparin demonstrated an even greater effect size (i.e., number needed to treat=7 vs. 13 for the overall population), 23 of 24 respondents (96%) selected dalteparin as their preferred treatment. this higher preference translated into a healthy month equivalence gain of 2.0 months, which corresponded to an additional gain of ~0.17 qalys (table 5). from the results in the itt clot population, dalteparin was associated with an incremental cost of ~$41,200 (95% ci : $ 34,300$46,200) per vte avoided (table 5). one of the limitations of only reporting the cost per vte avoided is that it does not incorporate the patient quality of life and utility gain associated with the avoidance of dvt or pe. therefore, the incremental cost of dalteparin (i.e., $ 3,202) was combined with the 0.14 qalys gained (a gain of 1.67 health months equivalence corresponds to a gain of 0.14 qaly). the findings revealed an incremental cost of ~$23,100 per qaly gained (95% ci : $ 19,200$25,800). the analysis was also done for the renally impaired patient subgroup. when used in patients with moderate - to - severe renal impairment, dalteparin was associated with an incremental cost per vte avoided and qaly gained of $ 16,400 and $ 14,000, respectively (table 5). a series of one - way sensitivity analyses were conducted using the 95% ci of the differences in healthy months equivalent scores (1.182.15) and cost ($ 2,668$3,588) in the full itt clot population. under the worst - case scenario for dalteparin where the highest cost difference between the two treatments was used (i.e., $ 3,588) along with the lowest gain in healthy months equivalence (i.e., 1.18), the cost per qaly gained with dalteparin increased to ~$36,500. under the best - case scenario for dalteparin where the lowest cost difference (i.e., $ 2,668) was used along with the highest gain in healthy months equivalence (i.e., 2.15), the cost per qaly gained with dalteparin was reduced to $ 14,900 in the itt clot population. repeating the analysis in patients with renal impairment, the cost per vte avoided and qaly gained changed to $ 23,500 and $ 6,800 under the two worst- and best - case scenarios, respectively. these findings imply that the base case cost per vte avoided and qaly gained estimates were stable. there have been a least six randomized trials comparing extended duration therapy with lmwh to vka for secondary prophylaxis of vte in patients with cancer.812 from these studies, the only agent to demonstrate a statistically significant reduction in the risk of recurrent vte has been dalteparin.12 following the publication of the clot trial, a patient - level pharmacoeconomic analysis from a canadian health care system perspective was initially conducted.14 deriving health state utilities from a sample of oncology nurses and pharmacists, the study determined that dalteparin as an alternative to vka was associated with an incremental cost of ~$13,800 (95% ci : $ 12,400$15,100) per qaly gained in 2005 canadian dollars.14 since that time, there have been changes in the cost of drugs, diagnostics tests, and in managing dvts and pe.26 in addition, recent data in patients with renal impairment prior to the start of treatment have also become available.13 hence, a reanalysis of the canadian economic evaluation of the clot trial was undertaken, which included the collection of new health state utility data from members of the general public. the results from this reanalysis using 2015 costs maintain that secondary dalteparin prophylaxis in cancer patients with prior symptomatic vte is a cost - effective alternative to vka therapy, with an incremental cost of $ 23,100 (95% ci : $ 19,200$25,800) per qaly gained. furthermore, the economic value of dalteparin was especially good in patients with moderate - to - severe renal impairment, with the cost per qaly gained being $ 14,000. such low incremental cost - effectiveness ratios are rare in oncology today, as most drugs used in cancer patients have cost per qaly ratios in excess of $ 100,000.27 therefore, dalteparin continues to be an economically attractive drug. the main factors responsible for the differences in cost per qaly gained between the original study and the current analysis are changes in the cost of drugs, treatment of major bleeds, and the management of recurrent vte. however, despite these changes, dalteparin remained a cost - effective therapy. one of the strengths of this study was the availability of patient - level health care resource use data. because the data source is a randomized trial, some of the hospital resources were mandated by the study protocol and may not completely reflect standard practice. in addition, not all the health care resources needed to manage recurrent vte were available in the trial database. therefore, we had to rely on literature reported cost estimates for managing such events in both the hospitalized and outpatient settings. another limitation was that indirect costs such as lost productivity and care giver costs were not included. finally, members of the general canadian public instead of cancer patients with vte were used in the utility assessment. using general public as a utility source always presents with challenges in terms of ensuring their understanding of the health states under investigation. dalteparin used for the secondary prevention of vte in cancer patients remains cost effective, with an incremental cost of $ 23,100 per qaly gained. the economic value was further enhanced in cancer patients with moderate - to - severe renal impairment, with the cost per qaly gained being reduced to $ 14,000. in addition to the practical advantages of dalteparin over vka, which include less monitoring and improved efficacy, long - term dalteparin therapy remains an economically attractive alternative to vka for the prevention of recurrent vte in patients with cancer, especially in those with renal impairment. | backgroundpatients with cancer are at increased risk of venous thromboembolism (vte) and the risk is further elevated after a primary vte. to reduce the risk of recurrent events, extended prophylaxis with vitamin k antagonists (vka) is available for use. however, in a large randomized trial (comparison of low - molecular - weight heparin versus oral anticoagulant therapy for the prevention of recurrent venous thromboembolism in patients with cancer [clot ] ; lee), extended duration dalteparin reduced the relative risk of recurrent vte by 52% compared to vka (p=0.002). a recent subgroup analysis of patients with moderate - to - severe renal impairment also revealed lower absolute vte rates with dalteparin (3% vs. 17% ; p=0.011). to measure the economic value of dalteparin as an alternative to vka, a patient - level cost utility analysis was conducted from a canadian perspective.methodsresource use data captured during the clot trial were extracted and linked to 2015 canadian unit cost estimates. health state utilities were then measured using the time - trade - off technique in 24 randomly selected members of the general canadian public to estimate the gains in quality - adjusted life years (qalys).resultsfor the entire clot trial population (n=676), the dalteparin group had significantly higher mean costs compared to the vka group ($ can5,771 vs. $ can2,569 ; p<0.001). however, the utility assessment revealed that 21 of 24 respondents (88%) selected dalteparin over vka, with an associated gain of 0.14 (95% confidence interval [ci ] : 0.100.18) qalys. when the incremental cost of dalteparin was combined with the qaly gain, dalteparin had a cost of $ can23,100 (95% ci : $ can19,200$can25,800) per qaly gained. the analysis in patients with renal impairment suggested even better economic value with the cost per qaly gained being < $ 14,000.conclusionextended duration dalteparin is a cost - effective alternative to vka for the prevention of recurrent vte in patients with cancer, especially in those with renal impairment. |
dental caries is one of the causes of tooth loss for all human beings across age and gender. numerous studies have been carried out, which have helped to increase our knowledge of dental caries and reduce the prevalence of dental caries. however, according to the world oral health report, dental caries still remains a major dental disease. the caries process is well understood as a process of alternating demineralization and remineralization of tooth mineral (featherstone 1999). the major shortcoming of currently available anti - caries products is the fact that their ability to remineralize enamel is limited by the low concentration of calcium and phosphate ions available in saliva. this has led to the research of many new materials that can provide essential elements for remineralization. some of them are bioactive glass (bag), casein phosphopeptide - amorphous calcium phosphate (cpp - acp). bag is an unique material has numerous novel features ; the most important feature is its ability to act as a biomimetic mineralizer, matching the body 's own mineralizing traits. cpp - acp is also one of the novel calcium phosphate remineralization technology, that shows to promote remineralization of enamel subsurface lesions in various in - vitro and in - vivo studies. in - vitro ph - cycling technique was introduced, over 20 year ago, to study the effect of caries - preventive regimens and treatments. many of the researchers have utilized and modified this ph - cycling model to suit their own studies to test different caries - preventive agents. hence the aim of this study was to evaluate and compare the remineralization potential of bag and cpp - acp on early enamel carious lesion. the null hypothesis for the present study was that there is no significant difference in the mean microhardness values of the two groups. a total of 30 healthy human premolars, which were extracted for orthodontic purposes, were collected. teeth were sectioned 1 mm below the cemento - enamel junction with a slow speed diamond disc. the roots were discarded and the crowns were used for the study. to remove variability in samples, 200 m of surface enamel was removed from the buccal surface of all teeth with help of 600 grit abrasive paper and confirmed with digital caliper. a 4 mm 4 mm working window was marked on the buccal surfaces of all the samples. group a : bag containing dentifrice (shy - nm ; group pharmaceuticals ; india) and group b : cpp - acp (gc tooth mousse ; recaldent ; gccorp ; japan) containing dentifrice. the area of the crown other than the working window was covered with nail varnish. b - smh was checked with vicker 's microhardness testing machine (vmt) for all the tooth samples in the area of the working window. the indentations were made with vmt at the rate of 25 gram load for 5 seconds. the average microhardness of the specimen was determined from 3 indentations to avoid any discrepancy. the buffered de-/re - mineralizing solutions were prepared using analytical grade chemicals and deionized water. the demineralizing solution contained 2.2 mm calcium chloride, 2.2 mm sodium phosphate, and 0.05 m acetic acid ; the ph was adjusted with 1 m potassium hydroxide to 4.4. the remineralizing solution, which contained 1.5 mm calcium chloride, 0.9 mm sodium phosphate, and 0.15 m potassium chloride, had a ph of 7.0. samples were kept in demineralizing solution for 96 h to produce the artificial carious lesion in the enamel. d - smh was checked with vmt, similarly as done for b - smh. dentifrice supernatants were prepared by suspending 12 g of the respective dentifrice in 36-ml deionized water to create a 1:3 dilution. the suspensions were thoroughly stirred with a stirring rod and mechanically agitated by means of a vortex mixer for 1 min. the suspensions were then centrifuged at 3500 rpm for 20 min at room temperature, once daily before starting the ph - cycling. the specimens were placed in the ph - cycling system on a cylindrical beaker for 10 days. each cycle involved 3 h of demineralization twice a day with a 2 h immersion in a remineralizing solution in between [figure 1 ]. a 1-min treatment with a toothpaste solution of 3:1 deionized water to toothpaste, after centrifugation (5 ml / section), was given before the first demineralizing cycle and both before and after the second demineralizing cycle and sections were placed in a remineralizing solution overnight. r - smh was checked with vmt, similarly as done for b - smh. statistical analysis of data were conducted using anova and multiple comparisons within groups was done using bonferroni method (post - hoc tests) the decision criterion was to reject the null hypothesis if the p 0.05) and after demineralization (p > 0.05). this indicates that the tooth samples were demineralized to almost the same level of hardness after demineralization, hence giving non - significant result on statistical analysis. mean microhardness between the groups however, the difference in mean microhardness between the groups after remineralization, was found to be statistically significant after remineralization (p 0.05) before the in - vitro ph cycling commenced. it was therefore reasonable to disregard such variations when analyzing the data after ph cycling. after the treatment regime with the respective dentifrices, increase in mean microhardness was observed in both groups [table 1 ] ; this is in accordance with various previous studies carried out for determining the remineralization potential of bag, cpp - acp, and when compared group a and group b showed statistically significant values after ph - cycling regimen [table 2 ]. bag is a ceramic material consisting of amorphous sodium - calcium - phosphosilicate which is highly reactive in water and as a fine particle size powder can physically occlude dentinal tubules. in the aqueous environment around the tooth, i.e., saliva in the oral cavity, sodium ions from the bag particles rapidly exchange with hydrogen cations (in the form of h3o) and this brings about the release of calcium and phosphate (po4) ions from the glass. a localized, transient increase in ph occurs during the initial exposure of the material to water due to the release of sodium. this increase in ph helps to precipitate the extra calcium and phosphate ions provided by the bag to form a calcium phosphate layer. as these reactions continue, this layer crystallizes into hydroxycarbonate apatite (hca). unlike other calcium phosphate technologies, the ions that bag release form hca-(a mineral that is chemically similar to natural tooth mineral) directly, without the intermediate acp phase. these particles also attach to the tooth surface and continue to release ions and re - mineralize the tooth surface after the initial application. these particles have been shown, in in - vitro studies, to release ions and transform into hca for up to 2 weeks. a study was carried out to determine the remineralizing effects of bag on bleached enamel. it concluded that bag deposits were found on the enamel surface of all the specimens, suggesting that they may act as a reservoir of ions available for remineralization at sites of possible demineralization. the present study also revealed that cpp - acp remineralized enamel lesion in human enamel in - vitro. cpp - acp is calcium phosphate - based delivery systems containing high concentrations of calcium phosphate. the roles of cpp - acp has been described as - localization of the acp on the tooth surface and buffer the free calcium and phosphate ion activity, thereby helping in maintaining the role of super saturation. the cpp stabilizes the calcium and phosphate in a metastable solution facilitating high concentration of the ca and po4 which diffuses in the enamel lesion when cpp - acp comes in contact with the lesion. however the lower hardness values for cpp - acp may be due to its amorphous nature ; which does not adhere to the enamel surface, unlike bag gets attached to tooth, hence not remineralizing the tooth surface for a longer period of time to enhance its hardness. a study investigated the enamel remineralization potential of cpp - acp and bag, showed that, after scanning electron microscope analysis it was clearly seen that although both group samples had plugs that sealed the fissures formed by demineralization, bag plug appeared to be more compact and intimately attached to the enamel surface. the deposits formed by cpp - acp were smaller and amorphous, while bag created larger, more angular deposit. this may also explain the high values of hardness for bag as compared to cpp - acp in the current study ; as bag attaches more intimately and compactly to the tooth surface. under the current in - vitro experimental conditions it can be concluded that both bag and cpp - acp are capable to remineralize early carious lesion and also that bag has a better remineralization potential than cpp - acp. | aims : the aim of this study was to evaluate and compare the remineralization potential of bioactive - glass (bag) (novamin/calcium - sodium - phosphosilicate) and casein phosphopeptide - amorphous calcium phosphate (cpp - acp) containing dentifrice.materials and methods : a total of 30 sound human premolars were decoronated, coated with nail varnish except for a 4 mm 4 mm window on the buccal surface of crown and were randomly divided in two groups (n = 15). group a bag dentifrice and group b cpp - acp dentifrice. the baseline surface microhardness (smh) was measured for all the specimens using the vickers microhardness testing machine. artificial enamel carious lesions were created by inserting the specimens in de - mineralizing solution for 96 h. smh of demineralized specimens was evaluated. 10 days of ph - cycling regimen was carried out. smh of remineralized specimens was evaluated.statistical analysis : data was analyzed using anova and multiple comparisons within groups was done using bonferroni method (post - hoc tests) to detect significant differences at p < 0.05 levels.results:group a showed significantly higher values (p < 0.05) when compared with the hardness values of group b.conclusions:within the limits ; the present study concluded that ; both bag and cpp - acp are effective in remineralizing early enamel caries. application of bag more effectively remineralized the carious lesion when compared with cpp - acp. |
it initially presents with difficulty swallowing and is a chronic progressive condition that usually does not resolve. although the specific cause is unknown, the primary pathology involves the inability of the lower esophagus to relax, resulting in delayed passage of food and liquid into the stomach. clinical symptoms range from initial dysphagia to heartburn, a feeling of fullness in the chest and sometimes weight loss. the objective work - up includes barium radiography, esophagoscopy, and esophageal manometry. in patients with achalasia barium radiography often reveals a classic 1) in which the dilation tapers into the lower esophagus, because of the chronic buildup of food contents. long - term sequelae include an increased risk of the development of esophageal cancer in patients diagnosed with achalasia. 1bird s beak appearance of lower esophagus during an upper gastrointestinal x - ray swallow study bird s beak appearance of lower esophagus during an upper gastrointestinal x - ray swallow study the objective of treatment is to resolve symptoms in this usually progressive disease. conservative maneuvers have included the use of muscle relaxing medications or injections and dilation of the lower esophagus with endoscopically guided balloons. these usually result in temporary relief only rather than achieving long - term resolution of the problem. surgical intervention involving splitting of the restrictive external muscle of the lower esophagus and upper stomach with subsequent wrapping of the upper stomach partially around the lower esophagus results in symptom relief in 85% of people 10 years after surgery and in approximately 65% of people 20 years after surgery. achalasia was first described, with its initial treatment, by thomas willis in 1679. treatment and instrumentation have rapidly evolved since his first description of dilation of the esophagus using a tapered whale bone. in 1887, russel described initial balloon dilatation using a silk bag over a rubber balloon. this surgical technique involved splitting the outer muscle of the lower esophagus to relieve internal narrowing and associated swallowing difficulties. this procedure was subsequently popularized by approaches through the abdomen or through the chest. in the early 1990s, during the beginning of the era of minimally invasive surgery, thoracoscopic and laparoscopic heller myotomy were described. prompt recovery and good outcomes quickly enabled clinicians to conclude that surgical treatment was the optimum primary treatment of choice for amelioration of symptoms in patients with achalasia. laparoscopic heller myotomy using standard laparoscopic techniques has evolved into an extremely safe and efficient technique of reducing symptoms in the treatment of achalasia. preliminary case series demonstrated that early outcomes were favorable and the perioperative course was much improved compared with an open laparotomy or thoracotomy. perioperative complications were reduced and the complications secondary to the trauma of the incision were largely eliminated. laparoscopic heller myotomy has now been widely reported with large case series with relatively good outcomes in both early and late results [68 ]. in addition, favorable outcomes have been achieved in some series in which laparoscopic heller myotomy was offered as the first - line therapy. although patient satisfaction, relief of symptoms, and recovery time were categorized as favorable, these large case series still reported a significant number of intraoperative esophageal perforations ranging between 1 and 15%. in these series, the mucosal perforations were identified to have occurred as a technical failure during the course of the division of the muscle fibers from the underlying esophageal mucosa. the clinical relevance of these complications may be uncertain, and when the perforation is recognized at the time of surgery and repair performed immediately the outcomes are still usually good. these perforations expose the patients to a prolonged hospital stay and the risk of an esophageal leak, however. delayed diagnosis of an esophageal perforation is a potential fatal complication and is one of the most significant and serious risks of heller myotomy. management in this situation requires broad - spectrum antibiotic coverage, proximal bowel decompression and rest, potential operative drainage, debridement and reconstruction of the injured tissues, and sometimes intravenous nutritional support for a prolonged period of time. different surgical techniques have been described to avoid the perforation of the underlying mucosa and have included the injection of epinephrine into the submucosal plane, blunt dissection, sharp dissection, and intraoperative esophagoscopy to evaluate the esophageal mucosa and detect underlying perforations. the da vinci surgical system (intuitive surgical, inc ; sunnyvale ca, usa) is currently the only robotic telesurgical device marketed and approved for general surgery in the united states. the device is a computer interface that interacts with the operating surgeon who sits at a control panel connected to a bedside actuator with instrument holders and a camera manipulator (figs. 2, 3). motion is enhanced by filtering fine motor tremor and providing motion scaling between the hand - piece and the activator arm, enabling up to 5-to-1 scaling. innovations have now incorporated 5 mm instrumentation enabling reduced abdominal wall trauma and addition of a fourth working arm for enhanced mobility, tissue handling, and retraction. 3the da vinci surgical robot in use with head surgeon operating from the console the da vinci surgical robot in use with assistant surgeons alongside operative table the da vinci surgical robot in use with head surgeon operating from the console the robotic device has the unique ability to enhance surgical technique and, therefore, optimize surgical outcome. these advantages specifically are fine motor control, three - dimensional magnification, and motion scaling. these may be significant in operative procedures such as heller myotomy or in cases when optimum completion of the operation requires these specific surgical techniques. it is this enhancement of surgical performance that may have improved the surgical outcomes mentioned above regarding recent case series comparing robotic telesurgery with historic laparoscopic approaches to heller myotomy. using this model it may be reasonable to identify some surgical procedures and interventions, for example heller myotomy, that can specifically benefit from this type of therapy. at the author s institution robotic telesurgery for achalasia preoperative preparation of patients with achalasia may include a prolonged period of fasting to completely empty the esophagus. clear liquids for 48 h preoperatively are routine, especially in cases of severe esophageal dilatation. preoperative hospital admission may be necessary for patients to enable them to maintain fasting with intravenous hydration. preoperative antibiotics and oral anti - fungal medications should be included to help reduce the amount of contamination within the esophagus. the patient is positioned on a split leg table or in a modified lithotomy (legs apart and gently angled down) position after the induction of general anesthesia. the bedside component of the robot is positioned over the patients left shoulder after accessing the abdomen through four ports. the patient is placed with the head of the bed angled upwards and the patient s motion must be completed before docking to the robot ports. two working ports are used with downward traction of the upper part of the stomach by the assistant or the fourth arm of the robot. the esophageal hiatus of the diaphragm is identified and the crura are circumferentially dissected free of peritoneal attachments. the distal esophagus is dissected, and a soft rubber penrose drain is usually placed around the distal esophagus for traction, enabling dissection up into the lower mediastinum. the esophageal myotomy is created using the electrocautery hook of the robotic device (fig. the articulation of this arm provides precise control and helps keep the end of the hook directly parallel to the esophageal mucosal wall. this dissection is aided by a curved dissector on the opposite arm, to help completely separate the muscle fibers. when the submucosal is plane is developed between the outer esophageal musculature and the inner mucosa, the division of the muscle fibers is carried 810 cm proximal and at least 2 cm distally from the junction of the esophagus and stomach. the gastric dissection is clearly identified by the disordered character of the vasculature of the gastric submucosa. the mucosa is carefully inspected for injury and for complete division of all muscle fibers. the muscular bundles bordering the esophageal diaphragmatic hiatus are reapproximated behind the esophagus using non - absorbable sutures (fig. 5). a partial gastric fundoplication is then created using a 90 anterior dor or a 270 posterior toupet fundoplication (fig. patients are allowed liquids post operatively and then are maintained on a soft diet for several days. the rubber penrose drain is used for retraction of the abdominal esophagus during this stepfig. note : the abdominal esophagus is coursing through the diaphragm in the top of the photographfig. 6operative completion of the heller myotomy and posterior 270 toupet fundoplication operative step of longitudinal esophageal myotomy using electrocautery. the rubber penrose drain is used for retraction of the abdominal esophagus during this step operative step of diaphragmatic posterior crural repair using robotic instrumentation. note : the abdominal esophagus is coursing through the diaphragm in the top of the photograph operative completion of the heller myotomy and posterior 270 toupet fundoplication postoperative patients should receive routine follow - up consisting of surveillance endoscopy every 3 years in anticipation of early detection of asymptomatic pathology. patients who complain of recurrent symptoms should undergo formal evaluation and work - up as outlined for initial patients suspected of having achalasia. long - term postoperative results for achalasia patients are good, particularly when patients are selected with preoperative lower esophageal sphincter pressures greater than 35 mmhg. for a group of 174 patients reported overall symptomatic relief of dysphagia in 87% of patients 15 years after heller myotomy and dor fundoplication with 9% of patients requiring medical / surgical treatment of symptomatic reflux and 4% of patients requiring esophagectomy for malignant disease or recurrent disease not amenable to repeat myotomy and fundoplication. in patients who require re - operation for recurrent dysphagia following esophagomyotomy symptoms often present within the first postoperative year and the root cause is usually inadequate or healed myotomy. although success can usually be achieved, results from repeat esophagomyotomy are less impressive than adequate initial operations. it is indicated after failed medical therapy, pneumatic dilation, non - resecting surgical, and redo procedures. although laparoscopic heller myotomy has emerged as the treatment of choice for achalasia, the intraoperative esophageal perforation rate of 115% has remained a significant problem that has prompted investigation of improvements of this operative technique. in 2000, the da vinci surgical system (intuitive surgical, inc ; sunnyvale ca, usa) a robotic telesurgical device, entered the market. the advantages reported include a stable work platform, a magnified three - dimensional image, fine motor control of articulated instruments, and motion scaling, which are uniquely designed to facilitate surgery requiring fine tissue manipulation. they provide a narrower field of view of the operative site and they provide the surgeon with essentially no tactile feedback. with these advantages and hurdles in mind, a variety of surgical procedures have been reported using robotic telesurgical devices. initial data obtained before food and drug administration approval was obtained in clinical trials performing cholecystectomy and gastroesophageal fundoplication. after food and drug administration approval brief clinical reports described cholecystectomy, fundoplication, colon resection, pancreatic resection, heller myotomy, and other general surgery procedures [19, 22 ]. with experience, case series were presented which eventually demonstrated the safety and feasibility of robotic general surgery procedures [22, 23 ]. reports have also been presented that look at traditional laparoscopic procedures compared with robotic telesurgical assistance. reported a comparison case series of gastroesophageal fundoplication performed by standard laparoscopy or with robotic telesurgical assistance. both techniques were safe and associated with low occurrence of complications ; no clear benefit was seen in the robotic group, however. use of the da vinci robotic telesurgical device to perform a heller myotomy was first reported in 2001 and a subsequent small case series was then reported [23, 25 ]. the academic robotic surgery group, comprising surgeons from ohio state university, columbus, oh, usa, johns hopkins university, baltimore, md, usa, and the university of illinois at chicago, chicago, il, usa, has prospectively collected data on 104 robotic telesurgical heller myotomies, the largest reported case series, which were performed without a single esophageal perforation. similar results favoring the robotic telesurgical approach were obtained in three subsequent published series comparing robotic telesugical heller myotomy with laparoscopic heller myotomy [2628 ]. this improvement over standard laparoscopic studies which reflected the esophageal perforation rate of 115% may be related to the advantages of the robotic telesurgical technique and surgical enhancement the device offers compared with laparoscopic techniques. one pilot study evaluated the technique of pancreaticojejunostomy after pancreatic head resection using the robotic surgical device. five patients were enrolled and underwent a computer - enhanced pancreaticojejunostomy using a 60 suture in an open abdomen after pancreatic head resection. robotic assistance also has the potential to contribute to the evolving technology of natural orifice transluminal endoscopic surgery (notes) in which therapeutic approaches to abdominal surgical diseases are being pursued via an endoscope that is advanced from an intraluminal position within the stomach, vagina, bladder, or rectum into the abdominal cavity to perform a variety of diagnostic and therapeutic maneuvers. this was first attempted with simple peritonoscopy and has recently advanced to organ retrieval, for example appendectomy [30, 31 ]. although no formal treatments for achalasia by a notes approach have been published, a treatment involving transgastric peritonoscopy with retroflexion and endoscopic myotomy with potential intraluminal gastroesophageal plication may prove to be feasible for treatment of achalasia with end results similar to heller myotomy and fundoplication. | the craft of surgery has always relied on the use of instruments. innovations in surgery have paralleled innovations in instrumentation. advances in surgical instrumentation continue today and have enabled huge strides in surgical procedures and outcomes during this generation. computers and related technology are now changing the interface between the surgeon and the patient, and are poised to improve patient outcomes by enhancing the surgeon s skills and training. the application of computer enhanced telemanipulators, or robots, may specifically enhance operations, for example heller myotomy, that require good visualization and precise careful dissection of delicate structures. this review covers the pathophysiology of achalasia and its history of medical and surgical treatment, leading to modern robotic telesurgical approaches. improvements in outcome from medical to standard surgical to robotic telesurgical approaches are discussed. current operative technique for robotic telesurgical treatment of achalasia is described and the authors conclude with a glimpse of where, in the future, current research endeavors will lead us in the treatment of achalasia. |
ribosomally synthesized and post - translationally modified peptides are a rapidly expanding class of natural products. they are typically biosynthesized by modification of a c - terminal segment of the precursor peptide (the core peptide). the precursor peptide also contains an n - terminal leader peptide that is required to guide the biosynthetic enzymes. for bioengineering purposes, the leader peptide is beneficial because it allows promiscuous activity of the biosynthetic enzymes with respect to modification of the core peptide sequence. however, the leader peptide also presents drawbacks as it needs to be present on the core peptide and then removed in a later step. we show that fusing the leader peptide for the lantibiotic lacticin 481 to its biosynthetic enzyme lctm allows the protein to act on core peptides without a leader peptide. we illustrate the use of this methodology for preparation of improved lacticin 481 analogues containing non - proteinogenic amino acids. |
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the syndrome of severe, acute, intensive care unit (icu)-acquired neuromuscular weakness poses a common and serious diagnostic, prognostic, and therefore management issue. it goes by various names, some of which presuppose a mechanism : acute necrotizing myopathy of intensive care, acute quadriplegic myopathy, critical care myopathy, critical illness myopathy (cim), critical illness neuromuscular disease, critical illness neuromyopathy, critical illness polyneuromyopathy, critical illness polyneuropathy (cip), icu - acquired paresis, quadriplegic and areflexic icu illness, rapidly evolving myopathy with myosin - deficient fibres and thick filament myopathy [1 - 6 ]. the problem affects at least 1.7% of children in paediatric icus, more than half of adult patients admitted to general icus for more than 1 week and more than 70% of those with sepsis and multiorgan failure [7 - 9 ]. neuromuscular weakness typically becomes apparent when an attempt is made to wean the patient from the ventilator, although there are earlier clues, which include grimacing without movement with noxious stimuli before recovery of consciousness, relative lack of movement after regaining consciousness, and (not inevitably) loss of deep tendon reflexes that had been present earlier. although most cases will turn out to be critical illness polyneuromyopathy a term that embraces cip, cim, or a combination of the two other potential causes should not be overlooked. 1. the algorithm illustrates the early ruling in or out of spinal cord disease (e.g. in cases of trauma, coagulation disturbance, west nile virus infection, acute disseminated encephalomyelitis, etc.), and then moving on to a clinical biochemical electromyographic assessment. a neuromuscular transmission defect (e.g. slow inactivation of neuromuscular blocking agents, unrecognized myasthenia gravis, or myasthenic [lambert eaton ] syndrome) is easily detected with repetitive nerve stimulation, revealing either a decremental or incremental response. neuropathies other than cip that may manifest after icu admission include guillain barr syndrome / acute immune - mediated demyelinating polyneuropathy (and its various subtypes), porphyria and recurrent chronic inflammatory demyelinating polyneuropathy. demyelinating inflammatory neuropathies usually cause slowing of conduction velocity and conduction block on electromyographic studies and produce increased protein in the cerebrospinal fluid. other conditions, such as inflammatory myopathies or unrecognized motor neurone disease, are not discussed here because they are usually diagnosed before admission to icu, although they sometimes present with respiratory weakness that requires icu admission. the last step, differentiating the most common causes of icu - acquired generalized weakness (myopathy, neuropathy, or a combination of the two), is practical because their prognoses differ considerably. some cases of myopathy appear to be merely ' membranopathies ', with normal histology and rapid recovery. presumably, the muscle membrane becomes dysfunctional, inexcitable and leaky, allowing myoglobin to leave the muscle. others, especially in those treated with corticosteroids and neuromuscular blocking agents, exhibit a relative loss of thick myosin filaments. many cases show variable panfascicular necrosis, which can sometimes be widespread and severe. in general, the prognosis for recovery from myopathies is favourable, with most patients recovering fully within 13 months. this takes many months and recovery is often incomplete, leaving patients with significant weakness, sensory loss, and absent reflexes distally in the lower limbs and variably more proximally. patients with cip who fail to show significant recovery by 4 weeks are often disabled with diminished quality of life. some older patients may fail to survive or wean from the ventilator because their recovery is so protracted and other complications ensue. electromyography is helpful in differentiating cip from cim but it has limitations. reduced or absent sensory nerve action potentials favours a neuropathy, but sensory potentials may be difficult to record if there is considerable oedema or a pre - existing polyneuropathy (e.g. from diabetes mellitus) may have clouded the issue. direct muscle stimulation (not commonly done) may reveal no or absent response in cim but normal responses in cip. unfortunately, needle electrode studies of muscle can show similar features in cip and cim ; both may exhibit spontaneous activity (fibrillations and positive sharp waves). often cip and cim coexist and their relative contributions to the weakness may vary considerably when this occurs. elevated serum creatine kinase may help to identify cim, but the peak may be missed in the membranopathy / necrotizing varieties or creatine kinase may not be significantly elevated in cases with loss of myosin filaments. to determine the relative contributions of nerve versus muscle disease in explaining weakness, muscle or both nerve and muscle biopsies have been utilized and recommended, most recently in this issue by kerbaul and colleagues. in most cases muscle biopsy will address the relative contribution of myopathy to the picture because the neuropathy can be adequately assessed electrophysiologically. percutaneous muscle biopsy, although providing limited tissue, has a number of advantages over operative biopsies : greater spatial sampling, minimal bleeding, negligible infection rate (we have had none in over 1000 biopsies), portability (done at the bedside), no general anaesthetic, speed of performance and ease of arrangement. nerve and muscle biopsy is seldom necessary in the icu, unless a vasculitis is suspected. being aware of the incidence and signs of icu - acquired weakness with ventilatory failure and having an approach to such disorders will prove valuable in management. further insights into the mechanisms of cip and cim may provide some preventive strategies that will ameliorate their severity, shorten the duration of icu stays and improve long - term outcomes. cim = critical illness myopathy ; cip = critical illness polyneuropathy ; icu = intensive care unit. a flow chart giving an approach to generalized weakness and/or ventilatory failure in the intensive care unit. ck, creatine kinase ; csf, cerebrospinal fluid ; gbs, guillain barr syndrome ; emg, electromyography ; mri, magnetic resonance imaging ; lems, lambert eaton (myasthenic) syndrome ; mg, myasthenia gravis ; ncv, nerve conduction velocity studies ; n - m, neuromuscular transmission ; snaps, sensory nerve action potentials. modified from bolton and young. | icu - acquired limb and respiratory muscle weakness is a common, serious icu syndrome, increasing in frequency with prolonged icu stay and sepsis. a systematic approach facilitates precise localization of the problem within central or peripheral nervous system. most cases relate to critical illness polyneuropathy or myopathy or a combination of both (critical illness neuromyopathy). within the latter entity, the relative contribution of neuropathy versus myopathy varies considerably among affected patients. muscle enzyme testing, electromyography - nerve conduction and muscle biopsy are valuable investigative tests. nerve biopsy is less commonly needed, but is useful when vascultis is suspected. |
case 1 involved a male neonate delivered by cesarean section in obstetric hospital a in taiwan in april 2013, after an uneventful pregnancy of 38 weeks. he was fed infant formula in the hospital s nursery. on postpartum day 7, he had a fever of 39c and tachypnea and was taken to a tertiary hospital. chest radiograph showed a ground - glass opacity in the middle lobe of his right lung, which subsequently developed into cavities. the case was reported to the unknown pathogen discovery / investigation group at the taiwan centers for disease control for extensive etiologic survey. multiplex real - time reverse transcription pcr showed sputum and blood specimens to be negative for adenovirus, respiratory syncytial virus, coronaviruses (229e, oc43, nl63, hku1, and middle east respiratory syndrome coronavirus), metapneumovirus, influenza virus, parainfluenza viruses 14, herpes simplex viruses 1 and 2, varicella - zoster virus, epstein - barr virus, cytomegalovirus, human herpesviruses 6 and 7, bocavirus, parvovirus, enterovirus, and rhinovirus. water specimens from all sources with which the patient had contact were tested for legionella by culture. l. pneumophila serogroup 4 was isolated from 3 tap water sources, and l. pneumophila serogroup 5 was isolated from the cold water source of the hot and cold water dispenser from which water for making infant formula was collected (table). the water dispenser was located in the room next to the nursery. the hot water faucet provided boiled water at 95100c, and the cold water faucet provided unboiled water that was treated by a built - in reverse osmosis device. the pulsed - field gel electrophoresis (pfge) profiles of the l. pneumophila serogroup 5 strains isolated from a sputum sample from the infant and those from a water sample from the water dispenser were indistinguishable (figure). both strains were sequence type 1032 (12). both case - patients were delivered by cesarean section and were not breast fed or placed in incubators. st, sequence type ; tmtc, too many to count ; nd, testing not done. the first samples from the environments of case - patients 1 and 2 were collected 33 d and 23 d after birth, respectively. the second samples were collected 34 d and 26 d after birth, respectively. concentration of legionella spp. detected in water specimens, attributed to the presence of sampling swabs. the pulsed - field gel electrophoresis profile of the strain was indistinguishable from that of the strain isolated from the patient s sputum. the st of the strain was the same as that of the strain isolated from the patient s sputum. pulsed - field gel electrophoresis patterns for legionella pneumophila isolates from neonates with legionella infection and hospital water sources. genomic dna was digested with sfii and separated in 1% agarose gel by bio - rad chef mapper. lane m, reference size maker (xbai - digested genomic dna fragments of salmonella enterica ser. braenderup h9812) ; lanes 1 and 2, clinical and environmental isolates of l. pneumophila serogroup 5 from case - patient 1 ; lanes 3 and 4, clinical and environmental isolates of l. pneumophila serogroup 1 for case - patient 2. parents of the 175 neonates that were delivered 3 months before the patient s birth in hospital a were interviewed by telephone ; 3 of the 175 infants had fever within the first month of life. serologic testing 3 weeks after their fever episodes was negative for igm or igg antibodies against l. pneumophila serogroups 16. the water dispenser in hospital a was replaced with one that provided only hot water. the point - of - use filters were mounted on the water faucets in the nursery (pall - aquasafe water filter, portsmouth, uk). no legionella infection was identified in any neonate who was born after the patient s birth in hospital a during the following 8 months. case 2 involved an asymptomatic male neonate delivered by cesarean section in obstetric hospital b in november 2013 after an uneventful pregnancy of 38 weeks. he remained asymptomatic and was fed infant formula throughout his 8-day stay in the hospital. at home on the day of discharge he had tachypnea and cyanosis of the lips during the following days, and on day 10 after birth, he was taken to the same tertiary hospital as case - patient 1. bilateral pulmonary infiltrates and mild right pneumothorax were identified. the patient s urine and sputum specimens tested positive for l. pneumophila serogroup 1. testing of related environmental water specimens for legionella spp. by culture was negative for all specimens except that from the cold water source of the hot and cold water dispenser in hospital b, which was positive for l. pneumophila serogroup 1 (table). the water dispenser in the nursery, which consisted of a tank containing boiled, hot water and another tank containing cool water pipelined from the boiled water tank, was used for making infant formula. the pfge profiles of the strains from the patient and those from the water dispenser were indistinguishable (figure). both strains were sequence type 1. the parents of the 79 neonates born 3 months before the patient s birth in hospital b were interviewed by telephone. the water dispenser in hospital b was replaced with one that provided only hot water. no legionella infection was identified in any neonate who was born after the patient s birth in hospital b during the following 3 months. we report 2 cases of neonatal legionellosis associated with infant formula prepared with contaminated water. only the cold water from both hospitals hot and cold water dispensers used for making infant formula was positive for l. pneumophila, with indistinguishable pfge profiles and the same sequence types as those isolated from the neonates. some environmental specimens were tested twice to compensate for inadequate sensitivity of the culture method and yielded the same results. no humidifier was used in either case. in the hospital where the first case - patient was identified, the water dispenser was not located in the nursery, which likely reduced the risk of transmitting legionella spp. through contaminated aerosol droplets generated by the water dispenser. aspiration is a major transmission mode in hospital - acquired legionella infection among adults (13,14). neonatal legionella infection through aspiration of contaminated water during water birth has been reported (9,10). our results also indicate the legionella infection described here was likely transmitted by aspiration of contaminated infant formula during the first days of life. both water dispensers were colonized with legionella spp. although the pervasiveness of these 2 problematic types of water dispenser is not known, hot and cold water dispensers are used in many hospitals in taiwan. we speculate the complicated pipeline system and inappropriate maintenance of water dispensers might increase the risk for legionella colonization, thus facilitating legionella infection. most cases were hospital acquired, and the patients had severe pneumonia that was associated with a high mortality rate (8,15). the manifestation of legionella infection is not pathognomonic (15), therefore, underdiagnoses or delayed diagnoses combined with inappropriate treatment likely contribute substantially to mortality rates and severity of neonatal legionellosis. the 2 neonates described here survived after prompt diagnosis and treatment enabled by an extensive etiologic investigation and high suspicion of legionella infection. our report underscores the importance of suspecting and testing for legionella infection when neonates have respiratory symptoms. | we report 2 cases of neonatal legionella infection associated with aspiration of contaminated water used in hospitals to make infant formula. the molecular profiles of legionella strains isolated from samples from the infants and from water dispensers were indistinguishable. our report highlights the need to consider nosocomial legionellosis among neonates who have respiratory symptoms. |
the spine creates a closed kinematic chain through segmental configuration of the upper and lower extremities and the pelvic girdle, influencing static and dynamic balance during sports activities and the activities of daily life1, 2, while also having a direct effect on postural alignment through musculoskeletal connections3. however, if a normal form can not be maintained due to internal and external forces, any force applied to the spine can cause misalignment, causing lateral imbalance and the accumulation of fatigue in the adjacent muscle, which often results in chronic low back pain4. treatment with a rigid thoracolumbosacral orthosis is the most common non - operative treatment for the prevention of curve progression. there are many different brace designs, but with all of them, the objective is to restore the normal contours and alignment of the spine by means of external forces and, in some designs, the stimulation of active correction as the patient moves the spine away from pressures within the brace5. a thoracolumbosacral orthosis (tlso), a brace that extends from the spine to the pelvis, has a direct impact on the movement of the spine during walking and hence has a significant influence on the movement of the pelvis and the lower limbs. spinal orthoses are commonly used in the treatment of various conditions that affect the spine. therefore, in this study, we examined the impact on the pattern of walking on both level ground and stairs after restricting the movement of the spine and the trunk of the body through the use of a spinal orthosis. in this study, 40 healthy males in their 20s were selected as a sample, which was randomly and evenly divided into two groups : (1) the wt group (with a tlso) and (2) the wot group (without a tlso). the inclusion criteria included (1) no neurologic disease history, (2) capability to maintain independent standing, walking, and landing, (3) no surgical history that could affect walking or landing, and (3) no inborn deformity or disease in the relevant areas, including the foot, pelvis, and spine. for the wt group, the average age was 24.9 2.4 years, the average height was 173.3 4.2 cm, and the average weight was 70.6 6.6 kg. for the wot group, the average age was 25.3 2.7 years, the average height was 175.6 4.4 cm, and the average weight was 69.1 6.8 kg. using levene s variance homogeneity test and a normality test on the general physical characteristics, no statistically significant difference was found between the two groups (p>0.05). all of the patients understood the purpose of this study and provided written informed consent prior to participation in accordance with the ethical principles of the declaration of helsinki. the spinal orthosis used in this study was a tlso called a plastic body jacket. it is made of lightweight polypropylene that has thermoplasticity, and is designed so that it is easy to put on and take off. it is a commercially available product that comes in four sizes (extra - large, large, medium, and small), and the appropriate size was used for the body figure of each research subject. more than any other tlso, this particular plastic body jacket is known to be effective for maintaining the body s alignment and for effectively restricting movement in three 3-dimensional planes : (1) flexion and extension of the body s trunk in the sagittal plane, (2) lateral flexion in the frontal plane, and (3) rotation of the trunk of the body in the horizontal plane. the steps of the staircase fabricated and used in this study were 17 cm high, 28 cm wide, 61 cm across, and were fabricated according to housing construction standards ; four steps were installed for this study. a motion analysis system composed of six infrared cameras (vicon motion capture system, vicon motion systems ltd., oxford, uk) was used for measurement of the spatiotemporal variables and for the kinematic variables of walking, with the sample collection rate of the infrared camera set at 120 hz. the subjects were told to walk comfortably and naturally down a 10-m long straight walking lane drawn on firm, even ground at their usual speed while looking straight ahead. walking on level ground was performed after oral instruction, and this was followed by ascending and descending the stairs. the measurement interval was not mentioned so that the subjects were not aware of when the measurements began. walking was measured after the first five footsteps, and a 30-second rest period was given after each motion. spss for windows 17.0 (ibm spss, chicago, il, usa) was used for the statistical data analysis. an independent t - test was used, given the homogenous nature of the subject ; the difference wt and wot group was calculated and comparisons were made using an independent t - test. table 1table 1.comparison of joint angle between with tlso group and without tiso group (unit :)gait conditionsagittal planefrontal planetransverse planewithout tlsowith tlsowithout tlsowith tlsowithout tlsowith tlsolevel walkingh147.1 9.151.9 5.9h52.9 1.42 1.6h93.6 1.33.4 1.1stair ascent64.9 10.268.2 9.14.9 1.63.8 1.5 7.3 1.76.6 2.1stair descent27.9 4.630.2 4.34.4 45.2 2.10.5 1.10.9 1.9level walkingh28.4 0.914.9 3.8h62.3 0.91.2 1h106 44.2 2.8stair ascent10.5 1.818.9 6.5 3.3 1.42.7 1.38.2 3.89.9 3stair descent30.2 7.335.4 5.9 4.6 1.55.1 1.54.2 3.83 2.3level walkingh318.9 5.217.7 5.1h78.5 1.77.7 2.3h113 1.72.2 2.8stair ascent10.9 2.516.5 47.8 2.05.9 2.15.6 26.6 2.4stair descent19.4 7.130.9 6.14.3 2.13.9 25.6 2.65.7 3.1level walkingh448.0 5.648.3 5.9h85.6 1.56.3 1.4h126.5 1.66.4 1.7stair ascent17.7 2.919.2 4.2 9.3 1.98 4.17.5 1.47.7 1.5stair descent40.6 8.142.2 9.45.6 2.64.6 1.85.9 1.85.6 1.6level walkingk116.1 4.319.2 5.4k52.4 13 1.9k98.4 1.87.9 2.2stair ascent60.8 9.166.6 8.4 5.7 1.95.9 2.110.3 1.811.2 2.9stair descent19.4 4.422.4 5.24.2 2.44.5 2.36.2 1.98 3.9level walkingk210.6 2.515.8 3.4k65.2 1.76.1 2.2k107.7 1.87.3 2.1stair ascent20.9 4.721.9 3.16.9 1.85.9 1.91.9 1.21.7 1.5stair descent11.5 3.217.7 3.5 7.3 2.35 25.1 2.25.5 2level walkingk332.6 11.733.4 8.2k71.8 11.3 0.9k113.1 1.83.2 1.9stair ascent28.7 6.925.7 8.13.7 0.73.4 0.95.5 1.55.7 1.9stair descent73.4 10.571.2 22.62.7 1.12.7 1.58 2.89.1 2.4level walkingk447.5 4.246.9 5.1k83.3 13.4 1.6k120.6 1.90.1 1.1stair ascent81.2 12.381.1 11.44.3 14.4 1.515.8 6.413.9 3.1stair descent84.8 9.279.6 11.24.3 1.34.2 1.16.2 29.2 3.8level walkinga13.4 1.92.7 1.2a53.9 1.63.1 0.9a912.6 4.412.4 3.5stair ascent14.3 3.912.7 3.96.4 1.45.6 1.527.5 3.926.6 6.2stair descent16.0 5.621.2 4.93.3 1.23.7 1.313 4.613.6 7.3level walkinga25.8 1.45.7 1.1a64.7 2.15.4 1.5a1011.8 3.610.5 2.9stair ascent6.6 3.26.6 3.65.2 1.74.3 1.320.9 7.517.9 4.3stair descent15.3 5.613.8 4.13.7 1.43.9 2.214.9 517.5 4.2level walkinga311.8 2.813.6 3.7a73.6 1.43.3 1a119.1 2.28.9 2.4stair ascent9.4 1.910.4 2.64.6 1.55.4 2.37.6 2.76.7 2stair descent2.3 1.32.3 1.14.1 1.84.3 213.8 4.816.3 5.2level walkinga43.8 1.43.8 0.6a86.2 1.95.5 1.4a1224.4 5.322.2 12.2stair ascent14.5 19.910.2 5.87.1 1.86.9 1.931.1 5.231.7 4.9stair descent12.4 3.511.8 3.16.6 1.96.4 2.225.4 6.828.5 5.8significantly different between with tlso and without tlso (p0.05), while a significant difference was found only at the k12 phase while descending the stairs. when the kinematic variables in the lower extremities were compared in the sagittal plane, the flexion angles of the hip joints were considerably increased in h2 during both the level ground and the stair walking, an increase that seemed to compensate for restricted pelvic movements due to the spinal orthosis. increase in the flexion angle at k1 and k2 and in the plantarflexion angle at a1 in the stair walking may serve as compensation for shock absorption against the ground reaction. a research by konz reported that pelvic obliquity and pelvic rotation were remarkably reduced when a tlso was applied and such pelvic reduction in the frontal and the horizontal planes affected the lower extremity movements during gait6. another research reported that plantar flexion of the ankle joints in the loading response and knee joint flexion in the stance phase decreased the shock from the ground reaction7. in this context, restriction in pelvic movements due to the spinal orthosis induced increased flexion in the ankle, knee, and hip joints in the sagittal plane in order to alleviate the vertical ground reaction. as for changes in the ankle joint angles in the sagittal plane in stair walking, dorsiflexion occurred between the initial contact and the initial phase of ascent, followed by gradual plantar flexion and then sudden plantar flexion at the middle phase, while maximal plantar flexion was shown at initial descent, followed by rapid dorsiflexion until the early part of the gaiting phase and then gradual dorsiflexion during the rest of the phase, a change that seemed to absorb the shock of the body load8. changes in the angles of the lower extremities in the sagittal plane in this study showed significant differences in stair gaiting rather than in level ground walking ; thus, the wt group showed increased flexion in their lower extremities. in the angles of the lower extremity motions in the frontal plane, the internal rotation was reduced at h6 during level ground walking, at h5, and at h7 during stair ascent, and at k6 during stair descent, a reduction that may be consistent with the results of the research by konz, which reported that changes in the motion angles in the thigh and the pelvic segment in the frontal plane due to spinal orthosis application were directly associated with reduction in the pelvic oblique. in the angles of the lower extremity motions in the horizontal plane, the internal rotation was increased at k12 only during stair descent, an increase that is consistent with the results of the research by konz, which reported that no differences were found in the angles of the lower extremity motions in the horizontal plane during level ground walking. in the present study, significant differences in the sagittal plane were shown in stair walking rather than in level ground walking. the increase in flexion in the lower extremities might be caused by an increase in the knee joint flexion and the plantar flexion of the ankle joints to compensate for reduced shock absorption against the ground reaction due to the restriction in trunk movements caused by the application of the spinal orthosis. it can be concluded that a spinal orthosis is useful in stabilizing the lower extremities during stair gaiting, and that appropriate application of the orthosis plays a supporting role in the activities of daily life and therapeutic intervention. | [purpose ] the purpose of this study was to investigate the effect of restricted spinal motion on kinematic changes in the lower extremities using a rigid thoracolumbosacral orthosis. [subjects and methods ] forty healthy males in their 20s were selected as the sample, which was randomly and evenly divided into two groups : (1) the wt group (with a thoracolumbosacral orthosis) and (2) the wot group (without a thoracolumbosacral orthosis). the spinal orthosis used in this study was a thoracolumbosacral orthosis called a plastic body jacket. [results ] the sagittal plane ; in the level ground walking measurements, significance differences were found at the h2 (hip maximum flexion / extension in midstance phase) and k2 (knee maximum flexion / extension in midstance phase) between the wt group and the wot group. [conclusion ] it can be concluded that a spinal orthosis is useful in stabilizing the lower extremities during stair gaiting, and that appropriate application of the orthosis plays a supporting role in the activities of daily life and therapeutic intervention. |
with explosive increase in knowledge, new technologies, and rapid changes in pattern of diseases, there is concern that nursing students are not provided with enough opportunities to learn required clinical skills. to meet this concern, finding effective strategies to improve students learning, especially clinical learning, shift in teacher - centered paradigm to student - centered paradigm and replacing the traditional and passive strategies such as mentoring by active strategies such as peer learning are counterparts in the same direction. although some benefits have been identified in mentoring students by clinical trainers, such as encouragement, advice, and feedback, the importance of balancing among clinical role, educational role, and scientific preparation has caused many difficulties in mentorship programs. on the other hand, sharing knowledge with others teaching skills has been recognized for registered nurses as core competency. however, peer learning as a strategy in which a group of students involve in the learning process and training the other students is increasingly considered in other disciplines of medicine. unfortunately, evidences indicate that in nursing education, there is less attention given to peer learning over recent years. a peer is a student of the same age, group, academic level, or experience level. the oxford dictionary (2009) defines a peer as someone of the same age or someone who was attending the same university. the term peer can also refer to people who have equivalent skills of different experiences. peer learning is also described as a two - way reciprocal learning activity which includes sharing knowledge, ideas, and experiences in a way that has some benefits for both groups of peer and student. as a benefit of peer learning, it seems necessary to appreciate friendship in clinical learning environments among nursing students. in other words, more flexibility with students at clinical learning environments in interacting with their peers, whom they trust as friends, can facilitate earlier integration into the students community and, hence, enable peer learning for support. in spite of these benefits, unfortunately, there is limited evidence indicating use of peer learning in clinical education in developing countries like iran. a review of studies has shown the use of peer learning method in nursing education. results of most of these studies that have been designed as a quantitative approach indicate that peer learning encourages interaction, facilitates engagement with learning, and increases personal development. at monash university in australia, mclelland., investigated the benefits of an interprofessional peer - assisted learning for both midwifery and paramedic students. also, both groups had a newly found respect and understanding for each other 's disciplines. besides, another study confirmed the existence of peer effects in a learning process, showing a partner motivational effect even before the actual cooperation took place. students can be sensitively encouraged to share their views on participating in peer learning programs, which may well provide important insights into the benefits and challenges presented by student support initiatives as well as offer an outlook onto some important interactional processes influencing learners educational journeys. it should be noted that there are some controversies regarding the outcomes of peer learning applicabilities. for example, brannagan., conducted a study evaluating the impact of peer learning on nursing students perception of learning environment, self - efficacy, and knowledge. learning did not decrease anxiety in the first year students, and, concerning self - efficacy and knowledge acquisition, no differences were found between the two groups receiving either peer tutoring (intervention group) or faculty instruction only (control group). in iran, limited studies have been conducted on nursing students peer learning. hemat., assessed the effect of conducting training programs for high school students on the performance of the peers with asthma. investigated the facilitators and barriers in the application of such a method in clinical settings. dehghani. examined the impact of peer educational program on the anxiety of multiple sclerosis (ms) patients. results indicated that peer group educational program reduced anxiety in patients suffering from ms. regarding the mentioned controversies and due to the shortage of studies in the peer learning realm, and based on the results of some studies on the peer experiences and peer learning / teaching processes, this study aimed to explore nursing students experiences of peer learning in clinical practice. to explore the nursing students experiences of peer learning, a study was conducted with a qualitative research design in 2010. the emphasis of the investigation on the concept of peer learning within the real life context of nursing students in clinical practice was best facilitated using a qualitative research approach, conventional content analysis. qualitative content analysis is a research methodology and a reasonable tool to describe the quality of a phenomenon. through purposeful and criterion - based sampling, 28 senior bsc nursing students were selected and assigned to four focus - group discussions. under the supervision of one of the researchers, participants had passed pediatric and neonatal field practices as a peer learning method of field training (just for medication and iv therapy). to ensure that correct information would be given by the peers, on the first day of their training programs, all the students were provided with enough opportunities to practice the mentioned skills (medication and iv therapy) and learn from the lecturer 's demonstration. the next day, the peer volunteers carried out some selected nursing cares under supervision, and on the third day, they acted as peer to help others. in order to collect data, researches, a focus group involves a number of people (often with common experiences or characteristics) interviewed by a researcher for the purpose of eliciting ideas, thoughts, and perceptions about a specific topic or certain issues linked to an area of interest. focus group interviews were undertaken with each group after finishing their field training at the end of their semesterand after the students grades had been given. an expert convener led the focus groups to ensure that all members could participate freely. the research questions focused on how the nursing students experiences of peer learning were compared to usual learning. what aspects of peer learning made the experience either positive or negative for the nursing students ? there were some other questions based on their answers. thereafter, participants were encouraged to express their opinions, and were provided with sufficient time to do so. in total, four focus group interviews were conducted, with seven participants in each. a research assistant was present throughout to help with organization, audio - recording, and to write field notes. data on demographic characteristics, including age, sex, and marital status, were extracted and recorded. following the focus group discussion, based on the students explanations and condensing the codes that emerged, saturation in the categories had been seemingly achieved. the principles of qualitative data analysis are similar to those of other non - structured or semi - structured interviews. the analysis stages include the following : finding meaning, condensing, abstracting, identifying content that addresses a specific topic in an interview, and identifying emerging codes, categories, and themes. audio - taped recordings of the focus group interviews were transcribed verbatim, and the transcripts were read and reread by the investigators. notes were made on the thematic and conceptual categories emerging from the transcripts and on the reasons why the categories emerged. transcripts were then re - examined independently, pursuing the themes and concepts, resulting in the emergence of several subthemes and themes. at each stage of the analysis process, groupings and subsequent themes of the two researchers were compared and contrasted and then independently reviewed by the other one. the researchers discussed the differences and deviations in detail till consensus was reached ; all relevant data were then categorizedby consensus. codes were used when presenting participants quotes. by extracting the essence of ideas and using labels, the interviewers coded paragraphs and sentences were put into the margin of the transcript. by reducing thesecodes into larger categories, themes were formed. to increase the trustworthiness and rigor, we used effective communication principles with the participants in this study, returned the coded information to them, and checked the accuracy of the interviews by using all our colleagues supplementary opinions to ensure that the interview responses were well understood. we also checked the research findings as peer reviews to increase the credibility and confirmability of the research. the research ethics committee of bushehr university of medical sciences approved this study for the participants protection. they were informed about their rights to withdraw from the study at any time with no consequences. the participants had a mean (sd) age of 22 (1.47) years ; majority of the participants were females (91.4%) and single. out of the focus group analysis, four major themes were obtained : paradoxical dualism, peer exploitation, first learning efficacy, and socialization practice[table 1 ]. nursing students experiences in peer learning a dual sense of peer learning experience referred to the acquired advantages and perceived disadvantages applied to the nursing students. thus, peer learning caused an increase in self - confidence, accuracy, and skill in the students performance and a decrease in stress and practical mistakes. while dependency on others in performing the activities reduces the opportunity to display individual capabilities, from the students viewpoints, restricted learning ways or making mistakes were the disadvantages of this method. it also seemed these two dual senses rooted from two dimensions : the educational environment and the amount of workload. from the students viewpoint, the advantages of peer learning were better learning with no stress and reduced anxiety in making mistakes. one of the students said, when the peer was our classmate, our stress got less ; it was easier to talk about our problems to him / her than to the teacher. the teacher could criticize us why we had nt learned such cases yet. naturally answering the student 's comment on accuracy of the work and self - confidence had two aspects : increasing the accuracy of the work and reducing the mistakes. but if the students did such things alone, they could increase their accuracy in order to avoid mistakes. the students explained that if peer learning was the only teaching method at clinical settings, there would not be any chance for them to show their capabilities. they added they could get more dependent on the peers if they did not find any opportunity to do the cares independently. moreover, if the peer is not exposed to independent learning, he / she may acquire the ideas in wrong or limited ways, thus not being able to solve the problems. another student said, although peer can be useful for better group working, this method brings about dependency, especially if one student is weak and the other peer does the duty for him / her ; this is not suitable for their future, for they need to do it independently. according to the students, these two dual senses rooted from two dimensions : the educational environment and the amount of workload in the educational environment. despite passing various hospital wards, it was their first time being a pediatric and infant trainee. from their point of view, due to the high sensitivity of nursing care the feeling of not having an opportunity to compensate for the mistakes and being very novice for some cares caused high stresses ; thus, they automatically referred to their peer groups for consultation and collaboration to get more support and precision in their cares. due to the stress and high sensitivity, if any of their peers was weak in performing the duties, they tended not to become his / her peer. one of the students said, we are dealing with infants and children ; therefore, we have to be careful about the quantity of their body liquid and calculating the volume of serum and the number of drops, because unlike the adult 's wards, we do nt have any opportunity to compensate the mistake. if i know that the person who is going to be my peer has done too many mistakes, i try not to be his / her peer anymore. they stressed that for better learning, staff nurses should be in a protective role instead of being as a source of stress for students. the amount of workload, in the students viewpoint, was another factor affecting the gained advantages or perceived disadvantages. another student said, if the workload gets less, we can act and work more accurately and give more tips to one another. the students maintained that by selecting appropriate peers (based on their scientific capabilities and some individual characteristics which improve the learning process) and training the peers to do their roles correctly, teachers could exploit the best results from peer teaching and learning. scientific and individual features were the important factors in students who played the role of peers. based on the experiences they had with their peers, students mentioned lots of factors that were important in the process of learning. having enough experience, information, and patience in education helped them correct mistakes, create learning opportunities, and being responsible in true teaching. among the scientific characteristics of the peers were having a role in monitoring performance and leadership mentoring to teach others. having speech ability, transferring contents, and showing self - confidence were the individual characteristics of a peer. one of the students said, speech ability is so important because someone may know the subject but ca nt express what s / he knows. another one said, the peer 's role should be more supervisory than duty performance. one of the problems of my peer was that instead of giving me a chance to do the work, he tried to do all the activities by himself. this subtheme pointed out to the importance of a lecturer 's role in the field. these aspects include teaching the features and characteristics of a peer to students, the supervisory role of the individual mentor in the learning process, teaching how to correct the peers mistakes, adopting active and capable peers in the first days of training, using reinforcement tools, proper warnings, and distinguishing the students with false high confidence and students with ingratiation and flattering manner. one of the students said, in my opinion, the teacher must supervise the students activities to see whether the information they have conveyed is really correct or not. according to the participating students in this research, the value and importance of peer learning is regarding to provide a less stressful and more respectful learning environment. because of the importance of independence in giving cares to the patients, most of the students advocated the early application of this teaching method in learning, after which the process of learning could be handled each student individually. thus, they consider this learning method to be more effective during the early days of learning. because of the students high collusion in giving high scores to each other, their inability to have a comprehensive approach, and their consideration of different aspects of the evaluation, they assumed that peer assessment roles were inefficient. they added that in the case of necessary assessment, it would be better that the peers give only a small percentage of the total evaluation score. the trainer bases both processes of education and training for the peers on predefined educational objectives.. however, it would be better if the teachers make a comparative assessment of the students work with that of their peers ; this is due to the fact that some students group work is better than their individual performance. most of the students mentioned this as teamwork learning which helped them identify their own and their peers characteristics much better. moreover, there were some points showing the socialization process of students, i.e. students awareness of their negative characteristics and the ways to control or overcome them while working with others, respectful training, and preserving the peers characteristics, condemning jealousy or humiliating peer groups mistakes. i believe we should train our peer students in a completely sympathetic friendly way to learn something, not teasing the peers for training them. because if they were to know everything, why would they need to have peers ? a dual sense of peer learning experience referred to the acquired advantages and perceived disadvantages applied to the nursing students. thus, peer learning caused an increase in self - confidence, accuracy, and skill in the students performance and a decrease in stress and practical mistakes. while dependency on others in performing the activities reduces the opportunity to display individual capabilities, from the students viewpoints, restricted learning ways or making mistakes were the disadvantages of this method. it also seemed these two dual senses rooted from two dimensions : the educational environment and the amount of workload. from the students viewpoint, the advantages of peer learning were better learning with no stress and reduced anxiety in making mistakes. one of the students said, when the peer was our classmate, our stress got less ; it was easier to talk about our problems to him / her than to the teacher. the teacher could criticize us why we had nt learned such cases yet. naturally answering the student 's comment on accuracy of the work and self - confidence had two aspects : increasing the accuracy of the work and reducing the mistakes. but if the students did such things alone, they could increase their accuracy in order to avoid mistakes. the students explained that if peer learning was the only teaching method at clinical settings, there would not be any chance for them to show their capabilities. they added they could get more dependent on the peers if they did not find any opportunity to do the cares independently. moreover, if the peer is not exposed to independent learning, he / she may acquire the ideas in wrong or limited ways, thus not being able to solve the problems. another student said, although peer can be useful for better group working, this method brings about dependency, especially if one student is weak and the other peer does the duty for him / her ; this is not suitable for their future, for they need to do it independently. according to the students, these two dual senses rooted from two dimensions : the educational environment and the amount of workload in the educational environment. despite passing various hospital wards, it was their first time being a pediatric and infant trainee. from their point of view, due to the high sensitivity of nursing care the feeling of not having an opportunity to compensate for the mistakes and being very novice for some cares caused high stresses ; thus, they automatically referred to their peer groups for consultation and collaboration to get more support and precision in their cares. due to the stress and high sensitivity, if any of their peers was weak in performing the duties, they tended not to become his / her peer. one of the students said, we are dealing with infants and children ; therefore, we have to be careful about the quantity of their body liquid and calculating the volume of serum and the number of drops, because unlike the adult 's wards, we do nt have any opportunity to compensate the mistake. if i know that the person who is going to be my peer has done too many mistakes, i try not to be his / her peer anymore. they stressed that for better learning, staff nurses should be in a protective role instead of being as a source of stress for students. the amount of workload, in the students viewpoint, was another factor affecting the gained advantages or perceived disadvantages. another student said, if the workload gets less, we can act and work more accurately and give more tips to one another. the students maintained that by selecting appropriate peers (based on their scientific capabilities and some individual characteristics which improve the learning process) and training the peers to do their roles correctly, teachers could exploit the best results from peer teaching and learning. scientific and individual features were the important factors in students who played the role of peers. based on the experiences they had with their peers, students mentioned lots of factors that were important in the process of learning. having enough experience, information, and patience in education helped them correct mistakes, create learning opportunities, and being responsible in true teaching. among the scientific characteristics of the peers were having a role in monitoring performance and leadership mentoring to teach others. having speech ability, transferring contents, and showing self - confidence were the individual characteristics of a peer. one of the students said, speech ability is so important because someone may know the subject but ca nt express what s / he knows. another one said, the peer 's role should be more supervisory than duty performance. one of the problems of my peer was that instead of giving me a chance to do the work, he tried to do all the activities by himself. this subtheme pointed out to the importance of a lecturer 's role in the field. these aspects include teaching the features and characteristics of a peer to students, the supervisory role of the individual mentor in the learning process, teaching how to correct the peers mistakes, adopting active and capable peers in the first days of training, using reinforcement tools, proper warnings, and distinguishing the students with false high confidence and students with ingratiation and flattering manner. one of the students said, in my opinion, the teacher must supervise the students activities to see whether the information they have conveyed is really correct or not. according to the participating students in this research, the value and importance of peer learning is regarding to provide a less stressful and more respectful learning environment. because of the importance of independence in giving cares to the patients, most of the students advocated the early application of this teaching method in learning, after which the process of learning could be handled each student individually. thus, they consider this learning method to be more effective during the early days of learning. because of the students high collusion in giving high scores to each other, their inability to have a comprehensive approach, and their consideration of different aspects of the evaluation, they assumed that peer assessment roles were inefficient. they added that in the case of necessary assessment, it would be better that the peers give only a small percentage of the total evaluation score. the trainer bases both processes of education and training for the peers on predefined educational objectives.. however, it would be better if the teachers make a comparative assessment of the students work with that of their peers ; this is due to the fact that some students group work is better than their individual performance. most of the students mentioned this as teamwork learning which helped them identify their own and their peers characteristics much better. moreover, there were some points showing the socialization process of students, i.e. students awareness of their negative characteristics and the ways to control or overcome them while working with others, respectful training, and preserving the peers characteristics, condemning jealousy or humiliating peer groups mistakes. i believe we should train our peer students in a completely sympathetic friendly way to learn something, not teasing the peers for training them. because if they were to know everything, why would they need to have peers ? paradoxical dualism, peer exploitation, first learning efficacy, and socialization practice have emerged from students experiences as concepts of peer learning. according to the participants, dual role of the environment, type of the work and peers in creating stress, or quite the opposite, maintaining a secure environment for students, brought about a kind of stress for them. there are so many sources of stress in the hospital or social health environments, such as too much workload, insufficient staff to support practitioners, inadequate communication, secrecy, lack of trust, and so on. the findings of a study reveal that peer practice learning undertaken in a safe controlled environment enhances the realism of the experience, and therefore, will increase the likelihood of students engaging in the learning process. one of the most positive outcomes due to the effectiveness of peer teaching and learning, according to some studies, was the students increasing confidence in clinical practice and improved learning in the psychomotor and cognitive domains. feedback from participants in near - peer teaching suggests that the program fulfills its aims of providing an effective environment for developing deeper learning. the students who participated in peer learning clinical teaching strategy claimed it to be mutually supportive, cooperative, and collaborative, and also to have grown in both the diligence and precision with which they approached their own practice and in the personal confidence with which they made clinical and practice decisions. the students who participated in our study were completely relying on learning from their peers and had taken this seriously as they expected an educational role from their peers like from their own teachers. one of the important points about role - playing is that students, after some self - consciousness about the role, quickly settle down to project their own character and values into the role. it is the role play element of peer practice learning that also appears to provide some of the wider benefits highlighted in the study, such as increased empathy, improved communication skills, and enhanced decision - making ability. the findings of another study reveal that the third year nursing students who play the role of peers for the first year students commented that the peer learning experience gave them an opportunity to review their skills, allowing them to evaluate their knowledge base, whereas the first year students focused on the personal attributes of the third year students, rather than their teaching ability, with comments such as my third year student was a friendly partner who was very patient with me. in some cases, it is thought that there are links between confidence and learning, as students who are confident are allowed more access to patients. from the students point of view, this educational process had some disadvantages also such as lack of any chance for them to show their capabilities, acquiring the ideas in wrong or limited ways from peers, dependence on peers, and so on. there can be some disadvantages in the form of competition, along with feelings of being misunderstood leading to hurt and making unhelpful comparisons with others in peer - assisted ways. there are also many serious barriers to mentors that include difficulty of role modeling care work in the context of nursing roles which are increasingly concerned with more technical work. for peer exploitation, teachers could exploit the best results from peer teaching and learning. despite expecting a mentor - like role from the peers moreover, it increases its importance and students expect their teachers to provide the background process of education of the peers. the students in our study pointed out to the need of clinical teacher for supervising the process of teaching by peers. a systematic review of peer teaching and learning in clinical education suggested that the students evaluated their own learning and reported increased confidence in leadership roles when working with a peer. it has also been emphasized that students adopting the peer mentor role get some benefits like leadership and teaching skills from peer teaching and learning experience. peer practice learning should only occur with a small number of students for one facilitator to enable the facilitators give the evident support required. as a limitation and negative aspect, personality and learning style of students should be appropriate to peer learning. most of the students regarded this method to be effective in learning and spoke with resistance against the evaluation process done by peer assessment. peer assessment is useful for assessing practical skills ; but one of the problems with any kind of peer assessment is the potential for collusion among the students to raise the level of marks. one of the solutions to this problem is to use peer assessments as feedback rather than as final grading, to ensure honesty of the feedback. findings of a focus group discussion on nursing students experiences of formative assessments indicate that nursing students are not being prepared for the critical feedback associated with peer review and they may, therefore, be vulnerable to the process and outcome of peer review. in fact, the students had gained a kind of respect to the values and received new social roles. the process by which an individual undergoes induction into these expected behaviors or roles is termed socialization. secondary socialization begins as the child commences school, influenced not only by teachers but also by peers ; occupational socialization involves induction into specific occupational roles after leaving school. in a study about exploration of reflective groups, it was revealed that being able to reflect on real life experiences helped the students to recognize that others had similar experiences to their own and these interconnected experiences made them realize that they were not on their own. students involved in the peer mentorship programs might offer important illustrations of the critical aspects of pastoral and social support. another group of students explained their friendship and peer learning in clinical practice as valuable sources of information, which was the result of asking questions about the culture and convergence of each other, particularly when they found themselves alone or when their mentors were busy elsewhere. finally, it seems students in our study were interested to change their clinical groups based on their changed friendship and the effects of group atmosphere and team work during their educational carrier. similarly, in many nursing programs, it has resulted in changed membership of the learning communities in every semester, allowing students to work with different peers during each clinical rotation. this study sought to explore the common reasonable perceptions of peer learning typically designed to support better learning in clinical settings. there is some evidence that students learning is facilitated if peer learning in clinical settings can be followed, as it may improve in - depth learning with less stress, role satisfaction, and create a positive environment in which students can learn appropriate practices. besides, the findings depict a general satisfaction among the participating students from peer learning in both direct learning outputs and indirect (hidden) learning outputs. while role transition to advance practice is a key priority for the development of effective health care programs around the world, our findings support the nursing students role transition to educational leadership and teaching others, along with socialization. based on our findings, it seems this method of learning can be utilized in learning practical and laboratory techniques in fields such as biochemistry and microbiology, or in the operating room for students of different disciplines such as medical, nursing, environmental health, biology, etc. it suggests the teachers to determine the level and amount of support of peers required in clinical settings by the students, based on their year of education and level of excellence in practice, and then assess its outputs on the students level of learning. it seems this will help to facilitate a student - centered method of learning in clinical settings, especially for disabled students or with students with a learning difference. | background : peer learning is an educational process wherein someone of the same age or level of experience level interacts with other students interested in the same topic. there is limited evidence specifically focusing on the practical use of peer learning in iran. the aim of this study was to explore nursing students experiences of peer learning in clinical practice.materials and methods : a qualitative content analysis was conducted. focus groups were used to find the students experiences about peerlearning. twenty - eight baccalaureate nursing students at bushehr university of medical sciences were selected purposively, and were arranged in four groups of seven students each. the focus group interviews were conducted using a semi - structured interview schedule. all interviews were tape - recorded, transcribed verbatim, and analyzed using conventional content analysis method.results:the analysis identified four themes : paradoxical dualism, peer exploitation, first learning efficacy, and socialization practice. gained advantages and perceived disadvantages created paradoxical dualism, and peer exploitation resulted from peer selection and peer training.conclusion:nursing students reported general satisfaction concerning peer learning due to much more in - depth learning with little stress than conventional learning methods. peer learning is a useful method for nursing students for practicing educational leadership and learning the clinical skills before they get a job. |
the prognosis for oesophageal cancer is poor with a median survival of 35 months and recurrences are frequent. the best chance of cure is successful surgery and pre - operative chemo - radiotherapy is used to try and improve outcomes with the aim of eradicating lymphatic and haematogenous metastases, not only to improve survival and decrease recurrences, but also to shrink the primary tumour. patients who achieve a complete response with the chemo - radiotherapy have a 5-year survival of 63% compared to 23% in the non - responders. however patients may either not respond or may progress during therapy and these groups may benefit from early surgical intervention and so it is important to differentiate the responders from the non - responders. clinical parameters such as weight gain and improvement in swallowing can be assessed but imaging is used in an attempt to improve outcomes but has rather variable results. endoscopic ultrasound (eus) is the most accurate method for staging the primary tumour and local lymph nodes at diagnosis, but has limitations following chemo - radiotherapy as eus can not differentiate between fibrosis and residual disease with over staging being reported in up to 69% of patients. however, using a reduction of 50% or greater in the maximum cross sectional area is relatively accurate in both predicting response (ppv 80%) and survival. the use of computed tomography (ct) has produced conflicting results. walker. found a wide discrepancy between the reported ct response and the pathological correlation (48% responding on ct with a 90% pathological response) and these authors suggested ct could predict a response, but lack of ct response did not preclude a pathological response. however, more recent studies found no correlation between the ct and the pathological response although a recent study by beer. using multidetector ct suggested that a ct scan performed 14 days after the initiation of chemotherapy could predict the final response (sensitivity 100%, specificity 53%) but using a volumetric method of measurement, rather than the tumour diameter. fdg - positron emission tomography (fdg - pet) is used to assess response in other tumours and appears to be the best method for identifying responders in oesophageal cancer. studies by ott. in assessing early response to therapy (within 14 days of commencement of chemo - radiotherapy) found a decrease in uptake (suv) of greater than 35% indicated a major pathological response with a 3-year survival of 70%, compared to a 3-year survival of only 35% in the non responders. fdg - pet can also be used at the end of treatment to predict response in both adenocarcinoma and squamous cell carcinoma. flamen. found fdg - pet was both sensitive and specific (71% and 82%) in identifying a major response, although in this study the response was both over and under estimated in 11% of patients. swisher. compared eus, ct and fdg - pet and found fdg - pet was more accurate (70%) than eus (68%) or ct (62%). in this study an suv of greater than 4 was an independent predictor of survival with a 2-year survival of 34%, compared to a 2-year survival of 64% with a suv of less than 4. however, none of the imaging modalities can differentiate a complete response (0% viable cells) from microscopic residual disease (110% viable cells) so patients with a complete metabolic response may still need further intervention. duong. found patients with a complete response on fdg - pet who did not undergo surgery had a comparable survival to those that did, perhaps therefore allowing a more conservative approach in selected patients. the recurrence rate after resection is high (3479%) with more than 50% occurring in the first year and most presenting within 2 years of surgery. recurrences may be either local (30%) or distant, with local or distant nodal deposits and haematogenous spread to the lungs common. there is no correlation between the site of the primary tumour and that of the recurrence presumably because of extensive lymphatic spread prior to the surgery. distant spread may occur without any local recurrence in 40% of patients and is commonest, in descending order to nodes, lung, liver, pleura and adrenals. a recent autopsy study found tumour in 63% of patients following curative surgery and in this study 43% of patients flamen. in a study of patients with clinical or radiological suspected recurrence compared fdg - pet with the conventional work up of ct and eus. in this study all equivocal lesions on any modality were called positive. the sensitivity for fdg - pet for peri - oesophageal recurrence was 100% with a specificity of 57% and accuracy of 74%, whereas the conventional work up was 100% sensitive, 93% specific and 96% accurate. a false positive result may occur with fdg - pet in patients who have undergone dilatations. the majority of recurrences were distant metastases and the sensitivity, specificity and accuracy of fdg - pet compared to conventional imaging was 94%, 82% and 87% vs 81%, 82% and 81%, respectively. although in this study there was no significant difference in the results between the methods of investigation, on a patient basis fdg - pet did provide additional information in 11 out of 41 patients (27%), identifying unsuspected recurrence in 5 and upstaging a further 5 patients. kato. looked at a group of post surgical patients, only 8% of whom were symptomatic, but 35% had recurrent disease. fdg - pet was 100% sensitive but only 75% specific for local recurrence compared to ct (84% and 86%, respectively). the false positives for fdg - pet were in physiological uptake in the gastric tube and in mediastinal lymph nodes probably related to chronic lung disease. for distant recurrence the diagnostic accuracy for fdg - pet and ct were similar for liver metastases, whereas fdg - pet was less sensitive than ct (50% vs 100%) for lung metastases. fdg - pet was more sensitive for bone metastases (100% vs 17% respectively). imaging provides important information for assessing response to therapy, predicting survival and identifying recurrent disease in oesophageal cancer. fdg - pet / ct would appear to be the most appropriate method for assessing response to therapy but all methods have limitations in identifying small volume disease. local recurrence is equally well demonstrated by conventional and functional imaging, although there are some advantages in using fdg - pet for distant recurrences. at the present time | abstractthe prognosis for oesophageal cancer is poor with a median survival of 35 months and recurrences are frequent. the best chance of cure is successful surgery and pre - operative chemoradiotherapy is used to try and improve outcomes. however, patients may either not respond or may progress during therapy and it is important to differentiate the responders from non - responders. clinical parameters such as weight gain and improvement in swallowing can be assessed but imaging is used in an attempt to improve outcomes. |
inflammatory myofibroblastic tumor (imt) is rare and characterized by spindle cell proliferation with inflammatory cell infiltration. because of its cytological features and infiltrative nature, it may be difficult to distinguish histologically from malignant sarcomatous lesions. a 36-year - old male was referred to our hospital for further examination of a bladder tumor. cystoscopy revealed a broad - based tumor 40 mm in diameter (fig 1). he had a medical history of hyperlipidemia, diabetes mellitus, asthma, and bipolar disorder. the hematological and biochemical data showed no abnormal findings without elevation of hba1c of 8.4%. the tumor markers were also within normal limits (cea 1.4 ng / ml, ca125 12 u / ml, ca199 7 u / ml). magnetic resonance imaging (mri) showed a 40 25-mm mosaic bladder mass of high intensity on diffusion images ; the tumor was suspected to have invaded the muscle (fig 2), but computed tomography revealed no distant metastasis. histologically, the urothelium showed no atypia. in the submucosa, spindle cells proliferated in interlacing fascicles or storiform arrays with inflammatory cell infiltration. immunohistochemically, the spindle tumor cells were strongly positive for anaplastic lymphoma kinase (alk) and smooth - muscle actin, weakly positive for cytokeratin ae1/ae3 and p53 but negative for desmin. based on these findings, the tumor was diagnosed as imt. the patient has not experienced recurrence in the 10 months since resection of the bladder tumor. the hematological and biochemical data showed no abnormal findings without elevation of hba1c of 8.4%. the tumor markers were also within normal limits (cea 1.4 ng / ml, ca125 12 u / ml, ca199 7 u / ml). magnetic resonance imaging (mri) showed a 40 25-mm mosaic bladder mass of high intensity on diffusion images ; the tumor was suspected to have invaded the muscle (fig 2), but computed tomography revealed no distant metastasis. histologically, the urothelium showed no atypia. in the submucosa, spindle cells proliferated in interlacing fascicles or storiform arrays with inflammatory cell infiltration. immunohistochemically, the spindle tumor cells were strongly positive for anaplastic lymphoma kinase (alk) and smooth - muscle actin, weakly positive for cytokeratin ae1/ae3 and p53 but negative for desmin. based on these findings, the tumor was diagnosed as imt. no adverse perioperative events were observed. the patient has not experienced recurrence in the 10 months since resection of the bladder tumor. imt also known as postoperative spindle cell nodule, pseudosarcomatous fibromyxomatous tumor, and reactive pseudosarcomatous response involve only inflammatory cells. these tumors have been observed in any organ, including the lung, liver, and kidney. the most frequently reported symptom is gross hematuria followed by urinary pain, dysuria, and increased urination frequency. the tumor has been observed in a wide range of ages, from around 30 to 50 years, with an equal balance of genders. although the causes of this tumor have long been believed to be urinary tract infection, surgical invasion, diabetes mellitus, and immune disorders, a number of idiopathic cases have also been reported [4, 5 ]. in our case, the patient had diabetes mellitus. in this case, the tumor size was around 4 cm in diameter. reported imts were around 25 cm in most cases, but 1 case showed 37.5 cm [1, 4 ]. due to the risk of local recurrence, complete resection is recommended. on the other hand, grossly, imt shows a variety of forms, but in most cases, the tumor adopts a nonpapillary form with a rounded surface. on mri, the tumor typically shows low intensity on t1-weighted imaging and high intensity at the surface with low intensity inside on t2-weighted imaging. histologically, imt is characterized by proliferation of myofibroblastic spindle cells and inflammatory cell infiltration. the most important entities in differential diagnosis are sarcomatoid urothelial carcinoma, leiomyosarcoma, and rhabdomyosarcoma because of the similarity of histological findings. finding cytological atypia and atypical mitotic figures and immunostaining with alk are useful to differentiate imt from other malignant spindle cell tumors [1, 9 ]. although there have been no reports of distant metastasis, intravesicle recurrence was reported after transurethral resection. partial bladder resection is required after transurethral resection. however, in cases of benign tumors, total cystectomy should not be performed due to the risk of misdiagnosing them as sarcomatoid carcinoma. | a 36-year - old male was referred to our department for further examination of asymptomatic gross hematuria emanating from a bladder tumor. cystoscopy revealed a broad - based tumor 40 mm in diameter. urinary cytology was negative. preoperative magnetic resonance imaging suggested a muscle invasive tumor. transurethral resection was performed, and the pathological findings revealed an inflammatory myofibroblastic tumor. we herein report a rare case of bladder inflammatory myofibroblastic tumor. |
the myelodysplastic syndromes (mds) are characterized by proliferation of hematopoietic stem cells with abnormalities of differentiation leading to peripheral blood cytopenia and a preleukemic state. the mechanism of transformation of mds to acute myeloid leukemia (aml) includes many factors, and prediction of the transformation is not yet clear. we report a case of leukemic transformation of mds with multi - muscle involvement and whole - body 2-[f18 ] fluoro-2-deoxy - d - glucose positron emission tomography (f18-fdg pet). a 52-year - old female, presenting with high fever for 10 days, chills, frequent micturition, and urgency, was admitted to the regional hospital on 10 april 2009. were given, and after a week, she was relieved of the above - mentioned symptoms. blood counts showed moderate pancytopenia (hemoglobin 92 g / l, wbc 1.57 10/l and platelets 92 10/l). on 20, 22 and 27 april, bone marrow aspiration biopsy was performed repeatedly, and the percentage of blast cells from bone marrow was 12.55%, 22.5%, and 26.5%, respectively. on 20 april, flow cytology detected 13.50% blast cells from bone marrow, which had cd34, cd2, and cd41phenotype, and bone marrow biopsy microscopically revealed that granulocyte and megakaryocyte series reduced, and a fraction of plasma cells diffusing encroached that were mpo, cd38, cd138, cd68, cd61, cd3, cd45ro, cd20, cd79a. however, 4.49% blast cells were detected that had cd34, cd2, cd7, cd117, cd71, hla - drphenotype on 27 april, and it microscopically disclosed scattering immature mononuclear cells which were mpo, cd68, and cd61. therefore, f18-fdg pet of whole body was carried out on 5 may, which manifested no evidence of malignancy, including that of bone marrow [figure 1 ]. the final diagnosis could not be made because of the patient 's resolution to give up treatment. she was intermittently transfused blood without any other treatment for about 10 months since she had been discharged. on 25 march, she was hospitalized with fever of 3 days duration accompanied by swelling and pain of the right upper leg and stern. on physical examination routine blood analysis showed serious pancytopenia (hemoglobin 81 g / l, wbc 0.67 10/l, platelets 35 10/l). bone marrow aspiration displayed that immature monocytes accounted for 25.0%, periodic acid - schiff stain (pas) (+), peroxidase stain (pox) (+), considering aml. flow cytometry detected 28.60% blast cells, which had cd34, cd13, cd64, cd11b, hla - dr phenotype, from bone marrow. whole - body lymphoimaging showed that lymph fluid circulation was unobstructed and soft tissues of the right lower limb were swollen [figure 2 ]. f18-fdg pet of whole body showed spotted and focal increased uptake in ribs, vertebrae, ilium, humerus, femur, tibia, and high intensity foci of fdg uptake in right axillary, mediastinal, bilaterally lung portal, peritoneal, right communistic and external iliac lymph nodes. moreover, a significant lesion of increased metabolism infiltrated muscles of right rear upper legs, right gluteus maximus and medius [figure 3 ]. according to the above outcomes and physical examination, the final diagnosis was inclined to be aml. as a result, treatment with cag chemotherapy regimen was started. during the course of chemotherapy, intumescence of right upper leg and stern conspicuously lessened. however, after less than a week, the patient died from cerebral hemorrhage and hernia. f18-fdg pet manifests no evidence of malignancy, including that of bone marrow whole - body lymphoimaging shows easy and smooth lymph fluid circulation and lymph node enlargement f18-fdg pet demonstrates focal increased uptake in ribs, vertebrae, ilium, humerus, femur, tibia, and high intensity foci of fdg uptake in right axillary, mediastinal, bilaterally lung portal, peritoneal, right communistic and external iliac lymph nodes. moreover, a chunk of lesion of increased metabolism infiltrated the muscles of right rear upper legs, right gluteus maximus and medius the case reported here was that of an old woman presenting with pancytopenia, chromosome clonal abnormality, fluctuation of the percent of blast cells at 20%, and negative evidence of malignancy in whole - body f18-fdg pet. after about 10 months, the blast cells accounted for about 25%, the morphology of which was similar to that of the previous ones, and f18-fdg pet demonstrated diffusing increased uptake in the right upper leg and lymph nodes, and patchy high uptake of bone marrow. in this case, the main differential diagnosis of pancytopenia was between leukemia and mds. the percent of blast cells of bone marrow is critical to differentiate leukemia and mds. in this patient, retrospectively, it is speculated that it was the transformation from mds to aml in april 2009, which might have led to inconsistent bone biopsies, aspirations and immunophenotypes. immunophenotyping demonstrated that blast cells were of myeloid lineage, which is consistent with the earlier reports.[13 ] therefore, close follow - up was recommended. to the best of our knowledge, few studies about multi - muscle of leg involvement have been reported, and obviously the diagnostic value of f18-fdg pet in the fields of mds and aml has been attributed to reduced f18-fdg pet uptake in the bone marrow. it was reported that f18-fdg pet performed in a patient with mds showed diffusing increased uptake of bone marrow of the whole body, which transformed into aml after 2 months, showing that f18-fdg pet may be an indicator for transformation of mds to aml. in this case, f18-fdg pet detected tumor burden and was positive for extramedullary infiltration in aml phase, which is compatible with the previous reports, but was negative for malignancy of bone marrow in the state of transformation phase, which is not concordant with the previous reports. agool and others[68 ] reported that homogeneous increased uptake of bone marrow was seen in eight mds patients in 3-deoxy-3-[f18]-fluorothymidine positron emission tomography (f18-flt pet), which, in comparison to f18-fdg pet, is more related to the rate of dna synthesis and cycling activity and provides visual and quantitative information on the entire bone marrow compartment. perhaps it is more sensitive than f18-fdg pet in forecasting transformation of mds to aml. the value of f18-fdg or f18-flt pet as an indicator in predicting the transformation of mds to aml needs to be explored in the future, and f18-fdg or f18-flt pet can reflect extramedullary infiltration and bone marrow cellularity of whole body, compared with invasive, regional biopsies and aspirations. | the case reported here was that of an old woman characterized by pancytopenia, chromosome clonal abnormality, fluctuation of the percent of blast cells at 20%, and negative evidence of malignancy in whole - body 2-[f18 ] fluoro-2-deoxy - d - glucose positron emission tomography (f18-fdg pet). after about 10 months, the blast cells accounted for about 25%, the morphology of which was similar to that of previous ones, and f18-fdg pet demonstrated diffusing increased uptake in the right upper leg and lymph nodes and patchy high uptake of bone marrow. 2-[f18]-fluoro-2-deoxyglucose can reflect extramedullary infiltration and bone marrow cellularity of the whole body, compared with invasive, regional biopsies and aspirations. the value of 2-[f18]-fluoro-2-deoxyglucose or 3-deoxy-3-[f18]-fluorothymidine positron emission tomography as an indicator in predicting the transformation of myelodysplastic syndrome to acute myeloid leukemia needs to be explored in the future. |
though corticosteroids are the most potent anti - inflammatory agents, yet they do not suppress inflammatory mediators like leukotrienes which have an important role in the pathogenesis of asthma. moreover, pharmacokinetic studies have shown the effects of systemic corticosteroids to be apparent only after 46 h of administration. as a consequence of these deficiencies, a need for additional therapeutic options exists and, hence, various drugs have been studied for their efficacy in acute asthma. however, the results of these studies have not been very encouraging, which has prompted investigators to probe the utility of leukotriene modifiers, such as montelukast (oral or intravenous) in acute asthma the usefulness of which in chronic asthma is well - established. in this context, the present study was carried out to compare oral montelukast with oral zileuton in acute asthma in patients who were already receiving standard treatment for acute asthma. the study assessed and compared the effects of these drugs on lung function (pefr) and the need for rescue medications. this was a randomized, prospective, placebo - controlled, double - blinded, single - center, comparative study conducted in the department of pulmonary medicine at a tertiary care hospital in south india. patients presenting to outpatient unit or emergency triage, from september 2012 to march 2014, with a primary diagnosis of acute exacerbation of asthma requiring hospitalization, were included in the study. the inclusion criteria were age between 18 and 65 years, previously diagnosed case of bronchial asthma, a primary diagnosis of acute exacerbation of asthma on presentation, peak expiratory flow rate (pefr) 75% of personal best within the last 12 months or of predicted value, and no other acute pathology complicating the present condition such as cardiac, metabolic, or other respiratory causes. patients with a history of smoking more than 10 pack years, pregnant females, breastfeeding females, patients on regular leukotriene receptor antagonists or 5-lipoxygenase inhibitors within 2 weeks of presentation, intake of oral or parenteral steroids for > 5 days within 1-month of presentation, intake of theophylline within 1-week of presentation, need of intubation before presentation, patients on regular rifampicin, phenytoin or phenobarbitone, and known allergic reaction to montelukast or zileuton were excluded from the study. informed written consent was taken from the patients in the language understandable to them prior to inclusion. history, findings of physical examination and laboratory investigations were noted and entered as per a predesigned proforma. the proforma was designed by the authors based on the study requirements, i.e., the data to be captured, taking into consideration earlier studies. however, the proforma was not validated. anticipating a minimum difference of 40 l / min in pefr with a standard deviation of 100 l / min for 80% power at 95% confidence level, 40 subjects were recruited, in each study arm. enrolled patients had a baseline measurement of pefr using a mini wright 's pef meter. after 5 min of baseline pefr, the patients were randomized into three groups using block randomization (chit method) : a total of 20 envelopes were created. in addition, 42 chits were prepared, with each bearing the letters p, m, and z (corresponding to placebo, montelukast, and zileuton, respectively) arranged in one of six possible sequences. the possible sequences with the letters pmz are pmz, pzm, mpz, mzp, zmp, and zpm. seven such sets were prepared (adding up to a total of 42 chits). two chits were placed in each of the twenty envelopes, and the remaining 2 chits were discarded. while allocating cases to a group, envelopes were opened one by one, chits were picked up and cases allocated to each group as per the letter sequence. patients in all the three groups received standard treatment for asthma exacerbation, i.e., nebulized salbutamol 2.5 mg 6 hourly, nebulized ipratropium bromide 500 mcg 6 hourly and intravenous methyl prednisolone 40 mg 8 hourly. rescue medications comprised nebulized salbutamol 2.5 mg, given on need basis. additional oxygen and methylxanthines were given, if indicated. patients and the investigators were kept blinded regarding the study medication, and a third observer distributed and administered the tablets from the packs montelukast, zileuton, and placebo to the patients as per the randomization code. patients randomized to group 1 received oral montelukast 10 mg tablet with a placebo at enrollment and two placebo tablets after 12 h. patients randomized to group 2 received two oral zileuton cr 600 mg tablets twice a day. the investigational medications were continued till the patient was discharged from the hospital or developed any adverse effects to these drugs. pefr values, details of rescue medication, and vital signs (pulse rate, blood pressure, respiratory rate, and spo2 by pulse oximetry) were recorded at 6 h, 12 h, 24 h, and 48 h of drug or placebo administration and at discharge. additional recording of these parameters was done in the morning (810 am) following admission. the primary endpoint was the mean pefr of each group at different measured time points following treatment. repeated measures anova with baseline pefr set as a covariate was used to analyze mean pefr of each group at different measured time points after initiation of treatment. univariate anova with baseline pefr as a covariate was used to analyze mean pefr at 810 am on the morning following admission. fisher 's exact test and chi - square test were used to analyze the need for rescue medications and the requirement of methylxanthines and oxygen in the various groups. analysis was performed using spss 15.0 (spss, south asia, bangalore) and the means and percentages were expressed in tables and graphs. repeated measures anova with baseline pefr set as a covariate was used to analyze mean pefr of each group at different measured time points after initiation of treatment. univariate anova with baseline pefr as a covariate was used to analyze mean pefr at 810 am on the morning following admission. fisher 's exact test and chi - square test were used to analyze the need for rescue medications and the requirement of methylxanthines and oxygen in the various groups. analysis was performed using spss 15.0 (spss, south asia, bangalore) and the means and percentages were expressed in tables and graphs. a total of 153 patients were screened for eligibility of inclusion into the study, of which 120 patients met the inclusion criteria. of the enrolled patients, 40 patients were randomized to enter the placebo arm, 40 patients to the montelukast arm and 40 patients to the zileuton arm. all 120 patients who were enrolled completed the study and results were analyzed at the end of the study. the montelukast group had more patients with severe exacerbation and lesser with mild - moderate exacerbation, but there was no significant difference between the groups so far as the severity of exacerbations was concerned (p = 0.07). baseline patient characteristics (n=120) the mean pefr for the three study groups at baseline and over the hospital stay was analyzed using repeated measures anova with post hoc analysis, with baseline pefr set as the covariate. on comparing with placebo, zileuton group had significantly higher mean pefr values (p = 0.007) whereas montelukast group did not differ significantly (p = 0.181) [figure 1 ]. the mean pefr for the three study groups at 810 am on the morning following admission was analyzed using univariate anova, with baseline pefr set as the covariate. again on comparing with placebo, zileuton group had significantly higher mean pefr values (p = 0.041) whereas montelukast group did not differ significantly (p = 0.651). mean peak expiratory flow rate at different time points after initiation of treatment (with baseline peak expiratory flow rate [167.92 l / min ] set as the covariate) mean peak expiratory flow rate at different time points after initiation of treatment (n=120) mean peak expiratory flow rate on the morning following admission (8 - 10 am) (n=120) the secondary end points were analyzed using chi - square test. five patients in the placebo group required rescue medications, one of them requiring thrice, three patients requiring it twice, and one patient requiring it once. one patient in the montelukast group required rescue medication once. however, no patients in the zileuton arm needed rescue medications [table 4 ]. this difference in the requirement for rescue medications in the montelukast and zileuton groups compared to the placebo group met statistical significance (p = 0.049). need for rescue medications in each group (n=120) the need for methylxanthines and oxygen in three groups is shown in table 5. the difference in the requirement for methylxanthines and oxygen in the montelukast and zileuton groups compared to the placebo group was not statistically significant (p = 0.237). need for additional methylxanthines and supplemental oxygen in each group (n=120) the patients were discharged based on clinical improvement, improvement in vital signs and improvement in pefr though there were no strict prescribed criteria for discharge. all patients, except one, showed a significant improvement in vital signs and pefr at the time of discharge. one patient from placebo group who did not exhibit clinically significant change from baseline values of vital signs and pefr was discharged based on significant symptomatic improvement. there was a significant difference in the time to discharge between the various groups (p = 0.022). the number of hospital days expressed as median (interquartile ranges) for placebo, montelukast, and zileuton groups were 5 (46.5), 6 (59), and 5 (46), respectively. the mean pefr for the three study groups at baseline and over the hospital stay was analyzed using repeated measures anova with post hoc analysis, with baseline pefr set as the covariate. on comparing with placebo, zileuton group had significantly higher mean pefr values (p = 0.007) whereas montelukast group did not differ significantly (p = 0.181) [figure 1 ]. the mean pefr for the three study groups at 810 am on the morning following admission was analyzed using univariate anova, with baseline pefr set as the covariate. again on comparing with placebo, zileuton group had significantly higher mean pefr values (p = 0.041) whereas montelukast group did not differ significantly (p = 0.651). mean peak expiratory flow rate at different time points after initiation of treatment (with baseline peak expiratory flow rate [167.92 l / min ] set as the covariate) mean peak expiratory flow rate at different time points after initiation of treatment (n=120) mean peak expiratory flow rate on the morning following admission (8 - 10 am) (n=120) five patients in the placebo group required rescue medications, one of them requiring thrice, three patients requiring it twice, and one patient requiring it once. one patient in the montelukast group required rescue medication once. however, no patients in the zileuton arm needed rescue medications [table 4 ]. this difference in the requirement for rescue medications in the montelukast and zileuton groups compared to the placebo group met statistical significance (p = 0.049). the difference in the requirement for methylxanthines and oxygen in the montelukast and zileuton groups compared to the placebo group was not statistically significant (p = 0.237). the patients were discharged based on clinical improvement, improvement in vital signs and improvement in pefr though there were no strict prescribed criteria for discharge. all patients, except one, showed a significant improvement in vital signs and pefr at the time of discharge. one patient from placebo group who did not exhibit clinically significant change from baseline values of vital signs and pefr was discharged based on significant symptomatic improvement. there was a significant difference in the time to discharge between the various groups (p = 0.022). the number of hospital days expressed as median (interquartile ranges) for placebo, montelukast, and zileuton groups were 5 (46.5), 6 (59), and 5 (46), respectively. leukotriene receptor antagonists like montelukast have a well - defined role in the management of chronic asthma ; and they have been subjected to a number of studies for evaluating their role in acute asthma. there has been some evidence in support of usefulness of montelukast in acute asthma. in a study done in the usa, camargo. demonstrated a rapid and significant improvement in fev1, noted at 10 min and over 2 h after administration of intravenous montelukast, when compared with placebo. later, these results were reproduced in a study with a larger sample size (n = 583). a recent japanese study by adachi. also found that intravenous montelukast was significantly more effective than placebo in the improvement of fev1 in the acute asthma setting. on comparing the effect of intravenous versus oral montelukast in a randomized placebo - controlled study in patients with acute asthma faster onset of action and better improvement in fev1 however, oral montelukast group also demonstrated significant improvement in fev1 when compared to placebo. investigators from the united kingdom, in a randomized, placebo - controlled, single - center study, involving a total of 73 patients with acute asthma randomized to receive oral montelukast or placebo in addition to standard treatment, reported significantly higher pef values recorded on the morning following admission (p = 0.046) in the montelukast arm. in contrast, the results of our study showed that oral montelukast, when used in addition to standard treatment, did not produce statistically significant improvement in pefr (p = 0.181) compared to placebo. however, the requirement for rescue medications in the montelukast group was significantly less compared to the placebo group (p = 0.049). there are other studies which have also questioned the role of montelukast in acute asthma. a study from portugal reported no significant differences between montelukast and placebo groups in terms of improvement in pefr and duration of stay in the emergency room in a study involving 20 adults with acute asthma but showed trends in favor of montelukast in terms of lesser requirement for systemic therapy with aminophylline or steroids (p = 0.03). the results of this study are identical to ours so far as lack of improvement in pefr is concerned. however, in our study, the need for additional requirement of methylxanthines, even though lesser among montelukast group, failed to show any statistical significance (p = 0.956). a study done in the tertiary care hospital in turkey randomized 70 patients with acute asthma to receive placebo alone, prednisolone alone or though the patients receiving both prednisolone and montelukast had significantly higher percentage change in pef from baseline over a 24 h period in comparison with placebo group (p < 0.05), the percentage change in pefr in comparison with patients receiving prednisolone alone did not show any statistical significance (p = 0.064). moreover, patients receiving both prednisolone and montelukast neither had significantly better borg dyspnea scores (p = 0.34) nor lesser need for rescue medications (0.064) compared to prednisolone group. the results of this study were similar to our study in terms of the primary endpoint (pefr trend), but the results of secondary endpoints need for rescue medications were contrasting. results obtained in a recent randomized, placebo - controlled study by zubairi., in which 100 patients were randomized to receive either montelukast or placebo, in addition to standard treatment with systemic steroids and nebulized bronchodilators, have raised further doubts there was no statistically significant difference between the treatment groups during hospital stay (p = 0.20 at day 2 and p = 0.47 at day 3) and at the time of discharge (p = 0.15), in terms of both fev1 and pef. the difference between the time to discharge in both groups was also not statistically significant (p = 0.90). in our study, the time to discharge in the montelukast group was greater than in placebo group. as is apparent from the contrasting results of the studies on montelukast, oral montelukast is not currently recommended for use in acute asthma ; however, to ascertain the role of intravenous montelukast in acute asthma, more research is required. however, in spite of these contrasting results, there may still be a case for the use of leukotriene modifiers in this setting, and this perhaps was underscored by the results of the zileuton arm of our study. acute bronchodilator effect has been observed with use of zileuton, but most of the earlier studies on zileuton have been carried out in patients with chronic stable asthma and not during episodes of acute asthma. in addition, studies comparing the efficacy of zileuton and montelukast are also few. in one such study, involving mild to moderate chronic stable asthmatics, the investigators reported significant improvement in pefr, reduction in the mean overall symptom intensity score, and also reduced but not significantly different adverse event rate in zileuton group when compared to the montelukast group. our study tried to address both the issues of zileuton use in acute setting and its comparison with montelukast. our results show that zileuton, when used in such a setting, produced statistically significant improvement in pefr (p = 0.007) compared to the placebo group, unlike montelukast which failed to demonstrate such an effect. moreover, the need for rescue medications in the zileuton group was also significantly less compared to the placebo group. with respect to this outcome measure, the need for methylxanthines and oxygen were also comparatively lesser among zileuton group, though it failed to reach statistical significance (p = 0.055). though the time to discharge in the zileuton group was shorter in comparison with the placebo group, it was not statistically significant (p = 0.187). these results will continue to stoke interest in the use of leukotriene modifiers in acute asthma, and perhaps the last word on this issue has not been said yet. the limitation of our study was the use of pefr as an indicator of airflow obstruction as many studies done in this context have used fev1 - a better indicator. we also did not assess biological markers like urinary cysteinyl leukotriene levels or subjective measures of clinical improvement like borg dyspnea score. there were no set criteria for discharging patients, and this could have affected the results regarding the length of hospital stay. as our study was a single center study on a localized population in south india, a larger multicenter study is required to generalize these results to a larger population. addition of oral zileuton to standard treatment of acute asthma results in significant improvement in lung function and reduction in the need for rescue medications when compared to standard treatment alone. however, addition of montelukast to standard treatment does not improve lung functions significantly but reduces the need for rescue medications. | background : leukotriene modifiers have an established role in the management of chronic asthma but their role in acute asthma is still under evaluation.objective:to study and compare the effects of oral montelukast with oral zileuton in acute asthma.materials and methods : this study included 120 asthmatics and was conducted from september 2012 to march 2014. patients were randomized into three different groups to receive montelukast or zileuton or placebo in addition to standard treatment for asthma exacerbation. peak expiratory flow rate (pefr) values, details of rescue medication and vital signs were recorded at 6 h, 12 h, 24 h, and 48 h of drug or placebo administration and at discharge. additional recording was done in the morning (810 am) following admission. the primary endpoint was the mean pefr of each group at these time points ; the secondary end point being the need for rescue medications.results:the mean pefr recordings of the three study groups placebo, montelukast, and zileuton respectively, at various time points were as follows : at 6 h (223.25 90.40, 199.00 82.52, 233.75 84.05 ; p = 0.240) ; at 12 h (271.00 109.38, 251.50 101.44, 309.50 129.63 ; p = 0.048) ; at 24 h (288.25 114.26, 269.00 107.51, 324.50 127.88 ; p = 0.080) ; and at 48 h (295.00 114.80, 293.50 113.24, 344.75 119.91 ; p = 0.015) ; discharge (305.00 118.56, 305.25 119.51, 361.25 119.70 ; p = 0.010). the mean pefr for the three study groups at 810 am on the morning following admission was 268.75 111.43, 252.50 99.99, 306.75 114.44 ; p = 0.047. total rescue doses needed were 10, 1, and 0, respectively (p = 0.049).conclusion : zileuton is better than montelukast as an additional drug in acute asthma and results in significant improvement in lung function, and reduction in the need for rescue medications. |
the term epigenetics was coined some 70 years ago by sir conrad waddington, who theorized the existence of a necessary layer of molecular complexity beyond the genome that must be responsible for producing distinct and variable cellular phenotypes from a singular genome. instincts and genetic programs as useful conceptualizations of the ontogeny of behavior and, importantly, have initiated an appreciation of the multitude of complex influences on phenotypic expression throughout development. epigenetics in its current formulation is more narrowly defined as the perpetuation of genetic information from a cell to its descendants without any necessary change to the genetic code itself, and has been posited as a molecular bridge between the information contained in the genotype and what emerges as a complex and evermodifiable phenotype. the revolution in molecular biology that began in the 1950s, following shortly after waddington 's theoretical formulation of epigenetics, has provided scientists with tools capable of characterizing and understanding the mechanisms that comprise this layer of complexity beyond the genome (ie, the epigenome). dna exists in a continuum of variably compacted states controlled by the structural state of chromatin, ie, the dna and the histone proteins around which it is wrapped. alterations to the structural state of the chromatin can have profound and persistent effects on gene expression. simply put, by establishing and maintaining the structural state of the chromatin, epigenetic processes regulate the ease in which transcription factors and other proteins can access their dna substrates. for example, the amino acid tails of histone proteins are subjected to various post - translational modifications (eg, acetylation, methylation, phosphorylation) that render the chromatin relatively compact and transcriptionally inactive (ie, heterochromatin) or less compact and transcriptionally active (ie, euchromatin). methylation at the 5-position of cytosine nucleotides within cpg dinucelotides is the only direct epigenetic modification of dna and is associated with transcriptional silencing. epigenetic processes have long been recognized as indispensable for appropriate embryonic and early postnatal development. more recently it has come to light that these same mechanisms that drive critical processes in development and in mitotic cells throughout the lifespan remain dynamic in neurons that, once differentiated, are incapable of mitosis. describes processes that utilize the same mechanisms classically defined as epigenetic, but are clearly for functionally distinct purposes. moreover, there is now recognition that dna methylation itself, once thought to be the most stable of epigenetic marks, may switch between methylated and unmethylated states that render stretches of the chromatin a dynamic canvas on which epigenetic and other mechanisms can work to promote forms of plasticity necessary for long - term information storage. the hypothesis that the processes underlying stable transmission of chromatin states in dividing cells remain active in neurons for the purpose of long - term information storage is gaining significant traction and interest within neuroscience, and a better understanding of these processes is necessary for a more complete conceptualization of neural plasticity and memory. alterations in the structural state of chromatin appear to be highly conserved mechanisms underlying information storage in invertebrate (eg, crab, honeybee) and vertebrate (eg, rat, mouse, human) central nervous systems. the primary focus of the current review is to highlight the accumulating data suggesting that dynamic dna methylation and demethylation, and the enzymes responsible for methylating and demethylating dna, are critically involved in memory formation and behavioral plasticity. while the recognition that the structures of chromatin and dna are rapidly modifiable in the brain adds a significant amount of complexity to our understanding of behavioral and neuronal plasticity, it also suggests a heretofore largely untapped therapeutic potential for alleviating a wide range of neurological, and other, disorders. dna methylation plays an essential role in several developmental processes (eg, genomic imprinting, x - chromosome inactivation), in the maintenance of genome stability by silencing repetitive elements, and in maintaining tissue - specific and appropriate patterns of gene expression through cell division. during embryonic and early postnatal development coordinated waves of methylation and demethylation ensure temporally specific patterns of gene expression that act to establish and perpetuate tissue appropriate cellular identities. once established in somatic cells, methylation patterns have traditionally been considered immutable. a seminal study in 2004 by meaney and colleagues showed that variable early postnatal levels of maternal care (eg, nursing, grooming) could alter dna methylation patterns in neurons and that these alterations persisted into adulthood and influenced behavioral and neural responses to stress. methylation of a cytosine nucleotide (5mc) is a thermodynamically very stable modification that is endowed with robust power to influence gene expression. for example, methylation of a single site in a brain - derived neurotrophic factor (bdnf) exon promoter can silence the gene. transcriptional silencing is thought to occur via one of two nonmutually exclusive mechanisms ; 5mc can physically restrict transcription factor and rna polymerase ii binding, or 5mc can recruit transcriptional repressor protein complexes. until recently, it was believed that 5mc occurred only in the context of cg dinucleotides ; however, new findings have demonstrated the existence of substantial levels of mch methylation where h represents an a, t, or c. like 5mc, mch is depleted in expressed genes and inversely proportional to the level of expressed transcript. the enzymes responsible for catalyzing the transfer of a methyl group to cytosine nucleotides, using dietary sources of s - adenosyl - l - methoinine as the methyl donor, are the dna methyltransferases (dnmts) and are broadly subdivided into two categories : the de novo dnmts, dnmt3a and dnmt3b, establish initial methylation patterns on unmethlyated dna ; and the maintenance dnmt, dnmt1, recreates already established methylation patterns on hemimethylated replicating dna. dnmts are essential to normal development as evidenced by the embryonic or early postnatal lethality of constitutive knockout of dnmt1 or dnmt3a. dnmt1, dnmt3a, and dnmt3b are all expressed in the postnatal developing rat brain. dnmt1 and dnmt3a are expressed in adult neurons and oligodendrocytes, and dnmt3b expression is detectable as well, although not to the extent of dnmt3a. dnmt mrna generally reaches its highest level at around 1 week postnatal and subsequently decreases in brain. conditional brain - specific dnmt knockouts have yielded insights into the roles of the dnmts in the central nervous system. a conditional dnmt1 knockout induced during embryonic development using the cre - lox system upregulated apoptotic genes and led to degeneration of the cortex and hippocampus, abnormal morphology of dendrites, and alterations in the resting electrophysiological properties of neurons. the conditional forebrain knockout mice survived to adulthood but, not surprisingly, evidenced severe learning and memory impairments, as they failed to show any learning curve in a spatial memory test following 13 days of training. another study that assayed neurological phenotypes in conditional dnmt1 knockout mice in which the knockout occurred at embryonic day 12 (e12) reported that dnmt1 deficiency led to hypomethylation in differentiated neurons and apoptosis of neurons prior to postnatal day 21 in mosaic animals. dnmt3a null mice are essentially normal at birth, but quickly deteriorate and die in early postnatal development. these mice exhibit impaired postnatal neurogenesis accompanied by dramatic alterations in gene expression profiles in neural stem cells with 1253 genes upregulated and 1022 downregulated, effects likely mediated by impaired polycomb repression of neurogenic genes. gene - specific dna methylation as well as neuronal expression of dnmt enzymes, fluctuates as a result of experiences ranging from intake of drugs of abuse, associative and nonassociative learning experiences, cellular insults, and prenatal stressors. furthermore, in several brain pathologies the expression of the dnmts, as well as methylation of specific gene promoters, appears aberrant. endres reported that an increase in dna methylation was associated with more robust brain lesions following induction of stroke using the middle cerebral artery occlusion (mcao) model. treatment with the nonspecific dnmt inhibitor 5-aza-2'-deoxycytodine (5aza) or mice heterozygous for dnmt evidenced a reduction in neuronal damage following mcao. a more recent study in gerbils determined that 5 minutes of ischemia induced by bilateral common carotid artery occlusion (2vo) significantly upregulated dnmt1 expression specifically in hippocampal ca1 gabaergic neurons as well as in astrocytes 4 days after the occlusion. ninety days of chronic brain hypoperfusion induced by 2vo promoted a decrease in global dna methylation accompanied by a decrease in expression of dnmt3a in parietal lobe cortex with no change in dnmt1 expression. these findings suggest that acute vs chronic ischemic insults may differentially affect dna methylation and expression of de novo and maintenance dnmts in adult brain, and that targeted dnmt inhibition may offer some therapeutic potential in restricting or preventing brain damage invoked by a cerebrovascular accident. temporal lobe epilepsy is associated with aberrant dna methylation of specific genes as well as increased expression patterns of dnmt isoforms in postmortem tissue from human epileptics and in animal models of the disorder. studies using postmortem tissue from patients with intractable epilepsy demonstrate that dnmt1 and dnmt3a protein expression are robustly increased in hippocampal tissue from epileptics, and hypermethylation of the reelin gene promoter, presumably mediated by a dnmt, has been reported. using a rat model, parrish recently found that kainic acidinduced epileptic activity in the hippocampus led to increased global dna methylation in the ca1 and ca3 and decreased methylation in the dentate gyrus 6 weeks after kainate treatment. interestingly, distinct patterns of dnmt1 and dnmt3a expression were observed in hippocampal subregions immediately after (1 hour) and 6 weeks after induction of epilepsy. decreased expression of dnmt3a persisted 6 weeks after induced seizure, whereas immediate decreases in dnmt1 expression normalized by 6 weeks. importantly, intrahippocampal treatment with the nonspecific dnmt inhibitor zebularine decreased the latency to seizure onset and prevented the changes in global methylation and promoter methylation of the grin2b / nr2b, a gene known to play a role in epilepsy. collectively, the results from ischemic and epileptic models suggest that wide - ranging insults can influence dna methylation in brain. in the case of epilepsy it is not yet clear if the changes in expression are involved in the etiology of epilepsy or result from the pathology. these findings nevertheless demonstrate that genomic methylation is indeed plastic and likely plays a key role in neurological disorders and neurological responses to insult. prenatal stressors as well as stress paradigms administered in adulthood have repeatedly been shown to influence neuronal dna methylation. indeed, differential methylation of the corticotropin - releasing factor gene promoter follows maternal deprivation stress, prenatal stress, and chronic mild stress. variation in the diet of mice during gestation or later in development can alter the methylation status of dna in a persistent fashion. an increase in dietary l - methionine or treatment with the nonspecific histone deacetylase inhibitor trichostatin a (tsa) reverses the effects of poor maternal care behavior on dna methylation and hypothalamic - pituitary - adrenal axis, and behavioral responses to stress, providing further evidence that dna methylation marks in neurons are modifiable. exposure to a cat increases bdnf gene methylation in the dorsal hippocampus in rats, while simultaneously favoring a decrease in methylation of the bdnf gene in the ventral hippocampus with no change in bdnf methylation in the basolateral amygdala or the prefrontal cortex. the functional significance of bidirectional methylation of the same gene in different brain regions, or how this is mediated, is not yet clear. prenatal exposure to the environmental toxin methyl mercury hypermethylates the bdnf promoter region in the hippocampal dentate gyrus, with an accompanying hypoacetylation of histone h3 and a decrease in bdnf mrna expression. these epigenetic alterations are associated with increased depressive - like behavior in adulthood, as assessed by the forced swim test. interestingly, bdnf expression in the hippocampal dentate gyrus is necessary for antidepressant efficacy in the forced swim test, thus suggesting a mechanism whereby various stressors experienced in the intrauterine environment may relate to deleterious behavioral phenotypes in adult animals. tian reported that conditioned place preference to cocaine led to global methylation in the prefrontal cortex, and that methionine supplementation prevented the establishment of conditioned place preference for cocaine, but not morphine or food reward. bodetto have confirmed a role for dna methylation in mediating the rewarding effects of cocaine. in their study, cocaine increased dna methylation at the protein phosphatase 1 (pp1) gene promoter, a memory suppressor gene, and increased expression of dnmt3a. dnmt3a overexpression specifically in the nucleus accumbens, a brain region often implicated in behavioral responses to drugs of abuse, has been shown to attenuate cocaine reward and increase dendritic spine density of thin spines in nucleus accumbens neurons. by contrast, nucleus accumbens - specific knockout of dnmt3a potentiated conditioned place preference for cocaine. acute vs chronic cocaine use has opposite effects on dnmt3a expression in nucleus accumbens as acute treatment increases, whereas chronic treatment decreases, dnmt3a expression in the accumbens. administration of the dnmt inhibitor rg108 blocks cocaine 's effect on spine density in the nucleus accumbens and enhances conditioned place preference for cocaine. ethanol exposure during all 3 trimesters of embryonic development similarly has been shown to upregulate the expression of dnmt3a as well as dnmt1 and the methyl - binding protein methyl - cpg - binding protein 2 (mecp2) in the hippocampus. importantly, cellular insults, prenatal stressors, or exposure to aversive stimuli are not the only experience - driven changes in dna methylation or dnmt expression. single running wheel exercise sessions or week - long access to a running wheel, known to be a rewarding activity in rodents, demethylate the bdnf exon iv promoter, increase bdnf mrna and protein in the hippocampus of sprague - dawley rats, and can elevate levels of phosphorylated mecp2, and subsequently silence the associated gene. phosphorylation of mecp2 can lead to its dissociation from chromatin, which may favor transcriptional activation of bdnf. single exercise sessions decreased dnmt3b and dnmt1 in hippocampus in young, but not old, rats. interestingly, long - term exercise is associated with improved learning in rodents and humans, and in enhanced hippocampal plasticity in rodent models, an effect that may be related to the increases in bdnf. in summary, highly variable and biologically relevant environmental experiences appear to alter the methylation state of specific regions of the genome and promote increases or decreases in the associated mrna and protein. long thought to be a paragon of biological stability, gene methylation, at least in the brain, may be a rather dynamic process that is altered as a result of environmental input. broadly speaking, epigenetic processes have been implicated in behavioral adaptations that rely on associative and nonassociative learning processes as well as in the subsequent storage of putative memory traces in the central nervous system. the notion that long - term memories are encoded in the methylation state of the dna was initially proposed by griffith and mahler in a theoretical paper published in nature (the dna ticketing theory of memory), in 1969. they proposed that the physical basis of memory could lie in the enzymatic modification of the dna of nerve cells. the events in the nervous system that are necessary for the formation of long - term memories are complex and not completely understood. memory formation requires the orchestration of precise and temporally coordinated changes in gene expression, transcription factor activation and inactivation, and bidirectional changes in the expression and activity of chromatin and dna modifying enzymes. these molecular events coalesce to produce de novo changes in the synaptic strength and connectivity within specific circuitry underlying the formation and long - term storage of new information. moreover, the specific neural pattern responsible for the storage of the memory is retrievable in the presence or absence of the stimuli that promoted its formation. importantly, the memory trace must be self - perpetuating, must persist in spite of the continual turnover of molecules involved in its genesis, and can potentially last the lifetime of an organism. the idea that dynamic changes in dna methylation are necessary for long - term memory formation was first given empirical support by sweatt and colleagues. initially the sweatt laboratory reported that treatment with nonspecific dnmt inhibitors impaired the formation of contextual fear associations. a follow - up study found that experience in associative fear learning to context, a paradigm in which an animal is exposed to contiguous presentations of a novel and initially innocuous environmental context paired with an aversive footshock, could rapidly (ie, within 30 minutes) increase the methylation of the memory suppressor gene protein phosphatase 1 (pp1), while concurrently demethylating the promoter region of the plasticity - related gene reelin. experience in fear learning upregulated the expression of dnmt3a and dnmt3b in areas of the brain necessary for learning (eg, hippocampus), with no effect on dnmt1. moreover, nonspecific inhibition of dnmts impaired memory and prevented the methylation of pp1, while enhancing the demethylation of reelin. following training, the methylation of reelin and pp1 returned to normal within 24 hours, leading the authors to conclude that dna methylation, while critical for memory formation, is not likely to be a mechanism of longterm storage, at least in the hippocampus. further studies have expanded on these initial findings and implicated methylation of the bdnf gene in associative fear learning. experience in a fear learning paradigm demethylates the bdnf exon iii and exon iv promoters in the hippocampus, and these effects are blocked by application of the nmda receptor antagonist mk801, indicating that they are activity - driven. in accordance with the aforementioned miller and sweatt study, the effects on methylation of bdnf in the hippocampus were relatively transient and observable 30 minutes and 24 hours after training. although methylation changes in the hippocampus are relatively transient, methylation of the memory suppressor calcineurin is increased in the prefrontal cortex 7 days after fear conditioning, and this hypermethylation migrates to the anterior cingulate cortex as long as 1 month following initial training. infusion of dnmt inhibitors directly into the anterior cingulate cortex blocks memory retrieval 30 days after training. these data are consistent with the known roles of these brain regions in memory formation vs storage, and suggest that transient and long - lasting methylation / demethylation in distinct brain circuits is important in establishing long - term memory of fearful stimuli. day have demonstrated that the importance of dna methylation / demethylation in memory formation is not restricted to aversive events (eg, fear conditioning, morris water maze). using a cued - sucrose delivery associative reward learning paradigm, the authors found that experience in the learning task increased the expression of the immediate early genes erg1 and c - fos, which were demethylated following learning. in a neuronal culture preparation, kc1 depolarization did not change dnmt3a or dnmt3b expression ; however, it did increase dnmt3a binding at the genomic sites (ergl and c - fos) that underwent de novo methylation in the in vivo reward - learning experiments. dnmt inhibition before kc1 treatment prevented depolarization - induced changes in dna methylation, and pharmacological inhibition of dnmts in the ventral tegmental area in vivo blocked reward learning without influencing motivation in general. interestingly, treatment with dnmt inhibitors can prevent the memory enhancing effects induced by other compounds, as dnmt inhibition has been shown to prevent estrogen - induced improvements in memory. estrogen treatments increase the hippocampal expression of dnmt3a and dnmt3b, but not dnmt1. using mice with conditional forebrain - specific double knockout of dnmt1 and dnmt3a in neurons, feng reported learning and ltp deficits that were not apparent with a single knockout of either dnmt1 or dnmt3a. therefore, although experience in associative learning tasks and other stimuli appear to differentially affect de novo vs maintenance dnmt expression, it seems that each of these distinct isoforms can compensate for the lack of the other. the culmination of the molecular events that promote long - term memory formation leads to structural changes at synapses in brain - region specific circuits that underlie learning and memory. therefore, it is not surprising that epigenetic modifications of chromatin have been implicated in regulating the forms of synapse plasticity believed to establish memory. initial studies implicating epigenetic processes in synaptic plasticity focused on histone modifications such as acetylation and deacetylation. subsequently, manipulation of dnmts in dissociated neuronal cultures and in acute brain slice experiments have implicated the process underlying addition or removal of 5mc in regulating basal neuronal function as well as plasticity within brain circuits. levenson found that treatment of a hippocampal slice preparation with the dnmt inhibitors 5aza or zebularine impaired the magnitude of long - term potentiation (ltp) at the schaeffer collateral - ca1 pathway. ltp, widely believed to be a cellular correlate of learning, is typically induced via electrical stimulation of brain slices with robust high - frequency stimuli, which causes a demonstrable increase in synaptic responses subsequent to the high - frequency induction protocol. -burst stimulation, a physiologically relevant means of inducing ltp, led to robust and enduring potentiation (3 hours) in vehicle - treated slices ; however, treatment with either dnmt inhibitor impaired ltp magnitude and maintenance. in a follow - up study, it was shown that dnmt - inhibitor induced ltp deficits were rescued by slice application of the histone deacetylase inhibitor sodium butyrate, suggesting a crosstalk between dna methylation and histone acetylation in the regulation of hippocampal synaptic plasticity. in accordance with the observation that nonspecific dnmt inhibition impairs ltp magnitude and maintenance, feng have shown that forebrain - specific conditional double knockout of both dnmt1 and dnmt3a led to similar ltp impairments. somewhat surprisingly, no effects on synaptic function were observed in dnmt1 or dnmt3a single knockouts. depolarization of hippocampal neuron cultures with 50 mm kc1 downregulates dnmt1 and dnmt3a expression, an effect that is prevented by the sodium - channel blockers tetrodotoxin and veratridine. kc1-induced increases in neuronal activity demethylate the regulatory region of the bdnf exon iv promoter, and promote the dissociation of a corepressor complex composed of mecp2, histone deacetylases, and sin3a from the bdnf promoter. bdnf has been broadly implicated in neuronal viability, synaptic plasticity, synaptogenesis, and memory, suggesting important activity - dependent functional consequences of bdnf demethylation. although basal synapse function is normal in acute hippocampal slices treated with dnmt inhibitors, dnmt inhibitors administered to dissociated hippocampal neuron cultures decrease the frequency of spontaneous miniature excitatory postsynaptic currents (mepscs) and demethylate the bdnf i promoter. these effects are activity - dependent, as the nmda receptor antagonist ap5 prevented bdnf promoter demethylation. although most studies assessing the role of histone modifications and dna methylation on ltp have examined the hippocampus, sui found that high frequency stimulation - induced ltp led to demethylation of the reelin and bdnf gene promoters in the prefrontal cortex and increased acetylation of histone 3 and histone 4, marks of active transcription. dnmt inhibitor treatment impaired ltp in prefrontal cortex and prevented the alterations in histone acetylation. in future experiments it will be interesting to determine if the electrophysiological correlates of memory formation (eg, ltp) migrate from brain regions necessary for memory formation (eg, hippocampus) to areas more involved in memory storage (eg, anterior cingulate cortex, prefrontal cortex) and if such changes require dna methylation status updates within this geography. any role for rapid dna methylation / demethylation in memory formation necessitates the existence of an active mechanism for removing 5mc from specific genes involved in plasticity and memory. in other words there switch on specific cytosine nucleotides that is responsive to environmental contingencies that promote associative and nonassociative forms of learning. among the strongest candidates as molecular agents of demethylation tet1, tet2, and tet.3 are known to be bona fide mediators of 5mc demethylation in plants, and in mammalian tissue exhibit a strong preference for cpg - rich motifs. the pathway responsible for conversion of 5mc to cytosine is thought to involve successive oxidation of 5mc to 5-hydroxymethylcytosine (5hmc) to 5-formylcytosine (5fc) to 5-carboxylcytosine (5cac). the presence of 5hmc in the brain is significantly diminished when tet proteins are inhibited. all three members of the tet protein family are capable of converting 5mc to 5hmc as well as subsequent oxidation of 5hmc to 5fc and 5cac. the modified bases can then be further subjected to deamination, glycosylation, and base excision repair to result in final conversion back to a cytosine base. reconversion back to cytosine may require the activity of base - excision repair mechanisms. 5hmc can be deaminated to 5hmu by activation - induced deaminase (aid), with subsequent removal of 5hmu by thymine dna glycosylase (tdg), methyl - binding domain protein 4 (mdb4), and single strand - specific monofunctional uracil dna glycosylase 1 (smug1). thymidine glycosylase can also directly target 5fc and 5cac ; however, any in vivo role of tdg in demethylation remains unclear. interestingly, it was recently shown that dnmts may also act as demethylases capable of converting 5hmc to c, possibly by a direct interaction with tdg. methyl - binding domain protein 2 (mdb2) can directly demethylate dna containing 5mc by a reaction that releases formaldehyde. guo have demonstrated activity - dependent demethylation of two plasticity - related genes, fibroblast growth factor 1 (fgf1) and bdnf following electrical stimuli capable of inducing epileptiform activity. in the hippocampus, mice with reduced levels of tet1 were incapable of demethylating bdnf and fgf genes following seizure - inducing stimuli. tet1 knockout mice exhibit downregulated expression of the neuronal activity - related genes npas4, c - fos, and arc and a reduction in 5hmc levels in the hippocampus and cortex with no change in 5mc. tet1 knockouts develop normally without any observable brain abnormalities, which may suggest that tet2 or tet3 can compensate for loss of tet1. tet1 knockouts also have impaired short - term spatial memory formation and abnormal neuro - genesis in neural precursor cells, but do not appear to have robust long - term memory impairments. the knockouts surprisingly have abnormally enhanced long - term depression elicited in the hippocampus and an impaired ability to extinguish previously acquired associative memories (ie, extinction). expression of several plasticity - related genes were shown to be diminished, however, including c - fos, arc, npas4, and erg2 in hippocampus, likely resulting from hypermethylation of those genes. tet1 knockout elevated promoter methylation of 478 genes and decreased methylation in 38 genes, with an overlap of 39 that were both hypermethylated and downregulated. by contrast, hippocampaltargeted overexpression of tet1 upregulated memory - associated genes including c - fos, arc, erg1, homer1, and nr4a2 and impaired contextual fear learning. interestingly, overexpression of a catalytically inactive form of tet1 also increased expression of those genes and impaired fear learning suggesting demethylase - dependent and -independent effects on learning and gene expression. recent studies have also characterized the role of other potential demethylation factors in brain function. the growth arrest and dna - inducible 45 (gadd45) family of enzymes may bind to and focus the enzymatic activity of cytidine deaminases and thymidine glycosylases to specific gene promoters, thereby tagging specific genes for active demethylation. ma suggested that neuronal activity may focus base excision repair mechanisms to cpg promoters and this may be mediated by gadd45 enzymes. leach reported that gadd45b knockout mice are impaired in contextual fear conditioning, whereas sultan and coworkers found improvements in contextual fear learning at 24 hours and 28 days post - training, and an enhanced late - phase ltp. the reasons behind the contrasting results are not yet clear ; however, in both cases the studies suggest that manipulations of putative demethylases can alter normal learning and memory. any role for rapid dna methylation / demethylation in memory formation necessitates the existence of an active mechanism for removing 5mc from specific genes involved in plasticity and memory. in other words there must exist a switch on specific cytosine nucleotides that is responsive to environmental contingencies that promote associative and nonassociative forms of learning. among the strongest candidates as molecular agents of demethylation tet1, tet2, and tet.3 are known to be bona fide mediators of 5mc demethylation in plants, and in mammalian tissue exhibit a strong preference for cpg - rich motifs. the pathway responsible for conversion of 5mc to cytosine is thought to involve successive oxidation of 5mc to 5-hydroxymethylcytosine (5hmc) to 5-formylcytosine (5fc) to 5-carboxylcytosine (5cac). the presence of 5hmc in the brain is significantly diminished when tet proteins are inhibited. all three members of the tet protein family are capable of converting 5mc to 5hmc as well as subsequent oxidation of 5hmc to 5fc and 5cac. the modified bases can then be further subjected to deamination, glycosylation, and base excision repair to result in final conversion back to a cytosine base. reconversion back to cytosine may require the activity of base - excision repair mechanisms. 5hmc can be deaminated to 5hmu by activation - induced deaminase (aid), with subsequent removal of 5hmu by thymine dna glycosylase (tdg), methyl - binding domain protein 4 (mdb4), and single strand - specific monofunctional uracil dna glycosylase 1 (smug1). thymidine glycosylase can also directly target 5fc and 5cac ; however, any in vivo role of tdg in demethylation remains unclear. interestingly, it was recently shown that dnmts may also act as demethylases capable of converting 5hmc to c, possibly by a direct interaction with tdg. methyl - binding domain protein 2 (mdb2) can directly demethylate dna containing 5mc by a reaction that releases formaldehyde. guo have demonstrated activity - dependent demethylation of two plasticity - related genes, fibroblast growth factor 1 (fgf1) and bdnf following electrical stimuli capable of inducing epileptiform activity. in the hippocampus, mice with reduced levels of tet1 were incapable of demethylating bdnf and fgf genes following seizure - inducing stimuli. tet1 knockout mice exhibit downregulated expression of the neuronal activity - related genes npas4, c - fos, and arc and a reduction in 5hmc levels in the hippocampus and cortex with no change in 5mc. tet1 knockouts develop normally without any observable brain abnormalities, which may suggest that tet2 or tet3 can compensate for loss of tet1. tet1 knockouts also have impaired short - term spatial memory formation and abnormal neuro - genesis in neural precursor cells, but do not appear to have robust long - term memory impairments. the knockouts surprisingly have abnormally enhanced long - term depression elicited in the hippocampus and an impaired ability to extinguish previously acquired associative memories (ie, extinction). expression of several plasticity - related genes were shown to be diminished, however, including c - fos, arc, npas4, and erg2 in hippocampus, likely resulting from hypermethylation of those genes. tet1 knockout elevated promoter methylation of 478 genes and decreased methylation in 38 genes, with an overlap of 39 that were both hypermethylated and downregulated. by contrast, hippocampaltargeted overexpression of tet1 upregulated memory - associated genes including c - fos, arc, erg1, homer1, and nr4a2 and impaired contextual fear learning. interestingly, overexpression of a catalytically inactive form of tet1 also increased expression of those genes and impaired fear learning suggesting demethylase - dependent and -independent effects on learning and gene expression. recent studies have also characterized the role of other potential demethylation factors in brain function. the growth arrest and dna - inducible 45 (gadd45) family of enzymes may bind to and focus the enzymatic activity of cytidine deaminases and thymidine glycosylases to specific gene promoters, thereby tagging specific genes for active demethylation. ma suggested that neuronal activity may focus base excision repair mechanisms to cpg promoters and this may be mediated by gadd45 enzymes. leach reported that gadd45b knockout mice are impaired in contextual fear conditioning, whereas sultan and coworkers found improvements in contextual fear learning at 24 hours and 28 days post - training, and an enhanced late - phase ltp. the reasons behind the contrasting results are not yet clear ; however, in both cases the studies suggest that manipulations of putative demethylases can alter normal learning and memory. the brain exhibits dynamic patterns of dna methylation and dnmt expression during aging, and the transcription of key memory - related genes declines in aging. consistent with this observation is that in aged animals impairments in ltp magnitude and maintenance are observed when weak ltp induction protocols (ie, near - threshold) are used. siegmund have shown changing patterns of dna methylation patterns at various gene loci in human brain, with a general trend for increasing methylation over the lifespan in the 50 loci examined. inappropriate methylation of the activity - related cytoskeleton - associated protein (arc), a known actor in synaptic plasticity and memory, may play a role in age - related memory impairments. old rats have higher levels of methylation of the arc promoter than do adult rats, and the transcription of arc is reduced in the aged hippocampus after learning events relative to younger animals. in addition, inhibition of arc interferes with maintenance of ltp, likely due to its role in synaptic -amino-3-hydroxy5-methyl-4 isoxazolepropionic acid (ampa) receptor trafficking. oliviera have demonstrated an aging - associated decrease in the expression of dnmt3a2, one of two transcripts from the dnmt3a gene, in the hippocampus of aged mice. dnmt3a2 is structurally identical to dnmt3al except it lacks 219 amino acids in the n- terminus, is associated with euchromatin, and appears to act as an immediate early gene. learninginduced activation of dnmt3a2 was shown to be impaired in aged mice and overexpression of dnmt3a2, which increased global dna methylation in the hippocampus, improved performance in fear learning and object location memory tasks. specific gene methylation alterations have been shown in postmortem brain tissue from alzheimer 's disease patients, and these patients exhibit an accelerated rate of age - related change in methylation. alzheimer's - related alterations in dna methylation may be complex and vary by brain region, as global hypomethylation has been shown in entorhinal cortex in postmortem alzheimer 's disease tissue, as well as hypermethylation in the dorsolateral prefrontal cortex. debilitating psychiatric disorders including schizophrenia, bipolar disorder, and major depressive disorder have been linked to aberrant dna methylation. mill and coworkers examined genomic dna from 125 postmortem brains of schizophrenics, bipolar, and nonpsychiatric patients and concluded that dna methylation is significantly altered in major psychiatric disorders. the reelin and gad1 promoter regions are hypermethylated in the brains of schizophrenic patients, and dnmt1 expression is upregulated. the gad1 gene may be of particular interest as it codes for glutamic acid decarboxylase, the enzyme responsible for synthesis of -aminobutyric acid (gaba), the major inhibitory neurotransmitter in the brain. noh demonstrated that an antisense - driven knock - down of dnmt1 in mouse cortical neuron cultures was accompanied by an increase in reelin expression and suggest that reduced reelin and gad67 protein may be due to dnmt1-mediated hypermethylation of their promoters. treatment with the histone deacetylase inhibitors tsa or valproate can decrease gad1 promoter methylation, decrease dnmt1 expression in mouse cortex, and increase the expression of reelin and gad67. increased reelin and gad67 expression were associated with the dissociation of mecp2containing corepressor complexes from their promoter regions. deficits in the ability to inhibit a startle response to an auditory stimulus when that stimulus is preceded by a smaller magnitude auditory stimulus are observed in human schizophrenic patients as well as in animal models of schizophrenia. our laboratory has recently found a dissociable impact of conditional forebrain knockout of dnmt1 and dnmt3a on prepulse inhibition in mice (unpublished data). dnmt1 knockout mice showed an enhanced inhibition of their startle response at all prepulse stimulus magnitudes tested, effects in opposition to the impairments in prepulse inhibition observed in schizophrenia models. significant stressors experienced in the prenatal environment may predispose an individual to the development of a psychiatric disorder in adulthood. restraint stress experienced by a pregnant mouse leads to increases in dnmt1 and mecp2 binding at the reelin and gad1 promoter regions, changes that resemble those observed in postmortem samples from schizophrenic brain. polymorphisms in the dnmt3b gene were recently found to be associated with suicide attempts in depressed patients. mcgowan reported increased methylation of the glucocorticoid receptor gene in postmortem brain tissue from suicide victims that had experienced child abuse, findings that are consistent with the behavioral abnormalities observed in animal models of insufficient postnatal care. overexpression of dnmt3a in the nucleus accumbens has been demonstrated to induce depressive - like behavior in mice, whereas inhibition of dnmts in the nucleus accumbens has antidepressantlike effects in a chronic social defeat model as well as in the forced swim test. one hurdle in the way of a better understanding of the role of dna methylation, and chromatin modification in general, in memory is the robust and dynamic interplay between the various enzymes and proteins capable of altering the chromatin. 5mc, by poorly understood signaling pathways, is able to recruit methyl - binding proteins and subsequently large chromatin remodeling complexes that are believed to stably mark stretches of the genome. the methyl - binding protein mecp2 recognizes single 5mc sites and is thought to further recruit transcriptional compressor complexes. interestingly, approximately 95% of cases of the neurodevelopmental mental retardation syndrome rett syndrome are caused by mutations of mecp2, with some similar phenotypes apparent in cases of mecp2 duplication syndrome, which involves a duplication of the xq28 chromosomal region harboring mecp2. using a mouse model of mecp2 duplication syndrome, our laboratory has shown that a 50% increase in mecp2 in brain promotes motor coordination deficits, anxiety, learning and memory, and ltp impairments. therefore, the molecular events proceeding from dna methylation followed by binding of mecp2 appear to be of critical importance for cognitive function and synaptic plasticity. dnmt inhibition can impair memory formation as well as ltp in hippocampal slices, and these effects are reversed by treatment with nonspecific histone deacetylase inhibitors. drugs that promote the acetylation of histones may facilitate the loosening of chromatin by leading to the release of methyl - binding proteins (eg, mecp2). our laboratory has shown that pharmacological inhibition of dnmts in cultured hippocampal neurons decreases mepscs, however this effect is occluded in mecp2 knockout neurons. another large gap remaining in the pursuit of a more complete understanding of the role of dna methylation in memory formation is determining the mechanisms both upstream and downstream of the epigenetic alterations as well as how individual epigenetic modifiers are activated in distinct environmental and cellular contexts. for instance how are dnmts or tet proteins directed in a sequence - specific manner ? although the factors that guide a dnmt to a specific methylated site are not known for certain, it is believed that interactions with transcription factors and other chromatin proteins play a critical role. heterochromatin is characterized by distinct epigenetic marks, eg, methylated lysines 9 (h3k9) and 27 (h3k27), and dna cpg methylation, which are associated with further recruitment of methyl - binding proteins, histone deacetylases, and other proteins. these histone modifications and the enzymes that catalyze their formation are known to interact with dnmts and methyl - binding proteins. for example, the histone protein hid recruits dnmt1 and dnmt3b, and the histone methyltransferases suv39h1 and g9a recruit dnmt3a. it has previously been shown that the mir-29 family of micro rnas is capable of targeting dnmt3a, dnmt3b, and the tet proteins, potentially establishing a balance between methylation and demethylation at specific genomic targets. vire have highlighted the importance of polycomb group proteins as links between the methylation of histones and dna methylation. trimethylation of histone 3 at lysine 9 (h3k9) and h4k20 appears to be a prerequisite for subsequent methylation of a gene. following h3k9 binding, heterochromatin protein 1 (hp1) can associate with dnmt3a by direct binding to its plant homeodomain (phd) motif. how dynamic are chromatin states and the molecular agents that confer specific states resulting from environmental experience ? are there distinct roles for different dnmt or tet isoforms in methylation and demethylation in specific tissues or in response to distinct signals ? in spite of the inherent complexity of the epigenome, great strides are being made to determine its role in the central nervous system. these advances are, and will continue to be, essential for understanding diverse processes including memory formation, responsiveness to stressors and neurological insults, and the etiology of psychiatric disorders. | dynamic regulation of chromatin structure in postmitotic neurons plays an important role in learning and memory. methylation of cytosine nucleotides has historically been considered the strongest and least modifiable of epigenetic marks. accumulating recent data suggest that rapid and dynamic methylation and demethylation of specific genes in the brain may play a fundamental role in learning, memory formation, and behavioral plasticity. the current review focuses on the emergence of data that support the role of dna methylation and demethylation, and its molecular mediators in memory formation. |
collagen cross - linking (cxl) has been employed for many years as a means of stabilizing cornea ectasia [1, 2 ]. among the multitude of treatments and technique variations applied, cxl has been well documented to almost invariably result in some central anterior corneal flattening [3, 4, 5, 6, 7 ]. disease regression. we present herein a custom application with a novel device, enabling the topographically customizable administration of very high - fluence cxl, applied in a specific toric pattern, in order to achieve, besides ectasia stabilization, predictable corneal astigmatic changes. this study received approval by the ethics committee of our institution, and adhered to the tenets of the declaration of helsinki. written informed consent we report herein a case of a 6-month postoperative evaluation of an innovative customized toric application of transepithelial cross - linking. the patient was a 37-year - old female with a preoperative clinical diagnosis of progressive moderate keratoconus of the left eye, minimum corneal pachymetry of 450 m, and uncorrected distance visual acuity (udva) between 20/32 and 20/25. the patient was enrolled due to keratoconus progression noted over the previous year. in the past, the patient 's other eye (right) had a more evident keratoconic manifestation and had been treated a few years ago with the athens protocol procedure [8, 9 ]. the left eye was not treated at that time due to the patient 's age and because it was less affected ; however, the progression, in combination with the patient 's desire for quick visual rehabilitation played a role in the decision to treat this keratoconic eye with this novel approach. the riboflavin penetration through the intact epithelium (transepithelial) into the stroma involved a two - step process. the manufacturer cautions against administering paracel for over 4 min to minimize the risk of epithelial sloughing. the second step was the application of vibex xtra (avedro inc.), 0.25% riboflavin isotonic saline solution. application time was 6 min, with a drop every 30 s. with this two - step soaking process, the riboflavin passage to the anterior chamber was approximately complete in 10 min. the kxl ii cross - linking system (avedro inc.) was employed for the uva exposure. following alignment and focusing by the surgeon, the toric treatment pattern and orientation relative to the astigmatic axes were investigated in preclinical computational modeling studies [10, 11 ]. as shown in fig. 1, the specific toric irradiation pattern had a butterfly - wing shape, extending to 5-mm, axis oriented at 129 (orthogonal to the steep axis of astigmatism), with a total energy of 14.0 j / cm applied for 10 min and 22 s. overlapped to this was a circular irradiation area extending to a 6-mm diameter, with a total energy of 4.0 j / cm applied for 2 min and 56 s. the uva power was 45 mw / cm, delivered with a pulsed mode of 1-second on and a 1-second off. during the uva irradiation, the cornea was moistened every couple of minutes with a drop of balanced salt solution. the patient was postoperatively treated with a combination of antibiotic / corticosteroid drops for 10 days. the patient was examined the first day, the first week, and every month for a total duration of 6 months. at each visit, the cornea was assessed with placido topography (topolyzer vario ; wavelight, erlangen, germany) by means of curvature maps and simulated keratometry, scheimpflug imaging (oculyzer ii ; wavelight) by means of curvature maps and simulated keratometry, anterior surface irregularity indices and keratoconus classification, autorefraction and keratometry (nidek ar 1 ; nidek co. ltd., tokyo, japan), anterior segment optical coherence tomography (oct) (rtvue-100 ; optovue inc., usa) providing corneal pachymetry and epithelial pachymetry maps, and endothelial cell counts measurement with a confocal specular microscopy device (cellchek xl ; konan, irvine, calif. the riboflavin penetration through the intact epithelium (transepithelial) into the stroma involved a two - step process. the first step was the application of a paracel solution (avedro inc., waltham, mass., usa), a slightly hypotonic specially formulated 0.25%-riboflavin solution with 0.02% benzalconium chloride. the manufacturer cautions against administering paracel for over 4 min to minimize the risk of epithelial sloughing. the second step was the application of vibex xtra (avedro inc.), 0.25% riboflavin isotonic saline solution. application time was 6 min, with a drop every 30 s. with this two - step soaking process, the riboflavin passage to the anterior chamber was approximately complete in 10 min. the kxl ii cross - linking system (avedro inc.) was employed for the uva exposure. following alignment and focusing by the surgeon, the toric treatment pattern and orientation relative to the astigmatic axes were investigated in preclinical computational modeling studies [10, 11 ]. as shown in fig. 1, the specific toric irradiation pattern had a butterfly - wing shape, extending to 5-mm, axis oriented at 129 (orthogonal to the steep axis of astigmatism), with a total energy of 14.0 j / cm applied for 10 min and 22 s. overlapped to this was a circular irradiation area extending to a 6-mm diameter, with a total energy of 4.0 j / cm applied for 2 min and 56 s. the uva power was 45 mw / cm, delivered with a pulsed mode of 1-second on and a 1-second off. during the uva irradiation, the cornea was moistened every couple of minutes with a drop of balanced salt solution. the patient was postoperatively treated with a combination of antibiotic / corticosteroid drops for 10 days. the patient was examined the first day, the first week, and every month for a total duration of 6 months. at each visit, the cornea was assessed with placido topography (topolyzer vario ; wavelight, erlangen, germany) by means of curvature maps and simulated keratometry, scheimpflug imaging (oculyzer ii ; wavelight) by means of curvature maps and simulated keratometry, anterior surface irregularity indices and keratoconus classification, autorefraction and keratometry (nidek ar 1 ; nidek co. ltd., tokyo, japan), anterior segment optical coherence tomography (oct) (rtvue-100 ; optovue inc., usa) providing corneal pachymetry and epithelial pachymetry maps, and endothelial cell counts measurement with a confocal specular microscopy device (cellchek xl ; konan, irvine, calif. the patient achieved udva of 20/32 at the first postoperative week and 20/25 at 6 months. the patient reported no pain (0 on a scale of 010) and minimal discomfort during the procedure. during the immediate postoperative follow - up visits, endothelial cell counts were stable, preoperatively at 2,430 cells / mm and postoperatively at 2,400 cells / mm. preoperative and postoperative visual acuity, pachymetry and keratometry average data are reported in table 1. 3 illustrate examples of placido disc and scheimpflug imaging sagittal curvature preoperative, 6-month postoperative and difference maps. a significant astigmatic reduction was noted (on average 0.8 d) as measured by all keratometric modalities employed in the study. topography (fig. 3), reaching a maximum of 2.5 d at the steepest preoperative location. based on the scheimpflug imaging (amsler - krumeich criteria) analysis, preoperatively, the cornea was classified for the topographic keratoconus classification of kc1 and the values for the anterior irregularity indices of height decentration (ihd) and surface variance (isv) were 0.039 and 43, respectively (fig. poss (possible kc), while the anterior surface irregularity indices of surface variance and height decentration were lower, with values of 32 and 0.024, respectively, indicating a trend towards a more normalized cornea. 4 illustrates oct - derived corneal and epithelial thickness pachymetry maps preoperatively, at 1-week and 6-month postoperatively. the significant refractive changes as a result of classic cxl [3, 4, 5, 6, 7, 14, 15, 16 ], when applying the dresden protocol (3 mw / cm for 30 min), as well as higher uva fluence, either superficially, via riboflavin placed within a femtosecond laser - created pocket or icrs channels can be established by a number of reports [18, 19 ]. such flattening, which constitutes a refractive effect resulting from differential cross - linking - induced stiffening effects, has been described by a number of clinicians as disease regression. we have reported on the use of higher fluence cxl as an agent for refractive stabilization in high - myopic and hyperopic lasik [20, 21, 22 ], and have also recently reported significant refractive changes in astigmatic keratotomy, when facilitated by the development of a customizable differential uv delivery system by avedro and the ability to perform feasibility studies with the kxl ii device, the current study provides additional evidence for the potential of patterned cxl in the deliberate treatment of refractive error. we have recently reported in vivo, topographically regular and predictable central cornea flattening effects consistent with a correction of myopia of about 2.5 d. the available interim data appear promising with regard to the potential for correcting low myopic refractive errors without tissue removal in an excimer - like fashion or other previously described thermal techniques combined with cxl. myopic, hyperopic and astigmatic corrections are other novel applications that are currently under study with this technology. the novel toric topographically customized application of cross - linking presented herein (photorefractive intrastromal cross - linking) employed preferential corneal flattening along the flat refractive axis. six months postoperatively, a very specific reduction of astigmatism of 0.8 d (reaching up to 2.5 d at certain loci) was observed. the epithelial remodeling maps in the first 6 months, when compared to normal eyes, suggest that there was some modest change in the epithelial distribution, with the treated area having initially a slight reduction in the average epithelial thickness, and recovering at the 6-month follow - up to a more regular distribution than the preoperative distribution. the epithelial data suggest that the achieved refractive effect is not a result of epithelial remodeling, and thus may be attributed to direct stromal remodeling. as shown in the oct pachymetry data illustrated in fig. 4, preoperatively, the difference between the minimum corneal thickness of 503 m to the pupil center corneal thickness of 535 m was 32 m, while 6 months postoperatively, the minimum corneal thickness was 542 m and the center corneal thickness 557 m, with a difference of 15 m. additionally, the procedure has neither an obvious corneal thinning effect nor an apparent haze following treatment. since it is essentially performed through the intact epithelium, the procedure required minimal postoperative adjustment of the daily activities by the patient and produced essentially no pain or discomfort even within the first few hours after the procedure. the rapid recovery and relative safety of transepithelial cxl may additionally offer the possibility to titrate the effect through two or more treatments over time as needed. as the patient is under continuous follow - up, and as more patients are being enrolled for the procedure, we anticipate reporting on the stability of the procedure, as well as on further refinement of treatment patterns, diameter, and centration for specific topographic patterns. we have herein introduced a novel technique based on refractive cxl application on previously untreated keratoconic cornea as a conservative management option. at 6-month follow - up, a stable reduction in astigmatism this is a nonablative and nonincisional approach to treat irregular astigmatism in ectatic cornea with rapid visual rehabilitation. alcon / wavelight, allegran, avedro, i - optics ; optovue (a.j.k.). | purposeto report a novel application of toric topographically customized transepithelial collagen cross - linking (cxl) aiming to achieve refractive astigmatic changes in a keratoconic cornea.methodsspecially formulated riboflavin transepithelial administration and delivery of high - fluence uva in a topographically customized pattern was applied in an eye with progressive keratoconus. visual acuity, cornea clarity, keratometry, topography, and pachymetry with a multitude of modalities, as well as endothelial cell counts were evaluated for > 6 months.resultsuncorrected distance visual acuity changed from preoperative 20/40 to 20/25 at 6 months. a mean astigmatic reduction of 0.8 d, and significant cornea surface normalization was achieved 6 months postoperatively. there was some mild change in the epithelial distribution, with the treated area having a slight normalization in the average epithelial thickness.conclusionswe introduce herein the novel application of a topographically customizable transepithelial cxl in progressive keratoconus in order to achieve an astigmatic refractive effect and ectasia stabilization. this novel technique offers a nonablative and nonincisional approach to treat irregular astigmatism in ectatic cornea with rapid visual rehabilitation. |
at least two overlapping signaling pathways are regulated by the family of secreted glycoproteins known as wnts. the highly conserved canonical wnt/-catenin signaling pathway is activated by the binding of wnt ligand to the receptors frizzled (fzd) and low - density lipoprotein receptor related protein 5/6 (lrp5/6), triggering a series of downstream events that culminate in the cytosolic accumulation and nuclear translocation of the multifunctional protein -catenin. interaction of -catenin with transcription factors of the tcf and lef family results in the regulation of certain target genes that mediate the ultimate effects of this pathway on cellular processes including cell fate, proliferation, and migration. there are also one or more non - canonical or -catenin independent wnt signaling pathways that are less well understand, and that act in a -catenin independent manner leading to changes to cytoskeletal dynamics, adhesion, and motility. interestingly, -catenin independent wnt signaling can antagonize wnt/-catenin signaling in development, regeneration, and cancer [4, 5 ], highlighting the complex interplay between downstream effectors of wnt signaling. wnt pathways have been intimately linked to cancer ever since the original realization that the mouse mammary oncogene int-1 is a homologue of the drosophila wingless (wg) gene, resulting in the portmanteau family designation of wnt. subsequent studies have implicated wnt signaling in almost every major disease and cancer model, reflecting the importance of major developmental pathways in the pathogenesis of adult disease processes [7, 8 ]. most dramatically, almost all colorectal carcinomas harbor inactivating mutations in the gene for adenomatous polyposis coli (apc), which forms a complex with axin and glycogen synthase kinase 3- (gsk3b) that normally phosphorylates -catenin to target the protein for proteasomal degradation. mutations or loss of apc in colorectal carcinoma therefore prevent degradation of -catenin and consequently lead to constitutive activation of the pathway. further studies using both cell - based models and transgenic animal models have validated the essential role of wnt dysregulation in the formation of colorectal cancer, establishing this disease paradigm as a primary model for studying the molecular mechanisms of wnt/-catenin signaling in oncogenesis [9, 10 ]. since the initial demonstration that wnt signaling regulates the stability and translocation of -catenin, the immunohistochemical detection of nuclear -catenin in both laboratory models and in patient tumors has been widely employed as a surrogate for demonstrating activation of the wnt/-catenin pathway. in several cancer models including colorectal carcinoma, breast cancer, and esophageal carcinoma, the presence of nuclear -catenin in cancer tissue compared to normal tissue has implicated this signaling pathway in cancer biology. further studies have observed that the presence of nuclear -catenin can predict decreased survival in these cancers, solidifying the importance of this pathway in oncogenesis and in cancer progression. not surprisingly, wnt/-catenin signaling has also been implicated in a broad variety of noncancerous medical conditions. genetic polymorphisms in lrp5/6 that decrease wnt/-catenin signaling have been linked to altered bone density, metabolic syndrome, and to alzheimer s disease. in normal tissues and organs, without genetic polymorphisms or mutations, wnt/-catenin signaling is activated in every animal that displays regeneration, and -catenin signaling is also activated in traumatic brain injury, which does not display extensive regeneration. moreover, it is clear that attenuating -catenin signaling delays regeneration while augmenting -catenin signaling often enhances the rate of regeneration, as determined by analysis of tail fin regeneration in zebrafish, and liver regeneration in both mouse and zebrafish. given that regeneration employs progenitor cells it should come as no surprise that wnts regulate embryonic stem cells, though there is not a consensus on the precise roles. the increasing body of literature on wnt/-catenin signaling in disease has generated tremendous interest in the potential therapeutic targeting of this pathway. until recently, the only modulator of wnt/-catenin signaling approved by the us food and drug administration was lithium chloride, which prevents the degradation of -catenin by inhibiting its phosphorylation by gsk3b. more recent studies have identified small molecule activators as well as inhibitors of wnt/-catenin signaling that may eventually have therapeutic utility in patients [17, 18, 19, 20, 21, 22 ]. in parallel with studies on wnt/-catenin signaling in cancer and other diseases, substantial progress has also been made in understanding how this pathway regulates developmental processes such as melanocyte differentiation. wnt/-catenin signaling is a major regulator of the pigmented cell lineage, playing a major role in determining the fate of neural crest cells and its derivative pigment cell lineages. wnt/-catenin signaling directly regulates the expression of microphthalmia transcription factor (mitf), a major determinant of both melanocyte development and melanoma progression [2325 ]. wnt3a ligand is one of only three factors required to differentiate a pluripotent human embryonic stem cell into a functional melanocyte, further highlighting the critical role of this pathway in pigment cell biology. not surprisingly, the wnt/-catenin pathway has been implicated in the pathogenesis of both benign melanocytic nevi as well as in malignant melanoma. in the last two decades since the initial identification of activated wnt/-catenin signaling in the murine breast cancer model, the role of this pathway in promoting proliferation has fostered the prevailing view that wnt/-catenin signaling is uniformly the wnt/-catenin pathway would not fit the original definition of an oncogene as a gene or pathway that causes cancer when aberrantly activated, since the forced expression of a melanocyte - specific, nondegradable, constitutively active -catenin mutant in either transgenic or cre / lox systems is not enough to induce melanoma in mice. as our understanding of cancer has advanced, the term oncogenic has seemingly broadened to include any gene or pathway implicated in cancer progression, a benchmark that is more difficult to define. the finding that constitutive activation of wnt/-catenin signaling increases the proliferation of murine melanoma cells in vitro, accompanied by mitf - dependent increases in clonogenic growth, implicates this pathway as a promoter of melanoma progression. likewise, the activation of this pathway acts in concert with activation of ras to promote increased tumor formation in transgenic mice, further suggesting a role in tumor promotion. in contrast to these studies in cell culture and mouse - based models, there have been several recent reports that activation of wnt/-catenin signaling in patient tumors, as monitored by increased levels of nuclear -catenin, correlates with an improved rather than poorer prognosis [5, 3032 ]. consistent with the data seen in human patients, forced expression of wnt3a in b16 melanoma cells leads to decreased proliferation in vitro and in vivo, along with the upregulation of genes associated with melanocyte differentiation that are frequently lost with melanoma progression. furthermore, almost all benign nevi are positive for nuclear -catenin, and studies have observed a loss of nuclear -catenin with melanoma progression to metastases. since the rate of transformation of nevi to melanomas is estimated to be very low, these collective observations from patients bring up the possibility that wnt/-catenin signaling may not be oncogenic in any sense, but rather is required to maintain a homeostatic balance that, when disrupted or lost, can lead to early melanoma transformation. another interesting aspect of wnt/-catenin signaling in melanoma involves -catenin independent wnt signaling activated in many (but not all) contexts by wnt5a. wnt5a was first linked to disease as a gene that was more highly expressed in aggressive and late - stage melanomas, and the immunohistochemical detection of high wnt5a in tumors was subsequently correlated with decreased patient survival. studies on wnt5a in melanoma have focused largely on the effects of this ligand on cell motility, based on the precedent that wnt5a regulates the movement of cells during the convergent - extension phase of vertebrate gastrulation [2, 36 ]. interestingly, at least two studies have shown that wnt5a inhibits the transcription of wnt/-catenin target genes in melanoma, recapitulating the ability of -catenin independent wnt signaling to inhibit wnt/-catenin signaling that was first observed in developmental models [4, 5 ]. this finding suggests that the acquisition of increased wnt5a with later - stage tumors may be involved with the inhibition and/or loss of wnt/-catenin signaling seen during melanoma progression from benign nevi to metastases. if one looks at the available data from patient - based studies of tumor tissue, the notion that wnt/-catenin signaling may not be universally oncogenic is supported by studies in a growing number of diseases. in the case of medulloblastoma, all patients with extensive nuclear -catenin staining had a 5-year overall survival rate of 92.3% versus a rate of 65.3% in patients with nucleonegative tumors. furthermore, in a study of 72 children, all six children who had an activating mutation in the ctnnb1 gene were alive and free of disease 5 years following diagnosis, compared to a survival rate of 53.7% for the ctnnb1 wild - type group [38 ]. in addition, these favorable activating mutations of -catenin correlate with a gene expression profile that distinguishes it from other medulloblastoma subtypes. more limited studies in prostate cancer, ovarian cancer, and even in late - stage colorectal carcinoma also show that the presence of active wnt/-catenin signaling is correlated with improved patient outcomes. ultimately, debates on whether wnt/-catenin signaling is oncogenic are less relevant than understanding whether the therapeutic activation of this pathway could be beneficial for various diseases, including melanoma and other cancers. interestingly, forced activation of wnt/-catenin signaling in the setting of kras - driven murine pancreatic cancer models can antagonize the development of pancreatic intraepithelial neoplasia lesions, and drive the development of tumors that resemble more benign human solid pseudopapillary neoplasms rather than more malignant pancreatic ductal adenocarcinoma [43, 44 ]. this type of intriguing observation invites the hypothesis that wnt/-catenin signaling can be leveraged clinically to prevent aggressive tumors at the expense of potentially acquiring tumors that are more benign and treatable. similarly, the improved survival seen with activating -catenin mutations in medulloblastoma, which is largely a hedgehog - driven tumor, may also reflect the ability of wnt/-catenin signaling to promote cell fates that are less aggressive, a hypothesis supported by observations with genome - wide transcriptional profiling of patient tumors. although in theory the idea of activating wnt/-catenin signaling in cancer patients could be achieved, further studies will be needed to address the timing, strength, and duration of wnt/-catenin signaling required for the desired outcome. in melanoma, activation of wnt/-catenin signaling through the forced expression of wnt3a or through treatment of cells with soluble wnt3a ligand results in the decreased tumor cell proliferation in vitro and in vivo, as well as the transcriptional upregulation of genes associated with melanocyte differentiation. the ability of wnt/-catenin signaling to drive the expression of certain differentiation - related genes likely reflects its role as a major regulator of melanocyte development. further studies will ultimately address whether the therapeutic activation of wnt/-catenin signaling in human melanoma patients is either practicable or beneficial. perhaps the time has come when we should not categorize signaling pathways as either oncogenic or tumor - suppressive, but instead view them in the context of cellular homeostasis. most definitions of cellular homeostasis include the property of cells to maintain a viable and healthy state through the constant adjustment of various inputs such as biochemical pathways. in this context, it is not hard to envision a role for wnt/-catenin signaling in maintaining cell fate and regulating proliferation for both cancerous and noncancerous cells. the transcriptional response of wnt/-catenin signaling itself illustrates the importance of ongoing homeostasis, since many of the classical -catenin dependent target genes, including dkk1, axin2, tcf7, and lef1, can function as feedback inhibitors to modulate the pathway. there are several well - studied examples where wnt/-catenin signaling maintains normal adult tissue, including in intestinal crypts and in hair follicle units. benign melanocytic nevi are similar, since they are almost all positive by immunohistochemistry for nuclear -catenin. taking into consideration that nevi are thought (based on histology of patient tumors) to be precursors for a significant portion of melanomas, it is not so surprising that in some systems, the presence of active wnt/-catenin signaling may be needed for melanoma development. the dysregulation of wnt/-catenin homeostasis is reflected in the observation that nuclear -catenin decreases with melanoma progression. further studies will be needed to determine whether this loss of signaling represents a cause or effect of melanoma - genesis. unlike colon cancer, where mutation or loss of apc plays a primary role in constitutive activation of the pathway, studies have found that mutations leading to constitutive wnt/-catenin signaling in melanoma are rare. as a result, it is thought that the activation of wnt/-catenin signaling in benign nevi and melanomas is likely driven by wnt ligand, either secreted by tumor cells themselves or from cells in the surrounding environment. based on studies in cell - based systems, the signaling induced by secreted wnt ligand is more susceptible to regulation than constitutive activation of the pathway by downstream mutations, suggesting that from a therapeutic point of view, the cause of activated wnt/-catenin signaling in different cancer contexts is an essential consideration. additionally, in cases like melanoma where wnt/-catenin homeostasis is largely mediated by wnt ligand, the role of this pathway in cancer progression may be more difficult to dissect since it likely involves complex interactions between wnt/-catenin signaling and other pathways activated by genetic amplification, deletion, or mutation. accumulating evidence suggests that wnt/-catenin signaling is subject to regulation by cellular, temporal, and spatial contexts that make it difficult to generalize the end results of pathway activation or inhibition. to this end, it is overly simplistic to think of the pathway as oncogenic or tumor - suppressive. while it is easy to draw conclusions from experiments in cell culture dishes or in mouse models, these findings should be reconciled particularly with data on wnt/-catenin activation in human tumors. in studying the role of wnt/-catenin signaling in melanoma first, how well do the mouse models of melanoma recapitulate what is seen in patient tumors with regard to the loss of wnt/-catenin signaling during tumor progression ? second, how reliable is our evaluation of wnt/-catenin activation in patient tumors using nuclear -catenin as our sole measurement ? the context - specific nature of wnt/-catenin signaling may preclude the development of universally applicable assays based on gene targets, but further studies could identify context - specific readouts that could be used in melanoma or other diseases to validate the presence or absence of nuclear -catenin and provide a robust indicator of pathway activation in tumors. by developing reliable models and a faithful set of cell and tissue assays for human diseases, we can begin to fully grasp the cellular, spatial, and temporal contexts that determine the consequences of wnt/-catenin homeostasis and its dysregulation during disease. | in cancer, wnt/-catenin signaling is ubiquitously referred to as an oncogenic pathway that promotes tumor progression. this review examines how the regulation and downstream effects of wnt/-catenin signaling in cancer varies depending on cellular context, with a focus on malignant melanoma. we emphasize that the cellular homeostasis of wnt/-catenin signaling may represent a more appropriate concept than the simplified view of the wnt/-catenin pathway as either oncogenic or tumor - suppressing. ultimately, a more refined understanding of the contextual regulation of wnt/-catenin signaling will be essential for addressing if and how therapeutic targeting of this pathway could be leveraged for patient benefit. |
the development of comprehensive stroke centers within hub - and - spoke stroke networks has been recommended to improve stroke care and to increase the utilization of approved therapies.1 within these networks, eligible patients from community or primary stroke centers could be transferred to comprehensive stroke centers for acute management. the ministry of health and welfare designated 28 cerebrovascular - specified centers (cscs), major trauma - specified centers, and cardiovascular - specified centers in april 2010 as a result of preliminary designation in may 2009. with the designation of a hospital as a " center, " the government has started to support the resources and administrative changes necessary to establish a service for such time - sensitive conditions.2 high - level centers are equipped with the workforce, facilities, and instruments needed to diagnose and treat more complex patients. however, recently, there was social concern that emergency patients were left to wander among major hospitals. delays occurred in finding available facilities owing to a weak coordination system for interhospital transfer. as seen in a study on trauma centers, exhaustion of medical resources in high - level trauma centers owing to overcrowding is related to worse outcomes of patients and high social expenses.3,4 it seems that it is theoretically possible that the same situation could occur in the care of acute ischemic stroke (ais) patients. some ais patients admitted to small hospitals need to be transferred to highly qualified stroke centers. optimal matching of the patient 's needs with hospital capabilities relies on appropriate transfer to the high - level centers as well as the back - transport of patients from high - level centers to community hospitals. to get the right patient to the right hospital in the right time to improve patient outcome, an organized and coordinated approach is needed.5 in busan and ulsan metropolitan cities, the busan emergency medical information center (emic) has been performing the coordination for interhospital transfers since 2001. the purpose of this study was to evaluate changes in the flow of ais patients and the role of the emic in a regional stroke care system. this was a pre- and post- observational study designed to assess the effect of designation of a csc on the flow of ais patients and the coordination for interhospital transfer by busan emic. the study was deemed exempt from review and informed consent by the institutional review board at the hospital site because of the observational nature of the study. busan emic has been operating under the control of the busan wide regional emergency center by the ministry of health and welfare since 2001. the busan emic is in charge of busan and nearby ulsan metropolitan cities with a combined population of 4.7 million (busan : 3.6 million, 766 km ; ulsan : 1.1 million, 1,057 km). the busan emic has been coordinating inquiries of laypersons, ambulance crews, and medical providers to search for available hospitals on a 24/7 basis. most of the coordination is performed by emergency medical technicians under the supervision of board - certificated physicians. busan emic has data on the level of hospitals such as cscs and non - cscs. they had been using data on the availability of computed tomography, ventilators, and beds in the emergency room and intensive care unit through the internet to search for receiving hospitals. in the first step, the hospitals to contact were chosen by matching the medical need of the patient, the level of the receiving hospital, and the distance between transferring and receiving hospitals. the emic also has the cellular phone number of designated vascular neurologists and physicians in the emergency department on a volunteer basis. emergency medical providers are able to inquire about interhospital transfers through a single phone number of 1339. in the second step, permission for the interhospital transfer is granted by the vascular neurologist or physician in the emergency department of the receiving hospital under the coordination of the emic. the number, time, and the content of each call for interhospital transfer are automatically recorded in the electrical database of busan emic. data on interhospital transfers of ais patients by the emic were reviewed for 6 months before (from december 2008 to march 2009) and 6 months after (from december 2010 to march 2011) the designation of the cscs, respectively. definition of acceptance was the granting of permission for interhospital transfer by the accepting hospital. the data included the success or failure of coordination for interhospital transfer, the time consumed for coordination, and the name of the requesting and accepting hospitals. analyses for continuous and categorical variables were performed by using the student 's t- or mann - whitney u test and chi - square (or fisher 's exact) test, respectively. all tests for significance were two - tailed with an alpha level of 0.05. this was a pre- and post- observational study designed to assess the effect of designation of a csc on the flow of ais patients and the coordination for interhospital transfer by busan emic. the study was deemed exempt from review and informed consent by the institutional review board at the hospital site because of the observational nature of the study. busan emic has been operating under the control of the busan wide regional emergency center by the ministry of health and welfare since 2001. the busan emic is in charge of busan and nearby ulsan metropolitan cities with a combined population of 4.7 million (busan : 3.6 million, 766 km ; ulsan : 1.1 million, 1,057 km). the busan emic has been coordinating inquiries of laypersons, ambulance crews, and medical providers to search for available hospitals on a 24/7 basis. most of the coordination is performed by emergency medical technicians under the supervision of board - certificated physicians. busan emic has data on the level of hospitals such as cscs and non - cscs. they had been using data on the availability of computed tomography, ventilators, and beds in the emergency room and intensive care unit through the internet to search for receiving hospitals. in the first step, the hospitals to contact were chosen by matching the medical need of the patient, the level of the receiving hospital, and the distance between transferring and receiving hospitals. the emic also has the cellular phone number of designated vascular neurologists and physicians in the emergency department on a volunteer basis. emergency medical providers are able to inquire about interhospital transfers through a single phone number of 1339. in the second step, permission for the interhospital transfer is granted by the vascular neurologist or physician in the emergency department of the receiving hospital under the coordination of the emic. sometimes, several calls are needed to find an available hospital for interhospital transfer. the number, time, and the content of each call for interhospital transfer are automatically recorded in the electrical database of busan emic. data on interhospital transfers of ais patients by the emic were reviewed for 6 months before (from december 2008 to march 2009) and 6 months after (from december 2010 to march 2011) the designation of the cscs, respectively. definition of acceptance was the granting of permission for interhospital transfer by the accepting hospital. the data included the success or failure of coordination for interhospital transfer, the time consumed for coordination, and the name of the requesting and accepting hospitals. data were analyzed by using pasw 18.0 (spss inc., chicago, il, usa). analyses for continuous and categorical variables were performed by using the student 's t- or mann - whitney u test and chi - square (or fisher 's exact) test, respectively. all tests for significance were two - tailed with an alpha level of 0.05. during the pre- and post - designation study periods, there were 198 and 244 interhospital transfers of ais patients coordinated by the busan emic, respectively. the distribution of ages was as follows (in sequence) : 70s (25.9%), 60s (22.9%), 50s (20.6%), above 80s (13.4%), 40s (12.9%), and 30s (2.9%). we compared the median time used for coordination of interhospital transfers in the pre - designation study period with that of the post - designation study period. after the designation of the cscs, the time decreased from 8.0 minutes to 4.0 minutes (p<0.001, fig. the success rate of coordination for interhospital transfer increased from 88.9% to 96.7% (p<0.001, table 1). the requests for interhospital transfer increased with the designation of the cscs, but the proportion of cscs decreased from 3.5% to 0.4% (p=0.025, table 2). with the designation of the cscs, the proportion of acceptance for interhospital transfer by non - csc hospitals increased from 15.9% to 25.8% (p=0.015, table 2). construction of a regional stroke care system improves outcome.6 - 9 concepts for the development of a regional care system for time - sensitive conditions such as trauma, cardiovascular disease, and cerebrovascular disease are similar in many aspects. however, in contrast with a trauma system, which has a long history of evolution, the concept of a stroke care system is only recently developed and reflects the latest concepts of the trauma system.10,11 the concept of the trauma system has evolved from getting " injured patients to the nearest facilities quickly " to " severely injured patients to definitive care facilities quickly " to " the right patients to the right facilities in the right time."12 before the 1970s, trauma patients were transported to the nearest facilities without field triage. in the 1970s, patients with major injuries began to be concentrated in high - level trauma centers according to the exclusive trauma system. the system was based on the concept that a better outcome would come from more experienced facilities with a large volume of injured patients where trauma teams provide coordinated resuscitation, evaluation, and definitive treatment.2 therefore, acute care facilities need to be categorized according to their ability to provide care, and patients are distributed to facilities at each level of care according to severity. in reality, however, patients with minor injuries were concentrated at high - level centers owing to over - triage at the prehospital and interhospital transfer level. overcrowding of high - level centers resulted in exhaustion of medical resources, wasting of social resources, and worse outcomes. thus, some trauma centers did not want to receive more patients.13 - 15 in contrast with older statistics that compared high - level centers and small hospitals in a region, expanded statistics could compare hospitals among regions with different trauma systems. in the 1990s, these systems were designed to care for all injured patients in a given geographical area. therefore, all acute care hospitals were expected to participate in a trauma system as one of the multi - level centers from level i to iii or v.16 in the inclusive trauma system, collaboration should exist between the government, emergency medical services, and acute care hospitals.17 however, the inclusion system also has theoretical disadvantages. spreading the volume of trauma care among centers may diminish providers ' experience and efficiency of care in high - level centers. the inclusive system may also delay definitive care for patients who should have been triaged directly from the injury scene or should have been transferred to a high - level center after their initial evaluation at a small hospital. first is the infrastructure of the regional stroke care system. the internal change in designated hospitals second is the interhospital transfer system itself. in the well - organized stroke care system, any hospital in the stroke care system that provides emergency department services should be able to function as a primary stroke center or in the rapid transfer of appropriate patients through the use of prenegotiated interhospital protocols, transfer agreements, and transport protocols.10 theoretically, the receiving hospitals are decided by the system itself. in practice, however, there should be an agreement on interhospital transfer between the transferring and the receiving physicians and there may be several trials to find the final receiving hospital.18 recently, consensus has been reached that the formal transfer system is not enough. system - level coordination is needed.4 with respect to agreement, there can be 3 types of interhospital transfer when a hospital decides to transfer stroke patients. epley reported on an organized system combining a communications center with a formal interhospital transfer system of trauma patients.13 before the activation of the communication center, the interval from the transfer decision to the acceptance decision was 30.5 minutes conservatively. in fact, there were anecdotal cases of 6 to 12 hours. with the activity of a communication center, the interval decreased to 10.0 minutes. the necessity of coordination has been described in interhospital transfer of st elevation myocardial infarction. coordination with a single phone number has decreased the time from visiting former hospitals to balloon angioplasty at later hospitals.19,20 this has been described in other time - sensitive conditions as well. in ais patients, prior notice to the receiving vascular neurologist via the busan emic has decreased the door - to - needle time for intravenous thrombolysis in a hospital.21 in contrast with the lately developed concept of a stoke system based on a multi - level stroke center, our regional stroke care system is based on the earlier concept of a single high - level center. several components of the formal system for getting " the right patients to the right facilities in the right time " were short in our regional stroke care system. after our designation of a csc, the requests for interhospital transfer from the csc decreased. this reflects internal changes within the csc, which wants to treat more ais patients. however, the proportion of acceptance by the cscs decreased despite the increasing volume of ais patients. ais patients were dispersed more to non - csc hospitals. these findings might be explained by the fact that the cscs still do not have sufficient resources to cover the increasing volume of ais patients, and the designation of a csc might encourage non - csc hospitals to accept ais patients more easily so as not to lose some ais patients. the success rate of coordination for interhospital transfer increased and the time used for coordination decreased after designation of the cscs. the emic might have been able to arrange for faster transfers because competition for referrals from the coordinating center encourages hospitals to cooperate with the emic. these results also suggest that the busan emic has a properly coordinated network for interhospital transfer in our regional stroke care system. however, our study had several limitations. there could be regions where large, overcrowded designated cscs do not want to treat more ais patients. furthermore, dispersion of ais patients to csc or non - csc hospitals could have positive or negative effects on the patients ' outcome. in fact, even in the trauma system with its long history, interhospital transfer is called a " curse " of registry.22 uniform data templates and data input on a regional basis would be other problems.23 also, the stroke system has just began to evolve. there would be a long journey of evolution to relieve the scientific gap. at any rate, our study is not related to patient outcomes but to stroke center designation and patient flow. another limitation is that internationally, there is no single, simple way in which to enact a regional stroke care system for ais patients. like other studies of such systems, our results also can not be generalized. in conclusion, with the designation of cscs, however, we also found that ais patients were dispersed to both csc and non - csc hospitals in our region. these results might be explained by the fact that the cscs still do not have sufficient resources to cover the increasing volume of ais patients, or that not only cscs but also some non - csc hospitals wanted to treat more patients. further studies based on patients ' outcomes are needed to determine the adequate type of interhospital transfer for ais patients. | the ministry of health and welfare of korea recently designated cerebrovascularspecified centers (cscs) to improve the regional stroke care system for acute ischemic stroke (ais) patients. this study was performed to evaluate the changes in the flow of ais patients between hospitals and to describe the role of the emergency medical information center (emic) after the designation of the cscs. data for coordination of interhospital transfers by the emic were reviewed for 6 months before and after designation of the cscs. the data included the success or failure rate, the time used for coordination of interhospital transfer, and the changes in the interhospital transfer pattern between transfer - requesting and transfer - accepting hospitals. the total number of requests for interhospital transfer increased from 198 to 244 after designation of the cscs. the median time used for coordination decreased from 8.0 minutes to 4.0 minutes (p<0.001). the success rate of coordination increased from 88.9% to 96.7% (p<0.001). the proportion of requests by cscs decreased from 3.5% to 0.4% (p=0.017). however, the proportion of acceptance by non - csc hospitals increased from 15.9% to 25.8% (p=0.015). with the designation of cscs, the emic could coordinate interhospital transfers more quickly. however, ais patients are more dispersed to csc and non - csc hospitals, which might be because the cscs still do not have sufficient resources to cover the increasing volume of ais patients and non - csc hospitals have changed their policies. further studies based on patients ' outcome are needed to determine the adequate type of interhospital transfer for ais patients. |
malaria remains an important public health concern in countries where transmission occurs regularly as well as in areas where transmission has been largely controlled or eliminated. it was estimated that there are 39 million children under 5 years of age who experience 33.7 million malaria episodes and 152,000 childhood deaths from malaria each year in areas suitable for seasonal malaria chemoprevention. factors such as drug pressure, strain variation, or approaches to blood collection affect the morphological appearance of malaria species which have created diagnostic problems that invariably had a negative effect on malaria control. with the introduction of high cost antimalarial (artemisinin based therapies) the need for accurate diagnostic tools for monitoring malaria elimination / eradication successes becomes a task that must be achieved [3, 4 ]. in most endemic countries malaria diagnosis depends mainly on clinical evidence and in some cases thick film microscopy (tfm) and rapid diagnostic technique (rdt) may be used for laboratory confirmation. microscopy remains the gold standard for malaria diagnosis and it is less costly with a threshold sensitivity of 5 to 50 parasite/l (depending on the microscopist expertise). the major constraints of microscopy include the requirement of considerable technical expertise and the fact that it is time - consuming for optimal blood film preparation, examination and interpretation. rdt, an immunochromatographic capture procedure was developed to improve the timeless sensitivity, and objectivity of malaria diagnosis through less reliance on expert microscopy. preferred targeted antigens for rdts are those which are abundant in all asexual and sexual stages of the parasite. currently the focus of rdt is on the detection of histidine - rich protein2 (hrp-2) from plasmodium falciparum and parasite - specific lactate dehydrogenase (pldh) or plasmodium aldolase from the parasite glycolytic pathway found in all species. however, several factors in the manufacturing process as well as environmental conditions may affect rdt performance, and these include suboptimal sensitivity at low parasite densities, inability to accurately identify parasites to the species level or quantify infection density, and a higher unit cost relative to microscopy. polymerase chain reaction (pcr), another diagnostic technique, detects specific nucleic - acid sequence and its values lie in its sensitivity, with the ability to detect five parasites or less/l of blood. pcr is useful both for initial parasite diagnosis and for followup during drug efficacy study. it is also useful as a sensitive standard against which other non - molecular methods can been evaluated. however it is expensive and time - consuming and because of the amount of resources needed in the running of the pcr laboratory, it is used more for research purposes. clinical diagnosis is imprecise but remains the basis for therapeutic care for the majority of febrile patients in malaria endemic areas, where laboratory support is often out of reach. clinical diagnosis also referred to as presumptive diagnosis is the least expensive and most commonly used method and is the basis for self - treatment in endemic countries. overlap of malaria symptoms with other tropical diseases like typhoid fever, respiratory tract infections and viral infections impairs the specificity of presumptive diagnosis thereby encouraging indiscriminate use of antimalarials in endemic areas. accuracy of clinical diagnosis varies with the level of endemicity, malaria season, and age group. therefore no single clinical algorithm can be regarded as a universal predictor. this paper reports the comparative performance of clinical diagnosis, tfm, rdt, and pcr in the diagnosis of p. falciparum malaria in nigeria. osogbo is the state capital of osun state, nigeria, and it represents a typical urban setting in nigeria. patients (ages 4 months to 20 years) who were clinically diagnosed for malaria at the outpatient departments of general hospital asubiaro and lautech health centre in osogbo were recruited into the study. all the patients that were clinically diagnosed were subsequently confirmed using tfm, rdt, and pcr before treatment. ethical approval was obtained from the ethical committee of osun state hospital management board, osogbo. clinical diagnosis based on fever (temperature 37.5c) and/or history of fever other symptoms considered for clinical diagnosis include headache, joint pains, body weakness, cough, diarrhea, loss of appetite / refusal of feeds, abdominal pain, and generalized body weakness. 5 ml of blood was collected aseptically from antecubital vein of consenting febrile patients, into edta bottle. rdt was performed on about 5 l of blood using paracheck (orchid biomedical system, verna, goa, india) according to manufacturer 's instruction. a drop of blood was used for microscopic examination of malaria parasites using thick films method stained with 5% giemsa for 30 minutes. parasites were counted against 200 white blood cells (wbcs) from the thick film. the parasite density was obtained by assuming a total wbc count of 8000/ml and 4.5 million rbc / ml and at least 200 fields were examined before being taken as a negative result. 10 l of blood was dotted on whatman 3 mm filter paper and air - dried at room temperature for pcr. parasite genomic dna was extracted from blood samples collected on filter paper using methanol extraction method as previously described. pcr was carried out using primer pairs that target the multicopy p. falciparum stevor gene. primary amplification was performed with reaction mixture of 25 l containing 2.5 l 10x reaction buffer, 5 l of magnesium chloride, 0.75 l of each primers (p5, p18, p20, p19), 0.2 l of dntps, 9.05 l of water, 0.25 l of taq polymerase, and 5 l of dna extract. the pcr programme was as follows : 93c for 3 minutes, 22 cycles of 30 seconds at 93c, 50 sec at 50c, and 30 sec at 72 and final extension period of 3 minutes at 72c. 2.0 l of the first pcr product was used in the second round amplification which was performed with a reaction mixture of 25 l containing 2.5 l 10x reaction buffer, 2.5 l of magnesium chloride, 0.4 l of each dntps, 0.25 l of taq polymerase, 1.0 l of each primers (p24, p17), and 15.35 l of water. dna extracted from fcr p. falciparum laboratory adapted strain was used as positive control and water as negative control. pcr products were subjected to electrophoresis on 1.5% agarose gels and visualized using syngene gel documentation system (syngene, cambridge, uk) after staining with ethidium bromide. the sensitivity, specificity, and predictive values of each of the three test methods were calculated by comparing to a composite reference gold standard generated from the three methods. the composite reference method was defined as a method that is positive for malaria parasites by all the three methods (tfm, rdt, and pcr) and also negative for malaria parasites by all the three methods. this gives the method 100% hypothetical sensitivity, specificity, and positive and negative predictive values. the sensitivity, specificity, and predictive values of each of the 3 methods were then calculated using the formulas (1)sensitivity = tp(tp+fn)100specificity = tn(tn+fp)100ppv = tp(tp+fp)100npv = tn(tn+fn)100, where tp = true positive, fp = false positive, tn = true negative, and fn = false negative. sensitivity was defined as the probability that a truly infected individual will test positive and specificity as the probability that a truly uninfected individual will test negative. we compared the diagnostic value of 3 methods (tfm, rdt, and pcr) for the detection of malaria parasites in nigeria. a total of 217 individuals clinically diagnosed for malaria were recruited into the study. of these, the mean age of the patients was 8 years 3.04 and the mean axillary temperature was 38.2c 0.96. one hundred and six (48.8%) individuals were positive for malaria by tfm, 84 (38.7%) by rdt, and 125 (57.6%) by pcr. there were significant differences (p = 0.0005) when the prevalence of 3 methods (tfm, rdt, and pcr) was compared (table 1). using a composite reference (gold standard) method generated from the three diagnostic methods, only 71 (32.7%) patients were found to be truly infected, with p. falciparum 90 (41.5%) truly uninfected while 56 (25.8%) were misidentified as infected or noninfected by the three methods. when each of the 3 diagnostic methods was compared with the composite reference method, pcr had sensitivity of 97.3%, specificity of 62.5%, positive predictive value (ppv) of 56.8%, and negative predictive value (npv) of 97.8% ; microscopy had sensitivity of 77.2%, specificity of 72%, ppv of 66.9%, and npv of 81.1%, while rdt had sensitivity of 62.3%, specificity of 87.4%, ppv of 67.7%, and npv of 84.5% (table 2). correlation of rdt and pcr to parasite density observed by microscopy is shown in table 3. out of 109 patients that were negative by microscopy 22 and 29 out of this 81 microscopy positive patients, 47 and 73 patients were detected by rdt and pcr, respectively (table 3). this study provides a dataset for judging the performance of clinical diagnosis against tfm, rdt, and pcr for the detection of p. falciparum in a malaria endemic area. clinical diagnosis, for instance, is commonly used because it is cheap and allows for prompt treatment of the patient. nonspecific symptoms like fever, headache, weakness, myalgia, chills, dizziness, abdominal pain, diarrhea, nausea, vomiting, anorexia, and pruritus and other malaria related symptoms are used as the basis for clinical diagnosis. microscopy remains the gold standard for malaria diagnosis ; it is less expensive compared to other laboratory methods but has a low sensitivity. it requires well trained microscopist and when this is not present the result will not be reproducible, there will be variable sensitivity and unacceptably high false - positive rates. rdts are antigen capture tests that have been shown to be capable of detecting > 100 parasites/l (0.002% parasitemia) and of giving rapid results (15 to 20 min). they are commercially available in kit form and the ease of performance of the procedures does not require extensive training, equipment, or difficulty in result interpretation. the main drawback is in its specificity as parasite antigen could persist in the blood of the patient after parasite clearance by chemotherapy thereby producing false positive. pcr values lie in its high sensitivity, with the ability to detect five parasites or less/l of blood [15, 17 ] ; however it is expensive and time - consuming. our results show that the continuous practice of using clinical diagnosis as the basis for antimalarial treatment in endemic area is by far not an effective diagnostic method in our study area. out of the 217 (100%) patients that were clinically diagnosed for malaria, 104 (49.8%), 83 (38.2%), and 123 (56.7%) were positive by tfm, rdt, and pcr, respectively. invariably irrespective of the laboratory method, about half of the patients who were diagnosed as having malaria through clinical diagnosis (syndrome approach) and who should have received antimalarial turned out to be parasite - negative. there is therefore an urgent need to review the clinical diagnosis procedure. although it may be argued that in some cases especially in children, promptness of malaria treatments reduces the progression of simple malaria to severe malaria, which still encourages syndromic approach to malaria diagnosis. nevertheless malaria over diagnosis is still a major public health problem in africa with studies suggesting between 50% and 99% of those prescribed antimalarial to be test negatives depending on endemicity of the clinical setting [5, 18, 19 ]. the ability to rule out malaria can help to better diagnose and treat other causes of fever such as acute respiratory infection, typhoid fever, and meningitis and also avoid exposing those without malaria to drug and restricting antimalarial use to true test - positives. till date our study confirmed that continual dependence on this method will lead to overdiagnosis of malaria which will result into drug wastage and encourage antimalarial drug resistance. in this study routine microscopic examination of giemsa - stained blood smears which is considered as the gold standard for malaria diagnosis had a sensitivity of 77.2% and was able to detect more parasites than the rdt (sensitivity 62.3%). though the specificity of microscopy (72%) was not as high as that of rdt (87.4%) ; nevertheless, it has high sensitivity, possibility for quantification of parasitemia, and easy handling which is a good advantage. detection of parasites depends on many factors including the amount of blood processed and the competence of the microscopist, among others. also the information obtained by microscopy is limited when parasite levels are very low or when parasite morphology is altered. the development of rapid diagnostic assays has attempted to address some of these shortcomings of microscopy. rdts have the potential to improve the accuracy and time needed for malaria diagnosis particularly for laboratories in low or nonendemic countries, where expertise with microscopy may be limited. major advantages of rdts include the fact that it can be performed close to home in settings with no sophisticated infrastructure, and they do not require much skill although some level of training is needed in order for rdts to be used properly. different pcr based methods have been constantly shown to be powerful tools for malaria diagnosis with better sensitivity than conventional microscopy and antigen - based diagnostic tests [18, 22 ]. most positive cases were detected by the stevor pcr in this study and this method has been reported to be at least 100-fold more sensitive than other pcr assays [15, 23 ]. generally, pcr has proven to be a sensitive method for diagnosis of all four species of human malaria parasites. the detection of < 5 parasite/l and identification to the species level make this an excellent technique against which to compare the sensitivity and specificity of other nonmolecular methods. greater percentage of children presented at general outpatient department of the hospital in our study with fever were diagnosed for malaria (pcr56.7%, microscopy 49.8%, and rdt 38.2%). available records also show that at least 50% of the population of nigeria suffer from at least one episode of malaria each year accounting for over 45% of all out - patient visits. the implication of this is that malaria is still a public health problem in this area. more concerted effort is needed by government and all stake holders involved in malaria control if the goal of eradicating malaria by 2015 is to be achieved. in conclusion our study revealed the need for complete shift from symptom - based diagnosis to parasite - based diagnosis. this can bring significant improvement to tropical fever management and reduce drug wastage and also help to curtail development of malaria drug resistance. | this study compares the performance of clinical diagnosis and three laboratory diagnostic methods (thick film microscopy (tfm), rapid diagnostic test (rdt), and polymerase chain reaction (pcr)) for the diagnosis of plasmodium falciparum in nigeria. using clinical criteria, 217 children were recruited into the study out of which 106 (48.8%) were positive by tfm, 84 (38.7%) by rdt, and 125 (57.6%) by pcr. using a composite reference method generated from the three diagnostic methods, 71 (32.7%) patients were found to be truly infected and 90 (41.5%) truly uninfected, while 56 (25.8%) were misidentified as infected or noninfected. when each of the 3 diagnostic methods was compared with the composite reference, pcr had sensitivity of 97.3%, specificity of 62.5%, positive predictive value (ppv) of 56.8%, and negative predictive value (npv) of 97.8% ; microscopy had sensitivity of 77.2%, specificity of 72%, ppv of 66.9%, and npv of 81.1%, while rdt had sensitivity of 62.3%, specificity of 87.4%, ppv of 67.7%, and npv of 84.5%. pcr test performed best among the three methods followed by tfm and rdt in that order. the result of this study shows that clinical diagnosis can not be relied upon for accurate diagnosis of p. falciparum in endemic areas. |
mycosis fungoides (mf) is a cutaneous t cell lymphoma (ctcl) characterized by infiltration of skin with patches, plaques and nodules composed of t - lymphocytes. it has various stages, premycotic, patch, plaques, nodules, tumours and erythroderma. tumour demblee is a variant of tumour stage, which develops from normal skin without prior patch or plaque stage. a 55-year - old hindu male, tobacco farmer by occupation, presented with multiple infiltrated plaques and nodules of 3 months duration over the face and scalp. the nodules started from ears and spread all over the face and scalp, increasing in size and number. patient had a history of severe itching episodes for past three years which were not relieved by antihistaminics. there was history of episodes of fever, weight loss, anorexia, and nausea for the past 2 months. multiple infiltrated plaques and nodules with few erosions and foul smelling superficial ulcers were present over face and scalp. the infiltrated skin over forehead, nose and ear lobules and loss of eyebrows gave a leonine face appearance [figures 1 and 2 ]. the nodes were discrete, non - tender, mobile, and firm in consistency. loss of hair was seen on scalp, eyebrow, and axillary region. nodules plaques and erosions over face investigations revealed a hemoglobin level of 8.6% gm and the erythrocyte sedimentation rate (esr) of 110 mm / hour. chest x - ray, lymph node aspiration cytology and bone marrow examination did not reveal anything abnormal. ultrasonography of abdomen and pelvis and computed tomography (ct) scan of head and neck, chest, abdomen, and pelvis were normal. histopathological examination of the excision biopsy from a nodule over face showed a lymphocytic infiltrate in the papillary dermis and around the hair follicle and pilosebaceous unit. epidermotropism was noted with formation of well defined pautrier 's microabscess at places [figure 4 ]. biopsy showing epidermotropism (h and e, 40) biopsy showing clusters of atypical lymphocytes within the epidermis (pautrier microabscesses) (h and e, 400) immunohistochemistry showed positive cd3 and lca markers as and cd 30 and cd 20 negativity. he termed it mycosis fungoides because of the resemblance of the lesions to mushrooms. in 1885, vidal and brocq described mycosis fungoides d emblee for a patient presenting with skin tumours not preceeded by patch or plaques. in this type of mf mf is the most common type of ctcl and accounts for almost 50% of all primary cutaneous lymphomas. however other lymphoproliferative disease also involve the skin including ki-1 + anaplastic large cell lymphoma, peripheral t - cell lymphoma, cutaneous b - cell lymphoma, adult t - cell leukaemia/ lymphoma, t - cell lymphoid leukaemia and cutaneous hodgkin 's disease. incidence of mf has been estimated to range from 0.06 to 0.1 per 10,000 cancer cases per year in the usa. the term tumour d emblee is now falling into disrepute and these tumors may, in fact, be pleomorphic cd 30 negative cutaneous t - cell lymphoma (peripheral t - cell lymphoma), which have undergone large cell transformation. many of these cases are likely to be classified by immunophenotyping as various types of non - mf t - cell lymphoma or even b - cell lymphoma of the skin. such type of mf d emblee has been reported rarely in past.[1214 ] many cases described as the demblee variant in the past may have represented other types of lymphomas. the cd 30 negative large ctcl and small/ medium sized pleomorphic ctcl have been described in literature to be presenting with tumors without prior or concurrent patches or plaques along with histological presentation sometimes similar to that of mf. the cd 30 negative large ctcl (5 year survival of 15%) has a poor prognosis compared with small/ medium sized pleomorphic ctcl (5 year survival of 60%). usually the mean interval between appearance of skin lesions and definite diagnosis by histopathology is approximately 6 years, however, in our case it was only 3 - 4 months. the patient was treated with chop regimen [cyclophosphamide, hydroxydaunorubicin (doxorubicin), oncoverin (vincristine), prednisone ] plus methotrexate. taking into account that this case of tumour d emblee also showed typical histopathological changes along with cd30 negativity and the eventual death of the patient within short span of time after the diagnosis, it is possible that the patient had cd 30 negative large ctcl, which could not be confirmed owing to limited resources in our hospital set up. this case is reported because of an acute and masquerading presentation of mycosis fungoides reiterating the fact that ctcl can pose an enormous diagnostic challenge. | mycosis fungoides is a cutaneous t - cell lymphoma characterized by infiltration of skin with patches, plaques, and nodules composed of t - lymphocytes. it is the most common type of cutaneous t - cell lymphoma and accounts for almost 50% of all primary cutaneous lymphoma. tumour d emblee is the term used for the patient presenting with skin tumors not preceded by patches or plaques. we report a rare case of mycosis fungoides d emblee variant with tumors of only 3 months duration without any preceding skin lesions. |
cullin proteins are molecular scaffolds that have crucial roles in the post - translational modification of cellular proteins involving ubiquitin. the mammalian cullin protein family comprises eight members (cul1 to cul7 and parc), which are characterized by a cullin homology domain. cul1 to cul7 assemble multi - subunit cullin - ring e3 ubiquitin ligase (crl) complexes, the largest family of e3 ligases with more than 200 members. although cul7 and parc are present only in chordates, other members of the cullin protein family are found in drosophila melanogaster, caenorhabditis elegans, arabidopsis thaliana and yeast. a cullin protein tethers both a substrate - targeting unit, often through an adaptor protein, and the ring finger component in a crl. the cullin - organized crl thus positions a substrate close to the ring - bound e2 ubiquitin - conjugating enzyme, which catalyzes the transfer of ubiquitin to the substrate. in addition, conjugation of cullins with the ubiquitin - like molecule nedd8 modulates activation of the corresponding crl complex, probably through conformational regulation of the interactions between cullin 's carboxy - terminal tail and crl 's ring subunit. genetic studies in several model organisms have helped to unravel a multitude of physiological functions associated with cullin proteins and their respective crls. crls target numerous substrates and thus have an impact on a range of biological processes, including cell growth, development, signal transduction, transcriptional control, genomic integrity and tumor suppression. moreover, mutations in cul7 and cul4b genes have been linked to hereditary human diseases. cullins, containing an evolutionarily conserved cullin homology domain, are a family of structurally related proteins required for ubiquitin - dependent protein degradation (box 1). kipreos. identified cullins as a novel gene family involved in cell cycle regulation in nematodes. isolated cdc53 (cell division control protein 53), a cullin homolog in budding yeast, and elucidated its role in ubiquitin - dependent proteolysis of cell - cycle regulators. thus, cullins are named after their role in ' culling ', sorting or ' selecting ' cellular proteins for ubiquitin - mediated proteasomal degradation (e kipreos, personal communication). the cullin gene family is evolutionarily conserved. table 1 presents a list of the cullin family genes from a range of representative species with respect to their gene organization and expression. there are seven cullins in mammals (cul1 to cul3, cul4a, cul4b, cul5, cul7 and the closely related p53-associated parkin - like cytoplasmic protein (parc) in homo sapiens, mus musculus and rattus norvegicus), six in c. elegans (cul-1 to cul-6) and five in drosophila (cul1 to cul5). arabidopsis has five cullins (cul1, cul2, cul3a, cul4 and cul5), and yeast genomes encode three cullin proteins (cul1, cul3, cul8 in saccharomyces cerevisiae ; cul1, cul3 and cul4 in schizosaccharomyces pombe). in total, 490 cullin domains in 490 proteins are described in the smart nrdb database, which estimates 60% conservation in the cullin homology domains. chromosomal localization of the cullin genes from several representative species gene ids are as listed in entrez gene. the intron number and transcript information are indicated for the cullin genes in human and mouse. only the variants in entrez gene the presence of the cul1 to cul5 genes in the early - branching metazoans trichoplax adhaerens and nematostella vectensis indicates that cullin genes are ancient and originated before the separation of the different animal lineages. an extensive genome - wide analysis of the cullin family has suggested that three ancestral cullin genes, termed ' cul ', ' cul ' and ' cul ', appeared in early eukaryotic evolution, from which the cullin genes evolved after the split of the unikonts (which include animals and fungi) and the bikonts (which include plants). in this model, the human cul1, cul2, cul5, cul7 and parc genes were derived from one common ancestral gene (cul), whereas the cul3 and cul4a/4b genes evolved from two distinct ancestors, the cul and cul gene, respectively. notably, cul7 and parc, found only in chordates, are highly similar in sequence and both contain a cph (conserved in cul7, parc and herc2) domain and a doc domain (similar to the doc1 of the anaphase - promoting complex / cyclosome) of unknown functions (figure 2). in human and mouse, cul7 and parc are located on the same chromosome in close proximity (260 kb apart). based on these findings, marin. suggested that parc originated from a gene fusion of a duplicate of cul7 and an ariadne gene, which encodes a putative e3 ubiquitin ligase sharing structural similarity with parkin fungal species contain only three cullin genes : cdc53, cul3 and cul8 (also known as rtt101) in s. cerevisiae and cul1, cul3 and cul4 in s. pombe. however, cul8/rtt101 in saccharomycotina differs significantly from the cul4-like genes of other fungi (such as ascomycetes and basidiomycetes). it was postulated that cul8/rtt101 originated from the cul4 gene that underwent an accelerated evolution, or that saccharomycotina has lost their cul4-like gene and the cul8/rtt101 gene arose in parallel as a result of gene duplication. phylogenetic tree based on the alignment of cullins of homo sapiens (hs), mus musculus (mm), rattus norvegicus (rn), drosophila melanogaster (dm) and caenorhabditis elegans (ce). gene ids are as listed in table 1. the bar indicates the proportion of amino acid sites at which two compared sequences are different. cullin repeat 1 (cr1) anchors the cognate adaptor proteins, and the cullin homology domain (ch) at the carboxyl terminus is critical for binding of the ring - finger protein. size and location of the individual domains are schematic representations and do not depict the exact proportions. abbreviations : aa, amino acids ; ch, cullin homology domain ; cph, conserved domain in cul7, parc and herc2 ; cr, cullin repeat ; doc, a domain similar to the doc1 domain of the anaphase - promoting complex / cyclosome but of unknown function ; ibr, in between ring ; ring, really interesting new gene. ring (really interesting new gene)-type e3 ubiquitin ligases orchestrate ubiquitination by simultaneously binding to a protein substrate and anchoring an e2 ubiquitin - conjugating enzyme through the ring domain (for detailed description of the actions of e2 and e3 in ubiquitination, see boxes 1 and 2 and figure 3). cullins are molecular scaffolds that organize the largest class of ring e3 ligases, known as the cullin - ring ligase complexes (crls). below the ubiquitin - proteasome system. the conserved 76-amino - acid polypeptide ubiquitin (ub) is activated in an atp - dependent reaction by an e1 ubiquitin - activating enzyme and transferred to an e2 ubiquitin - conjugating enzyme. an e3 ubiquitin ligase binds both the substrate protein and a ubiquitin - charged e2 enzyme for ubiquitin transfer, resulting in the mono-, multi- (not shown) or polyubiquitination of the substrate. the mode of ubiquitination determines whether the substrate protein is degraded by the 26s proteasome or altered in a non - proteolytic manner. the structural properties of cullins have been revealed in the context of crls by high - resolution structural studies and biochemical reconstitution experiments (table 2). although all the cullin (and crl) structures that have been solved so far have been mammalian proteins (table 2), the interactions between cullins and their crl components have been analyzed using biochemical assays in systems from yeast to humans, highlighting an extraordinary conservation regarding the scaffolding functions of cullins. thus, the cullin and crl structural models discussed in this section are generally applicable to counterparts of all origins. cullin structures and cullin - ring e3 complex assembly crystal structures of crl complexes were solved with recombinant human proteins. cul1 to cul5 have a long stalk - like amino - terminal domain (ntd), consisting of three cullin repeats (cr1 to cr3), and a globular carboxy - terminal domain (ctd), which harbors a signature cullin homology domain (ch), a highly conserved stretch of about 200 amino acids (figure 2). the cullin ctd binds to its ring partner, regulator of cullins 1 (roc1) or roc2 (also called ring box protein (rbx)1 and rbx2, respectively), which recruits the ubiquitin - loaded e2 enzymes for catalysis. the cul1-roc1 association is established by multiple interface interactions, primarily involving cul1 's / domain and the amino - terminal s1 -strand of roc1, which enable the formation of an intermolecular / hydrophobic core that essentially renders cul1-roc1 physically inseparable. the cullin - ring interaction creates a catalytic core and is the most characteristic structural feature that defines crls. on the basis of structural studies of the human s - phase kinase - associated protein 1 (skp1)-cul1-f - box (scf) complex and the cul4a ring (crl4a) complex, cullins organize crls by forming two distinct modules : a substrate - targeting unit, composed of a substrate - recognition protein and an adaptor protein that links the module to the cullin, and the ring component that is active in recruiting an e2 ubiquitin - conjugating enzyme (figure 4). for instance, scf contains a substrate recognition subunit known as the f - box protein, which is characterized by a 40-amino - acid f - box domain. the skp1 adaptor protein mediates binding of the f - box protein to the amino terminus of cul1. in crl4a, the substrate - targeting unit is composed of damage - specific dna binding protein 1 (ddb1) as the adaptor protein, and a member of the ddb1 and cul4 associated factor (dcaf) family that recognizes a substrate. instead, it incorporates btb (bric - a - brac, tramtrack, broad - complex), a dual function molecule capable of binding to cul3 and targeting a substrate (figure 4). (scf, also called crl1, is historically the prototype of all crls and thus the name scf remains commonly used in the current literature.) cullin proteins are molecular scaffolds that assemble multi - subunit cullin - ring e3 ubiquitin ligase (crl) complexes. the mammalian cullin protein family comprises eight members (cul1 to cul7 and parc). in crl, a cullin protein tethers both a substrate - recognition subunit, often through an adaptor protein, and the ring finger component. the cullin - organized crl thus positions a substrate in close proximity to the ring - bound e2 ubiquitin - conjugating enzyme (not shown), which catalyzes the transfer of ubiquitin to the substrate. btb, bric - a - brac, tramtrack, broad - complex domain ; dcaf, ddb1-cul4 associated factor ; ddb1, dna damage - binding protein 1 ; fbw8, f - box and wd repeat domain containing protein 8 ; parc, p53-associated parkin - like cytoplasmic protein ; socs, suppressors of cytokine signaling ; skp1, s - phase kinase - associated protein 1 ; vhl - box, von hippel - lindau box. accumulating evidence points to a common mechanism by which cul1 to cul5 build a substrate - targeting unit. cul1 to cul5 use amino - terminal helices h2 and h5 of cr1 to anchor their cognate adaptors. there are two distinct types of recognition fold in the adaptor (table 2). in the scf, crl2, crl3 and crl5 e3s, different adaptors (skp1, elongin c (eloc) or btb) share a similar structural motif termed the skp1/btb / pox virus and zinc finger (poz) fold, which is a primary determinant for affinity interactions with the cullin amino terminus. by contrast, the ddb1 adaptor of crl4 lacks the skp1/btb / poz fold and instead uses its bpb domain to interact with the cul4a h2 and h5 helices, as well as the amino - terminal extension. in this regard, it is worth commenting on the enormous structural complexity of ddb1, a 127 kda protein containing three large propeller folds, which potentially enable multiple interactions with cellular proteins. indeed, ddb1 was found to form a complex with de - etiolated 1 (det1), ddb1 associated 1 (dda1) and the e2 ubiquitin - conjugating enzyme ube2e. initial efforts to isolate the human cul4a - containing complexes resulted in very large complexes that contained the constitutive photomorphogenesis 9 (cop9) signalosome. the role of the cop9 signalosome in crl function is discussed in the next section. structural and biochemical analyses have revealed additional protein - protein interactions that contribute to the cullin - mediated crl assembly. in addition to the skp1-cul1 interactions, skp2 's f - box domain also binds to cul1, thus contributing to the assembly of the scfcomplex. although cul3 mediates interactions to btb proteins through the skp1/btb / poz fold, it binds to a conserved helical structure carboxy - terminal of the btb domain, which was named ' 3-box ' for cul3-interacting box (table 2). the cul3 - 3-box association strengthens the cul3-btb protein interactions. despite sharing the identical adaptor protein elongin c (eloc), cul2 and cul5 direct the assembly of distinct e3 complexes : crl2 with von hippel - lindau (vhl) or related bc box proteins, and crl5 containing suppressors of cytokine signaling (socs)-box proteins (figure 4). however, it is unclear whether cul2 and cul5 recognize specific determinants within vhl and socs - box proteins, respectively. a recent study provided some insight into how cul5 assembles into a crl5 complex with the hiv protein virion infectivity factor (vif ; a socs - box protein), thereby yielding an e3 ligase that targets the human antiviral protein apobec3 g for proteasomal degradation. it seems that loops 2 and 5 of cul5 are engaged in interactions with vif 's socs - box and zinc finger motif (h-(x)5-c-(x)17 - 18-c-(x)3 - 5-h), respectively. in summary, it seems that to assemble crls, cullins not only bind to a common recognition fold in the adaptor, such as the skp1/btb / poz motif (table 2), but also form interface interactions with structural determinants within the substrate - recognition molecules (srms) that include f - box, btb, vhl and socs proteins. however, future structural and biochemical studies, using a larger set of substrates, are required to more rigorously define the ' srm determinants '. to understand the differential ability of cul2 and cul5 to assemble crl2 and crl5, respectively, it is critical to solve their structures, especially the amino terminus. there are no structures available that reveal the three - dimensional organization of either cul7 or parc. although cul7 resembles cul1 in using the skp1 adaptor, it remains to be determined how cul7 selects f - box and wd - repeat - domain - containing protein 8 (fbw8). at present, it is also unclear whether parc forms a multi - subunit complex. cullin family proteins are involved in a diverse array of functions, including cell - cycle control, dna replication and development. the major physiological functions of the cullin family proteins have been revealed by genetic ablation experiments in a variety of metazoan model organisms, including mouse, c. elegans and drosophila (table 3). in arabidopsis, crls regulate hormonal signaling, light responses, circadian rhythms and photomorphogenesis (for a recent review, see). the cullin family proteins seem to be widely expressed and to locate both to the nucleus and cytoplasm, but there are no compelling data suggesting that cullin activity is controlled by subcellular localization or by differential expression in a tissue - specific manner. major physiological roles of cullins revealed by deletion studies with model organisms e, embryonic day ; ko, knockout. cul1 and cul3 mouse knockout experiments have revealed their indispensable roles in cell cycle progression and early embryogenesis (table 3). it was the pioneering work using the budding and fission yeast systems that led to the discovery of cullins and other crl components and their role in cell cycle control (reviewed in). work in c. elegans and drosophila has demonstrated the requirement of cul1 for cell cycle progression (table 3). mechanistically, it is believed that the cul1-based scf regulates the mammalian cell cycle, at least in part, by using the skp2 f - box protein, which directs the ubiquitin - dependent degradation of p27 and p21 (inhibitors of cyclin - dependent kinases), thereby activating cyclin - dependent kinases. scf malfunction has been linked to malignancy, as mutations in the fbw7 f - box protein are frequently found in a variety of human cancers. studies with cul4 deletion in c. elegans have established a crucial role for cul4 in dna replication (table 3). it is well accepted that the crl4 complex with the dcaf protein cdt2 as substrate - recognition molecule (referred to as crl4) targets the replication initiation factor cdt1 for degradation, thereby preventing dna re - replication. in mammals, cul4a and cul4b are believed to be functionally redundant, as deletion of cul4a in mice resulted in viable animals and relatively subtle phenotypes (table 3). clearly, complete understanding of the physiological functions of cul4 in mouse development requires future studies with animals lacking cul4b and cul4a / cul4b.. however, crl2has a critical role in control of oxygen homeostasis, acting by targeting the subunit of hypoxia - inducing transcription factor (hif) for degradation (reviewed in). tissue - specific gene targeting of vhl in mice has demonstrated that efficient execution of crl2-mediated hif-1 proteolysis under normal levels of oxygen is fundamentally important for survival, proliferation, differentiation and normal physiology of many cell types (reviewed in). these studies have explained the tumor suppressor function of vhl, whose germline mutations inactivate its ability to form the crl2 complex or bind to hif-, leading to the formation of highly vascularized tumors such as renal clear - cell carcinomas. two hereditary human diseases have been linked to genes encoding members of the cullin protein family. mutations in the cul7 gene were linked to 3-m syndrome (online mendelian inheritance in man (omim) i d 273750), an autosomal - recessive disorder characterized by pre- and postnatal growth retardation (final height 3 to 4 standard deviations below the mean for the population), facial dysmorphism, large head circumference, normal intelligence, and skeletal anomalies that include long slender tubular bones and tall vertebral bodies. identified cul7 gene mutations in 52 out of 62 cases (84%), arguing for cul7 as the major disease gene of 3-m syndrome. the mutations were located throughout the cul7 gene and most are predicted to cause premature termination of translation. reverse transcriptase (rt)-pcr analysis of patient fibroblast mrna detected a cul7-specific transcript, but at reduced levels, arguing that cul7 mrna is expressed at least in a subset of 3-m syndrome patients. approximately 50% of the mutations identified by huber. are located within the cullin homology domain (exons 19 to 24), which is responsible for roc1 binding. biochemical characterization of the cul7 nonsense and missense mutations arg1445x (where x indicates a stop codon) and his1464pro, respectively, were shown to render cul7 deficient in recruiting roc1. arg1445x was predicted to yield a truncated cul7 polypeptide (lacking the 254 carboxy - terminal amino acids), and his1464pro was predicted to introduce a structural alteration in the cullin homology domain. a study by maksimova. identified 43 patients from 37 yakut families, a geographically isolated ethnic group in russia, with a short stature syndrome similar to 3-m syndrome. a common mutation in the cul7 gene, insertion t at position 4582 in exon 25, was identified that is predicted to cause a frameshift and subsequent premature stop codon at position 1553 (q1553x). given that cyclin d1 and insulin receptor substrate 1 (irs-1) are potential proteolytic targets of the cul7 e3 ligase, it is tempting to speculate that either disturbed cyclin d1-dependent mechanisms or dysregulated irs-1-mediated signaling pathways might contribute to the pathomechanism of 3-m syndrome. altogether, studies with 3-m and yakut patients, combined with proliferative defects observed in cul7 knockout mice (table 3), have strongly suggested a prominent role for cul7 in growth regulation (reviewed by). several familial mutations in the cul4b gene were associated with x - linked mental retardation syndrome (xlmr ; omim 300639). the authors reported three truncating, two splice - site and three missense variants at conserved amino acids in the cul4b gene on xq24 in 8 of 250 families (3%) with xlmr. during adolescence of these patients a syndrome emerged with delayed puberty, hypogonadism, growth retardation, foot abnormalities, relative macrocephaly, central obesity, aggressive outbursts and seizures. the complex phenotype of patients with cul4b mutations argues for pleiotropic roles of cul4b that remain to be determined. the cullin - based crls function through their cognate substrate - recognition molecules, such as the f - box, socs, btb and dcaf proteins (figure 4 and table 2). the f - box, socs, btb and dcaf protein families each contain a distinct motif that is recognized by an adaptor molecule, which is linked to a cognate cullin (figure 4). for example, humans contain about 61 f - box proteins, all of which can bind to skp1 through the f - box domain. through skp1, which binds to cul1 (table 2), most of the f - box proteins can thus be assembled into the scf e3 complex (figure 4). bioinformatics studies have identified hundreds of human genes that are predicted to encode f - box, socs, btb and dcaf proteins, thereby potentially forming over 200 crls (figure 5). although the extent of the crl family requires experimental verification, it is nonetheless reasonable to assume that crls target numerous cellular protein substrates and hence have an impact on all biological processes. in addition, studies with f - box proteins, including -trcp, skp2 and fbw7, demonstrate an ability of one substrate recognition protein to bind multiple substrates, thereby expanding the functional range of crls. for recent reviews on the diverse targeting functions of these proteins, see (f - box family), (btb family) and (dcaf family). pie chart of the numbers of human e3 ubiquitin ligases, estimated from the numerical distribution of genes predicted to encode e3 ring or hect polypeptides or, in the case of crls, the substrate - recognition molecules that include f - box, socs, btb with 3-box and dcaf proteins. numbers refer to human gene numbers for f - box, socs, hect, u - box and non - crl ring proteins, derived from li.. kay hofmann has kindly provided the tally of human genes encoding btb and 3-box proteins (personal communication). the hofmann estimation is based on the structural work from schulman and colleagues, which identified a critical role for the 3-box in the assembly of crl3 with the spop btb protein. 72 btb - only (without 3-box) genes have not been counted for the estimation of crl3s. most, if not all, cullins are found covalently conjugated with an ubiquitin - like molecule, nedd8. this modification, termed neddylation, activates the e3 ligase activity of crls by promoting substrate polyubiquitination (reviewed by). recent studies have suggested conformation - based mechanisms that explain the activating role of neddylation. in vitro mutagenesis experiments have suggested that the interactions between human roc1 and cul1 's carboxy - terminal tail in the unmodified state render scf inactive. it was proposed that the conjugation of nedd8 to the residue k720 of cul1 induces drastic conformational changes in cul1 that liberate roc1, thereby driving scf into an active state. this hypothesis was supported by recent structural studies by schulman and colleagues that revealed extensive conformational changes in cul5 when conjugated with nedd8. another activating mechanism, proposed by schulman and colleagues, suggests that the neddylation - mediated conformational changes in cullin enabled the repositioning of the ring - tethered ubiquitin - loaded e2 to a bound substrate for catalysis. in support of this model, in vitro cross - linking experiments have revealed that neddylation brought a scf substrate into a close proximity to an e2 ubiquitin - conjugating enzyme. neddylation is reversed by the cop9 signalosome, which enzymatically removes nedd8 from a cullin molecule. cop9 is an eight - subunit complex that was originally identified as a suppressor of plant photomorphogenesis. it was shown that cop9 's jab1/csn5 subunit contains a jab1/mpn domain metalloenzyme (jamm) motif critical for cop9 's nedd8 isopeptidase activity. it is thus believed that crl activities are dynamically controlled by cullin neddylation - deneddylation cycles. it was observed that an scf complex bound to a substrate contained higher levels of neddylated cul1, suggesting that substrate - e3 interactions may trigger neddylation. crl is also regulated by cullin - associated and neddylation - dissociated-1 (cand1), which inhibits the e3 ligase activity of crls by binding to all cullins in their un - neddylated forms. the cul1-cand1 interaction is understood at the structural level, as the human cul1-rbx1-cand1 complex showed that cand1 binds both the cul1 amino and carboxyl termini. however, a recent study has revealed that only a small fraction of cullin is bound to cand1 in human cells. by organizing crls that presumably direct numerous substrates to ubiquitin - dependent degradation, the cullin family proteins build a cellular regulatory network of fundamental importance in controlling protein homeostasis, thereby altering a wide range of biological processes, from cell cycle regulation to signal transduction. although bioinformatics predicts a large number of crls (figure 5), we still have knowledge on only a handful of substrates. proteomics - based approaches and newly developed global protein stability profiling technology have proven effective in the identification of novel substrates. however, it remains to be seen whether these methods, coupled with agents that affect cell signaling, could lead to the isolation of substrates, whose turnover rates are dictated by often transient post - translational modifications, such as phosphorylation, prolyl hydroxylation and glycosylation. although the assembly of a majority of crls is understood in considerable detail (table 2 and figure 4), little is known about the control of the assembly in cells. it has long been suggested that the substrate - crl interactions dictate the cullin neddylation - deneddylation cycle, which turns on and off the crl 's e3 ubiquitin ligase activity. however, the mechanism by which this is achieved is elusive. given the intricate role of cullins and crls in a multitude of biological processes, it is likely that cullin dysfunction will emerge as a pathogenetic factor in diseases. indeed, cul7 and cul4b mutations have been identified in human disorders, but further studies are required to determine the underlying pathomechanisms. genetic studies in organisms from yeast to mouse have revealed a prominent role for cullins and crls in cell - cycle progression (table 3). recent advances have validated efforts in drug - targeting the ubiquitin - proteasome system, with the proteasome inhibitor bortezomib now approved for the treatment of patients with multiple myeloma or mantle cell lymphoma (reviewed by). a small molecule inhibitor (mln4924) suppressing the nedd8 activating pathway is currently in clinical trials, having demonstrated success in tumor suppression in animal model studies, and two small molecule inhibitors have been identified recently to inhibit scf activities by different mechanisms. the ubiquitin - proteasome system is a selective protein degradation pathway in which a substrate is first tagged with a chain of ubiquitin and the resulting modified protein is then recognized by the 26s proteasome, where proteolysis of the substrate takes place. first, the chemically inert ubiquitin molecule is activated in an atp - dependent reaction by forming a thioester bond between the carboxy - terminal carboxyl group of ubiquitin and the catalytic cysteine of an e1 activating enzyme. second, in a trans(thio)esterification reaction ubiquitin is transferred to the active site cysteine of an e2 ubiquitin - conjugating enzyme. in the last step, an e3 ubiquitin ligase functions to orchestrate the transfer of ubiquitin to a substrate protein, forming an isopeptide bond between the ubiquitin carboxy - terminal glycine residue and substrate lysine -amino group. the action of an e3 typically involves recognition of a specific degradation motif (degron) on the substrate. the human genome encodes two e1 activating enzymes, 37 e2 conjugating enzymes and more than 500 e3 ubiquitin ligases. a substrate protein can be conjugated with just one ubiquitin (monoubiquitination), one ubiquitin molecule at different lysines residues (multiubiquitination) or by a chain of several ubiquitin moieties (polyubiquitination). ubiquitin contains seven lysine residues (lys6, lys11, lys27, lys29, lys33, lys48 and lys63) that can act as acceptors and result in entirely different chain conformations. for instance, although lys48-linked chains are the canonical recognition motif for the proteasome, lys63-linked chains have important non - degradative roles in cell signaling, dna - damage response and endocytosis. monoubiquitination typically has non - proteolytic functions, such as the internalization of cell - surface receptors. e3 ubiquitin ligases are a diverse group of enzymes that recognize both a substrate protein and an e2 ubiquitin - conjugating enzyme. e3 ubiquitin ligases can be subdivided into two major classes : hect - type and ring - type e3 ligases. the single - molecule hect - type e3 ligases are characterized by a homologous to the e6-ap carboxyl terminus (hect) domain that forms a thioester intermediate with ubiquitin as a prerequisite for ubiquitin transfer to the substrate protein. in contrast, ring - type e3 ligases use ring (really interesting new gene)-zinc finger domains to recruit and allosterically activate an ubiquitin - charged e2 enzyme for direct ubiquitin transfer to the substrate. ring finger domains have a characteristic architecture of three strands, one -helical domain and two free loops that are arranged by znions. the loops are stabilized by a cluster of cysteine residues and up to two histidines. u - box e3 ligases are a subgroup of the ring - type e3s and contain a structurally modified ring - motif (the u - box) that lacks the ability to chelate znions. of about 300 ring proteins expressed in human cells, the multi - subunit cullin - ring ligase (crl) complexes constitute the major group and are characterized by two signature components : a cullin (cul) scaffold protein and the ring - finger protein roc1 or roc2 (also known as rbx1/hrt1 or rbx2, respectively). we thank r deshaies for helping in the preparation of figure 5 and k hofmann for sharing his bioinformatics information on estimation of the number of btb proteins with a 3-box. we thank maren hieber, kathleen meyer, wiebke schulze and benjamin wolf for critical reading of the manuscript. as was supported by the german research foundation (dfg) grant sa 1706/3 - 1 and the marie curie international reintegration grant 256584. | summarycullin proteins are molecular scaffolds that have crucial roles in the post - translational modification of cellular proteins involving ubiquitin. the mammalian cullin protein family comprises eight members (cul1 to cul7 and parc), which are characterized by a cullin homology domain. cul1 to cul7 assemble multi - subunit cullin - ring e3 ubiquitin ligase (crl) complexes, the largest family of e3 ligases with more than 200 members. although cul7 and parc are present only in chordates, other members of the cullin protein family are found in drosophila melanogaster, caenorhabditis elegans, arabidopsis thaliana and yeast. a cullin protein tethers both a substrate - targeting unit, often through an adaptor protein, and the ring finger component in a crl. the cullin - organized crl thus positions a substrate close to the ring - bound e2 ubiquitin - conjugating enzyme, which catalyzes the transfer of ubiquitin to the substrate. in addition, conjugation of cullins with the ubiquitin - like molecule nedd8 modulates activation of the corresponding crl complex, probably through conformational regulation of the interactions between cullin 's carboxy - terminal tail and crl 's ring subunit. genetic studies in several model organisms have helped to unravel a multitude of physiological functions associated with cullin proteins and their respective crls. crls target numerous substrates and thus have an impact on a range of biological processes, including cell growth, development, signal transduction, transcriptional control, genomic integrity and tumor suppression. moreover, mutations in cul7 and cul4b genes have been linked to hereditary human diseases. |
echo - endoscopes are much stiffer and thicker than regular endoscopes. during the procedure of endoscopic ultrasound (eus) procedure, water irrigation is a very useful for imaging but very dangerous if the patient receive the procedure under anesthesia without intubation. in this study, we evaluated the safety and effectiveness of nitrous oxide (n2o) (70%) for eus. among 4230 patients who underwent eus from december 2012 to december 2013, 347 out patients who need irrigation during the procedure were enrolled and divided into group a. group a (n2o sedation group, n = 162) and group b (no sedative group, n = 185). for group the mixture rate of n2o and o2 was 45 - 65% and controlled by nurses according to individual effect. the cardiorespiratory functions, procedure duration, complications, and visual analog scale (vas) of the patient and examiner satisfaction were compared. both group had comparable diagnostic accuracy, but vas of the patients who received n2o sedation was higher than that in group b (7.5 vs. 4.5, p < 0.05) and the examiner procedural satisfaction ratings of group a was also better than that of group b (8.1 vs. 5.1, p < 0.05) ; most patients in group a reported willingness to reconsider n2o for a future eus. n2o for eus is comfortable, safe and feasible, especially for the procedures that need irrigation. | objective : echo - endoscopes are much stiffer and thicker than regular endoscopes. during the procedure of endoscopic ultrasound (eus) procedure, water irrigation is a very useful for imaging but very dangerous if the patient receive the procedure under anesthesia without intubation. in this study, we evaluated the safety and effectiveness of nitrous oxide (n2o) (70%) for eus.methods:among 4230 patients who underwent eus from december 2012 to december 2013, 347 out patients who need irrigation during the procedure were enrolled and divided into group a. group a (n2o sedation group, n = 162) and group b (no sedative group, n = 185). for group a, patients received n2o by inhalation during the procedure. the mixture rate of n2o and o2 was 45 - 65% and controlled by nurses according to individual effect. the cardiorespiratory functions, procedure duration, complications, and visual analog scale (vas) of the patient and examiner satisfaction were compared.results : both group had comparable diagnostic accuracy, but vas of the patients who received n2o sedation was higher than that in group b (7.5 vs. 4.5, p < 0.05) and the examiner procedural satisfaction ratings of group a was also better than that of group b (8.1 vs. 5.1, p < 0.05) ; most patients in group a reported willingness to reconsider n2o for a future eus. no pulmonary aspiration occurred in this study.conclusions:n2o for eus is comfortable, safe and feasible, especially for the procedures that need irrigation. |
injury to superficial nerves in the foot and ankle can lead to painful sequelae that have a profound impact on patient quality of life. these nerve injuries are often the consequence of trauma or iatrogenic injury during operative interventions. in a recent report, deng found neurologic injury to be the most common complication following ankle arthroscopy. despite the relative prevalence of these injuries operative strategies range from neurectomy or neurolysis to neuroma excision with mobilization and burial of the proximal nerve. neurectomy and neurolysis have failed to yield durable pain relief, and extensive nerve mobilization for transposition and burial necessitates greater operative morbidity. an ideal approach would limit the operative field to the site of injury, simplify intraoperative decision making, provide durable pain relief, and offer the potential to restore sensation. experience with the use of nerve transfers to decrease neuroma pain in amputees has encouraged a novel approach to pain caused by injured nerves in the intact lower extremity. we attempted to repair injured nerves rather than to hide the painful neuroma, based on the concept that a reinnervation target is the key to achieving reduced sprouting and increased axon size, we resisted the sacrifice of an uninjured sensory nerve to serve as a donor graft. we hypothesized that pain associated with chronic neuromas and neuromas - in - continuity in the lower extremity could be effectively managed with resection of the involved nerve segment followed by nerve allograft reconstruction of the resulting gap. the purpose of this study was to evaluate the efficacy of this approach based on a comparison of prospectively collected pain assessments. after obtaining a complete history and review of any prior operative reports, physical examination consisted of a peripheral pulse exam and documentation of all prior operative scars. inclusion criteria for this study included lower extremity patients with a physical examination consistent with the point tenderness of localized nerve pain. typically, patients had decreased sensation distal to the area of greatest tenderness, though the extent and character of this sensory loss was variable. all patients in this series responded to a 1 ml injection of lidocaine just proximal to the area of greatest tenderness. lack of improvement would lead to a repeat office examination for reinjection in the area of the same nerve or the injection of an adjacent nerve with overlapping territory. exclusion criteria for the study included a lower extremity without documented pulsatile blood flow or lack of symptom improvement with local anesthetic injection. imaging with ultrasound or magnetic resonance imaging was not a critical component of the diagnostic evaluation. overall, 26 patients met inclusion criteria for the study. mean age in the cohort was 46 (range 18 - 75). typically, with the aid of a tourniquet, a 4- to 5-cm long incision was made to identify the target nerve. neuromas had a rounded feel like a soft marble, whereas neuromas - in - continuity were palpable as a mildly swollen and firm change in the character of the nerve immediately under the area of greatest tenderness. dissection typically began a few centimeters proximal and distal to the area of greatest tenderness, and then went in each direction toward the damaged nerve segment (figure 1). the neuroma was sharply resected back to healthy fascicles both proximally and distally. in most cases, the gap created was under 3 cm, with the nearest joint positioned in full extension. the gap was then reconstructed using an appropriately sized nerve allograft (avance nerve graft, axogen inc, alachua, fl). the allograft was placed as an interposition graft and coapted end - to - end in a tension - free manner using 2 to 3 epineurial 7 - 0 polypropylene sutures placed under loupe magnification (figure 2). the nerve repair was typically performed after any associated orthopedic procedures such as ankle arthroscopy, hardware removal, or achilles tendon lengthening. neuroma - in - continuity of the sural nerve at the level of the lateral malleolus. nerve allograft placed as an interposition graft following complete resection of the involved sural nerve segment. a splint was used to immobilize the nerve allograft in its soft tissue bed for 2 weeks. preoperative neuroma pain was typically improved shortly after neuroma resection, and instead replaced with operative pain that was readily controlled with oral narcotics. the northwestern university institutional review board approved this retrospective review of a prospectively maintained database of consecutive patients who underwent reconstruction with a processed nerve allograft for management of a painful lower extremity nerve by the senior author (g.a.d.) between may 2010 and june 2015. patient demographic and operative information was reviewed with particular emphasis on identification of the involved nerve, location of the neuroma, and the length of nerve allograft used. the electronic medical record was queried for preoperative information, medication use, complications, and follow - up information. a standardized pain assessment was obtained for all patients on their initial presentation to the clinic with pain as a predominant symptom. the pain assessment included both an evaluation of maximum pain intensity on an accepted 0 - 10 ordinal scale (0 is no pain and 10 is the worst), as well as a validated assessment of pain behaviors and pain - related impairment of social and physical function (pain interference) using promis (patient reported outcomes measurement information system) instruments. the pain score and promis assessments were performed at each postoperative visit. for patients with multiple postoperative pain reports, the postoperative pain scores obtained at the longest duration since surgery were used for comparison with preoperative scores. promis is a validated, nih - supported set of instruments for measuring health status. the promis physical function item bank has previously been reported to be superior to conventional outcome measurements for assessment of physical function in the foot and ankle patient. in the case of the promis pain behavior and pain interference item banks, questions delivered via an iterative algorithm rapidly assess the effect of pain on a patient s behavior and daily function, respectively. promis outcomes are reported as t - scores, which have a population mean of 50 and standard deviation (sd) of 10. importantly, the raw score can be easily translated into a percentile corollary, which enables comparison between the subject s score and those harvested from a 21 000-person control population representative of the us general population. as such, the promis tools provide an easy means for comparison not only across an intervention, but also against age and sex - matched controls. while the minimum clinically important difference (mcid) for the pain behavior and interference instruments have not been determined, the mcid for the majority of promis instruments has been set at 0.5 sd or t - score value of 5. the 2 instruments utilized in this study determined different aspects of pain on a patient s life. the promis pain behavior instrument is designed to assess behaviors that would indicate to others that a patient is experiencing pain, such as wincing, crying, or verbal reports of pain. the pain interference instrument assessed the effect of pain interfering with social and recreational activities, as well as sleep. data analysis was performed with spss statistics (ibm corp, armonk, ny). pre- and postoperative promis behavior and interference scores, as well as ordinal pain scores, were compared using the wilcoxon rank - sum test. regression analysis was performed to determine if there were any significant predictors of decreased pain score of any type. after obtaining a complete history and review of any prior operative reports, physical examination consisted of a peripheral pulse exam and documentation of all prior operative scars. inclusion criteria for this study included lower extremity patients with a physical examination consistent with the point tenderness of localized nerve pain. typically, patients had decreased sensation distal to the area of greatest tenderness, though the extent and character of this sensory loss was variable. all patients in this series responded to a 1 ml injection of lidocaine just proximal to the area of greatest tenderness. lack of improvement would lead to a repeat office examination for reinjection in the area of the same nerve or the injection of an adjacent nerve with overlapping territory. exclusion criteria for the study included a lower extremity without documented pulsatile blood flow or lack of symptom improvement with local anesthetic injection. imaging with ultrasound or magnetic resonance imaging was not a critical component of the diagnostic evaluation. overall, 26 patients met inclusion criteria for the study. mean age in the cohort was 46 (range 18 - 75). typically, with the aid of a tourniquet, a 4- to 5-cm long incision was made to identify the target nerve. neuromas had a rounded feel like a soft marble, whereas neuromas - in - continuity were palpable as a mildly swollen and firm change in the character of the nerve immediately under the area of greatest tenderness. dissection typically began a few centimeters proximal and distal to the area of greatest tenderness, and then went in each direction toward the damaged nerve segment (figure 1). the neuroma was sharply resected back to healthy fascicles both proximally and distally. in most cases, the gap created was under 3 cm, with the nearest joint positioned in full extension. the gap was then reconstructed using an appropriately sized nerve allograft (avance nerve graft, axogen inc, alachua, fl). the allograft was placed as an interposition graft and coapted end - to - end in a tension - free manner using 2 to 3 epineurial 7 - 0 polypropylene sutures placed under loupe magnification (figure 2). the nerve repair was typically performed after any associated orthopedic procedures such as ankle arthroscopy, hardware removal, or achilles tendon lengthening. neuroma - in - continuity of the sural nerve at the level of the lateral malleolus. nerve allograft placed as an interposition graft following complete resection of the involved sural nerve segment. a splint was used to immobilize the nerve allograft in its soft tissue bed for 2 weeks. preoperative neuroma pain was typically improved shortly after neuroma resection, and instead replaced with operative pain that was readily controlled with oral narcotics. the northwestern university institutional review board approved this retrospective review of a prospectively maintained database of consecutive patients who underwent reconstruction with a processed nerve allograft for management of a painful lower extremity nerve by the senior author (g.a.d.) between may 2010 and june 2015. patient demographic and operative information was reviewed with particular emphasis on identification of the involved nerve, location of the neuroma, and the length of nerve allograft used. the electronic medical record was queried for preoperative information, medication use, complications, and follow - up information. a standardized pain assessment was obtained for all patients on their initial presentation to the clinic with pain as a predominant symptom. the pain assessment included both an evaluation of maximum pain intensity on an accepted 0 - 10 ordinal scale (0 is no pain and 10 is the worst), as well as a validated assessment of pain behaviors and pain - related impairment of social and physical function (pain interference) using promis (patient reported outcomes measurement information system) instruments. the pain score and promis assessments were performed at each postoperative visit. for patients with multiple postoperative pain reports, the postoperative pain scores obtained at the longest duration since surgery were used for comparison with preoperative scores. promis is a validated, nih - supported set of instruments for measuring health status. the promis physical function item bank has previously been reported to be superior to conventional outcome measurements for assessment of physical function in the foot and ankle patient. in the case of the promis pain behavior and pain interference item banks, questions delivered via an iterative algorithm rapidly assess the effect of pain on a patient s behavior and daily function, respectively. promis outcomes are reported as t - scores, which have a population mean of 50 and standard deviation (sd) of 10. importantly, the raw score can be easily translated into a percentile corollary, which enables comparison between the subject s score and those harvested from a 21 000-person control population representative of the us general population. as such, the promis tools provide an easy means for comparison not only across an intervention, but also against age and sex - matched controls. while the minimum clinically important difference (mcid) for the pain behavior and interference instruments have not been determined, the mcid for the majority of promis instruments has been set at 0.5 sd or t - score value of 5. the 2 instruments utilized in this study determined different aspects of pain on a patient s life. the promis pain behavior instrument is designed to assess behaviors that would indicate to others that a patient is experiencing pain, such as wincing, crying, or verbal reports of pain. the pain interference instrument assessed the effect of pain interfering with social and recreational activities, as well as sleep. data analysis was performed with spss statistics (ibm corp, armonk, ny). pre- and postoperative promis behavior and interference scores, as well as ordinal pain scores, were compared using the wilcoxon rank - sum test. regression analysis was performed to determine if there were any significant predictors of decreased pain score of any type. mean preoperative pain score on the ordinal pain scale was 7.5. in terms of the promis pain assessment, the mean preoperative pain behavior t score was 63, with a mean pain interference t score of 68. this translates to mean population percentile scores of the 88th and 92nd percentiles, respectively. it follows that these patients had multiple interventions for pain. at the time of the preoperative pain assessment, 22 patients (85%) were actively seeing a pain specialist for management of their pain. seventeen patients (65%) were taking over - the - counter pain medications and 13 patients (50%) were taking prescription medications for pain control. thirteen patients (50%) had undergone a previous operative procedure intended to address their pain, with 2 patients having undergone multiple procedures. of these 13 patients, the remaining 7 patients had undergone nonneurologic procedures in an attempt to address their pain. all patients in the study underwent excision of the involved nerve segment and immediate nerve repair with a processed nerve allograft. nerves repaired were sural (10), superficial peroneal (9), common digital nerve (5), deep peroneal (1) and lateral plantar nerve (1) (figure 3). there were 8 end - neuromas and 18 neuromas - in - continuity. length of graft used was most commonly 3 cm (14 patients), with a mean length of 3.3 cm and a range of 1 to 6 cm. concurrent procedures were performed in 9 patients, including a tarsal tunnel release in 3 patients, 3 ankle arthroscopies, 1 midfoot arthrodesis, 1 achilles tendon lengthening, and 1 transposition flap for soft tissue coverage. twenty - six nerves were treated, but only the 22 patients had follow - up data sufficient to be included in the final analysis. overall, 22 patients had adequate follow - up (defined as 6 months or more) for postoperative ordinal and promis pain assessments (table 1). postoperatively, there were significant decreases in ordinal pain scores (figure 4), as well as pain behavior and interference scores (figure 5). ordinal pain scores decreased by a mean of 2.6 points (range + 2 to 8). pain behavior t score decreased by 7.3 (range + 2 to 22), a mean percentile decrease of 24%. pain interference t score decreased by a mean of 11.3 (range + 2 to 27) with a mean percentile change of 30.7% (figure 5). no single variable was a significant predictor of decrease in any type of pain score on regression analysis. there were no significant differences in the treatment of end neuromas and neuromas - in - continuity. there were also no differences in outcomes between patients who had prior operative treatment or no previous surgeries. mean postoperative pain scores were an ordinal pain score of 5, behavior raw score of 56 (64th percentile), and interference raw score of 57 (percentile 62th). individual patient data : nerve location, neuroma type, prior nerve surgery, graft length, and change in pain outcomes. abbreviations : dpn, deep peroneal nerve ; lpn, lateral plantar nerve ; nic, neuroma - in - continuity ; promis, patient reported outcomes measurement information system ; spn, superficial peroneal nerve. change in mean ordinal pain score after neuroma excision and gap repair with processed allograft (p =.016 ; n = 22 patients). change in promis pain behavior and interference t scores after neuroma excision and gap repair with processed allograft. all differences significant, p <.006 ; n = 22 patients. one patient, with a neuroma - in - continuity of the tibial side digital nerve of the great toe had a repeat procedure to treat a recurrence of local neuroma pain. in this patient, a new neuroma developed at the proximal allograft coaptation site, and no nerve fascicles were found on histology within the allograft. it was suspected that the previous neuroma resection had not extended sufficiently proximal to enable allograft coaptation with healthy nerve fascicles. in the revision, an autogenous motor nerve branch to the vastus lateralis muscle was used as a graft to span the 5-cm gap of the re - resected neuroma and failed nerve allograft. follow - up in this patient was not sufficient to be included in this manuscript. mean preoperative pain score on the ordinal pain scale was 7.5. in terms of the promis pain assessment, the mean preoperative pain behavior t score was 63, with a mean pain interference t score of 68. this translates to mean population percentile scores of the 88th and 92nd percentiles, respectively. it follows that these patients had multiple interventions for pain. at the time of the preoperative pain assessment, 22 patients (85%) were actively seeing a pain specialist for management of their pain. seventeen patients (65%) were taking over - the - counter pain medications and 13 patients (50%) were taking prescription medications for pain control. thirteen patients (50%) had undergone a previous operative procedure intended to address their pain, with 2 patients having undergone multiple procedures. of these 13 patients, the remaining 7 patients had undergone nonneurologic procedures in an attempt to address their pain. all patients in the study underwent excision of the involved nerve segment and immediate nerve repair with a processed nerve allograft. nerves repaired were sural (10), superficial peroneal (9), common digital nerve (5), deep peroneal (1) and lateral plantar nerve (1) (figure 3). length of graft used was most commonly 3 cm (14 patients), with a mean length of 3.3 cm and a range of 1 to 6 cm. concurrent procedures were performed in 9 patients, including a tarsal tunnel release in 3 patients, 3 ankle arthroscopies, 1 midfoot arthrodesis, 1 achilles tendon lengthening, and 1 transposition flap for soft tissue coverage. twenty - six nerves were treated, but only the 22 patients had follow - up data sufficient to be included in the final analysis. overall, 22 patients had adequate follow - up (defined as 6 months or more) for postoperative ordinal and promis pain assessments (table 1). postoperatively, there were significant decreases in ordinal pain scores (figure 4), as well as pain behavior and interference scores (figure 5). ordinal pain scores decreased by a mean of 2.6 points (range + 2 to 8). pain behavior t score decreased by 7.3 (range + 2 to 22), a mean percentile decrease of 24%. pain interference t score decreased by a mean of 11.3 (range + 2 to 27) with a mean percentile change of 30.7% (figure 5). no single variable was a significant predictor of decrease in any type of pain score on regression analysis. there were no significant differences in the treatment of end neuromas and neuromas - in - continuity. there were also no differences in outcomes between patients who had prior operative treatment or no previous surgeries. mean postoperative pain scores were an ordinal pain score of 5, behavior raw score of 56 (64th percentile), and interference raw score of 57 (percentile 62th). individual patient data : nerve location, neuroma type, prior nerve surgery, graft length, and change in pain outcomes. abbreviations : dpn, deep peroneal nerve ; lpn, lateral plantar nerve ; nic, neuroma - in - continuity ; promis, patient reported outcomes measurement information system ; spn, superficial peroneal nerve. change in mean ordinal pain score after neuroma excision and gap repair with processed allograft (p =.016 ; n = 22 patients). change in promis pain behavior and interference t scores after neuroma excision and gap repair with processed allograft. one patient, with a neuroma - in - continuity of the tibial side digital nerve of the great toe had a repeat procedure to treat a recurrence of local neuroma pain. in this patient, a new neuroma developed at the proximal allograft coaptation site, and no nerve fascicles were found on histology within the allograft. it was suspected that the previous neuroma resection had not extended sufficiently proximal to enable allograft coaptation with healthy nerve fascicles. in the revision, an autogenous motor nerve branch to the vastus lateralis muscle was used as a graft to span the 5-cm gap of the re - resected neuroma and failed nerve allograft. follow - up in this patient was not sufficient to be included in this manuscript. localized nerve pain has received increased attention as a source of chronic disability after trauma. it has also been identified as the most common complication following orthopedic and podiatric interventions in the foot and ankle. the mixed training backgrounds of providers managing these conditions and the lack of a single reliable treatment method has led to significant variability in management. all management strategies are challenged by the superficial location of the injured nerves and the constant pressure applied by shoe wear. the lack of a simple and standardized treatment algorithm has often relegated these patients to pain management clinics. end neuromas have traditionally been treated with excision and burial of the nerve ending in tissues thought to either inhibit new neuroma formation or to render the recurrent neuroma less susceptible to movement and pressure. in comparison, neuromas - in - continuity represent a quandary in management, as resection requires sacrifice of any residual distal innervation. many advocate neurolysis or nerve wrapping in these situations, but neither technique effectively ameliorates dysfunction due to intraneural scarring. the treatment of painful neuromas outlined in this study is guided by laboratory evidence demonstrating cessation of sprouting and evidence of coordinated regeneration when the proximal nerve is provided with a reinnervation target. in contrast, there is histologic evidence of neuroma recurrence with techniques that fail to provide the proximal nerve with this management strategy was also encouraged by a recent clinical report demonstrating improved pain outcomes when nerve grafting was performed in favor of neurectomy or nerve burial. unlike other treatments for foot and ankle neuroma pain, only nerve graft repair offers the potential for coordinated axon regeneration into a denervated bed. although there have been sporadic case reports citing favorable pain outcomes after autologous nerve grafting, our use of nerve allografts avoids the potential for new painful loci from autogenous nerve graft harvest. to simplify intraoperative decision making and effectively eliminate intraneural scarring, neuromas in continuity are resected back to healthy nerve fascicles, in comparable fashion to the management of end neuromas. comprehensive resection is justifiable, on account of the non - critical nature of the involved nerves. though sensory outcomes were not recorded in this study, the available evidence suggests that allograft reconstruction yields reliable sensory recovery when performed for small to moderate nerve gaps. likewise, the morbidity of resection is further diminished by sensory recovery because of collateral sprouting from adjacent nerves. the reconstructive component of this approach required an additional direct cost for the nerve allograft material. however, this cost was largely offset by avoidance of the additional operating room time, physician time, and anesthesia time that accompany techniques that require more extensive dissection or nerve autograft harvest. resection of the scarred nerve segment with allograft reconstruction was fast and straightforward, as evidenced by a mean operative time of 95 minutes. the procedure did not require special instrumentation or the use of a microscope. furthermore, preoperative evaluation minimized the use of costly imaging and relied chiefly on patient history, physical examination, knowledge of anatomy, and the use of diagnostic nerve blocks. this review of prospectively collected pain data suggests that use of the described nerve allograft technique yields a statistically significant and clinically important improvement in pain symptoms. the promis data collected also provides important insights into the degree of impairment experienced by patients with neuroma pain in the foot and ankle. it is noteworthy that at the time of presentation to our clinic, the patients in this cohort experienced a degree of impairment that ranked in the 88th percentile for pain behavior and 92nd percentile for pain interference. as a point of comparison, patients presenting to the same senior surgeon for carpal tunnel surgery over a similar time period exhibited a mean pain behavior score in the 64th percentile and pain interference score in the 62nd percentile. on presentation to our outpatient clinic the majority of patients were taking prescription pain medications, and half had previously undergone at least 1 operative procedure for management of their pain. given the relative novelty of the promis pain assessments, we included a commonly used ordinal scale to serve as a legacy measure of pain intensity. our technique yielded a significant improvement in pain intensity, which lends credence to the benefits identified by promis. while promis offers many insights, it is not a disease - specific instrument. as a result, pain outcomes can be negatively affected by confounding sources of pain that are unrelated to the nerves managed with this procedure. all of the patients in this series sustained nerve injuries as a result of trauma or iatrogenic causes, and thus had a high incidence of additional lower extremity pathology. the fact that promis provides a holistic measure of pain, rather than isolating nerve - related pain, makes the significant reduction in pain behaviors and pain - related impairment in this group of patients all the more impressive. although the data were collected prospectively, the study design and chart review were retrospective and thus susceptible to the inherent biases introduced by a retrospective study. likewise, the retrospective design of this study means that potentially useful preoperative data, such as a standardized preoperative sensory evaluation, could not be performed. early in the study period, the senior surgeon s efforts to measure sensory thresholds yielded such marked variability that thorough sensory examination was abandoned as a component of the preoperative evaluation. as a result, the extent of sensory recovery following allograft reconstruction was not studied. as mentioned above, collateral still, it can not be excluded that pain improvement is in some way related to the material properties of the nerve allograft rather than to coordinated nerve regeneration, as we hypothesized. additionally, while lengthy follow - up duration is the ideal, the moderate size of the nerve gaps reconstructed in this series make a mean follow - up of almost 16 months sufficient to enable identification of neuroma recurrence. perhaps the greatest limitation of this study is the absence of an alternative technique for comparison. unfortunately, these patients pre - dated the standardized pain assessments used in this study. in summary, in properly selected patients, painful sensory nerve neuromas of the lower extremity were improved with excision of the damaged nerve and grafting using processed nerve allograft. we found a statistically significant decrease in patient - reported pain at an average of 15.5 months after operative intervention. | background : localized nerve pain in the foot and ankle can be a chronic source of disability after trauma and has been identified as the most common complication following operative interventions in the foot and ankle. the superficial location of the injured nerves and lack of suitable tissue for nerve implantation make this pain refractory to conventional methods of neuroma management. we describe a novel strategy for management using processed nerve allografts to bridge nerve gaps created by resection of both end neuromas and neuromas-in-continuity.methods:a retrospective review of a prospectively maintained database was performed of all patients who received a processed nerve allograft for treatment of painful neuromas in the foot and ankle between may 2010 and june 2015. patient demographic and operative information was obtained, as well as preoperative and postoperative pain assessments using a conventional ordinal scale and promis (patient reported outcomes measurement information system) pain behavior and pain interference assessments. twenty - two patients were identified, with postoperative pain assessments occurring at a mean of 15.5 months after surgery.results:neuromas of the sural and superficial peroneal nerves were the most common diagnoses, with 3-cm nerve allografts being used as the interposition graft in the majority of cases. eight patients had end neuromas and 18 patients had neuromas in continuity. analysis of paired data demonstrated a mean ordinal pain score decrease of 2.6, with 24 and 31 percentage - point decreases in promis pain behavior and pain interference measures, respectively. all changes were significant (p <.002).conclusion : the painful sequelae of superficial nerve injuries in the foot and ankle was significantly improved with complete excision of the involved nerve segment followed by bridging of the resulting nerve gap with a processed nerve allograft. this approach limits surgery to the site of injury and reconstitutes the peripheral nerve anatomy.level of evidence : level iv, retrospective case series. |
peroxisome proliferator - activated receptors (ppars) are involved in the regulatory response processes of lipid and glucose metabolisms, adipocyte differentiation, and inflammatory response. the three peroxisome proliferator - activated receptors (ppars), (), and constitute a distinct subfamily in the superfamily of nuclear receptors. ppars are ligand - regulated transcription factors that control gene expression by binding to specific response elements within promoters. studies have shown that ppars are involved in the physiopathology of complex metabolic disorders, such as those related to metabolic syndrome, resulting in atherosclerosis and cardiovascular diseases. pparg regulates the expression of numerous genes involved in controlling cellular energy homeostasis, lipid metabolism, insulin action, and adipocyte differentiation. in a study using cell culture, uruno. showed the inhibitory effect of pparg agonists, controlling blood pressure, on angiotensin - ii induced aldosterone synthase expression and aldosterone secretion. an association of pparg gene variants with insulin resistance, type 2 diabetes, obesity, and hypertension has been shown [5, 6 ]. additionally, pparg activation is associated with increased consumption of free fatty acids by skeletal muscle and adipose tissue, inhibition of the expression of proinflammatory genes (tnf) that trigger atherosclerosis, and increased insulin sensitivity. atherosclerosis is usually preceded by endothelial dysfunction, whereas agonists have been reported to improve the function of these cells in type 2 diabetic patients and nondiabetic patients with coronary artery disease. besides the role played by pparg activation in regulating the metabolism of glucose and lipids, a possible regulatory role in the expression of proinflammatory adipokines has also been suggested [1, 6 ]. additionally, the renal protective effect of pparg agonists against nondiabetic renal disease and anticancer activities of pparg agonist was recently reported. it has been also shown that pparg variant (pparg c1a) is related to effects on the effectiveness of aerobic exercise training to increase aerobic fitness and insulin sensitivity. moreover, results suggest that the mitochondrial function associated with aerobic fitness and insulin resistance is deeply affected by the expression of coactivators (pgc-1 and pgc-1) of the pparg. therefore, pparg may be a promising candidate for regulation by maximal oxygen uptake. the cardiorespiratory fitness as defined by maximal oxygen uptake (vo2max) is modulated largely by physical activity level and genotype. a low vo2max has been shown to be associated with glucose intolerance, insulin resistance, type-2 diabetes mellitus [15, 16 ], and downregulation of the pparg mrna expression from skeletal muscle and peripheral mononuclear cells [17, 18 ]. however, it has not been demonstrated whether association between vo2max and pparg mrna expression is influenced by familial or genetic background. nevertheless, studies involving the analysis of the interaction between physical activity and genetic aspects have increased considerably in recent years, but knowledge about the impact or effect of physical exercise on genome or highlighting the limitations of genetic background in physical performance is preliminary. it has been argued that the human genome has evolved over a period when high levels of physical activity were essential for survival, which supports the view that there is a link between exercise and gene expression regulation. the direct influence of vo2max on pparg expression has not been previously examined, regardless of genetic background, but it can be evaluated using the discordant monozygotic (mz) twin model (cotwin case control study design). this experimental model allows estimating the effect of one discordant environmental factor [20, 21 ]. mz twins have the same inherited genes, and if they differ in a particular trait, the difference can be considered to be due to environmental factors. using this model, the influence of vo2max can be estimated, isolated from additional covariates that usually confound other studies. the aim of this study was to investigate the influence of vo2max on pparg mrna expression using mz twin model case control. ninety - eight twin pairs who attended private and public schools of rio claro, so paulo, were invited to participate in this study. among the twin pairs, 53 were girls and 45 were boys aged from 11 to 18 years. in 31 twin pairs, one or both twins declined participation and 13 pairs were not found. fifty - four twin pairs (35 girls and 19 boys) attended clinical and evaluation sessions. parental testing by microsatellite analysis was performed as previously described and revealed that the sample evaluated had 38 mz twin pairs and 16 dizygotic (dz) twin pairs. in the study, only mz twin pairs were included (24 girls and 14 boys, 14.3 2.1 years old ; 51.4 12.9 kg ; 158.2 10.6 cm). the cotwin control design is a powerful research methodology for studying the effects of environmental risk factors on the development of disease. in the case - control study, twin pairs, their parents, and/or guardians were previously informed about the experimental procedures and provided written consent for participation. 5093) of the so paulo state university (unesp), according to the norms of resolution 196/96 of the national health council on research involving humans. body mass and height were measured barefoot in light clothing to the nearest 100 g and 0.1 cm, respectively. body mass index (bmi) was calculated by dividing body mass (kg) by height (m). waist circumference was measured in triplicate, midway between the lowest rib and the superior border of the iliac crest, with a flexible tape. skinfold thicknesses were measured at the triceps (tr) and subscapular (sb) on the right side of the body with a harpenden skinfold calipers (holtain ltd., bryberian, uk). three sets of measurements were taken to the nearest 0.2 mm at each site and the mean of the three values was used. work - conducted maximal exercise test with gas exchange analysis (spiroergometry) was performed using treadmill model atl super (inbrasport, porto alegre, rs, brazil) with 1% inclination, in the morning and afternoon periods from 9:00 a.m. to 11:30 a.m. and from 2:00 p.m. to 6:00 p.m. in a room temperature chamber maintained between 20 and 25c. after a 5-minute adaptation period with treadmill at different speeds (4 to 7 km / h), the individuals remained five minutes on the treadmill at rest in standing position. at rest and during exercise test, respiratory minute volume (ve), oxygen intake (vo2), and carbon dioxide production (vco2) were continuously recorded by assessing pulmonary gas exchanges (metabolic analyzer vo2000 medgraphics medical graphics corp., st. paul, mn). the equipment was calibrated prior to the beginning of each individual test. then, the test was performed using protocol that provided initial speed of 4 km / h with progressive increase in workload of 1 km / h per minute until exhaustion (19 - 20/20 on the borg scale for perceived exertion or respiratory quotient (rq = vco2/vo2) of at least 1.1). vo2max was collected breath by breath and the value adopted for analysis of data was recorded as the average oxygen uptake during 30 s that preceded test interruption. heart rate was obtained by the polar s810 heart rate monitor (polar electro, finland). to apply the model of studies with mz twin pairs (case - control model), the absolute intrapair difference in vo2max 10 mlkgmin was used to define twin pairs discordant for vo2max. blood samples were collected from an antecubital vein using vacutainer tubes (vacutainer becton dickinson company, plymouth, uk) exactly one week after cardiorespiratory fitness assessment (vo2max), from the twin pairs under their parents ' care and authorization, at 7.30 a.m. after overnight fasting (10 to 12 h) and 30 min resting. approximately 150 l of blood was transferred to qiacard fta spots (qiagen, valencia, usa) for dna extraction and parental testing by microsatellite analysis. for mrna expression, 4 ml of blood was transferred to paxgene tubes (qiagen, valencia, usa) to further total rna extraction. total rna was isolated immediately after blood collection according to manufacturer 's instructions with minor modifications using paxgene blood rna kit (qiagen, valencia, usa). rna purity was determined by spectrophotometry (a260/a280 ratio) using the equipment nanodrop (nanodrop technologies, inc.,, santa clara, usa) and rna was regarded as intact when showing two distinct bands for 28s and 18s rna. the pparg mrna expression was measured by quantitative real - time pcr (q - pcr). cdna was synthesized using the high capacity reagent (applied biosystems, foster city, usa), according to manufacturer 's protocol using the thermocycler veriti 96 well thermal cycler (applied biosystems, foster city, usa), and it was stored at 20c. the cdna from total rna of peripheral blood leukocytes was amplified by qpcr using taqman amplifying system and abi 7500 equipment (applied biosystems, foster city, usa). primers and fluorophore - labeled probes specific for pparg and reference gene glyceraldehyde-3 phosphate dehydrogenase (gapd) (table 1) were designed using primer express (applied biosystems, foster city, usa) and primer premier 5.0 (premier biosoft, palo alto, usa) software. qpcr was performed under the following conditions : 20 l of final reaction volume containing taqman master mix 1 x (amplitaq dna polymerase, dntps with dutp, amperase ung) (applied biosystems, foster city, usa), 300 nm of primers, and 200 nm of labeled probes. the pcr amplification program consisted of (1) one cycle of 2 min at 50c (ung activation), (2) one cycle of 5 minutes at 95c (ung inactivation) ; (3) 40 cycles of 15 sec at 95c (denaturation) ; (4) 1 min at 60c (hybridization and extension). the fluorescence signals emitted by fluorophores of taqman probes were detected by abi prism 7500 (applied biosystems, foster city, usa). data were analyzed using the system 7500 sds software (applied biosystems, foster city, usa) that generates half - log curves of amplification signals and ct values. the ct values were used to calculate the relative mrna expression of pparg compared to gapd (reference gene) using the ct. it is a calculated formula : ct = (cttarget gene ctreference gene). total dna was extracted from whole blood samples in qiacard fta spots with qiaamp 96 dna blood kit (qiagen, valencia, usa) according to manufacturer 's recommendations. microsatellite analysis was carried out by pcr using the identifiler commercial kit, according to the manufacturer 's instructions (applied biosystems, foster city, eua). this assay allows the genotyping of 15 multiple informative genetic markers (csf1po, d2s1338, d3s1358, d7s820, d8s1179, d13s317, d16s539, d18s51, d19s433, d21s11, d5s51, fga, th01, tpox, vwf, and amelxloci), which allows for the identification of monozygotic and dizygotic twins. for normally distributed variables, one - way analysis of variance for multiple comparisons followed by a bonferroni 's post hoc test was used to compare the means (anthropometric traits) between twin pairs with high and low vo2max and wilcoxon 's signed rank test for intrapair comparison of nonnormally distributed data (gene expression). spearman 's rank coefficient was used to investigate correlations between vo2max - discordant and concordant mz twins. all statistical analyses were conducted using spss software version 20 (ibm spss), and alpha was set at p < 0.05. twin pairs were distributed into discordant (10 girls and 8 boys aged 13.9 2.2 years old) and concordant (38 girls and 20 boys aged 14.2 2.1 years old) for vo2max, and within each group, cotwins were divided into high and low vo2max. comparison of anthropometric characteristics for vo2max - discordant and concordant pairs is shown in table 2. there were no differences (between and within group) in age and anthropometry characteristics (body mass, height, bmi, waist circumference, and sum of skinfold tr + sb). as expected, cotwins from the discordant group (d - high versus d - low) showed difference in vo2max values (32.4 10.6 versus 45.9 10.0 mlkgmin, p = 0.025), while cotwins from the concordant group (c - high versus c - low) did not show difference in vo2max (38.8 9.8 versus 42.4 9.2 mlkgmin, p = 0.952). the analysis also revealed that discordant low cotwins (d - low) showed marginal difference in vo2max values compared for concordant high twins (c - high) (32.4 10.6 versus 42.4 9.2 mlkgmin, p = 0.050) (table 2). correlation analysis for vo2max and pparg mrna expression with anthropometric characteristics in mz twin 's pairs discordant and concordant for vo2max is shown in table 3. the vo2max was negatively correlated with the sum of skinfolds (0.540, p = 0.021) and it had a strong positive correlation with pparg mrna expression (r = 0.952, p = 0.001) in discordant mz twins. additionally, pparg expression was positively correlated with body mass (r = 0.714, p = 0.047) and height (r = 0.762, p = 0.028). the concordant mz twins demonstrated negative correlation of the vo2max with body mass (r = 0.290, p = 0.027), bmi (r = 0.472, p = 0.001), waist circumference (r = 0.426, p = 0.001), and sum of skinfolds (r = 0.739, p = 0.001). however, in mz group, there was no correlation between pparg mrna expression with anthropometric characteristics and vo2max (table 3). regarding the pparg mrna expression, it was observed that discordant cotwins with the high vo2max values (d - high) had 1.78-fold greater pparg mrna expression when compared to cotwins with the low vo2max values (d - low) (p = 0.048) (figure 1). in the concordant group, cotwins with low vo2max values (c - low) had 1.22-fold greater pparg mrna expression when compared to cotwins with high vo2max (c - high). however, there was no significant difference (p = 0.374) (figure 1). the major finding from this study indicates that vo2max is capable of affecting pparg mrna expression in vo2max - discordant mz twins regardless of genetic background. the results showed 30.6% higher vo2max values in cotwins discordant for vo2max (d - high) compared with cotwins with low vo2max values (d - low), but this difference is not associated with differences in anthropometric variables between twin 's pars discordant and concordant. the correlation analysis was performed for anthropometric characteristics with vo2max and pparg mrna expression in mz twin 's pairs with discordant and concordant for vo2max. according to results for vo2max - discordant mz twins, the vo2max values were negatively correlated with sum of skinfolds and strong positive correlation with pparg mrna expression. the pparg mrna expression was positively correlated with body mass and height. for vo2max - concordant mz twins, the vo2max values were negatively correlated with body mass, bmi, waist circumference, and sum of skinfolds. interestingly for this group, there was no correlation between pparg mrna expression with anthropometric characteristics and vo2max. in previous studies, there was correlation between obesity (bmi) and vo2max (r = 0.88, p < 0.05) in young subjects and a strong positive correlation between the ratio of pparg (splice variants y2/y1) mrna and the bmi (r = 0.70, p < 0.001) in obese patients. the expression of pparg mrna (splice variants y2) is increased in adipose tissue in obese men and women, and their direct correlation with bmi is unknown but it may be related to the expansion of adipose tissue mass. the pparg mrna expression in circulating monocytes from healthy individuals was increased after participating in a program of cycling exercise that caused a significant increase in vo2max. significant increase in leukocyte mrna expression for pparg was observed in healthy but previously sedentary individuals that participated in an 8-week low - intensity exercise program consisting of walking 10,000 steps, three times a week. we have demonstrated that children and adolescents with high vo2max showed higher pparg mrna expression. although the cardiorespiratory fitness of the mz twin pairs was evaluated only once (cross - sectional study), the method used to measure vo2max (indirect calorimety based in gas exchange) is more accurrate than the physical activity evaluation using questionnaires and surveys, which have been questioned. the absolute value of vo2max is one of the indices of an individual 's cardiorespiratory fitness to transport oxygen to working muscles. additionally the evidence suggests that physically active children have significantly better cardiorespiratory fitness levels than inactive children [30, 31 ]. in an animal study, the effect of acute and endurance exercise training on mrna expression pattern of the different ppars and ppara coactivator-1 alpha (pgc-1) in muscles that largely rely on either glycolytic (plantaris) or oxidative (soleus) metabolism was analyzed. moreover, ppar mrna levels were the lowest in skeletal muscle which is the most responsive to changes in physical activity levels. in spite of the important contribution of this study, there are some limitations such as the cut - off values of the discordant cardiorespiratory fitness and timing of blood sampling for rna extraction. the cardiorespiratory fitness (based on studies of familial aggregation and twins) shows significant genetic effects (5067%) [32, 33 ] ; our results should be interpreted with caution. among the assumptions of the study with twins, one of them admits that the phenotype carries great genetic background when showing low variance between mz twins. in classical studies with mz twin pairs, discordance is defined using criteria based on literature in which phenotype is a condition usually acquired. however, even in studies in which variance allows establishing discordance between identical twins (i.e., one twin is diabetic / obese / hypertensive / sedentary and the other is not), environmental and/or genetic factors that may have significantly influenced this discrepancy are still questioned [35, 36 ]. in addition, it was observed that the behavior of the cardiorespiratory fitness is unstable during life, being influenced by several factors such as age, gender, and physical activity level. it should be emphasized that, besides the improvement promoted by physical exercise on vo2max, the period corresponding to childhood and adolescence is marked by growth and gradual development of the cardiorespiratory system components that determine vo2max (lungs, heart, and muscles). in this sense, possible discrepancies in vo2max in this age group in genetically identical individuals would be more likely to be due to environmental factors (physical activity and/or exercise). in other words, the determinants that distinguish aerobic fitness of a mz twin pair compared to his cotwin could be due to environmental factors because, in this model, the changes observed in vo2max as a function of growth and development of the various systems would be under genetic control. considering the increase in vo2max caused by growth, reducing aerobic capacity at this stage is less likely than increasing it. thus, ignoring errors from evaluation, evaluator, and measurement equipment, it is possible that discordant cotwin pair with higher vo2max in this study is actually more active than his correspondent cotwin. we should also highlight that differences in the pparg mrna expression observed between discordant mz twins (d - high versus d - low) may be influenced by the timing of blood sample collection. however it is noteworthy that concordant twins (c - high versus c - low) underwent the same protocol, but did not show variability in pparg expression. there is evidence showing that the pparg mrna expression increases significantly (1.5- to 2.5-fold) up to 3 hours after exercise, but returning to basal levels within 24 h of exercise. reinforcing this information, it was observed that the mrna of the ppar family of transcription factors is sensitive to exercise training in skeletal muscle but is not particularly sensitive to acute bouts of endurance exercise. these findings would suggest that changes in the ppar mrna expression are most likely mediated by the accumulation of repeated bouts of exercise as opposed to single bouts of activity. regarding the cut - off value of 10 mlkgmin adopted to establish discordance between mz twin pairs in this study. in a longitudinal study with mz and dz twin pairs discordant for cardiorespiratory fitness (vo2 peak of 26.4 4.9 versus 32.5 5.5 mlkgmin), the researchers used lower cut - off values (6 mlkgmin) and emphasized that the differences within twin pairs were sufficient to identify metabolic disorders. in another study, researchers adopted 9% as cut - off value to determine discordances (mean of 18 10%) in the vo2max values of mz twins. the results revealed that the more and less active twins showed, respectively, vo2max values of 50.9 5.1 versus 43.4 6.7 mlkgmin. it is noteworthy that the relative difference between twins with higher and lower vo2max (discordant) in this study ranged from 16.9% to 42.1%, with mean of 30.6 9.5% (data not shown). it is also noteworthy that the absolute average of these differences was 13.5 3.7 mlkgmin for discordant pairs and approximately 7.5 mlkgmin for the study of hannukainen.. in conclusion, the results of this study suggest that the cardiorespiratory fitness evaluated by vo2max parameter may modulate the pparg mrna expression in childhood and adolescence, independently of the genetic background. considering that the pparg expression regulates lipid and glucose metabolism, insulin action and adipocyte differentiation the effect of cardiorespiratory fitness on pparg may contribute to the prevention of future metabolic diseases in adults such as type 2 diabetes. | the influence of cardiorespiratory fitness (vo2max) on anthropometric variables and pparg mrna expression was investigated. monozygotic twin pairs aged 1118 years were grouped into discordant (d) and concordant (c) high and low vo2max groups. vo2max was determined by progressive maximal exercise test on treadmill with gas exchange analysis. body mass (bm), bmi, waist circumference (wc), triceps (tr), and subscapular (sb) skinfold thicknesses were measured. twins from the discordant group had differences in vo2max values (d - high = 45.9 10.0 versus d - low = 32.4 10.6 mlkg1min1, p = 0.025), while no differences were found in the concordant group (c - high = 42.4 9.2 versus c - low = 38.8 9.8 mlkg1min1, p = 0.952). in discordant group, vo2max was negatively correlated with tr + sb (r = 0.540, p = 0.021) and positively correlated with pparg expression in leukocytes (r = 0.952, p = 0.001). moreover, pparg expression was directly correlated with bm (r = 0.714, p = 0.047) and height (r = 0.762, p = 0.028). in concordant twins, vo2max was inversely correlated with bm (r = 0.290, p = 0.027), bmi (r = 0.472, p = 0.001), wc (r = 0.426, p = 0.001), and tr + sb (r = 0.739, p = 0.001). twins d - high had 1.78-fold greater pparg expression when compared with twins d - low (p = 0.048). in conclusion, the cardiorespiratory fitness may modulate pparg expression in childhood and adolescence, independently of the genetic background. |
timed - pregnant (second parity) female sprague - dawley rats (charles river, kingston, ny) were received on gestation day 2. animals were individually housed in conventional tub cages with ad libitum access to food and water in an environmentally controlled room maintained on a 12-h light / dark cycle with light onset at 0600. all animal procedures were approved by the animal care and use committee of the johns hopkins university school of medicine. pregnant rats were assigned to two diet groups : standard chow diet (harlan teklad 2018, 17% kcal from fat ; n = 21) or high - fat diet (research diets, d12492, 60% kcal from fat, n = 21). all dams remained on their respective diets from gestation day 2 throughout gestation and lactation. beginning on gestation day 14, 10 dams from each diet group (n = 10, standard chow stress group and high - fat stress group) were subjected to an 8-day schedule of variable stress (table 1) (20). each stressor was applied during the light cycle unless noted otherwise. we selected a variable stress paradigm for our studies to prevent the animals from habituating to the stress, as has been documented using predictable stressors such as repeated restraint stress (29). stress was restricted to the 3rd week of gestation because the neural circuits that regulate the hpa axis and energy homeostasis, including the hypothalamus, cortex, and hippocampus, are rapidly developing during this period (30). schedule of variable stress during gestation the remaining control dams were exposed to normal animal room husbandry practices in the animal facility (n = 11, standard chow control group and high - fat control group). tail blood samples were collected from all dams on the morning of gestation days 14 (prestress) and 21 (poststress) for measurement of basal plasma corticosterone, leptin, and insulin. the day a litter was found was designated postnatal day 0. on postnatal day 1, litters were culled to 10 pups each (5 male and 5 female). pups were weaned on postnatal day 21 and housed in groups of two to three by sex and treatment group. half of each group (n = 45 pups) were weaned onto either standard chow or high - fat diet. on postnatal day 22 and postnatal day 70, rats were food - deprived overnight for 16 h with only water available. a baseline blood sample was taken via a small tail nick for determination of plasma insulin. baseline fasted blood glucose was determined at the same time by a handheld glucose meter (freestyle ; therasense, alameda, ca). an oral gavage of glucose (2.0 g / kg body wt, 20% glucose in sterile water solution) was then administered. blood samples were collected at 15, 30, 45, 60, and 120 min after glucose gavage to determine plasma insulin levels. male pups (n = 1 per litter) were killed on postnatal day 1, 7, 14, 21, and 90 by decapitation. blood was collected into a heparinized microcentrifuge tube, centrifuged at 4c to collect plasma, and stored at 80c for hormone analysis. fat pads (dorsosubcutaneous, inguinal, and retroperitoneal) were unilaterally dissected and weighed. the entire subscapular brown adipose tissue fat pad was removed and weighed. plasma hormone concentrations were determined by commercially available radioimmunoassay kits for corticosterone (for rats and mice ; mp biomedicals, solon, oh) and for leptin and insulin (both for rat ; millipore, billerca, ma). inter- and intra - assay variability for each assay, respectively, were as follows : corticosterone, 6.57.1% and 4.410.3% ; leptin, 3.05.7% and 2.04.6% ; and insulin, 8.59.4% and 1.44.6%. data were analyzed by statistica 7.0 (systat, tulsa, ok) by anova, repeated - measures anova, or student 's t tests for independent samples as appropriate. beginning on gestation day 14, 10 dams from each diet group (n = 10, standard chow stress group and high - fat stress group) were subjected to an 8-day schedule of variable stress (table 1) (20). each stressor was applied during the light cycle unless noted otherwise. we selected a variable stress paradigm for our studies to prevent the animals from habituating to the stress, as has been documented using predictable stressors such as repeated restraint stress (29). stress was restricted to the 3rd week of gestation because the neural circuits that regulate the hpa axis and energy homeostasis, including the hypothalamus, cortex, and hippocampus, are rapidly developing during this period (30). schedule of variable stress during gestation the remaining control dams were exposed to normal animal room husbandry practices in the animal facility (n = 11, standard chow control group and high - fat control group). tail blood samples were collected from all dams on the morning of gestation days 14 (prestress) and 21 (poststress) for measurement of basal plasma corticosterone, leptin, and insulin. the day a litter was found was designated postnatal day 0. on postnatal day 1, litters were culled to 10 pups each (5 male and 5 female). pups were weaned on postnatal day 21 and housed in groups of two to three by sex and treatment group. half of each group (n = 45 pups) were weaned onto either standard chow or high - fat diet. on postnatal day 22 and postnatal day 70, rats were food - deprived overnight for 16 h with only water available. a baseline blood sample was taken via a small tail nick for determination of plasma insulin. baseline fasted blood glucose was determined at the same time by a handheld glucose meter (freestyle ; therasense, alameda, ca). an oral gavage of glucose (2.0 g / kg body wt, 20% glucose in sterile water solution) was then administered. blood samples were collected at 15, 30, 45, 60, and 120 min after glucose gavage to determine plasma insulin levels. male pups (n = 1 per litter) were killed on postnatal day 1, 7, 14, 21, and 90 by decapitation. blood was collected into a heparinized microcentrifuge tube, centrifuged at 4c to collect plasma, and stored at 80c for hormone analysis. fat pads (dorsosubcutaneous, inguinal, and retroperitoneal) were unilaterally dissected and weighed. the entire subscapular brown adipose tissue fat pad was removed and weighed. plasma hormone concentrations were determined by commercially available radioimmunoassay kits for corticosterone (for rats and mice ; mp biomedicals, solon, oh) and for leptin and insulin (both for rat ; millipore, billerca, ma). inter- and intra - assay variability for each assay, respectively, were as follows : corticosterone, 6.57.1% and 4.410.3% ; leptin, 3.05.7% and 2.04.6% ; and insulin, 8.59.4% and 1.44.6%. data were analyzed by statistica 7.0 (systat, tulsa, ok) by anova, repeated - measures anova, or student 's t tests for independent samples as appropriate. there were no significant differences in maternal body weight among the groups before stress exposure (table 2). however, compared with beginning body weight on gestation day 2, dams maintained on high - fat diet gained significantly more weight during the first 2 weeks of gestation compared with dams fed standard chow diet (p < 0.05). the greater weight gain in high - fat dams may be attributable to significantly higher caloric intake of dams fed high - fat diet during gestation days 214 (p < 0.05). there were no significant differences in plasma corticosterone among the groups before stress. maternal body weight, food intake, and endocrine measures during gestation p < 0.05 vs. standard chow control group ; p < 0.05 vs. standard chow control group and standard chow stress group. stress during the last week of gestation (gestation days 1421) resulted in less body weight gain in stress groups (table 2). post hoc analysis revealed that dams in the high - fat stress group gained significantly less body weight compared with standard chow control group. however, body weight on the final day of gestation (gestation day 21) was not different among the groups. caloric intake was not affected by stress (gestation days 1521). to assess the effect of stress and consumption of high - fat diet, we measured plasma corticosterone, leptin, and insulin on gestation day 21. at this time point, plasma corticosterone was significantly higher in the variable stress groups compared with nonstressed controls (p < 0.05), indicating that exposure to variable stress resulted in hpa axis activation and increased circulating glucocorticoid levels. there was an overall effect of diet on plasma leptin levels after 20 days of high - fat diet feeding (p < 0.05). leptin was significantly elevated in the high - fat control group compared with standard chow fed groups. because leptin is secreted by adipocytes and is highly correlated to adiposity, these data suggest that even though they were not significantly heavier, the high - fat diet dams had greater body adiposity compared with the standard chow fed dams. blood glucose (data not shown) and plasma insulin were not different among the groups, suggesting that high - fat feeding did not result in symptoms of gestational diabetes in high - fat fed dams. among the groups, there were no significant differences in litter size (standard chow stress, 15.3 0.9 ; high - fat control, 14.6 0.9 ; and high - fat stress, 14.5 0.7) or male - to - female ratios (standard chow stress, 0.48 0.03 ; high - fat control, 0.45 0.04 ; and high - fat stress, 0.50 0.03). there was an overall effect of maternal stress resulting in higher birth weight of both males (p < 0.05) and females (p < 0.05). by postnatal day 21, there were significant effects of both maternal diet and stress on body weight of male and female pups (fig. fat pad (retroperitoneal and subcutaneous pads) weight as a percentage of body weight was reliably higher in both male (p < 0.01) and female (p < 0.001) pups from dams that were fed high - fat diet during gestation and lactation (fig. thus, these data suggest that the increase in body weight on postnatal day 21 in pups from dams on high - fat diet is attributable, at least in part, to increased adiposity. subscapular brown adipose tissue was not different among the groups (data not shown). a and b : body weight and fat as a percentage of body weight (inset graphs) of offspring through postnatal day 21. a : male offspring in each litter were weighed on postnatal days 1, 7, 14, and 21. b : female offspring in each litter were weighed on postnatal days 1, 7, 14, and 21. fat as a percentage of body weight was determined on postnatal day 21 (inset graphs). groups shown are : standard chow control (chow - con ; n = 11 litters), standard chow stress (chow - stress ; n = 10 litters), high - fat control (hf - con ; n = 11 litters), and high - fat stress (hf - stress ; n = 10 litters). main effect of stress, p < 0.05 vs. control group ; main effect of high - fat diet, p < 0.05 vs. standard chow ; main effect of high - fat diet and stress, p < 0.05. c - c, standard chow control ; c - s, standard chow stress group ; h - c, high - fat control group ; h - s, high - fat stress group. plasma leptin was elevated at birth in male pups from dams in the high - fat stress group (p < 0.05) (fig., maternal high - fat diet resulted in significantly higher plasma leptin levels compared with pups of standard chow fed dams, and this effect persisted throughout the preweaning period (p < 0.05). pups from dams in the high - fat stress group had significantly greater blood glucose levels on postnatal days 7 and 14, and by postnatal day 21, both high - fat control and high - fat stress groups had elevated blood glucose compared with the standard chow control group (p < 0.05) (fig. there was no difference in plasma insulin from postnatal day 1 through 14 ; however, by postnatal day 21, insulin was significantly greater in pups from high - fat diet fed dams (high - fat control group, p < 0.01 ; high - fat stress group, p < 0.05) (fig. 2c). plasma corticosterone was significantly lower on postnatal day 1 in pups from stressed dams (p < 0.05), regardless of maternal diet, but there were no differences among the groups for the remainder of the preweaning period (fig. groups shown are : standard chow control (chow - con ; n = 11), standard chow stress (chow - stress ; n = 10), high - fat control (hf - con ; n = 11), and high - fat stress (hf - stress ; n = 10). p < 0.05 vs. standard chow control groups ; p < 0.05 vs. standard chow control and standard chow stress groups ; p < 0.05 vs. standard chow control group. at weaning, there were no significant differences between males and females within each group in any of the measures associated with the gtt ; therefore, the data have been combined in fig., there was an overall effect of high - fat diet in elevating baseline blood glucose (time 0). although there was no difference in blood glucose at 15 min, it remained elevated in the high - fat control and high - fat stress pups at 30, 45, 60, and 120 min. the glucose area under the curve (auc) was higher in high - fat diet pups compared with standard chow pups, indicating that pups from dams fed high - fat diet cleared the glucose load slower than pups from dams fed standard chow diet (p < 0.001) (fig. there was a main effect of maternal high - fat diet on insulin secretion in response to the glucose load. at 30, 45, 60, and 120 min, plasma insulin remained higher in both high - fat control (p < 0.05) and high - fat stress (p < 0.01) pups compared with standard chow pups. insulin auc was higher in both high - fat control (p < 0.05) and high - fat stress (p < 0.01) pups compared with pups from standard chow dams (fig., these data suggest that maternal high - fat diet resulted in offspring that cleared glucose more slowly and required greater insulin to do so. a and b : glucose tolerance test (2.0 g / kg, oral gavage) for male and female pups on postnatal day 23. groups shown are : standard chow control (chow - con ; n = 8), standard chow stress (chow - stress ; n = 8), high - fat control (hf - con ; n = 8), and high - fat stress (hf - stress ; n = 8). p < 0.05 standard chow stress, high - fat control, and high - fat stress vs. standard chow control groups ; p < 0.05 high - fat control and high - fat stress vs. standard chow control and standard chow c - c, standard chow control ; c - s, standard chow stress ; h - c, high - fat control ; h - s, high - fat stress. on postnatal day 21, half of the male and female offspring were weaned onto standard chow diet and half on to high - fat diet. when pups were weaned onto standard chow diet, there were no longer any significant differences in body weight among the groups in either males or females at postnatal day 70 (fig. body composition was not different among males, but there was a significant increase in percent body fat in high - fat stress females compared with the standard chow control group (p < 0.05) (fig. plasma insulin after an overnight fast was elevated in the high - fat stress offspring (p < 0.05), but there were no significant differences among the groups in plasma leptin or fasted blood glucose (table 3). when adult male offspring (postnatal day 70) were again challenged with a gtt 5a). offspring from high - fat fed dams had higher fasting insulin and showed a prolonged elevation of insulin at 60 and 120 min, although there was no overall effect on insulin auc (fig. 5c). however, among females, the high - fat stress group cleared glucose slower and had greater glucose auc compared with standard chow control and standard chow stress groups (p < 0.05) (fig. 6a). the standard chow stress group had lower insulin auc compared with the standard chow control group, suggesting that this group may be more efficient in clearing the glucose load (fig. d : body weight and fat as a percentage of body weight for adult male (a) and female (b) offspring. (d) is expressed as the weight of dorsosubcutaneous, inguinal, and retroperitoneal fat pads as a percentage of body weight. males weaned on standard chow (chow wean) are : standard chow control (chow - con ; n = 6), standard chow stress (chow - stress ; n = 4), high - fat control (hf - con ; n = 4), and high - fat stress (hf - stress ; n = 4). males weaned on high - fat diet (hf wean) are : standard chow control (n = 4), standard chow stress (n = 4), high - fat control (n = 5), and high - fat stress (n = 4). females weaned on standard chow are : standard chow control (n = 5), standard chow stress (n = 4), high - fat control (n = 4), and high - fat stress (n = 4). females weaned on high - fat diet are : standard chow control (n = 4), standard chow stress (n = 4), high - fat control (n = 5), and high - fat stress (n = 4). p < 0.05 vs. weaned on standard chow ; p < 0.05 vs. standard chow control group. endocrine measures in adult male offspring p < 0.05 vs. standard chow control group ; p < 0.05 vs. corresponding weaned on standard chow. blood glucose (a and b) and plasma insulin (c and d) were determined for 2 h after oral administration of glucose for male offspring weaned onto standard chow (a and c) or high - fat diet (b and d). males weaned on standard chow are : standard chow control (chow - con ; n = 6), standard chow stress (chow - stress ; n = 4), high - fat control (hf - con ; n = 4), and high - fat stress (hf - stress ; n = 4). males weaned on high - fat are : standard chow control (n = 4), standard chow stress (n = 4), high - fat control (n = 5), and high - fat stress (n = 4). high - fat diet main effect, p < 0.05 ; stress main effect, p < 0.05 ; high - fat diet and stress interaction, p < 0.05. a d insets : the integrated auc was determined for glucose and insulin using the trapezoidal method. p < 0.05 vs. corresponding standard chow weaned group ; p < 0.05 vs. standard chow control and standard chow stress groups ; p < 0.05 vs. standard chow control and high - fat control groups. c - c, standard chow control ; c - s, standard chow stress ; h - c, high - fat control ; h - s, high - fat stress. a blood glucose (a and b) and plasma insulin (c and d) were determined for 2 h after oral administration of glucose for female offspring weaned onto standard chow (a and c) or high - fat diet (b and d). females weaned onto standard chow are : standard chow control (chow - con ; n = 5), standard chow stress (chow - stress : n = 4), high - fat control (hf - con ; n = 4), and high - fat stress (hf - stress ; n = 4). females weaned onto high - fat are : standard chow control (n = 4), standard chow stress (n = 4), high - fat control (n = 5), and high - fat stress (n = 4). high - fat diet main effect, p < 0.05 ; stress main effect, p < 0.05. a d insets : the integrated auc was determined for glucose and insulin using the trapezoidal method. p < 0.05 vs. corresponding standard chow wean group ; p < 0.05 vs. standard chow control and standard chow stress groups ; p < 0.05 vs. standard chow control and high - fat control groups. c - c, standard chow control ; c - s, standard chow stress ; h - c, high - fat control ; h - s, high - fat stress. when male and female pups were weaned onto a high - fat diet on postnatal day 21, those pups that were from dams in the high - fat diet or stress groups or combination high - fat stress group gained more weight compared with pups from the standard chow the increase in body weight could be attributed to significantly greater subcutaneous and retroperitoneal fat pad weights (p < 0.05) (fig. 4c and d), and this was associated with elevated plasma leptin levels in standard chow stress and high - fat control groups (p < 0.05) (table 3). when male offspring were challenged with a gtt at postnatal day 70 after being weaned onto a high - fat diet, those from dams in the high - fat diet or stress groups or combination high - fat stress group cleared glucose more slowly relative to male offspring from the same groups weaned onto standard chow diet (fig. 5b) (p < 0.05). overall, male offspring from the stress group required greater insulin to clear the glucose load, regardless of maternal diet, compared with those pups weaned onto a standard chow diet (p < 0.05) (fig. 5d). among female offspring that were weaned onto high - fat diet, the standard chow control and standard chow stress groups cleared glucose slower than high - fat control and high - fat stress groups (p < 0.05) (fig. although the offspring weaned onto high - fat diet had greater insulin responses compared with those weaned onto standard chow, there was no significant difference among the high - fat weaned groups in insulin auc (fig. there were no significant differences in maternal body weight among the groups before stress exposure (table 2). however, compared with beginning body weight on gestation day 2, dams maintained on high - fat diet gained significantly more weight during the first 2 weeks of gestation compared with dams fed standard chow diet (p < 0.05). the greater weight gain in high - fat dams may be attributable to significantly higher caloric intake of dams fed high - fat diet during gestation days 214 (p < 0.05). there were no significant differences in plasma corticosterone among the groups before stress. maternal body weight, food intake, and endocrine measures during gestation p < 0.05 vs. standard chow control group ; p < 0.05 vs. standard chow control group and standard chow stress group. stress during the last week of gestation (gestation days 1421) resulted in less body weight gain in stress groups (table 2). post hoc analysis revealed that dams in the high - fat stress group gained significantly less body weight compared with standard chow control group. however, body weight on the final day of gestation (gestation day 21) was not different among the groups. caloric intake was not affected by stress (gestation days 1521). to assess the effect of stress and consumption of high - fat diet, we measured plasma corticosterone, leptin, and insulin on gestation day 21. at this time point, plasma corticosterone was significantly higher in the variable stress groups compared with nonstressed controls (p < 0.05), indicating that exposure to variable stress resulted in hpa axis activation and increased circulating glucocorticoid levels. there was an overall effect of diet on plasma leptin levels after 20 days of high - fat diet feeding (p < 0.05). leptin was significantly elevated in the high - fat control group compared with standard chow fed groups. because leptin is secreted by adipocytes and is highly correlated to adiposity, these data suggest that even though they were not significantly heavier, the high - fat diet dams had greater body adiposity compared with the standard chow fed dams. blood glucose (data not shown) and plasma insulin were not different among the groups, suggesting that high - fat feeding did not result in symptoms of gestational diabetes in high - fat fed dams. among the groups, there were no significant differences in litter size (standard chow stress, 15.3 0.9 ; high - fat control, 14.6 0.9 ; and high - fat stress, 14.5 0.7) or male - to - female ratios (standard chow stress, 0.48 0.03 ; high - fat control, 0.45 0.04 ; and high - fat stress, 0.50 0.03). there was an overall effect of maternal stress resulting in higher birth weight of both males (p < 0.05) and females (p < 0.05). by postnatal day 21, there were significant effects of both maternal diet and stress on body weight of male and female pups (fig. fat pad (retroperitoneal and subcutaneous pads) weight as a percentage of body weight was reliably higher in both male (p < 0.01) and female (p < 0.001) pups from dams that were fed high - fat diet during gestation and lactation (fig. 1a and b, inset). thus, these data suggest that the increase in body weight on postnatal day 21 in pups from dams on high - fat diet is attributable, at least in part, to increased adiposity. subscapular brown adipose tissue was not different among the groups (data not shown). a and b : body weight and fat as a percentage of body weight (inset graphs) of offspring through postnatal day 21. a : male offspring in each litter were weighed on postnatal days 1, 7, 14, and 21. b : female offspring in each litter were weighed on postnatal days 1, 7, 14, and 21. fat as a percentage of body weight was determined on postnatal day 21 (inset graphs). groups shown are : standard chow control (chow - con ; n = 11 litters), standard chow stress (chow - stress ; n = 10 litters), high - fat control (hf - con ; n = 11 litters), and high - fat stress (hf - stress ; n = 10 litters). main effect of stress, p < 0.05 vs. control group ; main effect of high - fat diet, p < 0.05 vs. standard chow ; main effect of high - fat diet and stress, p < 0.05. c - c, standard chow control ; c - s, standard chow stress group ; h - c, high - fat control group ; h - s, high - fat stress group. plasma leptin was elevated at birth in male pups from dams in the high - fat stress group (p < 0.05) (fig., maternal high - fat diet resulted in significantly higher plasma leptin levels compared with pups of standard chow fed dams, and this effect persisted throughout the preweaning period (p < 0.05). pups from dams in the high - fat stress group had significantly greater blood glucose levels on postnatal days 7 and 14, and by postnatal day 21, both high - fat control and high - fat stress groups had elevated blood glucose compared with the standard chow control group (p < 0.05) (fig. there was no difference in plasma insulin from postnatal day 1 through 14 ; however, by postnatal day 21, insulin was significantly greater in pups from high - fat diet fed dams (high - fat control group, p < 0.01 ; high - fat stress group, p < 0.05) (fig. 2c). plasma corticosterone was significantly lower on postnatal day 1 in pups from stressed dams (p < 0.05), regardless of maternal diet, but there were no differences among the groups for the remainder of the preweaning period (fig. d : endocrine parameters in male offspring postnatal days (pnd) 121. a : plasma leptin. groups shown are : standard chow control (chow - con ; n = 11), standard chow stress (chow - stress ; n = 10), high - fat control (hf - con ; n = 11), and high - fat stress (hf - stress ; n = 10). p < 0.05 vs. standard chow control, standard chow stress, and high - fat control groups ; p < 0.05 vs. standard chow control and standard chow stress groups ; p < 0.05 vs. standard chow control group. at weaning, there were no significant differences between males and females within each group in any of the measures associated with the gtt ; therefore, the data have been combined in fig., there was an overall effect of high - fat diet in elevating baseline blood glucose (time 0). although there was no difference in blood glucose at 15 min, it remained elevated in the high - fat control and high - fat stress pups at 30, 45, 60, and 120 min. the glucose area under the curve (auc) was higher in high - fat diet pups compared with standard chow pups, indicating that pups from dams fed high - fat diet cleared the glucose load slower than pups from dams fed standard chow diet (p < 0.001) (fig. there was a main effect of maternal high - fat diet on insulin secretion in response to the glucose load. at 30, 45, 60, and 120 min, plasma insulin remained higher in both high - fat control (p < 0.05) and high - fat stress (p < 0.01) pups compared with standard chow pups. insulin auc was higher in both high - fat control (p < 0.05) and high - fat stress (p < 0.01) pups compared with pups from standard chow dams (fig. 3b). together, these data suggest that maternal high - fat diet resulted in offspring that cleared glucose more slowly and required greater insulin to do so. a and b : glucose tolerance test (2.0 g / kg, oral gavage) for male and female pups on postnatal day 23. a : blood glucose after oral administration of glucose. b : plasma insulin after oral administration of glucose. the integrated auc was determined for glucose and insulin using the trapezoidal method. groups shown are : standard chow control (chow - con ; n = 8), standard chow stress (chow - stress ; n = 8), high - fat control (hf - con ; n = 8), and high - fat stress (hf - stress ; n = 8). p < 0.05 standard chow stress, high - fat control, and high - fat stress vs. standard chow control groups ; p < 0.05 high - fat control and high - fat stress vs. standard chow control and standard chow stress groups. c - c, standard chow control ; c - s, standard chow stress ; h - c, high - fat control ; h - s, high - fat stress. on postnatal day 21, half of the male and female offspring were weaned onto standard chow diet and half on to high - fat diet. when pups were weaned onto standard chow diet, there were no longer any significant differences in body weight among the groups in either males or females at postnatal day 70 (fig. body composition was not different among males, but there was a significant increase in percent body fat in high - fat stress females compared with the standard chow control group (p < 0.05) (fig. plasma insulin after an overnight fast was elevated in the high - fat stress offspring (p < 0.05), but there were no significant differences among the groups in plasma leptin or fasted blood glucose (table 3). when adult male offspring (postnatal day 70) were again challenged with a gtt 5a). offspring from high - fat fed dams had higher fasting insulin and showed a prolonged elevation of insulin at 60 and 120 min, although there was no overall effect on insulin auc (fig. 5c). however, among females, the high - fat stress group cleared glucose slower and had greater glucose auc compared with standard chow control and standard chow stress groups (p < 0.05) (fig. 6a). the standard chow stress group had lower insulin auc compared with the standard chow control group, suggesting that this group may be more efficient in clearing the glucose load (fig. d : body weight and fat as a percentage of body weight for adult male (a) and female (b) offspring. (d) is expressed as the weight of dorsosubcutaneous, inguinal, and retroperitoneal fat pads as a percentage of body weight. males weaned on standard chow (chow wean) are : standard chow control (chow - con ; n = 6), standard chow stress (chow - stress ; n = 4), high - fat control (hf - con ; n = 4), and high - fat stress (hf - stress ; n = 4). males weaned on high - fat diet (hf wean) are : standard chow control (n = 4), standard chow stress (n = 4), high - fat control (n = 5), and high - fat stress (n = 4). females weaned on standard chow are : standard chow control (n = 5), standard chow stress (n = 4), high - fat control (n = 4), and high - fat stress (n = 4). females weaned on high - fat diet are : standard chow control (n = 4), standard chow stress (n = 4), high - fat control (n = 5), and high - fat stress (n = 4). p < 0.05 vs. weaned on standard chow ; p < 0.05 vs. standard chow control group. endocrine measures in adult male offspring p < 0.05 vs. standard chow control group ; p < 0.05 vs. corresponding weaned on standard chow. blood glucose (a and b) and plasma insulin (c and d) were determined for 2 h after oral administration of glucose for male offspring weaned onto standard chow (a and c) or high - fat diet (b and d). males weaned on standard chow are : standard chow control (chow - con ; n = 6), standard chow stress (chow - stress ; n = 4), high - fat control (hf - con ; n = 4), and high - fat stress (hf - stress ; n = 4). males weaned on high - fat are : standard chow control (n = 4), standard chow stress (n = 4), high - fat control (n = 5), and high - fat stress (n = 4). high - fat diet main effect, p < 0.05 ; stress main effect, p < 0.05 ; high - fat diet and stress interaction, p < 0.05. a d insets : the integrated auc was determined for glucose and insulin using the trapezoidal method. p < 0.05 vs. corresponding standard chow weaned group ; p < 0.05 vs. standard chow control and standard chow stress groups ; p < 0.05 vs. standard chow control and high - fat control groups. c - c, standard chow control ; c - s, standard chow stress ; h - c, high - fat control ; h - s, high - fat stress. a blood glucose (a and b) and plasma insulin (c and d) were determined for 2 h after oral administration of glucose for female offspring weaned onto standard chow (a and c) or high - fat diet (b and d). females weaned onto standard chow are : standard chow control (chow - con ; n = 5), standard chow stress (chow - stress : n = 4), high - fat control (hf - con ; n = 4), and high - fat stress (hf - stress ; n = 4). females weaned onto high - fat are : standard chow control (n = 4), standard chow stress (n = 4), high - fat control (n = 5), and high - fat stress (n = 4). high - fat diet main effect, p < 0.05 ; stress main effect, p < 0.05. d insets : the integrated auc was determined for glucose and insulin using the trapezoidal method. < 0.05 vs. corresponding standard chow wean group ; p < 0.05 vs. standard chow control and standard chow stress groups ; p < 0.05 vs. standard chow control and high - fat control groups. c - c, standard chow control ; c - s, standard chow stress ; h - c, high - fat control ; h - s, high - fat stress. when male and female pups were weaned onto a high - fat diet on postnatal day 21, those pups that were from dams in the high - fat diet or stress groups or combination high - fat stress group gained more weight compared with pups from the standard chow the increase in body weight could be attributed to significantly greater subcutaneous and retroperitoneal fat pad weights (p < 0.05) (fig. 4c and d), and this was associated with elevated plasma leptin levels in standard chow stress and high - fat control groups (p < 0.05) (table 3). when male offspring were challenged with a gtt at postnatal day 70 after being weaned onto a high - fat diet, those from dams in the high - fat diet or stress groups or combination high - fat stress group cleared glucose more slowly relative to male offspring from the same groups weaned onto standard chow diet (fig. 5b) (p < 0.05). overall, male offspring from the stress group required greater insulin to clear the glucose load, regardless of maternal diet, compared with those pups weaned onto a standard chow diet (p < 0.05) (fig. 5d). among female offspring that were weaned onto high - fat diet, the standard chow control and standard chow stress groups cleared glucose slower than high - fat control and high - fat stress groups (p < 0.05) (fig. although the offspring weaned onto high - fat diet had greater insulin responses compared with those weaned onto standard chow, there was no significant difference among the high - fat weaned groups in insulin auc (fig. the intrauterine environment is critical to fetal development, and perturbations of this environment can have significant short- and long - term consequences on the offspring. a growing body of epidemiological data demonstrates that obesity and other metabolic disease may have developmental origins, and determination of mechanisms contributing to those conditions may lead to treatment strategies and early interventions to prevent these disorders. we hypothesized that exposure to prenatal stress or maternal high - fat diet in utero would predispose offspring to develop obesity and that weaning onto a high - fat diet would exacerbate this effect. our data suggest that exposure to maternal prenatal stress or high - fat diet feeding results in offspring that gain more weight and have greater adiposity than controls. consistent with their body composition, offspring of stressed or high - fat diet fed dams are hyperleptinemic and hyperinsulinemic at weaning. males and females are similarly affected by maternal prenatal stress and high - fat diet, resulting in impaired glucose tolerance at weaning. although weaning on standard chow diet appears to normalize early obesity, the combination of maternal prenatal stress and high - fat diet continued to have some effects on glucose tolerance in both male and female offspring. weaning onto high - fat diet resulted in obesity and impaired glucose tolerance in offspring exposed to prenatal stress, high - fat diet, or both. together, the data suggest that maternal prenatal stress or high - fat susceptibility of offspring to diet - induced obesity and its metabolic consequences. although high - fat diet fed dams were not heavier, their plasma leptin levels were elevated on gestation day 21, suggesting that they had greater adiposity. the offspring that were born to dams on high - fat diet were heavier at birth, remained heavier throughout the suckling period, and had impaired glucose tolerance at weaning, suggesting that they were developing insulin resistance. prior studies that have attempted to identify the effects of prolonged maternal high - fat diet consumption have been difficult to interpret because of concurrence of high - fat diet exposure and maternal obesity (31). maternal obesity is usually associated with other comorbid conditions, such as gestational diabetes, that also have a significant influence on the phenotype of the offspring independent of dietary fat consumption during gestation. indeed, offspring of rodents with gestational diabetes show increased adiposity, impaired pancreatic function, impaired glucose tolerance, and altered hypothalamic development (3234). for these reasons, the dams in our study were provided with high - fat diet only during gestation and lactation. although dams in the current study were hyperleptinemic, indicating that they had greater adiposity after 3 weeks on high - fat diet, there were no differences in plasma concentrations of insulin or glucose, suggesting that they had not developed signs of gestational diabetes. it is unclear from this experiment whether maternal high - fat diet consumption during gestation or lactation had a greater influence because high - fat dams remained on high - fat diet throughout these periods. future studies with cross - fostered control groups will be required to make this distinction. baseline corticosterone was elevated in dams in the stress group, suggesting that exposure to variable stress during the 3rd week of gestation was effective. maternal prenatal stress in our study resulted in higher birth weights in both male and female offspring, and this occurred in the absence of significant differences in litter size or male - to - female ratios. by postnatal day 21, both male and female offspring of the stress group had greater body weight and showed signs of impaired glucose tolerance, suggesting that prenatal stress has early consequences for the offspring. in contrast to the effect of high - fat diet on endocrine measures, the only hormone that was significantly different in the stress offspring was plasma corticosterone, which was lower in the stress group compared with control pups at birth. this may be a compensatory response resulting from the stressed dams ' elevated plasma corticosterone at parturition. it is not known whether negative feedback control of corticosterone secretion is operational at this early age. prior studies examining the effects of prenatal stress on birth weights have produced mixed results. some report decreased birth weights resulting from prenatal stress (26,35,36), and others show increased birth weights (27) or no difference compared with controls (25). one possible explanation to account for some of the difference is that those studies reporting lower birth weights used the immobilization stress paradigm, which also results in decreased food intake and body weight gain in the dams. because stress - induced maternal undernutrition and decreased body weight gain during pregnancy has consequences similar to those of intrauterine growth restriction, parceling out the direct effects of stress is difficult in these models. the variable stress schedule that we used did not significantly affect maternal food intake of dams during the stress period. epidemiological reports in humans indicate that the birth weight to adult fat mass picture has developed into a u - shaped curve, such that being very light or very heavy at birth may predispose offspring to cardiovascular disease, diabetes, obesity, and some types of cancer (15,37). therefore, although the findings regarding the effects of prenatal stress on birth weight are variable, the different models may operate through different mechanisms depending on the type, intensity, and timing of stress during gestation (38). maternal obesity and high - fat diet feeding have been associated with hyperphagia and obesity in offspring, particularly when challenged postnatally with a hypercaloric diet (39). we hypothesized that prenatal stress against a background of high - fat diet feeding would result in an additive or synergistic effect on the offspring. the data at the time points examined suggest that there is no increased effect of the combination of stress and high - fat diet. an alternative possibility was that maternal high - fat diet would have a beneficial effect in attenuating the stress response of the dam and, in turn, lessen its impact on the developing fetus. (40) have demonstrated that high - fat diet can attenuate the hpa axis response to stress. we did not note either a detrimental or beneficial effect of maternal high - fat diet on top of the effects of stress. in adulthood, offspring exposed to stress or high - fat diet or both gained significantly more body weight and adiposity, but only if they were weaned onto a high - fat diet. consistent with increased body weight and adiposity on high - fat diet in adulthood, animals from stressed or high - fat diet fed dams also continued to have impaired glucose tolerance. in contrast, when offspring were weaned on to standard chow, body weight, body composition, and glucose tolerance differences among the groups were no longer as pronounced. it is important to note that the data reported here are from young adult offspring and do not preclude the possibility that metabolic disorders may occur more slowly, emerging later in life. overall, our data suggest that prenatal conditions can alter the susceptibility of offspring to future metabolic challenge, in this case high - fat diet resulting in obesity. this concept has been proposed as the two - hit model and suggests that genetic or environmental factors disrupt early development and produce increased susceptibility to disease, including parkinson 's disease (41,42) and schizophrenia (43,44). first hit disruptions that occur during early development set the stage for long - term vulnerability to a thus, it appears that prenatal stress or maternal high - fat diet is not the direct cause of increased body weight or disturbances in energy homeostasis, but instead increases future susceptibility. identification of the pathways and mechanisms that produce long - term vulnerability in response to early environmental disruption will facilitate development of clinical intervention and prevention strategies to reduce the incidence of disease. using these animal models, we are now able to extend our studies to determine the potential mechanisms through which prenatal stress or high - fat diet may program systems that regulate body weight and control food intake. there is evidence that hormones such as corticosterone, insulin, and leptin can cross the placental - fetal barrier to potentially affect fetal development (4547), and thus it is likely that fetuses have hormone levels that mirror those of their dams. the significant changes in endocrine parameters in offspring throughout the early postnatal period reported here represent reasonable candidates for metabolic programming that occurs in response to high - fat diet consumption or maternal stress (48). both leptin and insulin are trophic factors that act during the pre- and postnatal periods and can significantly affect development of neural systems important in the maintenance of energy homeostasis (49). similarly, stress and excess glucocorticoids may have an effect prenatally by programming increased susceptibility to stress in adulthood. although placental 11-hydroxysteroid dehydrogenase (11-hsd) serves to buffer the developing fetus from excess glucocorticoids in response to acute stress, its upregulation of 11-hsd is often insufficient during chronic stress (50). future studies are required to explore the role of 11-hsd in the phenotypes we observed. the intrauterine period is a critical time of development, and evidence shows that disturbances to the fine balance in utero can have significant and persistent consequences for the offspring. the development of relevant animal models is required to examine the etiology of metabolic disorders and to determine the mechanisms for greater predisposition to those disorders. | objectiveperturbations to the prenatal environment have been associated with the development of adult chronic disease, findings that gave rise to the barker hypothesis or the developmental origins of adult disease concept. in this study, we used an animal model to determine the metabolic consequences of maternal prenatal stress and high - fat feeding on the developing offspring.research design and methodspregnant female sprague - dawley rats were maintained on standard chow or 60% high - fat diet throughout gestation and lactation. half of each group were exposed to a novel variable stress paradigm during the 3rd week of gestation, whereas control dams were left undisturbed. body weight, body composition, glucose tolerance, and endocrine parameters were measured in offspring through early adulthood.resultsmale and female pups from dams that experienced prenatal stress and/or were on a high - fat diet weighed more beginning on postnatal day 7 compared with standard chow control pups. access to high - fat diet at weaning increased the body weight effect through early adulthood and was attributable to greater adiposity. pups weaned onto standard chow diet showed no significant difference in glucose clearance or insulin secretion. however, pups weaned onto high - fat diet had impaired glucose tolerance if their dams were on a high - fat diet, experienced prenatal stress, or both.conclusionsour data demonstrate that prenatal stress and/or high - fat diet during the intrauterine or postnatal environment affects offspring in a manner that increases their susceptibility to diet - induced obesity and leads to secondary adverse metabolic consequences. |
spontaneous isolated abdominal aortic dissection (iaad) (not associated with trauma or with descending thoracic aortic dissection) is rare and accounts for less than 2% of all aortic dissections. spontaneous suprarenal iaads are even more rarely encountered as most appear from infrarenal intimal defects. this subgroup of iaad is often associated with poorer prognosis as the dissection may interfere with blood flow through the renal and mesenteric arteries predisposing to ischemia of their respective organs and consequent high mortality [2, 3 ]. we present a quite unusual case of spontaneous supraceliac iaad sparing both renal and mesenteric vessels, and manifesting atypically as chronic rather than the more typical acute limb ischemia in a patient with chronic paraplegia secondary to previous spinal surgery for chondrosarcoma. a 41-year - old paraplegic presented to the emergency department with a ten - day history of worsening pain in his left forefoot. his symptoms began after he injured his left hallux whilst swimming, and he had just completed three days of antibiotics for apparent cellulitis over the toe with no improvement in his symptoms. he was a nonsmoker and had no history of intermittent claudication, dvt / pe, calf tenderness, arrhythmia, diabetes, hypertension, or high cholesterol. he had spinal surgery 2 years earlier for chondrosarcoma, and this rendered him paraplegic. clinical examination revealed a dusky discoloration to his left hallux with a cold and tender forefoot. he had normal pulses with good doppler signals in the right leg but no palpable pulses from the left common femoral artery down the left leg. his abdomen was soft and nontender, and he had no other detectable clinical signs. a provisional diagnosis of left iliofemoral artery thrombosis with possible distal embolic disease was made. he proceeded to have an urgent right transfemoral angiogram which revealed normal - looking right lower limb vessels with no atherosclerotic lesions or changes. there was, however, a cutoff of contrast at the origin of the left common iliac artery (cia) extending into the left external iliac artery (eia). there was reconstitution of the left common femoral artery (cfa), profunda femoris artery (pfa), and superficial femoral artery (sfa). the above knee popliteal artery (pa) was occluded with reconstitution of a single vessel runoff at mid leg, most probably the posterior tibial artery (figure 1). the impression at this point was that of a thrombotic event at the left cia with showering of emboli to the distal sfa and pa. the patient proceeded to theatre the next day for a left iliac / femoral artery embolectomy + / stent insertion + / thrombolysis. a fogarty catheter passed through an arteriotomy of the left cfa was used to retrieve large amounts of thrombi, but there was little improvement to blood flow. further embolectomy was carried out until no more thrombus was retrievable, but flow through the left cfa remained poor. a strong right common femoral pulse, however, remained and as such an impression of probable undiagnosed aortic dissection was made. the left cfa was ligated, and a right - to - left femorofemoral crossover using an 8 mm polytetrafluorethylene (ptfe) graft was established with good results. abdominal computed tomography (ct) after surgery confirmed the presence of a left anterolateral abdominal aortic dissection from just above the celiac level of the celiac axis extending down into a ligated left common iliac artery. the dissection lies to the left of the celiac axis curving anteriorly to the left and sparing the left and right renal arteries which appeared patent behind the dissection (figures 2 and 3). a previous abdominal ct scan taken 1 year after his spinal surgery showed a normal aorta. this patient subsequently required amputation of his left forefoot but has otherwise made good progress and is now having regular outpatient surveillance of his aortic dissection and close blood pressure monitoring. spontaneous isolated abdominal aortic dissection is very rare. in the international registry of acute aortic dissection (irad) (the largest group of patients treated for acute aortic dissections), only about 1.3% of the enrolled patients were identified as having isolated dissections of the abdominal aorta. furthermore, a recent meta - analysis of all english language articles regarding abdominal aortic dissection found only 73 reported cases of spontaneous iaad. in these cases, the dissection flap predominantly originated below or at the level of the renal arteries ; very few had the intimal tear in the suprarenal aorta as illustrated in this case. the mean age at presentation of iaad is in the fifth or sixth decade of life. it is more common in caucasians, and men are affected twice as commonly as women. identified risk factors include hypertension, smoking, dyslipidemia, connective tissue disease, and trauma. the natural history of iaad is yet to be clearly defined, and in many cases, the presenting clinical symptoms are nonspecific. it, however, most commonly presents with sudden onset of abdominal pain radiating to the back and to the buttock but could also present with acute lower limb ischemia (or chronic lower limb ischemia as this case describes), or symptoms of visceral malperfusion and renal ischemia. the mortality rate of patients with renal ischemia is reported to be 50 to 70%, whilst mortality figures in mesenteric ischemia can be as high as 87%. this may be a result of obstruction to blood flow through the adamkiewicz artery (the largest anterior segmental medullary artery) that supplies the lower two - thirds of the spinal cord. his paraplegia was secondary to spinal surgery to remove a large chondrosarcoma that had invaded the 1st and 2nd sacral segments via the spinal canal and extended superiorly within the spinal canal to the level of the midbody of l5. although he admittedly had extensive spinal surgery with the institution of an internal fixation device to his lumbar spine making it difficult to completely exclude this as a contributory factor to his dissection, abdominal ct scan carried out one year after his spinal surgery revealed a normal abdominal aorta. accurate early diagnosis is paramount in spontaneous iaad, and contrast ct scan is the investigation of choice. whilst our case did present with a recognised clinical feature of this pathology albeit atypical, that is, chronic rather than acute lower limb ischemia, he had no abdominal symptoms or signs, and the nature of the onset of his symptoms coupled with his lack of discernible risk factors made the diagnosis difficult. indications for operative intervention include aortic rupture, unremitting pain, associated aortic aneurysm, prevention of future aneurysmal change, and visceral, renal, or lower extremity ischemia. asymptomatic patients with a nondilated aorta are treated with antihypertensive medication [10, 11 ]. operative management is via open (graft) or endovascular (stent) repair of the affected abdominal aorta. this decision is greatly influenced by anatomical conditions, the patient 's comorbidities, and the surgeon 's experience. where the dissection extends to the iliac arteries, aorto - bifemoral / monofemoral grafting is the operation of choice. described the first successful repair of a spontaneous suprarenal abdominal aortic dissection by graft insertion with obliteration of the entry tear. before this, operative intervention of spontaneous suprarenal abdominal aortic dissection met with generally poor results. percutaneous balloon aortic fenestration involves obtaining arterial access at the common femoral artery, and under intravascular ultrasound (ivus) or arteriographic guidance, fenestration is accomplished by puncturing the intimal flap with an intravascular needle followed by balloon dilatation (at least a 15 mm balloon) of the flap. this provides local blood flow between the true and false lumens by creating a tear in the intervening dissection septum thus achieving an equalization of pressures between the two lumens. the degree of obstruction of the branch vessel ostia is also examined, and stents may be added to optimize flow. a stent graft may or may not be required within the aorta. the technical success of this procedure is estimated to be about 90%, with visceral vessel involvement, more so than the renal vessels, predicting worse outcome. described complications of percutaneous fenestration include aneurysm formation, transmural perforation during the creation of the fenestration, and manipulation of the intimal flap, which can propagate the dissection or cause occlusion of previously patent vessels. furthermore, laboratory studies have shown that balloon fenestration tears are typically along a transverse orientation relative to the longitudinal axis of the aorta. as such, in some cases, the transverse tear could circumferentially transect an aortic septum (when a septal tube is present) and result in intimo - intimal intussusceptions with resultant occlusion of distal vessels. balloon fenestration and intraluminal stent procedures remain promising, but only small series have so far documented their use and long - term data are not yet available suspicion of our patient 's diagnosis was only arrived at on the operating table during which femorofemoral crossover revascularisation of the left system had to be established due to lack of adequate analysis of the position or extent of the dissection flap. spontaneous supraceliac iaad is a very rare but potentially life- or limb - threatening condition particularly if misdiagnosed. it may have a number of clinical presentations with potentially serious adverse effects and should thus be considered in the differential diagnosis of any patient with an acute onset of abdominal pain radiating to the back and the buttocks together with presence or absence of a pulsatile abdominal mass, signs of limb ischemia, or discernible risk factors. the unusual presentation of this case led to difficulty in early diagnosis culminating in loss of part of the patients ' limb. better illustration of the natural history of this ill - defined pathology is needed to aid understanding and improve patient care. | aortic dissections that originate from isolated tears in the abdominal aorta are uncommon. rarer still are cases of isolated abdominal aortic dissections arising in suprarenal locations, as most appear from infrarenal intimal defects. we present a quite unusual case of a spontaneous supraceliac isolated abdominal aortic dissection sparing the renal and mesenteric arteries and manifesting as chronic rather than acute limb ischemia. the atypical presentation of this case led to repeated misdiagnosis with consequent loss of part of the patient 's limb. better illustration of the natural history of this ill - defined pathology is needed to aid understanding and improve patient care. |
as a therapy for hypercholesterolaemia, statins have been used clinically for over two decades. however, over the last decade, immunosuppressive effects have also been demonstrated which are independent of their cholesterol - lowering properties. statins in vitro modulate cell adhesion through effects on endothelial cells and leukocytes, via blocking activation of lfa-1 and decreasing icam-1 and mcp-1 expression on activated leukocytes and endothelium [24 ]. statins have been shown in vitro and in vivo to reduce leukocyte motility, migration, and infiltration. as compared to cyclosporine, statins were effective in reducing leukocyte infiltration in a rat model of allograft rejection. they also inhibit the nf-b pathway, involved in transcriptional regulation of cytokines, chemokines, and adhesion molecules. statins have been shown to upregulate suppressor of cytokine secretion (socs) 3 and socs 7 which, in turn, downregulate il-23 and il-6 production, thus decreasing il-17 production. clinical studies of statins in patients with immune - mediated diseases such as rheumatoid arthritis (ra), multiple sclerosis, and organ rejection following transplantation have shown conflicting results due to different statins being used in those studies and often open or retrospective studies involving small patient numbers [9, 10 ]. immunomodulatory effects in experimental autoimmune encephalitis (eae) have been observed with atorvastatin attenuating murine eae, and it was found that atorvastatin upregulated il-4, il-5, and il-10 and downregulated il-2, il-12, ifn-, and tnf-. however, in another study investigating atorvastatin in murine experimental autoimmune uveitis (eau), atorvastatin was found not to modulate the immune response despite histological grading suggesting mildly decreased inflammation, whereas in murine eau attenuated by lovastatin, there was a decrease in ifn- production but no effect on th2 cytokines. in a lewis rat model, eau was decreased in severity by both atorvastatin and lovastatin, even when given after disease onset. with both of these statins, there were decreases in clinical and histological disease scores, antigen responsiveness, and ifn- production. simvastatin was reported to decrease cytokine production, including il-10, in a murine model of collagen - induced arthritis and in a murine lupus model, it decreased serum tnf- and ifn- levels but increased transcription of il-4 and il-10. overall, there appear to be a range of immune - related effects by statins depending on species, model, and cell type investigated. anti - inflammatory therapy for intraocular inflammation often requires use of corticosteroids, yet these can have severe side effects in the eye including raising intraocular pressure, cataracts, and glaucoma. to reduce these effects, steroid - sparing agents are also used including cyclosporin a (csa), mycophenolate, and rapamycin. however, these drugs all have systemic side effects which limit their use in the long - term management of chronic disease. in chronic immune - mediated conditions such as ra and systemic lupus erythematosus (sle), there is an associated increased premature atherogenesis and cardiovascular disease risk secondary to inflammatory processes [1719 ]. in addition, patients with uveitis who are treated with steroids and immunosuppressive agents such as cyclosporine and mycophenolate have an increased risk of developing cardiac disease. therefore, in these cases, the combined cholesterol - lowering and anti - inflammatory properties of statins may be clinically very useful. the aim of this study was to determine whether individual statins exert immunosuppressive effects on t cells equivalent to those of conventional immunosuppressive agents in vitro. the effects of atorvastatin, lovastatin, and simvastatin on normal human whole blood - derived t - cell viability, proliferation, and cytokine responses were studied and compared to dexamethasone, csa, mycophenolate, and rapamycin. all drugs were dissolved in either dimethylsulphoxide (dmso), rpmi 1640 (dutch modification), and/or 100% ethanol., ny, usa) was prepared as a 1 mm stock solution ; lovastatin (calbiochem, nottingham, uk) at 40 mm ; simvastatin (msd, nj, usa) at 10 m ; dexamethasone (chauvin, kingstonupon - thames, uk) at 400 g / l. ; mevalonate (sigma - aldrich company ltd, dorset, uk) at 0.5 m ; mycophenolate mofetil (roche products limited, hertfordshire, uk) at 10 mm ; rapamycin (wyeth, pfizer, berkshire, uk) at 0.1 mg / ml ; cyclosporin a (sandoz, novartis pharmaceuticals, surrey, uk) at 1 mg / ml. peripheral blood was obtained from 16 healthy donors (all working at the institute / moorfields) with informed consent (mean age (range), 34.3 (22.546.7) years ; six males). exclusion criteria included a history of autoimmune disease, atopy, haematological disorder, or current usage of systemic medication. the protocols used in this study were reviewed and approved by the local ethics committee. all studies involving human subjects were conducted according to the tenets of the declaration of helsinki. all assays were performed in rpmi 1640 (dutch modification) supplemented with 2 mm l - glutamine, 10 g / ml gentamycin, 20 m 2-me, nonessential amino acids, sodium pyruvate, and 10% human ab serum. peripheral blood mononuclear cells (pbmc) cells were incubated for 10 min in a 37c water bath with 7.5 m cfse (molecular probes, uk) in serum - free medium. 1 ml cold stop buffer (10% fcs in rpmi) was then added before incubation at room temperature for 30 min. cells were washed once with rpmi before being resuspended at 2 10/ml in t - cell medium for 5 days with or without atorvastatin, lovastatin, simvastatin, rapamycin, mycophenolate, csa, and dexamethasone. t cells were stimulated with anti - cd3 (10 ng / ml ; clone hit3a) and anti - cd28 (5 ng / ml ; clone cd28.2) antibodies (pharmingen). all drugs or vehicle controls were added at start of culture, except csa, which was added 2 h prior to stimulation since no effect was seen with csa if added at time 0. two - color flow cytometry was performed (facscan ; becton dickinson, oxford, uk ; bd). gates were set on viable lymphocytes according to forward (fsc) and side scatter (ssc). listmode data was generated using cellquest acquisition software on 1525,000 events (bd). for viability assays, all lymphocytes were gated to assess level of nonviable propidium iodide expressing (pi) cells. for proliferation, nonviable pi cells were excluded. the total numbers of events were determined by analyzing the data using dot plots and rectangular regions to define the cell populations. histograms were used to track the divisions of cfse - labeled cells enabling identification of the percentage of divided (proliferated) cells. concentrations of statins required to achieve maximal inhibition of proliferation whilst maintaining viability were identified and used in a multiplex bead array to determine the effects of three statins on cytokine production. 100 l of heparinised whole blood was cultured with or without simvastatin, lovastatin, atorvastatin, dexamethasone, csa, mycophenolate, and rapamycin and stimulated with pma (50 ng / ml) and ionomycin (1 g / ml) supernatants were harvested at 18 h, centrifuged to remove cells, and stored at 70c. multiplex bead cytokine arrays were conducted with a 10-plex kit (bender medsystems, ebiosciences, hatfield, uk) as per the manufacturer 's instructions. supernatants were analyzed simultaneously for ifn-, il-1, il-2, il-4, il-5, il-6, il-8, il-10, and tnf-. the lowest levels of detection were il-1 (6.2 pg / ml) ; il-2 (6.3 pg / ml) ; il-4 (5 pg / ml) ; il-5 (5.8 pg / ml) ; il-6 (14.8 pg / ml), il-8 (58.6 pg / ml) ; il-10 (7.3 pg / ml) ; ifn- (5.7 il-17a was assayed by elisa (r&d systems, abingdon, uk), with a minimum level of detection of 62.5 pg / ml. whole blood cells were stimulated with pma / ionomycin, and 10 g / ml brefeldin a was added prior to culture for 18 h in the presence or absence of drugs. cd3 t cells were stained intracellularly with mouse anti - human ifn--fitc (clone 4s.b3) and rat anti - human il-10-pe (clone jes3 - 9d7 ; both bd) as previously described. all intra - assay comparisons of means were analysed using student 's t - tests and for inter - assay data by mann - whitney u tests. kruskal - wallis tests were used for multiplex cytokine detection analysis for 6 donors, with nonparametric post hoc comparisons. in experiments with csa, only 5 donors were included due to lack of available donors. significance was reached when p < 0.05, and this was achieved for several assays, despite relatively small sample sizes. to determine whether the drugs had any effect on t - cell viability, cells were cultured in the presence of drugs, mevalonate, and vehicle controls for 72 h prior to staining for pi (figure 1). a range of concentrations were investigated, and viability was generally high with the lowest viability observed at the highest concentrations of lovastatin and atorvastatin. this was reversed with the addition of mevalonate, a downstream product of hmg - coa reductase (p < 0.05) relative to lovastatin alone, suggesting that these higher drug concentrations reduced cell viability by inhibiting the mevalonate pathway and not via direct toxicity. there was a dose - dependent inhibition of t - cell proliferation with all statins, with maximal inhibition at 50 m atorvastatin and lovastatin and 100 m simvastatin (p < 0.01 ; figure 2(a)). the addition of mevalonate, fully restored proliferation (figures 2(b)2(e) insert) confirming that these modulatory effects were statin - mediated. a dose - dependent inhibition of anti - cd3/28-induced t - cell proliferation was also seen for the standard immunosuppressive agents with maximal inhibition at 100 m mycophenolate mofetil, 100 m rapamycin, (figure 2(f)), 100 g / ml dexamethasone, and 100 ng / ml csa (data not shown). six human donors ' pbmc were included in this study, and the responses were highly variable. the mean background levels of cytokines secreted by unstimulated cells were subtracted from all wells. the levels secreted by stimulated cells in the absence of exogenous drugs were as follows : tnf- (30.31 13.74 pg / ml), ifn- (0.66 0.47 pg / ml), il-10 (337.04 374.8 pg / ml), il-4 (4.47 6.64 pg / ml), il-1 (447.46 236.07 pg / ml), il-5 (14.53 13.66 pg / ml), il-17 (1186.50 599.27), and il-2 (1.50 2.45 pg / ml). following stimulation, there was variability in the cytokines produced among the donors, not unexpected in a mixed donor population. nevertheless, for all six donors, simvastatin significantly increased il-1 (figure 3 ; p < 0.05), and all three statins inhibited il-17 production (p < 0.01). since not all donors ' cells exhibited the same cytokine response profile, trends were observed although these failed to reach significance. atorvastatin reduced il-1, il-4, il-5, and ifn- and increased il-10, tnf-, and il-2. lovastatin increased il-1 and il-5 production and decreased il-10, tnf-, and ifn- production. mean levels of ifn- were relatively low, suggesting a low frequency of ifn- secreting cells in the normal donor population. due to the variable cytokine response profiles seen in individual donors, we investigated whether there were any correlations between the cytokines that were common to the drugs in comparison with cells stimulated in the absence of drug (pma / ionomycin alone). stimulation in the absence of statins induced a strong correlation between il-2 and ifn- and between il-6 and tnf-. in contrast, following atorvastatin treatment, il-6 showed a good correlation with il-2, ifn-, and tnf-, and il-2 correlated strongly with ifn- and tnf-, as did ifn- with tnf-. in the simvastatin - treated group, il-6 correlated with il-2, ifn-, and il-1 and il-2 with ifn-. finally, the lovastatin - treated group showed correlations of il-6 with ifn-, il-5 with tnf-, and ifn- with tnf-. atorvastatin was selected to investigate its effects on cytokine responses in combination with dexamethasone. in the presence of either drug alone or when added in combination, il-6, il-8, ifn-, and il-1 levels were all decreased although these did not reach significance, suggesting that the immunosuppressive effects of each of the drugs were maintained when added in combination (figure 4). interestingly, the level of il-10 production was enhanced in the presence of dexamethasone, although this failed to reach significance. for the production of il-2, il-5, and tnf-, the effects of atorvastatin were reversed upon addition of dexamethasone, or with dexamethasone alone, suggesting that this drug combination was antagonistic with respect to some cytokine responses. for the cytokine correlations (table 1), there was a baseline correlation between il-6 and tnf- following addition of atorvastatin but not in the presence of dexamethasone alone or combined with atorvastatin. in contrast, the correlation of il-2 with ifn- was maintained in the presence of either atorvastatin or dexamethasone alone but was lost when the drugs were added in combination. this suggests differential modulatory effects, with the combination of dexamethasone and atorvastatin exerting a distinct pattern of cytokine responses. for a more sensitive method of detecting ifn-, there was a significant decrease in intracellular expression of ifn- in cd3 t cells in the presence of the statins (p < 0.05 for atorvastatin and lovastatin ; p < 0.01 for simvastatin). dexamethasone, csa, mycophenolate, and rapamycin also demonstrated significant decreases in intracellular ifn- expression as compared to stimulated controls (p < 0.01 ; figure 5). dexamethasone exerted the most significant reduction in expression of intracellular ifn-, whilst the combination of dexamethasone with atorvastatin also significantly reduced ifn- (p < 0.05). statins have been demonstrated to exert anti - inflammatory effects in vitro : reduction in cd11b expression ; inhibition of ifn--induced mhc - ii expression on endothelial cells, macrophages, and therefore t - cell activation ; disruption of lipid rafts essential for t - cell activation. considering the use of statins as an adjunctive therapy for relapsing remitting multiple sclerosis, the 2010 cochrane review on statins for multiple sclerosis concluded that data from double - blinded randomized trials would be needed before licensing for treatment of multiple sclerosis. in spite of the large amount of in vitro data indicating a potential anti - inflammatory role of statins, there is little evidence for their direct role in modulating human t - cell cytokines. the concentrations of statins required to inhibit t - cell responses to anti - cd3/28 stimulation used for this in vitro study were selected from titration experiments, and these concentrations have been used in other in vitro studies [2527 ]. due to the potency of the in vitro stimulants, higher, nonphysiological concentrations of statins were required, and therefore, we can not extrapolate the data to statin doses used clinically. nevertheless, these studies can inform on the effects of statins on human t - cell responses. comparing the effects of drugs on t - cell proliferation, the statins inhibited proliferation to a comparable level to that achieved with conventional immunosuppressive drugs. the viability of the cells in our study was reduced in the presence of these statin concentrations, but, following the addition of mevalonate, there was a recovery both in viability and in proliferation, confirming that the inhibitory effect was reversible, and a direct result of statin - mediated inhibition of the hmg - coa pathway. of the conventional immunosuppressive drugs investigated, dexamethasone exerted the most potent immunomodulatory effect, and its ability to decrease production of il-1, il-2, il-6, il-8, ifn-, and tnf- is in agreement with previous studies [29, 30 ]. interestingly, dexamethasone treatment upregulated il-10 production, as reported by others. of course the study design does not permit us to determine the cellular source of the cytokines, and monocytes, which were likely to be present at low numbers, have previously been observed to respond to simvastatin by upregulating ifn- production which, in turn, led to a decrease in il-17 production. the downregulatory effect of other immunosuppressive drugs (csa, rapamycin, and mycophenolate) on ifn- expression is in agreement with other studies [29, 3234 ]. these drugs also differentially inhibited other cytokines, with csa significantly decreasing levels of il-2, il-4, and il-5 ifn- but not il-10 production (data not shown). in contrast, a decrease in il-10 mrna expression has been reported in a study on csa - treated normal human whole blood following anti - cd3/cd28 t cell costimulation. however, changes in il-10 mrna expression do not necessarily correlate with the level of protein production, which could explain the contradictory findings. mycophenolate has recently been demonstrated to have a profound inhibitory effect on th17 cells, and we have not yet investigated this drug for its effects on th17 cells in our model (in progress). other discrepancies in cytokine responses observed across the literature are easily explained by the different genetic backgrounds of donors used in different studies. all three statins in this study significantly reduced intracellular ifn- expression, as previously reported for mouse and human t cells [15, 36, 37 ]. for the other cytokines investigated in this study, intracellular expression levels of il-2, il-4, and il-10 were not significantly altered (data not shown) due to the variable responses between the individual donors and very low levels of il-4 and il-10 expression. atorvastatin, when given to hypercholesterolemic patients, has been found to decrease pbmc production of tnf- il-1, il-6, il-2, and ifn- in vitro. decreased serum il-8 and il-6 levels have been observed with atorvastatin postcoronary artery bypass grafts [40, 41 ]. more recently, atorvastatin has been reported to reduce the pathogenic production of il-6 and il-10 by activated t cells from sle patients thereby readjusting them toward a more tolerant phenotype. in our study, atorvastatin decreased production of il-17 and intracellular expression of ifn-, in agreement with data from a lewis rat model of experiment autoimmune neuritis and in balb / c mice with experimental colitis, in which tnf- was also downregulated. in murine eae treated with lovastatin, reduced levels of il-6, tnf-, and ifn- with upregulation of il-4, il-5, and il-10 were found, suggesting a th2 polarization [44, 45 ]. we detected significant decreases in production of il-17 and in ifn- expression in response to lovastatin. simvastatin decreased il-17 and ifn- expression and increased il-1. it has previously been found that simvastatin decreased tnf- in hypercholesterolemic patients but had inconsistent effects on plasma il-6 levels. simvastatin was not found to influence tnf-, il-6, and il-1 receptor antagonist levels in an endotox in induced human in vivo model of low - grade inflammation but decreased il-8 production by pbmcs of cad patients after 6 months of systemic treatment. this contrasts with in vitro effects of simvastatin on anti - cd3/anti - cd28-stimulated pbmcs from ra patients with decreased ifn- and il-10 unaffected. simvastatin also decreased levels of serum il-6 in mouse collagen - induced arthritis and il-17 gene expression in human ms patients cd4 t cells. due to the clinical use of atorvastatin for controlling cholesterol levels, it was selected in this study for possible combinatory effects when added with dexamethasone. however, by comparing atorvastatin, dexamethasone, and dexamethasone / atorvastatin together, no significant differences were found in overall levels of cytokines produced. however, the combination did affect cytokine correlations, suggesting that there might be an additive anti - inflammatory effect. there is little in the literature to suggest contraindications in terms of side effects, and the combination has been used experimentally in a rat model to reverse dexamethasone - induced hypertension. in conclusion, different statins, with their distinct effects on cytokine responses, could have clinical applications in specifically targeting those key cytokines relevant to each inflammatory disease process, perhaps as a corticosteroid - sparing therapy, while providing effective disease control. | hmg - coa reductase inhibitors (statins) have been demonstrated to be immunomodulatory for human immune - mediated disease and in experimental models. the aim of this study was to compare statin - mediated immunosuppressive effects on human t - cell responses in vitro with those of conventional immunosuppressives (dexamethasone, cyclosporin a (csa), mycophenolate, and rapamycin). statins (atorvastatin, lovastatin, and simvastatin) were investigated for their modulatory effects on human pbmc viability, cytokine profiles, and t - cell proliferation. at concentrations that inhibited anti - cd3/28-stimulated t - cell proliferation (p < 0.01), simvastatin significantly decreased intracellular cd4 + t - cell expression of ifn- (p < 0.01) to levels similar to those induced by conventional immunosuppressives. atorvastatin and lovastatin also decreased ifn- expression, although to a lesser degree (p < 0.05). all three statins reduced levels of il-17 production (p < 0.01). however, in response to anti - cd3/28 stimulation, simvastatin significantly upregulated il-1 production (p < 0.05). the profile of cytokines produced in response to anti - cd3/28 stimulation was similar when both atorvastatin and dexamethasone were added as compared with dexamethasone alone, suggesting that atorvastatin can synergise with dexamethasone with respect to immunomodulation of cytokines. this data supports the hypothesis of selective statin - mediated immunomodulatory effects on human immune cells. |
endovascular treatment of intracranial aneurysms is rapidly becoming the preferred method of aneurysm treatment over open neurosurgical clipping.1 2 this is in large part due to advances in coil and stent technology that have improved safety and outcomes of the procedure. however, complex and broad - necked aneurysms continue to pose a challenge with a higher incidence of recurrence.3 4 these aneurysms also require advanced techniques such as balloon remodeling, dual microcatheter or stent - assisted coiling.510 aneurysms located in a terminal or bifurcation anatomy create an even further difficulty as the anatomic and hematologic mechanical forces further increase the chance of coil compaction and aneurysm recurrence. when these aneurysms have broad necks, they often incorporate the adjacent branch vessels into the aneurysm neck which can create difficulty in treating the aneurysm without either occluding one of the outflow branches or leaving residual aneurysm.11 12 early results suggest that more durable results may be obtained by using stents in conjunction with coils ; however, current stents are engineered for sidewall aneurysm morphologies.13 14 the use of stents in bifurcation or terminal morphologies requires creative techniques such as y - stenting where one stent passes through the interstices of the other stent, or side - by - side (kissing) stents.14 15 these techniques are associated with increased difficulty of the procedure and peri - procedural risk.15 we present our early experience with a novel device designed for bifurcation aneurysm morphology in an experimental canine aneurysm model. the study was performed under an institutional animal committee approved protocol. using a previously described surgical technique, all animals were treated with 81 mg aspirin and 75 mg clopidogrel daily beginning a week before the procedure. at least 3 weeks before evaluation of the device, vein pouch bifurcation aneurysms were surgically created using a well - described technique.16 all procedures were performed using standard endovascular techniques through femoral artery access. the dogs were heparinized with 1250 units heparin before commencing the procedure. the pulsar vascular aneurysm neck reconstruction device (pvanrd) is a novel approximately 0.002-inch thick laser cut nitinol self - expanding device specifically shaped to fit within bifurcated arteries. it is designed to be deployed through a 0.027-inch microcatheter at the bifurcation and abut the aneurysm ostium while remaining outside the aneurysm. saddle is oriented by opposing struts that align with the outflow branches to ensure preservation of those branches (figure 1). this results in a saddle - shaped web across the aneurysm neck that supports coils in the aneurysm while maintaining patency of the outflow branches. the proximal end of the device tapers to two struts that are anchored in the proximal parent vessel. four radiopaque markers are present at the tips and mid - portion of the saddle to ensure appropriate orientation of the device at the aneurysm neck. there are four additional radiopaque markers oriented orthogonal to the saddle markers along the anchor legs to ensure appropriate expansion and alignment of the device. at the proximal end of the anchor legs is the electrolytic detachment zone, which is visible within the microcatheter prior to detachment. the pulsar vascular aneurysm neck reconstruction device (pvanrd) is a novel approximately 0.002-inch thick laser cut nitinol self - expanding device specifically shaped to fit within bifurcated arteries. it is designed to be deployed through a 0.027-inch microcatheter at the bifurcation and abut the aneurysm ostium while remaining outside the aneurysm. saddle is oriented by opposing struts that align with the outflow branches to ensure preservation of those branches (figure 1). this results in a saddle - shaped web across the aneurysm neck that supports coils in the aneurysm while maintaining patency of the outflow branches. the proximal end of the device tapers to two struts that are anchored in the proximal parent vessel. four radiopaque markers are present at the tips and mid - portion of the saddle to ensure appropriate orientation of the device at the aneurysm neck. there are four additional radiopaque markers oriented orthogonal to the saddle markers along the anchor legs to ensure appropriate expansion and alignment of the device. at the proximal end of the anchor legs is the electrolytic detachment zone, which is visible within the microcatheter prior to detachment. standard digital subtraction angiography was performed to characterize the aneurysm and obtain a working projection that best delineated the aneurysm neck and outflow branches and relation to the parent artery. the dimensions of the parent vessel, outflow branches and aneurysm were recorded. through a 6 f sheath, a 0.027-inch microcatheter was advanced to the aneurysm neck and the pvanrd deployed by unsheathing the device at the bifurcation. the device was placed so that the wings of the saddle were outside the aneurysm, aligned along the aneurysm neck and engaged with the proximal outflow branches. to position the arms of the saddle properly and to evaluate performance, the device was recaptured or repositioned several times. a technique deploying the device inside the aneurysm and then pulling back to engage the aneurysm neck was also used on some occasions. all operators have significant clinical experience with y - stenting to allow for educated comparison. the technical performance of the pvanrd was evaluated as follows : overall performance : graded as acceptable or unacceptable. microwire traversal : a 0.010-inch or 0.014-inch guidewire was then advanced across the stent multiple times to assess for microwire interaction with the device. this was graded on a 3-point scale as same, better, or worse than that expected with y - stenting in the operator 's judgment. microcatheter traversal : a microcatheter of 0.017-inch internal diameter was manipulated over a 0.014-inch microwire through the interstices of the device and into the aneurysm. this was graded on a 3-point scale as same, better or worse than that expected with y - stenting in the operator 's judgment. coil retention : coil(s) were placed into the aneurysm to determine the ability of the device to maintain the coils within the aneurysm as well as to evaluate the stability of the device when oversized coils were placed (table 1). device performance, vessel and aneurysm dimensions, and coils placed anrd, aneurysm neck reconstruction device. the goal of this project was not to achieve maximum packing density but rather to determine the performance of the device in stabilizing the framing and filling coils within the aneurysms. at the conclusion of the procedure twelve devices were placed in eight animals, each with a single experimental vein pouch bifurcation aneurysm. the devices were successfully delivered and deployed in all cases. specific device performance, vessel and aneurysm while the devices could have been removed at that time, the devices were detached to better understand their behavior in suboptimal conditions. the first device was eventually pushed into the aneurysm, but this required some effort with a curved wire against the saddle. a second device was more stable but, as expected due to its undersizing, poor apposition to the wall was found. nonetheless, a 15 cm30 mm coil was supported in the 13 mm12 mm aneurysm by the device. the second animal similarly was found to have vessels larger than indicated for the test device. however, due to the design of the device, the wings of the saddle prevented distal migration into the aneurysm. although poorly positioned, it did adequately support a 15 mm30 cm and a 24 mm40 cm coil placed into a 10 mm11 mm aneurysm despite the undersized device. design enhancements were subsequently made to the device to allow for use in a broader vessel size range for the subsequent studies. in the remaining six animals in the series it was easily manipulated so that appropriate positioning of the saddle across the aneurysm neck was possible in all cases. in most cases the device was resheathed and the orientation was rotated and changed several times to ensure design robustness. in all cases the device was stable and unable to be displaced with a guidewire or by navigating a microcatheter over a guidewire through it. the device was able to support placement of multiple significantly oversized coils into the aneurysm without prolapse of coil loops (figure 2). it was universally felt to be significantly easier to traverse than a y - stent. the last device was found to be situated slightly caudal and in the bifurcation after its detachment. subsequently, it could be advanced into an optimal position across the aneurysm ostium by careful engagement and manipulation with a curved guidewire, suggesting some degree of support by the wings of the saddle in the outflow branches. (a) native digital subtraction angiography (dsa) image showing device deployed across aneurysm neck and microcatheter in aneurysm. (b) native dsa image showing placement of a 14 mm30 cm and two 13 mm30 cm coils with stable device position at aneurysm neck and no coil loop prolapse. the treatment of broad - necked aneurysms, especially those with a terminal or bifurcation morphology, continues to pose challenges for endovascular embolization. these lesions often require advanced techniques that can increase the risk of treatment and are also prone to increased risk of aneurysm recurrence.38 this is probably related to adapting tubular endovascular devices created for standard sidewall anatomy and using them in a creative fashion to treat bifurcation aneurysms. in this study we describe our experience with a novel device designed for bifurcation aneurysms which was found to offer superior performance, excellent stability and ease of use with robust coil support in experimental canine bifurcation aneurysms. the primary challenge faced in treating broad - necked bifurcation aneurysms is the ability to achieve sufficient aneurysm occlusion with coils without compromising the outflow vessels. dual or multiple microcatheter techniques are commonly used in many parts of the world, most prevalently in countries that do not have ready access to balloons.810 the foundation of the technique relies on complexing multiple coils together to form a stable basket by independently varying the orientation and placement of the complex shaped coils through multiple microcatheters. this allows the operator to optimize the features of complex coils to form a stable and ultra - complex basket that can then be filled in from the microcatheters at various points within the aneurysm. this technique is quite effective, but requires significant skill and does not provide definitive structural protection of the aneurysm neck or individual outflow branches. balloon remodeling allows the operator to expand the balloon and have it asymmetrically expand in a bifurcation to protect the aneurysm neck.7 11 12 for more complex aneurysms, kissing balloons can be used with a balloon in each outflow vessel, but this requires significant skill to manage two balloons simultaneously and coil the aneurysm.6 there are also challenges with access, as two guide catheters are often required for this procedure. this technique can be especially challenging in the posterior circulation where broad - necked basilar artery aneurysms may be the most prevalent. while balloons provide definitive vessel protection while they are inflated, there is a chance of coil loop prolapse when the balloon is deflated which could compromise the bifurcation or outflow branches. most recently, y - stenting of these difficult lesions has shown the most promise for durable results but is associated with not insignificant procedural complications and technical challenges as well as increased cost.15 y - stenting results in multiple layers of stents in the parent artery which may delay the healing process and increase the risk of thrombosis. this technique requires catheterization of one of the outflow branches followed by placement of a stent from the outflow branch distal to the aneurysm neck proximally into the parent vessel, then traversing through the interstices of the first stent and into the other outflow branch and deploying a second stent from that branch back into the first stent and parent artery.13 this can be technically very challenging, especially in large aneurysms with acute angulation of the outflow branch vessels in relation to the aneurysm ostium. complications associated with this technique include prolapsing of the stent into the aneurysm while trying to advance a second stent for the final limb of the y. this has resulted in a technique where some operators simply deploy a stent from proximally within the aneurysm down into the parent artery that is, the waffle cone technique.14 this does provide some structure for coils to engage and complex at the aneurysm neck, but does not protect the outflow branches. there are challenges imposed by the study design, primarily related to the small number of aneurysms. however, this was a preliminary preclinical study for safety and device performance in a biologic model. the lack of long - term follow - up is also a limitation ; however, this is related to the extreme mechanical movement of the animal neck with a fixed aneurysm implant which could artificially influence the histologic and angiographic outcome. the grading of device performance is subjective, but this was mitigated as much as possible by using multiple operators, all with significant clinical experience in treating these types of aneurysms. the development of a true bifurcation aneurysm device may represent an important step forward in advancing the treatment of broad - necked bifurcation aneurysms. for a brief period the tri - span neck bridge device was available for human use outside the usa.17 this device similarly provided support at the aneurysm neck for coil retention in wide - necked aneurysms ; however, the device was designed to be deployed inside the aneurysm and to be cohesive with the coil mass. this feature limited the use of the device only to those aneurysms that were taller than wide. the location of the device within the aneurysm is attractive in that antiplatelet drugs would not be required for its use ; however, the structural support preventing compaction and recurrence may not be as robust as extra - aneurysmal device location. the current pvanrd device provides extra - aneurysmal structural support at the aneurysm neck with minimal intravascular surface exposure. the surface area of the pvanrd is approximately 85% less than the currently available tubular stents. this reduced intravascular exposure could potentially reduce the duration of clopidogrel and aspirin required for prophylaxis against thrombosis. the present study demonstrates the feasibility of using the pvanrd to support coil embolization of bifurcation aneurysms. the device has some unique characteristics which may provide some advantages over conventional stents for the treatment of experimental bifurcation aneurysms | introductiontreatment of wide - necked bifurcation aneurysms often poses procedural and long - term outcome challenges. the initial preclinical experience with the pulsar vascular aneurysm neck reconstruction device (pvanrd) in a canine bifurcation model is described.methodsexperimental bifurcation vein pouch aneurysms were surgically created in the carotid arteries of eight dogs. endovascular coiling of the aneurysms with assistance of the pvanrd was performed in all cases with acute performance compared with y-stenting.resultstwelve devices were deployed in the eight cases. deployment of the devices was straightforward and successfully protected the parent artery and maintained patency of the bifurcation in all cases, despite the use of oversized coils.conclusionthe pvanrd is a novel bifurcation stent that facilitates treatment of wide - necked bifurcation aneurysms compared with currently available adjunctive devices. |
studies related to anti - aging are currently being conducted actively around the world, and the related markets are growing fast. in particular, many studies are being conducted on delaying the skin aging process, as people have an increasing interest in changing their external form as well as the functional aspects of their bodies to increase their self - esteem1. furthermore, with the development of industry, interest in the effects of air contamination and environmental pollution on the skin and the body is rising, which has led to rising interest in scientific skin management and the introduction of various new skin care devices2. skin aging can be classified into two categories : intrinsic aging which develops naturally with age, and aging caused by the external environment3. intrinsic aging appears as wrinkles, sagging, and pigmentation, whereas aging caused by the external environment, e.g. ultraviolet rays, wind, coldness, dryness, and tobacco, appears as skin roughness, vasodilatation, actinic keratosis, and solar lentigo, which are generated only in exposed regions4. in particular, skin photo - aging by uv rays leads to acute reactions of erythema and edema, which increase the incidence of skin cancer and chronic reactions such as skin pigmentation. such pigmentation appears when melanin pigments are synthesized excessively in melanin cells exposed to uv rays or when the physiological functions of the skin deteriorate. when this happens, pigments are deposited in the horny cells of the epidermis, which appear as melasma and freckles5. various therapies to improve skin aging are being researched in dermatology, and various laser treatments, chemical peels, surgical treatments, fillers, and botox are being used6,7,8,9. the effects of low - level laser therapy (lllt) include reduced inflammation, improved lymphedema, relief of pain, and accelerated cell metabolism and cell division, which promote skin regeneration and help facilitate wound healing. it is also effective in collagen generation, acne treatment, wrinkle improvement, and skin laxity10,11,12,13,14,15,16. electroacupuncture (ea) has been reported to accelerate the healing of wound tissues, and to elicit anti - inflammation effects, wrinkle improvements, and the reduction of face edema17,18,19. radiofrequency (rf) has been reported to be effective at reducing of wrinkles and in the treatment of acne scars by increasing blood circulation and promoting collagen generation, as well as reducing abdominal obesity11, 20,21,22. this study was conducted to observe the effects of lllt, ea, and rf, which are used in physical therapy, on the pigmentation and skin tone of adult women s faces to provide basic data on skin interventions. the subjects of this study were 30 adult women between 30 and 55 years of age who visited m dermatology hospital in yongin, gyeonggi - do, south korea and who voluntarily agreed to participate after listening to an explanation of the purpose of this study. the subjects were assigned to a lllt group, (n=10), an ea group (n=10), and an rf group (n=10). inter - group homogeneity testing for general characteristics before treatment revealed no significant differences. the characteristics of the subjects are shown in table 1table 1.general characteristicsvariableslllt (n=10)ea (n=10)rf (n=10)age (yrs)42.8 4.943.4 7.142.9 3.7height (cm)163.0 7.9158.0 4.3159.8 4.3weight (kg)56.7 7.558.8 7.252.4 lllt : low - level laser therapy group, rf : radiofrequency group, ea : electroacupuncture group. lllt : low - level laser therapy group, rf : radiofrequency group, ea : electroacupuncture group for the lllt group, redpulsar (sometech inc., korea), which is a diode laser, was used the treatment sessions. it is a class iiib laser with a wavelength of 635 nm, and power output of 10 mw23. for the rf group, prix iii (shenb co., ltd, korea) was used at a frequency of 1 mhz (rf energy, maximum 450 w / cm) the treatment sessions24. for the ea group, disposable stainless steel acupuncture with a size of 0.20 15 mm was used. for the ea stimulator, an ots h-306 (hanil tm co., ltd., korea) was used with operating conditions of a frequency of 3 hz, 5 seconds of on time, and 2 seconds of off time. ea was performed at a clinical practice by a therapist with 10 years experience. the intensity of the ea was increased until the subject felt simulation and a little muscular contraction and was then maintained19. for the skin measurement, janus (psi co., ltd. after the face was fixed at a certain position and external light sources were blocked, the facial skin conditions under irradiation by white light, polarized light and uv light were captured with a canon dslr camera with 10-million pixelresolution. for the measurement of pigmentation and skin tone, the measurement areas were set and measured ratios of each variable. to minimize the measurement errors, the same rater performed measurements from the beginning to the end in similar indoor environments (room temperature 2021 c and humidity 5060%). all data were coded and analyzed using the statistical analysis program spss (ver.18). to determine the post - intervention changes in pigmentation and skin tone in each group, a test of normality was conducted. if subjects passed the test, a paired samples t - test was conducted ; otherwise, the wilcoxon rank - sum test was performed. to determine the inter - group differences in pigmentation and skin tone, one - way anova was used when the data satisfied normality ; otherwise, the significance of differnces was verified by the kruskal - wallis test. the lllt group showed no significant changes in post - intervention pigmentation (p>0.05). the ea group showed significant decreases in pigmentation in the left and right eye rims (p0.05) (table 3table 3.comparison of the intra - group and inter - group changes in skin tone among the lllt, ea, and rf groupsskin toneinterventionprepostforeheadlllt57.2 2.660.2 2.0ea55.9 3.055.9 3.2rf56.9 3.655.6 3.8noselllt52.8 2.852.7 2.5ea51.8 2.051.4 2.9rf52.6 3.355.6 3.8under eyesrightlllt46.2 3.847.1 3.9ea47.8 2.847.9 3.1rf47.0 3.048.3 3.9leftlllt47.2 3.848.1 3.6ea48.3 3.048.4 3.4rf47.6 3.249.0 3.5eye rimsrightlllt48.7 5.251.1 5.3ea49.5 3.750.1 3.9rf50.8 3.351.8 3.5leftlllt49.9 4.651.5 4.9ea50.2 3.650.4 4.1rf50.6 3.751.7 4.0cheeksrightlllt50.0 3.650.2 3.2ea49.7 2.248.9 3.5rf50.0 3.350.5 4.1leftlllt51.7 3.951.8 3.9ea50.70 2.750.2 3.5rf51.3 3.551.6 4.03values are shown as the mean sd. significant difference between (p<0.05) the pre- and post - tests within each group : paired samples t - test, wilcoxon rank - sum test. lllt : low - level laser therapy, ea : electroacupuncture, rf : radiofrequency, 1 : lllt, 2 : ea, 3 : rf). values are shown as the mean sd. significant difference between (p<0.05) the pre- and post - tests within each group : paired samples t - test, wilcoxon rank - sum test. lllt : low - level laser therapy, ea : electroacupuncture, rf : radiofrequency, 1 : lllt, 2 : ea, 3 : rf aging refers to the physiological phenomenon in which structural and functional changes naturally appear in entities over time. in particular, skin aging can be classified into two categories : intrinsic aging caused by genetic programming and aging caused by external environmental conditions, e.g. exposure to uv rays, wind, heat, and tobacco3, 25. the categories of skin aging appear in various forms depending on the region and psychological stress. to overcome this process, various efforts to delay the progress of skin aging are being made worldwide, and anti - aging - related markets are growing rapidly1. in this study, the effects of lllt, ea, and rf on the pigmentation and skin tone of adult women s faces were investigated. lee.23 found that the melanin level significantly decreased after the irradiation by blue o red led light (p<0.05). weiss.12 also found that erythema and pigmentation decreased in 90% of subjects. in their study, however, even though the values decreased, the differences were not significant. in an ea experiment with diabetic mice by zhai.26, both the erythema index and melanin index of the back skin decreased significantly. in a study by budamakuntla.27, the melasma area severity index (masi) significantly decreased after microneeding, which was identical to the finding of this study. kim.28 reported that after the application of fractional rf, the melanin index and erythema index improved significantly. in the present study, pigmentation significantly decreased under the left eye, and in the right eye rim, the left eye rim, and the right cheek. budamakuntla.27 which compared a tranexamic acid microinjection group and a tranexamic acid with microneedling group, and found the masi of the microneedling group significantly decreased from 9.11 4.09 to 5.06 2.14 post - treatment (p<0.001), but the intergroup differences were not significant. choi.24 compared rf and ultrasound and reported, the rf group showed a significant reduction in pigmentation compared to the ultrasound group. in a study by park.29, the pigmentation of subjects who received microneedle therapy system (mts) treatment significantly decreased compared to those receiving rf. in the present study, the ea group showed better effects in terms of nose pigmentation, and the ea and rf groups showed better results the left eye rim than the lllt group. lee. reported that 14 out of 24 patients experienced an increase in skin tone after being irradiated by red led light23. similarly, in the present study, the skin tone of the lllt group significantly increased in the forehead, the right eye rim, and the left eye rim. fitzpatrick.20 reported improved skin tone after the application of rf. in the present study, the rf group showed significant increases in skin tone under the left and right eyes. according to tobin and paus, skin tone is generated by a mixture of melanin, hemoglobin, and carotene, with melanin having the greatest effect. in the present study, barrett19 also reported that skin tone improved after the application of ea, and fabbrocini.31 reported that skin tone significantly increased in 17.4% of a group who received skin needling and depigmenting serum treatment and in 11.2% of a group who received depigmenting serum treatment only. in the present study, the ea group did not show significant changes in skin tone post intervention. the reason for this seems to be the differences between previous studies and the present study in terms of the electrical variables of the ea and in the application of serum, which can affect skin tone. first, the number of subjects was small and only included adult women between 30 and 55 years of age. therefore, the findings of this study can not be generalized. second, this study was conducted for a short period of only six weeks and the long - term effects were not evaluated. in the future, adult women of a broader age group should be studied, and long - term effects of more than six months should be verified. | [purpose ] in this study, the effects of low - level laser therapy (lllt), electroacupuncture (ea), and radiofrequency (rf), which are used in physical therapy, on the pigmentation and skin tone of adult women s faces were investigated to provide basic data for skin interventions. [subjects and methods ] thirty adult females were assigned to either an lllt group (n=10), an ea group (n=10), or an rf group (n=10). the intervention was performed in two 15-minute sessions per week for six weeks. subjects skin tone and pigmentation were observed before and after the intervention. [results ] the ea group showed significant reductions in pigmentation in the left and right eye rims, as well as in the left cheek. the rf group showed significant post - intervention reductions in pigmentation under the left eye, as well as in the left and right eye rims and the left cheek. the lllt group showed significant increases in skin tone in the forehead and both eye rims. the rf group showed significant increases in skin tone under both eyes. [conclusion ] the application of lllt, ea, and rf had positive effects on pigmentation and skin tone of adult women s faces. |
inflammatory bowel disease (ibd) is a group of inflammatory intestinal disorders mainly comprising 2 types of disease : crohn 's disease (cd) and ulcerative colitis (uc). traditionally, the incidence of this disease is relatively stable in western countries ; however, the ibd incidence has been increasing in asian countries in the past few years. peripheral arterial disease (pad) is the narrowing of arteries other than those that supply blood to brain or heart, causing a considerable burden on health care systems worldwide. the prevalence of peripheral vascular disease in the general population is 12% to 14%, affecting up to 20% of people > 70 years. according to the taiwan national health insurance (nhi) data analysis, the incidence of invasive pad treatment is increasing. traditional risk factors for pad are an older age, male sex, hypertension, diabetes, hyperlipidemia, obesity, smoking, and family history of vascular disease. chung observed that ibd patients exhibited a high risk of developing deep vein thrombosis and pulmonary embolism. in addition, recent studies have revealed an association between ibd and arterial thromboembolic events. pad is a type of arterial disease, but epidemiological studies on the relationship between ibd and the development of pad are scant. therefore, we conducted a nationwide population - based cohort study to investigate whether ibd increases the risk of developing pad. the national health insurance program was implemented in taiwan on march 1, 1995 and covered > 99% of the 25 million residents in taiwan. in this study, data were accessed from the national health insurance research database (nhird), which is managed and released for research purposes by the national health research institutes. the bureau of national health insurance (bnhi) scrambled the identification of insurants in the nhird to protect their personal information before releasing the data to researchers. a committee of the bnhi is responsible for randomly selecting and verifying the accuracy of claims. the international classification of disease, ninth revision, clinical modification (icd-9-cm) was used to code diagnoses. this study was approved to fulfill the condition for exemption by the institutional review board (irb) of china medical university (cmuh-104-rec2 - 115). patients in the inpatient claims with newly diagnosed ibd, including crohn 's disease (icd-9-cm codes 555) and ulcerative colitis (icd-9-cm codes 556) from 2000 to 2010 were selected as the ibd cohort. the ibd diagnosis is made based on clinical history, physical examination, and objective findings from endoscopic, radiological, laboratory, and histological studies. the comparison cohort was randomly selected from inpatient claims without the history of ibd. for each ibd case, 4 comparisons were identified, frequency matched by sex, age (within 5 years), index year of patients in the ibd cohort, and comorbidities including diabetes, hypertension, hyperlipidemia, copd, heart failure, cad, stroke, obesity, chronic kidney disease, autoimmune disease, surgery of the intestine, laparotomy, and colorectal cancer. the control - to - case ratio was set at 4:1 to enhance the power of statistical tests. in both cohorts, the patients who were diagnosed with pad (icd-9-cm codes 440.2, 440.3, 440.8, 440.9, 443, 444.22, 444.8, 447.8, and 447.9) and deep vein thrombosis (icd-9-cm codes 453.8) before the index date, histories of coagulopathies (icd-9-cm codes 286, 289.8), sickle - cell disease (icd-9-cm codes 282.6), age 99% of the 25 million residents in taiwan. in this study, data were accessed from the national health insurance research database (nhird), which is managed and released for research purposes by the national health research institutes. the bureau of national health insurance (bnhi) scrambled the identification of insurants in the nhird to protect their personal information before releasing the data to researchers. a committee of the bnhi is responsible for randomly selecting and verifying the accuracy of claims. the international classification of disease, ninth revision, clinical modification (icd-9-cm) was used to code diagnoses. this study was approved to fulfill the condition for exemption by the institutional review board (irb) of china medical university (cmuh-104-rec2 - 115). patients in the inpatient claims with newly diagnosed ibd, including crohn 's disease (icd-9-cm codes 555) and ulcerative colitis (icd-9-cm codes 556) from 2000 to 2010 were selected as the ibd cohort. the ibd diagnosis is made based on clinical history, physical examination, and objective findings from endoscopic, radiological, laboratory, and histological studies. the comparison cohort was randomly selected from inpatient claims without the history of ibd. for each ibd case, 4 comparisons were identified, frequency matched by sex, age (within 5 years), index year of patients in the ibd cohort, and comorbidities including diabetes, hypertension, hyperlipidemia, copd, heart failure, cad, stroke, obesity, chronic kidney disease, autoimmune disease, surgery of the intestine, laparotomy, and colorectal cancer. the control - to - case ratio was set at 4:1 to enhance the power of statistical tests. in both cohorts, the patients who were diagnosed with pad (icd-9-cm codes 440.2, 440.3, 440.8, 440.9, 443, 444.22, 444.8, 447.8, and 447.9) and deep vein thrombosis (icd-9-cm codes 453.8) before the index date, histories of coagulopathies (icd-9-cm codes 286, 289.8), sickle - cell disease (icd-9-cm codes 282.6), age < 20 years, or missing information, were excluded. the main outcome was hospitalization with a new diagnosis of pad during follow - up. person - years of the follow - up were calculated for each patient until hospitalization for pad or censored for loss to follow - up, withdrawal from the insurance program, or the end of 2011. comorbidities were identified according to hospital admissions prior to the index date as potential confounding factors. these comorbidities included diabetes (icd-9-cm code 250), hypertension (icd-9-cm codes 401- 405), hyperlipidemia (icd-9-cm code 272), chronic obstructive pulmonary disease (copd) (icd-9-cm codes 491, 492, and 496), heart failure (icd-9-cm code 428), coronary artery disease (cad) (icd-9-cm codes 410414), stroke (icd-9-cm codes 430438), obesity (icd-9-cm code 278.0), chronic kidney disease (icd-9-cm codes 580589), autoimmune disease (icd-9-cm codes 710.0, 710.1, 710.2, 714), surgery of the intestine (icd-9-cm procedure codes 45.045.9, 46.046.9, 48.048.9), laparotomy (icd-9-cm procedure codes 54.11, 54.19), and colorectal cancer (icd-9-cm codes 153154). chi - squared tests were used to examine the differences in the distribution of sex, age, and comorbidities between the ibd and comparison cohorts. student 's t test was used to examine the mean ages and mean follow - up period between both cohorts. the sex-, age-, and comorbidity - specific incidence density rates of pad per 10,000 person - years of follow - up were calculated for each cohort. univariable and multivariable cox proportional hazard models were applied to measure the hazard ratios (hrs) and 95% confidence intervals (cis) for pad in the ibd cohort compared with those in the comparison cohort. the multivariable cox models were simultaneously adjusted for sex, age, and comorbidities of diabetes, hypertension, hyperlipidemia, copd, heart failure, cad, stroke, obesity, chronic kidney disease, autoimmune disease, surgery of the intestine, laparotomy, and colorecal cancer. response effect of ibd on the risk of pad according to the average frequency of hospitalizations per year for ibd. the cumulative incidence curves of pad were determined using the kaplan meier method between the ibd cohort and the comparison cohort. all analyses were performed using sas, version 9.3, computer software (sas institute inc, cary, nc). a 2-tailed p the distributions of gender, age, and comorbidity were similar in the both cohorts, except colorectal cancer. the mean age of the comparison cohort was 53 years and that of the ibd cohort was 53.1 years, with 37% of the patients in the age group of 40 to 64 years (table 1). as shown in figure 1, the cumulative incidence of pad was higher in the ibd cohort than in the comparison cohort by the end of follow - up. overall, the incidence of pad was 1.24-fold higher in the ibd cohort compared with the comparison cohort (23 vs 18.6 per 10,000 person - years). after age, sex, and comorbidities were adjusted, the adjusted hr for developing pad was 1.29-fold higher (95% ci = 1.221.37) for the ibd patients compared with the comparison cohort. the sex - specific adjusted hrs for pad in the ibd cohort were statistically significant for both the women (hr : 1.25, 95% ci = 1.141.36) and men (hr : 1.35, 95% ci = 1.241.46) relative to those of the comparison cohort. the age - specific adjusted hrs for pad in the ibd cohort were significantly higher for all age groups, relative to those of the comparison cohort. a comorbidity - specific analysis revealed a high incidence of pad for patients with comorbidities in both cohorts (table 2). comparison of demographics and comorbidity between inflammatory bowel disease patients and controls cumulative incidence of peripheral arterial disease for patients with (dashed line) or without (solid line) inflammatory bowel disease. incidence and adjusted hazard ratio of peripheral arterial disease stratified by sex and age compared between ibd cohort and comparison cohort table 3 presents the risk of pad according to annual mean hospitalizations for the ibd patients, compared with the comparison cohort. the ibd patients presenting with 2 or more hospitalizations per year exhibited a 27.5-fold increased risk of pad development compared with the comparison cohort (95% ci = 18.740.4). table 4 shows the incidence and hrs for pad associated with ulcerative colitis and crohn 's disease in the ibd patients. the patients with uc exhibited a 32% higher risk of pad compared with the comparison cohort (adjusted hr = 1.32, 95% ci = 1.221.43). the cd patients also showed a 27% higher risk of pad compared with the comparison cohort (adjusted hr = 1.27, 95% ci = 1.181.38), which was significant. the risk of peripheral arterial disease related to annual times of hospitalization in patient with ibd and comparison cohort using the cox proportional hazard regression incidence and adjusted hazard ratio of peripheral arterial disease between different entities ibd this is the first study to investigate the long - term risk of pad in patients with ibd based on nationwide data. we adjusted for several classical pad risk factors in this population - based cohort study and determined that ibd is an independent risk factor for pad. we observed that adults with ibd had 1.29-fold risks of developing pad compared with the comparison cohort. in addition, by using the frequency of hospitalization as a surrogate index of ibd activity, we determined that ibd severity was significantly associated with the risk of pad (table 3). although the patients with ibd in this study exhibited a higher prevalence of comorbidities and coexistent conditions associated with the development of pad than the comparison cohort, ibd remained an independent risk factor for developing pad after covariates were adjusted. long - term inflammation is known to predispose patients to vascular endothelial dysfunction and atherosclerosis. similarities between the pathophysiological processes in the colonic wall of ibd patients and the process in the arterial wall exist during the progression of atherosclerosis, subsequently resulting in atherosclerotic plaque rupture and thrombotic vascular events. several reports have indicated that patients with ibd tend to exhibit lipid profile derangement, which promotes atherosclerosis. most of the ibd patients in this study were men, and pad risks increased in both sexes with ibd. the age - stratified effect of ibd on pad development was the highest in the patients < 40 years. however, the incidence of pad increased with age in the patients with ibd and those in the non - ibd cohorts, which is consistent with previous studies. as people age recent studies have shown that frailty, which is commonly associated with aging, can cause subclinical peripheral vascular disease to develop. in our study, study cohort with comorbidities were shown to further increase the risk of pad compared with the comparison group (adjusted hr = 1.45). among the comorbidities, there were several well - known traditional risk factors for pad such as hypertension, diabetes, and hyperlipidemia. this study revealed a strong association between an increased number of annual ibd - related medical admissions and the risk of developing pad. ibd patients with high admission rates may represent increasing ibd flare - ups and severity of the active periods. the results showed that the systemic inflammatory burden with ibd patients may be a key determinant of atherosclerosis risk. thus, aggressively controlling the inflammatory activity in ibd patients may reduce the vascular complications. first, the insurance data do not contain detailed information on smoking habits, body mass index, or family history of pad, all of which might be confounding factors in this study. consequently, as with previous publications, we adjusted for several comorbidities of smoking - related diseases (including cad, copd, and stroke) to make the potential confounding effect of smoking on our study results minimally. in addition, to adjust for the influence of body mass index, we included several traditional metabolic syndrome comorbidities such as hypertension, diabetes, and hyperlipidemia. second, the evidence derived from a retrospective cohort study is generally lower in statistical quality than that from randomized trials because of potential biases related to adjustments for confounding variables. despite our meticulous study design and control measures for confounding factors, bias resulting from unknown confounders might have affected our results. in conclusion, patients hospitalized for ibd exhibited an increased risk of pad compared with the comparison cohort. | abstractstudies on the association between inflammatory bowel disease (ibd) and peripheral arterial disease (pad) are scant. this nationwide population - based cohort study assessed the relationship between ibd and further risk of pad.this nationwide population - based cohort study was based on data obtained from the taiwan national health insurance database from 2000 to 2010, with a follow - up period extending to the end of 2011. we identified inpatients with newly diagnosed ibd by using international classification of diseases, ninth revision, clinical modification (icd-9-cm) codes. in addition, we selected a comparison cohort from the inpatient claims that was randomly frequency - matched according to age, sex, and index year. we analyzed the risks of pad by using cox proportional hazards regression models, including sex, age, and comorbidities.a total of 11,067 ibd patients and 43,765 controls were enrolled in this study. the risk of developing pad was 1.29-fold in the patients with ibd compared with the comparison cohort, after age, sex, and comorbidities were adjusted. the patients with ibd who required 2 or more hospitalizations per year were nearly 27.5-fold more likely to have pad compared with the comparison cohort.this nationwide population - based cohort study demonstrated that pad risks are higher in patients with ibd compared with those inpatients without ibd. careful follow - up observation and aggressive effective treatment should be sought for patients with ibd to reduce the risk of pad. |
we describe one patient who was affected by an and presented high level increase of serum liver enzymes, along with sever thrombocytopenia. a 14-year - old boy with an was admitted in the pediatric psychiatric emergency department of alzahra hospital with impaired electrolyte levels, bradycardia, hypotension, liver dysfunction, and thrombocytopenia. a ten - time increase in liver enzymes and thrombocytopenia were observed on admission. after two months of treatment, improvement of initial clinical symptoms and recovery of liver enzymes and thrombocytopenia after the treatment suggested that liver dysfunction and thrombocytopenia may be observed in an patients and should be taken care of by physicians. a 14-year - old boy with an 8-month history of an referred to our outpatient clinic (department of pediatrics, alzahra hospital). because of his physical condition (weight loss, bradycardia, imbalanced electrolytes, hypothermia, hypotension, and lung and liver dysfunction) we decided to hospitalize him (figure 1). a male patient with anorexia nervosa. on admission to the psychiatric unit, his body weight and body mass index (bmi) were 31 kg and 13.2, respectively. in addition, his liver enzymes were high, and his platelet decreased as shown in table 1. other causes of acute liver dysfunction such as hepatotoxic drugs, alcohol, cocaine and viral hepatotoxic diseases were evaluated and then ruled out. we started to treat the patient with conservative management including stabilizing electrolytes by serum therapy and nourishment using a high calorie diet. a marked decrease in serum transaminase and an increase in platelet count were observed with a gradual normalization of all the biochemical parameters within one months of hospitalization. he started eating and his electrolytes got balanced and his family became happy about his recovery. therefore, he was discharged after one months of hospitalization and successfully continued his rehabilitation program on an outpatient basis. this was one of the uncommon cases of an with a high level increase (10 times more) of liver enzymes and sever thrombocytopenia. minor degrees of liver injury have been reported in up to 40% of patients with an.13 although the mechanism of liver injury in an has thought to be due to protein - calorie malnutrition of the kwashiorkor type accompanied with fatty changes, this has not been rigorously demonstrated and the mechanism is still unknown.1314 in an, malnutrition has been reported to cause thrombocytopenia along with liver dysfunction.2 one report has described temporal changes in liver enzyme levels, platelet count, and thyroid peroxidase (tpo) levels in a patient with an.3 acute liver failure with a massive increase of serum liver enzyme may be a sever, though relatively uncommon, complication caused by severe malnutrition such as that secondary to an.46 as reported in the literature, serum transaminases do not reach such high values during the refeeding syndrome.7 to avoid complications of refeeding syndromes,7 nutritional rehabilitation is started with a low - energy oral and parental nutrition (25 kcal / kg body weight). therefore, follow up of serum liver enzymes should be part of our evaluation to prevent the uncommon but serious risk of acute liver failure. our case report showed an an patient with increased level of liver enzymes about 10 times more than normal range, as well as severe thrombocytopenia, which is uncommon. therefore, it is concluded that physicians should be aware of high level increase of liver enzymes and severe thrombocytopenia which may occur in an. | background : anorexia nervosa (an) is a difficult - to - treat psychosomatic disease. very few cases of acute liver failure associated with an have been described. we describe one patient who was affected by an and presented high level increase of serum liver enzymes, along with sever thrombocytopenia. then, we discuss the possible etiopathogenic factors.methods:a 14-year - old boy with an was admitted in the pediatric psychiatric emergency department of alzahra hospital with impaired electrolyte levels, bradycardia, hypotension, liver dysfunction, and thrombocytopenia.results:a ten - time increase in liver enzymes and thrombocytopenia were observed on admission. after two months of treatment, the levels were within the normal range.conclusions:improvement of initial clinical symptoms and recovery of liver enzymes and thrombocytopenia after the treatment suggested that liver dysfunction and thrombocytopenia may be observed in an patients and should be taken care of by physicians. |
aggressive angiomyxoma was first reported as a distinct variant of myxoid neoplasms in the female vagina and pelvis by steeper and rosai. these tumors mostly occurs in 3 to 5 decade of life and 95% in females. these lesions have a predilection for female pelvic soft parts, slow growth, frequent recurrences, and are characterized histologically by a predominantly myxoid stroma and an abundance of thin and thick walled vascular channels. this is a deep soft tissue tumor, which as the name suggests, may have a locally aggressive course. most tumors occur in women and are large, usually greater than 10 cm, slowly growing, and painless. the most common location is in the perineal region and may exert pressure on adjacent organs. no. a 8332 came to our opd on 05.08.05 with chief complaints of growth in the vulva - 2 month, bleeding from the growth - 1 month, irregular period - 6 month. no history of htn, dm, tb. tubectomy was done a year ago. on examination, gc is fair, pallor +, no palpable ln. no organomegaly, others nad, groin- nad. local examination of the vulva revealed a 8 5 cms pedunculated growth [figure 1 ] having its thick pedicle of about 1.5 cm thickness hanging from the clitoreal region slightly on the left side [figure 2 ]. p / r- nad. clinical presentation growth coming out from the clitoreal region on investigation, hb % - 9.8 gm%, abo gr.- o + ve, rbs- 101 mg / dl, rft - wnl, lft - wnl. pap smear - nad, cxr - nad, usg (w / a) nad. she was planned for wide excision of the vulval growth ; accordingly it was done under sa on 18.08.05 [figure 3 ]. on sectioning, the mass had focally infiltrating margins and a rubbery glistening grey / white surface [figure 4 ]. post op hpe revealed aggressive angiomyxoma [figures 5 and 6 ], cut margins negative. ihc studies revealed desmin and vimentin + ve, er, pr + ve and s100 protein negative. she is under regular follow - up, and doing well for the last 5 years. hpe shows thin epidermis and underlying hypo cellular myxoid tissue containing blood vessels of various calibers high power view showing hyaline thickening of blood vessels aggressive angiomyxoma is a rare, benign neoplasm that can be mistaken both clinically and on microscopy for several other conditions, it should be included as a differential diagnosis for any vaginal mass. it is important to diagnose this condition because the tumour is locally infiltrative and requires wide excision and follow up. it occurs mainly in the female pelvis, vulva, or perineum, though some cases are described in men. some authors believe that it is the only possible treatment, but surgery is often radical and can be mutilating, with massive blood loss. it also requires postoperative intensive care monitoring, and the recurrence rate after surgery remains high even if negative margins are obtained at the initial resection. a retrospective review has shown that patients having positive margins were as likely to have recurrence as those with negative margins. immunohistochemistry of the tumor cells revealed diffuse immunoreactivity for estrogen receptors, progesterone receptors, vimentin, and cd34. no expression of s-100 protein, bcl-2 protein, cd117 (c - kit gene product), epithelial membrane antigen, desmin, or h - caldesmon could be demonstrated. | aggressive angiomyxoma is a rare, benign neoplasm occurring in 3rd to 5th decade of life that can be mistaken both clinically and on microscopy for several other conditions, it should be included as a differential diagnosis for any vaginal mass. these lesions have a predilection for female pelvic soft tissues, slow in growth, and are characterized histologically by a predominantly myxoid stroma and an abundance of thin and thick walled vascular channels. this is a deep soft tissue tumor, which as the name suggests, may have a locally aggressive course. most tumors occur in women and are large, usually greater than 10 cm, slowly growing, and painless. standard of care treatment for angiomyxoma has been surgery. some authors believe that it is the only possible treatment, but surgery is often radical and can be mutilating, with massive blood loss. |
the electron phonon interaction in semiconductor heterostructures is of greater interest in comparison to bulk materials. this is due to the fact that the quasi - particle space localization leads to the modifications of the energy spectrum. the all - important factor is the rise of new vibration branches of optical spectrum, namely, the interface optical phonon. this interaction is of considerable importance in the understanding of the properties of heterostructures based on material with high ionicity. it can lead to self - consistent bond state of a charge particle and phonons, that is, the large radius polaron. currently, an investigation on the part played by interface phonons has attracted considerable interest in polaron state formation study. the contributions to polaron binding energy both of interface and of bulk optical phonons are the same value order in the quantum dots [3 - 5 ]. taking into account, alternatively, the interface phonon role dominates in polaron binding energy for quantum well case. in response to this fact, the strong electron phonon interaction can be realized in the quantum wells based on non - polar material with high iconicity barrier material. in addition, from the results, it follows that profound polaron effects should be expected, e.g., in the si / sio2 compounds. although there are no polar optical phonons in the material of such quantum well, these may be produced at the heteroboundary. as a result, the strong interaction of charged particles with interface phonons becomes possible. phonon interaction is possible when the quantum well is made of polar material and for the barriers is taken non - polar material. in recent years, varied technologies of semiconductor quantum wire growth with assorted barriers are progressing rapidly. the most success has been achieved for the quantum wires based on iii v compounds [8 - 12 ]. it is in these structures that the polaron states can arise. at the same time, no extended theoretical study of the polaron states in such structures is available. proper allowance must be made for the interaction of charge particles with interface optical phonons for an understanding of this problem. in this paper, we develop a theory of polarons in the quantum wires, taking into account the interaction of charged particles with all branches of the optical phonon spectrum. the general equations have been obtained to describe the phonon spectrum taking into account the interaction of polarization and deformation potentials. in materials with high ionicity degree, the charge particle interaction with polar optical phonons is of crucial importance in polaron state formation. this has led us to use the model which takes into account this phonon type in the quantum wire. optical - phonon modes in the quantum wire are determined using the classical electrostatics equations : together with conventional boundary conditions at heterointerfaces, where is the polarization field, the electric field, the scalar potential, the total charge density, and is the dielectric susceptibility of the material i (i = 1, 2). the dielectric function is given by : where and are the frequencies of longitudinal - optical (lo) phonons and transverse - optical (to) phonons, respectively, and is the high - frequency dielectric constant. 1) for the cylindrical quantum wire leads to the equation defining the dispersion law for interface optical phonons : here, i m is the m - th order modified bessel function of the first kind, km is the m - th order modified bessel function of the second kind, k is the wave vector, 0 is the quantum wire radius. 3. in fig. 1 is shown the wave - vector dependence of the interface phonon frequencies. this dependence is calculated for the quantum wire based on cdse surrounded by znse barriers with m = 0 in eq. the wave - vector dependence of interface optical phonon frequencies for znse / cdse / znse quantum wire the hamiltonian operator for phonon subsystem is conveniently written in terms of the phonon creation and annihilation operators : where the operators describe the creation of bulk phonons localized inside the quantum wire, are the interface phonon creation operators. phonon interaction for the cylindrical quantum wire can be represented by the method supposed in : here, the coefficients are defined as : here, n(m) is n - th order root of the equation jm() = 0, jm is the m - th order bessel function of the first kind. the interaction parameters m(k) have the form : the expressions (6), (7) do not require in the region. the reason is that we suppose the total electron localization within the quantum wire. in eq the general equations have been obtained to describe the phonon spectrum taking into account the interaction of polarization and deformation potentials. in materials with high ionicity degree, the charge particle interaction with polar optical phonons is of crucial importance in polaron state formation. this has led us to use the model which takes into account this phonon type in the quantum wire. optical - phonon modes in the quantum wire are determined using the classical electrostatics equations : together with conventional boundary conditions at heterointerfaces, where is the polarization field, the electric field, the scalar potential, the total charge density, and is the dielectric susceptibility of the material i (i = 1, 2). the dielectric function is given by : where and are the frequencies of longitudinal - optical (lo) phonons and transverse - optical (to) phonons, respectively, and is the high - frequency dielectric constant. 1) for the cylindrical quantum wire leads to the equation defining the dispersion law for interface optical phonons : here, i m is the m - th order modified bessel function of the first kind, km is the m - th order modified bessel function of the second kind, k is the wave vector, 0 is the quantum wire radius. 3. in fig. 1 is shown the wave - vector dependence of the interface phonon frequencies. this dependence is calculated for the quantum wire based on cdse surrounded by znse barriers with m = 0 in eq. the wave - vector dependence of interface optical phonon frequencies for znse / cdse / znse quantum wire the hamiltonian operator for phonon subsystem is conveniently written in terms of the phonon creation and annihilation operators : where the operators describe the creation of bulk phonons localized inside the quantum wire, are the interface phonon creation operators. phonon interaction for the cylindrical quantum wire can be represented by the method supposed in : here, the coefficients are defined as : here, n(m) is n - th order root of the equation jm() = 0, jm is the m - th order bessel function of the first kind. the interaction parameters m(k) have the form : the expressions (6), (7) do not require in the region. the reason is that we suppose the total electron localization within the quantum wire. in eq. let the quantum wire be surrounded with compositionally identical barriers. in order to separate the effect of exactly dielectric irregularities, we assume that the potential well for electrons is rather deep, so that the penetration of the wave functions under the barrier can be disregarded. in this case, the interaction of charged particles with barrier phonons is weak. we write the hamiltonian of the system as here, is the electron hamiltonian for which the interaction of the electron with phonons is disregarded. the hamiltonian is given by where is the quantum wire potential and m is the electron effective mass. if the interaction of an electron with polar optical phonons is strong, the polaron binding energy can be determined with the use of adiabatic approximation. in so doing, the adiabatic parameter here is the ratio of the quantum wire radius 0 to the polaron radius a0 : the exact expression for polaron radius a0 is obtained below. the condition (eq. 13) implies that the main contribution to the polaron binding energy is given by small values of the wave vector k such that if condition (eq. 13) is satisfied, the wave function of an electron localized in the n - th size - quantization level can be represented as : where the wave function describes the two - dimensional electron motion not disturbed by electron the wave function represents the electron localization in the self - consistent potential well created by phonons. the quantum numbers n, m define not disturbed electron state in the quantum wire. in the case of total electron localization in the cylindrical quantum wire, the wave function has the form : here is n - th root of m - th order bessel function. the wave function is to be obtained by solving self - consistent problem. in so doing, the total wave function from eq. 12) takes the form after this procedure : here is the energy of an electron on relevant size - quantization level, m is the electron mass inside the quantum wire. phonon interaction can be written as : here, and are the coefficients and m(k) from eq. we obtain average hamiltonian it can be brought to the form diagonal in phonon variables by the unitary transformation where the unitary transformation application gives the following equation : from expression (eq. 21), we can see that, in the adiabatic approximation used here, the bulk phonon spectrum and the interface phonon spectrum remain unchanged. 21) presents the energy of a large radius polaron. in the general case, the energy involved in (eq. 21) depends on the dielectric properties of the materials of both the quantum wire and the barriers. in the general case, the polaron binding energy ee depends on electron size - quantization level number and on optical - phonon spectrum properties. after the procedure of angle averaging which is expressible in explicit form, we obtain this energy ee as : the energy (eq. 22) is defined by the electron interaction with phonon modes correspond to m = 0 only. this equation (eq. this contribution is caused by the interaction of localized electron with confined and interface phonons. however, the electron energy and wave function can be obtained analytically on condition the unequality (eq. the most significant contribution to the polaron binding energy in the parameter (eq. 14) gives the interaction of an electron with interface phonon mode of the frequency close to barrier frequency the largest contribution to the energy in the parameter (eq. this quantity comes about from taking into account the interaction of an electron with interface optical phonons. 23 that the quantum wire material properties have no effect on the polaron state formation. the part of quantum wire material dielectric properties can be obtained in higher orders in the parameter (eq. 23 that the characteristic values of the phonon wave vector k which describe the value of electron phonon interaction is of the order reciprocal to polaron radius a0 the logarithmic function changes weakly in this region. therefore, we can consider with the same accuracy in parameter (14) that the energy is equal to : the substitution of the energy from eq. 19 leads to the expression for polaron binding energy as the functional of unknown yet wave function (z). it can be written as : the following equation is obtained by variational method using wave functions (z) : this nonlinear eq. 26 has the solutions which can be written in the form with any energy values epol : the polaron binding energy is found by substitution of eq. 27 to 26 : the polaron radius a0 is obtained by solving the transcendental equation. it has the form : it is this quantity from eq. 29 which contains the adiabatic parameter (eq. 13). substituting material parameters into eq. 29 for the quantum wire znse / cdse / znse leads one to expect that the strong polaron effects for these structure should be observed when the quantum wire radius 0 < 40. it might be well to point out that both the polaron binding energy (eq. 29) depend on effective electron mass inside the quantum wire and barrier dielectric properties. this clearly demonstrates the prevailing role of the interaction of an electron with interface optical phonons. the availability of the surface phonons leads to widening the range of materials in which the strong polaron effect should be expected. phonon interaction may exist near the interface between polar and non - polar materials. among other things phonon interaction is determined significantly by interface optical phonons in narrow quantum wires corresponding to the condition (eq. is meant that the interface phonons can produce the effective canal of excitation transfer in the structures with several quantum wires. this work was supported by russian foundation for basic research, grant 09 - 02 - 00902-a and the program of presidium of ras the fundamental study of nanotechnologies and nanomaterials no. 27. this article is distributed under the terms of the creative commons attribution noncommercial license which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. | the theory of large radius polaron in the quantum wire is developed. the interaction of charge particles with interface optical phonons as well as with optical phonons localized in the quantum wire is taken into account. the interface phonon contribution is shown to be dominant for narrow quantum wires. the wave functions and polaron binding energy are found. it is determined that polaron binding energy depends on the electron mass inside the wire and on the polarization properties of the barrier material. |
type 2 diabetes, while being initially characterized by decreased insulin sensitivity of peripheral tissues, ultimately results in the loss of -cell mass. islet transplantation has the potential to rescue the -cell deficiency observed in both forms of diabetes. a major obstacle to greater use of islet transplantation various studies have demonstrated that most -cell proliferation occurs prior to adolescence [26 ]. interestingly, -cell proliferation has been observed in obese and pregnant human populations [79 ]. these data suggest that while -cell proliferation is tightly controlled the pathway responsible for proliferation is nonetheless intact and given the proper stimuli can be engaged to induce pancreatic islet -cell expansion. various groups have sought to delineate the molecular pathways responsible for inducing proliferation of adult primary -cells as a potential therapeutic intervention for diabetes. recent studies have shown that the -cell transcription factor nkx6.1 induces -cell proliferation by upregulating expression of the orphan nuclear receptors nr4a1 and nr4a3 [10, 11 ]. these data begin to delineate a molecular pathway capable of inducing -cell proliferation that may be leveraged as a therapeutic for enhancing functional -cell mass in diabetic patients. the cdk5/cdk5r1 complex is highly expressed in neurons and is required for proper central nervous system development and neuronal migration. furthermore, deregulation of the cdk5 kinase has been associated with progression of alzheimer 's disease, breast cancer, and prostate cancer [1618 ]. studies have illustrated that activated cdk5 promotes insulin exocytosis and that cdk5 protects against -cell apoptosis [2023 ]. here we report that cdk5r1 is upregulated by the orphan nuclear receptors nr4a1 and nr4a3 in primary rat islets. we demonstrate that overexpression of cdk5r1, and not cdk5, is sufficient to induce -cell proliferation in the 832/13 ins-1 -cell line and primary rat islets while maintaining insulin content and glucose stimulated insulin secretion. furthermore, we show that cdk5r1 mediated proliferation is dependent on the cdk5 complex kinase activity. finally, we demonstrate that cdk5r1 mediated proliferation correlates with increased phosphorylation of prb, which results in activation of -cell cell cycle machinery through release of e2f transcription factors. we propose that the cdk5r1 mediated -cell proliferation pathway may be harnessed to increase -cell numbers for ultimate islet transplantation therapy. wistar rat breeding pairs were purchased from harlan and maintained on standard chow diet (teklad 7001 ; harlan). male rats were allowed to feed ad libitum and were maintained on a 12-hour light - dark cycle. pancreatic islets were isolated from 5-week - old male rats as previously described [2426 ]. all animal studies were approved and performed in accordance with brigham young university 's iacuc guidelines. primary rat islets were cultured in rpmi 1640 and supplemented with 10% fbs, 1% fungizone antimycotic (life technologies), and 1% hepes., islets were cultured for 48 hours with 10 mol / l roscovitine (santa cruz) or dmso (vehicle, sigma). for all islet experiments using adenoviral transduction, viral treatments occurred within four hours of completing the islet isolation procedure as previously published [10, 11, 26, 2832 ]. islets were transduced with adenovirus for 18 hours, after which media and adenoviral vectors were removed. all measurements on primary rat islets (proliferation, survival, and insulin secretion) occurred 96 hours after islet isolation. adenoviruses expressing nkx6.1, nr4a1, nr4a3, and gfp have been described elsewhere [10, 3439 ]. all recombinant viruses were shown to be replication incompetent by being e1a deficient, using an rt - pcr screen, as previously described. for studies involving adenovirus mediated gene manipulation, pools of 200 islets were transduced with ~2 10 ifu / ml adenovirus (moi~100200) for 18 hours, as previously described [10, 11, 29, 32, 41, 42 ]. media were changed after 18 hours and islets were cultured for up to 96 hours. assays were completed at 48, 72, or 96 hours after harvest depending on the experiment. for apoptosis assays, 832/13 cells were plated at a concentration of 2 10 cells / ml in 24-well plates at 1 ml per well. following 24 hours of incubation, the cells were transduced with ~2 10 ifu / ml adenovirus (moi~100200) of adcmv - gfp, adcmv - cdk5, or adcmv - cdk5r1 or left untransduced. following 48 hours of culture, the cells were treated with etoposide (0.9 m), camptothecin (2.0 m), thapsigargin (0.32 mm), or dmso for 18 hours. cell viability was determined by comparing the percentage of cells remaining after drug treatment to the percentage of cells after dmso treatment. total caspase-3 and cleaved caspase-3 were measured by western blot analysis of cells transduced with adcmv - gfp or adcmv - cdk5r1 treated with dmso (no drug), camptothecin, etoposide, or thapsigargin. ratio of cleaved caspase-3 versus total caspase-3 was measured and quantified. 832/13 cells were transfected using lipofectamine rnaimax (life technologies) with sirna duplexes against rat cdk5, 5-guucagcccuccgggagautt-3, or a previously described scrambled control, 5-gagaccctatccgtgatta-3 [32, 34 ]. following an overnight incubation, cells were transduced with adcmv - gfp or adcmv - cdk5r1 or left untreated. rna was harvested to measure extent of cdk5 knockdown by rt - pcr, and proliferation was determined by cell counts. [methyl - h]-thymidine was added at a final concentration of 1 ci / ml to groups of ~200 islets for the final 48 hours of culture. groups of 20 islets were picked in triplicate and washed twice in rpmi-1640 with unlabeled thymidine and twice in pbs and then the dna was precipitated with 500 l cold 10% trichloroacetic acid and solubilized with addition of 80 l of 0.3 n naoh. [h]-thymidine incorporation was measured by liquid scintillation counting and normalized to total cellular protein. insulin secretion was measured in triplicate in groups of 20 islets per condition as previously described in secretion assay buffer (sab) containing 2.5 mm glucose for 1 hour at 37c (basal) followed by incubation in sab containing 16.7 mm glucose for 1 hour (stimulatory). insulin was measured in sab using the coat - a - count kit (siemens). islets were lysed in ripa buffer and total protein was determined by bca (pierce), and insulin content was measured as described. clarified cell lysates were run on 412% nupage gels (invitrogen) and transferred to polyvinylidene fluoride (pvdf) membranes. membranes were probed with diluted antibodies raised against nkx6.1 (iowa developmental hybridoma bank, f55a10), gfp (abcam, ab13970), -tubulin (sigma, t5326), cdk5r1 (cell signaling, 2680), cdk5 (cell signaling, 2506), caspase-3 (cell signaling, 9665), prb (cell signaling, 9313), or phosphorylated prb (cell signaling, 9309). sheep anti - mouse (1 : 10,000) and goat anti - rabbit (1 : 10,000) antibodies (ge healthcare) coupled to horseradish peroxidase were used to detect the primary antibodies. rna was harvested using the rneasy microkit (qiagen) and cdna was synthesized in an iscript reaction (biorad). real - time pcrs were performed using the life technologies one step plus sequence detection system and software (life technologies). taqman - based assay on demand primers and probes (life technologies) were used to detect rat cdk5, cdk5r1, and peptidylprolyl isomerase a (ppia, internal control). islets were cultured with edu at a concentration of 10 m, with daily media changes, for 96 hours. pools of 50 islets were dispersed and plated on poly - d lysine coated coverslips (bd). five sections containing greater than 400 nuclei were evaluated from edu signal evaluated using imagej software for each experimental condition. previously published microarray data were queried for genes upregulated by nr4a1 and nr4a3 48 hours after adenoviral transduction that were not regulated by nkx6.1 at 24, 48, or 96 hours after adenoviral transduction time points. genes were selected by being induced by nr4a1 and nr4a3, not by nkx6.1, with 1.5-fold or greater induction and a p value less than 0.05. gene ontology (go) analysis was completed using panther pathway analysis, as described. for statistical determinations, data were analyzed by the paired student 's t - test or by one - way anova with bonferroni post hoc analysis for multiple group comparisons. we have previously shown that overexpression of the -cell transcription factor nkx6.1 is sufficient to induce -cell proliferation. nkx6.1 mediated -cell proliferation is dependent on expression of the nr4a1 and nr4a3 orphan nuclear receptors. using microarray analysis, we have shown that many of the nr4a1 and nr4a3 upregulated genes are also upregulated by nkx6.1 at a later time point. furthermore, knockout of nr4a1 or nr4a3 is sufficient to impede expression of nkx6.1 upregulated genes and abrogate nkx6.1 mediated -cell proliferation, demonstrating nkx6.1 dependence on the nr4a1 and nr4a3 transcription factors. we sought to determine if any of the nr4a1 or nr4a3 upregulated genes is independent of the nkx6.1 proliferation pathway. to address this question, we used data mining techniques to compare the previous published microarray data to define genes that are induced by nr4a1 and nr4a3 but are not included in the nkx6.1 mediated -cell proliferation pathway. using our previously published microarray data we defined genes upregulated by nr4a1 and nr4a3 after 48 hours of expression which are not induced by nkx6.1 at the 24-, 48-, or 96-hour time points. from this analysis, we determined that 114 genes were upregulated by nr4a1 and nr4a3 by 1.5-fold or greater which are not regulated by nkx6.1. using the panther pathway analysis software, we determined the most highly represented biological functions from our selected genes, based on go terms. from this analysis, we determined that the strongest upregulated go biological functions at 49.5% of our genetic subset (54 genes) were metabolic process (which consists of 3 genes in biosynthetic process, 9 genes in catabolic process, 1 gene in coenzyme metabolic process, 1 gene in generation of precursor metabolites and energy, 12 genes in nitrogen compound metabolic process, 14 genes in phosphate - containing compound metabolic process, 43 genes in primary metabolic process, and 2 genes in sulfur compound metabolic process) and at 35.2% (42 genes) were cellular process (which consists of 13 genes in cell communication, 11 genes in cell cycle, 1 gene in cell proliferation, 6 genes in cellular component movement, 3 genes in chromosome segregation, and 1 gene in cytokinesis) (figure 1(a)). as our goal was to determine genes involved in -cell proliferation which are unique to the nr4a nuclear hormone receptors, we queried the cellular processes category for genes associated with the cell cycle. with 43.2% of the subset of genes, the cell cycle go biological function was the second largest category, just behind cell communication at 54.1% (figure 1(b)). within the cell cycle set of genes, 16 genes were associated with various parts of the cell cycle including cytokinesis, centromere formation, and microtubule formation. among these genes, the cdk5 ligand cdk5r1 was found to be upregulated 1.7-fold by nr4a1 and nr4a3 but not upregulated by nkx6.1. the binding partner of cdk5r1, cdk5, was unregulated by nr4a1, nr4a3, and nkx6.1 (table 1). cdk5r1, due to its key role as a cdk regulator and the activity of the cdk5 complex in diabetes, was chosen as a potential candidate for further studies. to verify the microarray analysis results, we measured cdk5r1 mrna from primary rat islets. islets were transduced with adcmv - gfp, adcmv - nkx6.1, adcmv - nr4a1, or adcmv - nr4a3 or left untreated. total rna was isolated 48 hours after viral transduction. while untreated islets, islets expressing gfp, and islets expressing nkx6.1 had no increased cdk5r1 mrna levels, islets transduced with nr4a1 or nr4a3 had 13.5- and 10.1-fold increase, respectively (figure 2(a)). in contrast, cdk5 levels do not change when comparing untreated primary rat islets or islets expressing gfp, nk6.1, nr4a1, or nr4a3 (figure 2(b)). overexpression of nr4a1 or nr4a3 is sufficient to induce increased cdk5r1 protein levels (figure 2(c)). finally, overexpression of nr4a1 and nr4a3 in the same islet population demonstrated no synergistic increase in cdk5r1 protein levels, which is consistent with the highly overlapping transcriptional profiles induced by these transcription factors in primary rat islets (figure 2(d)). these data demonstrate that cdk5r1 upregulation is unique to nr4a1 and nr4a3 induction and not induced in the nkx6.1 mediated pathway. to determine if cdk5r1 is sufficient to induce islet proliferation, adenoviral vectors expressing cdk5r1 and cdk5 were designed. primary rat islets were cultured without adenoviral transduction or were transduced with adcmv - gfp, adcmv - cdk5, or adcmv - cdk5r1 at isolation for 24 hours. islets were cultured in the presence of h - thymidine beginning 48 hours after adenoviral transduction and were harvested after 96 hours of culture. islets transduced with adcmv - cdk5r1 demonstrated sixfold induction in cdk5r1 protein level (figure 3(a)). this resulted in a 2.5-fold induction in islet proliferation rate (figure 3(b)). in addition, islets transduced with adcmv - cdk5r1 and adcmv - nkx6.1 demonstrated an additive effect in terms of proliferation, suggesting that cdk5r1 overexpression results in the activation of factors that are left unchanged by nkx6.1 alone, potentially demonstrating that different portions of replication competent pathways are in effect (figure 3(c)). furthermore the inability of cdk5 overexpression to induce proliferation in primary rat islets suggests that either cdk5r1 is acting in a cdk5 independent manner or sufficient cdk5 protein levels are present in the islet and that addition of cdk5r1 is necessary to activate the cdk5-cdk5r1 proliferation pathway resulting in induction of proliferation. our data demonstrate that cdk5r1 overexpression drives proliferation in primary rat islets. to determine if -cells specifically replicate in response to cdk5r1 overexpression, we cultured primary rat islets transduced with adcmv - gfp or adcmv - cdk5r1 with the thymidine analog edu. islets were cultured in the presence of edu for 96 hours, after which islets were dispersed and the percentage of insulin+edu+ and insulinedu+ cells were calculated. our findings demonstrate that untransduced islets and islets transduced with adcmv - gfp had less than 1% of cells positive for edu in the insulin positive and insulin negative populations (figures 4(a)4(c)). in contrast, islets transduced with adcmv - cdk5r1 had 8.57% insulin+edu+ cells and 2.80% insulinedu+ cells (figures 4(a) and 4(c)). furthermore, given that the majority of proliferation is observed in -cells and not -cells suggests that -cells may express a higher level of cdk5 than the other islet endocrine cells. knockdown of cdk5 and cdk5r1 has been shown to result in enhanced caspase-3 cleavage and induction of apoptosis in the 832/13 ins-1 cell line. based on these findings, we hypothesized that overexpression of cdk5r1 would result in protection against apoptotic stimuli such as etoposide, thapsigargin, and camptothecin. to determine the effect of cdk5r1 overexpression on these inducers of apoptosis, untreated cells were compared to cells transduced with adcmv - gfp, adcmv - cdk5, or adcmv - cdk5r1. following a 48-hour culture, the cells were exposed to doses of etoposide, thapsigargin, or camptothecin for 18 hours. cell viability assays demonstrated that cdk5r1 overexpression protected 832/13 ins-1 cells treated with thapsigargin or etoposide as compared to untreated cells or cells transduced with adcmv - gfp or adcmv - cdk5 (figure 5(a)). this demonstrates that cdk5r1 overexpression protects -cells from ca induced er stress and topoisomerase ii inhibitor induced apoptosis. interestingly, no protection was observed with cdk5r1 or cdk5 in cells treated with camptothecin, an inhibitor of topoisomerase i (figure 5(a)). in addition to measuring cell viability through cell counts, we also measured total and cleaved caspase-3 levels. we demonstrated that cells treated with adcmv - gfp had significantly higher levels of cleaved caspase-3 than cells transfected with adcmv - cdk5r1 when both cell types were treated with thapsigargin or etoposide. cells treated with camptothecin showed no decrease in cleaved caspase-3 levels, supporting our cell viability data for cdk5r1 and this compound. taken together, these data demonstrate that overexpression of cdk5r1 can protect -cellsagainst selected apoptotic stimuli and inhibits caspase-3 activation. various studies have demonstrated that induction of -cell proliferation is frequently accompanied by decreased insulin secretion [45, 46 ]. furthermore, recent studies have demonstrated a connection between the cdk5 kinase complex and insulin secretion rates. therefore, we sought to determine the effect of cdk5r1 overexpression on -cell insulin content and secretion. islets were transduced overnight with adcmv - gfp, adcmv - cdk5, or adcmv - cdk5r1 and subsequently cultured for 96 hours. total insulin content of islet populations was measured, and no significant difference was observed between the three treated populations and the untreated controls (figure 6(a)). similarly, secreted insulin was measured from adenoviral treated or untreated control islets cultured in the presence of low (2.8 mm) and high (16.7 mm) glucose concentrations. these data also demonstrate that the adenoviral treated islets have similar insulin secretion to the untreated controls (figure 6(b)). taken together, these data demonstrate that cdk5r1 overexpression in primary rat islets, following a 96-hour incubation period, results in maintenance of insulin content and insulin secretion. the primary function of cdk5r1 is to serve as the binding partner of cdk5, thus activating the cdk5 : cdk5r1 kinase activity. we therefore hypothesized that cdk5r1 mediated proliferation is dependent on cdk5 kinase activity. to test this hypothesis, islets were transduced with adcmv - gfp, adcmv - cdk5, or adcmv - cdk5r1 at isolation and compared to untreated islets. islets were cultured in the presence of h - thymidine 48 hours after isolation and were harvested 96 hours after isolation. 72 hours after isolation, the cdk5 inhibitor roscovitine was added for a 24-hour period. roscovitine, while being able to inhibit other cdks such as cdk2 (ic50 of 0.7 m) and cdc2 (ic50 of 0.65 m), has a high affinity for cdk5 (ic50 of 0.16 m). uninfected adcmv - gfp and adcmv - cdk5 treated islets showed no changes in proliferation rate when treated with roscovitine. islets transduced with adcmv - cdk5r1 had the expected induction in proliferation, which was decreased to control levels when cultured with roscovitine (figure 7(a)). these data demonstrate that cdk5r1 mediated proliferation is dependent on the cdk5 : cdk5r1 kinase activity, and that cdk5r1 is essential for this kinase to be activated in primary rat -cells. to determine if knockdown of cdk5 is sufficient to inhibit the proliferative capacity of cdk5r1, we used sirna mediated cdk5 knockdown in the ins-1 832/13 -cell line. using a sicdk5 sequence that has previously been shown to be sufficient to knockdown cdk5 expression in the ins-1 cell line, we were able to successfully decrease cdk5 mrna expression by 75% (figure 7(b)). cells were transfected with sicdk5 or a scrambled sirna sequence (sictrl), and 24 hours later the cells were transduced with adcmv - gfp or adcmv - cdk5r1. cells transduced with adcmv - cdk5r1 and transfected with sictrl had a significant increase in proliferation (figure 7(c)). interestingly, cells that were transfected with sicdk5 and transduced with adcmv - cdk5r1 had a significant decrease in the proliferation rate. these data support the roscovitine data in demonstrating using a genetic ablation model that cdk5r1 mediated proliferation is dependent on the cdk5 kinase activity. cdk5 : cdk5r1 has been shown to phosphorylate prb, which is sufficient to engage the cell cycle machinery through release of e2f1 and family members in various cell types [48, 49 ]. we hypothesized that the cdk5r1 mediated proliferation may be dependent on prb phosphorylation, which would then engage the cell cycle machinery and permit -cell proliferation. first, we measured the phosphorylated prb and total prb levels in untreated islets and islets transduced with adcmv - gfp, adcmv - cdk5, and adcmv - cdk5r1. our data demonstrate that while prb levels do not significantly change with transduction of any of the adenoviral constructs, the phosphorylated level of prb significantly increased in islets transduced with adcmv - cdk5r1 (figure 8(a)). interestingly, culture of islets in the presence of roscovitine significantly decreased the phosphorylation level of prb in the adcmv - cdk5r1 transduced islets (figure 8(b)). this data demonstrates that cdk5r1 increases the p - prb level in a cdk5 dependent manner. these data suggest that cdk5 : cdk5r1 mediated phosphorylation of prb may be essential for cdk5r1 mediated -cell proliferation. control of -cell replication is a highly regulated process, with the majority of -cell replication occurring during embryogenesis and during the neonatal period. studies have shown, however, that adult -cell expansion does occur during obesity and pregnancy [8, 9, 23 ]. these data demonstrate that this tightly controlled process is intact in -cells and suggest that defining the molecular components of the -cell proliferation cascade may be used to determine entry points, whereby -cell replication can be induced. these pathways could be leveraged to treat diabetes by inducing -cell replication ex vivo for islet transplantation or in vivo for expansion of endogenous -cell mass. therefore, it is imperative to define the molecules involved in known -cell proliferation pathways. we have previously shown that the nkx6.1 mediated -cell proliferation pathway is dependent on the orphan nuclear receptors nr4a1 and nr4a3 [10, 11 ]. many of the genes upregulated in the nkx6.1 pathway are directly regulated through the transcriptional activity of nr4a1 and nr4a3. interestingly, upon comparing the gene expression profiles for nkx6.1 treated islets with nr4a1 or nr4a3 treated islets, it is clear that there is a significant subset of genes induced by nr4a1 and nr4a3 that are not regulated in the defined nkx6.1 proliferation pathway. from this subset of genes we have demonstrated that the unique cell cycle activator cdk5r1 is upregulated by nr4a1 and nr4a3 and that cdk5r1 is sufficient to induce proliferation of primary rat -cells and 832/13 ins-1 cells while maintaining gsis. furthermore, we have shown that overexpression of cdk5r1 in 832/13 ins-1 cells protects against apoptosis induced by thapsigargin and etoposide but not camptothecin. finally, we have shown that the cdk5r1 dependent increase in -cells proliferation is dependent on the cdk5 : cdk5r1 kinase activity. the increase in proliferation corresponds with increased phosphorylation of the cell cycle inhibitor prb, which results in release and activation of e2f family members. these data demonstrate that expression of cdk5r1 is sufficient to induce -cells proliferation by releasing a cell cycle braking mechanism (figure 9). expression of cdk5 and cdk5r1 was first observed in neuronal cells [14, 50 ]. recent studies have clearly demonstrated that both factors are expressed in primary -cells [13, 27 ]. since the observation that both cdk5 and cdk5r1 are expressed in -cells, much work has been done to address their function in normal and diseased -cells [23, 44, 46 ]. to date, while other groups have looked at the effect of the cdk5 complex on glucose stimulated insulin secretion and cellular apoptosis, we are the first to show that cdk5r1 overexpression and activation of the cdk5 : cdk5r1 complex result in increased -cell proliferation. our data demonstrates that overexpression of cdk5r1 in primary rat islets results in increased -cell proliferation and that there is no significant increase in proliferation of other non - insulin expressing islet cells. given that cdk5r1 activates the cdk5 kinase complex, these data suggest that -cells and other islet endocrine cells may not express appreciable levels of cdk5. while no studies have been published to determine the cdk5 expression level in the non - insulin expressing endocrine cells, our data supports the hypothesis that cdk5 expression may be sequestered to -cells, thus explaining why overexpression of cdk5r1 induces proliferation in only the insulin expressing islet population. furthermore, endogenous levels of cdk5r1 appear to be very low in -cells (figure 2(c)). we propose that the low levels of endogenous cdk5r1 keep the cdk5 complex in an inactive state. this is typical of cyclin - dependent kinase family, of which cdk5 is a member. several groups have studied the role of the cdk5 kinase complex on insulin secretion. within these studies, there are some contradictory findings. early findings demonstrated that cdk5 overexpression enhanced insulin secretion from -cells by facilitating vesicle fusion. latter studies presented data that cdk5 kinase activity is increased in -cells after culturing with elevated glucose and cytokine levels and that this increase results in decreased insulin gene expression and secretion [22, 44, 51 ]. our findings demonstrate that overexpression of cdk5r1 in primary rat islets maintains glucose stimulated insulin secretion and that total insulin levels are maintained. our studies were in primary rat islets that were cultured for 96 hours after adenoviral transduction. many of the other studies were completed primarily in cell lines or used short - term culture conditions [22, 27, 44, 51 ]. we did not culture our islets under damaging conditions, such as long - term elevated glucose, or in the presence of cytokines. while our studies were not conducted in a way to determine the effect of endogenous cdk5r1 in a diseased -cell state, they do clearly demonstrate that cdk5r1 overexpression is sufficient to induce -cell proliferation while maintaining insulin content and the glucose stimulated insulin secretion response. in addition to studying the effect of the cdk5 complex on insulin secretion, other groups have studied the effect on protection from apoptosis. an early study showed in the min-6 cell line that overexpression of cdk5r1 and culture with high glucose resulted in increased apoptosis. a subsequent study using iapp and -1 integrin signal blockade as induction for -cell apoptosis demonstrated that cdk5 and cdk5r1 were necessary for protection against apoptosis. our data, using cdk5r1 overexpression, demonstrates that while no protection was observed for apoptosis induced by camptothecin, a significant level of protection was observed in cells overexpressing cdk5r1 that were treated with etoposide or thapsigargin. while the studies with min-6 cells measured cdk5r1 induced apoptosis, our studies indicated no increase in basal death rates. furthermore, our studies indicated protection against apoptotic stimuli, which was not measured in the previous study. these data demonstrate that cdk5r1 overexpression is sufficient to confer protection to select apoptotic stimuli in addition to enhancing cellular replication. each of the apoptotic inducing compounds used in this study has a unique mechanism by which it induces apoptosis. thapsigargin blocks the serca pumps, thus increasing cytosolic ca levels and inducing er stress. neuroblastoma cells treated with camptothecin had attenuated apoptosis levels when also treated with the cdk5 inhibitor roscovitine. these data demonstrate that cdk5 kinase activity is needed for camptothecin - induced apoptosis, which correlates with our findings that cdk5r1 overexpression fails to confer protection. studies using etoposide have demonstrated that cdk5r1 sequestration by c53 results in enhanced apoptosis and c53 deletion resulted in protection to etoposide induced apoptosis. finally, a recent study demonstrated that pc12 cells treated with the serca inhibitor thapsigargin had potentiation of apoptosis when coupled with roscovitine treatment, demonstrating the role of the cdk5 complex to inhibit ca flux. our data do not define a mechanism by which cdk5 kinase activity confers a protective effect against thapsigargin or etoposide induced apoptosis ; however a recent study demonstrated that the proapoptotic protein noxa is phosphorylated by the cdk5 kinase complex, which results in decreased apoptosis. future studies will address the mechanism by which cdk5r1 overexpression protects against these apoptotic stimuli. various studies have shown that the activated cdk5 complex can phosphorylate prb [48, 55, 56 ]. e2f family members, such as e2f1, e2f2, and e2f3, are critical for engaging the cell cycle by upregulating expression of key genes such as cyclin e, cyclin a, and cyclin b. studies using neuronal cell lines have shown that cdk5 mediated phosphorylation of prb results in reengagement of the cell cycle and induction of neuronal apoptosis [48, 55 ]. this is in contrast to our findings that prb phosphorylation is correlated with increased proliferation rates. previous studies that overexpressed e2f family members in primary -cells have failed to observe enhanced apoptosis rates [57, 58 ]. these previous findings and our presented data demonstrate that while the default for cdk5 mediated cell cycle entry in postmitotic neurons may be induction of apoptotic pathways, the same pathway in -cells results in cell cycle reentry and cellular proliferation. our data clearly demonstrate that cdk5r1 has relatively low expression in -cells under normal conditions. our data demonstrate that overexpression of nr4a1 or nr4a3 is sufficient to induce expression of cdk5r1 and that overexpression of cdk5r1 is sufficient to induce -cell proliferation. studies from other groups have demonstrated that cdk5r1 expression is elevated under high glucose culture conditions [44, 51 ]. these data are consistent with a proposed role for nr4a1 and nr4a3 as cellular nutrient sensors. expression and activity of the nr4a orphan nuclear receptors have been shown to increase in various tissues when cultured with elevated glucose, free fatty acid, cholesterol, and amino acid levels [59, 60 ]. these findings suggest that cdk5r1 expression may increase during the early stages of type 2 diabetes, presumably through glucose mediated activation of the nr4a orphan nuclear receptors. during this pathological period, increases in -cell proliferation have been observed [79, 23 ], presumably due to the elevated blood glucose level. our data may suggest a mechanism by which the initial elevated blood glucose levels increase nr4a nuclear receptor expression and activity, thus leading to induction of cdk5r1 expression and the initial proliferation observed during this period of disease progression. our findings demonstrate for the first time that overexpression of cdk5r1 is sufficient to induce -cell proliferation. this is a novel finding that suggests potential uses for ex vivo expansion of -cell mass for islet transplantation or in vivo induction of endogenous -cell proliferation. defining the complete cadre of cdk5 kinase -cell targets will be essential for understanding the activity of this kinase complex for inducing -cell proliferation. in conclusion, our findings demonstrate that the orphan nuclear receptors nr4a1 and nr4a3 induce expression of the novel cell cycle activator cdk5r1 independent of the nkx6.1 signaling pathway. we demonstrate that cdk5r1 is sufficient to induce -cell proliferation in the 832/13 ins-1 derived cell line and in primary rat islets. we show that cdk5r1 results in -cell specific proliferation and that this proliferation is dependent on the cdk5 : cdk5r1 kinase complex activity. finally, we demonstrate that cdk5r1 induced -cell proliferation corresponds with increased phosphorylation of the cell cycle inhibitor prb (figure 9). these data demonstrate that expression of cdk5r1 results in activation of endogenous levels of cdk5 in -cells resulting in prb phosphorylation and activation of cell cycle progression. therefore, our results demonstrate that activation of the novel cdk5 : cdk5r1 cell cycle kinase may be used as a potential therapeutic entry point for increasing -cell mass ex vivo for islet transplantation therapy or expansion of endogenous -cell mass in vivo as a treatment for diabetes. this is the first time that overexpression of cdk5r1 has been shown to be sufficient to induce primary -cell proliferation. future studies will address how overexpression of nr4a family members results in activation of cdk5r1 and other phosphotargets of the cdk5-cdk5r1 kinase complex. | decreased -cell mass is a hallmark of type 1 and type 2 diabetes. islet transplantation as a method of diabetes therapy is hampered by the paucity of transplant ready islets. understanding the pathways controlling islet proliferation may be used to increase functional -cell mass through transplantation or by enhanced growth of endogenous -cells. we have shown that the transcription factor nkx6.1 induces -cell proliferation by upregulating the orphan nuclear hormone receptors nr4a1 and nr4a3. using expression analysis to define nkx6.1-independent mechanisms by which nr4a1 and nr4a3 induce -cell proliferation, we demonstrated that cyclin - dependent kinase 5 regulatory subunit 1 (cdk5r1) is upregulated by nr4a1 and nr4a3 but not by nkx6.1. overexpression of cdk5r1 is sufficient to induce primary rat -cell proliferation while maintaining glucose stimulated insulin secretion. overexpression of cdk5r1 in -cells confers protection against apoptosis induced by etoposide and thapsigargin, but not camptothecin. the cdk5 kinase complex inhibitor roscovitine blocks islet proliferation, suggesting that cdk5r1 mediated -cell proliferation is a kinase dependent event. overexpression of cdk5r1 results in prb phosphorylation, which is inhibited by roscovitine treatment. these data demonstrate that activation of the cdk5 kinase complex is sufficient to induce -cell proliferation while maintaining glucose stimulated insulin secretion. |
these tumors tend to occur in characteristic locations like interhemispheric (approximately 50%), quadrigeminal or superior cerebellar cistern (10 - 25%), suprasellar or interpeduncular cistern, cerebellopontine angle, and sylvian fissure. almost half of the intracranial lipomas are asymptomatic, and frequently manifest in young adult age group. quadrigeminal cistern, also known as superior cistern or cistern of the great cerebral vein is one of the subarachnoid cisterns located between the splenium of the corpus callosum and the superior surface of the cerebellum. we describe a rare case of quadrigeminal cistern lipoma in a 10 years male child and discuss the role of squash preparation as an alternative to frozen sections for intraoperative diagnosis. a 10 years male child presented with headache since 2 years of age along with recurrent vomiting and drooping of left eyelid during the attack. there was no history of seizures, fever, bladder or bowel disturbances and an unremarkable physical examination. cranial computed tomography (ct) scan revealed a mass lesion in the quadrigeminal cistern with homogeneously low attenuation, lacking calcification or contrast enhancement. t1 weighted magnetic resonance imaging (mri) showed a 33 22 26 mm hyperintense lesion in the quadrigeminal cistern, displacing the midbrain anteriorly [figure 1a and b ]. the lesion revealed suppression on fat saturation sequences [figure 1c and d ], suggesting the presence of adipose tissue. a midline suboccipital craniotomy through supracerebellar approach was undertaken for near total removal of the lesion. intraoperatively the mass was firm, non - suckable, moderately vascular and partially adherent to the midbrain on the left side. the pressure on the midbrain was relieved, and a tiny residual tumor mass [figure 2a and b ] in close proximity to the superior cerebellar artery was left behind. the excised tissue measured 2.5 2 1.5 cm with a firm, pinkish white fibrofatty texture. cryostat sections were technically difficult, and few poor quality sections obtained were not adequate for interpretation. smears stained by toluidine blue and rapid hematoxylin and eosin (h and e) revealed lobules of fibroadipose tissue admixed with few blood vessels [figures 3 and 4 ]. with radiological correlation, a diagnosis of lipoma of the quadrigeminal cistern was suggested. the histology sections corroborated with the squash smears, bearing similar lobules of adipose tissue intimately admixed with fibrocollagenous tissue, blood vessels and gliotic white matter [figure 5 ]. (a and b) t1 weighted mri scans reveals homogenous intensely hyperintense lesion in the quadrigeminal cistern (arrow), pushing the midbrain anteriorly ; (c and d) suppression of the lesion (arrow) on fat saturation sequence, suggesting presence of adipose tissue (a) t2 weighted sagittal mri reveals homogenously hyperintense lesion occupying the quadrigeminal cistern (arrow) ; (b) post operative t1 weighted mri showing a small residual lesion (arrow) ; path of the surgical approach is visible as hyperintensity (vacant arrow), representing reactive gliosis squash smears showing fibroadipose tissue fragments (toluidine blue 100) squash smears showing fibroadipose tissue fragments (h and e, 100) histological section showing lobules of adipose tissue, fibrocollagenous tissue, blood vessels and gliotic white matter (h and e, 40) follow up mri scans did not show any increase in size of the residual lesion, and the patient remains symptom free at 1 year of follow up. intracranial lipomas comprise 0.1 to 0.5% of all primary brain tumors, and quadrigeminal lipomas form 10 - 25% of these intracranial lipomas.[24 ] quadrigeminal lipomas are common in young adults, though can occur in any age group without any gender predilection. patients may present with headache, dizziness, psychomotor retardation, epilepsy, sometimes with complex partial seizures, and visual disturbances. this 10 year old male child had a 33 22 26 mm lipoma in the quadrigeminal cistern at the time of detection. he presented with episodes of headache, recurrent vomiting and drooping of left eyelid of long duration, from 2 years of age, indicating that it was a slowly growing lesion. hamartomas are rare congenital malformations, characterized by benign proliferation of components unique to the tissue of origin. intracranial lipomas may be included within the spectrum of developmental malformations rather than a hamartoma or true neoplasm. these lesions originate from mesodermal germ plaque beneath the leptomeninges, or may be a result of abnormal persistence and mal - differentiation of meninx primitiva during the development of the subarachnoid cisterns. heterotopic components like cartilage, bone described in these lipomas, along with its association with other brain anomalies in almost half of the cases lends support to a developmental malformation. midline anomalies like mal - development of corpus callosum, partial absence of the septum pellucidum, microgyria, hemispheric atrophy and other malformations such as aneurysms has been found to be associated with these lipomas. quadrigeminal lipomas may be associated with agenesis of corpus callosum or hypoplasia of inferior colliculus. however, imaging studies in this child did not reveal any associated intracranial developmental malformation. on ct scan, intracranial lipomas appear as well demarcated mass lesions with homogeneous attenuation and usually not enhancing on contrast.. intense signal on t1 weighted mri and fluid attenuated inversion recovery (flair), and a relatively low intensity signal on t2-weighted images, supplemented by fat suppression sequences suggests the possibility of a lipoma.[2579 ] the lesion in this child also on imaging showed features suggesting the possibility of a quadrigeminal lipoma. differential diagnoses of such lesions include subacute hematomas, thrombosed berry aneurysm, epidermoid or dermoid tumors, or lipomatous transformation of neoplasms like primitive neuroectodermal tumors (pnets), gliomas or ependymomas. sometimes the lesions may be vascular, have specks of calcification, and traversed by vital structures like cranial nerves and cerebral vessels. quadrigeminal lipomas may occur in close relation to the superior cerebellar arteries, similar to the case reported here. although surgical excision is seldom required, it is useful in situations causing mass effect. complete excision is usually attempted, though at times close proximity to or dense adhesions with vital structures allow only partial removal. intraoperatively, a tiny residual tumor mass in close proximity to the superior cerebellar artery was left behind. follow up mri scans one year later did not reveal any increase in size of this lesion. although known to have a favorable outcome, complete removal on occasion is not possible due to proximity to vital structures, and recurrences are known. intraoperative squash smear cytology is an economical and convenient diagnostic modality for quadrigeminal cistern lipoma compared to frozen sections with subsequent histopathological confirmation to exclude other differentials. additionally, careful radiological evaluation is necessary to detect associated cns malformations in the cns, common in these lesions. | quadrigeminal lipoma is a rare tumor that has been categorized as developmental malformation rather than a hamartoma or true neoplasm, due to its origin from abnormal persistence and mal - differentiation of meninx primitiva during the development of the subarachnoid cisterns. reported admixture of adipose tissue with heterotopic elements also supports a developmental origin. quadrigeminal lipomas are frequently asymptomatic and detected incidentally. though a favorable clinical course is usually expected, recurrences may occur due to partial removal of lesions in close relation to vital structure. we describe the role of intraoperative squash smear cytology as a diagnostic aid in quadrigeminal cistern lipoma and an alternative to frozen sections that are technically difficult to obtain due to presence of lobules of fibro - adipose tissue. with radiological correlation, squash cytology can be an economical method for intraoperative diagnosis, pending subsequent histopathological confirmation. |
calcitonin is a 32-residue peptide hormone produced by specialized c - parafollicular cells of the thyroid glands in mammals or by cells of the ultimobranchial glands in reptiles and fish. the nucleotide sequence and translation of human calcitonin are shown in figure 1 [1, 2 ]. calcitonin plays an important role in regulating calcium and phosphorus metabolism, decreasing blood calcium concentrations and inhibiting bone resorption. natural calcitonin and synthesized analog are widely used in clinical practice for the treatment of postmenopausal osteoporosis, paget 's disease of bone, bone pain, spinal stenosis, acute pancreatitis, and gastric ulcer [3, 4 ]. following the increase of the proportion of the elderly people in the world, osteoporosis has become a major threat to the public health due to its high morbidity and mortality [59 ]. low bone mass and deterioration of bone microarchitecture are the major characteristics of osteoporosis, which results in increased bone brittleness and thus is associated with an increased risk for fracture. calcitonin is one of the effective and safe agents for the treatment of osteoporosis, along with its antalgic effect [3, 4 ]. calcitonins that are used in clinical practice are mainly extracted from the gills of salmon and pig thyroid glands. however, these heterologous products are short of resources and thus expensive. in addition, the heterologous calcitonins are weak antigens and antibody - related resistance occurs in 40%70% of patients. reported that transient or persistent nausea was experienced in about one third of patients, with diarrhea, vomit and local pain at the injection site being other side effects. in addition, currently recommended nasal administration of calcitonin only reaches low concentrations in blood, but may cause squamous metaplasia in the nasal mucous membrane, which greatly restricts its long - term application. therefore, gene engineering techniques with human calcitonin (hct) gene as the objective gene may provide solutions overcoming the above drawbacks seen in clinical practice. however, although there have been some studies on the synthesis of calcitonin [14, 15 ], there is little information in literature on its application in gene therapy. for this purpose, we constructed a recombinant eukaryotic expression plasmid, pcdna3.0-ig-hct, which contains human calcitonin gene and murine ig-chain leader sequence. the recombinant plasmid was transferred into cells of murine fibroblast cell line, nih3t3, and the expression and secretion of recombinant hct in nih3t3 cells were observed in this study. the sequences of a murine ig-chain leader sequence and hct gene [1, 2 ] were divided into six fragments and used as pcr primers and templates for cdna synthesis. the primers were as follows : : 5-caagcttatggagacagacacactcctgctatgggtactg-3, : 3-tgaggacgatacccatgacgacgagacccaaggtccaaggtgaccactgcgc-5, : 5-caggttccactggtgacgcggcccagccggccaggcgcgccgtacgggatccgtg-3, : 3-gcgcggcatgccctaggcacgccattagactcatgaacgtacgacccgtgtatg-5, : 5-cttgcatgctgggcacatacacgcaggacttcaacaagtttcacac gttccccca-3, : 3-gttcaaagtgtgcaagggggtttgacgttaaccccaacctcgtggaattagatctg-5, the sequence to be synthesized was shown in figure 1(a). a dynamic template pcr technique was used to synthesize ig-hct (figure 1(a)) [17, 18 ]. the pcr program included predenaturation at 95c for 5 minutes, 30 amplification cycles each consisting of denaturation at 94c for 30 seconds, annealing at 55c for 60 seconds, and extension at 72c for 50 seconds ; followed by further extension at 72c for 5 minutes. the cloned cdna fragments were ligated into the pmd 18-t vector (promega, usa) to form pmd 18-t - ig-hct. the recombinant vector pmd18t - ig-hct was amplified in e. coli jm109 and then extracted by a dna purification system (promega) according to the manufacturer 's instructions. pmd18t - ig-hct was digested by hindiii and xbai and then evaluated by 1.5% agarose gel electrophoresis. the sequence of the recombinant ig-hct eukaryotic expression vector identified by the restriction analysis was determined by using fluorescent dideoxynuleotides on an automated dna sequencer (gene core technologies co. ltd, shanghai, china). the procedure of construction of the recombinant pcdna3.0-ig-hct eukaryotic expression vector was shown in figure 1(b). the cdna fragment of ig-hct from pmd 18-t vector was further subcloned into the pcdna3.0 (promega) to form a eukaryotic expression vector, pcdna3.0-ig-hct. the recombinant expression vector pcdna3.0-ig-hct was amplified in e. coli jm 109 and then extracted by a dna purification system (promega). the pcdna3.0-ig-hct retrieved from the gel was digested by hindiii and xbai as previously described. nih3t3 (atcc) cells were transfected with pcdna3.0-ig-hct by liposome, lipofectamine 2000 (invitrogen, usa). after 6-hours exposure, the normal culture medium, dulbecco 's modified eagle media (dmem), supplanted with 10% fetal bovine serum (gibco, usa) was added into cells. forty eight hours later, cells were exposed to 200, 400, 600, 800, 900, and 1000 g / ml g418 (gibco, usa), respectively, with 3 days for each concentration. after 18 days of selection, g418-resistant clones were randomly picked up, and cultured in the medium containing 800 g / ml g418. single clones were transferred into and cultured in the medium with g418 concentration being reduced to 0.5 g / ml. nih3t3 cells that were transfected with pcdna3.0 were used as controls. to demonstrate the mrna expression of ig-hct mediated by pcdna3.0-igk - hct vector in nih3t3 cells, first strand cdna was performed by an amv transcriptase kit (promega) according to the manufacturer 's instructions. the sequences of primers used for the detection of ig-hct were 5-taatacgactcactatagggaga-3 (5-sense) and 5-actcgtatcatccthccgaa-3 (3-antisense) with a product of 212 bp. for the detection of -actin mrna, we use a 5-sense primer (5-gggaaatcgtgcgtgacat-3) and a 3-antisense primer (5-tcaggaggagcaatgatcttg-3), with a product of 385 bp. pcr program was predenaturation at 95c for 5 minutes, and then 30 cycles each consisting of denaturation at 95c for 50 seconds, annealing at 55c for 50 seconds, extension at 72c for 50 seconds, and an extension at 72c for 7 minutes. pcdna3.0-igk - hct - transfected, nontransfect ed and pcdna3.0-transfected nih3t3 cells were cultured in 6-well plates (1 10/ml). one slide was put in each well of six - well plates for 24 hours to grow nih3t3 cells. then, immunohistochemistry was performed on ethyl alcohol / acetone - fixed slides by immunohistological kit for hct (zhongshan golden bridge, beijing, china). supernatant of cultured pcdna3.0-igk - hct - transfected nih3t3 cells were subjected to western blotting after 10 fold condensation by lyophilization, with pcdna3.0-transfected nih3t3 cell culture enriched supernatant being used a control. piscataway, usa), the blots were probed with the following antibodies : rabbit antihct (chemicon international inc.,temecula, usa) and goat antirabbit igg - hrp (chemicon international inc.). pcdna3.0-igk - hct - transfected, nontransfected and pcdna3.0-transfected nih3t3 cells were cultured in 6-well plates (1 10 cells / ml). after the cells were cultured for 24 hours, secretion of hct in the supernatant was measured with a commercial hct detection kit (cehmclin, beijing, china) by using the radioimmuniassay method. pcdna3.0-igk - hct - transfected, nontransfected and pcdna3.0-transfected nih3t3 cells were cultured in 6-well plates (1 10/ml) for 24 hours.then, primarily isolated osteoclasts were settled onto the 6-well plates and cocultured with above nih3t3 cells, respectively, on 15 mm plastic coverslips which were placed on a metallic bracket in plates, and incubated for 7 days at 37c. as a positive control, salmon ct (sct, novartis, basel, switzerland) was added, at concentration of 1 ng / ml, into the coculture of nontransfected nih3t3 cells and osteoclasts. to determine the identity of osteoclasts, cells were fixed and stained by using a tartrate - reisitant acid phosphatase (trap) stain kit (sigma, usa) according to the manufacturer 's protocol on day 7 of culture. trap - positive multinucleated cells with more than three nuclei were considered to be osteoclasts (figure 3). finally, the osteoclasts were observed under a converted microscope, and their numbers were counted and morphology observed. database was set up with spss 10.0 software package (spss inc, chicago, il) for analysis. the significance of differences was determined by anova and bonferroni 's modification of student 's t - test. a difference with a p value of less than.05 the pcr product of pmd18t - ig-hct had the same length with designed ig-hct gene. ig-hct - cdna was successfully inserted into pmd18 t as shown by hindiii and xbai digestion and electrophoresis (data not shown). it was demonstrated that the stop codon and endonuclease sites of ig-hct was successfully cloned into pmd18-t (data not shown). the ig-hct cdna was successfully ligated into pcdna3.0 by hindiii and xbai digestion and electrophoresis (figure 1(c)). the 210 bp fragment corresponding to the ig-hct cdna amplified by rt - pcr was present only from pcdna3.0-igk - hct - transfected nih3t3 cells, but not from nontransfected or pcdna3.0-transfected cells (figure 2(a)). moreover, immunocytochemistry staining showed that expression of hct protein was detected in pcdna3.0-igk - hct - transfected nih3t3 cells (figure 2(b)1), but not in pcdna3.0-transfected or nontransfected cells (figures 2(b)2 and 2(b)3). western blotting showed tha hct was positively stained in pcdna3.0-igk - hct - transfected cells, but not in pcdna3.0-transfected or nontransfected cell (figure 2(c)). in addition, radioimmunoassay displayed significantly higher secretion of hct protein in the culture supernatant (46 7.29 ng / ml) of pcdna3.0-igk - hct - transfected nih3t3 cells than those of nontransfected cells (5.2 2.36 ng / ml) and pcdna3.0-transfected cells (4.8 4.31 ng / ml) (both p <.05). trap staining showed that primarily isolated osteoclasts cocultured with nontransfected and pcdna3.0-transfected cells were multinucleate and spreading under a converted microscope (figures 3(a) and 3(b)). however, it was observed that osteoclasts became crumpled when cocultured with pcdna3.0-ig-hct - transfected nih3t3 cells and nontransfected nih3t3 cells plus sct group. the number of trap - positive multicleate cells was significantly fewer when the primarily isolated osteoclasts were cocultured with pcdna3.0-ig-hct - transfected nih3t3 cells or with nontransfected nih3t3 cells plus sct, than with nontransfected or pcdna3.0-transfected cells (table 1, p <.01). osteoporosis is characterized with low bone mass and deterioration of bone microarchitecture which can cause decreased bone strength and an increased risk for fracture [59 ]. calcitonin is one of the most effective reagents for osteoporosis with antalgic activities [3, 4 ]. it is believed that sct can inhibit bone resorption, reduce bone mass loss and relieve bone pain [20, 21 ]. but oral or nasal administration of calcitonin can cause many side effects in osteoporosis patients [12, 13 ]. otherwise, long - term application of animal calcitonins leads to a sharp activity decrease in clinical use of osteoporosis due to the accumulation of antibodies against these heterologous calcitonins. therefore, gene therapy with calcitonin as an objective gene may represent an optimal resolution to overcome the above problems. hct can not be secreted after synthesized in the cytoplasm because the nucleotide sequence of hct1 - 32 lacks of a signal peptide, which greatly limits its application in gene therapy. in the present study thus, hct can be secreted into the culture supernatant after it is synthesized in the cytoplasm. at the same time, the signal peptide is cleaved to avoid influencing the activities of hct. in this study, there are 19 or 20 complementary bases between p1 and p2, p2 and p3, p3 and p4, p4 and p5, and p5 and p6. in the first stage of pcr, p1 and p2, p3 and p4, and p5 and p6 were used as primers and templates to amplify p1 - 2, p3 - 4 and p5 - 6. in the second stage, p1 - 2, p3 - 4 and p5 - 6 were used as primers and templates to amplify p14 and p36. the sequence analysis and electrophoresis of pcdna3.0-ig-hct after digestion with hindiii and xbai demonstrated that the objective gene ig-hct was successfully cloned and ligated into the eukaryotic expression vector. the 210 bp fragment corresponding to the ig-hct cdna was amplified by rt - pcr, and was expressed only from pcdna3.0-igk - hct - transfected nih3t3 cells. the expression and secretion of hct were also detected in pcdna3.0-ig-hct - transfected nih3t3 cells by the immunohistological method, western blotting and the radioimmunoassay method. these observations suggest that the target fragment was successfully integrated into the genome of infected nih3t3 cells, which was expressed successfully. it has been shown that calcitonin inhibits bone resorption and induces osteoclast apoptosis by binding to calcitonin receptors on osteoclasts. we observed that primarily isolated osteoclasts cocultured with the supernatant of pcdna3.0-igk - hct - transfected nih3t3 cells became crumpled and their number was decreased compared with those cocultured in nontransfected and pcdna3.0-transfected nih3t3 cells, which suggests its profound bioactivities such as inhibition of the growth and alteration of morphology of osteoclasts. many in vivo active peptides are posttranslationally amidated at the c terminus, which is of great importance to their activities and stability. eukaryotes can be used to make some modifications to products, which make it possible to directly produce amidated calcitonin. in our future research, we will aim to couple express a rat amidating enzyme and hct - gly in nih3t3 cells, to obtain amidated bioactive hct. microencapsulation is a technique that encapsules an implant into a biocompatible selective membrane made from polymers, which can insulate the implant and the immune system of the recipient, but allow small molecules, electrolytes, oxygen, and metabolites to get through the membrane. indeed, microencapsulated transplantation of cells and tissues has been demonstrated to be effective in the treatment of many diseases. we propose that cell lines secreting hct encapsulated by alginate - polylysine - alginate membranes microcapsules and transplanted would constantly secret bioactive hct for a long perioid of time in vivo, and thus represent a new strategy for the treatment of osteoporosis. in conclusion the hct gene was effectively expressed in nih3t3 cells and secreted into the culture supernatant with the help of the murine ig-chain leader sequence. this study provides experimental data for potential gene therapeutic usage of the recombinant hct for osteoporosis. | aim. to construct a recombinant eukaryotic expression plasmid containing human calcitonin (hct) gene and express the gene in murine fibroblast nih3t3 cells. materials and methods. a murine ig-chain leader sequence and hct gene were synthesized and cloned into pcdna3.0 to form the pcdna3.0-ig-hct eukaryotic expression vector, which was transfected into nih3t3 cells. the mrna and protein expressions and secretion of hct were detected. primarily cultured osteoclasts were incubated with the supernatant of pcdna3.0-igk - hct - transfected nih3t3 cells, and their numbers were counted and morphology observed. results. the expression and secretion of hct were successfully detected in pcdna3.0-igk - hct - transfected nih3t3 cells. the number of osteoclasts was decreased and the cells became crumpled when they were incubated with the supernatant of pcdna3.0-igk - hct - transfected nih3t3 cells. conclusion. a recombinant eukaryotic expression vector containing hct gene was successfully constructed and expressed in nih3t3 cells. the secreted recombinant hct inhibited the growth and morphology of osteoclasts. |
atrial septal defects (asd) are congenital deficiencies of the atrial septum, most commonly occurring in the fossa ovalis and referred to as secundum defects as they result from failure of development of the septum secundum.1 failure of the development of the septum primum results in partial atrio - ventricular septal defects and these are the second commonest. the venosus defects are the least common and arise at the junction of the sinus venosus and the primitive atrium ; when situated close to the superior vena cava they are often associated with anomalous drainage of the right pulmonary veins and when the occur close to the inferior cava there is often override of the cava in relation to the atrial septum. large defects allow substantial left to right shunting, resulting in volume overload of the right heart and pulmonary vascular tree.2 untreated asds may cause a variety of complications. these include the eventual development of pulmonary hypertension that is initially reversible, but may later become irreversible, atrial arrhythmias and paradoxical embolisation with organ infarction. asds may close spontaneously in childhood.2 persistent defects with pulmonary to systemic flow ratios (qp / qs) of > 1.5 are operated before school age or whenever a diagnosis is made if this occurs later.3 significant residual asds after surgical closure have been documented in up to 17% of patients at catheterisation,4 and currently, this figure is down to 2%.3 a variety of devices for transcatheter closure of asds have been developed and offer an alternative to surgical treatment.5 we present a patient who had surgical closure of asd, with a second defect that was overlooked. she presented with atrial flutter ; electrophysiological studies showed that she had a slow - slow atrioventricular nodal re - entry tachycardia which degenerated into atrial flutter. slow pathway ablation was carried out at the base of the triangle of koch. during the procedure, an inferior atrial defect was diagnosed and successfully closed with an amplatzer aso (atrial septal occluder) device. the patient (female) had had surgery for asd at 34 years of age at a tertiary referral centre where a two by three cm defect was closed by direct suture. she represented four years later with palpitations due to atrial flutter and ablation was undertaken. atrioventricular nodal re - entry tachycardia was found and slow pathway ablation was carried out. during the procedure, the ablation catheters persistently passed from right to left atrium indicating the presence of an atrial communication. a left upper pulmonary angiogram showed a significant defect low in the interatrial septum very close to the junction of inferior vena cava and right atrium with no inferior margin (figure 1). balloon sizing using the aga medical sizing balloon measured the stretched diameter at 19 mm (figure 2). a 19 mm amplatzer aso device was successfully deployed across the defect under transoesophageal echocardiography and fluoroscopic guidance (figures 3 and 4). right atrial angiography (figure 5) and transoesophageal echocardiography confirmed complete occlusion of the defect and with no impairment of flow through the inferior cava. transthoracic echocardiography and cxr on the following day confirmed good device placement and ecg was also normal. the amplatzer device has been used to close a wide variety of asds, ranging from defects in children, to persistently patent foramen ovale with presumed paradoxical embolism in older individuals.6 the device has also been used for palliation in complex congenital heart disease.7 around 70% of asds are amenable to trans - catheter closure and the amplatzer device is the most commonly used because of ease to deliver and deploy, retrievability, and a wide range of size from 4 to 40 mm. those asds not suitable for transcatheter closure include venosus ones with anomalous pulmonary venous drainage, very large defects, insufficient septal length to accommodate the device and poor margins, especially posteriorly and inferiorly. although this case had no inferior margin, it was possible to secure the device straddling the top end of the inferior cava and the atrial wall. as the septum secundum had been surgically closed, this part of the septum was firm and the device could, therefore, be secured superiorly so that inferiorly the device could be stabilised where the septal margin was deficient. this report highlights the need to fully assess the atrial septum both before and during surgery or intervention as multiple asds can be missed particularly if situated in unusual positions. some cases generally considered unsuitable for catheter closure can still be dealt with by intervention under certain circumstances. although, atrial flutter or incisional atrial tachycardia occur after atrial surgery, it is important to remember that other arrhythmia substrates may be responsible. | we present a patient who had transcatheter closure of a low atrial septal defect which was overlooked during surgical closure of a secundum atrial septal defect. the residual defect was detected during ablation for atrial flutter, and was closed successfully during the same procedure with an amplatzer atrial septal occluder (aso) device. |
solution nuclear magnetic resonance (nmr) spectroscopy is a powerful technique for studying the structures of macromolecules under a variety of conditions and their internal dynamics on a wide range of time scales. sample preparation is often a key factor in being able to obtain high - quality nmr data on complex macromolecular systems. methods for encapsulating proteins within the aqueous core of a reverse micelle, and then dissolving the micelle in low viscosity fluid, were introduced in the late 1990s to help overcome the since then, reverse micelles have been used to study integral and anchored membrane proteins, as well as soluble proteins and nucleic acids of marginal stability. encapsulation has also enabled studies of various biophysical properties of protein including cold denaturation, hydration and internal motion. 10mag / ldao reverse micelle surfactant system for solution nmr studies of encapsulated proteins and nucleic acids. (a) chemical structures of the nonionic / zwitterionic surfactant system 10mag / ldao compared to the currently used ionic surfactants aot or ctab. (b) cut - away view of a space - filling model of flavodoxin encapsulated in a 10mag / ldao reverse micelle. (c) the direct injection method for sample preparation is illustrated using flavodoxin bound to its brightly yellow cofactor flavin mononucleotide. conditions : 300 m flavodoxin in 150 mm 10mag / ldao surfactant at a molar percent ratio of 65%:35%, 15 mm hexanol and water loading (w0) of 20. a critical issue when using reverse micelle techniques in nmr studies is that the surfactants must be able to efficiently encapsulate the macromolecule with high structural fidelity while also allowing the use of low viscosity solvents such as liquid pentane, propane or ethane. thus, the overall size of the surfactant and the electrostatic characteristics of the headgroup are critical for maintaining the native structure while also providing the desired reduction in molecular reorientation time for the encapsulated macromolecule. several studies have identified reverse micelle surfactant systems that meet the strict requirements of high - resolution reverse micelle nmr, but a surfactant system of general utility has not yet been described. currently, the two commonly used surfactants in reverse micelle nmr are the anionic bis(2-ethylhexyl)-sulfosuccinate (aot) and the cationic hexadecyltrimethylammonium bromide (ctab), where the latter uses hexanol as a cosurfactant (figure 1). applications of aot in this context are limited because few proteins have been encapsulated with high structural fidelity, presumably due to the denaturing effect of the anionic headgroup. possibly due to the help of the nonionic cosurfactant hexanol and has been used to encapsulate various proteins with high structural fidelity and high quality nmr spectra. nevertheless, the search for optimal encapsulation conditions for a given protein has been somewhat ad hoc, which has likely limited general application of this approach. thus, there is a need for a highly robust reverse micelle surfactant system that allows for efficient discovery of encapsulation conditions for a given macromolecule with high structural fidelity. here, we describe the development of a novel surfactant system that can be generally applied to proteins and nucleic acids thus allowing for high - resolution nmr studies of a wide - range of encapsulated biological macromolecules. the experimental requirements imposed by reverse micelle nmr spectroscopy led to identification of a binary surfactant system consisting of the nonionic 1-decanoyl - rac - glycerol (10mag) and the zwitterionic lauryldimethylamine - n - oxide (ldao). by combining a zwitterionic headgroup with a relatively short contour length tail, the 10mag / ldao surfactant systems provides both high biocompatibility and fast molecular tumbling for the encapsulated macromolecule. the performance of 10mag / ldao was evaluated by comparing the nmr properties of a variety of reverse micelle encapsulated biological macromolecules with their properties in free aqueous solution. it is also demonstrated that 10mag / ldao reverse micelles provide an excellent membrane mimetic for studying lipid - modified, membrane associated proteins by solution nmr. an improved procedure that greatly simplifies sample preparation by avoiding the high protein concentrations previously required for preparing reverse micelle samples proteins and trna were isotopically labeled and purified as described in the supporting information (si). mixtures of up to four different surfactants featuring nonionic, ionic, or zwitterionic head groups and different types of linear, branched, or cyclic hydrophobic tails were screened for their ability to form stable reverse micelle solutions in the absence of protein. hexanol, which is typically present as a stabilizing cosurfactant in reverse micelle preparations, was not included in this initial screen. the buffer for testing empty reverse micelle formation was 20 mm sodium phosphate (ph 7.0) with 50 mm nacl. bromophenol blue (0.2 mg / ml) was added to the buffer as a visual aid to detect reverse micelle formation. the molar ratio of water to surfactant, also known as the water loading or w0, was typically tested from 10 to 30. positive surfactant combinations were defined as those showing a single clear blue phase and the absence of any precipitate with a w0 of at least 20. the 10mag / ldao surfactants were used in a molar percent ratio of 65%:35% and at a total surfactant concentration of 75150 mm as noted. the concentration of the cosurfactant hexanol depended on the total 10mag / ldao surfactant concentration and on the type of bulk alkane solvent used (pentane or ethane). pentane samples were prepared with 1020 mm hexanol at 75 mm surfactant and 2030 mm hexanol at 150 mm surfactant. ethane samples were typically prepared with 3050 mm hexanol at 75 mm surfactant and 6070 mm hexanol at 150 mm surfactant. reverse micelle samples in liquid ethane were prepared using specialized apparatus from daedalus innovations, llc (aston, pa). ldao, 10mag, and hexanol were dissolved in 300 l of d12-pentane and transferred to the mixing chamber (1.65 ml nominal volume) of a daedalus innovations rm synthesizer unit. a volume of concentrated protein solution yielding the desired final w0 was added and the mixing chamber was sealed. pressurized liquid ethane was pumped into the mixing chamber with an isco 65d (lincoln, ne) syringe pump while the sample was being stirred. the sample pressure was increased until a distinct phase transition from a cloudy suspension to a clear solution was observed. this generally occurred in the range of 275450 bar though higher pressures were sometimes required for macromolecules of larger size and/or at higher concentrations. after completion of mixing, the sample pressure was adjusted to 1420 bar above the observed encapsulation (transition) pressure. the sample was then transferred to a high - pressure 3.6 mm i.d. nmr cell rated to 1 kbar (daedalus innovations, llc, aston, pa) for further analysis by nmr. the final sample contained 80% (v / v) ethane and 20% (v / v) pentane. for samples prepared by the injection evaporation method, water, alkane solvent, and to some degree volatile surfactants such as hexanol are removed under a gentle stream of n2 gas. the pentane solvent and volatile components other than water are replenished, as indicated by h nmr spectra, to rebalance the surfactant mixture. nmr spectra were obtained using bruker avance iii nmr spectrometers equipped with 5 mm cryogenically cooled triple resonance probes. standard two- and three - dimensional heteronuclear nmr pulse sequences were used to assess spectral perturbations. effective molecular reorientation correlation times (m) were estimated using the n - trosy - based (tract) relaxation experiment. to determine m, multiple portions of the amide (or imino) region were integrated and that which gave the highest measured m is reported. error was estimated from the variance in integrated area for duplicate relaxation delay points. this error was far smaller than that imposed by the rigid body approximation assumed in the tract analysis. in order to more rigorously compare the tumbling of encapsulated mbp to that in aqueous solution, n t1, t2 and heteronuclear noe experiments were collected at 750 and 600 mhz. for comparison of n t2 values, t2 relaxation of aqueous mbp was collected at 750 mhz using a trosy - based pulse sequence.t1 and t2 experiments were collected using six delay times with two collected in duplicate for error analysis. rotational correlation times were determined by fit to an isotropic tumbling model using in - house software. spectra were processed using felix (accelrys, san deigo, ca) or in - house nmr processing software (al nmr). the sparky software package was used for spectral analysis and plotting (goddard, t.d. and kneller, d.g. a systematic screen was undertaken to identify promising surfactant mixtures for reverse micelle nmr, where various mixtures were tested for their ability to form stable empty the screen consisted of mixtures of up to four different commercially available surfactants featuring nonionic, ionic, or zwitterionic headgroups and different types of linear, branched, or cyclic hydrophobic tails (si table s1). this screen identified a binary surfactant mixture of 1-decanoyl - rac - glycerol and lauryldimethylamine - n - oxide (10mag / ldao) as having highly desirable properties for encapsulation of proteins (si figure s1 and table s2). the nonionic monoacylglycerol head groups of 10mag were expected to suppress denaturing ionic interactions between the surfactant shell and the surface of an encapsulated macromolecule (figure 1b). these properties suggested that 10mag / ldao should faithfully encapsulate target macromolecules independent of their net surface charge. in addition, the relatively short linear hydrophobic tails of 10mag and ldao (10 and 12 carbons, respectively) were anticipated to favor fast molecular reorientation of the reverse micelle particle and thus to enhance their performance in nmr spectroscopy. in a secondary screen we fine - tuned the molar ratio of the components of the 10mag / ldao reverse micelle system for optimal nmr performance. the molar ratio of water to surfactant is particularly important because larger proteins require larger encapsulation volumes to form a stable reverse micelle. nevertheless, the total amount of water should be limited to minimize the size and achieve the fastest reorientation of the reverse micelle particle. optimization of both the 10mag / ldao molar ratio and the amount of hexanol was required to achieve the desired range of water loading (w0) (supplementary figure 2).. a molar ratio of 65%:35% of 10mag : ldao at a total surfactant concentration of 75 150 mm accommodated water loadings from 5 to 40, which is sufficient to fully hydrate proteins up to 100 kda. the required hexanol concentration ranged from 0 to 100 mm depending on w0 and the alkane solvent employed. a total of seven globular, water - soluble proteins were tested to determine the general utility of the 10mag / ldao surfactant system for reverse micelle nmr. all seven of these proteins were successfully encapsulated in 10mag / ldao mixtures in pentane with minimal optimization required. w0 and the total surfactant and hexanol concentrations were optimized to maximize encapsulation efficiency (highest encapsulated protein concentration). the seven tested proteins range from 8.5 to 81 kda and have isoelectric points between 4.5 and 11. five of these proteins, ubiquitin (ub, 8.5 kda, pi 6.8), cytochrome c (cyt c, 11.7 kda, pi 11), flavodoxin (flv, 18.8 kda, pi 4.5), maltose binding protein (mbp, 40.8 kda, pi 5.2), and malate synthase g from e. coli (msg, 81.4 kda, pi 5.9) have been previously encapsulated using the ionic surfactants aot or ctab, or a triple surfactant mixture also containing tetraethylene glycerol monododecyl ether (c12e4). in addition, two proteins were examined that are large by routine solution nmr standards and have not previously been studied by reverse micelle nmr : arginine kinase from the horseshoe crab l. polyphemus (ak, 40.2 kda, pi 6.3) and human aldo - keto reductase 1c2 (akr, also known as type iii 3-hydroxysteroid dehydrogenase, 36.7 kda, pi 7.1). reverse micelle samples were prepared by the direct injection method, where a specific volume of concentrated aqueous solution of protein is directly pipettd to an alkane solution of surfactants and mixed by vortexing (figure 1c). ub, cyt c, and flv were used to examine how the charge of the protein affects the efficiency of encapsulation. the ph of the reverse micelle water core was adjusted so that the net charge of the encapsulated protein was negative for flv and positive for ub and cyt c. setting the ph of the aqueous core of reverse micelles is critically important for achieving optimal encapsulation. the ph of the water core is dominated by the surfactants and only marginally influenced by the buffer employed since there are a hundred or more zwitterionic head groups and only a handful of buffer molecules per reverse micelle. methods for adjusting the effective ph inside the water core of a reverse micelle sample have been described elsewhere. all three proteins were encapsulated in pentane at final concentrations of 200 m with w0 = 10 (ub, cyt c) and w0 = 12 (flv). all reverse micelle solutions were clear, showed no visible precipitate, and gave excellent n hsqc spectra (si figure s3). comparison of the backbone chemical shifts for the protein in aqueous solution and encapsulated in the reverse micelle was used as a quantitative measure of structural integrity. for flavodoxin there are negligible differences between the amide h and n chemical shifts in aqueous solution and encapsulated in 10mag / ldao (histogram in figure 2a). a more extensive backbone chemical shift analysis (hn, c, c, c, n) showed that reduced cytochrome c is encapsulated in 10mag / ldao with little apparent structural perturbation (figure 2b). three proteins akr bound to nadp, mbp bound to maltose, and apo - ak were used to assess the ability of 10mag / ldao to encapsulate larger proteins. comparison of the resolved cross peaks of the n hsqc spectra obtained in pentane and in aqueous solution indicates that the three proteins were encapsulated in their native state (si figure s4). ak was more extensively examined using triple resonance spectroscopy of a sample prepared in ethane, which afforded better relaxation properties and narrower lines. an excellent chemical shift correspondence was found, demonstrating the high structural fidelity of the encapsulated protein (figure 2c). finally, msg (81 kda) was used with minimal optimization required to demonstrate the application of the 10mag / ldao mixture to even larger proteins. the successful encapsulation of seven globular proteins of diverse physical characteristics with only minimal specific optimization demonstrates that 10mag / ldao represents a major step forward in the general applicability of the reverse micelle encapsulation methodology. encapsulation of diverse proteins with high - structural fidelity in the 10mag / ldao surfactant system. proteins of distinctly different net surface charges and molecular weights retain their bulk solution structure as evidenced by minimal chemical shift () perturbations upon encapsulation. (a) n hsqc spectrum (left) of encapsulated n - flavodoxin (18.8 kda, pi 4.5) in pentane acquired at 500 mhz (h). histogram of gyromagnetic ratio weighted h and n chemical shift differences relative to flavodoxin in free aqueous solution (right). (b) n hsqc spectrum (left) of encapsulated horse c, n - cytochrome c (12 kda, pi 11) in pentane acquired at 600 mhz (h). histograms of weighted h and n (middle) and c, c, and c (right) chemical shift differences relative to the protein in free aqueous solution. (c) n hsqc spectrum (left) of encapsulated c, n - arginine kinase (ak) (40.2 kda, pi 6.3) in ethane acquired at 750 mhz (h). correlation of amide h and n and carbonyl c chemical shifts for ak. encapsulated cyt c and flv were dissolved in pentane, and ak was dissolved in ethane at 380 bar. a central advantage of the reverse micelle encapsulation strategy is the ability to manipulate the nmr relaxation properties of the macromolecule by actively decreasing the molecular reorientation time (m) through the use of low viscosity solvents. though n - pentane is often initially employed to explore sample conditions for encapsulation, the volume penalty imposed by the reverse micelle particle (water core plus surfactant shell) often outweighs gains in tumbling. lower viscosity liquid alkanes require preparation under pressure. reverse micelle solutions prepared with liquid butane or propane are often sufficient to overcome this volume penalty, thereby providing shorter molecular reorientation times for large proteins. although only moderate pressure is required to liquefy ethane at room temperature (47 bar), considerably higher pressures are needed to support homogeneous and stable nmr samples of encapsulated proteins in condensed gases (typically 250500 bar). the volume penalty is evident for small proteins even when encapsulated in the ultralow viscosity ethane, as shown by the higher m values for encapsulated ubiquitin, cytochrome c, and flavodoxin shown in figure 3. n - tract estimates of effective molecular reorientation times (m) of six proteins and trna encapsulated in 10mag / ldao reverse micelles in liquid ethane () or free in aqueous solution (). error bars, as propagated from the quality of the relaxation fits, are smaller than the size of the symbols shown. as addressed in the text, the primary source of error in the tract analysis is the inherent assumption of no internal motion. the optimal sample conditions obtained in pentane were generally transferable to ethane although higher concentrations of the cosurfactant hexanol were often required (see si for detailed sample conditions). hexanol significantly lowers the pressure required for forming stable reverse micelle samples, and the resulting reduced pressure lowers the viscosity and therefore reduces the molecular reorientation time. after the sample is fully formed inside the mixing chamber it is transferred to a specialized nmr cell. n hsqc spectra of these proteins show excellent agreement with aqueous samples (figures 2c, 4a, 5a, and si figure s1). the encapsulation pressures required for preparation in ethane ranged from 275 to 480 bar, which are well below that typically required to significantly perturb the structure and dynamics of folded proteins. long - term stability of the ethane samples at room temperature ranged from more than 4 months for flv, to 6 weeks for msg, mbp, cyt c, ub, and 1 week for ak. the samples of akr in ethane were not stable for more than a day. determination of optimal encapsulation conditions in ethane required optimization of hexanol concentration, w0, and encapsulation pressure for each of the proteins tested. in general, optimal conditions were identified after only a few (5 or less) tests. once optimal conditions were identified, these proved to be highly reproducible (m 2 ns). subsequent to these tests, several other soluble, globular proteins have been tested for encapsulation in 10mag / ldao mixtures. to date, only one has been identified that does not encapsulate in 10mag / ldao. encapsulation of integral membrane proteins in 10mag / ldao mixtures has not yet been examined. (a) n hsqc spectra are shown of mbp in aqueous solution and encapsulated in 10mag / ldao reverse micelles dissolved in liquid ethane. spectra were collected to have comparably high signal - to - noise for direct comparison of line widths. (b) one - dimensional normalized n cross sections (aqueous : dashed lines, rm : solid lines) of three cross peaks (red arrows in a) are shown to illustrate the narrowed line widths as a result of improved tumbling upon encapsulation. (c) n t2 values were measured for 278 and 291 sites in the encapsulated (black bars) and aqueous (white bars) mbp samples, respectively, at 750 mhz. a histogram illustrates the overall improvement in relaxation behavior of the encapsulated protein as a result of improved tumbling. the difference in average t2 values (35 and 46 ms for aqueous and encapsulated protein, respectively) corresponds to a 31% improvement and correlates well the 32% decrease in rotational correlation time. (a) n - trosy spectra are shown of msg in aqueous solution and encapsulated in 10mag / ldao reverse micelles in liquid ethane. these spectra were collected with the same total acquisition time and show comparable signal - to - noise despite the reverse micelle sample containing approximately one - third of the protein as that of the aqueous sample. (b) h cross sections of exemplary (red arrows in a) cross peaks are shown for aqueous (red) and encapsulated (black) msg. the n - tract experiment, which measures the relaxation rates of the and spin states of the amide n, was used to estimate the protein s effective molecular reorientation time (m). although this method systematically underestimates the true molecular reorientation time (m), it provides a rapid method for comparing the tumbling of proteins in reverse micelle and aqueous solutions. the error that results from the rigid body assumption inherent in the tract analysis is far greater than the error resulting from the fits to the relaxation profiles. in general, the errors resulting from the quality of the relaxation fits are on the order of the sizes of the symbols shown in figure 3. comparing the m values shows that macromolecules larger than 20 kda can be made to tumble faster than in aqueous solution when encapsulated and prepared in liquid ethane (figure 3). for example, encapsulated mbp and msg showed approximately 50% reduction in m. however, it is important to note that, because the tract experiment underestimates the true rotational correlation time, the actual advantage may be more modest. to examine this, the rotational correlation time of mbp encapsulated in 75 mm 10mag / ldao was determined with a full suite of n - relaxation experiments to be 15.5 ns. this corresponds to a markedly slower tumbling behavior than indicated by the tract analysis (8.9 ns), but a similar discrepancy was noted previously for the tract estimation of tumbling for aqueous mbp. based on this more rigorous measurement of encapsulated mbp tumbling in ethane, the true improvement in rotational correlation time (15.5 ns for rm versus 23 ns for aqueous solution) is 32%. it should also be noted that some trade - off exists between optimal encapsulation efficiency (i.e., concentration of protein in the final sample) and molecular tumbling. in general, increasing the amount of surfactant in the reverse micelle system permits encapsulation of higher concentrations of protein, but the tumbling improvement at high surfactant concentrations is slightly reduced and there is the potential for partial alignment of reverse micelle systems at high surfactant concentrations. as an example, the optimal sample conditions for mbp in ethane were determined to be at 75 mm total surfactant with 100 mm hexanol. because ak is more difficult to concentrate in aqueous solution, higher surfactant concentrations (100150 mm) and higher water loading (w0 = 20 versus 12 for mbp) were required for this protein in order to achieve appropriate encapsulated concentrations (80100 m) for efficient collection of nmr data. as shown in figure 3, the tumbling of mbp in ethane is better than that of ak due to this difference in surfactant conditions. the spectral improvements in tumbling are demonstrated in figure 4 for mbp encapsulated in ethane as compared to the aqueous protein. the n hsqc spectrum of encapsulated mbp shows improved resolution due to narrowing of the resonance lines. this narrowing can more clearly be seen in the one - dimensional spectra given. in addition, the distribution of n t2 values is shifted markedly toward longer relaxation times for the encapsulated protein, demonstrating a general improvement in the relaxation properties of the encapsulated protein. this t2 advantage not only translates to improved line widths but also provides substantial gains in coherence transfer efficiencies in multidimensional nmr experiments. it is also important to consider that the sensitivity of a modern cryoprobe with rm samples is markedly better than that with aqueous solutions due to the low conductivity of the reverse micelle solution. this results in improved signal - to - noise per unit of protein in the encapsulated sample. in combination with the improved tumbling and resultant line narrowing and improved coherence transfer, for example, the spectra shown in figure 5 demonstrate that comparable signal - to - noise is obtained for encapsulated msg as compared to the aqueous condition using the same amount of signal averaging (i.e., identical collection time) but less than half the protein concentration of the aqueous sample. in summary, the successful encapsulation of a diverse set of globular proteins up to 81 kda in ultralow viscosity ethane shows that 10mag / ldao is a general surfactant system for comprehensive reverse micelle nmr. the direct injection method (figure 1c) is the simplest approach for preparing homogeneous solutions of encapsulated macromolecules. such solutions are usually prepared under water - limited conditions to obtain the desired water loading (w0) and fast molecular tumbling. thus, the concentration of protein in the injected solution defines the final concentration of encapsulated protein. if the protein can not be sufficiently concentrated, the direct injection method will fail to provide a sufficiently high concentration of encapsulated protein to allow facile multidimensional nmr spectroscopy. for example, using a 1 mm protein stock solution would result in a final concentration of encapsulated protein of only 18 m with w0 of 10 and 100 mm surfactant. to address this limitation, a method was developed that allows fewer soluble macromolecules to be encapsulated by employing successive rounds of injection of a dilute solution followed by evaporation of excess water. the injection evaporation method is illustrated using a solution of 500 m flavodoxin and performing seven rounds of protein injection and water evaporation (figure 6). the injection evaporation method of encapsulation eliminates the requirement for millimolar protein concentrations. (a) reference n hsqc spectrum of 500 m flv in aqueous solution. (b) n hsqc spectrum of 200 m encapsulated flv dissolved in pentane prepared by direct injection using 6.3 mm flv (bottom) and prepared by seven rounds of injection evaporation using a 500 m flv stock solution (top). (c) the effective tumbling rate of flv can be greatly increased by subsequent solvent exchange to ethane. all data were recorded at 25 c and at 500 mhz (h) and processed identically. the first round of protein injection yielded an encapsulated flavodoxin concentration of 30 m. in this example, this allows for a 12-fold reduction in the starting protein concentration (6.3 mm). the n hsqc spectra of encapsulated flavodoxin prepared by injection evaporation or direct injection were identical (figure 6b). furthermore, the flavodoxin sample prepared by the injection evaporation method could be redissolved in liquid ethane after partial evaporation of the pentane solvent (figure 6). thus, the advantages of the injection evaporation method can be combined with the improved molecular tumbling offered by ultralow viscosity ethane. post - translational lipid modification of proteins, such as attachment of myristate or isoprene groups, is responsible for a variety of specialized functions including localization at membranes or hydrophobic molecular switch - like behavior. exposure of the hydrophobic lipid to water can lead to poor solution behavior that severely compromises structural studies. consequently, high - resolution structures of lipid - modified proteins in the putative membrane - anchored state obtained using standard nmr spectroscopy or crystallography are surprisingly rare. it was recently shown that myristoylated recoverin and myristoylated hiv matrix protein could be stably encapsulated in ctab / hexanol reverse micelles in the lipid - extruded state. however, ctab is cationic and has electrostatic properties different from those of natural lipid bilayers, and this mismatch may become limiting. in contrast, the 10mag / ldao surfactant system more closely resembles the lipids of biological membranes. the zwitterionic ldao headgroup mimics the phosphatidylcholine headgroup, and 10mag is a glycerol group esterified to a fatty acid. the 23 kda myristoylated human recoverin (rec[+myr ]), which acts as a ca sensor in the visual system, was used to test if 10mag / ldao could support lipid - anchored proteins. calcium - loaded rec[+myr ] was efficiently encapsulated at 100 m in 10mag / ldao in both pentane and ethane at a water loading of 12. the n hsqc spectra of encapsulated rec[+myr ] were very similar to spectra of natively folded, ca - bound, myristoyl - extruded protein (see refs (5 and 40) and figure 7). the n - tract measurements showed much faster molecular tumbling in ethane than in pentane (m = 11.7 and 18.8 ns, respectively). interestingly, the m values suggested that two rec[+myr ] molecules were encapsulated in occupied reverse micelles. a similar observation was made in the previous study of rec[+myr ] encapsulated in ctab / hexanol, suggesting that the protein in the lipid - extruded state is a dimer. 10mag / ldao provides a membrane mimetic environment for nmr spectroscopy of membrane - anchored proteins. the dashed line box highlights two cross peaks that are assigned to two gly residues involved in stabilizing the ca - bound, myristoyl - extruded protein conformation. sample prepared in ethane at 100 m rec[+myr ] and w0 = 12 in 100 mm 10mag / ldao at 450 bar. dnas and rnas represent a significant challenge to solution nmr spectroscopy because they are built from only four different nucleotides and the proton and carbon resonances are highly overlapped due to their narrow chemical shift ranges. the 27 kda natively modified valine trna (trna) from e. coli(46) was employed here to evaluate the 10mag / ldao surfactant system for nmr studies of nucleic acids. the structure and dynamics of trna have been extensively studied, and it therefore serves as a model system for medium - sized rnas. n - labeled trna was encapsulated at 100 m in 10mag / ldao reverse micelles in both pentane and ethane using the procedures developed above for proteins. the imino region of the n hsqc spectrum of encapsulated trna is indicative of a natively folded macromolecule using a buffer that included 5 mm mg (figure 8). interestingly, amino resonances were sharper and consequently better resolved for the trna encapsulated in ethane (at relatively low water loading w0 = 9) than in free solution (boxes in figure 8). this is partially related to the reduced effective molecular reorientation time (m = 7.0 and 9.0 ns, for the encapsulated and aqueous trnas, respectively). in addition, many a, c, and g amino protons are in the intermediate exchange regime in aqueous solution due to either rotation about the c n bond, which has partial double bond character, or through the effects of hydrogen exchange with water. hydrogen exchange chemistry is slowed in the reverse micelle, and the corresponding reduction in hydrogen exchange rates will tend to sharpen the amino hydrogen resonances. furthermore, the relatively high effective viscosity of the water core of reverse micelles would also tend to slow conformational exchange processes toward the slow exchange time regime. conversely, the imino protons are in the slow exchange regime in bulk solution, and their resonances are largely unaffected by the slowed hydrogen exchange chemistry or the increased viscosity of the reverse micelle water core n hsqc spectra of the imino region of trna acquired at 500 mhz (h) (a) aqueous solution at 200 m with 16 scans per fid and (b) encapsulated in 150 mm 10mag / ldao and dissolved in ethane at 450 bar at w0 = 9 at 100 m with 32 scans per fid. the dashed line boxes highlight amino cross peaks, folded in the n chemical shift dimension, showing somewhat improved signal - to - noise for the encapsulated rna compared to rna tumbling free in aqueous solution. the trna was prepared in 10 mm sodium phosphate (ph 6.8), 80 mm nacl, 5 mm mgcl2, 0.1 mm edta. the effect of slowed hydrogen exchange chemistry on the line shape of exchangeable hydrogens has been observed for encapsulated proteins before, particularly in the ability to resolve noe cross peaks to hydroxyl hydrogens of serine and threonine residues. these hydroxyl hydrogens exchange far too rapidly in bulk solution for such signals to be observed, but they are visible upon encapsulation. at water loadings less than 20, the water dynamics and hydrogen exchange rates are considerably slower than in bulk water. many rnas require divalent ions for proper folding and function, with mg generally being the physiologically relevant ion. the zwitterionic character of the 10mag / ldao surfactant system means that neither the rna nor the metal ions required for correct folding / function of the macromolecule are likely to interact to any significant extent with the surfactant. furthermore, since nucleic acids all carry a large number of negative charges, they will have similar overall electrostatic surface charges, and encapsulation conditions that work for one nucleic acid, such as trna studied here, should be readily applicable to other nucleic acids. a new binary surfactant system for high - resolution solution nmr studies of encapsulated biological macromolecules dissolved in low viscosity fluids has been developed. the performance of 10mag / ldao in reverse micelle nmr has been validated with respect to encapsulation efficiency (yield), fidelity, and molecular tumbling behavior using a set of seven proteins and a trna. 10mag / ldao therefore represents a single surfactant system that can encapsulate with high structural fidelity a broad range of different target macromolecules with molecular weights at least up to 81 kda. this simplifies the process of preparing reverse micelle nmr samples, which is often plagued by trial and error searches for optimal encapsulation conditions. it has been found that 10mag / ldao may be employed with minimal a priori assumptions about the properties of a given target protein or rna to obtain significant reductions in molecular reorientation times at ambient temperature. results presented here indicate that proteins in the 40 kda range are rendered completely accessible to the full battery of triple resonance spectroscopy without the limitations imposed by extensive deuteration or use of the trosy effect. it should also be noted that the dielectric properties of reverse micelle solutions are more optimal for cryogenic probe technology than standard aqueous samples and can provide a 50100% gain in sensitivity. the ability of 10mag / ldao to faithfully encapsulate a range of protein sizes and charge states clearly originates from the favorable physicochemical properties of the two surfactant headgroups a nonionic monoacylated glycerol group in 10mag and a zwitterionic amine - n - oxide group in ldao. the nonionic surfactant content (65 mol % 10mag) combined with the zwitterionic character of ldao mixture reduces charge charge interactions in much the same spirit as the triple surfactant system derived from mixtures of anionic, cationic, and neutral polyether surfactants. here, however, the relatively short contour length of the ldao and 10mag minimizes the size of the resulting particle and thereby maximizes the effective tumbling time of the encapsulated macromolecule. the surfactant combination of 10mag and ldao also provides a very favorable environment for encapsulating proteins of poor solution properties, such as lipid - modified proteins. because all nucleic acids have an overall negative electrostatic surface, it is likely that encapsulation by the 10mag / ldao surfactant system will generally not be sensitive to the precise sequence of the rna (or dna). finally, the injection evaporation method allows for the preparation of reverse micelle samples using relatively dilute protein solutions and represents a significant advantage as it avoids the need for concentrated aqueous solutions of the target macromolecule. in conclusion, 10mag / ldao provides many aspects of an optimal surfactant system for solution nmr of encapsulated proteins dissolved in low viscosity fluids. it relieves the experimental problems that exist with the currently employed surfactants aot, ctab, and triple surfactant mixtures. we anticipate that the 10mag / ldao surfactant system will find broad utility in structural and biophysical studies of encapsulated proteins by high - resolution reverse micelle nmr. | an optimized reverse micelle surfactant system has been developed for solution nuclear magnetic resonance studies of encapsulated proteins and nucleic acids dissolved in low viscosity fluids. comprising the nonionic 1-decanoyl - rac - glycerol and the zwitterionic lauryldimethylamine - n - oxide (10mag / ldao), this mixture is shown to efficiently encapsulate a diverse set of proteins and nucleic acids. chemical shift analyses of these systems show that high structural fidelity is achieved upon encapsulation. the 10mag / ldao surfactant system reduces the molecular reorientation time for encapsulated macromolecules larger than 20 kda leading to improved overall nmr performance. the 10mag / ldao system can also be used for solution nmr studies of lipid - modified proteins. new and efficient strategies for optimization of encapsulation conditions are described. 10mag / ldao performs well in both the low viscosity pentane and ultralow viscosity liquid ethane and therefore will serve as a general surfactant system for initiating solution nmr studies of proteins and nucleic acids. |
it is frequently asymptomatic but when being symptomatic the most presenting problems are flank pain and urinary tract infection with an atrophic upper pole which may also cause hypertension. the standard treatment for a duplex kidney with a poorly functioning upper pole moiety is an upper pole heminephrectomy. this procedure is usually performed by open surgery but with recent developments in endo - urology techniques and uro - laparoscopy, many attempts started to do this procedure laparoscopically,,. there are few reports in laparoscopic heminephrectomy in children that are mainly done with retroperitoneal approach,,, and there is just one large series in adults with acceptable results. in this article we describe our results and patients outcomes with laparoscopic transperitoneal heminephrectomy in our consecutive cases. from february 2001 to may 2007, 14 patients (10 females, 4 males) with complete duplex kidneys were presented with flank pain or uti leading to final diagnosis of non functioning upper moiety which became candidate for transperitoneal laparoscopic heminephrectomy. prior to the procedure, complete radiologic evaluation including abdominopelvic sonography, intravenous urography (ivu) and abdomino - pelvic ct scan were contemplated in all patients to delineate renal anatomy and hydronephrosis. by dmsa isotope scan, hypofunctioning of this moiety was confirmed. in patients suspected of vesico - ureteral reflux, voiding cystourethrography (vcug) all patients were admitted the day before operation ; received adequate bowel preparation and other needed medications. renal function tests as well as complete blood count, urine analysis and culture were all performed during that period. with the patients under general anesthesia, a foley catheter as well as a nasogastric tube was inserted and they were placed in a 60 degrees lateral decubitus position with minimal flexion of the operating table supported by adequate padding. the abdominal cavity was explored using 4 port transperitoneal approach for left side operation (a camera port at the level of umbilicus lateral to the rectus muscle, two 5 mm and one 10 mm trocars as working ports). for right side operation, the same trocar arrangement was employed but another 5 mm subxiphoid port was added for liver retraction. after incising the white line of toldt, superior retraction of spleen / liver and medial mobilization of colon (and duodenum : on right side), exposure to the renal pedicle was made possible. after complete dissection of both renal artery and vein, gerota 's fascia was incised and the kidney fully mobilized within the gerota 's fascia. afterwards, the dilated ureter of the corresponding nonfunctioning moiety was localized and carefully dissected to the renal hilum. care was taken not to induce ureteral devascularization of the other healthy ureter. the diseased ureter was clipped and divided at the level of lower pole and used as a handle and guide to facilitate the dissection of segmental branches of renal artery and vein which supplied the nonfunctioning moiety. such dissection was embarked as meticulous as possible trying not to induce any ischemia and/or injury to the healthy moiety. then, the corresponding segmental renal artery and vein were doubly clipped and divided ; the nonfunctioning renal moiety was incised and separated from the whole renal unit on the demarcated ischemic line, using a ureteric stamp as a handle which facilitated this part of the procedure. the retroperitoneum irrigated and suctioned, hemostasis of the renal cut surface was ensured and an external drainage was placed with a tubular drain. all patients received prophylactic peri - operative intravenous antibiotics as of our routine program. upon ambulation, complete blood count and renal function tests were rechecked and assigned 6 hours after the operation as post - procedural analysis. surgical demographics such as operative time, blood loss, early and late complications were registered. three - month follow - up ivp was performed in all patients to assess about the functional status of the remained ipsilateral renal moiety. ten cases (71.5%) were adults and 4 cases (28.5%) were children (ages 3, 3, 5, and 5 years). overall mean age of the patients was 24.216.8 years old (adult age range : 18 to 52 years ; pediatric age range (table 1 (tab. 1)) : 3 to 5 years). the main presenting symptoms were flank pain in 10 patients (71.4%) followed by urinary tract infection in three (21.4%). in one pediatric patient follow up of prenatal hydronephrosis confirmed the diagnosis of nonfunctioning upper pole moiety. the atrophic part was located in the left side and in six patients (42.86%) in the right side., sd 80 (range 95 to 360 min). estimated blood loss was negligible. differences in pre - operative and post - operative hemoglobin and creatinine were not significant. mean hospital stay was 4.1 days (range 2 to 7). in one patient mild bleeding occurred due to injured abdominal wall vessels in the trocar site. on the second postoperative day the patient 's hemoglobin level dropped from 11.1 g / dl to 9.2 the patient 's abdominal wall hematoma, observed by daily ultrasonography, resolved spontaneously within one week. on patients ' mean follow up of 32 months there was not any pain or episodes of recurrent urinary tract infection in any case. in all patients post - operative ivps were done after 3 months. follow - up ivps showed ipsilateral functioning moieties with an intact collecting system in all cases (figure 1 (fig. 2)) except our first patient in whom atrophic kidney was detected. in this patient and during the renal pedicle dissection the lower pole artery was injured because of vascular anomalies. no hypertension was detected in this case after long - term follow - up in spite of atrophic kidney. duplication of the ureter and renal pelvis is the most common anomaly of the upper urinary tract with an incidence of 1 in 125 or 0.8%,,. the upper moiety is frequently obstructed, poor functioning, and can become symptomatic due to urinary tract infection or flank pain. recent advances in laparoscopic techniques have led to the selection of laparoscopic surgery for many if not most urological procedures. the first case of a laparoscopic partial nephrectomy was reported by winfield. in 1992. with further development of the technique, since then several reports on laparoscopic heminephrectomy have been published describing advantages, disadvantages and complications of the procedure. based on the literature review, and regardless of applied techniques, the main complications of laparoscopic heminephrectomy are urinoma, urine leakage, recurrent urinary tract infections, postoperative hypertension, and functional loss of the remaining moiety,. in our series just one case of functional loss of the remaining moiety was seen due to inadvertent injury of lower pole renal artery. this complication was detected as ipsilateral renal atrophy at follow - up ivp, 3 months after operation. almost all of the published series reported the efficacy of the procedure in pediatric patients. series which have exclusively studied the efficacy of the procedure in adult cases have recently been published by abouassaly. and gao.. in his series of 18 cases of transperitoneal laparoscopic heminephrectomy no major complication or open conversion occurred. the mean operative time was 142 min and the mean estimated blood loss was 196 ml. only one case of postoperative urine leakage was reported in his series that was treated with prolonged urethral catheterization. castellan. published their experience in a series of 48 pediatric patients (mean age 4.08 years) who underwent transperitoneal and retroperitoneal laparoscopic heminephrectomy. mean hospitals stay was 2.6 days in the transperitoneal and 2.3 days in the retroperitoneal group. one retroperitoneal procedure required conversion to open surgery. in this largest series of laparoscopic heminephrectomy, complications were seen in 5 patients (10%), including urinary leak, urinoma, pneumothorax, recurrent urinary tract infection, and postoperative hypertension. the authors found that laparoscopic heminephrectomy can be performed with minimal morbidity, improved cosmesis and short hospital stay. laparoscopic approach to heminephrectomy offers the patient the typical benefits of laparoscopy including shorter hospital stay, minimal morbidity, improved cosmesis, and lower need for analgesic use,,,,,. improved preservation of the functioning pole of kidney could be an advantage of laparoscopy specifically mentioned in this procedure. it is of worth noting that the objective of heminephrectomy is the safe maximal removal of the nonfunctioning moiety and simultaneous maximal preservation of the functioning pole. laparoscopic approach enables an excellent differentiation of each hemirenal tissue, separating the ill kidney and preserving the maximal possible function for the other half - kidney. there was not any major or minor operative complication in our series except one in which blood transfusion was needed because of trocar site hemorrhage due to injured abdominal wall vessels. mean hospital stay was only 4 days which is much less than open series. in mean follow up period of 32 months, the remaining kidney functioned properly (figure 1 (fig. excellent visualization during laparoscopy helps better preservation of renal pedicle especially in pediatric age groups and offers a preferred approach for management of atrophic renal moieties in duplex systems. | purpose : the standard treatment for a duplex kidney with poorly functioning upper pole moiety is ipsilateral upper pole heminephrectomy. this procedure is usually performed by open surgery, but with recent developments in techniques of uro - laparoscopy, it can be done with it, safely. in this study we evaluated the results and safety of laparoscopic heminephrectomy in our consecutive cases.materials and methods : from february 2001 to may 2007 fourteen unilateral laparoscopic heminephrectomy were performed in our center. patients ' characteristics, presenting symptoms, operative time, and blood loss, early and late complications were all collected retrospectively. using pre - operative ultrasonography, intravenous pyelography (ivp) and ct scanning, unilateral upper pole hydronephrosis was detected in all cases. by dmsa isotope scan hypofunctioning of ipsilateral moieties was detected in all cases.results : mean operative time was 20380 minutes. no major intra - operative or early complications were identified. mean hospital stay was 4.1 days. on mean follow - up of 32 months no disturbing symptoms or episodes of urinary tract infections (utis) were detected. atrophic kidney was detected in one case in post - operative ivp.conclusion : laparoscopic heminephrectomy is a valuable minimal invasive procedure that can be performed safely in experienced hands without any important complication. perfect renal pedicles vascular system manipulation is important for the preservation of renal function post - operatively. |
human exposure to 2,3,7,8-tetrachlorodibenzo - p - dioxin (tcdd) and chlorinated analogs commonly results in pathological changes in the skin and its appendages characterized by thickening of the epidermis (acanthosis), hyperkeratosis and squamous metaplasia of the epithelial lining of the sebaceous glands. acneform lesions (chloracne) develop as hair follicles dilate and fill with keratin and sebaceous glands become cystic. in animal models it has been found that the chloracneogenic potential of the halogenated aromatic compounds examined corresponds with the relative affinity of these same compounds for the cytosolic tcdd receptor. this receptor controls the coordinate expression of a number of inducible enzyme activities and in certain cell targets can alter normal programs of proliferation and differentiation. in this report we describe some of our ongoing studies on the mechanisms of action of tcdd in normal human epidermal cells and squamous cell carcinoma (scc) lines. these systems permit detailed investigation of the molecular and biochemical events underlying pathologic changes in the skin and offer the potential of establishing a risk assessment model for halogenated aromatic compounds by using human target cells.imagesfigure 2. afigure 2. bfigure 2. cfigure 2. dfigure 3. |
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diverticula of the female urethra are rare lesions occurring predominantly in the distal two thirds of the urethra. they are thought to be derived from the periurethral glands as a result of recurrent infections. urethral diverticula (ud) have historically been described with the classic triad of three d 's e.g. dysuria, dysparunia, and dribbling, which are present only in about one third of cases. they can be asymptomatic and incidentally detected or may present with symptoms like painful vaginal mass, chronic pelvic pain, refractory lower urinary tract symptoms, and recurrent urinary tract infections (uti). they predominantly affect females in third to fifth decade of life with overall incidence of 1 6%. they tend to be acquired with age ; however, rarely they have been described in neonates and children. we herein describe a young female with a giant ud measuring 7.3 6.2 cm who presented with acute urinary retention (aur) and had successful outcome after surgery. female ud presenting with bladder outlet obstruction is very rare and should be kept in mind while evaluating any female with aur. this case is unique because of its age of occurrence, presentation, and largest size reported till date. a 20-year - old unmarried girl presented to emergency with painful urinary retention since 8 h. she had history of similar episode 2 months back for which she was catheterized for 1 week. she also complained of frequency, poor stream, straining, incomplete voiding, and dysuria for 2 years. there was no history of hematuria, flank pain, urinary incontinence, menstrual disturbances, or sexual contact. urinary bladder was palpable on abdominal examination and genital examination revealed a large non - tender cystic mass over anterior vaginal wall. urine examination revealed 5 - 8 pus cells per high power field and sterile culture. transvaginal sonography performed with broadband 9- to 5-mhz tightly curved array probe revealed a hypoechoic cystic structure posterior to urinary bladder and urethra with enhanced through - transmission and a small echogenic structure within it, possibly a stone. a magnetic resonance imaging (mri) of pelvis was done which showed a well - defined cystic structure posterior to urinary bladder measuring 7.3 6.2 cm displacing the uterus superiorly and vagina and cervix posteriorly with no obvious connection with urethra or urinary bladder seen [figure 1 ]. cystourethroscopy was performed using 15f flexible cystoscope with simultaneous digital compression over the bladder neck area and diverticulum. it revealed a large ud opening into the urethra in the right postero - lateral wall of the middle third. a rubber catheter tip measuring about 1 cm was seen inside the diverticulum, a retained foreign body from previous catheterization. (a) t1-weighted mri image showing the urethral diverticulum (ud) and urinary bladder (ub) in the sagittal plane. (b) t2-weighted cross - sectional image showing the diverticulum. (c, d) reconstructed t2-weighted mri images showing relationship of urethral diverticulum with urinary bladder trans - vaginal excision of diverticulum was done through an inverted u - shaped incision over the anterior vaginal wall. vaginal flaps were raised with careful preservation of the periurethral fascia and meticulous dissection of the diverticulum up to the neck. diverticulum was excised and repair was done over 14f catheter in four layers, e.g. urethra, periurethral fascia, martius flap, and vaginal tissue [figures 2a, b ]. suprapubic catheter was not placed and anticholinergics were given in the post - op period for three weeks. the urethral catheter was removed at 3 weeks, after voiding cystourethrogram (vcug) revealed no extravasation [figures 2c, d ]. since the first description of ud by william hey in 1805, increasing number of cases is being reported, especially in last two decades. this is partly because of increased awareness among physicians and increased use of improved radiological investigations. although usually single, globular, and small, uds may be multiple, may attain various shapes and grow to large sizes. in our patient, it was extending from the external urerthral meatus up to trigone of bladder and displacing adjacent structures. the ud described in our patient is the largest one reported till date [table 1 ]. female ud is a very rare cause of urinary retention and should always be considered in the differential diagnosis. about 4% of female ud cause urinary retention. groutz showed that ud contributed to 3% of cases of female bladder outlet obstruction defined as persistent noninvasive maximum flow rate less than 12 ml / s combined with a detrusor pressure at maximum measured flow rate of more than 20 cm h2o in a pressure - flow study. sizes of urethral diverticula in different case series strong clinical suspicion based on history, with focussed physical examination and appropriate radiological investigations is vital for the diagnosis of ud. it should be differentiated from vaginal leiomyoma, skene 's gland cysts, and abscesses, gartner 's duct cysts, vaginal wall cysts, and ectopic ureterocele. although transvaginal ultrasonography, vcug, and positive pressure urethrography have traditionally been used in the diagnosis of ud, mri is regarded as the investigation of choice by many with 100% reported sensitivity. moreover, it is non - invasive, free from ionising radiations, does not depend on voiding of the patient and gives an excellent anatomical picture of the ud and adjacent pelvic structures. uds appear as areas of decreased signal intensity on t1-weighted images compared with the surrounding soft tissues, and they have high signal intensity on t2-weighted images. although endoluminal coil modalities have been described, surface coil non - contrast mri is used in majority of the centers. pre - operative video urodynamics is required in patients with symptomatic stress urinary incontinence and may delineate the ud under fluoroscopy in many cases. although very rare, malignant tumors, calculi and foreign bodies are to be ruled out in all cases of ud. in our patient, a broken catheter tip was recovered from the diverticular cavity. surgery is the mainstay of treatment for ud and surgical excision and urethral reconstruction remains the most popular modality though selected patients may be managed by transurethral or open marsupialisation. total excision of the diverticulum, preservation of periurethral fascia, and watertight and tension - free closure with non - overlapping suture lines are vital for optimal surgical outcome. simultaneous anti - incontinence surgery may be done in patients with symptomatic stress urinary incontinence. | female urethral diverticulum is a rare entity with diverse spectrum of clinical manifestations. it is a very rare cause of bladder outlet obstruction and should be considered as a differential diagnosis in females presenting with acute urinary retention associated with a vaginal mass. strong clinical suspicion combined with thorough physical examination and focused radiological investigations are vital for its diagnosis. herein we report a case of giant urethral diverticulum presenting with acute urinary retention in a young female. it was managed by excision and urethral closure, and is the largest urethral diverticulum reported till date in the literature. |
sephacryl s-300 hr and [-p]gtp (400 ci / mmol) were obtained from ge healthcare europe gmbh (vienna, austria). co., hohenlockstedt, germany) containing 4.4 mm gtn in 250 mm glucose were obtained from a local pharmacy ; dilutions were made in 50 mm triethanolamine hcl buffer. dea / no and odq (alexis corp., lausen, switzerland) were purchased via eubio (vienna). stock solutions of odq (10 mm) were prepared in dimethyl sulfoxide and diluted with 25% dimethyl sulfoxide in h2o (v / v). oxyhemoglobin was prepared by reduction of bovine hemoglobin (sigma) with sodium dithionite as described previously (21). daidzin was synthesized as described (22) and dissolved in 50 mm triethanolamine hcl at maximally 160 m (because of limited solubility). aldh expression and purification human liver aldh1 and aldh2 were expressed in escherichia coli bl21(de3) as described previously (23), using pt7.7 as the host vector for both cdnas. aldh2 was purified by p - hydroxyacetophenone affinity chromatography as described (24). for final purification, the protein was loaded on a size - exclusion chromatography column (sephacryl s-300 ; 100 2.5 cm), which had been equilibrated in 100 mm mes buffer (ph 6.5) containing 0.2 mm mgcl2, and eluted with the same buffer at 1 ml / min. aldh1 was purified according to the same protocol but without the final size - exclusion chromatography step. active fractions eluted from the p - hydroxyacetophenone column were pooled and dialyzed against 100 mm mes buffer (ph 6.5) containing 0.2 mm mgcl2 and 1 mm dtt. aldh activity was measured as formation of nadh from nad in the presence of varying concentrations of acetaldehyde by monitoring the increase in absorbance at 340 nm (340 = 6.22 mm cm) at 25 c (25). the incubation buffer (ph 9.0) was either 100 mm sodium pyrophosphate (aldh1) or 50 mm sodium pyrophosphate containing 10 mm mgcl2 (aldh2). as reported previously (26), we observed that mg ions slightly reduced the dehydrogenase activity of aldh1 (by 20%) but increased the respective activity of aldh2 (by 20%). the esterase activities of both isoforms were increased by 2030% in the presence of mg ions (data not shown). esterase activity was measured by monitoring formation of p - nitrophenolate from varying concentrations of p - nitrophenyl acetate (400 = 16 mm cm) in the presence of 100 mm sodium pyrophosphate buffer (ph 7.5). enzyme kinetic parameters were calculated by fitting the data according to saturation kinetics using the kaleidagraph software. determination of 1,2- and 1,3-gdn formation by radio thin layer chromatography the rate of gtn metabolism yielding 1,2- and 1,3-gdn was determined according to a described protocol (9). briefly, purified aldh1 or aldh2 (4 g of each) was incubated with c - labeled gtn (2 m ; 50,000 dpm) at 37 c for 10 min in a final volume of 200 l of 50 mm phosphate buffer (ph 7.4) containing 3 mm mgcl2, 2 mm gsh, 2 mm dtt, 1 mm nad, 1 mm edta, 1 mm egta, and unlabeled gtn as required to obtain the indicated final concentrations. the amount of added radioactivity was increased 3-fold for determination of reaction rates at gtn concentrations 0.3 mm. enzyme inhibitors (chloral hydrate and the aldh2-selective compound daidzin) were present as indicated in the text and figure legends. reaction products were extracted twice with 1 ml of diethyl ether, separated by thin layer chromatography, and quantified by liquid scintillation counting. determination of sgc activity purified bovine lung sgc (50 ng) was incubated at 37 c for 10 min in a final volume of 100 l with the indicated concentrations of donor compounds (gtn, nano2, and dea / no) in the absence and presence of purified aldh1 or aldh2 or bsa as indicated in the text and figure legends. assay mixtures contained 50 mm triethanolamine hcl (ph 7.4), 5 mm mgcl2, 0.5 mm [-p]gtp (250,000 cpm), 1 mm cgmp, and 2 mm dtt. where indicated, chloral hydrate (1 or 10 mm), daidzin (0.1 mm), oxyhemoglobin (0.1 mm), flavin adenine dinucleotide (0.1 mm), or odq (0.1 mm) was additionally present. reactions were terminated by the addition of 0.45 ml of zinc acetate (120 mm) and 0.45 ml of sodium bicarbonate (120 mm), followed by isolation and quantification of [p]cgmp as described previously (27). determination of gtn - derived no in the presence of purified aldh isoforms no formation was measured with a clark - type electrode (world precision instruments, berlin, germany), calibrated daily with acidified nitrite as described previously (28), in 50 mm triethanolamine hcl (ph 7.4) containing 1,000 units of superoxide dismutase, 2 mm dtt, and gtn (1 mm and 0.1 mm with aldh1 and aldh2, respectively). the incubation volume was 1 ml. where indicated, measurements were performed in the presence of the nucleotide cofactors nad or nadh (2 mm each) or the aldh inhibitor chloral hydrate (10 mm). after equilibration at 37 c, purified aldh1 (0.5 mg) or aldh2 (0.25 mg), which had been adjusted to 37 c for 10 min, was added. the no scavenger oxyhemoglobin (10 m final concentration) was added in some experiments to confirm that the observed signals did indeed reflect formation of no radical. analysis of aldh - bound metals for the determination of metals in aldh1 and aldh2, protein solutions (100 l, 80 m) were mixed with nitric acid (1% (v / v) final concentration) and vigorously shaken for 10 min. the samples were screened for metals with an inductively coupled plasma mass spectrometer (agilent 7500ce, waldbronn, germany) in the semiquantitative mode. the concentrations of fe, cu, mn, mo, and zn were 95% pure as judged by polyacrylamide gel electrophoresis (data not shown). the enzyme kinetic parameters determined for the dehydrogenase and esterase activities of aldh1 and aldh2 (table 1), including the significantly lower km values for aldehyde and ester substrates and higher specific activity of aldh2, agree well with values reported previously (25). table 1enzyme kinetic parameters of purified recombinant human aldh1 and aldh2dehydrogenase and esterase activities were determined photometrically in the presence of increasing concentrations of acetaldehyde and p - nitrophenyl acetate, respectively, as described under data are mean values s.e. of the parameters determined for three different enzyme preparations.dehydrogenase activity esterase activity kmvmaxkmvmax mnmol min mg mnmol min mgaldh1 200 14 1,060 20 0.69 0.03 155 4 aldh2 0.33 0.005 4,000 30 0.18 0.01 210 9 enzyme kinetic parameters of purified recombinant human aldh1 and aldh2 dehydrogenase and esterase activities were determined photometrically in the presence of increasing concentrations of acetaldehyde and p - nitrophenyl acetate, respectively, as described under experimental procedures. data are mean values s.e. of the parameters determined for three different enzyme preparations. aldh - catalyzed gtn metabolism determination of 1,2- and 1,3-gdn formation by radio thin layer chromatography revealed that both aldh isoforms catalyze gtn metabolism (fig. 1a) converted 2 m gtn into 1,2- and 1,3-gdn with rates of 3.0 0.16 and 0.66 0.03 nmol min mg, respectively. in the presence of this low gtn concentration, aldh2 (fig. 1b) exhibited 3-fold higher activity with respect to 1,2-gdn formation (9.00 0.28 nmol min mg). formation of 1,3-gdn was very low (0.18 0.01 nmol min mg), confirming the selective conversion of gtn into the 1,2-isomer under these conditions (5, 9). as expected, chloral hydrate inhibited gtn metabolism by both isoforms, whereas daidzin showed marked selectivity for aldh2. 2, the two isoforms exhibited similar maximal 1,2-gdn - forming activities of 3040 nmol min 2a) and aldh2 (b) was half - maximal at 200 and 20 m gtn, respectively, apparently reflecting the lower km values of aldh2 measured with dehydrogenase and esterase substrates (see table 1). as reported previously (5, 9), specific 1,2-gdn formation by aldh2 was lost at increasing gtn concentrations, although formation of the 1,3-isomer did not exceed 1,2-gdn formation even at 1 mm gtn. in contrast, the aldh1-catalyzed reaction resulted in predominant formation of the 1,3-isomer at 1 mm gtn. in fact, the rate of 1,2-gdn formation was significantly decreased at the highest gtn concentration tested (2.8 mm). formation of 1,3-gdn was not affected by substrate - competitive inhibitors, indicating that it does not involve binding of gtn to the catalytic sites of the enzymes. purified aldh1 and aldh2 (4 g of each) were incubated at 37 c for 10 min in the presence of 2 m [c]gtn, 3 mm mgcl2, 2 mm gsh, 2 mm dtt, 1 mm nad, 1 mm edta, and 1 mm egta. 1,2- and 1,3-gdn were extracted and quantified by radio thin layer chromatography as described under experimental procedures. purified aldh1 and aldh2 (4 g of each) were incubated at 37 c for 10 min in the presence of 2 m [c]gtn, 3 mm mgcl2, 2 mm gsh, 2 mm dtt, 1 mm nad, 1 mm edta, and 1 mm egta. the enzyme inhibitors chloral hydrate (ch) and daidzin were present as indicated. 1,2- and 1,3-gdn were extracted and quantified by radio thin layer chromatography as described under figure 2.dependence on gtn concentration of 1,2- and 1,3-gdn formation by aldh1 (a) and aldh2 (b). purified aldh1 and aldh2 (4 g of each) were incubated at 37 c for 10 min in the presence of 3 mm mgcl2, 2 mm gsh, 2 mm dtt, 1 mm nad, 1 mm edta, 1 mm egta, and the indicated concentrations of [c]gtn. 1,2- and 1,3-gdn were extracted and quantified by radio thin layer chromatography as described under dependence on gtn concentration of 1,2- and 1,3-gdn formation by aldh1 (a) and aldh2 (b). purified aldh1 and aldh2 (4 g of each) were incubated at 37 c for 10 min in the presence of 3 mm mgcl2, 2 mm gsh, 2 mm dtt, 1 mm nad, 1 mm edta, 1 mm egta, and the indicated concentrations of [c]gtn. 1,2- and 1,3-gdn aldh - catalyzed gtn bioactivation assayed as activation of sgc purified sgc was used for highly sensitive detection of no - related bioactivity associated with aldh - catalyzed gtn metabolism. taking into account the different gtn affinities of the two aldh isoforms, gtn was present at final concentrations of 100 and 10 m in the assays with aldh1 and aldh2, respectively. 3, cgmp synthesis was not considerably increased over basal in the presence of increasing amounts of bsa that was used as a protein control (specific activity varied between 0.11 0.02 and 0.36 0.05 mol of cgmp min mg). in the absence of gtn, neither aldh1 nor aldh2 affected cgmp formation (see fig. 5 below for gtn dependence). however, the combined presence of gtn and aldh1 or aldh2 led to significant sgc activation. maximal rates of cgmp formation were observed with 50100 g of aldh1 or 25 g of aldh2 ; sgc activity reached a plateau or even decreased at higher aldh concentrations and never exceeded 30% of the rates measured with maximally no - stimulated sgc. purified sgc (50 ng) was incubated at 37 c for 10 min in the presence of [-p]gtp (0.5 mm, 250,000 cpm), 5 mm mgcl2, 1 mm cgmp, and 2 mm dtt with the indicated amounts of purified aldh1, aldh2, or bsa. gtn concentration was 10 m (aldh2 and bsa) or 100 m (aldh1). purified sgc (50 ng) was incubated at 37 c for 10 min in the presence of [-p]gtp (0.5 mm, 250,000 cpm), 5 mm mgcl2, 1 mm cgmp, and 2 mm dtt with the indicated amounts of purified aldh1, aldh2, or bsa. gtn concentration was 10 m (aldh2 and bsa) or 100 m (aldh1). [p]cgmp was isolated and quantified as described under experimental procedures. data are mean values s.e. of three independent experiments. to see whether aldh - catalyzed gtn bioactivation involves the catalytic site of the proteins, we tested the effects of the established aldh inhibitors chloral hydrate and daidzin (fig. 4a). to account for slight inhibition of no stimulation of sgc (10%), the effects of the added compounds are shown as the percent of maximal sgc activity determined under identical conditions in the presence of 10 m dea / no. although the non - selective compound chloral hydrate (1 mm) inhibited gtn - induced sgc activation mediated by both aldh1 and aldh2, daidzin selectively blocked the effect of aldh2, strongly suggesting that binding of gtn to the substrate sites of the aldh isoforms is essential for bioactivation. the potential involvement of no binding to the regulatory heme group of sgc was tested with the heme site sgc inhibitor odq (29, 30), the superoxide - generating compound flavin adenine dinucleotide (31, 32), and the established no scavenger oxyhemoglobin. as shown in fig. 5 shows the gtn concentration dependence of cgmp formation in the presence of aldh1 and aldh2. the effects of gtn were biphasic with apparent ec50 values of 42 2.9 m (aldh1) and 3.1 0.4 m (aldh2). the 10-fold higher potency of gtn to stimulate sgc in the presence of aldh2 agrees well with the metabolism experiments shown in fig. gtn (10300 m) had no effect in the presence of bsa, which served as a protein control. activation of sgc by 0.1 mm gtn was not affected by the trace metal chelator diethylenetriaminepentaacetic acid (data not shown). figure 4.effects of enzyme inhibitors and no scavengers on aldh - mediated sgc activation by gtn. purified sgc was incubated with purified aldh1 (50 g) or aldh2 (25 g) in the presence of 100 m (aldh1) or 10 m (aldh2) gtn as described in the legend to fig. 3. the aldh inhibitors chloral hydrate (ch) or daidzin (a), the sgc inhibitor odq, the superoxide - generating compound flavin adenine dinucleotide (fad), and the no scavenger oxyhemoglobin (oxyhb)(b) were present as indicated. data are expressed as percent of maximal sgc activity obtained with 10m dea / no (a) or specific sgc activity (b) and represent mean values s.e. of three independent experiments. effects of enzyme inhibitors and no scavengers on aldh - mediated sgc activation by gtn. purified sgc was incubated with purified aldh1 (50 g) or aldh2 (25 g) in the presence of 100 m (aldh1) or 10 m (aldh2) gtn as described in the legend to fig. 3. the aldh inhibitors chloral hydrate (ch) or daidzin (a), the sgc inhibitor odq, the superoxide - generating compound flavin adenine dinucleotide (fad), and the no scavenger oxyhemoglobin (oxyhb)(b) were present as indicated. data are expressed as percent of maximal sgc activity obtained with 10m dea / no (a) or specific sgc activity (b) and represent mean values s.e. purified sgc was incubated with aldh1 (50 g) or aldh2 (25 g) or bsa (50 g) as described in the legend to fig. 3 in the presence of the indicated concentrations of gtn. purified sgc was incubated with aldh1 (50 g) or aldh2 (25 g) or bsa (50 g) as described in the legend to fig. 3 in the presence of the indicated concentrations of gtn. [p]cgmp was isolated and quantified as described under experimental procedures. data are mean values s.e. aldh - catalyzed gtn bioactivation resulting in formation of no inhibition of aldh - mediated sgc activation by oxyhemoglobin and superoxide (generated via flavin adenine dinucleotide - triggered o2 reduction) suggested the involvement of free no radical in gtn bioactivity. therefore, we attempted to measure gtn - derived no with a clark - type electrode. in preliminary experiments, we observed relatively small, transient no signals that were markedly enhanced and prolonged in the presence of superoxide dismutase (data not shown). to effectively prevent this apparent superoxide - mediated no decay, all no assays were performed in the presence of 1,000 units / ml of superoxide dismutase. 6, addition of aldh1 (a) or aldh2 (b) to gtn - containing assay mixtures resulted in the appearance of pronounced no signals with initial rates of 1.68 0.06 and 1.76 0.012 m min the maximal no concentrations measured with aldh1 and aldh2 were 0.47 0.01 and 0.33 0.001 m, respectively. no detectable signals were observed in the presence of chloral hydrate (10 mm). chloral hydrate did not affect no signals derived from dea / no decomposition (data not shown). the apparent no formation mediated by both isoforms was enhanced by nad, which stimulates gtn metabolism. the nucleotide increased both initial rates of no formation (to 8.40 0.08 and 4.08 0.08 m min mg aldh1 and aldh2, respectively) and no peak concentrations (0.90 0.007 m aldh1 and 0.57 0.003 m aldh2). we reasoned that no formation may involve nitrite reduction (see below) and therefore tested nadh as a potential reducing cofactor. nadh enhanced no formation in terms of initial rates and peak concentrations, but the effects were less pronounced than those observed with nad. aldh - mediated bioactivation of inorganic nitrite because aldh2 reduces gtn to inorganic nitrite (5), we speculated that the observed no formation could be due to intrinsic nitrite reductase activity of the aldh isoforms. to test this hypothesis, we used nitrite instead of gtn in sgc assays as potential aldh substrate. to minimize thiol - mediated non - enzymatic bioactivation of nitrite, the assays were performed in the absence of dtt. as shown in fig. 7, sgc exhibited a specific activity of 0.87 0.014 mol min mg in the presence of 10 mm nitrite. this activity was increased 2- or 4-fold in the additional presence of aldh1 or aldh2, respectively. chloral hydrate (10 mm) had no effect on sgc activity in the presence of nitrite alone but completely inhibited the effects of aldh1 and aldh2, excluding nonspecific protein effects. the present study demonstrates that gtn metabolism catalyzed by purified aldh isoforms is associated with formation of free no radical, resulting in pronounced sgc activation. considering that enzymatic nitrite reduction by the respiratory chain does not appear to contribute significantly to mitochondrial gtn bioactivation, these results may provide a simple and conclusive explanation for previous reports showing that aldh2-catalyzed gtn metabolism triggers accumulation of cgmp in blood vessels (5, 9). the initial rates of no formation measured in the presence of nad (8.4 and 4.0 nmol min mg) suggest that no formation accounts for 10% of total gtn turnover under our experimental conditions (cf. however, the transient time course of the no signals that we observed even in the presence of 1,000 units / ml of superoxide dismutase indicates that the true rates might have been underestimated because of additional, unknown no consumption pathways. this is even more relevant for the degree of sgc activation, which had to be measured in the absence of superoxide dismutase and nad to avoid interference of these agents with the enzyme assay. thus, specific no - forming activity and, in particular, sgc activation measured under our experimental conditions most likely reflect the lower limit of the true efficiency of aldh - catalyzed gtn bioactivation. purified aldh1 (0.5 mg ; a) or aldh2 (0.25 mg ; b) was incubated at 37 c with gtn (1 mm and 0.1 mm, respectively) in the absence and presence of nad and nadh (2 mm each) or chloral hydrate (ch ; 10 mm). c shows the effect of oxyhemoglobin, added at the time points indicated by arrows to give a final concentration of 10 m, on the no signals obtained from aldh1 and aldh2 incubated with gtn as described for a and b in the absence of nucleotides. purified aldh1 (0.5 mg ; a) or aldh2 (0.25 mg ; b) was incubated at 37 c with gtn (1 mm and 0.1 mm, respectively) in the absence and presence of nad and nadh (2 mm each) or chloral hydrate (ch ; 10 mm). c shows the effect of oxyhemoglobin, added at the time points indicated by arrows to give a final concentration of 10 m, on the no signals obtained from aldh1 and aldh2 incubated with gtn as described for a and b in the absence of nucleotides. figure 7.activation of sgc by inorganic nitrite in the presence of purified aldh1 and aldh2. purified sgc (50 ng) was incubated at 37 c for 10 min with [-p]gtp (0.5 mm, 250,000 cpm), 5 mm mgcl2, 1 mm cgmp, and 10 mm nano2 in the absence (control) or presence of purified aldh1 and aldh2 (50 g each). purified sgc (50 ng) was incubated at 37 c for 10 min with [-p]gtp (0.5 mm, 250,000 cpm), 5 mm mgcl2, 1 mm cgmp, and 10 mm nano2 in the absence (control) or presence of purified aldh1 and aldh2 (50 g each).. the pronounced effect of superoxide dismutase on detectable gtn - derived free no (data not shown) suggests significant generation of superoxide in our assays. an unavoidable superoxide source may be gtn itself, which was shown to trigger superoxide formation in blood vessels, thereby limiting vascular no bioavailability (3335). in addition, we found that dtt, which was present to minimize mechanism - based oxidative aldh inactivation, caused rapid consumption of authentic (dea / no - derived) no that was prevented by superoxide dismutase.4 this apparent superoxide generation may be due to dtt autoxidation described in detail recently (36). generation of superoxide is certainly one important factor limiting the degree of sgc activation by gtn in our experiments and may partly explain the sharply biphasic gtn concentration - response curves shown in fig. as studied in detail previously (3), oxidation of sgc - bound heme by high concentrations of gtn may counteract no - mediated sgc activation. however, neither of these mechanisms appears to sufficiently explain limited sgc activation by a fixed concentration of gtn in the presence of increasing amounts of aldh (fig. 3). because the time course of no release from dea / no was not affected by addition of the proteins (data not shown), aldh - catalyzed gtn bioactivation may have been compromised under our experimental conditions by a mechanism - based pathway that remains to be identified. first, gtn - triggered sgc activation and no formation were blocked by established substrate - competitive aldh inhibitors. in line with the known selectivity profile of the compounds (22, 25), chloral hydrate attenuated gtn bioactivation by both aldh1 and aldh2, whereas daidzin selectively interfered with the aldh2-catalyzed reaction. second, gtn concentration - response curves revealed an 10-fold higher potency of the nitrate to activate sgc in the presence of aldh2 than with aldh1. this difference in the apparent gtn binding affinities is in line with the differences in the km values of the two isoforms for aldehyde and ester substrates (see table 1). unequivocally demonstrate that the catalytic conversion of gtn into 1,2- and 1,3-gdn is not a specific feature of aldh2, suggesting a basic reaction mechanism common to both aldh isoforms. despite similar characteristics of aldh1- and aldh2-catalyzed gtn metabolism with respect to stimulation by nad and preferential formation of 1,2-gdn at low gtn concentrations, there were also clear differences apparent. besides the 10-fold higher gtn concentrations required for half - maximal activity of aldh1, product distribution differed significantly between the two isoforms. in contrast to aldh2, aldh1 preferentially produced 1,3-gdn at saturating gtn concentrations, and the rate of 1,2-gdn formation was reduced by 50% at the highest gtn concentration tested. together with preliminary findings indicating that aldh1-catalyzed gtn metabolism is less dependent on the presence of thiols than the aldh2-catalyzed reaction,5 these results point to subtle differences in the catalytic properties of the two isoforms. future studies on the structure and function of wild - type and mutated aldh isoforms should clarify the molecular basis of these differences. the observation that catalysis of gtn bioactivation is not a specific feature of aldh2 is certainly interesting with respect to aldh enzymology, but the pharmacological relevance of this finding is unclear. the low apparent gtn affinity of aldh1 is expected to limit the contribution of this isoform to gtn bioactivation in patients treated with therapeutically relevant low doses of the nitrate. nevertheless, aldh1 or other isoforms capable of catalyzing this reaction could contribute to the so - called high km pathway of gtn action (17) that persists upon aldh2 inhibition (6, 9) or gene deletion (10). interestingly, we found that gtn bioactivation catalyzed by rat liver microsomes was sensitive to chloral hydrate but not to daidzin (9). collectively, the currently available data suggest that aldh1 and possibly other isoforms of the enzyme could contribute to the low affinity component of gtn action in blood vessels. at present we can only speculate about the mechanism underlying the observed formation of free no radical in the course of aldh - catalyzed gtn metabolism. the significantly enhanced activation of sgc by inorganic nitrite in the presence of the purified aldh isoforms shows that the enzymes are capable of converting nitrite to no that activates sgc. inhibition of nitrite bioactivation by chloral hydrate excludes nonspecific effects of the proteins and suggests that no formation takes place within the catalytic sites of the enzymes. however, aldh - mediated activation of sgc by nitrite was only partial and required extremely high nitrite concentrations, seriously questioning the relevance of this reaction for formation of gtn - derived no. nevertheless, because the sgc assays were performed under suboptimal conditions, i.e. in the absence of nad and superoxide dismutase because of interference of these agents with the assay we are currently establishing a cgmp reporter assay with rat lung fibroblasts (37) to circumvent those limitations of the sgc assay. the high nitrite concentration that was required for aldh - mediated sgc activation may reflect low affinity of exogenous nitrite to the catalytic site but does not exclude that gtn - derived nitrite becomes efficiently converted to no in situ in the course of the enzymatic reaction. thus, the available data are not sufficient to definitively rule out nitrite as an intermediate. if no were indeed formed from gtn - derived nitrite, this could occur either via intrinsic nitrite reductase activity of aldh or disproportionation of nitrite after protonation in the catalytic site. preliminary data obtained with mutated aldh2 indicate that the reactive cysteine residue in the active site (cys) is essential for no formation and sgc activation. therefore, the initial step of no formation may be a nucleophilic attack of gtn at cys, yielding a thionitrate intermediate. as shown in scheme 1, this intermediate could be converted to no through two hypothetical pathways. (i) rearrangement to a sulfenyl nitrite intermediate leads to homolysis to yield no and sulfenyl radical. however, this pathway would result in irreversible inactivation of the enzyme after one turnover because of expected dimerization of sulfenyl radicals, and it does not explain why gtn bioactivation is triggered by free cysteine but not by cysteine - containing peptides (e.g. gsh) or proteins (e.g. bsa). (ii) metal - catalyzed reaction of the thionitrate intermediate (38) with an adjacent cysteine residue would result in formation of no and a disulfide. this pathway accommodates reversible inactivation of aldh2 by gtn as well as the inactivity of proteins not containing vicinal cysteine residues in appropriate conformation, but the lack of effect of diethylenetriaminepentaacetic acid argues against the involvement of a redox - active trace metal. however, further work with various metal chelators is required to definitively settle this issue. it has been shown that vascular gtn metabolism results in production of hydroxylamine, suggesting that hno may be formed as an intermediate of gtn bioactivation (39). the no - like properties of hno donors such as angeli 's salt may be explained by 1-electron oxidation of nitroxyl to no by superoxide dismutase or transition metal ions (40). although the trace metal chelator diethylenetriaminepentaacetic acid had no significant effect on gtn - triggered sgc activation, and the purified aldh proteins did not contain significant amounts of bound trace metals, we can not rule out hno as an intermediate of gtn bioactivation. taken together, the present results demonstrate that aldh1 and aldh2 convert gtn into significant amounts of no. it is hard to judge whether this direct formation of no explains gtn - induced cgmp formation in vivo. of note, we found that < 5% of maximal (no - stimulated) cgmp accumulation in blood vessels is sufficient for maximal vascular relaxation (9). therefore, suboptimal activation of sgc by gtn as observed here in the presence of purified aldh isoforms may fully account for the aldh - dependent vascular effects of the nitrate in vivo. nevertheless, we can not exclude the existence of additional pathways linking aldh - catalyzed gtn metabolism to sgc activation in vascular smooth muscle cells. further studies are warranted to clarify the biological relevance of our findings and the molecular mechanism underlying aldh - catalyzed no formation. | metabolism of nitroglycerin (gtn) to 1,2-glycerol dinitrate (gdn) and nitrite by mitochondrial aldehyde dehydrogenase (aldh2) is essentially involved in gtn bioactivation resulting in cyclic gmp - mediated vascular relaxation. the link between nitrite formation and activation of soluble guanylate cyclase (sgc) is still unclear. to test the hypothesis that the aldh2 reaction is sufficient for gtn bioactivation, we measured gtn - induced formation of cgmp by purified sgc in the presence of purified aldh2 and used a clark - type electrode to probe for nitric oxide (no) formation. in addition, we studied whether gtn bioactivation is a specific feature of aldh2 or is also catalyzed by the cytosolic isoform (aldh1). purified aldh1 and aldh2 metabolized gtn to 1,2- and 1,3-gdn with predominant formation of the 1,2-isomer that was inhibited by chloral hydrate (aldh1 and aldh2) and daidzin (aldh2). gtn had no effect on sgc activity in the presence of bovine serum albumin but caused pronounced cgmp accumulation in the presence of aldh1 or aldh2. the effects of the aldh isoforms were dependent on the amount of added protein and, like 1,2-gdn formation, were sensitive to aldh inhibitors. gtn caused biphasic sgc activation with apparent ec50 values of 42 2.9 and 3.1 0.4 m in the presence of aldh1 and aldh2, respectively. incubation of aldh1 or aldh2 with gtn resulted in sustained, chloral hydrate - sensitive formation of no. these data may explain the coupling of aldh2-catalyzed gtn metabolism to sgc activation in vascular smooth muscle. |
the high dose rate (hdr) brachytherapy is a technique where small iridium-192 source (active length 3.5 mm and diameter 0.6 mm) can be programmed to dwell for a given time at any point in the treatment catheters. the success of this treatment is strongly dependent on accurate knowledge of the source strength, dwell positions, and dwell times. since the complete treatment may be given in just a few minutes with hdr remote after loading equipment, it is extremely important to perform adequate quality control (qc) checks on the equipment before use. for high - activity source most of these methods, however, depend on visual inspection of either a radiographic image or a closed circuit television image to quantify the accuracy of hdr unit source positioning. this can be achieved if the verification method is capable of resolving minute differences in source position. most of the currently used quality assurance (qa) tests concentrate on the verification of source position and dwell time. rickey, 2010 and song, 2009 verified the source position and dwell time with their own designed equipment. however, uses of most of the equipments other than film are limited only to brachytherapy. furthermore, designing equipment for brachytherapy qa is not practicable or economically viable to all radiotherapy departments. thus, equipments used in external radiotherapy qa may be tailored for use in brachytherapy. in this study, commercially available detector array imatrixx was used to verify the dwell position, reference isodose length (length of 100% isodose line in source direction), and step size in comparison with treatment planning system (tps). all the tests included in this study were performed on two - dimensional, monolithic, large area pixel - segmented ionization chamber (pxc) commercially available as imatrixx. this device consists of 1020 vented ion chambers each having active volume 0.08 cc, arranged in 32 32 grids. the current, which is proportional to the dose rate, is measured and digitalized by a nonmultiplexed 1020 channels current sensitive analog to digital converter. the signals are analyzed by the omipro - imrt version 1.7 (iba, gmbh). first the imatrixx detector array is calibrated to external beam radiation (6 mv), to get the response of all detectors uniform. three interstitial flexible catheters were fixed above the imatrixx detector array such that 300thsource position (distal end) corresponds with the center of the superior most chamber. the distance between the center of first and last detector along the catheters was 23.6 cm. the flexible plastic catheters were placed straight above the flat panel at 5.71, 0.38, and 4.95 cm, respectively, from the central axis as shown in figure 1. the catheters were placed along the detector line (row wise / column wise) to avoid any systematic shift in the catheter placement, hence in the large systematic error. flexiplan version 2.6 (isodose control b.v.) was used to create plans for this fixed geometry. plans were created to verify (i) dwell position, (ii) reference isodose length, and (iii) step size. position of catheters over the detector array all plans created for this study used 5 mm step size with 2 s dwell time. the dwell time was set as 2 s in all the plans with the reason that the detector response was adequate to resolve the peaks in dose profile, which otherwise resulted in a single dark image. the dose profiles obtained along the source line at 5.71, 0.38, and 4.95 cm from central axis were analyzed. since the dwell positions were set to 2 s, plans created to verify the reference isodose length were not prescribed to any particular distance. thus a method was adopted in which a point that is 5 mm superior to the first source position (300th position) was selected [figure 2 ], and the dose at this point was normalized to 100%. with reference to this point the reference isodose length was measured as maximum length of 100% isodose along catheter axis [figure 3 ]. treatment plans with different dwell positions were executed, and measured reference isodose length were compared with the tps calculated values, as tabulated in tables 2 and 3. the step size was tested for 2, 3, 4, and 5 cm step sizes. comparison of planned and measured dwell positions the position of the point where the dose was considered as 100% reference isodose length verification reference isodose length verification for same source loading of 7 cm reference isodose length verification for different source loadings it was not possible to calibrate the detector array for ir-192 energy as well as brachytherapy (4) geometry, thus only relative isodose could be verified. the maximum dwell position error was noted to be 1.8 mm with a mean 0.451.99 mm 95% confidence interval. total error divided as systematic or mean error of 0.45 mm and random component error or standard deviation was 1.01 mm [table 1 ]. when planned and measured dwell positions were plotted, a linear relationship was obtained [figure 4 ]. planned versus measured dwell position the source was loaded for 10 cm from 270th source position to 170th source position. the second plan was created with source loading length of 7 cm in all catheters from 270 position as tabulated in table 2. the center catheter was loaded from 270th position to 200th position for a length of 7 cm. the other two catheters were loaded only for 3 cm in which the first position started from 170th position, as indicated in table 3. the reference isodose length was measured for each catheter. a total of seven reference isodose lengths in three plans were compared with the tps planned lengths [tables 2 and 3 ]. less than 1 mm error was found in 57% of the tested reference isodose lengths (maximum error was 1.5 mm). planned versus measured reference isodose length step sizes less than 1 cm were not able to be resolved as peaks with imatrixx. however, step sizes 2, 3, 4, and 5 cm were verified [figure 6 ]. maximum error was 1.2 mm in a plan where the step size was 2 cm. the maximum dwell position error was noted to be 1.8 mm with a mean 0.451.99 mm 95% confidence interval. total error divided as systematic or mean error of 0.45 mm and random component error or standard deviation was 1.01 mm [table 1 ]. when planned and measured dwell positions were plotted, a linear relationship was obtained [figure 4 ]. planned versus measured dwell position the source was loaded for 10 cm from 270th source position to 170th source position. the second plan was created with source loading length of 7 cm in all catheters from 270 position as tabulated in table 2. the center catheter was loaded from 270th position to 200th position for a length of 7 cm. the other two catheters were loaded only for 3 cm in which the first position started from 170th position, as indicated in table 3. the reference isodose length was measured for each catheter. a total of seven reference isodose lengths in three plans were compared with the tps planned lengths [tables 2 and 3 ]. less than 1 mm error was found in 57% of the tested reference isodose lengths (maximum error was 1.5 mm). step sizes less than 1 cm were not able to be resolved as peaks with imatrixx. however, step sizes 2, 3, 4, and 5 cm were verified [figure 6 ]. maximum error was 1.2 mm in a plan where the step size was 2 cm. pxc was designed for use in external beam dosimetry and to perform two - dimensional verifications of fields with large gradients and complex shapes. one of the limiting factors for this instrument is volume averaging effect due to finite size of the ion chamber, measured to have full width at half maximum of about 5.8 mm, when a gaussian response function was used. if the volume of the beam data collecting chamber is comparable with that of the imatrixx chambers then due to similar volume averaging effect, imatrixx measured values shows a very high degree of match with tps - calculated values. the literatures can be found for intensity - modulated radiotherapy (imrt) dose verification by this instrument. generally, the imrt calculation grid resolution (voxel size) is 2 2 2 mm and dose distribution is verified with 3% dose difference 3 mm distance to agreement (dta). under this scenario comparable results were obtained between imatrixx and film. the use of pxc for brachytherapy dosimetry was intended due to two reasons, first its use in dosimetry of high - gradient fields (imrt dose profile) verification, which is encountered in brachytherapy. secondly, due to its high charge collection efficiency (0.985) for a dose rate of 3.5 gy / min of 6 mv photon beam, this dose rate falls under hdr brachytherapy range as indicated by task group report 56 (dose rate at 1 cm for 10 ci source is 6.85 gy / min). no published literature could be found for ir-192 energy and brachytherapy geometry for use of pxc. in this study, all tests performed for dwell position verification resulted with errors < 1.8 mm. in order to investigate the spatial resolving efficiency of the detector array, two plans were created with 1 mm dwell position differences ; the dwell position in one plan was related with other plan with only 1 mm difference in their value (270th dwell position in one plan corresponds to 269th position in another plan). the omnipro software was able to differentiate the peaks in the dose profile of both plans [figure 7 ]. reference isodose length verification was used to indirectly verify these step sizes to the extent of their agreement with the planned reference isodose lengths in tps. the mean dwell position variation measured by imatrixx was smaller than the value 0.7 mm that had been reported by song. it is clear from the graph that peaks were differentiated with each other for ir-192 hdr treatments, source position and step sizes are usually verified in clinic by visual inspection by autoradiograph. this autoradiograph is taken as a film by attaching two flexible implant tubes, one having the dummy source and other having the true source as programmed. the other disadvantage of film is blurring, as reported by rickey ; the blurring effect was more pronounced with metal applicators than with plastic catheters. the accuracy of dwell position evaluation in a blur film is subjective and depends on the investigator 's and instrument 's ability to resolve the peak. this problem was not encountered in imatrixx since peaks obtained in the dose profile provided the spatial information, which in turn was used to find the dwell positions. the reproducibility of dwell position measurement using imatrixx was appreciable ; (it measured the dwell position accuracy 0.45 1.99 mm with 95% confidence interval). imatrixx was able to measure the reference isodose lengths with 2 mm accuracy against tps calculated values. the reference isodose length of the central catheter was expected to be more than that of other catheters as given in tps. this is because dose distribution against central catheter will have a contribution from each of the lateral catheters, whereas the lateral two catheters will have contribution only from the central catheter because of the large distance between them (~10.5 cm). imatrixx was able to quantify the same nature of isodoses and mimic the tps values. the mean dwell positional accuracy measured was 0.45 mm, with a standard deviation of 1.01 mm (maximum error of 1.8 mm). this is clearly below the tolerance limit of 2 mm. until the completion of this study, only one other report describing brachytherapy qa using a prototype flat panel detector with gafchromic film was found. the commercially available flat panel detector imatrixx proved to be an effective tool in measuring important parameters in brachytherapy. these promising results build a foundation on which further investigations in brachytherapy routine qa can be achieved in future. | for high dose rate (hdr) brachytherapy, independent treatment verification is needed to ensure that the treatment is performed as per prescription. this study demonstrates dosimetric quality assurance of the hdr brachytherapy using a commercially available two - dimensional ion chamber array called imatrixx, which has a detector separation of 0.7619 cm. the reference isodose length, step size, and source dwell positional accuracy were verified. a total of 24 dwell positions, which were verified for positional accuracy gave a total error (systematic and random) of 0.45 mm, with a standard deviation of 1.01 mm and maximum error of 1.8 mm. using a step size of 5 mm, reference isodose length (the length of 100% isodose line) was verified for single and multiple catheters of same and different source loadings. an error 1 mm was measured in 57% of tests analyzed. step size verification for 2, 3, 4, and 5 cm was performed and 70% of the step size errors were below 1 mm, with maximum of 1.2 mm. the step size 1 cm could not be verified by the imatrixx as it could not resolve the peaks in dose profile. |
global estimates for 2008 indicate that colorectal cancer is the third most common cancer in the world. reports in several countries have described diverging incidence rates in colorectal cancer by subsite, including, in relative terms, an increasing proportion of proximal tumors [215 ], and thus a shift in absolute incidence from left- to right - sided colon cancers. the reasons for this trend are not well understood ; the subsites differ in physical function, artery supply, histology, and innervation, and they also derive from different segments in the primitive intestinal tract in the embryo. the proximal colon originates from the midgut, whereas the distal colon and the rectum derivate originate from the hindgut. comparisons have also shown that proximal colon tumors tend to have different molecular characteristics, with a higher proportion of microsatellite instability, and are more likely to have cpg island methylator phenotype and ki - ras mutations than distal colon and rectal tumors. it has been estimated that 45 percent of all colorectal cancer cases can be prevented in high - risk populations through modifications of diet, physical activity habits, and weight control. according to the recent report from the world cancer research fund / american institute for cancer research (wcrf / aicr), there is convincing evidence that dietary fiber protects against colorectal cancer and that red and processed meat and alcohol (particularly in men) increase the risk of the disease. further, it is stated that garlic, milk, and calcium probably protect against colorectal cancer. also, although meta- [2023 ] and pooled analyses [2426 ] have provided quantitative synthesis for several of the dietary risk factors, little emphasis has been placed on subsite risks. based on the biological differences in the colorectal segments and the reported differences in incidence, we may suggest differences across the segments in their association to lifestyle factors, such as diet. the aim of the present paper is to give an updated overview summarizing the etiological differences between the colorectal subsites with regard to the dietary factors considered to be convincing or probable risk factors for colorectal cancer. the specific risk factors studied were red meat, processed meat, fiber, garlic, milk, calcium, and alcohol, selected given an a priori assessment of their importance in colorectal cancer etiology following the wcrf / aicr report in 2011, and for which their modification could lead to a reduction in rates of colorectal cancer. the outcome was the risk of primary colorectal cancer according to subsite. a search for cohort studies published as original articles was conducted via a search of pubmed (http://www.ncbi.nlm.nih.gov), using a search strategy that combined the term colorectal neoplasms with the terms risk factors and cohort study with either the term diet, nutrition, or alcohol. the search was restricted to studies published or available online as of october 8, 2012, in the english language. a detailed description of the search strategy and the resulting papers retrieved is given in table 1, and the procedure is described in figure 1. the initial evaluation yielded 108 articles in the study database and underwent a second evaluation based on full - text review. a similar pubmed search for case - control studies nested within a cohort identified 46 articles, of which one underwent full - text review. in addition, further 62 articles were identified by scanning the reference lists of retrieved articles, reviews, meta-, and pooled analyses and underwent full - text review (figure 1). studies were included if they provided risk estimates (and corresponding confidence intervals) for both proximal and distal colon cancer. proximal colon (right sided) includes ceacum, ascending and transverse colon, while distal colon (left - sided) includes descending colon, sigmoid flexure, and sigmoideum. some studies have not followed the above - mentioned classification [2729 ], and this is specified in table 2. further, to be included, the cohorts had to be either population based, registry based, or obtained from censuses. studies on specific subpopulations (e.g., hospital - based cohorts) were not included nor were studies examining second cancers, metastasis, survival, or mortality. following the full - text evaluation, 139 articles were excluded for the reasons indicated in figure 1, and 32 articles were included in the review. of these, seven gave data on (red) meat [3036 ], five on processed meat [3135 ], eight on fiber [3744 ], one on garlic, three on milk [4547 ], seven on calcium [28, 45, 4751 ], and ten on alcohol [27, 29, 36, 5258 ]. the number adds up to more than 32 as several papers give data for more than one risk factor. if several papers on the same risk factor were published for a given cohort, all data was retrieved from the most recent paper. the relative risk (rr), hazard rate ratio (hrr), hazard ratio (hr), incidence rate ratio (irr) or odds ratio (or), and corresponding 95% confidence intervals (95% ci) for each risk factor are presented (as rr) in figures 28, sorted in ascending order of magnitude by sex. for one study the risk estimates were tabulated according to the lowest versus highest exposure category in the original paper and are presented as the inverse of the value in the corresponding figure (figure 7). meta - analyses were not performed as a consequence of the heterogeneity in factors such as exposure measurement, the categorization of risk factor levels, and the confounders adjusted for in the studies. overall, data from 21 cohorts with information on one or more of the risk factors were included in this review. table 2 gives a detailed description of the studies, with information on cohort size, number of cases, sex, age distribution, follow - up time, and factors adjusted for in the risk analyses. the european prospective investigation into cancer and nutrition (epic) study consists of subcohorts from 10 european countries. the other cohorts were from the usa (10 cohorts), sweden (2), denmark (2), japan (3), korea (1), uk (1), and the netherlands (1). the subsite - specific results (figures 28) are described according to the individual risk factors, as presented in the following. seven cohort studies were included in the review, of which five reported on red meat [3033, 35 ], one on beef and pork, and one on meat (not further specified). the five studies on red meat reported an increased risk of colorectal cancer with increasing intake [3033, 35 ] (table 2), although most of the risk estimates were not statistically different from unity. in figure 2, there appears to be somewhat more consistency with the observation of an increased risk of cancers of the rectum and distal colon than there is for proximal colon cancer. this picture remains when restricting the evaluation to studies with adequate statistical power. in a swedish study, women consuming 94 or more grams of red meat per day had an increased risk of distal colon cancer of 2.22 (95% ci 1.343.68) compared to women consuming less than 50 grams / day, and there was a significant trend of increasing risk with increasing consumption (ptrend < 0.001). a long - term high intake of red meat yielded an increased risk of rectal cancer of 43% (95% ci 1.002.05) relative to low intake in a us study, whereas in another us study there was a significant trend of increased risk of both proximal colon, distal colon and rectal cancer with increasing consumption of red meat (including both processed and nonprocessed red meat) (for all ptrend = 0.02). neither beef consumption nor pork consumption was significantly associated with risk of cancer of any colorectal subsite in a japanese study, while the frequency of meat consumption (not further specified) was positively associated with proximal and distal colon cancer in south korean men, and with proximal colon and rectal cancer in south korean women. four studies have reported on processed meat [3133, 35 ] and one has reported on ham and sausages (table 2). in the epic study, consumption of 80 grams or more of processed meat per day conferred a 62% (95% ci 1.042.50) increased risk of rectal cancer compared to an intake of less than 10 grams per day, and in a us study a high long - term intake of processed meat was associated with a 50% (95% ci 1.042.17) increased risk of distal colon cancer (figure 3). in a more recent american study, consuming 22.3 grams or more of processed meat per 1000 kcal increased the risk of rectal cancer with 30% (95% ci 1.001.68) compared to consuming 1.6 gram or less per 1000 kcal. eight cohort studies were included in the review ; six have provided estimates for the risk of the colonic subsites in relation to fiber consumption [37, 4042, 44 ] and two have provided estimates for the whole grain consumption [39, 43 ] (table 2). as for fiber, most of the risk estimates were not statistically different from unity [37, 41, 42 ] (figure 4). the epic study reported an inverse association between fiber intake and colorectal cancer with no strong evidence of different associations across the subsites : when fiber intake was analyzed as a categorical variable, a significant inverse association was seen for distal colon cancer only (ptrend = 0.02), whereas when fiber intake was analyzed as a continuous variable and corrected for measurement errors, significant inverse associations were seen for proximal colon (hr per 10 gram / day increase 0.83, 95% ci 0.750.92) and rectum cancer (hr per 10 gram day / increase 0.87, 95% ci 0.790.96), but not for distal colon cancer. the multiethnic cohort observed a reduced risk for distal colon and rectal cancer among those in the highest compared to the lowest fiber intake group, but the reduction was only significant in men (rr 0.56, 95% ci 0.350.90 and rr 0.52, 95% ci 0.320.84, resp.). two scandinavian papers have reported on associations between whole grain consumption and cancer risk of colorectal subsites [39, 43 ]. in a danish study, which is also included in the epic study, total consumption of whole grain products was associated with a significantly lower risk of proximal colon cancer and a borderline significantly lower risk of distal colon cancer and rectal cancer in men (irr per each increment in intake of 50 gram per day 0.78, 95% ci 0.660.92 for proximal colon, 0.90, 95% ci 0.791.02 for distal colon, and 0.90, 95% ci 0.801.01 for rectum) but not in women (data not given). no significant associations between whole grain consumption and cancer of any of the colorectal subsites were seen in the swedish mammography cohort (figure 4). the width of the confidence intervals for the risk estimates on fiber and whole grain varies markedly between the included studies. however, whether all studies are included or those with the lowest power are excluded, there are rather minimal differences in relative risk across the subsites. only one study on garlic is included in the review. the iowa women 's health study reported that having at least one serving per week of garlic was associated with a 48% reduced risk of distal colon cancer compared to zero servings of garlic (rr 0.52, 95% ci 0.300.93) (table 2, figure 5). two cohort studies have examined the relation between total consumption of milk and the risk of cancer in colorectal subsites [45, 47 ] (table 2, figure 6). no significant associations were seen for any of the colorectal subsites in a us study that combined both sexes (estimates not given for women). in a study of swedish men, a significantly reduced risk for distal colon cancer was seen for those consuming 1.5 glasses or more of milk per day compared to those consuming less than two glasses per week (rr 0.53, 95% ci 0.330.87). in another swedish study restricted to high fat dairy food and conducted among women only, a significant inverse trend was observed between consumption of full - fat cultured milk and risk of distal colon cancer (ptrend = 0.03), whereas a significant increased risk of proximal colon cancer was observed among women who consumed whole milk (1 or more servings per day compared to never / seldom consumers rr 1.58, 95% ci 1.152.16). seven papers from eight cohorts were included in the review [28, 45, 4751 ] (table 2, figure 7). in the analyses from the nurses ' health study and the health professionals follow - up study that studied colon cancer only, non - significant inverse associations were seen between total calcium and distal colon cancer for both men and women, and a pooled analysis of the two cohorts gave a significant inverse trend for distal colon cancer (ptrend = 0.01). likewise, a significant inverse trend was reported between total calcium and distal colon cancer in the swedish mammography cohort (ptrend = 0.02), while in a us female cohort, a significant inverse trend was seen between dietary calcium intake and proximal colon cancer (ptrend = 0.01). no association was seen for total calcium. in a us study examining both sexes, total calcium intake was significantly inversely associated with proximal colon cancer among men (ptrend = 0.04), but not dietary calcium, and there were no significant associations seen for women (estimates not given). in a study of swedish men, a significant inverse relation was found between total calcium and rectal cancer (ptrend = 0.02), whereas nonsignificant inverse associations were seen for cancer of the colon. no significant associations between dietary calcium and cancer of any colorectal subsite were reported in a japanese study either for men or for women (estimates not given for women). an earlier study of hawaiian - japanese men reported a significant inverse association between dietary calcium and sigmoid colon cancer (ptrend = 0.02). in addition to the eight cohorts included in this paper, an additional cohort study among american women states that their data provided little support for a protective effect of total calcium at either tumor subsite, but does not present risk estimates. ten articles [27, 29, 36, 5258 ] were included in the review (table 2). three analyses for both sexes combined consistently showed a higher risk of rectal cancer with increasing alcohol consumption and no significant associations for any of the colon subsites [27, 54, 57 ]. in the epic study an increased risk was reported both for rectal and distal colon cancer, whereas in the uk dietary cohort consortium (part of which is included in the epic study) a significantly increased risk was found for distal colon cancer only (figure 8). several sex - specific analyses have been done. among men, two older studies with limited statistical power reported that alcohol consumption was positively related to proximal colon cancer only [52, 53 ], whereas four more recent studies reported non - significant associations with proximal colon cancer [36, 55, 57, 58 ]. a study among japanese men reported increased risk for distal colon cancer, but the confidence interval for the risk estimate was very wide (consumption of 45.6 grams alcohol or more per day compared to never drinkers hr 4.17, 95% ci 1.6310.66). in terms of consumption of alcohol and rectum cancer among men, two of four studies have reported a positive association [55, 57 ]. for women, fewer significant associations have been reported. in the iowa women 's health study, alcohol was not significantly associated with either proximal colon or distal colorectal cancer, nor did further separation into distal colon and rectal cancer reveal any significant associations (estimates not given). furthermore, no significant associations were seen in an earlier study of american women or in the uk dietary cohort consortium. in the netherlands cohort study, however, women consuming 30 grams or more of alcohol per day had hrr of proximal colon cancer of 2.28 (95% ci 1.124.62) compared to abstainers, whereas analyses for the other subsites were not sufficiently robust to draw any conclusions. in a korean study, women who frequently consumed alcohol or who consumed greater amounts of alcohol had a higher risk of rectal cancer. this review provides an overall and updated synthesis of the results from cohort studies examining the association between dietary factors that are convincingly or probably related to the risk of colorectal cancer and subsite - specific colorectal cancer. our study indicates that consumption of alcohol is more strongly related to the risk of rectal cancer than to colon cancer, also that meat consumption tends to be somewhat more strongly related to the risk of distal colon cancer and rectal cancer than proximal colon cancer, that there are only minor differences in relative risk for colorectal cancer across the major subsites for fiber, milk, and calcium, and that no statement can be given for garlic due to limited data. it should be noted that for all exposures the majority of the analyses showed nonsignificant associations with cancer risk, the exception being the positive association between alcohol consumption and rectal cancer. the subsite etiology is rather poorly understood, and the mechanism for the various dietary factors is likely to differ. even for red and processed meat, both established risk factors for colorectal cancer, the underlying mechanisms are not well defined. one suggested mechanism for the somewhat stronger association for rectum and distal colon relative to proximal colon cancer relates to the enhanced endogenous formation of carcinogenic n - nitroso compounds with a high intake of meat. the level of markers of n - nitroso compounds appears to be higher in tissue from distal colon and rectum than in that of the proximal colon. this finding is in keeping with a previously meta - analyses which implicated processed meat consumption as a stronger risk factor for cancer occurrence at the distal colon relative to the proximal colon. a suggested mechanism by which dietary fiber may decrease the risk of colorectal cancer is linked to the fermentation of fiber. the fermentation produces short - chain fatty acids and, in particular, acetic, propionic, and butyric acids. butyrate is particularly of interest, as it has been shown to induce apoptosis and to be cytotoxic to both colorectal adenoma and carcinoma cells. studies on mice have shown that the concentration of butyrate is highest in the distal colon. in humans, fiber is fermented in the proximal colon, and the total amount of short - chain fatty acids has been estimated to be considerably higher in the proximal site compared to the distal. one study on proximal and distal colonocytes indicates, however, that butyrate is a more important source of energy for the distal, than for the proximal, colonic mucosa. if so, this could be a relevant biological mechanism explaining any differences in risk between the sites. in addition, fiber may dilute the concentration of carcinogenic substances in the distal colon. despite these findings, a pooled analysis on fiber reported no convincing differences in colorectal risk for the various anatomical subsites, in line with our own results published here. the reduced risk of colorectal cancer with increasing consumption of milk is likely to be at least partly mediated by calcium, which is thought to have a protective effect through its ability to bind bile acids, and its growth - restraining and differentiation- and apoptosis - inducing effect on colorectal cells. no convincing site - specific associations regarding milk and calcium were seen in our review. a former pooled analysis reported an inverse association for milk limited to cancer of the distal colon and rectum, while the results from two meta - analysis on site - specific impact of calcium have been conflicting [20, 21 ]. the consumption of alcohol is associated with increasing risk of cancer in several organs in the digestive tract, including the colorectum. alcohol is not a carcinogen itself, but acts as a tumor promoter and possibly as a co - carcinogen. alcohol also acts as a solvent and thus might increase the exposure to other carcinogens by enhancing the penetration of carcinogens into the cell. acetaldehyde is a metabolite of alcohol and may be the most important agent responsible for the carcinogenic effect as it is highly toxic, mutagenic, and carcinogenic. a pooled analyses on alcohol reported similar risk across all areas of the large bowel. however, a stronger association with alcohol for rectal cancer compared with colon cancer as seen in our paper could possibly be related to a higher degree of epithelial hyperregeneration in rectum. the number of sex - specific analysis is presently too low to suggest any significant interaction by sex at the subsite level. there are some methodological issues in this study which may have impacted on the findings. there is considerable variability in several key characteristics of the assembled cohort studies : the follow - up time across studies included in this paper varied from 4 to 22 years, while the total number of colorectal cancer cases analyzed ranged from 126 to 2974. short - term follow - up studies tend to accrue a lower number of cases, and the resulting estimates are subject to greater uncertainty. the substratification of cases by sex, tumor location, and exposure categories, as presented here, inevitably leads to smaller numbers and a greater degree of imprecision in the estimates, even for relatively large studies. however, restricting the evaluation to studies with adequate statistical power did not change the overall picture. long - term follow - up studies will commonly accrue a greater number of cases, but are more prone to measurement error given that an increasing follow - up time raises the possibility that exposure status of the participants will change, leading to misclassification of exposure and under- or overestimation of the risk estimates. however, the natural history of colorectal cancer is on average of long duration, and exposure in the more distant past may be the most relevant when estimating subsequent risk. another issue is the difference in risk factor dosages between studies and that the categories compared sometimes vary considerably between studies. for instance, for calcium, in the swedish study by larsson. daily intake of 1445 mg or more is compared to an intake of less than 956 mg / day, whereas in the japanese study by ishihara. daily intake of 661 mg or more is compared with an intake of less than 337 mg / day. careful reading of the exposure categories (given in the figures) is therefore necessary when evaluating the findings. in addition, the specific confounding factors adjusted for at the analysis stage differ between studies. of the studies that were full - text reviewed, 76 were excluded as they did not reveal information at the level of subsite location. given the high proportion of cohort studies failing to report subsite - specific estimates, the potential publication bias prohibited a formal meta - analysis. a further rationale for this decision is the lack of uniformity in exposure categories within each risk factor. in summary, the strength of the association between dietary components and cancer of the large bowel may partially depend on the anatomic location within the colorectum. the most consistent finding is the stronger association between alcohol and rectal cancer, compared with alcohol and proximal and distal colon cancer. meat (red and processed) is possibly more strongly associated with risk of distal colon cancer and rectal cancer than the risk of proximal colon cancer. for fiber, milk, and calcium there seem to be only minor differences in relative risk across the subsites. however, caution is required as the number of papers presenting risk estimates by colorectal subsite is limited, particularly for milk and garlic. | objective. a shift in the total incidence from left- to right - sided colon cancer has been reported and raises the question as to whether lifestyle risk factors are responsible for the changing subsite distribution of colon cancer. the present study provides a review of the subsite - specific risk estimates for the dietary components presently regarded as convincing or probable risk factors for colorectal cancer : red meat, processed meat, fiber, garlic, milk, calcium, and alcohol. methods. studies were identified by searching pubmed through october 8, 2012 and by reviewing reference lists. thirty - two prospective cohort studies are included, and the estimates are compared by sex for each risk factor. results. for alcohol, there seems to be a stronger association with rectal cancer than with colon cancer, and for meat a somewhat stronger association with distal colon and rectal cancer, relative to proximal colon cancer. for fiber, milk, and calcium, there were only minor differences in relative risk across subsites. no statement could be given regarding garlic. overall, many of the subsite - specific risk estimates were nonsignificant, irrespective of exposure. conclusion. for some dietary components the associations with risk of cancer of the rectum and distal colon appear stronger than for proximal colon, but not for all. |
during the a / h1n1 2009 global pandemic, adjuvanted influenza vaccines were widely used for the first time. along with many other countries, all canadians older than 6 mo were eligible to receive this vaccine, which accounted for over 96% of pandemic vaccine doses administered. this vaccine was used in a dose - sparing formulation (3.75 g hemagglutinin per adult dose), one dose of which was adequately immunogenic in nave adults. by the end of the mass campaign in canada, over 40% of the population had received this vaccine. it was not known at the time if adjuvant use would be associated with persistently elevated antibody responses or the rapid decay typically seen after seasonal inactivated vaccines. in studies launched while the pandemic vaccine was initially deployed in 2009, evidence of prior infection was already present in one - third of adults under 60 y of age. these primed individuals developed strong booster antibody responses to h1n1pdm09 vaccination, with greater potential for persistence. both natural infection and vaccination with adjuvant were expected to elicit greater cell - mediated immune responses than observed following standard trivalent seasonal vaccines. in 2010 the a / h1n1pdm09 virus continued to circulate with minimal antigenic change. the world health organization recommended that the same h1n1pdm09 vaccine strain be included in the trivalent inactivated vaccine (tiv) for the 20102011 influenza season. some candidates for re - vaccination with the h1n1pdm09 strain would have been strongly primed by prior infection, vaccination with adjuvant or both. it was certainly conceivable that such individuals might have greater residual cellular immunity and/or specific antibody levels than ordinarily encountered after seasonal influenza vaccination and that this might predispose them to increased vaccine reactions. vaccination in the presence of specific cellular immunity has been linked to increased injection site reactions after childhood booster doses of adjuvanted, pertussis - containing vaccines while vaccination in the presence of high titers of antitoxin increased reaction rates after repeated tetanus toxoid boosters. it was not known at the time if residual cellular or humoral immunity to h1n1 would be sufficient to cause reactions with a first booster dose of h1n1 antigen. the post - pandemic situation was unique in modern experience with influenza vaccines and warranted investigation, especially given the potential for unpredictable variations in tiv reactogenicity such as occurred in children in western australia in 2010 with a particular tiv formulation. we conducted a pre - season study to rapidly assess the safety of re - vaccination with h1n1pdm09 antigen. eligible adults had received the adjuvanted pandemic vaccine 910 mo previously, from community providers. adverse events were documented daily ; rates following placebo were subtracted from rates after vaccine to determine vaccine - attributable rates. secondary objectives were to measure residual antibody titers to h1n1pdm09 virus in previously vaccinated adults and the magnitude of the booster response to re - vaccination with this antigen, in comparison to a / h3n2 responses. enrollment totaled 326 subjects, who were predominantly female and caucasian / white (table 1). the rate of prior seasonal influenza vaccination was high as most subjects were employed by or affiliated with the participating health care institutions. centers commenced enrollment on the same day (9 august 2010) and completed it within 5 d. compliance with the protocol was high (fig. opposite assignment applied to second (crossover) injections ; refers to any ongoing medical condition, not just those posing increased risk with influenza. comment : one subject withdrew before the initial injection, a second withdrew shortly after the initial injection (placebo, with code break) and a third withdrew prior to the second injection. as table 2 shows, tiv vaccination was associated with higher than background (post - placebo) rates for myalgia, tiredness, headache, malaise, arthralgia, sleep disturbance and diarrhea during the week after vaccination. only myalgia, tiredness and headache occurred at vaccine - attributable rates (var) > 5.0%, with myalgia leading at 18.6%. most subjects who reported these symptoms rated them as mild / moderate (table 2). the peak var s for myalgia and headache were reached later during the day of vaccination, at 16.9% and 4.9%, respectively, while reports of tiredness peaked next day, with a var of 5.8%. reported rates of myalgia and headache then declined to match those following placebo from day 3 onwards, with tiredness rates normalizing one day later. a similar pattern was seen with reports of malaise, arthralgia, sleep disturbance and diarrhea. background rates during the week after placebo were > 5% for headache, tiredness, myalgia and malaise (table 2), illustrating the value of controlled observations for this age group. at the injection site, pain was reported by 63.2% of participants (204/323) during the week after tiv vaccine and by 8.0% (26/323) after placebo. tiv - related pain was rated as mild (186 subjects, 57.6%) or moderate (18 subjects, 5.6%) with no instances of severe pain. local redness followed tiv in 45 participants (13.9%), with 38 (11.8%) having mild (5.0%, with myalgia leading at 18.6%. most subjects who reported these symptoms rated them as mild / moderate (table 2). the peak var s for myalgia and headache were reached later during the day of vaccination, at 16.9% and 4.9%, respectively, while reports of tiredness peaked next day, with a var of 5.8%. reported rates of myalgia and headache then declined to match those following placebo from day 3 onwards, with tiredness rates normalizing one day later. a similar pattern was seen with reports of malaise, arthralgia, sleep disturbance and diarrhea. background rates during the week after placebo were > 5% for headache, tiredness, myalgia and malaise (table 2), illustrating the value of controlled observations for this age group. at the injection site, pain was reported by 63.2% of participants (204/323) during the week after tiv vaccine and by 8.0% (26/323) after placebo. tiv - related pain was rated as mild (186 subjects, 57.6%) or moderate (18 subjects, 5.6%) with no instances of severe pain. local redness followed tiv in 45 participants (13.9%), with 38 (11.8%) having mild (40.0c, injection site redness or swelling 100 mm diameter or any symptom that precluded normal daily activities or prompted medical attention. serologic responses were analyzed according to standard international (emea / chmp) criteria for adults < 60 y old given seasonal influenza vaccine. seroprotection was considered the primary serologic outcome measure, defined as an hai titer 40. geometric mean titers were calculated, with any test - negative sample assigned to value of 5. geometric mean fold rise was calculated as the within - subjects ratios of the post - vaccination hai titer to the baseline hai titer. the seroconversion rate was defined as either a 4-fold or greater titer rise or conversion from a negative baseline titer to one 40. a sample size of 300 evaluable subjects was desired to permit detection of severe or unusual adverse events at rates as low as 1% with 90% probability. event rate differences after vaccine and placebo 6% could be detected with 80% probability (= 0.05) with 300 observations after each treatment, using fisher s exact test. to allow for drop - outs, the intended enrollment was 320 subjects, 64 per center. the data from this study showed that adults given a low - dose, aso3-adjuvanted h1n1pdm09 vaccine in 2009 had substantially reduced rates of seroprotection (hai titers 40) 910 mo later. while the low - dose formulation was intended to be dose - sparing, the short duration of protection could have been problematic had the pandemic been more prolonged. however, individuals given a subsequent non - adjuvanted dose of tiv containing the h1n1pdm09 antigen boosted well, reflecting excellent immune memory. the second vaccination with the h1n1pdm09 antigen was generally well tolerated and did not generate unusually high anti - h1n1 titers. the data from this study showed that adults given a low - dose, aso3-adjuvanted h1n1pdm09 vaccine in 2009 had substantially reduced rates of seroprotection (hai titers 40) 910 mo later. while the low - dose formulation was intended to be dose - sparing, the short duration of protection could have been problematic had the pandemic been more prolonged. however, individuals given a subsequent non - adjuvanted dose of tiv containing the h1n1pdm09 antigen boosted well, reflecting excellent immune memory. the second vaccination with the h1n1pdm09 antigen was generally well tolerated and did not generate unusually high anti - h1n1 titers. | many canadians received a novel as03-adjuvanted vaccine during the 2009 influenza a / h1n1 pandemic. longer term implications of adjuvant use were unclear : would anti - h1n1 immune responses persist at high levels and, if so, could that result in increased or unusual adverse effects upon re - exposure to h1n1pdm09 antigen in the trivalent influenza vaccine (tiv) for 201011 ? to answer these questions, adults given as03-adjuvanted h1n1pdm09 vaccine (arepanrix, gsk canada) 910 mo earlier were enrolled in an evaluator - blinded, crossover trial to receive 20102011 non - adjuvanted tiv (fluviral, gsk canada) and placebo 10 d apart, in random order. adverse effects were monitored for 7 d after each injection. vaccine - attributable adverse event (vaae) rates were calculated by subtracting rates after placebo from those after vaccine. blood was obtained pre - vaccination and 2130 d afterward to measure hemagglutination inhibiting antibody titers. in total, 326 participants were enrolled and 321 completed the study. vaae rates were low except for myalgia (18.6%) and injection site pain (63.2%). at baseline, h1n1pdm09 titers 40 were present in 176/325 subjects (54.2%, 95% confidence interval 48.6, 59.7), with a geometric mean titer (gmt) of 37.4 (95% ci 32.8, 42.6). post - immunization, 96.0% (95% ci 92.3, 97.8) had h1n1pdm09 titers 40, with gmt of 167.4 (95% ci 148.7, 188.5). responses to both influenza a strains in tiv were similar, implying no lasting effect of adjuvant exposure. in summary, titers 40 persisted in only half the participants 910 mo after adjuvanted pandemic vaccine but were restored in nearly all after tiv vaccination, with minimal increase in adverse effects. |
gestational diabetes mellitus (gdm) is defined as impaired glucose tolerance appearing specifically during pregnancy (1). gdm develops by excessive insulin resistance, inadequate -cell compensation, reduced -cell function, or any combination of these (2). a large number of epidemiological studies demonstrated that diabetes in pregnant women is associated with an increased risk of maternal and neonatal morbidity (3). infants of a diabetic mother (idms) have been shown to be prone to the development of complex diseases, including obesity, and metabolic and cardiovascular complications, during childhood and adulthood (4, 5). in animal models of gdm, it has been shown that idms overtly develop diabetes throughout life (6, 7). furthermore, population - based studies have also demonstrated that idms have an increased risk for type 2 diabetes in later childhood and as adults (8). insulin - producing -cells in the endocrine pancreas play a pivotal role in maintaining glucose homeostasis. adult -cells can dynamically respond to systemic increases in insulin demand by expan - ding their functional mass. compensatory changes in -cells mass are controlled by increases in cell size (hypertrophy) or increase in number of cells (hyperplasia) (9, 10). recent findings have shown that defect in these mechanisms is a key feature of the pathogenesis of diabetes (11, 12). similarly, idm mature animals show -cells dysfunction and decreased insulin secretion in response to glucose (7). however, the mechanisms responsible for -cells malfunction in idms are unknown. therefore, understanding how -cells proliferate and function is important and may lead to development of new therapeutic strategies for diabetes. strikingly, recent studies have shown a link between the cell cycle regulators and the risk of type 2 diabetes (13, 14). cyclin - dependent kinases (cdk) a family of serine / threonine protein kinases phosphorylate a number of substrates like retino - blastoma protein (prb) mainly implicated in cell cycle progression. subsequent phosphorylation of prb by the cdk results in the release of e2f. upon activation, e2f1 is able to turn on genes required for progression through g1 into the s phase of cell cycle (15). in addition to activation of cell cycle, e2f directly contributes to insulin secretion through the regulation of kir6.2 (also referred as kcnj11) expression. it has been proven that kir6.2 channels play a pivotal role in the regulation of insulin secretion. so, cdk4-prb - e2f1 pathway directly contributes to both proliferation and regulation of insulin secretory capacity of -cells (16). indeed, no study has investigated the gene expre - ssion of kir 6.2 and cdk4-prb - e2f1 factors in adult offspring of diabetic rats. thus, the purpose of the present investigation was to evaluate the expression changes of these genes in pancreatic islands extracted from offspring of streptozotocin - induced mildly hyperglycemic rats. this experimental study was performed to evaluate the effect of gestational diabetes on expression of cdk4-prb - e2f1 pathway genes in pancreatic islets of rat offspring. all animal procedures followed the guidelines set by the institutional animal care and use committee at the golestan university of medical sciences, gorgan, iran. the rats were kept in a temperature - controlled environment (212 c) on 12-hr light / dark cycles and allowed free access to standard rat chow and water. vaginal plaque was mentioned daily as a positive sign of pregnancy and the day on which vaginal plaque was observed, considered as day 0 of pregnancy. a total of 20 dams were made diabetic by single ip injection of freshly prepared streptozotocin (stz) solution (40 mg / kg body weight) in sterile saline solution (0.85%) on day zero of gestation (17) ; 7 dams were injected with equivalent volume of normal saline as control group. four days later, blood glucose levels were checked using a glucometer (accu - chek active glucometer, roche diagnostics, germany). if glucose levels were between 120 - 250 mg / dl, the rats were selected and used as gdm. mildly hyperglycemic dams in the current series of experiments were ~40%, or 8 out of 20 stz- injected rats. totally six diabetic offspring from gdm mothers at the age of 12 and 15 weeks were selected. islets were isolated from pancreases of diabetic and control group rats by a modification of the collagenase digestion technique (18). this involved cannulation of the common bile duct and the sequential administration of 2 ml digest solution containing 0.2 mg / ml liberase tl (roche, cat # 05 401 020 001) and 10 pg / ml dnase (takara) in serum free rpmi 1640 medium. the organ was then incubated at 37 c for 15 min and dispersed using pipetting action. at the end of the incubation, the tubes moved to ice and 10 ml rpmi 1640 with 10% serum added. the islets were separated by centrifugation on a ficoll gradient (histopaque1077, sigma - aldrich10771) and collected from the histopaque / media interface with a disposable 10 ml serological pipette and resuspended in serum containing rpmi. the islets were then isolated by passage through a 100 m cell strainer (bd falcon) and handpicked with a pasteur pipette using a dissecting microscope. rna was isolated from islets using the genabioscience rna extraction kit according to the manufacturer s instructions. residual dna was digested with 10 u rnase - free dnase (dnase i, takara) in the presence of 20 units of rnase inhibitor at 37 c for 20 min. after heat inactivation for 10 min at 75 c in 2 mm edta, concentration and purity of the dnase i - treated samples was measur- ed using a nanodrop nd-1000 spectrophotometer (a260/a280>1.8 and a260/a230>1.6). the integrity and stability of the rnas was confirmed by demonstrating the intact 28s and 18s bands on gel electrophoresis. for real - time rt - pcr, the cdna was synthesized from 1 g of dnasei - treated total rna using prime script rt reagent kit (takara) with random hexamer and oligo dt primers following the manufacturer s protocol. the forward and reverse pcr primers for the 7 genes were designed in accordance to the real - time pcr conditions, using perlprimer software (bio - rad, usa), and the sequences are listed in table 1. for each genes, the cdna amplified by specific primers using taq polymerase kit (takara), and correct product was confirmed by running on gel electrophoresis. real - time pcr primer name, sequences, size, genbank accession number and pcr condition real - time rt - pcr was performed using the sybr - green pcr master mix kit (takara) in the thermo cycler (abi, 7300). the cycling conditions were 95 c for 30 sec followed by 40 cycles at 95 c for 5 sec, 55 c for 30 sec and 72 c for 1 min. we used rat -actin as internal control and non- diabetic offspring islets cdna as calibrator. amplification specificity was confirmed by gel electrophoresis. the relative expression level of mrna between the diabetic and non - diabetic samples was determined with the comparative ct (cycle threshold) method. first, both ct values of target gene from control islets cdna (n=3) and diabetic islets cdna (n=3) were normalized by ct value of the internal control (ct). then, the former was subtracted by the latter, namely ct. the value of 2, the fold change of gene expression, real - time serial data were statistically analyzed. every real - time pcr experiment was repeated with three samples and each sample was run in duplicate. relative target gene expression and blood glucose level was analyzed with one - way anova using spss 16.0 statistical analysis software. the differences between groups were compared using unpaired t test and p1.8 and a260/a230>1.6). the integrity and stability of the rnas was confirmed by demonstrating the intact 28s and 18s bands on gel electrophoresis. for real - time rt - pcr, the cdna was synthesized from 1 g of dnasei - treated total rna using prime script rt reagent kit (takara) with random hexamer and oligo dt primers following the manufacturer s protocol. the forward and reverse pcr primers for the 7 genes were designed in accordance to the real - time pcr conditions, using perlprimer software (bio - rad, usa), and the sequences are listed in table 1. for each genes, the cdna amplified by specific primers using taq polymerase kit (takara), and correct product was confirmed by running on gel electrophoresis. real - time pcr primer name, sequences, size, genbank accession number and pcr condition real - time rt - pcr was performed using the sybr - green pcr master mix kit (takara) in the thermo cycler (abi, 7300). the cycling conditions were 95 c for 30 sec followed by 40 cycles at 95 c for 5 sec, 55 c for 30 sec and 72 c for 1 min. we used rat -actin as internal control and non- diabetic offspring islets cdna as calibrator. amplification specificity was confirmed by gel electrophoresis. the relative expression level of mrna between the diabetic and non - diabetic samples was determined with the comparative ct (cycle threshold) method. first, both ct values of target gene from control islets cdna (n=3) and diabetic islets cdna (n=3) were normalized by ct value of the internal control (ct). then, the former was subtracted by the latter, namely ct. the value of 2, the fold change of gene expression, real - time serial data were statistically analyzed. every real - time pcr experiment was repeated with three samples and each sample was run in duplicate. data were presented as meanstandard deviation (sd). relative target gene expression and blood glucose level was analyzed with one - way anova using spss 16.0 statistical analysis software. the differences between groups were compared using unpaired t test and p<0.05 was chosen as the level of significance. fasting blood glucose concentration was significantly increased in idm rats (figure 1). by 12 weeks of age, about 40% of the idms developed mild hyperglycemia and at 15 weeks of age, glucose levels were markedly elevated in idms compared to controls (p<0.001). blood glucose concentrations in idms (infants of a diabetic mother) and control animals (week 12 and week 15). blood glucose level of offspring was obtained via tail vein and was measured with accu - chek glucometer. values are means sem. p<0.001 we assessed whether the expression levels of cell cycle regulator genes (cdk4-prb - e2f1) were affected by gestational diabetes in adult offspring. real - time pcr melting curve for e2f1, cdk4, kir6.2, prb and -actin as internal control (a). agarose gel electrophoresis of pcr products following real - time sybr green amplification (b) the expression levels of prb in isolated islets were not significantly different between the two groups, indicating that the expression of prb was not disturbed by gdm. but analysis of the mrna levels of cdk4 and e2f1 showed significant alterations in the diabetic group compared to controls (figure 3). on the other hand, kir6.2 mrna expression severely reduced in idms, which followed the expected decrease in e2f1 protein (figure 3). mrna levels were measured using gene specific primers (table 1) and the values were normalized to -actin. statistical significance was calculated using the t test (p<0.05, p<0.01, n=3). fasting blood glucose concentration was significantly increased in idm rats (figure 1). by 12 weeks of age, about 40% of the idms developed mild hyperglycemia and at 15 weeks of age, glucose levels were markedly elevated in idms compared to controls (p<0.001). blood glucose concentrations in idms (infants of a diabetic mother) and control animals (week 12 and week 15). blood glucose level of offspring was obtained via tail vein and was measured with accu - chek glucometer. we assessed whether the expression levels of cell cycle regulator genes (cdk4-prb - e2f1) were affected by gestational diabetes in adult offspring. real - time pcr melting curve for e2f1, cdk4, kir6.2, prb and -actin as internal control (a). agarose gel electrophoresis of pcr products following real - time sybr green amplification (b) the expression levels of prb in isolated islets were not significantly different between the two groups, indicating that the expression of prb was not disturbed by gdm. but analysis of the mrna levels of cdk4 and e2f1 showed significant alterations in the diabetic group compared to controls (figure 3). on the other hand, kir6.2 mrna expression severely reduced in idms, which followed the expected decrease in e2f1 protein (figure 3). mrna levels were measured using gene specific primers (table 1) and the values were normalized to -actin. statistical significance was calculated using the t test (p<0.05, p<0.01, n=3). maintenance of appropriate insulin - producing -cells growth and mass is critical for metabolic balance (19, 20) ; thus, the molecular mechanisms by which -cells proliferate are the focus of recent studies. the first years of life in humans and the first postnatal months of life in rodents are crucial periods of islet -cells growth that result in establishment of appropriate -cells mass (21 - 24). fetal hyperglycemia is a consequence of maternal mild hyperglycemia that may disturb -cells growth during early postnatal period and causes diabetes in offspring later in life (25). previous studies have demonstrated that cdk4-prb - e2f1 pathway plays a crucial role in the control of glucose homeostasis. as a cell cycle regulatory pathway, cdk4-prb - e2f1 genes not only regulate -cells proliferation, but also control the expression of genes implicated in insulin secretion, such as kir6.2 (15, 16). in spite of several studies regarding the effects of diabetes i and ii on pancreas structure and function, there is no investigation about the effect of gdm on cell cycle regulators expression in offspring s pancreatic islets. thus, this study was designed to investigate the effects of hyperglycemic intrauterine environment on the expression of cell cycle regulator genes in langerhans islets of adult diabetic offspring. consistent with some previous studies (6, 7), our offspring rats developed diabetes at 12 weeks of age. in this study, we observed a 51% decrease for cdk4 expression in langerhans islets of diabetic offspring (p<0.05). many studies implicate cdk4 as a crucial factor for successful expansion of -cells mass in various conditions. for instance, when cell cycle arrest is altered specifically in -cells by deletion of cdk4 postnatally, -cells mass decreases or fails to expand (13). also rieck showed that during the peak of -cells dna synthesis, cdk4 expression is induced in the islets of pregnant mice (26). also, it has been shown that pharmacological inhibition of cdk4 activity on glucose tolerance in mice drama - tically decreases the clearance of glucose in treated, compared to non - treated mice with idcx, which is a specific cdk4 inhibitor (15). correlated with previous studies, our observation links cdk to the risk of diabetes in offspring of gdm mothers. furthermore, we showed that gdm also causes reduction of prb and e2f1 gene expression in pancreatic islets of diabetic offspring by 35% and 86% (p<0.01), respectively. findings have shown that e2f1 mice have decreased pancreatic size and insulin secretion, as the result of impaired postnatal pancreatic growth. the results have also demonstra - ted that e2f1 was highly expressed in non - proliferating pancreatic -cells, suggesting that e2f1, besides the control of -cells number could have a role in pancreatic -cells function (27). these findings provided enough evidence to propose that cdk4-prb - e2f1 pathway genes are critical mediators of insulin secretion (16). regarding the down - regulation of cdk4-prb - e2f1 pathway genes in pancreatic islets of rat (idms), we can conclude that induction of diabetes in offspring of gdm is mediated by the reduction of cdk4 activity and subsequent e2f1 transcriptional activity. in addition to evaluation of genes involved in -cells proliferation, we also wanted to evaluate whether gdm affects -cells function. so, we analyzed the expression of kir6.2, a key component of the atp - sensitive potassium channel involved in the regulation of glucose - induced insulin secretion in -cells. our data indicated that gdm causes reduction in kir6.2 gene expression by 0.12 fold compare to control group. recent in vitro and in vivo investigations have shown that e2f1 directly controls the expression of kir6.2. several studies on pancreas of e2f1-/- mice have shown that expression of kir6.2 is downregulated that causes insulin secretion defects in these animals (15, 16, 27). our data are in agreement with other reports and suggest that uncontrolled gdm may cause diabetes in offspring by repression of cdk4-prb - e2f1 pathway in their pancreases. our data showed that down - regulation of cdk4-prb - e2f1 pathway genes is related with development of diabetes in offspring of gestational diabetic rats. furthermore, in this study, decreased kir6.2 mrna expression in diabetic offspring underscores a dual effect of the gdm on both proliferation, and function of -cells. taken together, this study may open up new ways for understanding the molecular basis of gdm and type 2 diabetes in the offspring. however, many advances must be made to fully appreciate the exact molecular mechanism of inducing diabetes in offspring by gestational diabetes. | objective(s):the link between a hyperglycemic intrauterine environment and the development of diabetes later in life has been observed in offspring exposed to gestational diabetes mellitus (gdm), but the underlying mechanisms for this phenomenon are still not clear. reduced -cells mass is a determinant in the development of diabetes (type 1 and type 2 diabetes). some recent studies have provided evidence that the cdk4-prb - e2f1 regulatory pathway is involved in -cells proliferation. therefore, we postulated that gdm exposure impacts the offspring s -cells by disruption in the cdk4-prb - e2f1 pathway.materials and methods : adult wistar rats were randomly allocated in control and diabetic group. the experimental group received 40 mg / kg / body weight of streptozotocin (stz) on day zero of gestation. after delivery, diabetic offspring of gdm mothers and control dams at the age of 15 week were randomly scarified and pancreases were harvested. langerhans islets of diabetic and control groups were digested by collagenase digestion technique. after rna extraction, we investigated the expressions of the kir 6.2 and cdk4-prb - e2f1 pathway genes by quantitative real - time pcr.results:gdm reduced the expression of cdk4-prb - e2f1 pathway genes in langerhans islets cells of offspring. cdk4, prb and e2f1 pathway genes were downregulated in diabetic islets by 51%, 35% and 84%, respectively. also, the expression of kir 6.2 was significantly decreased in diabetic islets by 88%.conclusion : we suggest that the effect of gestational diabetes on offspring s -cells may be primarily caused by the suppression of cdk4-prb - e2f1 pathway. |
pyridines and diazines are among the most prevalent heterocycles in biologically active compounds. they are found in 2 of the 5 top - selling pharmaceuticals and 6 of the 23 small molecules approved by the fda in 2013. of course, the groups appended to the heteroarene and the heteroarene core together affect the activity of the molecule, not just the heteroarene itself. thus, studies of the structure activity relationships (sar) require derivatives containing a range of substituents attached to pyridines and diazines. one potential method to create these derivatives is c h bond functionalization of the heteroarene. yet, few c h bond functionalization reactions are amenable to late - stage functionalization of heteroarenes. instead, multistep syntheses are typically conducted to study the effects of substituents on the heteroarene units. some c h bond functionalization reactions of heteroarenes have been developed that exploit a directing group, but methods for site - selective functionalization without the influence of such groups would allow modifications at different positions and without the need to install and remove such groups. the first class comprises minisci - type reactions, which occur by the addition of carbon - centered radicals to heteroarenes (scheme 1a). these reactions are versatile, but mixtures of isomers are formed in most cases, and the regioselectivity depends on the steric and electronic properties of both the heteroarene and the radical partner. a second class of heteroarene c h bond functionalization reactions includes additions of nucleophiles to pyridine n - oxides in combination with a reagent to activate and dehydrate the pyridine n - oxide (scheme 1b). reactions of this type have been developed to install br, cl, cn, amino, phenoxy, and nucleophiles derived from reagents containing acidic c h and n h bonds (pka range of 1020). in most cases, the scope of the pyridine n - oxides that undergo these reactions is limited to those containing ether, ester, and nitrile substituents or halides at positions not reactive toward snar. in general, functionalization occurs at the 2-position, but competing functionalization at the 4-position or on pendant alkyl groups is often observed. in addition to these issues of functional group compatibility and site selectivity, the need to prepare and isolate the n - oxides limits the use of these reactions. most closely related to the work reported here, c h functionalization reactions at the 2-position of pyridines and pyridine n - oxides have been developed with transition metal catalysts. however, these reactions are limited to the formation of c c bonds, and the reaction conditions and scope suggest that these reactions are not suitable for complex substrates. other functionalizations of heteroaryl c h bonds involve cross - coupling at the most acidic c h bond ; these methods occur most commonly with five - membered ring heteroarenes. finally, c h borylation reactions catalyzed by transition metal complexes provide a means to install substituents onto heteroarenes by conversion of the heteroarene to the corresponding heteroarylboronate ester and subsequent transformations of the c b bond. however, borylations of pyridines occur predominantly at the 3- and 4-positions and, therefore, are complementary to the reactions at the 2-position described here. we aimed to develop a method for the late - stage functionalization of pyridines and diazines that would address the limitations of the regioselectivity and scope of c h functionalizations of heteroarenes. our approach was inspired by the value of borylation reactions developed in the authors laboratory to create synthetic intermediates that can be converted to a variety of functionalized products. h fluorination of pyridines and diazines at the position to nitrogen with agf2 we developed recently could be used for the late - stage functionalization of medicinally relevant compounds because pyridines and diazines are contained in many such compounds and the 2-fluoro group could be replaced with a wide range of nucleophiles. herein, we report mild conditions for the snar reaction of fluoroheteroarenes, an assessment of the potential of the fluorination and snar reactions to be conducted with complex structures, and the application of these findings to the development of late - stage functionalizations of complex heterocyclic compounds by the combination of c h fluorination and snar reactions (scheme 1c). in addition to revealing the potential of this reaction for the functionalization of complex heteroarenes, we demonstrate how the combination of ch bond fluorination and snar creates routes to several active pharmaceutical ingredients that occur in higher yields and fewer steps than previously reported syntheses of these molecules. snar reactions of 2- or 4-halopyridines comprise a site - specific method to synthesize substituted pyridines. however, this approach requires initial synthesis and isolation of halogenated pyridines that are typically prepared from pyridine n - oxides or hydroxypyridines with neat pox3 (x = br, cl) at high temperatures. chloropyridines are more available commercially than other halopyridines, but the reactions of fluoropyridines are likely to be faster than those of chloropyridines. as for snar reactions of arenes, the snar reactions of pyridines and diazines are likely to be accelerated by the high electronegativity of fluorine. indeed, the reaction of 2-fluoropyridine with naoet in etoh is 320 times faster than the reaction of 2-chloropyridine. this higher reactivity of fluoropyridines could allow snar reactions to occur under conditions that are mild enough to allow this class of reaction to occur on complex molecules ; however, a more detailed assessment of the rates and yields for the snar reactions of fluoropyridines and diazines would be needed to predict the scope of the snar process and a method to create the 2-fluoropyridines and diazines that could be conducted in a typical laboratory on complex pyridines and diazines would be required. h bond fluorination and snar, we first evaluated conditions to conduct snar reactions of 2-fluoropyridines. although snar reactions of electron - deficient fluoroarenes and chloropyridines are commonplace, few studies have been performed on snar reactions of fluoropyridines. the majority of the published reactions of 2-fluoropyridines have been conducted with unsubstituted 2-fluoropyridine ; a few examples were conducted with 2-fluoropyridines containing a single bromide substituent. moreover, these reactions were performed under conditions involving strong nucleophiles and bases at high temperatures (up to 130 c), neat reagents, microwave heating, strong reducing agents (libh3nr2), or toxic solvents (hmpa), and these conditions are unlikely to tolerate the functional groups found in complex molecules relevant to medicinal chemistry. several snar reactions of 2-fluoropyridines have been reported in the patent literature on both simple and complex substrates. however, the reactions described in these documents for a given class of nucleophile occur under varying conditions. therefore, the scope of each set of reaction conditions is unclear, and the snar reactions were performed with activated 2-fluoropyridines in most cases. from this body of work, it was unclear whether mild and general snar reactions with fluoroheteroarenes could be developed and if selectivity could be obtained for substitution of fluoride over other halides. in fact, previous reports showed that the same forcing reaction conditions lead to substitution of f, cl, br, and i. to identify conditions for the snar reaction that would tolerate common functionality, we studied reactions of unactivated 2-fluoropyridines with nucleophiles derived from alcohols, phenols, amines, amides, n - heterocycles, cyanide, and thiols under relatively mild conditions. the reaction conditions we identified afforded quantitative conversion to the substitution products, as indicated by gc / ms and tlc (table 1). variation of the cyanide source, stoichiometry, temperature, and solvent did not increase the yield, but the initial conditions did form the cyanopyridine product in a synthetically useful yield. having developed a set of snar reactions that occur under mild conditions, we explored the tandem fluorination substitution process. for the snar reactions to occur in tandem with c h fluorination, the mecn and silver salts from the fluorination reaction needed to be removed. filtering the fluorination reactions through a short silica - filled pipet and evaporating the solvent was sufficient to perform the subsequent snar reactions. yields of the snar reactions conducted after filtering the fluorination reaction through celite were low, due to the presence of soluble ag salts. for the fluorination snar sequence to be used broadly, procedures for conducting the reaction without specialized equipment for excluding air and moisture are needed. therefore, during our initial study, the fluorination reactions were assembled in a glovebox with rigorously dried mecn. however, despite the water sensitivity of agf2, simple procedures can be followed for conducting the reactions without rigorous exclusion of air or moisture. to assess the impact of water and oxygen on the yield of the fluorination reaction of complex molecules, we performed a series of experiments with (co2me)-vismodegib, a drug recently approved for the treatment of basal - cell carcinoma (table 2). assembling the fluorination reaction in a glovebox (oxygen and water content < 1 ppm), in an oven - dried vial, with mecn that had been rigorously dried over cah2, afforded the fluorinated vismodegib derivative in 99% yield by f nmr spectroscopy. reactions were performed with 0.2 mmol of (co2me)-vismodegib with 2 ml of mecn in 4 ml vials. since the use of a glovebox is not practical for all chemists, we studied the effects of weighing the solid reagents in air and assembling the reaction using standard air - free techniques. a reaction was assembled by adding the pyridine substrate to a dry vial in air and adding mecn that had been dried over cah2. agf2 was weighed quickly in air, added to the pyridine solution, and the vial was sealed under an atmosphere of n2. the reaction assembled in this manner afforded the 2-fluoropyridine product in 84% yield, only a slight decrease in yield compared to the reaction assembled entirely in the glovebox. performing the reaction in a similar manner with a nondried vial and sealing the reaction under an atmosphere of air (2 ml of mecn in a 4 ml vial ; 2 ml headspace of air) resulted in a similar yield of the fluoropyridine product (79%). acetonitrile dried with 5 wt % of 3 molecular sieves for 24 h was a suitable solvent ; the reaction of (co2me)-vismodegib with agf2 assembled by weighing the solid reagents in air in a nondried vial sealed under an atmosphere of air afforded the fluorinated product in 85% yield. the water content in mecn dried over 5 wt % molecular sieves for 24 h is near 4 ppm, and the water content further decreases with time. mecn is below 10 ppm water and should be equally suitable for this reaction. finally, the same reaction was assembled in air with acs grade mecn directly from a commercial bottle that had been opened and used over the course of a year ; a noticeable decrease in yield to 65% was observed, but a substantial amount of product was still formed. together, these results demonstrate that these fluorination reactions can be conveniently assembled completely in air, without the use of a glovebox or air - free techniques, and with mecn dried over molecular sieves, even though agf2 is sensitive to water and should be stored under an inert atmosphere. reactions performed on the benchtop occur in yields that are comparable to those performed under rigorously anhydrous reaction conditions. agf2 is supplied as a black, microcrystalline solid, which should be stored under an inert atmosphere. because agf2 undergoes decomposition with moisture, a noticeable color change from black to yellow or brown is observed when it is stored in air. for all reactions reported here, and in our previous paper, agf2 was used as received from alfa aesar and stored under a nitrogen atmosphere in a plastic bottle. in our experience, reactions with agf2 supplied from strem occurred in comparable rates and yields to those performed with agf2 puchased from alfa aesar. having identified convenient methods for conducting both the fluorination and snar reactions and having developed a protocol to conduct the two reactions in sequence, we investigated the scope of the ch bond fluorination representative examples illustrating the scope of the combined reactions are shown in tables 3 and 4. yields given are for isolated products starting from the heteroarene. the yields for the fluorination step are also shown to illustrate how the values for the two - step process compare to those of the first step. isolated yields from the two - step sequence for reactions performed with 0.2 mmol of heteroarene. yields in parentheses were determined by f nmr spectroscopy for the independent fluorination reaction. isolated yields from the two - step sequence for reactions performed with 0.2 mmol of heteroarene. yields in parentheses were determined by f nmr spectroscopy for the independent fluorination reaction. the isolated product contained 5% of an inseparable impurity. a variety of pyridines that are sterically hindered (2, 4, 11, 12, 14, 15) and/or electronically deactivated (2, 9, 13, 15) toward snar reactions afforded the substitution products in good yields. substrates containing alkyl groups in the 2-position reacted selectively at the 6-position (2, 4, 9, 14), while analogous reactions with pyridine n - oxides are known to result in substitution of a c h bond on the alkyl group (scheme 1b). a wide range of functional groups were tolerated, including ethers, halides, ketones, acetals, esters, amides, ethyl and t - butyl carbamates, nitriles, and sulfones. it is notable that the azetidine in 11 (table 4) did not undergo ring opening, a competing reaction observed under acidic conditions. the reactions of chloropyridines 7 and 11 revealed a high selectivity for substitution of a fluoride over a chloride under conditions of the snar reactions shown in table 1. this high selectivity, along with the high functional group compatibility, is attributed to the milder reaction conditions we developed for the snar reaction at the 2-fluoro position, relative to the conditions typically used to conduct substitutions with 2-fluoropyridines. in sum, this work shows that fluoropyridines undergo substitution reactions under conditions much milder than previously reported and can be performed in the presence of a wide range of functional groups, including those that are electrophilic. six - membered heteroarenes containing two nitrogen atoms (diazines) are prevalent subunits in medicinal chemistry. radical addition reactions to diazines are commonplace (scheme 1a), but a single example of ch bond functionalization by nucleophilic addition of a heteroatom to a diazine n - oxide (scheme 1b) has been demonstrated. like pyridines, diazines react with agf2 with exclusive selectivity for fluorination adjacent to nitrogen. h bond fluorination and snar reactions could be conducted with these heterocycles to form functionalized diazine products. indeed, pyrimidines (5, 6) and pyrazines (8) reacted in the two - step sequence following the standard conditions we developed for the fluorination and snar reactions reported in table 1. because the conditions for both the fluorination and the snar reactions with diazines are the same as those for pyridines, the tolerance of the reactions toward functional groups on pyridines and diazines should be comparable. this c h fluorination snar sequence was also applied to the synthesis of a reverse transcriptase inhibitor containing a tetra - substituted pyrimidine (vide infra). with conditions established for the fluorination and snar reactions of pyridines and diazines, we evaluated this sequence for the late - stage functionalization of more complex molecules in medicinal chemistry. first, we used our tandem sequence to prepare several 2-substituted derivatives of (boc - protected) betahistine (9), a histamine agonist used in the treatment of mnire s disease. reaction of 9 with agf2 formed the corresponding 2-fluoropyridine in nearly quantitative yield (98%). this electronically deactivated fluoropyridine intermediate reacted with nucleophiles derived from butanol, morpholine, and indole to provide several 2-substituted analogues of betahistine. although betahistine is a relatively simple compound that can be prepared in one step from 2-vinylpyridine, the synthesis of derivatives that are similar to those we report here would require 2-substituted 6-vinylpyridines. thus, our ch bond fluorinationsnar strategy for late - stage functionalization avoids lengthy synthetic sequences to prepare derivatives of betahistine. we also conducted the fluorination of compound 10, the direct precursor to loratadine (claritin) and desloratadine (clarinex), two common antihistamines. compound 10 is prepared in two steps from 3-methylpicolinic acid under relatively harsh reaction conditions (buli, kobu for the first step ; socl2, alcl3 for the second step). therefore, the synthesis of 2-substituted derivatives of 10 would require access to the appropriately substituted 3-methylpicolinic acid, which could require several steps to prepare, and an additional two - step sequence to construct the tricycle for each derivative. we prepared various analogues of 10 more directly through fluorination and snar reactions to form the corresponding 2-alkoxy-, 2-amino-, and 2-pyrazolyl - substituted derivatives. it is worthy to note that the substituents we installed in 10a10c would be unlikely to tolerate the conditions of a de novo synthesis of similar analogues of 10. our c h functionalization method also gave access to a series of (boc - protected) derivatives of tebanicline (11), a potent non - opiod analgesic that is structurally related to several nicotinic acetylcholine receptor agonists. as mentioned above, no ring opening of the azetidine or substitution of the chloride was observed. de novo syntheses of compounds similar to 11a11c would require access to 2-substituted 3-hydroxy-6-chloropyridine substrates and an additional c o bond - forming reaction to complete the synthesis of each derivative. the sequence of c h bond fluorination and snar also led to a convenient synthesis of 2-alkoxy and 2-amino analogues of roflumilast (12). roflumilast is a recently approved pde-4 inhibitor used in the treatment of chronic obstructive pulmonary disease. thus, the syntheses of the 2-substituted analogues we report would require access to 2-alkoxy or 2-amino-3,5-dichloro-4-aminopyridine, for which none are commercially available. therefore, preparing derivatives of roflumilast would mandate multistep syntheses of the appropriate pyridine, in addition to performing the subsequent amide bond formation for each derivative. in contrast, the ch fluorinationsnar strategy we report allows rapid access to analogues that would otherwise require several synthetic steps to prepare. in a similar manner, analogues of the precursor to pirenzepine (13), a benzodiazepine - based m1-selective antagonist used for the treatment of ulcers, were prepared. the sequence was used to install alkoxy, thio, and aryloxy substituents at the 2-position in good overall yields. the synthesis of the core of 13 requires three steps from 2-chloro-3-aminopyridine. because many medicinally important compounds contain multiple heteroaryl rings, it would be valuable if the fluorination was selective for the functionalization of one type of ring system over another. the proposed mechanism for the fluorination of pyridines and diazines with agf2 (eq 1) is initiated by coordination of the basic nitrogen to silver. this coordination could cause a more basic heterocycle to be more reactive than a less basic heterocycle. however, the second step in the proposed mechanism is addition of fluoride to the system, which would be favored for a more electron - deficient heteroarene. finally, the third step, a formal oxidation of the heterocycle through hydrogen atom abstraction, would likely be favored for a more electron - rich substrate. our previous studies of the selectivity between pyridine and pyridine - d5 demonstrated that coordination of agf2 to pyridine is reversible and that cleavage of the c h bond is irreversible.1 to determine how the electronic properties of the heteroarene influence the relative rates of fluorination, we conducted a series of competition experiments. in these experiments, agf2 was allowed to react with a 1:1 mixture of two different pyridines and diazines. because the yield of the fluorination reactions conducted with a large excess of pyridine, relative to agf2, is low, reactions containing 1 equiv of each heteroarene (0.1 mmol each) and 2 equiv of agf2 (0.2 mmol) were run, and the reactions were quenched after 15 min so that the selectivities were being measured at low conversion (25 2%). competition experiments between 2-ethyl, 2-methoxy, and 2-chloropyridine were conducted ; the steric properties of these substrates are similar to each other, but the basicity of the heterocycles differ incrementally from each other by 2.6 pka units. competition experiments were also conducted between alkyl - substituted pyridines, pyrimidines, and pyrazines containing two available c these data show that more lewis basic pyridines undergo the c h fluorination reactions in preference to less lewis basic pyridines. moreover, exclusive selectivity for fluorination of a 4-alkylpyridine over two alkyl - substituted diazines was observed ; the competition between 2-methylpyrimidine and 2,3-dimethylpyrazine showed that the pyrimidine was the more reactive diazine by a factor of 3.3. product ratios were determined by f nmr spectroscopy of the crude reaction mixture after quenching with aqueous nahco3. the results of these competition experiments contrast with what would be predicted based only on the relative rates of independent reactions between each substrate and agf2. 2-ethylpyridine reacts with 2 equiv of agf2 to give 38% yield of the 6-fluoropyridine after 15 min. similarly, 2-methoxypyridine reacts in 36% yield, and 2-chloropyridine reacts in 9% yield after 15 min. because the rates for fluorination of 2-ethylpyridine and 2-methoxypyridine are comparable, and because agf2 is present in excess, little selectivity for 2-ethylpyridine over 2-methoxypyridine would be expected based on these data alone. however, agf2 has negligible solubility in mecn ; therefore, competitive binding of the two substrates to the limited amount of available agf2 likely results in fluorination of the more basic pyridine.2 to assess the relative reactivity of multiple pyridines in the context of a medicinally important compound, we conducted the fluorination and snar reaction of etoricoxib (eq 2), a selective cox-2 inhibitor used in the treatment of arthritis. the more electron - rich ring contains methyl and 2-pyridyl substituents, and the less electron - rich ring contains chloro, aryl, and 3-pyridyl substituents. this molecule reacted with agf2 with complete selectivity for fluorination of the more basic pyridine system, as predicted from the results in table 5. following this site - selective fluorination, several derivatives of etoricoxib containing pendant alkoxy, amino, cyano, and pyrazolyl units were prepared. many medicinally active pyridines contain two inequivalent c h bonds that could undergo fluorination with agf2. we previously demonstrated that several 3-substituted pyridines undergo fluorination with exclusive selectivity to form the 2-fluoro-3-substituted pyridine product. the 3-substituted pyridines that react selectively at the 2-position include those containing 3-halo, alkoxy, cyano, or cf3 groups. 3-substituted pyridines that give a mixture of 2-fluoropyridine isomers include those containing 3-alkyl, 3-co2r, and 3-c(o)nr2 substituents. it was unclear from these results if 3,5-disubstituted pyridines would undergo the fluorination selectively. a set of 3,5-disubstituted pyridines containing phenyl, cyano, benzyloxy, bromo, methyl, and cf3 substituents was prepared. the fluorination of the 15 unsymmetrical pyridines containing these substituents occurred with poor site selectivity (from 1:1 up to 6:1), with the exception of the benzyloxy - substituted pyridines. the 3-benzyloxy - substituted pyridines containing various substituents in the 5-position reacted with agf2 with modest to high selectivity (4.2:1 to 20:1) for fluorination adjacent to the ether substituent (table 6). having shown that an alkoxy group can lead to selective fluorination of a 3,5-disubstituted pyridine, we exploited this selectivity to conduct the late - stage fluorination of a medicinally relevant, 3,5-disubstituted pyridine. the core of crizotinib (eq 3), a drug used for the treatment of metastatic non - small cell lung cancer, contains such a heteroaromatic unit. reaction of 15 with agf2 gave products reflecting a 7.2:1 selectivity for fluorination adjacent to oxygen. this selectivity is lower than that observed for the fluorination of 3-bromo-5-benzyloxypyridine (table 6), likely due to the steric hindrance of the arylethoxy substituent. to determine if the lower selectivity of this substrate is due to the greater steric hindrance of the benzyloxy group of 15 than that of a simple benzyloxy substrate, the fluorination reaction was also performed with a 2,6-dimethylphenyl arylethoxy substrate. this compound reacted in 62% yield with similar 5.9:1 selectivity for fluorination adjacent to the ether substituent. thus, the selectivity is lower for reactions of pyridines containing more hindered benzyloxy groups but is still significant. the steric hindrance of the arylethoxy substrates disfavors both the second and third steps in our proposed mechanism (eq 1) 3leading to an increase in the relative amount of product from fluorination adjacent to the bromide. even though the 2-fluoro-3-benzyloxypydridine intermediate is both sterically and electronically deactivated toward snar, several 2-substituted derivatives were prepared under the standard conditions for the snar step shown in table 1. even a secondary amine reacted with the 2-fluoropyridine to form the sterically congested product 15c. snar reactions were not separable by standard silica gel chromatography, even though the 2-fluoropyridine isomers could be separated by hplc and gc. although we have demonstrated that the c h fluorination and snar reactions occur with broad scope and can be conducted on complex molecules, there are some limitations. as we reported previously, the fluorination reaction is not compatible with free amines or alcohols, carboxylic acids, aldehydes, or electron - rich five - membered heterocycles ; however, several protected derivatives of these groups are tolerated by agf2. in addition, we have found that pyridines or diazines containing multiple electron - withdrawing substituents undergo the fluorination reaction in lower yields than those containing electron - neutral or electron - donating groups. examples of substrates that reacted with agf2 in low yields (030%) are shown in table 7. although the boc - protected derivative of hg-10 - 102 - 01 reacted with agf2 in low yield, a similar tetra - substituted pyrimidine reacted in high yield for the synthesis of etravirine (vide infra). we have demonstrated that a simple set of snar reaction conditions can be employed for substitution reactions on a variety of 2-fluoroheteroarenes. however, we did find substrates that underwent competing side reactions faster than they underwent snar (table 7). fluorinated analogues of tropicamide underwent retro - michael addition under the basic reaction conditions. although we demonstrated that tert - butyl esters remain intact during the snar reactions (table 3, substrate 3), we found that aryl acetates are cleaved faster than substitution of fluoride. finally, very electron - rich and sterically hindered pyridines, such as the precursor to nexium, underwent the snar step in low yields ; more forcing conditions resulted in the formation of side products. snar sequence for the late - stage derivatization of medicinally important compounds, we sought to evaluate whether the same strategy could create shorter and higher - yielding synthetic routes to the same types of compounds. although this chemistry requires stoichiometric silver and is not designed for process - scale chemistry, the strategy of c h fluorination and snar can be useful in discovery chemistry. one example of a synthesis that could be simplified by the fluorination and substitution process is the synthesis of the simple compound 6-(methylamino)-2-pyridineethanol (scheme 2, 16). compound 16 is a precursor to several important compounds, including the integrin inhibitor sb-273005 (scheme 2c). although 16 is structurally simple, the two reported syntheses of 16 by medicinal chemists were conducted in five and seven steps from 2-amino-6-methylpyridine (scheme 2a). we considered that the 2-methylamino group could be installed through c h bond fluorination and snar from a derivative of 2-pyridineethanol. fluorination did not occur on the free alcohol, but it did occur after protecting the alcohol as an ester. an acetyl protecting group was chosen because it could be cleaved in concert with the snar reaction. the fluorination of acetylated 2-pyridineethanol occurred in 88% yield (by f nmr spectroscopy). treatment of the crude mixture from the fluorination step with aqueous menh2 in dmso led to formation of the methylaminopyridine unit and cleaveage of the acetyl group to reveal the free alcohol. this route gave 16 with only two isolations in 64% overall yield, a significantly shorter and higher - yielding approach than the reported syntheses requiring five or seven steps. although our route requires stoichiometric silver, it is worthy to note that an improved process used to prepare 30 g of 16 still required four steps and occurred in only 39% overall yield. the combination of c h bond fluorination and snar also shortened the synthesis of pf-1247324, a potent and selective nav1.8 inhibitor (scheme 3). the medicinal chemistry route to this compound comprised six steps and occurred in less than 1% yield ; an advanced intermediate was outsourced for the kilogram - scale synthesis of pf-1247324. the fluoropyridine intermediate contains two electrophilic sites for nucleophilic substitution : a methyl ester and a 2-fluoropyridine. we demonstrated (tables 3 and 4) that substitution at the fluorine of a 2-fluoropyridine can occur in the presence of auxiliary electrophilic functional groups, including a tert - butyl ester. however, we considered that conditions could be identified to transform the methyl ester to the n - methyl amide without substitution of the fluoride. the reaction between the fluoropyridine methyl ester with aqueous menh2 in thf formed a mixture of products resulting from substitution of both methoxide and fluoride. however, simply changing the reaction solvent from thf to meoh allowed for the selective transformation of the methyl ester to the n - methyl amide without substitution of the fluoride. after aqueous workup, the crude reaction mixture was treated with ammonium hydroxide in dmso to substitute an nh2 group for the fluoride. finally, the 3-bromopyridine was subjected to the reported suzuki cross - coupling reaction conditions to provide the title compound. by our route, the synthesis of pf-1247324 involving c h fluorination was completed with just three total isolations in 51% overall yield, a major improvement in yield and step count over the published synthesis. furthermore, the route performed by medicinal chemists required 120 h of total reaction time, while the route we report here required less than 18 h. finally, we used our chemistry to prepare the non - nucleoside reverse transcriptase inhibitor intelence (etravirine, scheme 4), a compound used in the treatment of hiv. the route to this compound developed by medicinal chemists occurred in five steps and 9% yield from n-(4-cyanophenyl)guanidine hydrochloride. an alternative route used to prepare etravirine on kilogram scale required four steps and occurred in 30% yield. our synthesis of etravirine began with substitution of the 4-chloro substituent in 2,4-dichloro-5-bromopyrimidine with 2,6-dimethyl-4-cyanophenol, from which the solid product was isolated after the addition of water. next, substitution with 4-aminobenzontirile and boc protection in situ was conducted, and the product was isolated in 88% yield. the installation of a protecting group for the n the synthesis of etravirine was then completed by c h fluorination of the pyrimidine (71% yield by f nmr spectroscopy), substitution with aqueous ammonia, and addition of hcl(aq) to cleave the boc group. through this route, etravirine was prepared with three total isolations in 45% overall yield in under 6 h of total reaction time. in summary, we have developed a broadly applicable strategy for the diverse, site - selective c h functionalization of pyridines and diazines. the reaction sequence occurs to provide alkoxy-, aryloxy-, amino-, amido-, heteroaryl-, thio-, and cyano - substituted heterocycles that can be difficult to access through traditional methods. this tandem sequence is attractive for the direct diversification of heteroarenes, due to the exquisite site selectivity for c in addition, high site selectivity for the fluorination of substrates containing more than one heteroarene or more than one reactive c h bond is possible using this chemistry. finally, the process of fluorination and snar can allow medicinal compounds containing substituted pyridines and diazines to be prepared by short synthetic routes. we anticipate that these reactions will find immediate use for both late - stage functionalization and efficient syntheses of complex molecules. | we report the late - stage functionalization of multisubstituted pyridines and diazines at the position to nitrogen. by this process, a series of functional groups and substituents bound to the ring through nitrogen, oxygen, sulfur, or carbon are installed. this functionalization is accomplished by a combination of fluorination and nucleophilic aromatic substitution of the installed fluoride. a diverse array of functionalities can be installed because of the mild reaction conditions revealed for nucleophilic aromatic substitutions (snar) of the 2-fluoroheteroarenes. an evaluation of the rates for substitution versus the rates for competitive processes provides a framework for planning this functionalization sequence. this process is illustrated by the modification of a series of medicinally important compounds, as well as the increase in efficiency of synthesis of several existing pharmaceuticals. |
generalized lipodystrophy (gl) is categorized as a rare congenital generalized lipodystrophy (cgl) or acquired generalized lipodystrophy (agl) disease characterized by widespread loss of adipocytes, leading to leptin deficiency, ectopic fat deposition, and severe metabolic abnormalities (insulin resistance, diabetes, and/or hypertriglyceridemia).1,2 in women with insulin resistance and hypoleptinemia, polycystic ovarian syndrome, hyperandrogenism, and amenorrhea are very common.3 conventional glucose- and lipid - lowering medications do not address the underlying leptin deficiency and are often inadequate at addressing severe metabolic abnormalities.1 metreleptin is a recombinant human leptin analog that binds to and activates the leptin receptor, and it is the only us food and drug administration - approved treatment for patients with cgl or agl as an adjunct to diet and as replacement therapy to treat complications of leptin deficiency.4 metreleptin is not currently indicated for the treatment of partial lipodystrophy, liver disease, human immunodeficiency virus - related lipodystrophy, or diabetes and hypertriglyceridemia without evidence of concurrent cgl or agl. the starting daily dose of metreleptin varies by weight and sex (table 1), and daily dosing may be increased or decreased according to clinical response (changes in glycated hemoglobin [a1c ], triglycerides, etc.). per the us prescribing label, metreleptin should be administered once daily (qd) at the same time every day. in clinical trial experience, the most common adverse reactions (10% incidence) included headache, hypoglycemia, decreased weight, and abdominal pain. there is limited experience using metreleptin to treat patients with gl in the clinic setting. herein, we present the metreleptin treatment experience in three patients with gl inadequately managed with conventional medications. patient 1 was a 19-year - old female of arabic descent and consanguineous parentage diagnosed with cgl at early infancy (serum triglycerides > 2000 mg / dl at two months) table 2. prior to metreleptin, she presented with generalized subcutaneous fat loss, muscular appearance, acanthosis nigricans, elevated alanine aminotransferase (alt ; 47 u / l), hepatomegaly (confirmed later by magnetic resonance imaging scan : moderate hepatomegaly with fatty infiltration), irregular menstrual cycles (three to four per year), increased abdominal girth, and elevated a1c and triglycerides (11.8% and 3675 mg / dl, respectively ; table 1). these metabolic abnormalities were noted despite ongoing therapy with metformin 500 mg three times daily (tid), pioglitazone 30 mg qd, insulin detemir 40 u twice daily (bid), insulin aspart 40 u tid, fenofibrate 145 mg qd, niacin 500 mg qd, omega-3 fatty acids 2 g tid, and orlistat 120 mg tid for four years. metreleptin was initiated at 5 mg qd subcutaneously by self - injection. a1c and triglycerides were markedly improved at four weeks from 11.8% to 9.7% and 3675 to 2118 mg / dl, respectively (fig. 1). at 12 weeks, while maintained at the same metreleptin dose, a1c and triglycerides dropped further to normal levels (5.7% and 121 mg / dl, respectively), and slightly elevated alt at baseline was within normal limits (23 u / l). the normalization of laboratory values occurred while the patient discontinued insulin aspart, pioglitazone, niacin, and fenofibrate (insulin detemir was decreased from 40 to 30 u bid, and the dosing frequency of omega-3 fatty acids was reduced to qd). weight remained stable, hepatomegaly and abdominal girth were reduced, and the patient reported resumption of regular menstrual cycles for the last two months. patient 2 was a 10-year - old female with cgl diagnosed from birth with a history of pancreatitis (four episodes requiring hospitalization), lytic bone lesions, and mesangial proliferative glomerulonephritis (table 2). prior to metreleptin treatment, she presented with generalized subcutaneous fat loss, acanthosis nigricans, elevated liver enzymes (alt : 132 u / l ; aspartate aminotransferase [ast ] : 80 u / l), prominent abdomen due to hepatosplenomegaly (abdominal ultrasound demonstrated enlarged liver, homogeneous parenchyma, and decreased echogenicity), decreased energy, proteinuria (2.5 g/24 hours), and elevated a1c and triglycerides (9.2% and 497 mg / dl, respectively ; table 1). her metabolic abnormalities were managed with metformin 500 mg / day, regular u100 insulin 1000 u / day, fenofibrate 200 mg / day, and enalapril 20 mg / day for 21 months. metreleptin was initiated at 4.2 mg / day while the patient was enrolled in research protocol 02-dk-0022 at the national institutes of health. at 24 weeks, a1c and triglycerides were reduced from 9.2% to 8.0% and 497 to 302 mg / dl, respectively (fig. 1), and the patient s proteinuria was resolved (0 g/24 hours). as a1c was still elevated, the patient s metreleptin and metformin doses were increased in a stepwise manner over consecutive visits to 10 mg / day and 2000 mg / day, respectively. at week 168, the patient s metabolic parameters had improved (a1c : 6.6% ; triglycerides : 84 mg / dl), and her total daily insulin requirement was reduced from 1000 to 75 u / day. alt and ast were within normal limits (20 and 18 u / l, respectively). the patient s weight increased by 10%, and she self - reported improved energy levels, a flat abdomen, and that she completed her pubertal development with her first menses. patient 3 was a 19-year - old male with agl manifesting between eight and nine years of age with panniculitis in the abdomen, legs, and face, followed later by generalized loss of subcutaneous fat (table 2). he presented with acanthosis nigricans, prominent abdomen, acromegaloid features, hyperphagia, asthenia, elevated liver enzymes (alt : 171 u / l ; ast : 81 u / l), hyperinsulinemia (fasting : 228 u / ml), and elevated a1c and triglycerides (11.0% and 861 mg / dl, respectively). abdominal ultrasound demonstrated an enlarged spleen and enlarged liver with smooth contour, hyperechoic texture, and no focal hepatic lesions. metformin 500 mg / day was initiated and titrated later to 2000 mg / day. high insulin levels persisted (< 300 u / ml) in parallel with worsening acanthosis, and he was started on regular u500 insulin 120 u / day (equivalent of regular u100 insulin 600 u / day). the patient was on this treatment regimen for seven years, and his last a1c measurement before metreleptin treatment was 11.8%. he was also managed with fenofibrate 200 mg / day, vitamin k 5 mg / month (due to vitamin k deficiency related to steatohepatitis), ranitidine 150 mg / day, ursodiol 300 mg / day, and enalapril 5 mg / day. a1c was further reduced to 5.1% and triglycerides were reduced to 36 mg / dl. at 96 weeks, his a1c increased to 5.8%. it was noted that the patient gained 6 kg of weight during this time, so the metreleptin dose was increased to 4 mg / day. as of the last measurement (week 140), a1c was 5.1% and triglycerides were 80 mg / dl. during treatment with metreleptin, the patient was able to discontinue the use of insulin and fenofibrate, remaining only on ursodiol 300 mg / day and enalapril 10 mg / day. alt levels were reduced from 171 to 120 u / l, while ast remained stable (from 81 to 79 follow - up hepatic ultrasonography indicated increased spleen size with homogeneous structure and liver with rounded edges, enlarged, homogeneous - increased echogenicity compatible with steatosis, no focal lesions, and dilated intra- and extrahepatic bile ducts. the patient s weight increased from 61 to 67 kg with improvements in lean muscle mass observed. his appetite normalized allowing him to adhere to the recommended low - glucose diet for diabetes control, and his acanthosis nigricans was noted to have improved while on metreleptin. no aes were noted, and the patient continues to remain on the same daily dose of metreleptin (4 mg / day). all three patients with gl had significant metabolic abnormalities that were inadequately addressed with conventional glucose- and lipid - lowering therapies. metreleptin was effective at normalizing very elevated a1c and triglycerides, with this response occurring as early as four to six weeks in patients 1 and 3. metreleptin dose was individualized to match changes in a1c and triglycerides, and concomitant medications were adjusted to avoid hypoglycemia. in parallel with improved metabolic parameters, all patients were able to either discontinue or decrease their daily doses of insulin an important point as patients with lipodystrophy sometimes require very high doses of insulin to maintain glycemic control. high pill burden in conditions requiring long - term treatment has been associated with decreased adherence and quality of life (qol).5 the success of metreleptin therapy is dependent on good adherence to metreleptin and concomitant medications besides dietary modifications and other lifestyle changes.6 patients with lipodystrophy who are nonadherent to metreleptin and/or concomitant medications often experience relapses to previous metabolic derangements.68 metreleptin does not cure the underlying disorder of lipodystrophy but addresses the associated leptin deficiency ; thus, most patients may require lifetime therapy. although difficult to quantify, patients with lipodystrophy may have decreased qol9 owing to the metabolic abnormalities that they experience, particularly hyperphagia. compliance with diet may be difficult because of hunger related to the leptin deficiency, implying that hypothalamic satiety is also inhibited. during metreleptin treatment, patients 1 and 3 reported marked reductions in hunger, and patients 2 and 3 reported improved energy levels. while similar subjective findings have been reported in at least one other cohort of patients with gl,10 hyperphagia response to metreleptin has not been quantified in any previous clinical trials to date. cosmetic disfigurement due to loss of adipose tissue and leptin deficiency can be very discouraging. in this case series, patients 1 and 2 reported a less prominent abdomen or decreased girth during metreleptin treatment. the same patients also reported more regular menstrual cycles (patient 1) or the achievement of first menses (patient 2). in addition to improved a1c and triglycerides, elevations of liver enzyme were numerically improved or normalized in all patients, with decreased size of hepatomegaly observed in patient 1. at least one study group has described significant improvements in liver pathology from baseline with metreleptin treatment11,12 ; however, improved liver enzymes do not always correlate with improved liver - related end points.13 patient 2 had proteinuria at baseline that resolved after metreleptin treatment. metreleptin has been shown in a prior cohort of patients with gl to reduce urinary protein excretion in 11 of 15 patients treated for four months.14 in the patients with gl presented here, metreleptin was effective at improving certain metabolic abnormalities in the clinic setting with no notable aes. subjective qol improvements (increased energy, normalization of appetite / menstrual function, improved appearance, etc.) are equally important to the patient treatment experience, as are improvements in laboratory parameters. | generalized lipodystrophy (gl) is a rare inherited or acquired disease characterized by widespread loss of subcutaneous fat, leading to leptin deficiency, ectopic fat deposition, and severe metabolic abnormalities. previous studies have shown the benefit of leptin replacement (metreleptin) in ameliorating metabolic complications, but little is known about the experience of metreleptin treatment outside of a research setting. we report on post - marketing clinical experience with metreleptin therapy in three patients with gl and marked hypoleptinemia, uncontrolled diabetes, and hypertriglyceridemia. after metreleptin treatment for 12168 weeks, the mean glycated hemoglobin decreased from 10.9% to 5.8%, and serum triglycerides were normalized (a mean decline of 90%). these benefits were observed within weeks of starting therapy, were durable, and were accompanied by subjective improvements in quality of life, decreased need for concomitant medications, and no significant adverse effects. metreleptin was safe and effective in normalizing certain severe metabolic abnormalities in the clinic setting. |
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