Retreat sites of rain forest stream frogs are not a reservoir for Batrachochytrium dendrobatidis in northern Queensland, Australia.
Chytridiomycosis is a potentially fatal disease of amphibians caused by Batrachochytrium dendrobatidis, and is implicated in declines and extinctions of amphibian populations and species around the world. To cause local host extinction, a disease organism must persist at low host densities. One mechanism that could facilitate this is the ability to persist in the environment. In the laboratory, B. dendrobatidis spreads by both frog-to-frog and environment-to-frog transmission, and can persist on a number of biotic substrates. In the field, B. dendrobatidis has been detected on environmental samples taken during an epidemic, but it is not known if it persists in the environment when endemic. Retreat sites of 2 species of Australian rain forest stream frogs Litoria lesueuri and L. nannotis were sampled 0 to 3 d after occupation during the wet and dry seasons in northern Queensland, Australia, where chytridiomycosis has been endemic for at least 10 yr. The intensity and prevalence of infection in frogs during sampling were comparatively low compared with epidemics. Diagnostic quantitative polymerase chain reaction did not detect B. dendrobatidis in any retreat site samples. It thus appears that retreat sites are not a major environmental source of infection when B. dendrobatidis occurs at low prevalence and intensity on frogs. This suggests that control efforts may not need to eliminate the organism from the environment, at least when prevalence and intensity of infection are low in frogs. Simply treating hosts may be effective at controlling the disease in the wild.


Psychiatric disorders and drug use among human immunodeficiency virus-infected adults in the United States.
There have been no previous nationally representative estimates of the prevalence of mental disorders and drug use among adults receiving care for human immunodeficiency virus (HIV) disease in the United States. It is also not known which clinical and sociodemographic factors are associated with these disorders. We enrolled a nationally representative probability sample of 2864 adults receiving care for HIV in the United States in 1996. Participants were administered a brief structured psychiatric instrument that screened for psychiatric disorders (major depression, dysthymia, generalized anxiety disorders, and panic attacks) and drug use during the previous 12 months. Sociodemographic and clinical factors associated with screening positive for any psychiatric disorder and drug dependence were examined in multivariate logistic regression analyses. Nearly half of the sample screened positive for a psychiatric disorder, nearly 40% reported using an illicit drug other than marijuana, and more than 12% screened positive for drug dependence during the previous 12 months. Factors independently associated with screening positive for a psychiatric disorder included number of HIV-related symptoms, illicit drug use, drug dependence, heavy alcohol use, and being unemployed or disabled. Factors independently associated with screening positive for drug dependence included having many HIV-related symptoms, being younger, being heterosexual, having frequent heavy alcohol use, and screening positive for a psychiatric disorder. Many people infected with HIV may also have psychiatric and/or drug dependence disorders. Clinicians may need to actively identify those at risk and work with policymakers to ensure the availability of appropriate care for these treatable disorders.


Effectiveness of telaprevir or boceprevir in treatment-experienced patients with HCV genotype 1 infection and cirrhosis.
We investigated the effectiveness of the protease inhibitors peginterferon and ribavirin in treatment-experienced patients with hepatitis C virus (HCV) genotype 1 infection and cirrhosis. In the Compassionate Use of Protease Inhibitors in Viral C Cirrhosis study, 511 patients with HCV genotype 1 infection and compensated cirrhosis who did not respond to a prior course of peginterferon and ribavirin (44.3% relapsers or patients with viral breakthrough, 44.8% partial responders, and 8.0% null responders) were given either telaprevir (n = 299) or boceprevir (n = 212) for 48 weeks. We assessed percentages of patients with sustained viral responses 12 weeks after therapy and safety. This observational study did not allow for direct comparison of the 2 regimens. Among patients given telaprevir, 74.2% of relapsers, 40.0% of partial responders, and 19.4% of null responders achieved SVR12. Among those given boceprevir, 53.9% of relapsers, 38.3% of partial responders, and none of the null responders achieved SVR12. In multivariate analysis, factors associated with SVR12 included prior response to treatment response, no lead-in phase, HCV subtype 1b (vs 1a), and baseline platelet count greater than 100,000/mm(3). Severe adverse events occurred in 49.9% of cases, including liver decompensation, severe infections in 10.4%, and death in 2.2%. In multivariate analysis, baseline serum albumin level less than 35 g/L and baseline platelet counts of 100,000/mm(3) or less predicted severe side effects or death. Relatively high percentages of real-life, treatment-experienced patients with HCV genotype 1 infection and cirrhosis respond to the combination of peginterferon and ribavirin with telaprevir or boceprevir. However, side effects are frequent and often severe. Baseline levels of albumin and platelet counts can be used to guide treatment decisions. ClinicalTrials.gov number: NCT01514890.


Identification of novel synthetic toll-like receptor 2 agonists by high throughput screening.
Toll-like receptors (TLRs) play a central role in host defense by inducing inflammatory and adaptive immune responses following infection. Drugs that target TLRs are of considerable interest as potential inflammatory regulators, vaccine adjuvants, and novel immunotherapeutics. TLR2, in cooperation with either TLR1 or TLR6, mediates responses to a wide variety of microbial products as well as products of host tissue damage. In an effort to understand the structural basis of TLR2 recognition and uncover novel TLR2 agonists, a synthetic chemical library of 24,000 compounds was screened using an IL-8-driven luciferase reporter in cells expressing these human receptors. The screening yielded several novel TLR2-dependent activators that utilize TLR1, TLR6, or both as co-receptors. These novel small molecule compounds are aromatic in nature and structurally unrelated to any known TLR2 agonists. The three most potent compounds do not exhibit synergistic activity, nor do they act as pseudoantagonists toward natural TLR2 activators. Interestingly, two of the compounds exhibit species specificity and are inactive toward murine peritoneal macrophages. Mutational analysis reveals that although the central extracellular region of TLR1 is required for stimulation, there are subtle differences in the mechanism of stimulation mediated by the synthetic compounds in comparison with natural lipoprotein agonists. The three most potent compounds activate cells in the nanomolar range and stimulate cytokine production from human peripheral blood monocytes. Our results confirm the utility of high throughput screens to uncover novel synthetic TLR2 agonists that may be of therapeutic benefit.


Ratiometric bioluminescence indicators for monitoring cyclic adenosine 3',5'-monophosphate in live cells based on luciferase-fragment complementation.
Bioluminescent indicators for cyclic 3',5'-monophosphate AMP (cAMP) are powerful tools for noninvasive detection with high sensitivity. However, the absolute photon counts are affected substantially by adenosine 5'-triphosphate (ATP) and d-luciferin concentrations, limiting temporal analysis in live cells. This report describes a genetically encoded bioluminescent indicator for detecting intracellular cAMP based on complementation of split fragments of two-color luciferase mutants originated from click beetles. A cAMP binding domain of protein kinase A was connected with an engineered carboxy-terminal fragment of luciferase, of which ends were connected with amino-terminal fragments of green luciferase and red luciferase. We demonstrated that the ratio of green to red bioluminescence intensities was less influenced by the changes of ATP and d-luciferin concentrations. We also showed an applicability of the bioluminescent indicator for time-course and quantitative assessments of intracellular cAMP in living cells and mice. The bioluminescent indicator will enable quantitative analysis and imaging of spatiotemporal dynamics of cAMP in opaque and autofluorescent living subjects.


The E2F-DP1 Transcription Factor Complex Regulates Centriole Duplication in Caenorhabditis elegans.
Centrioles play critical roles in the organization of microtubule-based structures, from the mitotic spindle to cilia and flagella. In order to properly execute their various functions, centrioles are subjected to stringent copy number control. Central to this control mechanism is a precise duplication event that takes place during S phase of the cell cycle and involves the assembly of a single daughter centriole in association with each mother centriole . Recent studies have revealed that posttranslational control of the master regulator Plk4/ZYG-1 kinase and its downstream effector SAS-6 is key to ensuring production of a single daughter centriole. In contrast, relatively little is known about how centriole duplication is regulated at a transcriptional level. Here we show that the transcription factor complex EFL-1-DPL-1 both positively and negatively controls centriole duplication in the Caenorhabditis elegans embryo. Specifically, we find that down regulation of EFL-1-DPL-1 can restore centriole duplication in a zyg-1 hypomorphic mutant and that suppression of the zyg-1 mutant phenotype is accompanied by an increase in SAS-6 protein levels. Further, we find evidence that EFL-1-DPL-1 promotes the transcription of zyg-1 and other centriole duplication genes. Our results provide evidence that in a single tissue type, EFL-1-DPL-1 sets the balance between positive and negative regulators of centriole assembly and thus may be part of a homeostatic mechanism that governs centriole assembly.


Polyclonality of BRAF mutations in acquired melanocytic nevi.
Melanocytic nevi are thought to be senescent clones of melanocytes that have acquired an oncogenic BRAF mutation. BRAF mutation is considered to be a crucial step in the initiation of melanocyte transformation. However, using immunomagnetic separation or laser-capture microdissection, we examined BRAF mutations in sets of approximately 50 single cells isolated from acquired melanocytic nevi from 13 patients and found a substantial number of nevus cells that contained wild-type BRAF mixed with nevus cells that contained BRAF(V600E). Furthermore, we simultaneously amplified BRAF exon 15 and a neighboring single nucleotide polymorphism (SNP), rs7801086, from nevus cell samples obtained from four patients who were heterozygous for this SNP. Subcloning and sequencing of the polymerase chain reaction products showed that both SNP alleles harbored the BRAF(V600E) mutation, indicating that the same BRAF(V600E) mutation originated from different cells. The polyclonality of BRAF mutations in acquired melanocytic nevi suggests that mutation of BRAF may not be an initial event in melanocyte transformation.


Structural arrest and dynamic localization in biocolloidal gels.
Casein micelles interacting via an entropic intermediate-ranged depletion attraction exhibit a fluid-to-gel transition due to arrested spinodal decomposition. The bicontinuous networked structure of the gel freezes shortly after formation. We determine the timescales of structural arrest from the build-up of network rigidity after pre-shear rejuvenation, and find that the arrest time as well as the plateau elastic modulus of the gel diverge as a function of the volume fraction and interaction potential. Moreover, we show using scaling from naïve mode coupling theory that their mechanical properties are dictated by their microscopic dynamics rather than their heterogeneous large scale structure.


Effect of mold diameter on the depth of cure of a resin-based composite material.
The purpose of this study was to examine the effect of mold diameter on depth of cure of a resin-based composite material for varying amounts of irradiation. A resin-based composite was light-cured for 10-80 s in stainless-steel molds of either 6 mm or 4 mm in diameter and then dark-stored for 24 h. Specimens were then scraped back and the length of the cured specimens was measured to provide depth of cure (DSB ). Radiant exposure to each of the mold diameters was determined by measuring the power. The DSB values using the 4-mm molds were lower than those of the 6-mm molds. The average difference between the two groups for each irradiation time was 0.45 ± 0.02 mm. A fixed depth of cure required about 39% more irradiation time for the 4-mm mold than for the 6-mm mold but 75% more radiant exposure. The difference in cure depth for a fixed radiant exposure was 0.79 mm. A better comparison of depth of cure is obtained by using identical radiant exposures for different mold diameters. It is believed that greater loss of light by absorption at the stainless-steel cylinder walls for the 4-mm-diameter cylinders accounts for the lower depth of cure when compared with the 6-mm molds.


Predicting volume responsiveness by using the end-expiratory occlusion in mechanically ventilated intensive care unit patients.
During mechanical ventilation, inspiration cyclically decreases the left cardiac preload. Thus, an end-expiratory occlusion may prevent the cyclic impediment in left cardiac preload and may act like a fluid challenge. We tested whether this could serve as a functional test for fluid responsiveness in patients with circulatory failure. Prospective study. Medical intensive care unit. Thirty-four mechanically ventilated patients with shock in whom volume expansion was planned. A 15-second end-expiratory occlusion followed by a 500 mL saline infusion. Arterial pressure and pulse contour-derived cardiac index (PiCCOplus) at baseline, during passive leg raising (PLR), during the 5-last seconds of the end-expiratory occlusion, and after volume expansion. Volume expansion increased cardiac index by >15% (2.4 +/- 1.0 to 3.3 +/- 1.2 L/min/m, p < 0.05) in 23 patients ("responders"). Before volume expansion, the end-expiratory occlusion significantly increased arterial pulse pressure by 15% +/- 15% and cardiac index by 12% +/- 11% in responders whereas arterial pulse pressure and cardiac index did not change significantly in nonresponders. Fluid responsiveness was predicted by an increase in pulse pressure >or=5% during the end-expiratory occlusion with a sensitivity and a specificity of 87% and 100%, respectively, and by an increase in cardiac index >or=5% during the end-expiratory occlusion with a sensitivity and a specificity of 91% and 100%, respectively. The response of pulse pressure and cardiac index to the end-expiratory occlusion predicted fluid responsiveness with an accuracy that was similar to the response of cardiac index to PLR and that was significantly better than the response of pulse pressure to PLR (receiver operating characteristic curves area 0.957 [95% confidence interval [CI:] 0.825-0.994], 0.972 [95% CI: 0.849-0.995], 0.937 [95% CI: 0.797-0.990], and 0.675 [95% CI: 0.497-0.829], respectively). The hemodynamic response to an end-expiratory occlusion can predict volume responsiveness in mechanically ventilated patients.


Local anaesthetic toxicity: are we prepared for the consequences in the Emergency Department?
Local anaesthetic agents are commonly encountered in the Emergency Department (ED). Local anaesthetic toxicity leading to cardiorespiratory arrest is a rare, but potentially fatal, complication of an overdose of these agents. A recent innovation in the treatment of severe local anaesthetic toxicity has been the introduction of intravenous lipid emulsion therapy (Intralipid 20%). The aim of this study was to gauge the current level of knowledge surrounding the administration and complications associated with commonly used local anaesthetic agents. Questionnaires were distributed amongst the training grade doctors working in four Emergency Departments. Results were divided into two groups for ease of analysis. Core Trainees (CT) and Foundation Year 2 (F2) doctors were placed in one group. Specialist Registrars (SPR), Speciality Registrars (StR) and Staff Grades (SG) form the other group. The results showed that less than half of the CT/F2 group knew the maximum dose of lignocaine 1%. 80% of these doctors were unable to calculate the maximum dose of lignocaine 1% for an 80 kg man, and nearly one-third would administer a toxic dose. In addition, only one out of 30 in the CT/F2 group were aware of lipid emulsion therapy. Those using local anaesthetic should also be able to recognise the signs and symptoms of toxicity should this occur and act accordingly. The lack of knowledge amongst the more junior staff, as demonstrated by this project, highlights failings in teaching the basics of safe practices in the ED.


The Role of MSC Therapy in Attenuating the Damaging Effects of the Cytokine Storm Induced by COVID-19 on the Heart and Cardiovascular System.
The global pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that causes coronavirus disease 2019 (COVID-19) has led to 47 m infected cases and 1. 2 m (2.6%) deaths. A hallmark of more severe cases of SARS-CoV-2 in patients with acute respiratory distress syndrome (ARDS) appears to be a virally-induced over-activation or unregulated response of the immune system, termed a "cytokine storm," featuring elevated levels of pro-inflammatory cytokines such as IL-2, IL-6, IL-7, IL-22, CXCL10, and TNFα. Whilst the lungs are the primary site of infection for SARS-CoV-2, in more severe cases its effects can be detected in multiple organ systems. Indeed, many COVID-19 positive patients develop cardiovascular complications, such as myocardial injury, myocarditis, cardiac arrhythmia, and thromboembolism, which are associated with higher mortality. Drug and cell therapies targeting immunosuppression have been suggested to help combat the cytokine storm. In particular, mesenchymal stromal cells (MSCs), owing to their powerful immunomodulatory ability, have shown promise in early clinical studies to avoid, prevent or attenuate the cytokine storm. In this review, we will discuss the mechanistic underpinnings of the cytokine storm on the cardiovascular system, and how MSCs potentially attenuate the damage caused by the cytokine storm induced by COVID-19. We will also address how MSC transplantation could alleviate the long-term complications seen in some COVID-19 patients, such as improving tissue repair and regeneration.


Frequent Involvement of the Duodenum with Lanthanum Deposition: A Retrospective Observational Study.
Objective This study aimed to investigate the endoscopic features of lanthanum-associated duodenal lesions and the prevalence of duodenal involvement among patients with pathologically proven lanthanum deposition in the gastrointestinal tract. Methods We retrospectively reviewed 24 patients with pathologically proven lanthanum deposition in the gastrointestinal tract. Patients were subdivided into three groups: Group A, patients with pathologically-proven lanthanum deposition in the duodenum; Group B, patients without lanthanum deposition in the duodenum; and Group C, patients without a biopsy of the duodenum. Results A biopsy examination of the duodenum was performed in 19 patients, and lanthanum deposition was detected in 17 patients (17/19, 89.5%). In group A (n=17), whitish duodenal villi were detected in 15 patients during esophagogastroduodenoscopy (15/17, 88.2%). While the other two patients showed no whitish villi, a biopsy of the duodenal mucosa revealed lanthanum deposition. The deposition of a white substance showing a clear margin was visible within multiple villi under magnified observation in some patients of group A. Group B patients (n=2) also showed whitish villi. However, the whitish color was faint in one case and sparse in the other case. Conclusion Lanthanum deposits in the duodenum may resemble white villi. However, in some cases, these deposits may be unrecognizable during esophagogastroduodenoscopy due to the subtle degree of deposition. Endoscopists should biopsy the duodenum as well as the stomach, regardless of the presence or absence of white villi, for an accurate determination of lanthanum deposition in the gastrointestinal tract.


Emphysematous Gastritis in the Setting of Chronic Abdominal Pain.
Emphysematous gastritis is a rare but fatal variant of gastritis. It is caused by gastric wall invasion by gas-forming organisms. It follows disruption of gastric mucosal integrity by a variety of factors, most commonly caustic ingestion and alcohol abuse. Patients typically present with abdominal symptoms with features of septic shock. Emphysematous gastritis carries a high mortality rate warranting early intervention with supportive measures and broad-spectrum antibiotics. It is essential to consider this rare entity in the differential diagnosis of a patient presenting with abdominal pain as timely intervention is crucial for survival. Emphysematous gastritis is rare but important to keep in mind when evaluating patients with acute abdominal pain.Emphysematous gastritis is a serious condition which has a high mortality rate if not diagnosed early.


A randomized controlled trial of venlafaxine extended release in generalized social anxiety disorder.
Generalized social anxiety disorder is a debilitating psychiatric illness characterized by maladaptive thoughts about social situations. This double-blind study evaluated the anxiolytic efficacy, safety, and tolerability of venlafaxine extended release (ER) in adult out-patients with generalized social anxiety disorder. Patients were randomly assigned to receive 12 weeks of treatment with a flexible dose of venlafaxine ER (75 to 225 mg/day) or placebo. The Liebowitz Social Anxiety Scale (LSAS) total score was the primary efficacy variable. Secondary efficacy variables included scores on the Clinical Global Impressions-Severity of Illness (CGI-S) and -Improvement (CGI-I) scales, Social Phobia Inventory (SPIN), and LSAS subscales. Response was defined as a CGI-I score of 1 or 2. Two definitions of remission were used: LSAS total score < or = 30 and CGI-I score of 1. Data from 271 patients (intent-to-treat population) were analyzed for efficacy; 279 patients were analyzed for safety. Overall, 173 patients completed the study. Improvement on the LSAS was significantly greater with venlafaxine ER treatment than with placebo at weeks 6 through 12 (p < .05, weeks 6 and 8; p < .01, week 10; and p < .001, week 12) and at weeks 8 through 12 based on CGI-S and SPIN scores. Week 12 response and remission (LSAS score < or = 30) rates were significantly greater in the venlafaxine ER group than in the placebo group (response: 44% vs. 30%, respectively, p = .018; remission: 20% vs. 7%, respectively, p < .01). Patients experienced no unexpected or serious adverse events. Venlafaxine ER is safe, well tolerated, and efficacious in the short-term treatment of generalized social anxiety disorder.


Limnospira indica PCC8005 growth in photobioreactor: model and simulation of the ISS and ground experiments.
The Arthrospira-B experiment is the first experiment in space ever allowing the online measurements of both oxygen production rate and growth rate of Limnospira indica PCC8005 in batch photobioreactors running on-board ISS. Four bioreactors were integrated in the ISS Biolab facility. Each reactor was composed of two chambers (gas and liquid) separated by a PTFE membrane and was run in batch conditions. Oxygen production was monitored by online measurement of the total pressure increase in the gas chamber. The experiments are composed of several successive batch cultures for each reactor, performed in parallel on ISS and on ground. In this work, a model for the growth of the cyanobacterium Limnospira indica PCC8005 (also known as Arthrospira or spirulina) in these space membrane photobioreactors was proposed and the simulation results obtained are compared to the experimental results gathered in space and on ground. The photobioreactor model was based on a light transfer limitation model, already used to describe and predict the growth and oxygen production in small to large scale ground photobioreactors. It was completed by a model for pH prediction in the liquid phase allowing assessment of the pH increase associated to the bicarbonate consumption for the biomass growth. A membrane gas-liquid transfer model is used to predict the gas pressure increase in the gas chamber. Substrate limitation is considered in the biological model. A quite satisfactory fit was achieved between experimental and simulation results when a suitable mixing of the liquid phase was maintained. The data showed that microgravity has no first order effect on the oxygen production rate of Limnospira indica PCC8005 in a photobioreactor operating in space in zero gravity conditions.


Nitric oxide modulates air embolism-induced lung injury in rats with normotension and hypertension.
1. Air embolism the in lungs induces microvascular obstruction, mediator release and acute lung injury (ALI). Nitrite oxide (NO) plays protective and pathological roles in ALI produced by various causes, but its role in air embolism-induced ALI has not been fully investigated. 2. The purpose of the present investigation was to elucidate the involvement of NO and pro-inflammatory cytokines in the pathogenesis of ALI following air infusion into isolated perfused lungs from spontaneously hypertensive rats (SHR) and normotensive Wistar Kyoto (WKY) rats. 3. The extent of ALI was evaluated by changes in lung weight, Evans blue dye leakage, the protein concentration in the bronchoalveolar lavage and pathological examination. We also measured nitrite/nitrate (NO(x)), tumour necrosis factor (TNF)-alpha and interleukin (IL)-1beta concentrations in lung perfusate and determined cGMP in lung tissue. 4. The NO synthase (NOS) inhibitors N(G)-nitro-l-arginine methyl ester (l-NAME) and l-N(6)-(1-iminoethyl)-lysine (l-Nil), as well as the NO donors sodium nitroprusside (SNP) and s-nitroso-N-acetylpenicillamine (SNAP), were administered 30 min before air embolism at a concentration of 10(-3) mol/L in the lung perfusate. 5. Air embolism-induced ALI was enhanced by pretreatment with l-NAME or l-Nil, but was alleviated by SNP or SNAP pretreatment, in both SHR and WKY rats. In both SHR and WKY rats, AE elevated levels of NO(x) (2.6 and 28.7%, respectively), TNF-alpha (52.7 and 158.6%, respectively) and IL-1beta (108.4 and 224.1%, respectively) in the lung perfusate and cGMP levels in lung tissues (35.8 and 111.2%, respectively). Pretreatment with l-LAME or l-Nil exacerbated, whereas SNP or SNAP abrogated, the increases in these factors, except in the case of NO(x) (levels were decreased by l-LAME or l-Nil pretreatment and increased by SNP or SNAP pretreatment). 6. Air embolism caused increases in the lung weight (LW)/bodyweight ratio, LW gain, protein concentration in bronchoalveolar lavage and Evans blue dye leakage. These AE-induced changes were less in lungs isolated from SHR compared with normotensive WKY rats. 7. The results suggest that ALI and associated changes following air embolism in lungs isolated from SHR are less than those in WKY rats. Nitric oxide production through inducible NOS isoforms reduces air embolism-induced lung injury and associated changes. Spontaneously hypertensive rats appear to be more resistant than WKY rats to air embolism challenge.


Familial pancreatic cancer.
For many years anecdotal case reports have suggested that pancreatic cancer aggregates in some families. Two recent advances have established that this is in fact the case. First, large registries, such as the National Familial Pancreas Tumor Registry (NFPTR) at Johns Hopkins, have identified a number of families in which multiple family members have been diagnosed with pancreatic cancer. As a result, the patterns of inheritance of pancreatic cancer can now be studied on a scale not possible before. Second, advances in molecular genetic techniques make it possible to test members of these families for germline mutations in known candidate cancer causing genes. As a result, some of the genetic alterations responsible for the familial aggregation of pancreatic cancer have been identified in some families. The NFPTR has enrolled 362 families in which at least one family member has been diagnosed with pancreatic cancer. These include 151 families in which at least two first-degree relatives have been diagnosed with pancreatic cancer. Analysis of these families has revealed that even second-degree relatives of patients from these families are at increased risk of developing pancreatic cancer. In addition, a number of kindreds which exhibit aggregation of cancer have been tested for germline mutations in known cancer causing genes. Germline mutations in BRCA2 have been shown to predispose to both breast and pancreatic cancer, germline mutations in p16 to melanoma and pancreatic cancer (the FAMMM syndrome), and genetic mutations in STK11/LKB1 to pancreatic cancer in patients with the Peutz-Jeghers Syndrome (PJS). Pancreatic cancer aggregates in some families, and relatives of patients with pancreatic cancer have an increased risk of developing pancreatic cancer themselves. The genetic basis for the familial aggregation of pancreatic cancer has been shown to be germline mutations in known cancer causing genes in some of these families.


Techniques for quantifying effects of dietary antioxidants on transcription factor translocation and nitric oxide production in cultured cells.
Dietary antioxidants can affect cellular processes relevant to chronic inflammatory diseases such as atherosclerosis. We have used non-standard techniques to quantify effects of the antioxidant soy isoflavones genistein and daidzein on translocation of Nuclear Factor-KB (NF-KB) and nitric oxide (NO) production, which are important in these diseases. Translocation was quantified using confocal immunofluoresecence microscopy and ratiometric image analysis. NO was quantified by an electrochemical method after reduction of its oxidation products in cell culture supernatants. Activation of the RAW 264.7 murine monocytel macrophage cell line increased the ratio of nuclear to cytoplasmic immunostaining for NF-kappaB. The increase was exacerbated by pre-treatment with genistein or daidzein. To show that decreases could also be detected, pre-treatment with the pine bark extract Pycnogenol(R) was examined, and found to reduce translocation. NO production was also increased by activation, but was reduced by pre-treatment with genistein or daidzein. In the EA.hy926 human endothelial cell line, constitutive production was detectable and was increased by thrombin. The confocal and electrochemical methods gave data that agreed with results obtained using the established electromobility shift and Griess assays, but were more sensitive, more convenient, gave more detailed information and avoided the use of radioisotopes.


Crystal structure of putidaredoxin, the [2Fe-2S] component of the P450cam monooxygenase system from Pseudomonas putida.
Stability of the [2Fe-2S]-containing putidaredoxin (Pdx), the electron donor to cytochrome P450cam in Pseudomonas putida, was improved by mutating non-ligating cysteine residues, Cys73 and Cys85, to serine singly and in combination. The increasing order of stability is Cys73Ser/Cys85Ser>Cys73Ser>Cys85Ser>WT Pdx. Crystal structures of Cys73Ser/Cys85Ser and Cys73Ser mutants of Pdx, solved by single-wavelength anomalous dispersion phasing using the [2Fe-2S] iron atoms to 1.47 A and 1.65 A resolution, respectively, are nearly identical and very similar to those of bovine adrenodoxin (Adx) and Escherichia coli ferredoxin. However, unlike the Adx structure, no motion between the core and interaction domains of Pdx is observed. This higher conformational stability of Pdx might be due to the presence of a more extensive hydrogen bonding network at the interface between the two structural domains around the conserved His49. In particular, formation of a hydrogen bond between the side-chain of Tyr51 and the carbonyl oxygen atom of Glu77 and the presence of two well-ordered water molecules linking the interaction domain and the C-terminal peptide to the core of the molecule are unique to Pdx. The folding topology of the NMR model is similar to that of the X-ray structure of Pdx. The overall rmsd of Calpha positions between the two models is 1.59 A. The largest positional differences are observed for residues 18-21 and 33-37 in the loop regions and the C terminus. The latter two peptides display conformational heterogeneity in the crystal structures. Owing to flexibility, the aromatic ring of the C-terminal Trp106 can closely approach the side-chains of Asp38 and Thr47 (3.2-3.9 A) or move away and leave the active site solvent-exposed. Therefore, Trp106, previously shown to be important in the Pdr-to-Pdx and Pdx-to-P450cam electron transfer reactions is in a position to regulate and/or mediate electron transfer to or from the [2Fe-2S] center of Pdx.


Concurrent use of benzodiazepines, antidepressants, and opioid analgesics with zolpidem and risk for suicide: a case-control and case-crossover study.
To evaluate whether the concurrent use of benzodiazepines, antidepressants, and opioid analgesics with zolpidem increases the risk of suicide or triggers suicide compared with the use of zolpidem alone. We conducted a case-control and case-crossover study using the Korean National Health Insurance Service-National Sample Cohort database. Cases were older than 20 years with a suicide record (International Codes of Disease 10th Revision codes: X-60-X84 and Y87.0 intentional self-harm) between January 1, 2004, and December 31, 2013. For case-control design, ten controls were matched to each case by age, sex, index year, region, income, and health insurance type. For case-crossover analysis, we set hazard period to 60 days and assigned five corresponding sets of control periods of equal length. Exposure was assessed during 60 days before suicide for combinations of benzodiazepines, antidepressants, opioid analgesics with zolpidem against zolpidem alone. We conducted a conditional logistic regression to estimate odds ratios (ORs) and their 95% confidence intervals (CIs). In the case-control study, the risk of suicide was 2.80-fold higher in cases taking benzodiazepines and antidepressants with zolpidem than in those taking zolpidem alone (adjusted OR [aOR], 2.80; 95% CI, 1.38-5.70). However, in the case-crossover study, suicide risk showed no significant difference (crude OR [cOR], 0.92; 95% CI, 0.55-1.52) and was underpowered. The results of the traditional case-control study confirmed that the concurrent use of benzodiazepines and antidepressants with zolpidem was associated with an increased risk of suicide compared with the use of zolpidem alone. However, there was no significant difference in the magnitude of risk in the within-person comparison design.


Interexaminer reliability of eight evaluative dimensions of lumbar segmental abnormality.
The interexaminer reliability of noninvasive methods of examining lumbar spinal segments is not well established. In this project the interexaminer reliability of three experienced chiropractic examiners, who evaluated 21 symptomatic and 25 asymptomatic subjects, was explored. Eight noninvasive segmental examination strategies (dimensions) were employed at each spinal segment from T11/T12 through L5/S1. Marginal to good agreement beyond chance was noted for palpatory pain over osseous structures and in paraspinal soft-tissues. Weaker and less frequently, significant concordance between examiners was noted for detection of temperature differences (greater than or equal to 1.5 degrees F) between adjacent segments and for visual inspection for segmental abnormality. Little significant agreement between examiner was found for active and passive motion palpation, muscle tension palpation and misalignment palpation. This study suggests that "subjective" findings (pain) may be among the most reliable of conservative spinal observations. Weak but significant correlations were found when positive findings for the eight dimensions at each lumbar segment were summed to form a composite joint abnormality index. When the multidimension index was developed using the four most reliable dimensions, slightly stronger correlations were found. The strongest agreement between examiners tended to be found in the lower lumbar spine.


Water-soluble green perylenediimide (PDI) dyes as potential sensitizers for photodynamic therapy.
[reaction: see text] A series of water-soluble green perylenediimide (PDI) dyes have been synthesized. On red light excitation, these dyes were shown to be efficient generators of singlet oxygen, and in cell culture media, they were shown to display significant light-induced cytotoxic effects on the human erythroleukemia cell line (K-562). It appears that highly versatile PDI dyes are likely to find new applications in photodynamic therapy.


Is being underweight associated with impairments in quality of life in the absence of significant eating disorder pathology?
Few studies have compared low-weight individuals with eating disorder (ED) pathology with similar-weight individuals without significant pathology despite the fact that body weight is often used as a key outcome within ED research. This study compared quality of life (QoL) in one group with high levels of ED pathology to a group with low ED pathology, matched by body mass index (BMI). The high ED group reported significantly lower ED-specific quality of life (EDQoL) than the low ED group. These findings suggest that young women with high levels of ED pathology report significantly more impaired QoL than comparable young women with no ED pathology, and that being underweight alone is not a primary contributor to poorer EDQoL.


Use of diffusion tensor imaging in glioma resection.
Diffusion tensor imaging (DTI) is increasingly used in the resection of both high- and low-grade gliomas. Whereas conventional MRI techniques provide only anatomical information, DTI offers data on CNS connectivity by enabling visualization of important white matter tracts in the brain. Importantly, DTI allows neurosurgeons to better guide their surgical approach and resection. Here, the authors review basic scientific principles of DTI, include a primer on the technology and image acquisition, and outline the modality's evolution as a frequently used tool for glioma resection. Current literature supporting its use is summarized, highlighting important clinical studies on the application of DTI in preoperative planning for glioma resection, preoperative diagnosis, and postoperative outcomes. The authors conclude with a review of future directions for this technology.


Internal electrolyte temperatures for polymer and fused-silica capillaries used in capillary electrophoresis.
Polymers are important as materials for manufacturing microfluidic devices for electrodriven separations, in which Joule heating is an unavoidable phenomenon. Heating effects were investigated in polymer capillaries using a CE setup. This study is the first step toward the longer-term objective of the study of heating effects occurring in polymeric microfluidic devices. The thermal conductivity of polymers is much smaller than that of fused silica (FS), resulting in less efficient dissipation of heat in polymeric capillaries. This study used conductance measurements as a temperature probe to determine the mean electrolyte temperatures in CE capillaries of different materials. Values for mean electrolyte temperatures in capillaries made of New Generation FluoroPolymer (NGFP), poly-(methylmethacrylate) (PMMA), and poly(ether ether ketone) (PEEK) capillaries were compared with those obtained for FS capillaries. Extrapolation of plots of conductance versus power per unit length (P/L) to zero power was used to obtain conductance values free of Joule heating effects. The ratio of the measured conductance values at different power levels to the conductance at zero power was used to determine the mean temperature of the electrolyte. For each type of capillary material, it was found that the average increase in the mean temperature of the electrolyte (DeltaT(Mean)) was directly proportional to P/L and inversely proportional to the thermal conductivity (lambda) of the capillary material. At 7.5 W/m, values for DeltaT(Mean) for NGFP, PMMA, and PEEK were determined to be 36.6, 33.8, and 30.7 degrees C, respectively. Under identical conditions, DeltaT(Mean) for FS capillaries was 20.4 degrees C.


3-D technology used to accurately understand equine ileocolonic aganglionosis.
Ileocolonic aganglionosis (ICA) is the congenital and hereditary absence of neurons that constitute the enteric nervous system and has been described in various species including humans - Hirschsprung's disease - and horses - overo lethal white syndrome (OLWS). Hirschsprung's disease affects circa 1 in 5,000 live births. At best, this disease means an inability to absorb nutrients from food (humans). At worse, in horses, it always means death. Despite our general understanding of the functional mechanisms underlying ICA, there is a paucity of reliable quantitative information about the structure of myenteric and submucosal neurons in healthy horses and there are no studies on horses with ICA. In light of these uncertainties, we have used design-based stereology to describe the 3-D structure - total number and true size - of myenteric and submucosal neurons in the ileum of ICA horses. Our study has shown that ICA affects all submucosal neurons and 99% of myenteric neurons. The remaining myenteric neurons (0.56%) atrophy immensely, i.e. 63.8%. We believe this study forms the basis for further research, assessing which subpopulation of myenteric neurons are affected by ileocolonic aganglionosis, and we would like to propose a new nomenclature to distinguish between a complete absence of neurons - aganglionosis - and a weaker form of the disease which we suggest naming 'hypoganglionosis'. Our results are a step forward in understanding this disease structurally.


Resveratrol inhibits nonalcoholic fatty liver disease in rats.
The prevalence of nonalcoholic fatty liver disease (NAFLD) is high. NAFLD is linked to obesity, diabetes mellitus, and hypertriglyceridemia. Approximately 20% of patients with NAFLD will eventually develop cirrhosis. Our purpose was to investigate whether resveratrol decreased hepatic steatosis in an animal model of steatosis, and whether this therapeutic approach resulted in a decrease in tumor necrosis factor alpha (TNF-alpha) production, lipid peroxidation and oxidative stress. Male Wistar CRL: Wi (Han) (225 g) rats were randomized into three groups. A control group (n = 12) was given free access to regular dry rat chow for 4 weeks. The steatosis (n = 12) and resveratrol (n = 12) groups were given free access to feed (a high carbohydrate-fat free modified diet) and water 4 days per week, and fasted for the remaining 3 days for 4 weeks. Rats in the resveratrol group were given resveratrol 10 mg daily by the oral route. All rats were killed at 4 weeks and assessed for fatty infiltration and bacterial translocation. Levels of TNF-alpha in serum, hepatic malondialdehyde (MDA), oxidative stress (superoxide dismutase, glutathione peroxidase, catalase and nitric oxide synthase) and biochemical parameters were measured. Fat deposition was decreased in the resveratrol group as compared to the steatosis group (Grade 1 vs Grade 3, P < 0.05). TNF-alpha and MDA levels were significantly increased in the steatosis group (TNF-alpha; 33.4 +/- 5.2 vs 26.24 +/- 3.47 pg/ml and MDA; 9.08 +/- 0.8 vs 3.17 +/- 1.45 muM respectively, P < 0.05). This was accompanied by increased superoxide dismutase, glutathione peroxidase and catalase and decreased nitric oxide synthase in the liver of resveratrol group significantly (P < 0.05 vs steatosis group). Bacterial translocation was not found in any of the groups. Glucose levels were decreased in the group of rats given resveratrol (P < 0.05). Resveratrol decreased NAFLD severity in rats. This effect was mediated, at least in part, by TNF-alpha inhibition and antioxidant activities.


Corpus callosum and cerebellar vermis size in very preterm infants: Relationship to long-term neurodevelopmental outcome.
The neonatal changes of corpus callosum or cerebellar volume in preterm infants have been shown to link with abnormal mentality and motor disability in early childhood. This study aims to predict the long-term neurological outcomes by measuring these changes on neonatal brain ultrasound in preterm infants. Our cohort consisted of infants aged below 32 weeks' gestation with very low birth body weights who completed neuro-assessments at 5 years of age. Corpus callosum or cerebellar vermis were measured at 28-30 weeks and at 37-40 weeks gestational age in premature infants with cerebral palsy (CP), mental retardation (MR) and normal control premature infants. There are 12 patients in MR group, 12 in CP group and 27 patients as controls for final analysis. There was no significant difference in other factors between study groups except lower gestational age (P = 0.043) in CP group. Respiratory distress syndrome was more common in MR group (P = 0.037) and cystic periventricular leukomalacia was more common in CP group (P < 0.001) than controls. After adjusting for sex and birth body weight, the MR group had smaller cerebellar vermis area at 37-40 gestational weeks (P = 0.002) than controls. They also reduced the growth of corpus callosum area (difference = -0.12 ± 0.16, P = 0.029) and cerebellar vermis area (difference = 1.10 ± 0.44, P = 0.020) from 28 to 30 gestational weeks to 37-40 gestational weeks compared with controls (difference = 0.03 ± 0.15, 1.92 ± 0.70, respectively). In contrast, the CP group had reduced the growth of corpus callosum body (difference = -0.02 ± 0.18, P = 0.034) compared with controls (difference = 0.03 ± 0.04). They subsequently had smaller body thickness of corpus callosum (0.10 ± 0.02, P = 0.015) at 37-40 gestational weeks than controls (0.14 ± 0.04). Serial monitoring corpus callosum and cerebellar vermis size in early life of very preterm babies may predict the motor or mentality neurological outcome at 5 years of age.


RDC derived protein backbone resonance assignment using fragment assembly.
Experimental residual dipolar couplings (RDCs) in combination with structural models have the potential for accelerating the protein backbone resonance assignment process because RDCs can be measured accurately and interpreted quantitatively. However, this application has been limited due to the need for very high-resolution structural templates. Here, we introduce a new approach to resonance assignment based on optimal agreement between the experimental and calculated RDCs from a structural template that contains all assignable residues. To overcome the inherent computational complexity of such a global search, we have adopted an efficient two-stage search algorithm and included connectivity data from conventional assignment experiments. In the first stage, a list of strings of resonances (CA-links) is generated via exhaustive searches for short segments of sequentially connected residues in a protein (local templates), and then ranked by the agreement of the experimental (13)C(α) chemical shifts and (15)N-(1)H RDCs to the predicted values for each local template. In the second stage, the top CA-links for different local templates in stage I are combinatorially connected to produce CA-links for all assignable residues. The resulting CA-links are ranked for resonance assignment according to their measured RDCs and predicted values from a tertiary structure. Since the final RDC ranking of CA-links includes all assignable residues and the assignment is derived from a "global minimum", our approach is far less reliant on the quality of experimental data and structural templates. The present approach is validated with the assignments of several proteins, including a 42 kDa maltose binding protein (MBP) using RDCs and structural templates of varying quality. Since backbone resonance assignment is an essential first step for most of biomolecular NMR applications and is often a bottleneck for large systems, we expect that this new approach will improve the efficiency of the assignment process for small and medium size proteins and will extend the size limits assignable by current methods for proteins with structural models.


Trans-channel interactions in batrachotoxin-modified skeletal muscle sodium channels: voltage-dependent block by cytoplasmic amines, and the influence of mu-conotoxin GIIIA derivatives and permeant ions.
External mu-conotoxins and internal amine blockers inhibit each other's block of voltage-gated sodium channels. We explore the basis of this interaction by measuring the shifts in voltage-dependence of channel inhibition by internal amines induced by two mu-conotoxin derivatives with different charge distributions and net charges. Charge changes on the toxin were made at residue 13, which is thought to penetrate most deeply into the channel, making it likely to have the strongest individual interaction with an internal charged ligand. When an R13Q or R13E molecule was bound to the channel, the voltage dependence of diethylammonium (DEA)-block shifted toward more depolarized potentials (23 mV for R13Q, and 16 mV for R13E). An electrostatic model of the repulsion between DEA and the toxin simulated these data, with a distance between residue 13 of the mu-conotoxin and the DEA-binding site of approximately 15 A. Surprisingly, for tetrapropylammonium, the shifts were only 9 mV for R13Q, and 7 mV for R13E. The smaller shifts associated with R13E, the toxin with a smaller net charge, are generally consistent with an electrostatic interaction. However, the smaller shifts observed for tetrapropylammonium than for DEA suggest that other factors must be involved. Two observations indicate that the coupling of permeant ion occupancy of the channel to blocker binding may contribute to the overall amine-toxin interaction: 1), R13Q binding decreases the apparent affinity of sodium for the conducting pore by approximately 4-fold; and 2), increasing external [Na(+)] decreases block by DEA at constant voltage. Thus, even though a number of studies suggest that sodium channels are occupied by no more than one ion most of the time, measurable coupling occurs between permeant ions and toxin or amine blockers. Such interactions likely determine, in part, the strength of trans-channel, amine-conotoxin interactions.


Identification of critical residues in the active site of porcine membrane-bound aminopeptidase P.
The membrane-bound form of mammalian aminopeptidase P (AP-P; EC 3.4. 11.9) is a mono-zinc-containing enzyme that lacks any of the typical metal binding motifs found in other zinc metalloproteases. To identify residues involved in metal binding and catalysis, sequence and structural information was used to align the sequence of porcine membrane-bound AP-P with other members of the peptidase clan MG, including Escherichia coli AP-P and methionyl aminopeptidases. Residues predicted to be critical for activity were mutated and the resultant proteins were expressed in COS-1 cells. Immunoelectrophoretic blot analysis was used to compare the levels of expression of the mutant proteins, and their ability to hydrolyze bradykinin and Gly-Pro-hydroxyPro was assessed. Asp449, Asp460, His523, Glu554, and Glu568 are predicted to serve as metal ion ligands in the active site, and mutagenesis of these residues resulted in fully glycosylated proteins that were catalytically inactive. Mutation of His429 and His532 also resulted in catalytically inactive proteins, and these residues, by analogy with E. coli AP-P, are likely to play a role in shuttling protons during catalysis. These studies indicate that mammalian membrane-bound AP-P has an active-site configuration similar to that of other members of the peptidase clan MG, which is compatible with either a dual metal ion model or a single metal ion in the active site. The latter model is consistent, however, with the known metal stoichiometry of both the membrane-bound and cytosolic forms of AP-P and with a recently proposed model for methionyl aminopeptidase.


Color updating on the apparent motion path.
When a static stimulus appears successively at two distant locations, we perceive illusory motion of the stimulus across them-long-range apparent motion (AM). Previous studies have shown that when the apparent motion stimuli differ in shape, interpolation between the two shapes is perceived across the AM path. In contrast, the perceived color during AM has been shown to abruptly change from the color of the first stimulus into that of the second, suggesting interpolation does not occur for color during AM. Here, we report the first evidence to our knowledge, that an interpolated color, distinct from the colors of either apparent motion stimulus, is represented as the intermediate percept on the path of apparent motion. Using carefully chosen target colors-cyan, pink, and lime-that are perceptually and neurally intermediate between blue and green, orange and magenta, and green and orange respectively, we show that detection of a target presented on the apparent motion path was impaired when the color of the target was "in-between" the initial and terminal stimulus colors. Furthermore, we show that this feature-specific masking effect for the intermediate color cannot be accounted for by color similarity between the intermediate color and the color of the terminal inducer. Our findings demonstrate that intermediate colors can be interpolated over the apparent motion trajectory as in the case of shape, possibly involving similar interpolation processes for shape and color during apparent motion.


An approach for calculating a confidence interval from a single aquatic sample for monitoring hydrophobic organic contaminants.
The use of passive sampling devices (PSDs) for monitoring hydrophobic organic contaminants in aquatic environments can entail logistical constraints that often limit a comprehensive statistical sampling plan, thus resulting in a restricted number of samples. The present study demonstrates an approach for using the results of a pilot study designed to estimate sampling variability, which in turn can be used as variance estimates for confidence intervals for future n = 1 PSD samples of the same aquatic system. Sets of three to five PSDs were deployed in the Portland Harbor Superfund site for three sampling periods over the course of two years. The PSD filters were extracted and, as a composite sample, analyzed for 33 polycyclic aromatic hydrocarbon compounds. The between-sample and within-sample variances were calculated to characterize sources of variability in the environment and sampling methodology. A method for calculating a statistically reliable and defensible confidence interval for the mean of a single aquatic passive sampler observation (i.e., n = 1) using an estimate of sample variance derived from a pilot study is presented. Coverage probabilities are explored over a range of variance values using a Monte Carlo simulation.


Type 2 diabetes TCF7L2 risk genotypes alter birth weight: a study of 24,053 individuals.
The role of genes in normal birth-weight variation is poorly understood, and it has been suggested that the genetic component of fetal growth is small. Type 2 diabetes genes may influence birth weight through maternal genotype, by increasing maternal glycemia in pregnancy, or through fetal genotype, by altering fetal insulin secretion. We aimed to assess the role of the recently described type 2 diabetes gene TCF7L2 in birth weight. We genotyped the polymorphism rs7903146 in 15,709 individuals whose birth weight was available from six studies and in 8,344 mothers from three studies. Each fetal copy of the predisposing allele was associated with an 18-g (95% confidence interval [CI] 7-29 g) increase in birth weight (P=.001) and each maternal copy with a 30-g (95% CI 15-45 g) increase in offspring birth weight (P=2.8x10-5). Stratification by fetal genotype suggested that the association was driven by maternal genotype (31-g [95% CI 9-48 g] increase per allele; corrected P=.003). Analysis of diabetes-related traits in 10,314 nondiabetic individuals suggested the most likely mechanism is that the risk allele reduces maternal insulin secretion (disposition index reduced by ~0.15 standard deviation; P=1x10-4), which results in increased maternal glycemia in pregnancy and hence increased offspring birth weight. We combined information with the other common variant known to alter fetal growth, the -30G-->A polymorphism of glucokinase (rs1799884). The 4% of offspring born to mothers carrying three or four risk alleles were 119 g (95% CI 62-172 g) heavier than were the 32% born to mothers with none (for overall trend, P=2x10-7), comparable to the impact of maternal smoking during pregnancy. In conclusion, we have identified the first type 2 diabetes-susceptibility allele to be reproducibly associated with birth weight. Common gene variants can substantially influence normal birth-weight variation.


Phenformin and lactic acidosis.
Thirty-eight patients who presented with diabetes and a changed state of consciousness satisfied the criteria for lactic acidosis. Sixteen patients were non-ketotic, and 15 of these were receiving phenformin on admission. In all but one of these 15 patients, however, additional renal or cardiovascular abnormalities, or both, could be identified, which supported a multifactorial aetiology for lactic acidosis. Advanced age and cardiovascular and renal disease are absolute contraindications to the use of phenformin in diabetics.


New approaches & technologies of venomics to meet the challenge of human envenoming by snakebites in India.
The direct estimate of 46,000 snakebite deaths in India in 2005 (1 for every 2 HIV/AIDS deaths), based on verbal autopsies, renders unrealistic the total of only 47,000 snakebite deaths in the whole world in 2010, obtained indirectly as part of the "Global Burden of Disease 2010" study. Persistent underestimation of its true morbidity and mortality has made snakebite the most neglected of all the WHO's "neglected tropical diseases", downgrading its public health importance. Strategies to address this neglect should include the improvement of antivenom, the only specific antidote to envenoming. To accommodate increased understanding of geographical intraspecific variation in venom composition and the range of snake species that are medically important in India, the design of antivenoms (choice of venom sources and species coverage) should be reconsidered. Methods of preclinical and clinical testing should be improved. The relatively new science of venomics involves techniques and strategies for assessing the toxin composition of snake venoms directly through proteomics-centred approaches or indirectly via high-throughput venom gland transcriptomics and bioinformatic analysis. Antivenomics is translational venomics: a proteomics-based protocol to quantify the extent of cross-reactivity of antivenoms against homologous and heterologous venoms. These approaches could revolutionize the preclinical assessment of antivenom efficacy, leading to a new generation of antivenoms that are clinically more effective.


Replicative senescence: implications for in vivo aging and tumor suppression.
Normal cells have limited proliferative potential in culture, a fact that has been the basis of their use as a model for replicative senescence for many years. Recent molecular analyses have identified numerous changes in gene expression that occur as cells become senescent, and the results indicate that multiple levels of control contribute to the irreversible growth arrest. These include repression of growth stimulatory genes, overexpression of growth inhibitory genes, and interference with downstream pathways. Studies with cell types other than fibroblasts will better define the role of cell senescence in the aging process and in tumorigenesis.


Development of the brain's functional network architecture.
A full understanding of the development of the brain's functional network architecture requires not only an understanding of developmental changes in neural processing in individual brain regions but also an understanding of changes in inter-regional interactions. Resting state functional connectivity MRI (rs-fcMRI) is increasingly being used to study functional interactions between brain regions in both adults and children. We briefly review methods used to study functional interactions and networks with rs-fcMRI and how these methods have been used to define developmental changes in network functional connectivity. The developmental rs-fcMRI studies to date have found two general properties. First, regional interactions change from being predominately anatomically local in children to interactions spanning longer cortical distances in young adults. Second, this developmental change in functional connectivity occurs, in general, via mechanisms of segregation of local regions and integration of distant regions into disparate subnetworks.


Celastrol Enhances the Anti-Liver Cancer Activity of Sorafenib.
BACKGROUND Sorafenib, a multiple-target-point kinase inhibitor, has been used as a standard treatment for advanced liver cancer and has shown therapeutic benefits. However, resistance often occurs, prompting the need for identification of synergizing agents. Celastrol is a major active ingredient of Tripterygium wilfordii, which can increase the antitumor effect of traditional antitumor drugs. This work focused on the sensitization of liver cancers in use of celastrol combined with sorafenib. MATERIAL AND METHODS The IC50 values of sorafenib and celastrol on cancer cells were determined through MTT assays. The effects of sorafenib on AKT signaling and VEGF levels in sorafenib-treated cancer cells were analyzed by Western blotting and ELISA, respectively. After combined treatment with celastrol and sorafenib, the survival rate of tumor cells was determined by MTT and clonogenic assays, and the apoptosis rate was also determined by flow cytometry. In addition, the in vivo antitumor activity of celastrol combined with sorafenib was evaluated in Hepa1-6 tumor-bearing mice. RESULTS Sorafenib treatment induced the compensatory activation of the AKT pathway and autocrine VEGF in hepatoma cells, which could be reversed by celastrol. Furthermore, celastrol enhanced the growth inhibition and apoptosis induction of cancer cells by sorafenib both in vitro and in vivo and reduced the dosage of sorafenib needed. CONCLUSIONS Celastrol enhances the antitumor activity of sorafenib in HCC tumor cells by suppressing the AKT pathway and VEGF autocrine system.


An integrated randomized intervention to reduce behavioral and psychosocial risks: pregnancy and neonatal outcomes.
While biomedical risks contribute to poor pregnancy and neonatal outcomes in African American (AA) populations, behavioral and psychosocial risks (BPSR) may also play a part. Among low income AA women with psychosocial risks, this report addresses the impacts on pregnancy and neonatal outcomes of an integrated education and counseling intervention to reduce BPSR, as well as the contributions of other psychosocial and biomedical risks. Subjects were low income AA women ≥18 years living in the Washington, DC, metropolitan area and seeking prenatal care. Subjects (n = 1,044) were screened for active smoking, environmental tobacco smoke exposure (ETSE), depression, or intimate partner violence (IPV) and then randomized to intervention (IG) or usual care (UCG) groups. Data were collected prenatally, at delivery, and postpartum by maternal report and medical record abstraction. Multiple imputation methodology was used to estimate missing variables. Rates of pregnancy outcomes (miscarriage, live birth, perinatal death), preterm labor, Caesarean section, sexually transmitted infection (STI) during pregnancy, preterm birth (<37 weeks), low birth weight (<2,500 g), very low birth weight (<1,500 g), small for gestational age, neonatal intensive care unit (NICU) admission, and >2 days of hospitalization were compared between IG and UCG. Logistic regression models were created to predict outcomes based on biomedical risk factors and the four psychosocial risks (smoking, ETSE, depression, and IPV) targeted by the intervention. Rates of adverse pregnancy and neonatal outcomes were high and did not differ significantly between IG and UCG. In adjusted analysis, STI during the current pregnancy was associated with IPV (OR = 1.41, 95% CI 1.04-1.91). Outcomes such as preterm labor, caesarian section in pregnancy and preterm birth, low birth weight, small for gestational age, NICU admissions and >2 day hospitalization of the infants were associated with biomedical risk factors including preexisting hypertension and diabetes, previous preterm birth (PTB), and late initiation of prenatal care, but they were not significantly associated with active smoking, ETSE, depression, or IPV. Neither the intervention to reduce BPSR nor the psychosocial factors significantly contributed to the pregnancy and neonatal outcomes. This study confirms that biomedical factors significantly contribute to adverse outcomes in low income AA women. Biomedical factors outweighed psychosocial factors in contributing to adverse pregnancy and neonatal outcomes in this high-risk population. Early identification and management of hypertension, diabetes and previous PTB in low income AA women may reduce health disparities in birth outcomes. Level of evidence I.


The potential neurological effect of the COVID-19 vaccines: A review.
The coronavirus disease 2019 caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has become a pandemic with people infected in almost all countries. The most efficient solution to end this pandemic is a safe and efficient vaccine. Classic platforms are used to develop vaccines including live-attenuated vaccine, inactivated vaccine, protein subunit vaccine, and viral vector. Nucleic acid vaccine uses next-generation platforms for their development. Vaccines are now rushing to the market. Eleven candidates are in advance development. These comprise inactivated vaccines, viral vector vaccine, nucleic acid vaccine, and the protein subunit vaccine platform, which are now quite advanced in trials in various geographic and ethnic populations. The reported severe adverse effects raised the worries about their safety. It becomes critical to know whether these vaccines will cause neurologic disorders like previously recognized vaccine-related demyelinating diseases, fever-induced seizure, and other possible deficits. We reviewed the most promising COVID-2 vaccines with a particular interest in mechanism(s) and adverse effect(s). We exemplify potential neurological problems these vaccines could cause by looking at previous studies. The current evidence indicated a minor risk of the acute neurological disorders after the application. The observation of the long-time effect is still needed.


An ER-directed gelsolin nanobody targets the first step in amyloid formation in a gelsolin amyloidosis mouse model.
Hereditary gelsolin amyloidosis is an autosomal dominantly inherited amyloid disorder. A point mutation in the GSN gene (G654A being the most common one) results in disturbed calcium binding by the second gelsolin domain (G2). As a result, the folding of G2 is hampered, rendering the mutant plasma gelsolin susceptible to a proteolytic cascade. Consecutive cleavage by furin and MT1-MMP-like proteases generates 8 and 5 kDa amyloidogenic peptides that cause neurological, ophthalmological and dermatological findings. To this day, no specific treatment is available to counter the pathogenesis. Using GSN nanobody 11 as a molecular chaperone, we aimed to protect mutant plasma gelsolin from furin proteolysis in the trans-Golgi network. We report a transgenic, GSN nanobody 11 secreting mouse that was used for crossbreeding with gelsolin amyloidosis mice. Insertion of the therapeutic nanobody gene into the gelsolin amyloidosis mouse genome resulted in improved muscle contractility. X-ray crystal structure determination of the gelsolin G2:Nb11 complex revealed that Nb11 does not directly block the furin cleavage site. We conclude that nanobodies can be used to shield substrates from aberrant proteolysis and this approach might establish a novel therapeutic strategy in amyloid diseases.


The fundamental role of competition in the ecology and evolution of mutualisms.
Mutualisms are interspecific interactions that yield reciprocal benefits. Here, by adopting a consumer-resource perspective, we show how considering competition is necessary in order to understand the evolutionary and ecological dynamics of mutualism. We first review the ways in which competition shapes the ecology of mutualisms, using a graphical framework based on resource flows rather than net effects to highlight the opportunities for competition. We then describe the known mechanisms of competition and show how it is a critical driver of the evolutionary dynamics, persistence, and diversification of mutualism. We argue that empirical and theoretical research on the ecology and evolution of mutualisms will jointly progress by addressing four key points: (i) the existence and shape of physiological trade-offs among cooperation, competition, and other life-history and functional traits; (ii) the capacity for individuals to express conditional responses to variation in their mutualistic and competitive environment; (iii) the existence of heritable variation for mutualistic and competitive traits and their potentially conditional expression; and (iv) the structure of the network of consumer-resource interactions in which individuals are embedded.


The role of preoperative ultrasonography for patients with dense cataract: a retrospective study of 509 cases.
Ocular ultrasonography is widely used for the evaluation of eyes with opaque ocular media. Although preoperative ultrasonography of the globe has been recommended for patients with dense cataracts, the value of such an examination is currently unknown. The purpose of this study was to evaluate the prevalence and nature of intraocular pathologies detected by preoperative ultrasound examination in patients with dense cataract. The authors retrospectively studied 509 patients referred for routine ultrasound examination of the globe due to dense cataract. Exclusion criteria included known posterior segment pathology, previous ocular surgery or penetrating trauma, and age of younger than 18 years. All the patients underwent ultrasound examination by B-scan and standardized A-scan. In 19.6% of the patients, a posterior segment pathology was revealed by the ultrasound examination. The most frequently disclosed abnormalities were posterior staphyloma (7.2%), retinal detachment (4.5%), and vitreous hemorrhage (2.5%). One patient with a large choroidal malignant melanoma was identified. The prevalence of posterior segment pathologies was slightly higher in patients with a history of ocular trauma, compared with the nontraumatic cataract group (29.6% versus 19.0%, respectively; P = .1). The prevalence of retinal detachment was increased in the traumatic cataract subgroup (14.8% compared with 3.9%), but this difference did not reach statistical significance. Preoperative ultrasound examination for patients with dense cataract can be used to detect pathologies that may influence the surgical strategy and the postoperative visual prognosis.


[Monitoring anti-osteoporotic therapy: bone mineral density or markers of bone turnover].
This review discusses two methods of monitoring the effect of anti-osteoporotic treatment: bone mineral density (BMD) and bone turnover markers (BTMs). Both monitoring strategies are to some extent able to predict the treatment-induced change in risk of fracture. The use of BMD is commonplace, and clinicians are experienced in the interpretation of data. The use and knowledge of BTMs among clinicians is limited, but it allows for early testing while being practical and cheap. Further knowledge and implementation of BTMs in the clinical practice may improve the monitoring capabilities.


Contextually enhanced ES-dRNN with dynamic attention for short-term load forecasting.
In this paper, we propose a new short-term load forecasting (STLF) model based on contextually enhanced hybrid and hierarchical architecture combining exponential smoothing (ES) and a recurrent neural network (RNN). The model is composed of two simultaneously trained tracks: the context track and the main track. The context track introduces additional information to the main track. It is extracted from representative series and dynamically modulated to adjust to the individual series forecasted by the main track. The RNN architecture consists of multiple recurrent layers stacked with hierarchical dilations and equipped with recently proposed attentive dilated recurrent cells. These cells enable the model to capture short-term, long-term and seasonal dependencies across time series as well as to weight dynamically the input information. The model produces both point forecasts and predictive intervals. The experimental part of the work performed on 35 forecasting problems shows that the proposed model outperforms in terms of accuracy its predecessor as well as standard statistical models and state-of-the-art machine learning models.


Demonstration of an optical isolator by use of a nonreciprocal phase shift.
The experimental study of an optical isolator by use of a nonreciprocal phase shift is demonstrated. The isolator has an optical interferometer composed of tapered couplers, nonreciprocal phase shifters, and a reciprocal phase shifter. The isolator, designed for a 1.55-microm wavelength, was fabricated to investigate the characteristics of each component. The branching and coupling characteristics of the tapered coupler were measured. The nonreciprocal and reciprocal phase shifts were also evaluated. By applying an external magnetic field to the interferometer, we confirmed the nonreciprocal phase shift in the interferometric isolator.


Patterns of outdoor exposure to heat in three South Asian cities.
Low socio-economic status has been widely recognized as a significant factor in enhancing a person's vulnerability to climate change including vulnerability to changes in temperature. Yet, little is known about exposure to heat within cities in developing countries, and even less about exposure within informal neighbourhoods in those countries. This paper presents an assessment of exposure to outdoor heat in the South Asian cities Delhi, Dhaka, and Faisalabad. The temporal evolution of exposure to heat is evaluated, as well as intra-urban differences, using meteorological measurements from mobile and stationary devices (April-September 2016). Exposure to heat is compared between low-income and other neighbourhoods in these cities. Results are expressed in terms of air temperature and in terms of the thermal indices Heat Index (HI), Wet Bulb Globe Temperature (WBGT) and Universal Thermal Climate Index (UTCI) at walking level. Conditions classified as dangerous to very dangerous, and likely to impede productivity, are observed almost every day of the measurement period during daytime, even when air temperature drops after the onset of the monsoon. It is recommended to cast heat warnings in terms of thermal indices instead of just temperature. Our results nuance the idea that people living in informal neighbourhoods are consistently more exposed to heat than people living in more prosperous neighbourhoods. During night-time, exposure does tend to be enhanced in densely-built informal neighbourhoods, but not if the low-income neighbourhoods are more open, or if they are embedded in green/blue areas.


Alternative approaches to modeling hereditary dystonias.
Dystonia is a movement disorder characterized by involuntary muscle contractions resulting in abnormal postures. Although common in the clinic, the etiology of dystonia remains unclear. Most dystonias are idiopathic and are not associated with clear pathological brain abnormalities. Attempts to genetically model these dystonias in rodents have failed to replicate dystonic symptoms. This is at odds with the fact that rodents can exhibit dystonia. Because of this discrepancy, it is necessary to consider alternative approaches to generate phenotypically and genotypically faithful models of dystonia. Conditional knockout of dystonia-related genes is 1 technique that may prove useful for modeling genetic dystonias. Lentiviral-mediated small or short hairpin RNA (shRNA) knockdown of particular genes is another approach. Finally, in cases in which the function of a dystonia-related gene is well-known, pharmacological blockade of the protein product can be used. Such an approach was successfully implemented in the case of rapid-onset dystonia parkinsonism, DYT12. This (DYT12) is a hereditary dystonia caused by mutations in the α₃ isoform of the sodium potassium adenosine triphosphatase (ATPase) pump (sodium pump), which partially hampers its physiological function. It was found that partial selective pharmacological block of the sodium pumps in the cerebellum and basal ganglia of mice recapitulates all of the salient features of DYT12, including dystonia and parkinsonism induced by stress. This DYT12 model is unique in that it faithfully replicates human symptoms of DYT12, while targeting the genetic cause of this disorder. Acute disruption of proteins implicated in dystonia may prove a generally fruitful method to model dystonia in rodents.


Genomic patterns of pathogen evolution revealed by comparison of Burkholderia pseudomallei, the causative agent of melioidosis, to avirulent Burkholderia thailandensis.
The Gram-negative bacterium Burkholderia pseudomallei (Bp) is the causative agent of the human disease melioidosis. To understand the evolutionary mechanisms contributing to Bp virulence, we performed a comparative genomic analysis of Bp K96243 and B. thailandensis (Bt) E264, a closely related but avirulent relative. We found the Bp and Bt genomes to be broadly similar, comprising two highly syntenic chromosomes with comparable numbers of coding regions (CDs), protein family distributions, and horizontally acquired genomic islands, which we experimentally validated to be differentially present in multiple Bt isolates. By examining species-specific genomic regions, we derived molecular explanations for previously-known metabolic differences, discovered potentially new ones, and found that the acquisition of a capsular polysaccharide gene cluster in Bp, a key virulence component, is likely to have occurred non-randomly via replacement of an ancestral polysaccharide cluster. Virulence related genes, in particular members of the Type III secretion needle complex, were collectively more divergent between Bp and Bt compared to the rest of the genome, possibly contributing towards the ability of Bp to infect mammalian hosts. An analysis of pseudogenes between the two species revealed that protein inactivation events were significantly biased towards membrane-associated proteins in Bt and transcription factors in Bp. Our results suggest that a limited number of horizontal-acquisition events, coupled with the fine-scale functional modulation of existing proteins, are likely to be the major drivers underlying Bp virulence. The extensive genomic similarity between Bp and Bt suggests that, in some cases, Bt could be used as a possible model system for studying certain aspects of Bp behavior.


Evaluation of the prevalence of infections in cats after renal transplantation: 169 cases (1987-2003).
To determine the prevalence of infections developing postoperatively, document the contribution of infection to increased risk of death, and identify risk factors associated with the development of infectious complications in cats after renal transplantation. Retrospective study. 169 cats that received renal allograft transplants. Medical records of cats receiving renal transplants at the University of California from January 1987 through December 2003 were reviewed. 47 infections developed in 43 of 169 cats. Bacterial infections were most common (25/47 cats), followed by viral (13/47), fungal (6/47), and protozoal (3/47) infections. The median duration from transplant surgery to development of infection was 2.5 months. Infection was the second most common cause of death after acute rejection of the transplant, accounting for 14% of deaths overall. Cats with concurrent diabetes mellitus had a significantly increased risk of developing an infection after renal transplantation. Sex, increasing age, concurrent neoplasia, and previous treatment for transplant rejection were not associated with development of infection. Infection was a common complication and an important cause of death or euthanasia in cats after renal transplantation. Development of diabetes mellitus after transplantation significantly increased the risk of infection.


The innate immunity protein IFITM3 modulates γ-secretase in Alzheimer's disease.
Innate immunity is associated with Alzheimer's disease1, but the influence of immune activation on the production of amyloid-β is unknown2,3. Here we identify interferon-induced transmembrane protein 3 (IFITM3) as a γ-secretase modulatory protein, and establish a mechanism by which inflammation affects the generation of amyloid-β. Inflammatory cytokines induce the expression of IFITM3 in neurons and astrocytes, which binds to γ-secretase and upregulates its activity, thereby increasing the production of amyloid-β. The expression of IFITM3 is increased with ageing and in mouse models that express familial Alzheimer's disease genes. Furthermore, knockout of IFITM3 reduces γ-secretase activity and the formation of amyloid plaques in a transgenic mouse model (5xFAD) of early amyloid deposition. IFITM3 protein is upregulated in tissue samples from a subset of patients with late-onset Alzheimer's disease that exhibit higher γ-secretase activity. The amount of IFITM3 in the γ-secretase complex has a strong and positive correlation with γ-secretase activity in samples from patients with late-onset Alzheimer's disease. These findings reveal a mechanism in which γ-secretase is modulated by neuroinflammation via IFITM3 and the risk of Alzheimer's disease is thereby increased.


New functional activities for the p21 family of CDK inhibitors.
The association of cdk4 with D-type cyclins to form functional kinase complexes is comparatively inefficient. This has led to the suggestion that assembly might be a regulated step. In this report we demonstrate that the CDK inhibitors p21(CIP), p27(KIP), and p57(KIP2) all promote the association of cdk4 with the D-type cyclins. This effect is specific and does not occur with other cdk inhibitors or cdk-binding proteins. Both in vivo and in vitro, the abundance of assembled cdk4/cyclin D complex increases directly with increasing inhibitor levels. The promotion of assembly is not attributable to a simple cell cycle block and requires the function of both the cdk and cyclin-binding domains. Kinetic studies demonstrate that p21 and p27 lead to a 35- and 80-fold increase in K(a), respectively, mostly because of a decrease in K(off). At low concentrations, p21 promotes the assembly of active kinase complexes, whereas at higher concentrations, it inhibits activity. Moreover, immunodepletion experiments demonstrate that most of the active cdk4-associated kinase activity also associates with p21. To confirm these results in a natural setting, we examine the assembly of endogenous complexes in mammary epithelial cells after release from a G(0) arrest. In agreement with our other data, cyclin D1 and p21 bind concomitantly to cdk4 during the in vivo assembly of cdk4/cyclin D1 complexes. This complex assembly occurs in parallel to an increase in cyclin D1-associated kinase activity. Immunodepletion experiments demonstrate that most of the cellular cyclin D1-associated kinase activity is also p21 associated. Finally, we find that all three CIP/KIP inhibitors target cdk4 and cyclin D1 to the nucleus. We suggest that in addition to their roles as inhibitors, the p21 family of proteins, originally identified as inhibitors, may also have roles as adaptor proteins that assemble and program kinase complexes for specific functions.


SplicingTypesAnno: annotating and quantifying alternative splicing events for RNA-Seq data.
Alternative splicing plays a key role in the regulation of the central dogma. Four major types of alternative splicing have been classified as intron retention, exon skipping, alternative 5 splice sites or alternative donor sites, and alternative 3 splice sites or alternative acceptor sites. A few algorithms have been developed to detect splice junctions from RNA-Seq reads. However, there are few tools targeting at the major alternative splicing types at the exon/intron level. This type of analysis may reveal subtle, yet important events of alternative splicing, and thus help gain deeper understanding of the mechanism of alternative splicing. This paper describes a user-friendly R package, extracting, annotating and analyzing alternative splicing types for sequence alignment files from RNA-Seq. SplicingTypesAnno can: (1) provide annotation for major alternative splicing at exon/intron level. By comparing the annotation from GTF/GFF file, it identifies the novel alternative splicing sites; (2) offer a convenient two-level analysis: genome-scale annotation for users with high performance computing environment, and gene-scale annotation for users with personal computers; (3) generate a user-friendly web report and additional BED files for IGV visualization. SplicingTypesAnno is a user-friendly R package for extracting, annotating and analyzing alternative splicing types at exon/intron level for sequence alignment files from RNA-Seq. It is publically available at https://sourceforge.net/projects/splicingtypes/files/ or http://genome.sdau.edu.cn/research/software/SplicingTypesAnno.html.


Characterization and charge distribution of the asparagine-linked oligosaccharides on secreted mouse thyrotropin and free alpha-subunits.
Mouse hemipituitaries in vitro secrete TSH, composed of an alpha-beta heterodimer, as well as excess (free) alpha-subunits. By dual metabolic labeling with [35S]sulfate and [3H]mannose, we have characterized oligosaccharides from secreted TSH alpha, TSH beta, and free alpha-subunits released from the apoprotein by enzymatic deglycosylation. Oligosaccharides from each subunit displayed a distinct anion exchange HPLC profile due to a specific pattern of sialylation and sulfation. Six species were obtained from TSH alpha (with two glycosylation sites), including neutral oligosaccharides as well as those with one or two negative charges. For TSH beta (with one glycosylation site) at least eight oligosaccharide species were noted, representing nearly every permutation of sialylation and sulfation; approximately 30% contained three or more negative charges. Analysis of [3H]mannose-labeled oligosaccharides on Concanavalin-A-agarose showed 85% binding for those from TSH alpha, 70% for free alpha, and 50% for those from TSH beta. These data demonstrate that oligosaccharides from secreted TSH beta were more sialylated and sulfated, consistent with a more complex branching pattern, than those from TSH alpha. Oligosaccharides from free alpha-subunit were more sialylated than those from TSH alpha, and the net negative charge was intermediate between those of TSH alpha and TSH beta. Although great microheterogeneity is present even at the single glycosylation site on the beta-subunit of secreted TSH, a pattern of sialylation and sulfation could be discerned. If one assigns probabilities of sialylation [p(N)] and sulfation [p(S)] based on the observed distribution within monoacidic (charge -1) species, the proportion of diacidic (charge -2) oligosaccharides could be predicted for each subunit by [p(N)]2, 2[p(N)] [p(S)], [p(S)]2, corresponding to species containing two sialic acid, one sialic acid and one sulfate, and two sulfate residues, respectively. This suggests that the probability of sialylation or sulfation at a second site on these oligosaccharides is similar to that at the first and that anionic oligosaccharides in secreted TSH and free alpha are distributed binomially with regard to sialic acid and sulfate residues.


Development and Internal Validation of a Risk Score to Detect Asymptomatic Carotid Stenosis.
Asymptomatic carotid stenosis (ACS) is associated with an increased risk of ischaemic stroke and myocardial infarction. Risk scores have been developed to detect individuals at high risk of ACS, thereby enabling targeted screening, but previous external validation showed scope for refinement of prediction by adding additional predictors. The aim of this study was to develop a novel risk score in a large contemporary screened population. A prediction model was developed for moderate (≥50%) and severe (≥70%) ACS using data from 596 469 individuals who attended screening clinics. Variables that predicted the presence of ≥50% and ≥70% ACS independently were determined using multivariable logistic regression. Internal validation was performed using bootstrapping techniques. Discrimination was assessed using area under the receiver operating characteristic curves (AUROCs) and agreement between predicted and observed cases using calibration plots. Predictors of ≥50% and ≥70% ACS were age, sex, current smoking, diabetes mellitus, prior stroke/transient ischaemic attack, coronary artery disease, peripheral arterial disease, blood pressure, and blood lipids. Models discriminated between participants with and without ACS reliably, with an AUROC of 0.78 (95% confidence interval [CI] 0.77-0.78) for ≥ 50% ACS and 0.82 (95% CI 0.81-0.82) for ≥ 70% ACS. The number needed to screen in the highest decile of predicted risk to detect one case with ≥50% ACS was 13 and that of ≥70% ACS was 58. Targeted screening of the highest decile identified 41% of cases with ≥50% ACS and 51% with ≥70% ACS. The novel risk model predicted the prevalence of ACS reliably and performed better than previous models. Targeted screening among the highest decile of predicted risk identified around 40% of all cases with ≥50% ACS. Initiation or intensification of cardiovascular risk management in detected cases might help to reduce both carotid related ischaemic strokes and myocardial infarctions.


Effect of a prior bout of preconditioning exercise on muscle damage from downhill walking.
This study investigated whether reduced-duration downhill walking (DW) would confer a protective effect against muscle damage induced by a subsequent bout of longer duration DW performed 1 week or 4 weeks later. Healthy young adults were allocated to a control or one of the preconditioning exercise (PRE-1wk or PRE-4wk) groups (10 men and 4 women per group). PRE-1wk and PRE-4wk groups performed 20-min DW (-28% slope, 5 km/h, 10% body mass added to a backpack) 1 week and 4 weeks before 40-min DW, respectively, and the control group performed 40-min DW only. Maximal voluntary contraction (MVC) knee extension torque, plasma creatine kinase (CK) activity, and muscle soreness (100-mm visual analog scale) were measured before, immediately after, and 24, 48, and 72 h after DW, and the changes in these variables were compared among groups. The control group showed symptoms of muscle damage (e.g., prolonged decrease in MVC: -14% ± 10% at 48 h post-DW) after 40-min DW. Changes in all variables after 40-min DW of PRE-1wk and PRE-4wk groups were 54%-61% smaller (P < 0.05) than the control group, without significant differences between PRE-1wk and PRE-4wk groups for MVC and plasma CK activity. Importantly, changes after the preconditioning exercise (20-min DW) were 67%-69% smaller (P < 0.05) than those after the 40-min DW of the control group. These findings suggest that 20-min DW resulting in minor muscle damage conferred a protective effect against subsequent 40-min DW, and its effect could last for more than 4 weeks.


Use of ear tags and injectable transponders for the identification and traceability of pigs from birth to the end of the slaughter line.
A total of 557 newborn piglets were used to compare eight identification devices, including one plastic ear tag as a control (C, n = 348) and two types of electronic ear tags (E1, n = 106; and E2, n = 103), and five types of injectable transponders (n = 557): small 12-mm (D12, n = 116; and S12, n = 110), medium 23-mm (T23, n = 108), and large (32-mm, T32, n = 115; and 34-mm, S34, n = 108). Injections were made s.c. in the auricle base (n = 248) and intraperitoneally (n = 309) using a new technique. All piglets were identified with two devices, but using electronic ear tags in conjunction with injection in the auricle was avoided on the same pig. Readability of devices was checked during fattening (until 110 kg BW) and slaughtering. On-farm losses were lower for control than for electronic ear tags (C = 1.1%; E1 = 8.8%; and E2 = 44.9%; P < 0.01); the latter also suffered electronic failures (E1 = 5.5%; and E2 = 55.1%; P < 0.001). On-farm losses of transponders injected in the auricle base were greater in large (S34 = 72.5%; and T32 = 46.3%; P < 0.05) than in small transponders (S12 = 19.4%; and D12 = 17.1%), but T23 (29.8%) only differed from S34. Transponder size did not affect on-farm losses for intraperitoneal injections in which only one loss was recorded (0.4%). All ear tags had similar losses during transportation to the slaughterhouse (1.2%), but no losses were observed in injectables. Slaughtering losses did not differ between ear tags (C = 11.2%; and E1 = 6.4%), but apart from losses, 12.8% of E1 failed electronically. Injection site affected losses and breakages during slaughtering (auricle base = 6.4%; and intraperitoneal = 0%), but recovery time did not significantly differ (auricle base = 28.6 s; and intraperitoneal = 18.9 s). Transponders in the auricle base were recovered by sight (30.2%), palpation (27.4%), or by cutting (42.5%). Intraperitoneal transponders were mainly recovered loose in the abdominal cavity (81.4%), whereas 18.6% fell on the floor. As a result, traceability varied significantly (P < 0.05) between control (86.7%) and electronic ear tags (0 to 68.1%) and injectable transponders, with the auricle base (17.8 to 75.0%) having lower values than intraperitoneal (98 to 100%). Intraperitoneal injection was a very effective tool for piglet identification and traceability, ensuring the transfer of information from farm to slaughterhouse. To warrant the use of this technique in practice, transponder recovery requires further investigation.


Stiff matrix instigates type I collagen biogenesis by mammalian cleavage factor I complex-mediated alternative polyadenylation.
Alternative polyadenylation (APA) is a widespread and important mechanism in regulation of gene expression. Dysregulation of the 3' UTR cleavage and polyadenylation represents a common characteristic among many disease states, including lung fibrosis. In this study, we investigated the role of mammalian cleavage factor I-mediated (CFIm-mediated) APA in regulating extracellular matrix production in response to mechanical stimuli from stiffened matrix simulating the fibrotic lungs. We found that stiff matrix downregulated expression of CFIm68, CFIm59 and CFIm25 subunits and promoted APA in favor of the proximal poly(A) site usage in the 3' UTRs of type I collagen (COL1A1) and fibronectin (FN1) in primary human lung fibroblasts. Knockdown and overexpression of each individual CFIm subunit demonstrated that CFIm68 and CFIm25 are indispensable attributes of stiff matrix-induced APA and overproduction of COL1A1, whereas CFIm did not appear to mediate stiffness-regulated FN1 APA. Furthermore, expression of the CFIm subunits was associated with matrix stiffness in vivo in a bleomycin-induced mouse model of pulmonary fibrosis. These data suggest that stiff matrix instigates type I collagen biogenesis by selectively targeting mRNA transcripts for 3' UTR shortening. The current study uncovered a potential mechanism for regulation of the CFIm complex by mechanical cues under fibrotic conditions.