Cellular retinol-binding protein type III is needed for retinoid incorporation into milk.
The physiologic role(s) of cellular retinol-binding protein (CRBP)-III, an intracellular retinol-binding protein that is expressed solely in heart, muscle, adipose, and mammary tissue, remains to be elucidated. To address this, we have generated and characterized CRBP-III-deficient (CRBP-III(-/-)) mice. Mice that lack CRBP-III were viable and healthy but displayed a marked impairment in retinoid incorporation into milk. Milk obtained from CRBP-III(-/-) dams contains significantly less retinyl ester, especially retinyl palmitate, than milk obtained from wild type dams. We demonstrated that retinol bound to CRBP-III is an excellent substrate for lecithin-retinol acyltransferase, the enzyme responsible for catalyzing retinyl ester formation from retinol. Our data indicated that the diminished milk retinyl ester levels arise from impaired utilization of retinol by lecithin-retinol acyltransferase in CRBP-III(-/-) mice. Interestingly, CRBP-I and CRBP-III each appeared to compensate for the absence of the other, specifically in mammary tissue, adipose tissue, muscle, and heart. For CRBP-III(-/-) mice, CRBP-I protein levels were markedly elevated in adipose tissue and mammary gland. In addition, in CRBP-I(-/-) mice, CRBP-III protein levels were elevated in tissues that normally express CRBP-III but were not elevated in other tissues that do not normally express CRBP-III. Our data suggested that CRBP-I and CRBP-III share some physiologic actions within tissues and that each can compensate for the absence of the other to help maintain normal retinoid homeostasis. However, under conditions of high demand for retinoid, such as those experienced during lactation, this compensation was incomplete.


Pleomorphomonas diazotrophica sp. nov., an endophytic N-fixing bacterium isolated from root tissue of Jatropha curcas L.
A novel aerobic, non-motile, pleomorphic, Gram-negative and nitrogen-fixing bacterial strain, designated R5-392(T), was isolated from surface-sterilized root tissue of Jatropha curcas. The organism grew optimally at 30 °C in media containing 1 % (w/v) NaCl and at pH 6.0-8.0. The predominant ubiquinone was Q-10 and the major fatty acids were C18 : 1ω7c/C18 : 1ω6c, C16 : 0 and C19 : 0 cyclo ω8c. The DNA G+C content was 63.2 mol%. Analysis of the 16S rRNA gene sequence suggested that strain R5-392(T) is affiliated with the order Rhizobiales within the class Alphaproteobacteria and is most closely related to Pleomorphomonas oryzae F-7(T) (98.8 % similarity) and Pleomorphomonas koreensis Y9(T) (98.3 % similarity). Analysis of partial nifH gene sequences also revealed a monophyletic lineage within the class Alphaproteobacteria, and strain R5-392(T) was most closely related to P. oryzae F-7(T) (98 %). Highest nitrogenase activity was detected in the presence of low-level organic nitrogen or in the presence of nitrogenase co-factors (Fe/Mo) in N-free media. Phenotypic and chemotaxonomic data suggest that strain R5-392(T) represents a novel species within the genus Pleomorphomonas, for which the name Pleomorphomonas diazotrophica sp. nov. is proposed. The type strain is R5-392(T) ( = KACC 16233(T) = DSM 25022(T)).


Potentiometric and spectroscopic studies on aluminium(III) complexes of some catechol derivatives.
The interactions of aluminium(III) ion with the triprotic catechol derivatives (H3L), 2,3-dihydroxybenzoic acid (2,3-DHBA), 3,4-dihydroxyphenylacetic acid (3,4-DHPA), 3,4-dihydroxybenzoic acid (3,4-DHBA), and 3,4-dihydroxyhydrocinnamic acid (3,4-DHHCA) were investigated in aqueous solution at 25.0 degrees C. The Calvin-Bjerrum titration method was adopted for the determination of formation constants of proton-ligand and aluminium(III)-ligand complexes. Potentiometric and spectroscopic results indicated that these catechol derivatives exhibit a true bidentate character. The chelation occurs via their catecholate sites, with the exception of 2,3-DHBA. In the case of 2,3-DHBA complexes, the dominant species are either the salicylate type (COO-, O-) or catecholate type (O-, O-) complex. The protonation constants of ligands and their formation constants of Al(III) complexes were also correlated. The order of decreasing stabilities of complexes is: 3,4-DHPA>3,4-DHBA>3,4-DHHCA>2,3-DHBA.


[Hemogram reference values for Lisbon metropolitan area population].
The haemogram is, probably, one of the most requested analysis for laboratorial evaluation not only for ill patients but also in periodic evaluation of healthy subjects, allowing us to foil the presence of eventual illness, since there is an enormous variety of pathologies that can induce haemogram changes. The haematological parameters of healthy subjects are influenced by numerous factors. The ideal would be each laboratory should determine and establish his own reference values for each parameter, having in account all the variables. Since the available data for haematological parameters in the Portuguese population is very insufficient, it becomes necessary to report to exterior data based in populations with different characteristics from ours, which makes reference values' reliability questionable. To overcome this problem, we felt the necessity to evaluate our own population (healthy adults from metropolitan Lisbon area) concerning haemogram reference values, comparing the obtained results with those from the bibliography. We analyzed 363 blood samples from blood-donors that fulfilled the previously established protocol. The samples were collected before blood donation and the haemograms were done on the same day. We proceeded to the informatic registration of haemogram results, to the gathering of important data, to the characterization of the target population and to the statistical analysis of the obtained results. The sample population was globally analyzed and several groups were compared, according to sex, age group and possible history of smoking habits. Although we did not find in the bibliography data concerning the quantitative analysis of similar groups compared to ours, globally we concluded that the obtained results are similar to those described by other authors, both in qualitative and quantitative terms. The obtained results can only be of reference for this population and created subgroups. The definition of reference values for the Portuguese population requires the study of a much larger sample, due to the populational diversity that characterizes our country.


First International Survey on Diabetes Providers' Assessment of Skin Reactions in Youth With Type 1 Diabetes Using Technological Devices.
Advances in diabetes technological devices led to optimization of diabetes care; however, long-lasting skin exposure to devices may be accompanied by an increasing occurrence of cutaneous reactions. We used an open-link web-based survey to evaluate diabetes-care providers' viewpoint on prevalence, management practices, and knowledge related to skin reactions with the use of diabetes technological devices. A post hoc analysis was applied to investigate differences in the level of awareness on this topic in relation to the experience in diabetes technology. One hundred twenty-five responses from 39 different countries were collected. Most respondents (69%) routinely examine patients' skin at each visit. All the preventive measures are not clear and, mainly, homogenously put into clinical practice. Contact dermatitis was the most frequently reported cutaneous complication due to diabetes devices, and its most common provocative causes are not yet fully known by diabetes-care providers. Almost half of the respondents (42%) had discussed the presence of harmful allergens contained in adhesives with device manufacturers. There is general agreement on the need to strengthen knowledge on dermatological complications. Although diabetes-care providers are quite aware of the chance to develop skin reactions in people with diabetes using technological devices, there are still some unmet needs. Large follow-up studies and further dissemination tools are awaited to address the gaps revealed by our survey.


Faraday cup dosimetry in a proton therapy beam without collimation.
A Faraday cup in a proton beam can give an accurate measurement of the number of protons collected by the cup. It is shown that the collection efficiency with a proper design can be close to unity. To be able to calibrate an ionization chamber from such a measurement, as is recommended in some dosimetry protocols, the energy spectrum of the proton beam must be accurately known. This is normally not the case when the lateral beam extension is defined by collimators. Therefore a method for relating an ionization chamber measurement in an uncollimated beam to the total number of protons in the beam has been developed and is described together with experimental results from calibrating an ionization chamber using this method in the therapeutic beam in Uppsala. This method is applicable to ionization chambers of any shape and the accuracy is estimated to be 1.6% (1 SD).


Physical and economic impacts of sea-level rise and low probability flooding events on coastal communities.
Conventionally flood mapping typically includes only a static water level (e.g. peak of a storm tide) in coastal flood inundation events. Additional factors become increasingly important when increased water-level thresholds are met during the combination of a storm tide and increased mean sea level. This research incorporates factors such as wave overtopping and river flow in a range of flood inundation scenarios of future sea-level projections for a UK case study of Fleetwood, northwest England. With increasing mean sea level it is shown that wave overtopping and river forcing have an important bearing on the cost of coastal flood events. The method presented converts inundation maps into monetary cost. This research demonstrates that under scenarios of joint extreme surge-wave-river events the cost of flooding can be increased by up to a factor of 8 compared with an increase in extent of up to a factor of 3 relative to "surge alone" event. This is due to different areas being exposed to different flood hazards and areas with common hazard where flood waters combine non-linearly. This shows that relying simply on flood extent and volume can under-predict the actual economic impact felt by a coastal community. Additionally, the scenario inundation depths have been presented as "brick course" maps, which represent a new way of interpreting flood maps. This is primarily aimed at stakeholders to increase levels of engagement within the coastal community.


Projectile, blunt, or sharp force trauma? A facial lesion on a Bronze Age skull from Uglemose, Denmark.
This study presents and discusses the differential diagnosis of a unique case of facial trauma. The adult male skull was excavated in 1948 from Uglemose (Owl Bog) outside of Birket, on the island of Lolland (Denmark). Carbon-14 samples from an adult tibia excavated with the skull dates to the end of the Danish Bronze Age and transition period into the pre-Roman Iron Age. The skull shows a traumatic ante-mortem lesion to the left maxilla, directly below the orbit, that opens into the maxillary sinus. The left margin of the injury is slightly rounded and angled into the lesion while the right margin is pulled out from the lesion and has a much sharper border, suggesting a directional force from left to right. A previous assessment of the lesion suggested that projectile trauma caused the injury, but the lesion does not conclusively demonstrate features of projectile trauma. CT scans and 3D visualization of the skull show no evidence of structural changes to the maxillary sinus to support a conclusion of projectile trauma. Differential diagnosis through macroscopic and microscopic analysis of the lesion would suggest rapid blunt force or slow sharp force trauma as a potential etiology. The aim of this case study is to discuss the potential mechanisms of injury, including type of trauma as well as accidental versus intentional etiologies.


[Interaction of S- and R-lipopolysaccharides of Francisella tularensis with lypopolysaccharide-binding protein of human serum].
Investigation of ability of Francisella tularensis S- and R-lypopolysaccharide (LPS) preparations as well as the live bacteria with different chemotypes to interact with human lypopolysaccharide-binding protein (LBP) was carried out. It was found that LPS preparations derived from virulent(S-LPS) or isogenic avirulent mutant (R-LPS) strains of F. tularensis had markedly lower affinity to LBP as compared with typical S-LPS of Salmonella abortus and R-LPS of Yersinia pestis. It was shown that R-LPS preparation from avirulent mutant binds LPB more effectively than S-LPS from F. tularensis virulent strain. Differences in S- and R-LPS affinity were also confirmed for LPS represented by the live cells. Thus, bacteria with S-chemotype of LPS (F. tularensis 15/10) bound only 20.3% of LBP, whereas cells with R-LPS (F. tularensis 543 cap(-)) bound 39.9%. Such pattern was observed in experiments with both normal non-immune human serum and sera from people immunized with live tularemia vaccine. The latter indicates that opsonization of LPS by specific antibodies does not change its affinity to LBP. The observed more efficient binding of avirulent strain R-LPS to LBP is likely determines the more intensive host response directed to destruction and rapid elimination of the causative agent. At the same time, weak affinity of the vaccine and virulent strains S-LPS to LBP probably allows the bacterium to avoid activation of host defense mechanisms thus contributing to its long-term persistence in microorganism and development of specific immunity against tularemia.


Apolipoprotein E gene ε4ε4 is associated with elevated risk of primary open angle glaucoma in Asians: a meta-analysis.
Epidemiological studies have evaluated the association between Apolipoprotein E (APOE) gene ε2/ε3/ε4 polymorphism and glaucoma susceptibility. However, the published data are still inconclusive. The aim of the present study is to evaluate the impact of APOE gene ε2/ε3/ε4 polymorphism on glaucoma risk by using meta-analysis. A comprehensive literature search of PubMed, EMBASE, Cochrane, Elsevier Science Direct and CNKI databases was conducted to identify relevant articles, with the last report up to January 5, 2014. Pooled odds ratio (OR) and 95% confidence interval (CI) were used to assess the strength of association by using the fixed or random effect model. Fifteen separate studies including 2,700 cases and 2,365 controls were included in the meta-analysis. We did not detect a significant association between APOE gene ε2/ε3/ε4 polymorphism and glaucoma in overall population (P > 0.0083). In Asians, we detected an association of the ε4ε4 genotype with elevated risk for glaucoma (OR = 5.22, 95% CI = 1.85-14.68, P = 0.002), mainly for primary open angle glaucoma (OR = 4.98, 95% CI = 1.75-14.20, P = 0.003). The meta-analysis suggests that APOE gene ε4ε4 may be associated with elevated risk for primary open angle glaucoma in Asians. However, more epidemiologic studies based on larger sample size, case-control design and stratified by ethnicity as well as types of glaucoma are suggested to further clarify the relationship between APOE gene ε2/ε3/ε4 polymorphism and genetic predisposition to glaucoma.


High-Flow Nasal Cannula Oxygen Therapy in Adults: Physiological Benefits, Indication, Clinical Benefits, and Adverse Effects.
High-flow nasal cannula (HFNC) oxygen therapy is carried out using an air/oxygen blender, active humidifier, single heated tube, and nasal cannula. Able to deliver adequately heated and humidified medical gas at flows up to 60 L/min, it is considered to have a number of physiological advantages compared with other standard oxygen therapies, including reduced anatomical dead space, PEEP, constant F(IO2), and good humidification. Although few large randomized clinical trials have been performed, HFNC has been gaining attention as an alternative respiratory support for critically ill patients. Published data are mostly available for neonates. For critically ill adults, however, evidence is uneven because the reports cover various subjects with diverse underlying conditions, such as hypoxemic respiratory failure, exacerbation of COPD, postextubation, preintubation oxygenation, sleep apnea, acute heart failure, and conditions entailing do-not-intubate orders. Even so, across the diversity, many published reports suggest that HFNC decreases breathing frequency and work of breathing and reduces the need for respiratory support escalation. Some important issues remain to be resolved, such as definitive indications for HFNC and criteria for timing the starting and stopping of HFNC and for escalating treatment. Despite these issues, HFNC has emerged as an innovative and effective modality for early treatment of adults with respiratory failure with diverse underlying diseases.


Policy implementation in wheelchair service delivery in a rural South African setting.
Wheelchairs allow users to realise basic human rights and improved quality of life. South African and international documents guide rehabilitation service delivery and thus the provision of wheelchairs. Evidence indicates that rehabilitation policy implementation gaps exist in rural South Africa. The aim of this article was to explore the extent to which wheelchair service delivery in a rural, remote area of South Africa was aligned with the South African National Guidelines on Provision of Assistive Devices, The United Nations Convention on the Rights of Persons with Disabilities and The World Health Organization Guidelines on Provision of Wheelchairs in Less-Resourced Settings. Qualitative methods were used. Data were collected through semi-structured interviews with 22 participants who were identified through purposive sampling. Content analysis of data was preformed around the construct of wheelchair service delivery. Study findings identified gaps between the guiding documents and wheelchair service delivery. Areas where gaps were identified included service aspects such as referral, assessment, prescription, user and provider training, follow up, maintenance and repair as well as management aspects such as staff support, budget and monitoring. Positive findings related to individual assessments, enthusiastic and caring staff and the provision of wheelchairs at no cost. The gaps in policy implementation can have a negative impact on users and the service provider. Inappropriate or no wheelchairs limit user function, participation and quality of life. In addition, an inappropriate wheelchair will have a shorter lifespan, requiring frequent repairs and replacements with cost implications for the service provider.


Isolation and detection of extended spectrum β-lactamase (ESBL)-producing enterobacteriaceae from meat using chromogenic agars and isothermal loop-mediated amplification (LAMP) assays.
The aim of this work was to develop a molecular method using loop-mediated isothermal amplification (LAMP) for detection of extended spectrum β-lactamase (ESBL)-producing Enterobacteriaceae from meat, and to compare it with different isolation agars and microarrays. LAMP assays were developed for CTX-M groups 1, 2, and 9 and OXA-10-like genes. Chicken, lamb, beef, pork, and turkey samples were spiked with 10, 100, and 1,000 cfu/gram using 8 strains of ESBL-producing Enterobacteriaceae (CTX-M sequence types 1, 2, 3, 14, 15, OXA-11, SHV-2, TEM-52) +/- a mix of competitor organisms. Samples were enriched overnight in buffered peptone water (BPW) +/- antibacterials before plating to CHROMagar CTX, OXOID ESBL Brilliance agar, and MacConkey agar with 1 mg/L cefotaxime. Selected BPW broths were also tested using LAMP assays, microarrays and using cefpodoxime discs on agar. For isolation/detection of ESBL producers from beef, pork, lamb, and turkey spiked with 10 or 100 cfu/gram ESBL (natural flora only), all agars and the LAMP assays showed 100% sensitivity and specificity for ESBL spike strains. For chicken samples, both LAMP and chromogenic agars showed improved sensitivity and specificity for isolation of ESBLs compared with MacConkey agar, particularly with competitor bacteria added. In comparison, the cefpodoxime disc method and microarray showed reduced sensitivity.


[Inhibition of demand pacemakers by myopotentials and its clinical significance].
Unipolar demand pacemaker myopotential inhibition by 5 different provocative maneuvers was studied in 48 patients with stable pacing rhythm out of 82 patients with unipolar pacemakers from 3 manufacturers implanted over the right pectoralis major to evaluate the incidence of inhibiting phenomenon and its clinical significance. Transient pacing inhibition occurred in 19 (39.6%) of the 48 patients. Among them 3 (30.0%) of 10 patients with Model 28 (EDWARDS), 6 (31.6%) of 19 patients with Model 30 (EDWARDS), 10 (71.4%) of 14 patients with Model 7550 (ELAMEDICAL), and none of the 5 patients with Model P101 (QINMING). The effective maneuvers were the right hand-to-left shoulder press (63.2% positivity), the uprising from a supine position (21.0% positivity) and the hand-to-hand press (15.8% positivity). No pacemaker myopotential inhibition had been induced by Valsalva maneuver and the left hand-to-right shoulder press. 19 patients with positive myopotential inhibition had pauses of pacemaker function lasting from 1.2 to 3.9 seconds, 13 of them with pauses more than 2 seconds. Of all 48 patients, 9 (18.7%) experienced symptoms of cerebral ischemia due to pacemaker myopotential inhibition during test, among whom 8 had ventricular standstill more than 2 seconds. This showed a close relation between symptoms and duration of the pacing inhibition. The causes of the phenomenon and its prevention were briefly discussed.


Suffering caused by care-elderly patients' experiences in community care.
Growing old involves many changes in life and implies an increased risks of illness and different forms of disabilities. Life may change in a radical way when a person gets a disease like dementia or moves to a nursing home due to disabilities or needs. In both cases, it often leads to an increased dependency on care where the patient becomes exposed and vulnerable and thereby at a higher risk for experiencing different forms of suffering. The aim of this study was to elucidate and gain a deeper understanding of elderly patients' experiences of suffering in relation to community care in nursing homes and home care services. A lifeworld hermeneutical approach was used. Phenomenological interviews and conversations with an open approach were conducted and analysed with a focus on meanings. The findings were presented in four main themes; an absence of the other in care, an absence of dialogues, a sense of alienation and a sense of insecurity. The findings in this study revealed that persons who were cared for in nursing homes and home care services sometimes were exposed to an unnecessary suffering. The suffering sometimes was caused by various caring actions, that is, unnecessary suffering. The suffering caused by care that aroused was due to caregiver's inability to be present, to show their face, and truly meet the patient. Suffering from care increased the elderly patients' feelings of insecurity, loneliness, and alienation; this seemed to be the foundation for patients' experiences of being outside a human community. There was a lack of knowledge and understanding about the patient's lifeworld.


Open-access echocardiography to general practitioners for suspected heart failure.
Echocardiography is now considered to be the key investigation when heart failure is suspected, and should improve clinical management. An open-access echocardiography service was piloted to 24 general practitioners and the service was audited after 250 cases. The impact on clinical management was assessed by reviewing general practice notes 2 months after the echocardiogram. Significant impairment of left ventricular function was found in 49 patients (20%). Out of these subjects, 38 had been started on an ACE inhibitor. Twenty patients were considered to have a significant valve lesion by echocritiera, of whom 14 had been referred for a cardiological opinion. The provision of an open-access echocardiography service was popular with general practitioners and the information resulted in appropriate management decisions being made.


pH and kinetic studies of chloroplast sedoheptulose-1,7-bisphosphatase from spinach (Spinacia oleracea).
The aim of this paper is to study some steady-state kinetic properties of sedoheptulose-1,7-bisphosphatase, its pH-dependence and the effect of a substrate analogue, fructose 2,6-bisphosphate. Studies were carried out with sedoheptulose 1,7-bisphosphate and with fructose 1,6-bisphosphate, an alternative substrate. The pK values are identical for both substrates, and fructose 2,6-bisphosphate behaves like a competitive inhibitor. These results suggest that there exists a unique active site for either sedoheptulose 1,7-bisphosphate or fructose 1,6-bisphosphate on the enzyme molecule. Increasing Mg2+ concentrations shifted the optimum pH. As for fructose-1,6-bisphosphatase, we believe that this shift is due to the neutralization of negative charges near the active centre [Cadet, Meunier & Ferté (1987) Eur. J. Biochem. 162, 393-398]. The free species of sedoheptulose 1,7-bisphosphate and fructose 1,6-bisphosphate are not the usual substrates of enzyme, nor is Mg2+. But the kinetics relative to the (Mg2+-substrate4-)2- complex is not consistent with this complex being the substrate. An explanation of this discrepancy is proposed, involving both the negative charges near the active centre and the positive charges of Mg2+. The observed Vmax. of the reduced enzyme is 65% of the theoretical Vmax. for both substrates, but the observed Vmax. relative to sedoheptulose 1,7-bisphosphate is 3 times the one relative to fructose 1,6-bisphosphate. The specificity constant (kcat./Km), 1.62 x 10(6) M-1.s-1 with respect to sedoheptulose 1,7-bisphosphate compared with 5.5 x 10(4) M-1.s-1 with respect to fructose 1,6-bisphosphate, indicates that the enzyme specificity towards sedoheptulose 1,7-bisphosphate is high but not absolute.


Effect of repeated L-DOPA, bromocriptine, or lisuride administration on preproenkephalin-A and preproenkephalin-B mRNA levels in the striatum of the 6-hydroxydopamine-lesioned rat.
Abnormal involuntary movements, or dyskinesias, plague current symptomatic approaches to the treatment of Parkinson's disease. The neural mechanisms underlying the generation of dyskinesia following repeated l-3,4-dihydroxyphenylalanine (L-DOPA) or dopamine agonist administration in Parkinson's disease remain unknown. However, de novo administration of bromocriptine or lisuride to either l-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-lesioned primates or patients can alleviate parkinsonian symptoms without the development of dyskinesia. In this study, we have investigated behavioral responses and alterations in the expression of opioid neuropeptide precursors preproenkephalin-A (PPE-A, encoding methionine- and leucine-enkephalin) and preproenkephalin-B (PPE-B), the precursor encoding dynorphins (dynorphin A1-17 and B1-13, leucine-enkephalin, and alpha-neoendorphin) in striatal output pathways of the 6-hydroxydopamine (6-OHDA)-lesioned rat model of Parkinson's disease. Expression was assessed following repeated L-DOPA, bromocriptine, or lisuride administration. Given the functional organization of basal ganglia circuitry into anatomically discrete parallel circuits, we investigated alterations in peptide expression with reference to the detailed topography of the striatum. Following repeated L-DOPA administration (6.5 mg/kg, b.d., 21 days) in the 6-OHDA-lesioned rat a rotational response was observed. This became markedly enhanced with repeated treatment. We have previously characterized the pharmacology of this enhanced response and have suggested that it is a useful model for the elucidation of the cellular and molecular mechanisms underlying L-DOPA- and dopamine agonist-induced dyskinesia. In contrast to l-DOPA, de novo administration of bromocriptine (1 or 5 mg/kg, b.d., 21 days) or lisuride (0.01 or 0.1 mg/kg, b.d., 21 days) did not lead to an enhanced behavioral response. In vehicle-treated, 6-OHDA-lesioned animals, PPE-A expression was elevated rostrally and dorsally, while PPE-B expression was reduced in the striatum at all rostrocaudal levels. Repeated l-DOPA administration was accompanied by elevations in striatal PPE-B mRNA levels and a further elevation, above lesion-induced levels, in PPE-A expression. This further elevation was restricted to the dorsolateral striatum. However, following repeated bromocriptine or lisuride administration no increase in PPE-B expression was observed and the lesion-induced increase in PPE-A expression was normalized to prelesion levels. Increased PPE-A and PPE-B levels may, through decreasing GABA and glutamate release, respectively, in output nuclei of the basal ganglia, play a role in the development of L-DOPA- and dopamine-agonist induced dyskinesia in Parkinson's disease. These studies suggest that anti-parkinsonian treatments which are not associated with an elevation in PPE-B and/or normalize elevated PPE-A precursor expression, such as NMDA-receptor antagonists or long-acting dopamine D2 receptor agonists, e.g., cabergoline or ropinirole, may reduce dyskinesia in Parkinson's disease.


Developing an evidence-based guideline for the management of exposure to hepatitis B at a Victorian tertiary hospital.
To develop a guideline for the management of potential exposures to hepatitis B virus (HBV) at The Alfred Hospital, based on results of clinical audit, database analysis and literature review. i) Retrospective record review of all histories of patients who received HBV immunoglobulin (HBIG) at The Alfred between 1/1/2007 and 30/9/2011. ii) Analysis of HBV serological results of men who have sex with men (MSM) on Victorian NPEP Service (VNPEPS) database between 10/8/2005 and 31/12/2011. iii) Literature review to determine risks of HBV transmission and best practice for prevention of HBV transmission. A total of 48 patients were potentially exposed to HBV and given HBIG, with sexual exposure the most common indication (n=20). The source was reported to be HBsAg positive in one case only. Of the MSM on the VNPEPS database, 63% were immune to HBV, and only 0.5% of patients tested had evidence of chronic HBV infection. The recommendations for use of HBIG in The Australian Immunisation Guidelines are ambiguous and differ from other international guidelines. This audit at a tertiary referral hospital identified problems with the management of those potentially exposed to HBV. In those non-immune patients exposed to HBV, the combination of HBIG plus vaccination provides the best protection against infection. The risk of transmission of HBV is highly variable; rates of chronic HBV in PWID and MSM in Australia are low and do not warrant use of HBIG unless the source is known to be HBsAg positive.


Comparison of Deep Learning With Multiple Machine Learning Methods and Metrics Using Diverse Drug Discovery Data Sets.
Machine learning methods have been applied to many data sets in pharmaceutical research for several decades. The relative ease and availability of fingerprint type molecular descriptors paired with Bayesian methods resulted in the widespread use of this approach for a diverse array of end points relevant to drug discovery. Deep learning is the latest machine learning algorithm attracting attention for many of pharmaceutical applications from docking to virtual screening. Deep learning is based on an artificial neural network with multiple hidden layers and has found considerable traction for many artificial intelligence applications. We have previously suggested the need for a comparison of different machine learning methods with deep learning across an array of varying data sets that is applicable to pharmaceutical research. End points relevant to pharmaceutical research include absorption, distribution, metabolism, excretion, and toxicity (ADME/Tox) properties, as well as activity against pathogens and drug discovery data sets. In this study, we have used data sets for solubility, probe-likeness, hERG, KCNQ1, bubonic plague, Chagas, tuberculosis, and malaria to compare different machine learning methods using FCFP6 fingerprints. These data sets represent whole cell screens, individual proteins, physicochemical properties as well as a data set with a complex end point. Our aim was to assess whether deep learning offered any improvement in testing when assessed using an array of metrics including AUC, F1 score, Cohen's kappa, Matthews correlation coefficient and others. Based on ranked normalized scores for the metrics or data sets Deep Neural Networks (DNN) ranked higher than SVM, which in turn was ranked higher than all the other machine learning methods. Visualizing these properties for training and test sets using radar type plots indicates when models are inferior or perhaps over trained. These results also suggest the need for assessing deep learning further using multiple metrics with much larger scale comparisons, prospective testing as well as assessment of different fingerprints and DNN architectures beyond those used.


Associations of common chronic non-communicable diseases and medical conditions with sleep-related problems in a population-based health examination study.
A cross-sectional population-based survey, the National FINRISK 2012 Study, designed to monitor chronic diseases and their risk factors in Finland. A random sample of 10,000 adults aged 25-74 years, and of them, 64% (n=6424) participated the study. Participants subjectively reported the total durations for sleep and naps (n=6238), sleep quality (n=5878), bedtimes and wake-up times separately for working days and weekends yielding the amount of sleep debt (n=5878), and the seasonal variation in sleep duration (n=4852). The participants were asked whether they were diagnosed or treated for common chronic diseases in the past 12 months. Logistic regression models were adopted to analysis and adjusted for a range of covariates as potential confounding factors. Total sleep duration and nap duration prolonged in depression and other mental disorder (p<.001 for all). Seasonal variation in sleep duration was associated with depression (p=.014), hypertension (p=.018) and angina pectoris (p=.024). Participants with gallstones, cardiac insufficiency, depression, or degenerative arthritis had poor sleep quality (odds ratios of 1.6-6.3, p=.001 or less for each). Those with degenerative arthritis had sleep debt less (p<.05) and those with angina pectoris more (p<.05) than individuals without these medical conditions. Depression is significantly associated with sleep problems, albeit no sleep debt. Cardiovascular diseases, degenerative arthritis, and gallstones had significant associations with one or more sleep problems. There is therefore a need for more successful management of sleep problems in chronic diseases to improve the quality of life, to reduce treatment relapses, and to increase health and longevity in a population.


LSD1-mediated epigenetic modification contributes to ovarian cancer cell migration and invasion.
Lysine-specific demethylase 1 (LSD1) has been implicated in the process of tumor progression at various steps, but its role in epithelial-messenchymal transition (EMT) and the migration of ovarian cancer cells remains obscure. In this study, we demonstrated the effect of LSD1 on ovarian cancer cell migration and the regulatory role of LSD1 in the expression of EMT markers. Inhibition of LSD1 expression impaired the migration and invasion of HO8910 ovarian cancer cells. In contrast, overexpression of LSD1 enhanced the cell migration and invasion of HO8910 cells. Mechanistic analyses showed that LSD1 promoted cell migration through induction of N-cadherin, vimentin, MMP-2 and inhibition of E-cadherin. Furthermore, LSD1 interacted with the promoter of E-cadherin and demethylated histone H3 lysine 4 (H3K4) at this region, downregulated E-cadherin expression, and consequently enhanced ovarian cancer cell migration. These data indicate that LSD1 acts as an epigenetic regulator of EMT and contributes to the metastasis of ovarian cancer.


Inactivation of Escherichia coli DnaA protein by DNA polymerase III and negative regulations for initiation of chromosomal replication.
Genetic and biochemical evidence indicates that initiation of chromosomal replication in Escherichia coli occurs in a nucleoprotein complex at the replication origin (oriC) formed with DnaA protein. The frequency of initiation at oriC is tightly regulated to only once per chromosome per cell cycle. To prevent untimely, extra initiations, negative control for initiation is indispensable. Recently, we found that the function of the initiator protein, DnaA, is controlled by DNA polymerase III holoenzyme, the replicase of the chromosome. The ATP-bound form of DnaA protein, an active form for initiation, is efficiently converted to the ADP bound form, an inactive form, since a subunit of the polymerase loaded on DNA (beta subunit sliding clamp) stimulates hydrolysis of ATP bound to DnaA protein. Comparison of this system, RIDA (regulatory inactivation of DnaA), with other systems for negative regulation of initiation is included in this review, and the roles of these systems for concerted control for initiation during the cell cycle are discussed.


Heat stress is a potent stimulus for enhancing rescue efficiency of recombinant Borna disease virus.
Recently developed vector systems based on Borna disease virus (BDV) hold promise as platforms for efficient and stable gene delivery to the central nervous system (CNS). However, because it currently takes several weeks to rescue recombinant BDV (rBDV), an improved rescue procedure would enhance the utility of this system. Heat stress reportedly enhances the rescue efficiency of other recombinant viruses. Here, heat stress was demonstrated to increase the amount of BDV genome in persistently BDV-infected cells without obvious cytotoxicity. Further analyses suggested that the effect of heat stress on BDV infection is not caused by an increase in the activity of BDV polymerase. More cells in which BDV replication occurs were obtained in the initial phase of rBDV rescue by using heat stress than when it was not used. Thus, heat stress is a useful improvement on the published rescue procedure for rBDV. The present findings may accelerate the practical use of BDV vector systems in basic science and the clinic and thus enable broader adoption of this viral vector, which is uniquely suited for gene delivery to the CNS.


Identification of Salmonella typhi in faecal specimens by an antiserum-agar method.
An antiserum-agar technique was evaluated as a method for detecting Salmonella typhi in faeces. Thirty-one laboratory strains of S. typhi produced immunoprecipitate haloes during overnight growth on SS agar and blood-agar-base infusion agar (BAB) containing donkey antiserum to a vaccine strain of S. typhi. Other salmonella species sharing O serogroup antigens with S. typhi also produced haloes when streaked in pure culture on SS-antiserum agar but not on BAB-antiserum agar. One hundred and forty-one consecutive faecal specimens were cultured on SS-antiserum agar. Results with this method were concordant with those of established isolation techniques on specimens from six of seven suspected carriers of S. typhi. Ten other salmonellas were isolated from the faecal specimens but only S. javiana, like S. typhi a serogroup-D organism, yielded false-positive haloes on antiserum agar. The antiserum-agar technique offers promise as a means of screening for S. typhi in faecal cultures.


BK Polyomavirus Infection and Renourinary Tumorigenesis.
BK polyomavirus (BKPyV) infection represents a major problem in transplantation, particularly for renal recipients developing polyomavirus-associated nephropathy (PyVAN). The possibility that BKPyV may also be oncogenic is not routinely considered. Twenty high-grade renourinary tumors expressing polyomavirus large T antigen in the entirety of the neoplasm in 19 cases, including the metastases in six, have been reported in transplant recipients with a history of PyVAN or evidence of BKPyV infection. Morphological and phenotypical features consistent with inactivation of the tumor suppressors pRB and p53 were found in the bladder tumors, suggesting a carcinogenesis mechanism involving the BKPyV large tumor oncoprotein/antigen. The pathogenesis of these tumors is unclear, but given the generally long interval between transplantation and tumor development, the risk for neoplasms after BKPyV infections may well be multifactorial. Other elements potentially implicated include exposure to additional exogenous carcinogens, further viral mutations, and cell genomic instability secondary to viral integration, as occurs with the Merkel cell PyV-associated carcinoma. The still scarce but increasingly reported association between longstanding PyVAN and renourinary neoplasms requires a concerted effort from the transplant community to better understand, diagnose, and treat the putative association between the BKPyV and these neoplasms.


Discriminative stimulus effects of self-administered ethanol.
The conditions under which a drug is administered often alter the behavioral effects of that drug. The present study examined the effect of changes in response dependence on the discriminative stimulus effects of ethanol. Six Long-Evans rats were trained to discriminate 1000 mg/kg, interperitoneal (i.p.) ethanol from saline. A dose-effect curve was then obtained using i.p. doses of 100, 320, 560, 1000, 1320 and 1560 mg/kg ethanol. Ethanol doses of 1000 mg/kg and greater produced more than 80% ethanol-lever selection. The rats were then trained to orally self-administer 10% weight/volume ethanol and tested to determine if self-administered oral ethanol would substitute for experimenter administered i.p. ethanol. A mean self-administered ethanol intake of 1114 mg/kg (+/-156 mg/kg) produced 83% ethanol-lever responding. Restricted access to 560 mg/kg of self-administered ethanol resulted in 33% i.p. ethanol-lever responding. Doses of 100 and 320 mg/kg ethanol did not substitute for i.p. ethanol. These data show that orally self-administered ethanol can produce discriminative stimulus effects that are similar to i.p. experimenter-administered ethanol and that orally self-administered ethanol produces centrally-mediated discriminative stimulus effects.


Detection of human papillomavirus type 16 DNA and evidence for integration into the cell DNA in cervical dysplasia.
The presence of human papillomavirus (HPV) type 16 DNA in biopsies from precancerous lesions and from early lesions of human cervical cancer, and the integration of virus DNA into host cell DNA were analysed by dot blot and Southern blot hybridizations. HPV 16 DNA was detected in 23% of mild dysplasias, 32% of moderate dysplasias, 55% of severe dysplasias and 62% of carcinomas in situ by dot blot hybridization. Digestion of the DNA with restriction enzymes PstI and BamHI followed by Southern blot analysis revealed the presence of some typical restriction fragments of HPV 16 DNA in most virus-positive samples. In addition, we detected submolar fragments which might represent virus-cell junction sequences in 86% of dysplasias, suggesting that the integration of HPV 16 DNA could occur in the precancerous stage.


Regulation of ornibactin biosynthesis and N-acyl-L-homoserine lactone production by CepR in Burkholderia cepacia.
The CepR-CepI quorum-sensing system has been shown to regulate production of the siderophore ornibactin, extracellular proteases, and N-octanoyl-homoserine-L-lactone (OHL) in Burkholderia cepacia strain K56-2. To examine the effect of cepIR on production of other siderophores, cepR mutants were constructed in strains that produce pyochelin in addition to salicylic acid and ornibactins. Pc715j-R1 (cepR::tp) hyperproduced ornibactin but produced parental levels of pyochelin and salicylic acid, suggesting that CepR is a negative regulator of ornibactin synthesis but not pyochelin or salicylic acid. Pc715j-R1 was also protease deficient and OHL negative. The effects of cepR on ornibactin biosynthetic genes were examined by constructing cepR pvdA-lacZ and cepR pvdD-lacZ mutants and monitoring beta-galactosidase activity. There was an increase in expression of pvdA in the cepR mutant compared to the level in its parent strain in both low- and high-iron media during stationary phase. When the outer membrane protein profiles of a cepR mutant and the wild-type strain were compared on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, there did not appear to be any difference in levels of expression of the ornibactin receptor. Experiments with cepI-lacZ and cepR-lacZ transcriptional fusions indicated that cepI was not expressed in the cepR mutant and that cepR acts as a negative regulator of its own expression. By a thin-layer chromatography assay for N-acyl homoserine lactones, OHL and N-hexanoyl-L-homoserine lactone (HHL) were detectable in K56-2 and Pc715j, both wild-type strains. OHL was not detectable and HHL was only weakly detectable in the cepI and cepR mutants. These results suggest that CepR is both a positive and negative transcriptional regulator and that CepR may influence the expression of ornibactin biosynthetic genes in addition to the expression of the cepIR quorum-sensing system.


Comparative proteomics of Mn(II)-oxidizing and non-oxidizing Roseobacter clade bacteria reveal an operative manganese transport system but minimal Mn(II)-induced expression of manganese oxidation and antioxidant enzymes.
Manganese (Mn) is an essential nutrient and precipitates as minerals with technological and environmental relevance. To gain a proteomic understanding of how bacteria respond to Mn(II) and its connection to oxidation, a comparative examination of the proteomic response of Mn(II)-oxidizing (Roseobacter sp. AzwK-3b) and non-oxidizing (Ruegeria sp. TM1040) alphaproteobacteria was conducted. Both bacteria show an operative Mn(II) transport system. In the absence of Mn(II), both bacteria have higher expression of proteins that were homologous to SitA and SitB, known proteins in the Mn(II) transport system of other alphaproteobacteria. Overall, each bacterium demonstrated a varied response to Mn(II). Ru. TM1040 had a greater number of proteins differentially expressed in response to Mn(II) and also had a group of proteins related to chemotaxis at higher concentrations of Mn(II), suggesting a potential stress response. While both bacteria are able to generate extracellular superoxide and Mn(II) is a known antioxidant, the presence of Mn(II) did not significantly alter the expression of proteins related to antioxidant activity. Heme peroxidases, previously connected to Mn(II) oxidation, were found in the soluble protein extract of R. AzwK-3b, but only minor differential expression was observed as a function of Mn(II), indicating that their expression was not induced by Mn(II).


Wound healing efficacy of a polymeric spray film solution containing Centella asiatica leaf extract on acute wounds.
To study the clinical efficacy of a polymeric spray film containing Centella asiatica extract to heal acute wounds. A polymeric spray film solution for wound healing was formulated using Centella asiatica extract, which contained triterpenes, including asiatic acid, madecassic acid, asiaticoside and madecassoside. The stability and physicochemical properties of the formulation were evaluated, and a multicentre, randomised, controlled trial was conducted to assess its clinical wound-healing efficacy. The Pressure Ulcer Scale for Healing (PUSH Tool) score was used to evaluate wound healing on days 0, 3, 5 and 7. The cohort consisted of 60 volunteers with clean-contaminated wounds (class 1), randomly assigned to the Control (n=30) and Testing (n=30) groups. The spray product contained asiatic acid, madecassic acid, asiaticoside and madecassoside at 0.20±0.02mg/ml, 0.16±0.01mg/ml, 0.32±0.03mg/ml and 0.10±0.00mg/ml, respectively. The pH value was 5.5±0.01, and the viscosity was 33±4cP. The product was stable for six months when stored at 30±2°C and at 40±2°C, in 75±5% relative humidity. The tested product significantly reduced the total PUSH and exudate scores, indicating that the polymeric spray film solution containing Centella asiatica improved wound healing. The average healing recovery times for the Testing and Control groups were 4.6±1.1 days and 4.87±1.0 days, respectively. In this study, Centella asiatica extract-containing polymeric spray film solution was beneficial as an acute wound medication, which could shorten healing time with no adverse effects.


Impact of implant depth on hydrodynamic function of the ALLEGRA bioprosthesis in valve-in-valve interventions.
We aimed to assess the impact of implant depth on hydrodynamic function following valve-in-valve (VIV) intervention using the ALLEGRA transcatheter heart valve (THV) in three different surgical valve designs. Multiple implantation depths (+2 mm, -2 mm and -6 mm) were tested using a 23 mm ALLEGRA THV for VIV intervention in 19 mm, 21 mm, 23 mm, and 25 mm Epic, Mitroflow and Magna Ease bioprosthetic valves. Multimodality imaging and hydrodynamic evaluation was performed at each implantation depth. The 23 mm ALLEGRA valve had gradients <20 mmHg in the Mitroflow and Epic valves sized ≥21 mm, and in all sizes of the Magna Ease valve. Gradients did not increase significantly at lower implantation depths. The 19 mm Epic (+2 mm: 20.1±0.6 mmHg, -2 mm: 18.8±0.5 mmHg, -6 mm: 22.8±0.3 mmHg) and 19 mm Mitroflow (+2 mm: 24.1±0.2 mmHg, -2 mm: 31.5±0.3 mmHg, -6 mm: 25.6±0.2 mmHg) valves had elevated mean gradients. In larger sized surgical valves (≥23 mm) the regurgitant fraction was higher at low implantation depths. Pinwheeling was significantly worse in the smaller sized (≤21 mm) surgical valves and also at low (<-2 mm) implantation depth. The 23 mm ALLEGRA valve had favourable (<20 mmHg) gradients in all surgical valves sized ≥21 mm, even when the THV was implanted low. In 19 mm sized Mitroflow and Epic valves, gradients were elevated (>20 mmHg). While there was no major difference in mean transvalvular gradients, leaflet pinwheeling was worse at lower implantation depths.


Responses of midbrain auditory neurons to two different environmental sounds-A new approach on cross-sound modeling.
When modeling auditory responses to environmental sounds, results are satisfactory if both training and testing are restricted to datasets of one type of sound. To predict 'cross-sound' responses (i.e., to predict the response to one type of sound e.g., rat Eating sound, after training with another type of sound e.g., rat Drinking sound), performance is typically poor. Here we implemented a novel approach to improve such cross-sound modeling (single unit datasets were collected at the auditory midbrain of anesthetized rats). The method had two key features: (a) population responses (e.g., average of 32 units) instead of responses of individual units were analyzed; and (b) the long sound segment was first divided into short segments (single sound-bouts), their similarity was then computed over a new metric involving the response (called Stimulus Response Model map or SRM map), and finally similar sound-bouts (regardless of sound type) and their associated responses (peri-stimulus time histograms, PSTHs) were modelled. Specifically, a committee machine model (artificial neural networks with 20 stratified spectral inputs) was trained with datasets from one sound type before predicting PSTH responses to another sound type. Model performance was markedly improved up to 92%. Results also suggested the involvement of different neural mechanisms in generating the early and late responses to amplitude transients in the broad-band environmental sounds. We concluded that it is possible to perform rather satisfactory cross-sound modeling on datasets grouped together based on their similarities in terms of the new metric of SRM map.


Phase 1 Trial of Amnion Cell Therapy for Ischemic Stroke.
There is increasing interest in stem cell therapy as another treatment modality in stroke, particularly for patients who are unable to receive endovascular clot retrieval or thrombolysis therapies, or for whom standard treatment has failed. We have recently shown that human amniotic epithelial cells (hAECs) are effective in reducing infarct volume in different animal models of ischemic stroke, including in non-human primates. hAEC therapy attenuated infarct growth and/or promoted functional recovery, even when administered 1-3 days after the onset of stroke. We now propose an open label Phase 1 dose escalation trial to assess the safety of allogeneic hAECs in stroke patients with a view to providing an evidence platform for future Phase 2 efficacy trials. We propose a modified 3 + 3 dose escalation study design with additional components for measuring magnetic resonance signal of efficacy as well as the effect of hAECs on immunosuppression after stroke. The trial will commence in 2018. The findings will be published in a peer-reviewed journal. The trial is registered with ANZCTR (ACTRN12618000076279p).


On the reproduction of influenza virus; quantitative studies with procedures which enumerate infective and hemagglutinating virus particles.
Procedures which make possible the enumeration of both infective and hemagglutinating influenza A virus particles have been developed and used in a quantitative investigation on the reproduction of the agent. Infective particles were found to be highly unstable and their half-life was only 147 minutes in allantoic fluid at 35 degrees C. both in vitro and in vivo. The instability of infective particles provides an explanation for the rapid accumulation of non-infective particles which retained the hemagglutinating property. The number of non-infective (N) particles was determined from the difference between the number of hemagglutinating (H) particles and the number of infective (I) particles as indicated by the relation: [N] = [H]- [1]. When the half-life of infective particles was taken into account, both infective and hemagglutinating particles were found to disappear from the allantoic fluid; i.e., were adsorbed by the allantoic membrane, at the same logarithmic rate after inoculation. Inoculation of any number of particles up to 3 x 10(7) was followed by a constant and progressive decrease in the proportion of unadsorbed particles from 0 to 4 hours. Approximately 20 per cent of particles were unadsorbed at 2 hours and about 5 per cent at 4 hours. Inoculation of 3 x 10(8) or more particles led to a larger proportion of unadsorbed particles at 4 hours. The maximum number of particles adsorbed was computed to be about 1.6 x 10(9). The concentration of both infective and hemagglutinating particles increased rapidly in the allantoic fluid after 4 hours when any number of infective particles up to 3 x 10(7) was inoculated. With such inocula, the rate of increase during the logarithmic period was constant and the time to double the concentration of infective or hemagglutinating particles was 46 minutes. With larger inocula, i.e. 3 x 10(8) particles, the concentrations of infective and hemagglutinating particles did not increase until after 8 hours and the rate of increase was much slower. The time to double the concentration of either then became 92 minutes. The number of infective particles was approximately equal to the number of hemagglutinating particles during the logarithmic increase period when any number of infective particles up to 3 x 10(6) was inoculated and no more than 10(6) non-infective particles were included in the inoculum. This finding was taken to indicate that all or almost all particles produced and released under these conditions were infective. That such particles became inactivated rapidly and led to the accumulation of an increasing number of non-infective particles after the logarithmic period can be explained by the short half-life of infective particles. The number of infective particles was no larger than one-tenth the number of hemagglutinating particles during the logarithmic increase period after 3 x 10(7) or more infective particles had been inoculated or when smaller inocula were used which also contained 3 x 10(7) or more non-infective particles. Non-infective particles prepared in vitro at 35 degrees or 22 degrees C. were as effective as those which accumulated in vivo in diminishing the proportion of infective particles in the yield. The extent of the reduction in the proportion of infective particles was directly related to the number of non-infective particles included in the inoculum. The yield of hemagglutinating particles was diminished when the inoculum contained 3 x 10(7) or more non-infective particles. The rate of increase was reduced so that the time to double the concentration became 92 minutes when the inoculum contained 3 x 10(8) non-infective particles. It appears from these findings that the single condition which will lead to the emergence of non-infective particles during the logarithmic period is a high initial particle-cell ratio. Because non-infective particles are equally as effective as infective particles in producing this result, it seems probable that the appearance of non-infective but hemagglutinating particles is not a necessary accompaniment of the reproductive process.


The mobility of two kinase domains in the Escherichia coli chemoreceptor array varies with signalling state.
Motile bacteria sense their physical and chemical environment through highly cooperative, ordered arrays of chemoreceptors. These signalling complexes phosphorylate a response regulator which in turn governs flagellar motor reversals, driving cells towards favourable environments. The structural changes that translate chemoeffector binding into the appropriate kinase output are not known. Here, we apply high-resolution electron cryotomography to visualize mutant chemoreceptor signalling arrays in well-defined kinase activity states. The arrays were well ordered in all signalling states, with no discernible differences in receptor conformation at 2-3 nm resolution. Differences were observed, however, in a keel-like density that we identify here as CheA kinase domains P1 and P2, the phosphorylation site domain and the binding domain for response regulator target proteins. Mutant receptor arrays with high kinase activities all exhibited small keels and high proteolysis susceptibility, indicative of mobile P1 and P2 domains. In contrast, arrays in kinase-off signalling states exhibited a range of keel sizes. These findings confirm that chemoreceptor arrays do not undergo large structural changes during signalling, and suggest instead that kinase activity is modulated at least in part by changes in the mobility of key domains.


Food for thought: Reinforced learning and recall of physical activity calorie equivalent (PACE) and numerical calorie content in an associative learning task.
Calorie overconsumption has been proposed as a critical contributing factor to rising obesity rates. To combat this health issue, governments and policymakers have suggested implementing numerical caloric content labels. Alternatively, physical activity calorie equivalent (PACE) labels are being proposed as an easier-to-understand metric, representing the amount of physical activity required to burn off calorie content. This study examined individuals' ability to correctly estimate either the numerical caloric content or the PACE values of food images in an associative learning task. Moreover, it assessed whether this knowledge was learned and retained over time. One hundred and ninety-one participants were instructed to estimate either the numerical caloric content or PACE values of thirty food images. To facilitate learning, feedback on the correct number of calories or PACE values was provided during the first session (Time 1). To assess retention, people re-estimated numerical caloric content or PACE values of the same food pictures three days later (Time 2) and seven days later (Time 3), where feedback was not provided. Results showed that participants in both groups improved their estimations using feedback, with people being consistently more accurate when estimating numerical calorie content. Yet, our results also suggest that participants consolidated their knowledge of PACE values over time. Finally, our findings show that hunger moderates individuals' estimation ability, where hungrier people are less accurate than satiated ones. The results contribute to our understanding of how consumers process, estimate, and learn PACE labels versus numerical caloric content, and provide valuable information for researchers and policymakers to develop and implement nutritional labels as a health strategy.


An improved dual-expression concept, generating high-quality antibodies for proteomics research.
A novel, improved dual bacterial-expression system, designed for large-scale generation of high-quality polyclonal antibody preparations intended for proteomics research, is presented. The concept involves parallel expression of cDNA-encoded proteins, as a fusion with two different tags in two separate vector systems. Both systems enable convenient blotting procedures for expression screening on crude bacterial cell cultures and single-step affinity purification under denaturing conditions. One of the fusion proteins is used to elicit antibodies, and the second fusion protein is used in an immobilized form as an affinity ligand to enrich antibodies with selective reactivity to the cDNA-encoded part, common for the two fusion proteins. To evaluate the system, four cDNA clones from putative nuclear proteins from the non-biting midge Chironomus tentans were expressed. Antibodies to these cDNA-encoded proteins were generated, enriched and used in blotting and immunofluorescence procedures to determine expression patterns for the native proteins corresponding to the cDNAs. The four antibody preparations showed specific reactivity to the corresponding recombinant cDNA-encoded proteins, and three of the four antibodies gave specific staining in Western-blot analysis of nuclear cell extracts. Furthermore, two of the antibody preparations gave specific staining in immunofluorescence analysis of C. tentans cells. We conclude that the dual-vector concept presented offers a highly stringent strategy for the generation of monospecific polyclonal antibodies, which are useful in proteomics research.


Does chemotherapy or radiotherapy affect the postoperative complication in breast cancer patients who underwent immediate breast reconstruction with tissue expander?
Immediate breast reconstruction with tissue expander in breast cancer patients who were expected to receive adjuvant therapy, such as chemotherapy or radiotherapy, has been a topic of debate. Postoperative complications from tissue expander procedures can delay the timing of adjuvant treatment and subsequently increase the probability of recurrence. The purpose of this study was to identify the impact of chemotherapy and radiotherapy on postoperative complications in patients who underwent immediate reconstruction (IR) using tissue expander. We conducted a retrospective study of 1081 breast cancer patients who underwent mastectomy and IR using tissue expander insertion between 2012 and 2017 in Samsung Medical Center. The patients were divided into two groups based on complications (complication group vs. no complication group). Complication group was regarded to have surgical removal or conservative treatment based on clinical findings such as infection, capsular contracture, seroma, hematoma, rupture, malposition, tissue viability, or cosmetic problem. The complication group had 59 patients (5.5%) and the no complication group had 1022 patients (94.5%). In univariate analysis, adjuvant radiotherapy and adjuvant chemotherapy were significantly associated with postoperative complications. In multivariate analysis, however, only higher pathologic N stage was significantly associated with postoperative complications (p < 0.001). Chemotherapy (p = 0.775) or radiotherapy (p = 0.825) were not risk factors for postoperative complications. IR with tissue expander after mastectomy may be a treatment option even when the patients are expected to receive adjuvant chemotherapy or radiotherapy. These results will aid patients who are concerned about the complications of IR caused by chemotherapy or radiotherapy determine whether or not to have IR. Patients were selected and registered retrospectively, and medical records were evaluated.


Susceptibility of clinical Moraxella catarrhalis isolates in British Columbia to six empirically prescribed antibiotic agents.
Moraxella catarrhalis is a commensal organism of the respiratory tract that has emerged as an important pathogen for a variety of upper and lower respiratory tract infections including otitis media and acute exacerbations of chronic bronchitis. Susceptibility testing of M catarrhalis is not routinely performed in most diagnostic laboratories; rather, a comment predicting susceptibility based on the literature is attached to the report. The most recent Canadian report on M catarrhalis antimicrobial susceptibility was published in 2003; therefore, a new study at this time was of interest and importance. To determine the susceptibility of M catarrhalis isolates from British Columbia to amoxicillin-clavulanate, doxycycline, clarithromycin, cefuroxime, levofloxacin and trimethoprimsulfamethoxazole. A total of 117 clinical M catarrhalis isolates were isolated and tested from five Interior hospitals and two private laboratory centres in British Columbia between January and December 2012. Antibiotic susceptibility of M catarrhalis isolates was characterized using the Etest (E-strip; bioMérieux, USA) according to Clinical Laboratory Standards Institute guidelines. All isolates were sensitive to amoxicillin-clavulanate, doxycycline, clarithromycin, levofloxacin and trimethoprimsulfamethoxazole. One isolate was intermediately resistant to cefuroxime, representing a 99.15% sensitivity rate to the cephem agent. Cefuroxime minimum inhibitory concentrations (MICs) inhibiting 50% and 90% of organisms (MIC50 and MIC90) were highest among the antibiotics tested, and the MIC90 (3 μg/mL) of cefuroxime reached the Clinical Laboratory Standards Institute breakpoint of susceptibility. The antibiotic susceptibility of M catarrhalis isolates evaluated in the present study largely confirms the findings of previous surveillance studies performed in Canada. Cefuroxime MICs are in the high end of the sensitive range and the MIC50 and MIC90 observed in the present study are the highest ever reported in Canada. Although cefuroxime MICs in M catarrhalis are high, all agents tested showed antimicrobial activity, supporting their continued therapeutic and empirical use.


Butyrophilin-2A1 Directly Binds Germline-Encoded Regions of the Vγ9Vδ2 TCR and Is Essential for Phosphoantigen Sensing.
Vγ9Vδ2 T cells respond in a TCR-dependent fashion to both microbial and host-derived pyrophosphate compounds (phosphoantigens, or P-Ag). Butyrophilin-3A1 (BTN3A1), a protein structurally related to the B7 family of costimulatory molecules, is necessary but insufficient for this process. We performed radiation hybrid screens to uncover direct TCR ligands and cofactors that potentiate BTN3A1's P-Ag sensing function. These experiments identified butyrophilin-2A1 (BTN2A1) as essential to Vγ9Vδ2 T cell recognition. BTN2A1 synergised with BTN3A1 in sensitizing P-Ag-exposed cells for Vγ9Vδ2 TCR-mediated responses. Surface plasmon resonance experiments established Vγ9Vδ2 TCRs used germline-encoded Vγ9 regions to directly bind the BTN2A1 CFG-IgV domain surface. Notably, somatically recombined CDR3 loops implicated in P-Ag recognition were uninvolved. Immunoprecipitations demonstrated close cell-surface BTN2A1-BTN3A1 association independent of P-Ag stimulation. Thus, BTN2A1 is a BTN3A1-linked co-factor critical to Vγ9Vδ2 TCR recognition. Furthermore, these results suggest a composite-ligand model of P-Ag sensing wherein the Vγ9Vδ2 TCR directly interacts with both BTN2A1 and an additional ligand recognized in a CDR3-dependent manner.


Associations between diet and cancer, ischemic heart disease, and all-cause mortality in non-Hispanic white California Seventh-day Adventists.
Results associating diet with chronic disease in a cohort of 34192 California Seventh-day Adventists are summarized. Most Seventh-day Adventists do not smoke cigarettes or drink alcohol, and there is a wide range of dietary exposures within the population. About 50% of those studied ate meat products <1 time/wk or not at all, and vegetarians consumed more tomatoes, legumes, nuts, and fruit, but less coffee, doughnuts, and eggs than did nonvegetarians. Multivariate analyses showed significant associations between beef consumption and fatal ischemic heart disease (IHD) in men [relative risk (RR) = 2.31 for subjects who ate beef > or =3 times/wk compared with vegetarians], significant protective associations between nut consumption and fatal and nonfatal IHD in both sexes (RR approximately 0.5 for subjects who ate nuts > or =5 times/wk compared with those who ate nuts <1 time/wk), and reduced risk of IHD in subjects preferring whole-grain to white bread. The lifetime risk of IHD was reduced by approximately 31% in those who consumed nuts frequently and by 37% in male vegetarians compared with nonvegetarians. Cancers of the colon and prostate were significantly more likely in nonvegetarians (RR of 1.88 and 1.54, respectively), and frequent beef consumers also had higher risk of bladder cancer. Intake of legumes was negatively associated with risk of colon cancer in nonvegetarians and risk of pancreatic cancer. Higher consumption of all fruit or dried fruit was associated with lower risks of lung, prostate, and pancreatic cancers. Cross-sectional data suggest vegetarian Seventh-day Adventists have lower risks of diabetes mellitus, hypertension, and arthritis than nonvegetarians. Thus, among Seventh-day Adventists, vegetarians are healthier than nonvegetarians but this cannot be ascribed only to the absence of meat.


Personalized approach for type 2 diabetes pharmacotherapy: where are we and where do we need to be?
Cluster analysis has identified distinct groups of type 2 diabetes (T2D) subjects with distinct metabolic characteristics. Thus, personalizing pharmacologic therapy to individual phenotypic and pathophysiologic characteristics has potential to improve metabolic control and reduce risk of microvascular and macrovascular complications. The authors review the classification of T2D, genetic markers, pathophysiology and natural history of T2D, the ABCDE approach to therapy, the ADA/EASD stepwise approach to therapy, available antidiabetic agents, and provide a more rational therapeutic approach based upon pathophysiology and cardiovascular and renal outcome trials. Although insulin resistance is the earliest detectable abnormality, overt T2D does not occur in the absence of progressive beta cell failure. Because of the complex etiology of T2D (Ominous Octet), initiation of therapy with combined agents that (i) target both insulin resistance and beta cell dysfunction and (ii) prevent macrovascular, as well as microvascular, complications will be required. The ratio of C-peptide at 120 minutes (OGTT) to baseline C-peptide predicts with high sensitivity who will respond to metformin, the response to glucose-lowering agents and provides a useful tool to guide optimal glucose lowering therapy.


A 90-day toxicity study of GmTMT transgenic maize in Sprague-Dawley rats.
GmTMT transgenic maize is a genetically modified maize plant that overexpresses the γ-tocopherol methyltransferase (γ-TMT) from Glycine max (Gm). The γ-TMT gene was introduced into maize line Zhen58 to encode the GmTMT2a protein which can convert γ-tocopherol into α-tocopherol. Overexpression of GmTMT2a significantly increased the α-tocopherol content in transgenic maize. The present study was designed to investigate any potential effects of GmTMT maize grain in a 90-day subchronic rodent feeding study. Maize grains from GmTMT or Zhen58 were incorporated into rodent diets at low (12.5%), medium (25%) or high (50%) concentrations and administered to Sprague-Dawley rats (n = 10/sex/group) for 90 days. The negative control group of rats (n = 10/sex/group) were fed with common maize diets. Results from body weights, feed consumption, clinical chemistry, hematology, absolute and relative organ weights indicated no treatment-related side effects of GmTMT maize grain on rats in comparison with rats consuming diets containing Zhen58 maize grain. In addition, no treatment-related changes were found in necropsy and histopathology examinations. Altogether, our data indicates that GmTMT transgenic maize is as safe and nutritious as its conventional non-transgenic maize.


Development and validation of a novel nomogram predicting clinically significant prostate cancer in biopsy-naive men based on multi-institutional analysis.
Prediction of clinically significant prostate cancer (csPCa) is essential to select biopsy-naive patients for prostate biopsy. This study was to develop and validate a nomogram based on clinicodemographic parameters and exclude csPCa using prostate-specific antigen density (PSAD) stratification. Independent predictors were determined via univariate and multivariate logistic analysis and adopted for developing a predictive nomogram, which was assessed in terms of discrimination, calibration, and net benefit. Different PSAD thresholds were used for deciding immediate biopsies in patients with Prostate Imaging-Reporting and Data System (PI-RADS) 3 lesions. A total of 932 consecutive patients who underwent ultrasound-guided transperineal cognitive biopsy were enrolled in our study. In the development cohort, age (odds ratio [OR], 1.075; 95% confidence interval [CI], 1.036-1.114), PSAD (OR, 6.003; 95% CI, 2.826-12.751), and PI-RADS (OR, 3.419; 95% CI, 2.453-4.766) were significant predictors for csPCa. On internal and external validation, this nomogram showed high areas under the curve of 0.943, 0.922, and 0.897, and low Brier scores of 0.092, 0.102, and 0.133 and insignificant unreliability tests of 0.713, 0.490, and 0.859, respectively. Decision curve analysis revealed this model could markedly improve clinical net benefit. The probability of excluding csPCa was 98.51% in patients with PI-RADS 3 lesions and PSAD <0.2 ng/ml2 . This novel nomogram including age, PSAD, and PI-RADS could be applied to accurately predict csPCa, and 44.08% of patients with equivocal imaging findings plus PSAD <0.2 ng/ml2 could safely forgo biopsy.


Differential transcription attenuation of rabies virus genes by intergenic regions: generation of recombinant viruses overexpressing the polymerase gene.
Gene expression of nonsegmented negative-sense RNA viruses involves sequential synthesis of monocistronic mRNAs and transcriptional attenuation at gene borders resulting in a transcript gradient. To address the role of the heterogeneous rabies virus (RV) intergenic regions (IGRs) in transcription attenuation, we constructed bicistronic model RNAs in which two reporter genes are separated by the RV N/P gene border. Replacement of the 2-nucleotide (nt) N/P IGR with the 5-nt IGRs from the P/M or M/G border resulted in attenuation of downstream gene transcription to 78 or 81%, respectively. A severe attenuation to 11% was observed for the 24-nt G/L border. This indicated that attenuation in RV is correlated with the length of the IGR, and, in particular, severe downregulation of the L (polymerase) gene by the 24 nt IGR. By reverse genetics, we recovered viable RVs in which the strongly attenuating G/L gene border of wild-type (wt) RV (SAD L16) was replaced with N/P-derived gene borders (SAD T and SAD T2). In these viruses, transcription of L mRNA was enhanced by factors of 1.8 and 5.1, respectively, resulting in exaggerated general gene expression, faster growth, higher virus titers, and induction of cytopathic effects in cell culture. The major role of the IGR in attenuation was further confirmed by reintroduction of the wt 24-nt IGR into SAD T, resulting in a ninefold drop of L mRNA. The ability to modulate RV gene expression by altering transcriptional attenuation is an advantage in the study of virus protein functions and in the development of gene delivery vectors.


Integrated approach to the assessment of long range correlation in time series data
To assess whether a given time series can be modeled by a stochastic process possessing long range correlation, one usually applies one of two types of analysis methods: the spectral method and the random walk analysis. The first objective of this work is to show that each one of these methods used alone can be susceptible to producing false results. We thus advocate an integrated approach which requires the use of both methods in a consistent fashion. We provide the theoretical foundation of this approach and illustrate the main ideas using examples. The second objective relates to the observation of long range anticorrelation (Hurst exponent H < 1/2) in real world time series data. The very peculiar nature of such processes is emphasized in light of the stringent condition under which such processes can occur. Using examples, we discuss the possible factors that could contribute to the false claim of long range anticorrelations, and demonstrate the particular importance of the integrated approach in this case.


Complications in the use of the halo fixation device.
The medical records of 179 patients were reviewed to identify complications related to the use of the halo external skeletal-fixation device. The complications that were identified included pin-loosening in 36 per cent of the patients, pin-site infection in 20 per cent, pressure sores under either a plastic vest or a plaster cast in 11 per cent, nerve injury in 2 per cent, dural penetration in 1 per cent, dysphagia in 2 per cent, cosmetically disfiguring scars in 9 per cent, and severe pin discomfort in 18 per cent. One hundred and eighty (25 per cent) of the 716 pins used had become loose at least once, and an infection had developed at sixty-seven pin sites (9 per cent). Two-thirds of the pins that were loose or associated with infection required change or removal. These complication rates, particularly of pin-loosening and infection, are exceedingly high. Attention to details in pin application, pin maintenance, and proper pin-site care may minimize the number of complications.


Directed Evolution of a Designer Enzyme Featuring an Unnatural Catalytic Amino Acid.
The impressive rate accelerations that enzymes display in nature often result from boosting the inherent catalytic activities of side chains by their precise positioning inside a protein binding pocket. Such fine-tuning is also possible for catalytic unnatural amino acids. Specifically, the directed evolution of a recently described designer enzyme, which utilizes an aniline side chain to promote a model hydrazone formation reaction, is reported. Consecutive rounds of directed evolution identified several mutations in the promiscuous binding pocket, in which the unnatural amino acid is embedded in the starting catalyst. When combined, these mutations boost the turnover frequency (kcat ) of the designer enzyme by almost 100-fold. This results from strengthening the catalytic contribution of the unnatural amino acid, as the engineered designer enzymes outperform variants, in which the aniline side chain is replaced with a catalytically inactive tyrosine residue, by more than 200-fold.


Social Media and Total Joint Arthroplasty: An Analysis of Patient Utilization on Instagram.
The purpose of this study was to analyze the nature of shared content of total joint arthroplasty patients on Instagram. Specifically, we evaluated social media posts for: (1) perspective and timing; (2) tone; (3) focus (activities of daily living [ADLs], rehabilitation, return-to-work); and (4) the comparison between hip and knee arthroplasties. A search of the public Instagram domain was performed over a 6-month period. Total hip and knee arthroplasties (THA and TKA) were selected for the analysis using the following terms: "#totalhipreplacement," "#totalkneereplacement," and associated terms. 1287 individual public posts of human subjects were shared during the period. A categorical scoring system was utilized for media format (photo or video), time (preoperative, perioperative, or postoperative) period, tone (positive or negative), return-to-work, ADLs, rehabilitation, surgical site, radiograph image, satisfaction, and dissatisfaction. Ninety-one percent of the posts were shared during the postoperative period. Ninety-three percent of posts had a positive tone. Thirty-four percent of posts focused on both ADLs and 33.8% on rehabilitation. TKA patients shared more about their surgical site (14.5% vs 3.3%, P < .001) and rehabilitation (58.9% vs 8.8%, P < .001) than THA patients, whereas THA patients shared more about ADLs than TKA patients (60.5% vs 7.6%, P < .001). When sharing their experience on Instagram, arthroplasty patients did so with a positive tone, starting a week after surgery. TKA posts focused more on rehabilitation and wound healing than THA patients, whereas THA patients shared more posts on ADLs. The analysis of social media posts provides insight into what matters to patients after total joint arthroplasty.


Circulation of Crimean-Congo Hemorrhagic Fever Virus in the former Yugoslav Republic of Macedonia revealed by screening of cattle sera using a novel enzyme-linked immunosorbent assay.
There are only few assays available for the detection of Crimean-Congo Hemorrhagic Fever Virus (CCHFV)-specific antibodies in animals, and data about diagnostic sensitivity and specificity are incompletely documented for most of these tests. This is unfortunate since CCHFV antibodies in animals can be used as indicator for virus circulation in a geographic area and therewith potential risk of human exposure. This paper therefore reports on a novel ELISA for the detection of CCHFV-specific antibodies in cattle and on its application for testing ruminant sera from the Former Yugoslav Republic of Macedonia. A highly sensitive and specific ELISA was developed to detect CCHFV-specific IgG antibodies in cattle. The assay was validated by using 503 negative serum samples from a country where CCHFV has never been detected until now, and by using 54 positive serum samples. The positive sera were verified by using two commercially available assays (for testing human serum) which we have adapted for use in animals. The sensitivity of the novel ELISA was 98% and its specificity 99%. The presence of Hyalomma ticks was demonstrated in the Former Yugoslav Republic of Macedonia and depending on the region antibody prevalence rates up to 80% were detected in the cattle population. This article describes a fully validated, highly sensitive and specific ELISA for the detection of CCHFV-specific IgG antibodies in cattle. Using this assay, CCHFV-specific antibodies were detected for the first time in cattle in the Former Yugoslav Republic of Macedonia, giving evidence for an active circulation of this virus in the country. Supporting this conclusion, the occurrence of the main vector of CCHFV was demonstrated in the present work for the first time in Former Yugoslav Republic of Macedonia.


Effect of psychrotrophic growth on the milk fat fraction at different temperatures of storage.
The use of cooling, without using adequate hygienic practices in primary milk production, allows for the growth of psychrotrophic microorganisms that produce the thermoresistant lipases that give milk a rancid flavor. This study aimed to verify how the variation in temperature influences the lipolytic metabolism of the psychrotrophic organisms. Samples of raw milk were collected and submitted to laboratorial analysis as follows: psychrotrophic bacteria count, lipolytic bacteria count, and free fatty acids dosage. Each sample was divided into 3 aliquots and then incubated at 4, 8, and 12 °C, respectively. For each temperature, analyses were repeated after 12, 24, and 48 h of storage. Despite the psychrotrophs growth increase, according to temperature rise, the lipolytic metabolism was not consistent and presented the lower index at 8 °C, suggesting an intensification of the proteolytic compensatory activity at this temperature.


Anxiety and depressive symptom identification using the Duke Health Profile.
Duke Health Profile (DUKE) subscales were compared for their ability to identify anxiety and depressive symptoms as measured by the State Anxiety Inventory (SAI) and the Center for Epidemiologic Studies Depression Scale (CES-D) in 413 primary care patients. The seven-item Duke Anxiety-Depression Scale (DUKE-AD) was the best symptom identifier, with sensitivities and specificities greater than 70% for high scores on both the SAI and CES-D. Also, baseline DUKE-AD scores predicted five clinical outcomes during an 18-month follow-up period, with receiver operating characteristic (ROC) curve areas ranging from 57.1 to 58.7%. Patients shown by DUKE-AD scores to be at high risk (>30, scale 0-100) for symptoms of anxiety and/or depression were more often women, less well-educated, not working, and with lower socioeconomic status. The severity of illness was higher than that of low-risk patients. Although the providers did not know which patients were at high risk, they made a clinical diagnosis of anxiety or depression more often in high-risk patients.


[The use of L-tryptophan for correction of disbalance of central neurotransmitters in morphine withdrawal syndrome].
The influence of tryptophan on the content of central neurotransmitters (free amino acids and biogenous amines) in various structures of the rat brain under the conditions of morphine withdrawal has been studied. It is established that the chronic morphine intoxication followed by withdrawal lead to activation of the dopaminergic system and to an increase in the content of exciting amino acids in the rat brain. The morphine withdrawal syndrome is accompanied by inhibition of the serotoninergic activity in all rat brain structures studied. The administration of L-tryptophan (100 mg/kg, i.p.) on the morphine withdrawal syndrome activates the central serotoninergic structures and prevents from further increase in the content of exciting amino acids in the brain.


Directional preference of airway smooth muscle mass increase in human asthmatic airways.
Airway smooth muscle (ASM) orientation and morphology determine the ability of the muscle to constrict the airway. In asthma, ASM mass is increased, but it is unknown whether ASM orientation and morphology are altered as well or whether the remodeling at the source of the mass increase is ongoing. We dissected human airway trees from asthmatic and control lungs. Stained, intact airway sections were imaged in axial projection to show ASM bundle orientation, whereas cross-sectional histological slides were used to assess ASM area, bundle thickness, and ASM bundle-to-basement membrane distance. We also used these slides to assess cell size, proliferation, and apoptosis. We showed that ASM mass increase in cartilaginous airways is primarily the result of an increase of ASM bundle thickness (as measured radially in an airway cross section) and coincides with an increased distance of the ASM bundles to the airway perimeter. ASM orientation was unchanged in all airways. Apoptosis markers and cell size did not show differences between asthmatics and controls. Our findings show that ASM mass increase likely contributes to the airway-constricting capacity of the muscle. Both the increased bundle thickness and increased thickness of the airway wall inwards of the ASM bundles could further enhance this capacity. Turnover of ASM appears to be the same in airways and biopsies, but the lack of correlation between different markers of proliferation casts doubt on the specificity of markers generally used to assess proliferation.


[The evidence-based neurointensive care: what's next?].
The work is devoted to the analysis of modern trends of process of cognition in neurointensive care. In particular, considered the emergence and development of the method of "evidence-based medicine" in the treatment of patients with diseases and injuries of the brain. Demonstrated the positive features of the method. However, the authors are focused on the limitations of evidence-based approach, the main of which are the lack of an individualized treatment plan and the paucity of information obtained from materials of randomized controlled trials. The authors propose to combine the "evidence" data and the "classical style" of treatment, which involves individual clinical judgment based on knowledge of the pathophysiology and pathomorphology, intelligent use of technology, retrospective analysis of the beneficial and adverse effects of certain methods, creation of a special data bank of "natural" course of events, the development of individual prognostic model. The authors believe that the use of standards is more appropriate during acute illness and trauma, and the classic style is better for chronic situation.


Gene expression profiles of gliomas in formalin-fixed paraffin-embedded material.
We have recently demonstrated that expression profiling is a more accurate and objective method to classify gliomas than histology. Similar to most expression profiling studies, our experiments were performed using fresh frozen (FF) glioma samples whereas most archival samples are fixed in formalin and embedded in paraffin (FFPE). Identification of the same, expression-based intrinsic subtypes in FFPE-stored samples would enable validation of the prognostic value of these subtypes on these archival samples. In this study, we have therefore determined whether the intrinsic subtypes identified using FF material can be reproduced in FFPE-stored samples. We have performed expression profiling on 55 paired FF-FFPE glioma samples using HU133 plus 2.0 arrays (FF) and Exon 1.0 ST arrays (FFPE). The median time in paraffin of the FFPE samples was 14.1 years (range 6.6-26.4 years). In general, the correlation between FF and FFPE expression in a single sample was poor. We then selected the most variable probe sets per gene (n=17,583), and of these, the 5000 most variable probe sets on FFPE expression profiles. This unsupervised selection resulted in a better concordance (R(2)=0.54) between expression of FF and FFPE samples. Importantly, this probe set selection resulted in a correct assignment of 87% of FFPE samples into one of seven intrinsic subtypes identified using FF samples. Assignment to the same molecular cluster as the paired FF tissue was not correlated to time in paraffin. We are the first to examine a large cohort of paired FF and FFPE samples. We show that expression data from FFPE material can be used to assign samples to intrinsic molecular subtypes identified using FF material. This assignment allows the use of archival material, including material derived from large-randomised clinical trials, to determine the predictive and/or prognostic value of 'intrinsic glioma subtypes' on Exon arrays. This would enable clinicians to provide patients with an objective and accurate diagnosis and prognosis, and a personalised treatment strategy.


COX-2-specific inhibitors and the kidney: effect on hypertension and oedema.
Recent studies have investigated the renal and cardiovascular safety of celecoxib and rofecoxib. Both agents have been studied in long-term safety trials: the Celecoxib Long-Term Safety Study (CLASS) and Vioxx Gastrointestinal Outcomes Research Study (VIGOR). Renal safety was investigated in CLASS and the results indicated that celecoxib (even at the supratherapeutic 800 mg daily doses used in CLASS) is associated with lower rates of renal side effects than conventional non-steroidal anti-inflammatory drugs (NSAIDs). The renal safety of celecoxib was also assessed in healthy elderly subjects. In these subjects, celecoxib administration was associated with minimal effects on glomerular filtration rate compared with naproxen, while urinary sodium and prostaglandin E2 excretion were similar. Celecoxib and rofecoxib were compared with each other in two studies of elderly hypertensive patients with osteoarthritis (OA). These patients were treated with usual therapeutic doses of celecoxib (200 mg daily) or rofecoxib (25 mg daily). In the first study, celecoxib treatment had a significantly lower incidence of peripheral oedema than rofecoxib (4.9% versus 9.5%, P = 0.014). The second study confirmed the results of the initial study: the incidence of oedema was again lower with celecoxib than with rofecoxib (4.7% versus 7.7%, P < 0.05). Rofecoxib use was also associated with significantly greater mean and clinically relevant increases in systolic blood pressure than celecoxib. The CLASS study, which evaluated cardiovascular safety, showed that celecoxib treatment was not associated with an increase in myocardial infarction (MI) compared with non-selective NSAIDs (incidence in all patients was 0.3% for celecoxib and 0.3% for conventional NSAIDs). In a 12-week study of celecoxib (the Successive Celecoxib Efficacy and Safety Studies, SUCCESS-1) in more than 13000 patients with OA, combined doses of celecoxib (200 mg and 400 mg daily) and conventional NSAIDs (naproxen 1000 mg daily and diclofenac 100 mg daily) were again associated with similar rates of cardiovascular adverse events, including MI. However, in the VIGOR trial, treatment with rofecoxib was associated with a significantly higher rate of MI compared with naproxen (0.4% compared with 0.1% in the naproxen group, P < 0.05). In summary, celecoxib may have safety and tolerability advantages compared with non-selective NSAIDs studied and may have some cardiorenal benefits compared with rofecoxib.


Simulating flow induced migration in vascular remodelling.
Shear stress induces directed endothelial cell (EC) migration in blood vessels leading to vessel diameter increase and induction of vascular maturation. Other factors, such as EC elongation and interaction between ECs and non-vascular areas are also important. Computational models have previously been used to study collective cell migration. These models can be used to predict EC migration and its effect on vascular remodelling during embryogenesis. We combined live time-lapse imaging of the remodelling vasculature of the quail embryo yolk sac with flow quantification using a combination of micro-Particle Image Velocimetry and computational fluid dynamics. We then used the flow and remodelling data to inform a model of EC migration during remodelling. To obtain the relation between shear stress and velocity in vitro for EC cells, we developed a flow chamber to assess how confluent sheets of ECs migrate in response to shear stress. Using these data as an input, we developed a multiphase, self-propelled particles (SPP) model where individual agents are driven to migrate based on the level of shear stress while maintaining appropriate spatial relationship to nearby agents. These agents elongate, interact with each other, and with avascular agents at each time-step of the model. We compared predicted vascular shape to real vascular shape after 4 hours from our time-lapse movies and performed sensitivity analysis on the various model parameters. Our model shows that shear stress has the largest effect on the remodelling process. Importantly, however, elongation played an especially important part in remodelling. This model provides a powerful tool to study the input of different biological processes on remodelling.


Quantitative subsurface imaging in strongly scattering media.
We present a method to obtain quantitatively accurate images of small obstacles or inhomogeneities situated near the surface of a strongly scattering medium. The method uses time-resolved measurements of backscattered light to form the images. Using the asymptotic solution of the radiative transfer equation for this problem, we determine that the key information content in measurements is modeled by a diffusion approximation that is valid for small source-detector distances, and shallow penetration depths. We simplify this model further by linearizing the effect of the inhomogeneities about the known background optical properties using the Born approximation. The resulting model is used in a two-stage imaging algorithm. First, the spatial location of the inhomogeneities are determined using a modification of the multiple signal classification (MUSIC) method. Using those results, we then determine the quantitative values of the inhomogeneities through a least-squares approximation. We find that this two-stage method is most effective for reconstructing a sequence of one-dimensional images along the penetration depth corresponding to null source-detector separations rather than simultaneously using measurements over several source-detector distances. This method is limited to penetration depths and distances between boundary measurements on the order of the scattering mean-free path.