The combination of amoxicillin-clavulanic acid and ketoconazole in the treatment of Madurella mycetomatis eumycetoma and Staphylococcus aureus co-infection.
Eumycetoma is a chronic progressive disabling and destructive inflammatory disease which is commonly caused by the fungus Madurella mycetomatis. It is characterized by the formation of multiple discharging sinuses. It is usually treated by antifungal agents but it is assumed that the therapeutic efficiency of these agents is reduced by the co-existence of Staphylococcus aureus co-infection developing in these sinuses. This prospective study was conducted to investigate the safety, efficacy and clinical outcome of combined antibiotic and antifungal therapy in eumycetoma patients with superimposed Staphylococcus aureus infection. The study enrolled 337 patients with confirmed M. mycetomatis eumycetoma and S. aureus co-infection. Patients were allocated into three groups; 142 patients received amoxicillin-clavulanic acid and ketoconazole, 93 patients received ciprofloxacin and ketoconazole and 102 patients received ketoconazole only. The study showed that, patients who received amoxicillin-clavulanic acid and ketoconazole treatment had an overall better clinical outcome compared to those who had combined ciprofloxacin and ketoconazole or to those who received ketoconazole only. In this study, 60.6% of the combined amoxicillin-clavulanic acid/ketoconazole group showed complete or partial clinical response to treatment compared to 30.1% in the ciprofloxacin/ketoconazole group and 36.3% in the ketoconazole only group. The study also showed that 64.5% of the patients in the ciprofloxacin/ketoconazole group and 59.8% in the ketoconazole only group had progressive disease and poor outcome. This study showed that the combination of amoxicillin-clavulanic acid and ketoconazole treatment is safe and offers good clinical outcome and it is therefore recommended to treat eumycetoma patients with Staphylococcus aureus co-infection.


The effect of CD4 receptor downregulation and its downstream signaling molecules on HIV-1 latency.
HIV-1 can establish a latent infection in memory CD4+T cells to evade the host immune response. CD4 molecules can act not only as the HIV-1 receptor for entry but also as the trigger in an intracellular signaling cascade for T-cell activation and proliferation via protein tyrosine kinases. Novel chronic HIV-1-infected A3.01-derived (NCHA) cells were used to examine the involvement of CD4 downstream signaling in HIV-1 latency. CD4 receptors in NCHA cells were dramatically downregulated on its surface but were slightly decreased in whole-cell lysates. The expression levels of CD4 downstream signaling molecules, including P56(Lck), ZAP-70, LAT, and c-Jun, were sharply decreased in NCHA cells. The lowered histone modifications of H3K4me3 and H3K9ac correlated with the downregulation of P56(Lck), ZAP-70, and LAT in NCHA cells. AP-1 binding activity was also reduced in NCHA cells. LAT and c-Jun suppressed in NCHA cells were highly induced after PMA treatment. In epigenetic analysis, other signal transduction molecules which are associated with active and/or latent HIV-1 infection showed normal states in HIV-1 latently infected cells compared to A3.01 cells. In conclusion, we demonstrated that the HIV-1 latent state is sustained by the reduction of downstream signaling molecules via the downregulation of CD4 and the attenuated activity of transcription factor as AP-1. The HIV-1 latency model via T-cell deactivation may provide some clues for the development of the new antireservoir therapy.


Long-term multimodal imaging in acute posterior multifocal placoid pigment epitheliopathy and association with coxsackievirus exposure.
The purpose of this study was to evaluate potential insights into the pathogenesis of acute posterior multifocal placoid pigment epitheliopathy (APMPPE) using multimodal diagnostic imaging and laboratory evaluation in long-term follow-up. A retrospective, single-center case series was conducted on seven consecutive patients (14 eyes) who were given a diagnosis of APMPPE from March 1, 2011, through June 30, 2019 with at least three months of follow-up. Clinical characteristics (age, symptoms, visual acuity [VA]), laboratory testing including coxsackievirus titers, and multimodal imaging from fundus photography, spectral-domain optical coherence tomography (SD-OCT), fundus autofluorescence (FAF), fluorescein angiography (FA), and indocyanine green angiography (ICG) were analyzed for each patient. The initial median VA was 20/71 and final median VA was 20/22. Coxsackievirus B (CVB) titers were elevated (≥ 1:80) in six of seven patients, with a four-fold increase in convalescent titers seen in two patients suggestive of recent infection. All patients were treated with oral corticosteroids, and five patients underwent corticosteroid-sparing immunomodulatory therapy. Initially, multifocal deep choroidal lesions were observed in the posterior pole corresponding to patches of hypocyanescence on ICG. Overlying retinal pigment epithelium (RPE) disease was observed on FAF, although this finding was not universally observed, suggesting that RPE disease may occur as a sequelae to unchecked choroidal inflammation. SD-OCT architectural changes confirmed outer retina and ellipsoid zone disruption. FA of active lesions showed early hypofluorescence and late hyperfluorescence with surrounding leakage while inactive disease showed areas of staining. Long-term follow-up of multimodal diagnostic imaging in APMPPE revealed that choroidal inflammation likely precedes RPE change and photoreceptor damage. Elevation of coxsackievirus titers with seroconversion may be associated with an infectious trigger in concert with immune-mediated disease in this posterior uveitis syndrome.


Characterization of Arabica and Robusta Coffees by Ion Mobility Sum Spectrum.
Aroma is one of the main characteristics of coffee specimens. Different mixtures of Arabica and Robusta coffees are usually found in the market to offer specific aroma or flavor profiles to consumers. However, the mixed samples or their proportions are not always identified in the product labels. Since the price of Arabica is much higher than that of Robusta, this lack of information is not only an economical issue but a possible fraud to consumers, besides the potential allergic reaction that these mixtures may trigger in some individuals. In this paper, two sample preparation techniques were compared before the analysis of the total volatile organic compounds (VOCs) found in Robusta, Arabica, and in the mixture from both coffee types. The comparison of the signals obtained from the analyses showed that the VOCs concentration levels obtained from the headspace (HS) analyses were clearly higher than those obtained from the pre-concentration step where an adsorbent, an active charcoal strip (ACS + HS), was used. In the second part of this study, the possibility of using the headspace gas-chromatography ion mobility spectrometry (HS-GC-IMS) for the discrimination between Arabica, Robusta, and mixed coffee samples (n = 30) was evaluated. The ion mobility sum spectrum (IMSS) obtained from the analysis of the HS was used in combination with pattern recognition techniques, namely linear discrimination analysis (LDA), as an electronic nose. The identification of individual compounds was not carried out since chromatographic information was not used. This novel approach allowed the correct discrimination (100%) of all of the samples. A characteristic fingerprint for each type of coffee for a fast and easy identification was also developed. In addition, the developed method is ecofriendly, so it is a good alternative to traditional approaches.


Using expert models in human reliability analysis-a dependence assessment method based on fuzzy logic.
In human reliability analysis (HRA), dependence analysis refers to assessing the influence of the failure of the operators to perform one task on the failure probabilities of subsequent tasks. A commonly used approach is the technique for human error rate prediction (THERP). The assessment of the dependence level in THERP is a highly subjective judgment based on general rules for the influence of five main factors. A frequently used alternative method extends the THERP model with decision trees. Such trees should increase the repeatability of the assessments but they simplify the relationships among the factors and the dependence level. Moreover, the basis for these simplifications and the resulting tree is difficult to trace. The aim of this work is a method for dependence assessment in HRA that captures the rules used by experts to assess dependence levels and incorporates this knowledge into an algorithm and software tool to be used by HRA analysts. A fuzzy expert system (FES) underlies the method. The method and the associated expert elicitation process are demonstrated with a working model. The expert rules are elicited systematically and converted into a traceable, explicit, and computable model. Anchor situations are provided as guidance for the HRA analyst's judgment of the input factors. The expert model and the FES-based dependence assessment method make the expert rules accessible to the analyst in a usable and repeatable way, with an explicit and traceable basis.


Major groove substituents and polymerase recognition of a class of predominantly hydrophobic unnatural base pairs.
Expansion of the genetic alphabet with an unnatural base pair is a long-standing goal of synthetic biology. We have developed a class of unnatural base pairs, formed between d5SICS and analogues of dMMO2 that are efficiently and selectively replicated by the Klenow fragment (Kf) DNA polymerase. In an effort to further characterize and optimize replication, we report the synthesis of five new dMMO2 analogues bearing different substituents designed to be oriented into the developing major groove and an analysis of their insertion opposite d5SICS by Kf and Thermus aquaticus DNA polymerase I (Taq). We also expand the analysis of the previously optimized pair, dNaM-d5SICS, to include replication by Taq. Finally, the efficiency and fidelity of PCR amplification of the base pairs by Taq or Deep Vent polymerases was examined. The resulting structure-activity relationship data suggest that the major determinants of efficient replication are the minimization of desolvation effects and the introduction of favorable hydrophobic packing, and that Taq is more sensitive than Kf to structural changes. In addition, we identify an analogue (dNMO1) that is a better partner for d5SICS than any of the previously identified dMMO2 analogues with the exception of dNaM. We also found that dNaM-d5SICS is replicated by both Kf and Taq with rates approaching those of a natural base pair.


Effects of Conjugated Linoleic Acid Intake in the Form of Dietary Supplement or Enriched Food on C-Reactive Protein and Lipoprotein (a) Levels in Humans: A Literature Review and Meta-Analysis.
C-reactive protein (CRP) and lipoprotein (a) (Lp[a]) play essential roles in cardiovascular disease incidence. This study aimed to review the association between the intake of conjugated linoleic acid (CLA) in the form of dietary supplement or enriched food with different treatment durations and the levels of Lp(a) and CRP in human studies. All the articles published in Cochrane Library, ProQuest, Scopus, and Google Scholar from November 2014 to October 2015 were searched and the clinical trials on the effects of CLA on Lp(a) and CRP levels were assessed. Of the 2249 articles initially retrieved, 21 eligible randomized clinical trials were enrolled in this systematic review. The publication dates of the eligible articles ranged from 2005 to 2013. The mean difference and the standard deviation of changes in CRP and Lp(a) levels in intervention and control groups were used as effect-size measures for meta-analysis. The obtained data from the eligible randomized controlled trials were meta-analyzed using Stata, version 13. The intake of CLA as a dietary supplement led to a significant increase in CRP levels (standardized mean difference [SMD]=0.41, 95% CI: 0.28 to 0.54; P=0.001). Subgroup analysis based on the duration of CLA consumption showed that CLA consumption more than 24 weeks resulted in a significant increase in the levels of CRP (SMD=0.52, 95% CI: 0.36 to 0.68; P=0.001) and Lp(a) (SMD=0.24, 95% CI: 0.01 to 0.47; P=0.04). The current systematic review and meta-analysis showed that the long-term consumption of CLA increases the levels of CRP and Lp(a).


The quick response initiative in the emergency department: who benefits?
This collaborative project between two community hospitals, a Metropolitan Home Care Program and the University, was designed to quantify the applicability (who is eligible for) and acceptability (who will likely comply with) Home Care services, provided through a Quick Response Program (QRP) initiative as compared to usual hospital care services, to patients, families and physicians. During a 6 week period of sampling (5 days per week, 8 hours per day) in two Emergency Departments (ED) in moderately sized community hospitals in a major metropolitan city, all patients triaged to the urgent category were assessed for eligibility for QRP services by on-site Home Care Coordinators using specific criteria. Patients meeting the criteria initially were reviewed by the ED physician for approval for QRP services and then randomized to experimental and control conditions. Patients not meeting the eligibility criteria were managed by the usual ED services procedures. Demographic and clinical data were obtained on all urgent category patients at presentation to the ED. Additionally, the nature and cost of all health care services used by the ED patients during the ED event and 10 days follow up, were obtained through hospital and Home Care record abstraction and compared among the different sample groups. The QRP Initiative was applicable to 2% of the total ED patient population and 5% of the urgent category of patients triaged in the ED. It was acceptable to 97% of this eligible group. One hundred and fifty-five patients who initially qualified for QRP were excluded from eligibility at a subsequent assessment. Ninety of these patients were admitted to hospital and 65 were discharged home. In the total "exclusion" group, 37 refused Home Care services including the QRP. HEALTH CARE PRACTICE IMPLICATIONS: The sampling results raise important questions about broader system issues concerning the role of the hospital and community in providing health care services and the social value or utility that guides the allocation of health care funds. What level of applicability and acceptability would justify priority services for certain target groups. In the future, policy makers will need to be able to show that it is in the best interest of patients and society to prioritize mixtures of services to certain target groups.


Autoclaved, previously used intravaginal progesterone devices induces estrus and ovulation in anestrous Toggenburg goats.
Intravaginal progesterone devices are used worldwide for estrus induction in goats. Reused devices are able to induce estrus; however, this can be a health risk within a flock. The objective was to compare new and previously used (and autoclaved) progesterone-releasing intravaginal devices for induction of estrus and ovulation in seasonally anestrous Toggenburg goats. Anestrous goats (n=42) received new intravaginal devices containing 0.3g progesterone (CONTROL), or similar devices previously used for either 6 (USED6) or 12d (USED12) and subsequently autoclaved. All goats received 5mg dinoprost at device insertion and 200 IU eCG 5d later, and all devices were removed after 6d. After device removal, estrus was monitored and females displaying signs of estrus were mated by fertile bucks. Transrectal ovarian ultrasonography was performed after device removal until detection of ovulation. Blood samples were collected for determination of plasma progesterone concentration at different times. There was no difference (P>0.05) among groups CONTROL, USED6 or USED12 for: estrus response (87, 100 or 100%, respectively); duration of estrus (32.3±2.3, 25.2±3.4 or 27.3±4.1h); ovulation rate (100, 88 or 100%); number of ovulations (1.5±0.2, 1.9±0.3 or 1.7±0.3); and pregnancy rate (60, 58 or 67%). Plasma progesterone (P4) concentrations were greater (P<0.05) in CONTROL than in USED6-treated and USED12-treated goats (7.2±1.2, 4.7±0.7 and 4.3±0.6 ng/mL, respectively) at 6h after device insertion; these differences were maintained until 4d after device insertion (3.4±0.4, 2.3±0.2, and 2.5±0.2 ng/mL). Overall, plasma progesterone concentrations were greater (P<0.05) in nulliparous than in lactating goats (3.1±0.8 compared to 2.4±0.6 ng/mL, respectively). In conclusion, autoclaved, previously used intravaginal progesterone-releasing devices resulted in significant lesser plasma progesterone concentrations than new devices, but were similarly effective in inducing estrus and ovulation in anestrous goats.


Mycoplasma pneumoniae CARDS toxin exacerbates ovalbumin-induced asthma-like inflammation in BALB/c mice.
Mycoplasma pneumoniae causes a range of airway and extrapulmonary pathologies in humans. Clinically, M. pneumoniae is associated with acute exacerbations of human asthma and a worsening of experimentally induced asthma in mice. Recently, we demonstrated that Community Acquired Respiratory Distress Syndrome (CARDS) toxin, an ADP-ribosylating and vacuolating toxin synthesized by M. pneumoniae, is sufficient to induce an asthma-like disease in BALB/cJ mice. To test the potential of CARDS toxin to exacerbate preexisting asthma, we examined inflammatory responses to recombinant CARDS toxin in an ovalbumin (OVA) murine model of asthma. Differences in pulmonary inflammatory responses between treatment groups were analyzed by histology, cell differentials and changes in cytokine and chemokine concentrations. Additionally, assessments of airway hyperreactivity were evaluated through direct pulmonary function measurements. Analysis of histology revealed exaggerated cellular inflammation with a strong eosinophilic component in the CARDS toxin-treated group. Heightened T-helper type-2 inflammatory responses were evidenced by increased expression of IL-4, IL-13, CCL17 and CCL22 corresponding with increased airway hyperreactivity in the CARDS toxin-treated mice. These data demonstrate that CARDS toxin can be a causal factor in the worsening of experimental allergic asthma, highlighting the potential importance of CARDS toxin in the etiology and exacerbation of human asthma.


Quantifying the impact of molecular defects on polymer network elasticity.
Elasticity, one of the most important properties of a soft material, is difficult to quantify in polymer networks because of the presence of topological molecular defects in these materials. Furthermore, the impact of these defects on bulk elasticity is unknown. We used rheology, disassembly spectrometry, and simulations to measure the shear elastic modulus and count the numbers of topological "loop" defects of various order in a series of polymer hydrogels, and then used these data to evaluate the classical phantom and affine network theories of elasticity. The results led to a real elastic network theory (RENT) that describes how loop defects affect bulk elasticity. Given knowledge of the loop fractions, RENT provides predictions of the shear elastic modulus that are consistent with experimental observations.


LY294002 and metformin cooperatively enhance the inhibition of growth and the induction of apoptosis of ovarian cancer cells.
The phosphoinositide 3 kinase (PI3K)/v-akt murine thymoma viral oncogene homolog (AKT)/mammalian target of rapamycin (mTOR) pathway is frequently aberrantly activated in ovarian cancer and confers the chemoresistant phenotype of ovarian cancer cells. LY294002 (PI3K inhibitor) and metformin (5'-adenosine monophosphate [AMP]-activated protein kinase [AMPK] activator) are 2 drugs that were known to inhibit mTOR expression through the AKT-dependent and AKT-independent pathways, respectively. In this study, we explored the effectiveness of LY294002 and metformin in combination on inhibition of ovarian cancer cell growth. Western blotting was used to detect the changes of PI3K/AKT/mTOR and AMPK/acetyl-CoA carboxylase (ACC) signaling activities, cell cycle control, and apoptosis. Cell growth was evaluated by cell proliferation, colony formation, and soft agar assays. Flow cytometry was used to study cell cycle distribution and cell death upon drug treatment. Our study showed that LY294002 and metformin in combination could simultaneously enhance the repression of the PI3K/AKT/mTOR pathway and the activation of the AMPK/ACC pathway. The downstream target of AKT and AMPK, mTOR, was cooperatively repressed when the drugs were used together. The cell cycle regulatory factors, p53, p27, and p21, were up-regulated. On the other hand, caspase 3 and poly (ADP-ribose) polymerase activities involved in apoptosis were also activated. Cell growth assays indicated that LY294002 and metformin could effectively inhibit ovarian cancer cell growth. Flow cytometry analysis showed that the treatment of the 2 drugs mentioned above induced cell cycle arrest at G1 phase and increased sub-G1 apoptotic cells. The combinational use of LY294002 and metformin can enhance inhibition of the growth and induction of the apoptosis of ovarian cancer cells. Our results may provide significant insight into the future therapeutic regimens in ovarian cancer.


[The unexpectedly difficult airway -- a plea for the oxford-non-kinking-tube].
The skill to safely manage the unexpectedly difficult airway is expected from every anaesthetist. The strategies to safely overcome this severe problem have to be adapted to the given equipment and the individual aptitude and skills of the respective colleague. The algorithms for management of the difficult airway should be as simple as possible, and one cannot assume that devices for fibre-optic intubation are available at every site. Indispensable, however, is the availability of face masks, naso- and oropharyngeal airways and laryngeal mask airways in different sizes at each induction site. This paper is especially devoted to recalling the Oxford non-kinking tube and its specific way of handling, as a lot of cases of unexpectedly difficult airway can be safely managed with this tool. Alternatives to safeguarding the difficult airway are the intubation laryngeal mask airway or the esophago-tracheal combitube. For managing the worst case, the "cannot ventilate - cannot intubate" disaster, instruments for percutaneous punction of the trachea and devices for oxygen insufflation must be readily available in every theatre.


Localization of cyclic nucleotide phosphodiesterase in the multicellular stages of Dictyostelium discoideum.
Cyclic 3',5'-adenosine monophosphate (cAMP) is secreted as the chemotactic signal by aggregating amoebae of the cellular slime mold Dictyostelium discoideum. We have used ultramicrotechniques in the biochemical analysis of cyclic nucleotide phosphodiesterase (PD) distribution in individual aggregates at various stages of development. With handmade constriction pipettes in microliter volumes, sections of lyophilized individuals weighing 20-100 ng could be assayed in a reaction coupled to 5'-nucleotidase. Phosphodiesterase activity was measured at pH 7.5 with 12 microM cAMP, cAMP-PD activity in aggregates ranged from 20-40 mmol/h/kg. In the pseudoplasmodium it had dropped to 5-10 mmol/h/kg and a difference in activity between the anterior prestalk cells and posterior prespore cells began to appear. The utmost posterior sections showed elevated phosphodiesterase from this stage onward. During culmination, activity rose to 40-60 mmol/h/kg associated with the developing stalk, while it declined in the spore mass. The papilla remained constant at 5-10 mmol/h/kg. The pattern of localization in the stalk was the same when cGMP was used as substrate. Extracellular phosphodiesterase inhibitor produced at the aggregation stage was found to reduce the localized activity in the culmination stage by 50-80%, with the most marked inhibition occurring in the center of the papilla. We found no evidence of endogenous heat-stable phosphodiesterase inhibitor within the culminating sorocarp.


Activation of Epstein-Barr virus replication in Hodgkin and Reed-Sternberg cells.
Recent evidence has shown that Hodgkin's disease (HD) is associated with Epstein-Barr virus (EBV) in a substantial number of cases and that in these cases EBV DNA is localized exclusively to Hodgkin and Reed-Sternberg (RS) cells. The virus genome is not silent in RS cells because two EBV latent gene products, latent membrane protein (LMP) and EB early region (EBER) transcripts, have recently been reported to be expressed in RS cells. However, little information is available about the possible activation of EBV replicative genes in HD. This prompted us to investigate HD biopsies from 96 patients for expression of replicative gene products. Cryostat sections were immunostained with monoclonal antibodies to protein BZLF1, which controls the switch between EBV latency and replication, and also to LMP. LMP was demonstrated in RS cells in 47 cases (49%). Three of the LMP-positive cases (6%), but none of the LMP-negative cases, expressed the BZLF1 protein. BZLF1 positively was confined to rare RS cells. These three cases showed no detectable early, virus capsid, or membrane antigens. Our findings show that activation of EBV immediate early genes occurs only infrequently in RS cells, indicating that control of viral latency is not severely impaired in HD patients.


Resveratrol and its synthetic derivatives exert opposite effects on mesothelial cell-dependent angiogenesis via modulating secretion of VEGF and IL-8/CXCL8.
We examined the effect of resveratrol (RVT) and its two derivatives (3,3',4,4'-tetrahydroxy-trans-stilbene and 3,3',4,4',5,5'-hexahydroxy-trans-stilbene) on human peritoneal mesothelial cell (HPMC)-dependent angiogenesis in vitro. To this end, angiogenic activity of endothelial cells (HUVEC, HMVEC, and HMEC-1) was monitored upon their exposure to conditioned medium (CM) from young and senescent HPMCs treated with stilbenes or to stilbenes themselves. Results showed that proliferation and migration of endothelial cells were inhibited in response to indirect (HPMC-dependent) or direct RVT activity. This effect was associated with decreased secretion of VEGF and IL-8/CXCL8 by HPMCs treated with RVT, which confirmed the experiments with recombinant forms of these angiogenic agents. Angiogenic activity of endothelial cells treated with CM from HPMCs exposed to RVT analogues was more effective. Improved migration was particularly evident in cells exposed to CM from senescent HPMCs. Upon direct treatment, RVT derivatives stimulated proliferation (but not migration) of HUVECs, and failed to affect the behaviour of HMVEC and HMEC-1 cells. These compounds stimulated production of VEGF and IL-8/CXCL8 by HPMCs. Studies with neutralizing antibodies against angiogenic factors revealed that augmented angiogenic reactions of endothelial cells exposed to CM from HPMC treated with RVT analogues were related to enhanced production of VEGF and IL-8/CXCL8. Collectively, these findings indicate that RVT and its synthetic analogues divergently alter the secretion of the angiogenic factors by HPMCs, and thus modulate HPMC-dependent angiogenic responses in the opposite directions. This may have implications for the attempts of practical employment of the stilbenes for treatment of pathologies proceeding with abnormal vascularisation of the peritoneal tissue.


LAMP-based assay can rectify the diagnosis of Yersinia pseudotuberculosis infections otherwise missed by serology.
Despite being a well-known but seldom encountered zoonotic pathogen, diagnosis of Yersinia pseudotuberculosis is not necessarily easy. Infected patients occasionally present with various symptoms resembling Kawasaki disease; thus discriminating the two in the acute phase is challenging. In addition to bacterial culture and serology, novel detection methods based on loop-mediated isothermal amplification (LAMP) are reported in the literature. However, the clinical utility of LAMP-based methods in comparison with the other methods is scarcely documented in the literature. To clarify the clinical utility of a LAMP-based method in the diagnosis of Yersinia pseudotuberculosis infection. Inpatients admitted due to suspected Yersinia pseudotuberculosis infection during April 2008 through March 2015 were enrolled. Results of the LAMP-based method as well as culture and serology were collected and compared. Among 16 eligible cases, serology proved positive in 13 (81.3 %) cases, LAMP in eight (50 %) cases, and bacterial culture in four (25 %) cases. No significant difference among the three methods could be proved statistically. Although serology was the most sensitive method, it is known to miss cases such as young patients, whereas LAMP could complement all three cases missed by serology. Furthermore, LAMP can return the test result within a few hours from specimen receipt, whereas serology and bacterial culture requires days to weeks of time. Although second to serology in sensitivity, the LAMP-based method proved its utility in making rapid diagnosis, and serving a complementary role to serology.


Anisakidosis: Perils of the deep.
Anisakidosis, human infection with nematodes of the family Anisakidae, is caused most commonly by Anisakis simplex and Pseudoterranova decipiens. Acquired by the consumption of raw or undercooked marine fish or squid, anisakidosis occurs where such dietary customs are practiced, including Japan, coastal regions of Europe, and the United States. Severe epigastric pain, resulting from larval invasion of the gastric mucosa, characterizes gastric anisakidosis; other syndromes are intestinal and ectopic. Allergic anisakidosis is a frequent cause of foodborne allergies in areas with heavy fish consumption or occupational exposure. Diagnosis and treatment of gastric disease is usually made by a compatible dietary history and visualization and removal of the larva(e) on endoscopy; serologic testing for anti-A. simplex immunoglobulin E can aid in the diagnosis of intestinal, ectopic and allergic disease. Intestinal and/or ectopic cases may require surgical removal; albendazole has been used occasionally. Preventive measures include adequately freezing or cooking fish.


Locus coeruleus spiking differently correlates with S1 cortex activity and pupil diameter in a tactile detection task.
We examined the relationships between activity in the locus coeruleus (LC), activity in the primary somatosensory cortex (S1), and pupil diameter in mice performing a tactile detection task. While LC spiking consistently preceded S1 membrane potential depolarization and pupil dilation, the correlation between S1 and pupil was more heterogeneous. Furthermore, the relationships between LC, S1, and pupil varied on timescales of sub-seconds to seconds within trials. Our data suggest that pupil diameter can be dissociated from LC spiking and cannot be used as a stationary index of LC activity.


A fast and frugal algorithm to strengthen diagnosis and treatment decisions for catheter-associated bacteriuria.
Guidelines for managing catheter-associated urinary tract infection (CAUTI) and asymptomatic bacteria (ASB) are poorly translated into routine care due in part to cognitive diagnostic errors. This study determines if the accuracy for CAUTI and ASB diagnosis and treatment improves after implementation of a fast and frugal algorithm compared with traditional education methods. A pre and post-intervention with contemporaneous comparison site involving inpatient and long term care wards at two regional Veterans Affairs Systems in United States. Participants included 216 internal medicine residents and 16 primary care clinicians. Intervention clinicians received training with a fast and frugal algorithm. Comparison site clinicians received guidelines education. Diagnosis and treatment accuracy compared with a criterion standard was assessed during similar three-month, pre- and post-intervention periods. Sensitivity, specificity, and likelihood ratios were compared for both periods at each site. Bacteriuria management was evaluated against criterion standard in 196 cases pre-implementation and 117 cases post-implementation. Accuracy of bacteriuria management among intervention participants was significantly higher, post-implementation, than those at the comparison site (Intervention: positive likelihood ratio (LR+) = 8.5, specificity = 0.89, 95% confidence interval (CI) = 0.78-1.00; comparison: LR+ = 4.62, specificity (95%CI) = 0.79 (0.63-0.95). Further, improvements at the intervention site were statistically significant (pre-implementation: LR+ = 2.1, specificity (95%CI) = 0.60 (0.50-0.71); post-implementation: LR+ = 8.5, specificity (95%CI) = 0.89 (0.78-1.00). At both sites, there were similar improvements in negative LR from pre- to post-implementation: [Intervention site = 0.28 to 0.08; comparison site = 0.13 to 0.04]. Inappropriate management of ASB declined markedly from 32 (40%) to 3 (11%) cases at the intervention site. A fast and frugal algorithm improves diagnosis and treatment accuracy for CAUTI and reduces inappropriate treatment of ASB. Fast and frugal algorithms that realign diagnostic intuitions and treatment norms can enhance translation of evidence into practice.


Ontogenetic braincase development in Psittacosaurus lujiatunensis (Dinosauria: Ceratopsia) using micro-computed tomography.
Ontogenetic sequences are relatively rare among dinosaurs, with Ceratopsia being one of the better represented clades, and especially among geologically earlier forms, such as Psittacosaurus. Psittacosaurus is a small, bipedal basal ceratopsian abundant in the Lower Cretaceous deposits of Asia, whose cranial and endocranial morphology has been well studied, but only cursory details have been published on the bones surrounding the brain. Using reconstructions created from micro-computed tomography scans of well-preserved skulls from the Barremian-Aptian Yixian Formation, China, we document morphological changes in the braincase of Psittacosaurus lujiatunensis through three growth stages, hatchling, juvenile, and adult, thus providing the first detailed study of ceratopsian braincase morphology through ontogeny. Notable ontogenetic changes in the braincase of P. lujiatunensis include a dramatic relative reduction in size of the supraoccipital, an increase in the lateral expansion of the paroccipital processes and a decrease in the angle between the lateral semicircular canal and the palatal plane. These ontogenetic morphological changes in the braincase relate to expansion of the cranium and brain through growth, as well as reflecting the switch from quadrupedal juveniles to bipedal adults as documented in the changing orientation of the horizontal semicircular canal through ontogeny. Recognition of these patterns in a basal ceratopsian has implications for understanding key events in later ceratopsian evolution, such as the development of the parieto-squamosal frill in derived neoceratopsians.


Adaptive phenotypic plasticity and genetics of larval life histories in two Rana temporaria populations.
Phenotypic plasticity provides means for adapting to environmental unpredictability. In terms of accelerated development in the face of pond-drying risk, phenotypic plasticity has been demonstrated in many amphibian species, but two issues of evolutionary interest remain unexplored. First, the heritable basis of plastic responses is poorly established. Second, it is not known whether interpopulational differences in capacity to respond to pond-drying risk exist, although such differences, when matched with differences in desiccation risk would provide strong evidence for local adaptation. We investigated sources of within- and among-population variation in plastic responses to simulated pond-drying risk (three desiccation treatments) in two Rana temporaria populations originating from contrasting environments: (1) high desiccation risk with weak seasonal time constraint (southern population); and (2) low desiccation risk with severe seasonal time constraint (northern population). The larvae originating from the environment with high desiccation risk responded adaptively to the fast decreasing water treatment by accelerating their development and metamorphosing earlier, but this was not the case in the larvae originating from the environment with low desiccation risk. In both populations, metamorphic size was smaller in the high-desiccation-risk treatment, but the effect was larger in the southern population. Significant additive genetic variation in development rate was found in the northern and was nearly significant in the southern population, but there was no evidence for genetic variation in plasticity for development rates in either of the populations. No genetic variation for plasticity was found either in size at metamorphosis or growth rate. All metamorphic traits were heritable, and additive genetic variances were generally somewhat higher in the southern population, although significantly so in only one trait. Dominance variances were also significant in three of four traits, but the populations did not differ. Maternal effects in metamorphic traits were generally weak in both populations. Within-environment phenotypic correlations between larval period and metamorphic size were positive and genetic correlations negative in both populations. These results suggest that adaptive phenotypic plasticity is not a species-specific fixed trait, but evolution of interpopulational differences in plastic responses are possible, although heritability of plasticity appears to be low. The lack of adaptive response to desiccation risk in northern larvae is consistent with the interpretation that selection imposed by shorter growing season has favored rapid development in north (approximately 8% faster development in north as compared to south) or a minimum metamorphic size at the expense of phenotypic plasticity.


The specificity of extracellular signal-regulated kinase 2 dephosphorylation by protein phosphatases.
The extracellular signal-regulated protein kinase 2 (ERK2) is the founding member of a family of mitogen-activated protein kinases (MAPKs) that are central components of signal transduction pathways for cell proliferation, stress responses, and differentiation. The MAPKs are unique among the Ser/Thr protein kinases in that they require both Thr and Tyr phosphorylation for full activation. The dual phosphorylation of Thr-183 and Tyr-185 in ERK2 is catalyzed by MAPK/ERK kinase 1 (MEK1). However, the identity and relative activity of protein phosphatases that inactivate ERK2 are less well established. In this study, we performed a kinetic analysis of ERK2 dephosphorylation by protein phosphatases using a continuous spectrophotometric enzyme-coupled assay that measures the inorganic phosphate produced in the reaction. Eleven different protein phosphatases, many previously suggested to be involved in ERK2 regulation, were compared, including tyrosine-specific phosphatases (PTP1B, CD45, and HePTP), dual specificity MAPK phosphatases (VHR, MKP3, and MKP5), and Ser/Thr protein phosphatases (PP1, PP2A, PP2B, PP2C alpha, and lambda PP). The results provide biochemical evidence that protein phosphatases display exquisite specificity in their substrate recognition and implicate HePTP, MKP3, and PP2A as ERK2 phosphatases. The fact that ERK2 inactivation could be carried out by multiple specific phosphatases shows that signals can be integrated into the pathway at the phosphatase level to determine the cellular response to external stimuli. Important insights into the roles of various protein phosphatases in ERK2 kinase signaling are obtained, and further analysis of the mechanism by which different protein phosphatases recognize and inactivate MAPKs will increase our understanding of how this kinase family is regulated.


Large intestine permeability is increased in patients with compensated liver cirrhosis.
Intestinal barrier dysfunction, facilitating translocation of bacteria and bacterial products, plays an important role in the pathophysiology of liver cirrhosis and its complications. Increased intestinal permeability has been found in patients with liver cirrhosis, but data on small and large intestine permeability and tight junctions (TJs) in patients with compensated cirrhosis are scarce. We aimed to investigate both small and large intestine permeability in patients with stable compensated cirrhosis compared with healthy controls and evaluated the expression of TJ proteins in mucosal biopsies at duodenal and sigmoid level. Intestinal permeability was assessed in 26 patients with compensated cirrhosis and 27 matched controls using a multisugar test. Duodenal and sigmoid biopsies were available from a subgroup for analyses of gene transcription and expression of key TJ proteins by qRT-PCR and ELISA, respectively. Median 0-5-h urinary sucrose excretion and lactulose/rhamnose ratio were comparable between patients with compensated cirrhosis and controls, whereas 5-24-h urinary sucralose/erythritol ratio was increased in these patients. Downregulation of gene transcription was found for claudin-3 in duodenal biopsies and claudin-4 in sigmoid biopsies, and at the protein level occludin expression was significantly increased in both duodenal and sigmoid biopsies. This study shows that gastroduodenal and small intestine permeability are not altered, whereas large intestine permeability is increased in patients with stable compensated cirrhosis. Only limited alterations were found regarding the expression of TJ proteins in both the small and large intestine.


Double feature selection and cluster analyses in mining of microarray data from cotton.
Cotton fiber is a single-celled seed trichome of major biological and economic importance. In recent years, genomic approaches such as microarray-based expression profiling were used to study fiber growth and development to understand the developmental mechanisms of fiber at the molecular level. The vast volume of microarray expression data generated requires a sophisticated means of data mining in order to extract novel information that addresses fundamental questions of biological interest. One of the ways to approach microarray data mining is to increase the number of dimensions/levels to the analysis, such as comparing independent studies from different genotypes. However, adding dimensions also creates a challenge in finding novel ways for analyzing multi-dimensional microarray data. Mining of independent microarray studies from Pima and Upland (TM1) cotton using double feature selection and cluster analyses identified species-specific and stage-specific gene transcripts that argue in favor of discrete genetic mechanisms that govern developmental programming of cotton fiber morphogenesis in these two cultivated species. Double feature selection analysis identified the highest number of differentially expressed genes that distinguish the fiber transcriptomes of developing Pima and TM1 fibers. These results were based on the finding that differences in fibers harvested between 17 and 24 day post-anthesis (dpa) represent the greatest expressional distance between the two species. This powerful selection method identified a subset of genes expressed during primary (PCW) and secondary (SCW) cell wall biogenesis in Pima fibers that exhibits an expression pattern that is generally reversed in TM1 at the same developmental stage. Cluster and functional analyses revealed that this subset of genes are primarily regulated during the transition stage that overlaps the termination of PCW and onset of SCW biogenesis, suggesting that these particular genes play a major role in the genetic mechanism that underlies the phenotypic differences in fiber traits between Pima and TM1. The novel application of double feature selection analysis led to the discovery of species- and stage-specific genetic expression patterns, which are biologically relevant to the genetic programs that underlie the differences in the fiber phenotypes in Pima and TM1. These results promise to have profound impacts on the ongoing efforts to improve cotton fiber traits.


Gill erosion and herpesvirus in Crassostrea gigas cultured in Baja California, Mexico.
Recurrent episodes of mortality of Crassostrea gigas cultured in northwestern Mexico have been occurring since 1997. Previous studies on bacteria, protozoans, and metazoans as presumptive causal agents have been inconclusive. However, erosions in the marginal indentation of gills have been frequently observed in oysters from areas affected by mortality events, and in 2000 those lesions were associated with the detection of a herpes-like virus. The present study aimed to describe the histological alterations of eroded gills and to determine whether ostreid herpesvirus 1 (OsHV-1) or a related virus was associated with them using in situ hybridization (ISH). Histology showed that gill filaments were fused. In severe cases, deformation of the interlamellar junctions, swelling, and the loss of water channels was observed. ISH analysis revealed the presence of OsHV-1 DNA or a related virus in cells of the gills. Some labeled cells were large with dark granules inside their cytoplasm. These cells were surrounded by infiltrating hemocytes. Some cells interpreted as hemocytes were labeled and observed in eroded and non-eroded areas of the gill. Large cells detected by ISH were also observed by conventional histology with hematoxylin-eosin staining. Whether the virus produces the erosions in the gills, or the erosions in the gills are produced by an unknown condition and favor the presence of the virus, remains unresolved. It is also not clear whether the lesions contribute to mortality.


Tillage and field scale controls on greenhouse gas emissions.
There is a lack of understanding of how associations among soil properties and management-induced changes control the variability of greenhouse gas (GHG) emissions from soil. We performed a laboratory investigation to quantify relationships between GHG emissions and soil indicators in an irrigated agricultural field under standard tillage (ST) and a field recently converted (2 yr) to no-tillage (NT). Soil cores (15-cm depth) were incubated at 25 degrees C at field moisture content and 75% water holding capacity. Principal component analysis (PCA) identified that most of the variation of the measured soil properties was related to differences in soil C and N and soil water conditions under ST, but soil texture and bulk density under NT. This trend became more apparent after irrigation. However, principal component regression (PCR) suggested that soil physical properties or total C and N were less important in controlling GHG emissions across tillage systems. The CO2 flux was more strongly determined by microbial biomass under ST and inorganic N content under NT than soil physical properties. Similarly, N2O and CH4 fluxes were predominantly controlled by NO3- content and labile C and N availability in both ST and NT soils at field moisture content, and NH4+ content after irrigation. Our study indicates that the field-scale variability of GHG emissions is controlled primarily by biochemical parameters rather than physical parameters. Differences in the availability and type of C and N sources for microbial activity as affected by tillage and irrigation develop different levels and combinations of field-scale controls on GHG emissions.


Prenatal diagnosis and management of fetuses with liver hemangiomata.
To study the relationship between prenatal appearance and perinatal outcome of fetuses with hepatic hemangiomata with special emphasis on criteria that may help to improve perinatal management. In a tertiary referral center six fetuses with hepatic hemangiomata were evaluated by gray-scale, color, and pulsed wave Doppler ultrasound between 1994 and 2000. Fetal blood sampling was performed in four cases. All data (computerized files and video tapes) were analyzed retrospectively. Two fetuses showed very similar sonographic findings. They had an isolated large ('giant') round hepatic hemangioma (diameter 43 and 68 mm, respectively) supplied by one hepatic artery and drained by one hepatic vein, both of them showing high velocity and low pulsatility blood flow. Fetal blood count and coagulation parameters were normal in one case, whereas the other fetus showed a Kasabach-Merritt sequence with severe thrombocytopenia (10 platelets/nL) and mild disseminated intravascular coagulation. Intrauterine platelet transfusion was performed immediately prior to planned Cesarean delivery. Rapid platelet consumption continued postnatally, requiring several thrombocyte transfusions. Platelet counts stabilized only after tumor resection on the second day of life. One fetus with diffuse neonatal hemangiomatosis developed high-output cardiac failure with hydrops in addition to Kasabach-Merritt sequence (15 platelets/nL), and died following premature delivery. Three fetuses, however, showing an isolated small hyperechogenic hepatic hemangioma (5, 5, and 6 mm in diameter, respectively) did not develop any perinatal complications. Large fetal liver hemangiomata and diffuse hemangiomatosis may cause severe perinatal complications, particularly high-output cardiac failure and/or Kasabach-Merritt sequence with severe consumption of platelets and clotting factors and hemolytic anemia. Fetal blood sampling enables the prenatal detection of these potential complications, allowing critical modification of perinatal management such as intrauterine platelet transfusion, especially directly before delivery. In contrast, isolated small hyperreflexic hepatic hemangiomata do not appear to be associated with any of these fetal and postnatal sequelae.


Child abuse and neglect.
It is important for surgeons to be able to accurately assess and diagnose child abuse. While they are generally not the only medical professionals involved in the care of children with suspected abuse, they are highly regarded by hospital and social service staff, and their input regarding the cause of injury is unlikely to be ignored. Appropriate diagnosis of abusive injury can prevent future morbidity and mortality in these vulnerable patients, and can also reduce the trauma to families from false accusations of abuse. A sensitive and open-ended history, thorough physical examination, appropriate diagnostic workup with consideration of child abuse as an underlying cause, appropriate documentation of findings, and determination of safety for discharge can greatly improve an abused child's current and future health and safety.


The rhythmic expression of clock genes attenuated in human plaque-derived vascular smooth muscle cells.
Acute myocardial infarction and stroke are more likely to occur in the early morning. Circadian pacemakers are considered to be involved in the process. Many peripheral tissues and cells also contain clock systems. In this study, we examined whether the primary cultured human plaque-derived vascular smooth muscle cells (VSMCs) process circadian rhythmicity; furthermore, we investigated the expression difference of clock genes between normal human carotid VSMCs and human plaque-derived VSMCs. Fifty-six human carotid plaques provided the atherosclerotic tissue, and 21 samples yielded viable cultured primary VSMCs. The normal carotid VSMCs were cultured from donors' normal carotids. The mRNA levels of the target genes were measured by Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR). After serum shock, both types of cells showed clear circadian expressions of Bmal1, Cry1, Cry2, Per1, Per2, Per3 and Rev-erbα mRNA; meanwhile the Clock mRNA show a rhythmic expression in plaque-derived SMCs but not in normal carotid VSMCs. The expression levels of these main clock genes were significantly attenuated in human plaque-derived VSMCs compared with normal human carotid VSMCs. The rhythm of Bmal1 mRNA in plaque-derived VSMCs was changed. The present results demonstrate that the human plaque-derived VSMCs possess different circadian rhythmicity from that of normal carotid VSMCs. The rhythm changes of clock genes in plaque-derived VSMCs may be involved in the process of atherosclerosis and finally promote the rupture of plaque.


Developing a comprehensive career development scale for public health nurses in Japan.
This study aims to develop a scale that can comprehensively measure public health nurses'(PHNs) career development and to verify the validity and reliability of that scale. This study is a cross-sectional investigation. The participants were 1,009 PHNs working in a government facility in Japan. A total of 586 participants who had been working for more than 3 years as PHNs were analyzed. Anonymous self-response questionnaires included items on professional awareness, practical competency, and job satisfaction. We conducted exploratory factor analysis and confirmatory factor analysis to clarify the construct validity of the scale. The reliability was examined by the Cronbach's α coefficient. The validity was examined by an external reference relevant validity and the known-group method. The scale was structured with 32 items covering three factors: "community activities, policy, and management," "PHN identity," and "foundations as a PHN." The Cronbach's α coefficients were all greater than 0.9. In confirmatory factor analysis, the scale showed acceptable goodness of fit. The comprehensive career development scale for PHNs proved its reliability and validity. This scale may be useful to promote PHN identity and competency comprehensively in basic education and in-charge education.


Comparison of different methods used in balance evaluation in children with diparetic cerebral palsy.
In this study, we aimed to investigate the validity and reliability of Single Leg Stance (SLS), Tandem Stance (TS) and Pediatric Reach Tests (PRT) in children with Cerebral Palsy (CP). Fifty-three children (31 boys, 22 girls, age:3-15 years, Gross Motor Function Classification System (GMFCS) Level I- II with diparetic CP were included the study. SLS, TS, PRT test used for balance evaluations. SLS test was made by standing on dominant (D) and non-dominant (ND) extremity both eyes opened (EO) and closed (EC). The mean age of childrens were 7.88±3.43 years. The Cronbach's - coefficients of all tests used to evaluate the internal consistency were found to excellent to acceptable level. SLS-D with EO/ EC, SLS-ND with EO/ EC and TS with EO had strong correlation, TS with EC and PRT-Forward moderate correlation, PRT-Right and eft low correlation with GMFCS. All tests except PRT-Right and PRT-Left were discriminate balance ability between GMFCS level I and level II (AUC>0.800). In this study, we used SLS, TS, PRT for balance evaluations and we investigated reliability and validity (construct and predictive) of each balance tests in childeren with diparetic CP. All tests were sensitive in distinguishing between GMFCS levels I and II, except PRT-Right and PRT-Left. As a result of the study, it was determined that the tests were valid and reliable, suggesting that physiotherapists will provide clinical advantage in order to quickly learn about the balance of children with CP.


The mutational spectrum in Waardenburg syndrome.
One hundred and thirty-four families or individuals with auditory-pigmentary syndromes such as Waardenburg syndrome (WS) or probable neurocristopathies were screened for mutations in the PAX3 and MITF genes. PAX3 mutations were found in 20/25 families with definite Type 1 WS and 1/2 with Type 3 WS, but in none of 23 with definite Type 2 WS or 36 with other neurocristopathies. The PAX3 mutations included substitutions of conserved amino acids in the paired domain or the homeodomain, splice-site mutations, nonsense mutations and frame-shifting insertions or deletions. No phenotype-genotype correlations were noted within WS1 families. With MITF, mutations likely to affect protein function were found in seven families, five of which had definite Type 2 WS. We conclude that Type 1 and Type 3 WS are allelic and are normally caused by loss of function mutations in PAX3; that Type 2 WS is heterogeneous, with about 20% of cases caused by mutations in MITF, and that individuals with auditory, pigmentary or neural crest syndromes which do not fit stringent definitions of Waardenburg syndrome are unlikely to have mutations in either the PAX3 or MITF genes. The molecular pathology of MITF/microphthalmia mutations appears to be different in humans and mice, with gene dosage having more significant effects in humans than in the mouse.


Functional role for Tyr 31 in the catalytic cycle of chicken dihydrofolate reductase.
Despite much work, many key aspects of the mechanism of the dihydrofolate reductase (DHFR) catalyzed reduction of dihydrofolate remain unresolved. In bacterial forms of DHFR both substrate and water access to the active site are controlled by the conformation of the mobile M20 loop. In vertebrate DHFRs only one conformation of the residues corresponding to the M20 loop has been observed. Access to the active site was proposed to be controlled by residue 31. MD simulations of chicken DHFR complexed with substrates and cofactor revealed a closing of the side chain of Tyr 31 over the active site on binding of dihydrofolate. This conformational change was dependent on the presence of glutamate on the para-aminobenzoylamide moiety of dihydrofolate. In its absence, the conformation remained open. Although water could enter the active site and hydrogen bond to N5 of dihydrofolate, indicating the feasibility of water as the proton donor, this was not controlled by the conformation of Tyr 31. The water accessibility of the active site was low for both conformations of Tyr 31. However, when hydride was transferred from NADPH to C6 of dihydrofolate before protonation, the average time during which water was found in hydrogen bonding distance to N5 of dihydrofolate in the active site increased almost fivefold. These results indicated that water can serve as the Broensted acid for the protonation of N5 of dihydrofolate during the DHFR catalyzed reduction.


On the Analysis of Regularized Fuzzy Systems of Uncertain Differential Equations.
This article analyzes a regularized set of fuzzy differential equations with respect to an uncertain parameter. We provide sufficient conditions for the correctness of a new regularization scheme. For the resulting family of regularized fuzzy differential equations, the following properties are analyzed, and efficient criteria are proposed: successive approximations, continuity, global existence of solutions, existence of approximate solutions, existence of solutions in the autonomous case. In addition, we develop stability criteria for the regularized family of fuzzy differential equations on the basis of the comparison technique and the method of nonlinear integral inequalities. We expect that the derived results will inspire future research work in this direction.


[The fundamental and clinical studies on PT-122M (doxycycline) in the otorhinolaryngological field (author's transl)].
As the results of laboratory and clinical investigation with a new tetracycline derivate, PT-122M (doxycycline) was performed with the results which may lead to the following conclusions. 1) In vitro antibacterial activity: The minimal inhibitory concentration of doxycycline (DOTC) was measured by an agar plate dilution method. The MIC of DOTC against 60 strains of coagulase-positive Staphylococcus aureus isolated from otorrhoea was distributed over a range of 0.2 approximately 1.56 mcg/ml and 12.5 approximately 50 mcg/ml. Furthermore, Escherichia coli, Proteus mirabilis and Klebsiella pneumoniae was 3.13 to 100 mcg/ml. 2) Concentration in blood: The blood level of DOTC in healthy adults who were given 100 mg of DOTC intravenous reached maximum of 1.97 mcg/ml on the average 15 minutes after injection. Even at 12 hours after intravenous injection clinically effective serum DOTC concentration of 0.47 mcg/ml was still demonstrable. 3) CLINICAL RESULTS: PT-122M (DOCT) was intravenously injected into 30 cases with typical infection in the field of otorhinolaryngology. It was excellent in 15 cases, good in 10 cases, fair in 3 cases, and poor in 2 cases. When the cases in which it was excellent and good were considered together, good results were obtained in 25 cases, a ratio of effectiveness being 83 per cent. 4) Side effect: The comparative examination of hepatic function, electrolyte and auditory acuity before and after injection showed no significant disturbance. No side effect was shown with the intravenous injection.


Factors associated with the development of extrahepatic malignancy in patients with nonalcoholic fatty liver disease: a single-center longitudinal study.
The aim of the present study was to determine incident cases of extrahepatic malignancy in patients with nonalcoholic fatty liver disease (NAFLD) and to identify whether the factors are associated with cancer development. Between 15 January 2001 and 14 March 2021, a total of 1365 patients had been diagnosed with NAFLD were enrolled in the study. The median follow-up period was 59.5 months. The mean age was 50.9 ± 10.9 years. The female gender was predominant (57%). During the follow-up period, 62 extrahepatic malignancies and 11 hepatocellular carcinomas were identified. Of all extrahepatic malignancies, 51 were solid organ malignancies and 11 were hematological malignancies. Female breast cancer was the most frequent (25.8%), followed by thyroid cancer (19.4%), lymphoma (12.9%), and lung cancer (9.7%). In univariate and multivariable analyses, after adjusting for age and sex, the presence of diabetes and high initial baseline gamma glutamyl transpeptidase (GGT) levels were significantly associated with the development of extrahepatic malignancies [hazard ratio (HR) = 1.82, 95% confidence interval (CI): 1.04-3.20, P = 0.036] and HR = 1.96, 95% CI: 1.14-3.38, P = 0.015, respectively). In 424 biopsy-proven NAFLD patients, the development of extrahepatic cancer was significantly associated with the severity of hepatic fibrosis (HR = 3.31, 95% CI: 1.36-8.07; P = 0.008). Extrahepatic malignancies are frequently seen in patients with NAFLD. Diabetes mellitus, high baseline GGT levels, and significant hepatic fibrosis are associated with the development of extrahepatic cancer in patients with NAFLD.


Accurate Digital Polymerase Chain Reaction Quantification of Challenging Samples Applying Inhibitor-Tolerant DNA Polymerases.
Digital PCR (dPCR) enables absolute quantification of nucleic acids by partitioning of the sample into hundreds or thousands of minute reactions. By assuming a Poisson distribution for the number of DNA fragments present in each chamber, the DNA concentration is determined without the need for a standard curve. However, when analyzing nucleic acids from complex matrixes such as soil and blood, the dPCR quantification can be biased due to the presence of inhibitory compounds. In this study, we evaluated the impact of varying the DNA polymerase in chamber-based dPCR for both pure and impure samples using the common PCR inhibitor humic acid (HA) as a model. We compared the TaqMan Universal PCR Master Mix with two alternative DNA polymerases: ExTaq HS and Immolase. By using Bayesian modeling, we show that there is no difference among the tested DNA polymerases in terms of accuracy of absolute quantification for pure template samples, i.e., without HA present. For samples containing HA, there were great differences in performance: the TaqMan Universal PCR Master Mix failed to correctly quantify DNA with more than 13 pg/nL HA, whereas Immolase (1 U) could handle up to 375 pg/nL HA. Furthermore, we found that BSA had a moderate positive effect for the TaqMan Universal PCR Master Mix, enabling accurate quantification for 25 pg/nL HA. Increasing the amount of DNA polymerase from 1 to 5 U had a strong effect for ExTaq HS, elevating HA-tolerance four times. We also show that the average Cq values of positive reactions may be used as a measure of inhibition effects, e.g., to determine whether or not a dPCR quantification result is reliable. The statistical models developed to objectively analyze the data may also be applied in quality control. We conclude that the choice of DNA polymerase in dPCR is crucial for the accuracy of quantification when analyzing challenging samples.


Direct evidence for GABAergic activity of Withania somnifera on mammalian ionotropic GABAA and GABAρ receptors.
Withania somnifera (WS) has been traditionally used in Ayurvedic medicine as a remedy for debility, stress, nervous exhaustion, insomnia, loss of memory, and to enhance cognitive function. This study provides an empirical evidence to support the traditional use of WS to aid in mental process engaging GABAergic signaling. We evaluated the effect of aqueous WS root extract (aqWS), and its two main components, withaferin A and withanolide A, on the main inhibitory receptors in the central nervous system: ionotropic GABAA receptors. The pharmacological activity of aqWS, withaferin A and withanolide A, was tested on native rat brain GABAA channels microtransplanted into Xenopus oocytes and GABAρ1 receptors heterologously expressed in oocytes. The GABAergic activity of aqWS compounds was evaluated by the two-electrode voltage-clamp method and the fingerprint of the extract was done by LC-MS. Concentration-dependent inward ion currents were elicited by aqWS in microtransplanted oocytes with an EC50 equivalent to 4.7 mg/mL and a Hill coefficient (nH) of 1.6. The GABAA receptor antagonist bicuculline blocked these currents. Our results show that aqWS activated inotropic GABAA channels but with lower efficacy compared to the endogenous agonist GABA. We also demonstrate for first time that aqWS is a potent agonist of GABAρ1 receptors. GABAρ1 receptors were 27 fold more sensitive to aqWS than GABAA receptors. Furthermore, aqWS activated GABAρ1 receptors eliciting maximum currents that were no significantly different to those produced by GABA (paired t-test; p=0.533). The differential activity on GABAA and GABA ρ1 receptors and the reported lack of significant GABA presence in WS root extract indicates that the GABAergic activity of aqWS is not mediated by GABA. WS main active components, witaferin A and withanolide A, were tested to determine if they were responsible for the activation of the GABA receptors. Neither compound activated GABAA nor GABAρ1 receptors, suggesting that other constituent/s in WS are responsible for GABAA receptor mediated responses. Our results provide evidence indicating that key constituents in WS may have an important role in the development of pharmacological treatments for neurological disorders associated with GABAergic signaling dysfunction such as general anxiety disorders, sleep disturbances, muscle spasms, and seizures. In addition, the differential activation of GABA receptor subtypes elucidates a potential mechanism by which WS accomplishes its reported adaptogenic properties.


Medevac programs must face financial scrutiny.
Hospital-based air medical (medevac) programs underwent rapid expansion during the past decade, but the financial incentives for operating the programs may no longer exist. Today, as some medevac programs are being consolidated or are ceasing operations altogether, a successful program requires careful attention to charge structures, lease negotiations, and efficiency studies.


Correlation of human neutrophil secretion, chemoattractant receptor mobilization, and enhanced functional capacity.
Studies were performed to elucidate further the phenomenon of secretagogue-mediated enhancement in the binding of the chemoattractant f-met-leu-[3H]phe to human neutrophils (PMN). Specific f-met-leu-[3H]phe binding to unstimulated PMN reached maximum levels after 10 to 15 min of incubation at 0 degrees C with a saturating concentration of peptide, and consisted of a readily displaceable and a nondisplaceable component. PMN, preexposed to A23187 (2.5 X 10(-8) M) or PMA (0.5 ng/ml) for 30 min at 37 degrees C to stimulate limited and preferential release of specific (secondary) granules (10 to 20% of total lysozyme, no beta-glucuronidase), demonstrated an approximate doubling in the displaceable component of f-met-leu-["3H]phe binding, accompanied by an increasing nondisplaceable component that could not be explained by bulk pinocytosis of extracellular fluid (assessed by [3H]sucrose uptake). The increase in f-met-leu-[3H]phe binding was not affected by inhibitors of protein synthesis, could not be attributed to the secreted products lysosyme or lactoferrin acting on the cell, and, on the basis of studies with PMN from patients with chronic granulomatous disease, could not be attributed to the effects of reactive oxygen species generated in low concentration during stimulation. Functional studies on PMN indicated that preexposure to secretagogues at concentrations demonstrated to increase receptor availability also enhanced subsequent f-met-leu-phe-mediated superoxide and hydrogen peroxide generation. The present data demonstrate that secretagogues may activate PMN to enhance their subsequent responses in f-met-leu-phe-mediated processes, and, combined with previous reports, support the concept that specific granules provide a source of preformed membrane and receptor material that is translocated to the cell surface during the secretion associated with directed locomotion.


Herpes zoster myelitis.
We studied the clinical (10 patients) and pathological (9 patients) findings in 13 patients with herpes zoster myelitis, all of whom had systemic illnesses associated with immunosuppression. The median interval between the onset of the herpes zoster rash and myelopathic symptoms was 12 days, and the subsequent median interval to maximal deficit was 10.5 days. Presenting neurological symptoms were characteristically ipsilateral to the rash, with motor dysfunction predominating, followed by a spinothalamic and, less often, posterior column sensory deficit. Pathological involvement was most severe in the dorsal root entry zone and posterior horn of the spinal cord segment corresponding to the involved dermatome. There was variable spread both horizontally and vertically in the spinal cord. Direct varicella-zoster virus (VZV) infection of neuroectodermal cells, particularly oligodendrocytes, was demonstrated by immunostaining viral antigens (8 cases), and by the presence of Cowdry type A intranuclear inclusions (7 cases) and often was associated with focal demyelination (6 cases). In 4 patients a VZV vasculitis was associated with leptomeningitis and haemorrhagic necrosis (spinal cord in 1; brainstem or cerebellum in 3). The protracted evolution in many cases and the pathologically documented direct viral infection of the spinal cord provide a rational basis for the use of antiviral therapy in preventing or attenuating the evolving myelopathy.


Immunohistochemical analysis of bcl-2 family proteins in benign and malignant ameloblastomas.
To study the possible roles of bcl-2 family proteins in oncogenesis and cytodifferentiation of odontogenic epithelium, the expression of bcl-2, bcl-x and bax proteins was examined immunohistochemically in tooth germs and various types of ameloblastoma. In ameloblastomas, bcl-2 and bcl-x proteins were expressed intensely in peripheral columnar cells and weakly in central polyhedral cells, while bax protein reactivity was low. In variants of ameloblastoma, no expression of these proteins was found in the keratinizing areas or granular cell clusters. Basal cell ameloblastomas showed intense reactivity for bcl-2 and bcl-x proteins, but slight or no reactivity for bax protein. These results indicate that in ameloblastomas the bcl-2 family proteins might function primarily as anti-apoptotic factors, reflecting proliferative activity. Malignant ameloblastomas showed decreased reactivity for bcl-2 protein and increased levels of bax protein as compared with benign ones.


[The potential of prophylaxis and optimization of the treatment of rhinosinusitis in the children presenting with stenosing laryngotracheitis].
The objective of the present work was to estimate the clinical, prophylactic, and microbiological effectiveness of fusafungine applied for the treatment of acute rhinosinusitis (ARS) in the children that develops as a consequence of acute stenosing laryngotracheitis. The study included 61 children presenting with ARS and concomitant acute stenosing laryngotracheitis (ASLT) that were treated with fusafungine (Bioparox). Both tolerance and safety of this preparation were evaluated. Fusaferine was prescribed after reduction of pharyngeal stenosis. The children were divided into two groups. Group 1 was comprised of the patients with the respiratory symptoms and rhinosinusitis (n = 36), group 2 consisted of the children with the respiratory symptoms in the absence of rhinosinusitis (n = 25). Subgroups of the children treated with fusafungine and without it were distinguished to estimate the clinical, prophylactic, and microbiological effectiveness of fusafungide. Within the first days after hospitalization, 59% of the children with diagnosis ASLT developed bilateral rhinosinusitis, in all probability of viral etiology. Fusafungine produced the clinically apparent effect in the patients with ASLT regardless of the presence of ARS. Specifically, this preparation decreased the degree of hypertrophy of pharyngeal tonsils three times faster than standard therapy; moreover, it reduced the requirement for systemic antibiotics by 1.9 times. The treatment with fusaferine prevented the development of acute bilateral rhinosinusitis in the children with ASLT and promoted compete decontamination of the nasopharynx from M. catarrhalis, Str. pneumonia, Str. pyogenes, H. influenza, Cor. s the nasopharynx pecies, E. faecalis, and C. albicans. The frequency of adverse reactions of organoleptic character was estimated at 16.6%.


The Fab and Fc fragments of IgA1 exhibit a different arrangement from that in IgG: a study by X-ray and neutron solution scattering and homology modelling.
Human immunoglobulin A (IgA) is an abundant antibody that mediates immune protection at mucosal surfaces as well as in plasma. The IgA1 isotype contains two four-domain Fab fragments and a four-domain Fc fragment analogous to that in immunoglobulin G (IgG), linked by a glycosylated hinge region made up of 23 amino acid residues from each of the heavy chains. IgA1 also has two 18 residue tailpieces at the C terminus of each heavy chain in the Fc fragment. X-ray scattering using H2O buffers and neutron scattering using 100 % 2H2O buffers were performed on monomeric IgA1 and a recombinant IgA1 that lacks the tailpiece (PTerm455). The radii of gyration RG from Guinier analyses were similar at 6.11-6.20 nm for IgA1 and 5.84-6.16 nm for PTerm455, and their cross-sectional radii of gyration RXS were also similar. The similarity of the RG and RXS values suggests that the tailpiece of IgA1 is not extended outwards in solution. The IgA1 RG values are higher than those for IgG, and the distance distribution function P(r) showed two distinct peaks, whereas a single peak was observed for IgG. Both results show that the hinge of IgA1 results in an extended Fab and Fc arrangement that is different from that in IgG. Automated curve-fit searches constrained by homology models for the Fab and Fc fragments were used to model the experimental IgA1 scattering curves. A translational search to optimise the relative arrangement of the Fab and Fc fragments held in a fixed orientation resembling that in IgG was not successful in fitting the scattering data. A new molecular dynamics curve-fit search method generated IgA1 hinge structures to which the Fab and Fc fragments could be connected in any orientation. A search based on these identified a limited family of IgA1 structures that gave good curve fits to the experimental data. These contained extended hinges of length about 7 nm that positioned the Fab-to-Fab centre-to-centre separation 17 nm apart while keeping the corresponding Fab-to-Fc separation at 9 nm. The resulting extended T-shaped IgA1 structures are distinct from IgG structures previously determined by scattering and crystallography which have Fab-to-Fab and Fab-to-Fc centre-to-centre separations of 7-9 nm and 6-8 nm, respectively. It was concluded that the IgA1 hinge is structurally distinct from that in IgG, and this results in a markedly different antibody structure that may account for a unique immune role of monomeric IgA1 in plasma and mucosa.


A review of the evidence for the medical home for children with special health care needs.
The receipt of health care in a medical home is increasingly touted as a fundamental basis for improved care for persons with chronic conditions, yet the evidence for this claim has not been systematically assessed. Our goal was to determine the evidence for the federal Maternal and Child Health Bureau recommendation that children with special health care needs receive ongoing comprehensive care within a medical home. We searched the nursing and medical literature, references of selected articles, and requested expert recommendations. Search terms included children with special health care needs, medical home-related interventions, and health-related outcomes. Articles that met defined criteria (eg, children with special health care needs, United States-based, quantitative) were selected. We extracted data, including design, population characteristics, sample size, intervention, and findings from each article. We selected 33 articles that reported on 30 distinct studies, 10 of which were comparison-group studies. None of the studies examined the medical home in its entirety. Although tempered by weak designs, inconsistent definitions and extent of medical home attributes, and inconsistent outcome measures, the preponderance of evidence supported a positive relationship between the medical home and desired outcomes, such as better health status, timeliness of care, family centeredness, and improved family functioning. The evidence provides moderate support for the hypothesis that medical homes provide improved health-related outcomes for children with special health care needs. Additional studies with comparison groups encompassing all or most of the attributes of the medical home need to be undertaken.


[To the prevention of recurrent dacryocystitis after surgical treatment].
The efficacy of collagenase KK was evaluated in the treatment of 121 patients with lacrimal duct diseases. Collagenase KK used to prevent recurrent lacrimal duct disease contributed to a significant relief of an inflammatory process (by an average of 2.04+/-0.2 days as rapidly as in the controls), recovery of nasal lacrimation, shorter treatment (by an average of 1.8+/-0.11 bed/days), a reduction in the number of complications (by 2.1 times). The findings enable the authors to recommend the agent for wide clinical application in combination with other treatments.


Nephrogenic Diabetes Insipidus.
Body fluid homeostasis is essential for normal life. In the maintenance of water balance, the most important factor and regulated process is the excretory function of the kidneys. The kidneys are capable to compensate not only the daily fluctuations of water intake but also the consequences of fluid loss (respiration, perspiration, sweating, hemorrhage). The final volume and osmolality of the excreted urine is set in the collecting duct via hormonal regulation. The hormone of water conservation is the vasopressin (AVP), and a large volume of urine is produced and excreted in the absence of AVP secretion or if AVP is ineffective in the kidneys. The aquaporin-2 water channel (AQP2) is expressed in the principal cells, and it plays an essential role in the reabsorption of water in the collecting ducts via type 2 vasopressin receptor (V2R)-mediated mechanism. If neural or hormonal regulation fails to operate the normal function of AVP-V2R-AQP2 system, it can result in various diseases such as diabetes insipidus (DI) or nephrogenic syndrome of inappropriate diuresis (NSIAD). The DI is characterized by excessive production of hyposmotic urine ("insipidus" means tasteless) due to the inability of the kidneys to concentrate urine. In this chapter, we focus and discuss the pathophysiology of nephrogenic DI (NDI) and the potential therapeutic interventions in the light of the current experimental data.


Partition behavior of CD133(+) stem cells from human umbilical cord blood in aqueous two-phase systems: In route to establish novel stem cell primary recovery strategies.
Aqueous two-phase systems (ATPS) represent a promising strategy for the recovery of CD133(+) stem cells. This particular type of stem cells has great potential for research and clinical applications. Traditional [polyethylene glycol (PEG), dextran (DEX), and ficoll] and novel (Ucon) polymer-polymer ATPS were exploited to study the partitioning behavior of CD133(+) stem cells and contaminants from human umbilical cord blood (HUCB). The aim of the study was to select conditions under which the product of interest and the contaminants concentrate in opposite phases. To accomplish this, three independent samples were tested: (1) enriched CD133(+) sample, (2) whole HUCB (contaminants), and (3) complex sample (CD133(+) stem cells and contaminants). The objective of this research was to evaluate the partition behavior of CD133(+) in ATPS in route to establish the basis for the development of a novel and scalable purification bioprocess. In conclusion, the partitioning behavior of CD133(+) stem cells and contaminants from complex samples was as follows: 59% of CD133(+) stem cells fractionated to the top phase when employing ficoll 400,000-DEX 70,000 or 100% to the bottom phase with Ucon-DEX 75,000 and PEG 8,000-DEX 500,000 ATPS. In average, 35% of the contaminants partitioned to the top phase of the ficoll 400,000-DEX 70,000 ATPS, 99% to the dextran rich phase of the Ucon-DEX 75,000 systems and 97% to the bottom phase of the PEG 8,000-DEX 500,000. Cell viability was at least 98% after ATPS recovery.


Identification of human papillomavirus type 58 lineages and the distribution worldwide.
Human papillomavirus type 58 (HPV-58) accounts for a much higher proportion of cervical cancers in East Asia than other types. A classification system of HPV-58, which is essential for molecular epidemiological study, is lacking. This study analyzed the sequences of 401 isolates collected from 15 countries and cities. The 268 unique concatenated E6-E7-E2-E5-L1-LCR sequences that comprised 57% of the whole HPV-58 genome showed 4 distinct clusters. L1 and LCR produced tree topologies that best resembled the concatenated sequences and thus are the most appropriate surrogate regions for lineage classification. Moreover, short fragments from L1 (nucleotides 6014-6539) and LCR (nucleotides 7257-7429 and 7540-52) were found to contain sequence signatures informative for lineage identification. Lineage A was the most prevalent lineage across all regions. Lineage C was more frequent in Africa than elsewhere, whereas lineage D was more prevalent in Africa than in Asia. Among lineage A variants, sublineage A2 dominated in Africa, the Americas, and Europe, but not in Asia. Sublineage A1, which represents the prototype that originated from a patient with cancer, was rare worldwide except in Asia. HPV-58 can be classified into 4 lineages that show some degree of ethnogeographic predilection in distribution. The evolutionary, epidemiological, and pathological characteristics of these lineages warrant further study.


Improved metabolic stability for 18F PET probes rapidly constructed via tetrazine trans-cyclooctene ligation.
The fast kinetics and bioorthogonal nature of the tetrazine trans-cyclooctene (TCO) ligation makes it a unique tool for PET probe construction. In this study, we report the development of an (18)F-labeling system based on a CF3-substituted diphenyl-s-tetrazine derivative with the aim of maintaining high reactivity while increasing in vivo stability. c(RGDyK) was tagged by a CF3-substituted diphenyl-s-tetrazine derivative via EDC-mediated coupling. The resulting tetrazine-RGD conjugate was combined with a (19)F-labeled TCO derivative to give HPLC standards. The analogous (18)F-labeled TCO derivative was combined with the diphenyl-s-tetrazine-RGD at μM concentration. The resulting tracer was subjected to in vivo metabolic stability assessment, and microPET studies in murine U87MG xenograft models. The diphenyl-s-tetrazine-RGD combines with an (18)F-labeled TCO in high yields (>97% decay-corrected on the basis of TCO) using only 4 equiv of tetrazine-RGD relative to the (18)F-labeled TCO (concentration calculated based on product's specific activity). The radiochemical purity of the (18)F-RGD peptides was >95% and the specific activity was 111 GBq/μmol. Noninvasive microPET experiments demonstrated that (18)F-RGD had integrin-specific tumor uptake in subcutaneous U87MG glioma. In vivo metabolic stability of (18)F-RGD in blood, urine, and major organs showed two major peaks: one corresponded to the Diels-Alder conjugate and the other was identified as the aromatized analog. A CF3-substituted diphenyl-s-tetrazine displays excellent speed and efficiency in (18)F-PET probe construction, providing nearly quantitative (18)F labeling within minutes at low micromolar concentrations. The resulting conjugates display improved in vivo metabolic stability relative to our previously described system.


Effect of brown rice and soybean dietary fiber on the control of glucose and lipid metabolism in diabetic rats.
We have studied the effect of brown rice and soybean dietary fiber on the oral glucose tolerance test, plasma-insulin, -glucagon, -triglycerides, and cholesterol levels in control and streptozotocin-induced diabetic rats. Plasma glucose level after glucose loading in diabetic rats fed soybean fiber was considerably lower (60 and 120 min) than the corresponding group not receiving the fiber. Soybean fiber given to control rats decreased the plasma glucose at 60 min after the oral glucose test. Rice fiber decreased the plasma glucose of diabetic rats only at 60 min and the control rats fed with or without fiber elicited a comparable glycemic response. While soybean fiber significantly lowered the glucagon and triglyceride levels in both control and diabetic rats, rice fiber had no effect on these blood parameters. In conclusion, this study demonstrated the potential benefit of soybean dietary fiber over rice fiber in diabetes treatment with additional advantages resulting from its ease in usage either in a mixture of water or milk products and cooking. As well, being devoid of a disagreeable taste so characteristic of other fibers, patient acceptance is more forthcoming.


Self-organized nitrogen and fluorine co-doped titanium oxide nanotube arrays with enhanced visible light photocatalytic performance.
Self-organized nitrogen and fluorine co-doped titanium oxide (TiONF) nanotube arrays were created by anodizing titanium foil in a fluoride and ammoniate-based electrolyte, followed by calcination of the amorphous nanotube arrays under a nitrogen protective atmosphere for crystallization. TiONF nanotube arrays were found to have enhanced visible light absorption capability and photodegradation efficiency on methylene blue under visible light illumination over the TiO(2) nanotube arrays. The enhancement was dependent on both the nanotube structural architecture and the nitrogen and fluorine co-doping effect. TiONF nanotube arrays promise a wide range of technical applications, especially for environmental applications and solar cell devices.


A novel machine learning algorithm picks proteome signature to specifically identify cancer exosomes.
Non-invasive early cancer diagnosis remains challenging due to the low sensitivity and specificity of current diagnostic approaches. Exosomes are membrane-bound nanovesicles secreted by all cells that contain DNA, RNA, and proteins that are representative of the parent cells. This property, along with the abundance of exosomes in biological fluids makes them compelling candidates as biomarkers. However, a rapid and flexible exosome-based diagnostic method to distinguish human cancers across cancer types in diverse biological fluids is yet to be defined. Here, we describe a novel machine learning-based computational method to distinguish cancers using a panel of proteins associated with exosomes. Employing datasets of exosome proteins from human cell lines, tissue, plasma, serum and urine samples from a variety of cancers, we identify Clathrin Heavy Chain (CLTC), Ezrin, (EZR), Talin-1 (TLN1), Adenylyl cyclase-associated protein 1 (CAP1) and Moesin (MSN) as highly abundant universal biomarkers for exosomes and define three panels of pan-cancer exosome proteins that distinguish cancer exosomes from other exosomes and aid in classifying cancer subtypes employing random forest models. All the models using proteins from plasma, serum, or urine-derived exosomes yield AUROC scores higher than 0.91 and demonstrate superior performance compared to Support Vector Machine, K Nearest Neighbor Classifier and Gaussian Naive Bayes. This study provides a reliable protein biomarker signature associated with cancer exosomes with scalable machine learning capability for a sensitive and specific non-invasive method of cancer diagnosis.


GATA factors efficiently direct cardiac fate from embryonic stem cells.
The GATA4 transcription factor is implicated in promoting cardiogenesis in combination with other factors, including TBX5, MEF2C and BAF60C. However, when expressed in embryonic stem cells (ESCs), GATA4 was shown to promote endoderm, not cardiac mesoderm. The capacity of related GATA factors to promote cardiogenesis is untested. We found that expression of the highly related gene, Gata5, very efficiently promotes cardiomyocyte fate from murine ESCs. Gata5 directs development of beating sheets of cells that express cardiac troponin T and show a full range of action potential morphologies that are responsive to pharmacological stimulation. We discovered that by removing serum from the culture conditions, GATA4 and GATA6 are each also able to efficiently promote cardiogenesis in ESC derivatives, with some distinctions. Thus, GATA factors can function in ESC derivatives upstream of other cardiac transcription factors to direct the efficient generation of cardiomyocytes.


Practice Patterns in the Management of Primary Pterygium: A Survey Study.
To characterize cornea specialists' current practice preferences in the management of primary pterygium. A 25-item survey regarding indications for surgery, surgical technique, use of adjuvant therapy, type and duration of postoperative therapy, and treatment of early recurrences was designed and sent to members of the Cornea Society through the kera-net listserv. In total, 199 cornea specialists completed the questionnaire. More than 90% considered that surgery should be performed when there is proximity of the pterygium to the visual axis, pain or redness, eye movement restriction, or induction of astigmatism. Cosmesis was considered as an indication by 41.7% of the participants. The most frequent technique for pterygium excision was complete resection including the base and a moderate quantity of Tenon capsule followed by autologous conjunctival or limbal-conjunctival graft. The preferred graft fixation method in this survey was fibrin glue (61.2%). Most respondents reported a recurrence rate of less than 5% and no use of adjuvant agents to prevent recurrence. When early recurrence did occur, the preferred agents were corticosteroids. This study reflects the preferences of cornea experts regarding primary pterygium treatment and may serve as a guide for the management of this pathology.


In vitro selection and characterization of influenza A (A/N9) virus variants resistant to a novel neuraminidase inhibitor, A-315675.
With the recent introduction of neuraminidase (NA) inhibitors into clinical practice for the treatment of influenza virus infections, considerable attention has been focused on the potential for resistance development and cross-resistance between different agents from this class. A-315675 is a novel influenza virus NA inhibitor that has potent enzyme activity and is highly active in cell culture against a variety of strains of influenza A and B viruses. To further assess the therapeutic potential of this compound, in vitro resistance studies have been conducted and a comparative assessment has been made relative to oseltamivir carboxylate. The development of viral resistance to A-315675 was studied by in vitro serial passage of influenza A/N9 virus strains grown in MDCK cells in the presence of increasing concentrations of A-315675. Parallel passaging experiments were conducted with oseltamivir carboxylate, the active form of a currently marketed oral agent for the treatment of influenza virus infections. Passage experiments with A-315675 identified a variant at passage 8 that was 60-fold less susceptible to the compound. Sequencing of the viral population identified an E119D mutation in the NA gene, but no mutations were observed in the hemagglutinin (HA) gene. However, by passage 10 (2.56 microM A-315675), two mutations (R233K, S339P) in the HA gene appeared in addition to the E119D mutation in the NA gene, resulting in a 310-fold-lower susceptibility to A-315675. Further passaging at higher drug concentrations had no effect on the generation of further NA or HA mutations (20.5 microM A-315675). This P15 virus displayed 355-fold-lower susceptibility to A-315675 and >175-fold-lower susceptibility to zanamivir than did wild-type virus, but it retained a high degree of susceptibility to oseltamivir carboxylate. By comparison, virus variants recovered from passaging against oseltamivir carboxylate (passage 14) harbored an E119V mutation and displayed a 6,000-fold-lower susceptibility to oseltamivir carboxylate and a 175-fold-lower susceptibility to zanamivir than did wild-type virus. Interestingly, this mutant still retained susceptibility to A-315675 (42-fold loss). This suggests that cross-resistance between A-315675- and oseltamivir carboxylate-selected variants in vitro is minimal.


Structural Aspects of Heparan Sulfate Binding to Robo1-Ig1-2.
Roundabout 1, or Robo1, is a cell surface signaling molecule important in axon guidance. Its interaction with heparan sulfate (HS) and members of the Slit protein family is essential to its activity, making characterization of these interactions by structural methods, such as NMR, highly desirable. However, the fact that Robo1 is a glycosylated protein prevents employment of commonly used bacterial hosts for expression of properly glycosylated forms with the uniform 15N, 13C, and 2H labeling needed for NMR studies. Here, we apply an alternative methodology, based on labeling with a single amino acid type and high structural content NMR data, to characterize a two-domain construct of glycosylated Robo1 (Robo1-Ig1-2) interacting with a synthetic HS tetramer (IdoA-GlcNS6S-IdoA2S-GlcNS6S-(CH2)5NH2). Significant chemical shift perturbations of the crosspeak from K81 on titration with the tetramer provide initial evidence for the location of a binding site and allow determination of a 255 μM disassociation constant. The binding epitopes, bound conformation, and binding site placement of the HS tetramer have been further characterized by saturation transfer difference (STD), transferred nuclear Overhauser effect (trNOE), and paramagnetic perturbation experiments. A model of the complex has been generated using constraints derived from the various NMR experiments. Postprocessing energetic analysis of this model provides a rationale for the role each glycan residue plays in the binding event, and examination of the binding site in the context of a previous Robo-Slit structure provides a rationale for modulation of Robo-Slit interactions by HS.


Anxiety and depression in children and adults: influence of serotonergic and neurotrophic genes?
There are two major hypotheses regarding the etiology of anxiety and depression: the mono-amine hypothesis and the hypothesis of an abnormal stress response acting partly via reduced neurogenesis. Association studies have focused on genes involved in these processes, but with inconclusive results. This study investigated the effect of 45 single nucleotide polymorphisms (SNPs) in genes encoding for serotonin receptors 1A, 1D, 2A, catechol-O-methyltransferase (COMT), tryptophane hydroxylase type 2 (TPH2), brain derived neurotrophic factor (BDNF), PlexinA2 and regulators of G-protein-coupled signaling (RGS) 2, 4, 16. Anxious depression (A/D) symptoms were assessed five times in 11 years in over 11 000 adults with 1504 subjects genotyped and at age 7, 10, 12 and during adolescence in over 20 000 twins with 1078 subjects genotyped. In both cohorts, a longitudinal model with one latent factor loading on all A/D measures over time was analysed. The genetic association effect modeled at the level of this latent factor was 60% and 70% heritable in the children and adults, respectively, and explained around 50% of the total phenotypic variance. Power analyses showed that the samples contained 80% power to detect an effect explaining between 1.4% and 3.6% of the variance. However, no SNP showed a consistent effect on A/D. To conclude, this longitudinal study in children and adults found no association of SNPs in the serotonergic system or core regulators of neurogenesis with A/D. Overall, there has been no convincing evidence, so far, for a role of genetic variation in these pathways in the development of anxiety and depression.