Emricasan to prevent new decompensation in patients with NASH-related decompensated cirrhosis.
Non-alcoholic steatohepatitis is a leading cause of end-stage liver disease. Hepatic steatosis and lipotoxicity cause chronic necroinflammation and direct hepatocellular injury resulting in cirrhosis, end-stage liver disease and hepatocellular carcinoma. Emricasan is a pan-caspase inhibitor that inhibits excessive apoptosis and inflammation; it has also been shown to decrease portal pressure and improve synthetic function in mice with carbon tetrachloride-induced cirrhosis. This double-blind, placebo-controlled study randomized 217 individuals with decompensated NASH cirrhosis 1:1:1 to emricasan (5 mg or 25 mg) or placebo. Patients were stratified by decompensation status and baseline model for end-stage liver disease-sodium (MELD-Na) score. The primary endpoint comprised all-cause mortality, a new decompensation event (new or recurrent variceal hemorrhage, new ascites requiring diuretics, new unprecipitated hepatic encephalopathy ≥grade 2, hepatorenal syndrome, spontaneous bacterial peritonitis), or an increase in MELD-Na score ≥4 points. There was no difference in event rates between either of the emricasan treatment groups and placebo, with hazard ratios of 1.02 (95% CI 0.59-1.77; p = 0.94) and 1.28 (95% CI 0.75-2.21; p = 0.37) for 5 mg and 25 mg of emricasan, respectively. MELD-Na score progression was the most common outcome. There was no significant effect of emricasan treatment on MELD-Na score, international normalized ratio, total serum bilirubin, albumin level or Child-Pugh score. Emricasan was generally safe and well-tolerated. Emricasan was safe but ineffective for the treatment of decompensated NASH cirrhosis. However, this study may guide the design and conduct of future clinical trials in decompensated NASH cirrhosis. Patients with decompensated cirrhosis related to non-alcoholic steatohepatitis are at high risk of additional decompensation events and death. Post hoc analyses in previous pilot studies suggested that emricasan might improve portal hypertension and liver function. In this larger randomized study, emricasan did not decrease the number of decompensation events or improve liver function in patients with a history of decompensated cirrhosis related to non-alcoholic steatohepatitis. CLINICALTRIALS. NCT03205345.


Prolonged Morphine Exposure Induces Increased Firm Adhesion in an in Vitro Model of the Blood-Brain Barrier.
The blood-brain barrier (BBB) has been defined as a critically important protective barrier that is involved in providing essential biologic, physiologic, and immunologic separation between the central nervous system (CNS) and the periphery. Insults to the BBB can cause overall barrier damage or deregulation of the careful homeostasis maintained between the periphery and the CNS. These insults can, therefore, yield numerous phenotypes including increased overall permeability, interendothelial gap formation, alterations in cytokine and chemokine secretion, and accelerated cellular passage. The current studies expose the human brain microvascular endothelial cell line, hCMEC/D3, to prolonged morphine exposure and aim to uncover the mechanisms underlying alterations in barrier function in vitro. These studies show alterations in the mRNA and protein levels of the cellular adhesion molecules (CAMs) intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and activated leukocyte cell adhesion molecule that correlate with an increased firm adhesion of the CD3⁺ subpopulation of peripheral blood mononuclear cells (PBMCs). Overall, these studies suggest that prolonged morphine exposure may result in increased cell migration into the CNS, which may accelerate pathological processes in many diseases that involve the BBB.


Physical and cognitive function in midlife: reciprocal effects? A 5-year follow-up of the Whitehall II study.
Cognitive and physical functions are closely linked in old age, but less is known about this association in midlife. Whether cognitive function predicts physical function and whether physical function predicts cognitive function were assessed in middle-aged men and women. Data were from Whitehall II, an ongoing large-scale, prospective occupational cohort study of employees from 20 London-based white-collar Civil Service departments. The participants, 3446 men and 1274 women aged 45-68 years at baseline (1995-1997), had complete data on cognitive performance and physical function at both baseline and follow-up (2002-2004). A composite cognitive score was compiled from the following tests: verbal memory, inductive reasoning (Alice Heim 4-I), verbal meaning (Mill Hill), phonemic and semantic fluency. Physical function was measured using the physical composite score of the short form (SF-36) scale. Average follow-up was 5.4 years. Poor baseline cognitive performance predicted poor physical function at follow-up (beta = 0.08, p<0.001), while baseline physical function did not predict cognitive performance (beta = 0.01, p = 0.67). After full adjustment for sociodemographic, behavioural and biological risk factors, baseline cognitive performance (beta = 0.04 p = 0.009) remained predictive of physical function. Despite previous work indicating that the association between physical and cognitive performance may be bidirectional, these findings suggest that, in middle age, the direction of the association is predominantly from poor cognition to poor physical function.


Fermented soybeans by Rhizopus oligosporus reduce femoral bone loss in ovariectomized rats.
Soy isoflavones are structurally similar to estrogen and bind to estrogen receptors, suggesting that they exhibit estrogenic activities; therefore, they are referred to as phytoestrogens. Fermentation may affect the bioavailability of isoflavones altering soy isoflavone glycosides in the form of aglycones. Thus, this study investigated the effects of fermented soybeans by Rhizopus oligosporus on bone metabolism in both young rats as a pilot test and in ovariectomized (ovx) old rats as a model of menopause. In the pilot test, a total of 24 seven-week-old female Sprague-Dawley (SD) rats were fed one of three diets for a period of four weeks: casein, unfermented soybean product, or fermented soybean product by R. oligosporus. In the ovx rat model, 20-week-old SD rats weighing 260-290 g underwent either sham-operation (n = 10) or bilateral ovariectomy (n = 30) and were then fed the AIN-93M diet for one week. Thereafter, rats were fed sham-casein, ovx-casein, ovx-soybean, or ovx-fermented soybean diet for five weeks. After decapitation, femoral bones were isolated and preserved in 9% formalin for assessment of bone mineral density (BMD), bone mineral content (BMC), and bone-breaking strength (BBS). Ovx rats showed significantly increased weight gain and decreased uterine wet weight. Of particular interest, ovx rats fed fermented soybeans showed increased uterine wet weights compared to control rats. Fermented soybean diet caused a significant increase in plasma 17-β estradiol concentrations in young rats, and 17-β estradiol levels were enhanced in ovx rats to match those of sham-operated ones. Significantly lower femoral BMD and BMC were observed in ovx rats compared to sham-operated controls, whereas bone areas did not differ statistically among the groups. In addition, BBS tended to be increased in ovx rats fed soybeans and fermented soybeans. Supplementation of fermented soybeans could have preventive and therapeutic effects against osteoporosis in postmenopausal women.


Cell therapy using allogeneic bone marrow mesenchymal stem cells prevents tissue damage in collagen-induced arthritis.
Mesenchymal stem cells (MSCs) are precursors of tissue of mesenchymal origin, but they also have the capacity to regulate the immune response by suppressing T and B lymphocyte proliferation in a non-major histocompatibility complex-restricted manner. Use of MSCs as immunosuppressant agents in autoimmune diseases has been proposed and successfully tested in animal models. We explored the feasibility of using allogeneic MSCs as therapy for collagen-induced arthritis, a mouse model for human rheumatoid arthritis. DBA/1 mice were immunized with type II collagen in Freund's complete adjuvant, and some of the animals received an intraperitoneal injection of allogeneic MSCs. A single injection of MSCs prevented the occurrence of severe, irreversible damage to bone and cartilage. MSCs induced hyporesponsiveness of T lymphocytes as evidenced by a reduction in active proliferation, and modulated the expression of inflammatory cytokines. In particular, the serum concentration of tumor necrosis factor alpha was significantly decreased. MSCs exerted their immunomodulatory function by educating antigen-specific Tregs. Our results suggest an effective new therapeutic approach to target the pathogenic mechanism of autoimmune arthritis using allogeneic MSCs. However, further studies are required before these results can be translated to clinical settings.


Dysarthria and other-initiated repair in everyday conversation.
Dysarthria is commonly understood as a motor speech disorder characterized by symptoms that are framed as physiologically or acoustically measurable. The effects of dysarthria on social interaction through conversation have been reported but, in comparison with physical measures, remain relatively unexplored. Other-initiated repair sequences are particularly common in dysarthria-in-interaction, drawing attention to the actions of both participants in managing (un)intelligibility, rather than the behavior(s) of the person with dysarthric speech in isolation. These sequences merit detailed and ongoing investigation as they enable us to understand how dysarthria impacts on conversation and, critically, how participants attempt to manage difficulties when they arise. This study explores the organization of other-initiated repair sequences in a dyad where one participant has severe dysarthria arising from amyotrophic lateral sclerosis (ALS) also known as MND. Two hours of recordings were collected on four dates over a 12-month period with the data presented here from recording two. The evidence shows that the participants were able to resolve their troubles, but it required extensive work to both identify the trouble sources and to unravel the problems to reach a satisfactory understanding. The interactions presented in this paper reveal an important limitation of other initiation of repair. Physical restrictions were seen to play an important part in the dysarthric speaker's ability to position his talk in sequential context and successfully accomplish self-repair; particularly, third-turn repair. The present study has offered a depiction of layered conversational problems that other-initiation of repair may not completely resolve or, in some cases, multiply.


SNP-Based Linkage Mapping for Validation of QTLs for Resistance to Ascochyta Blight in Lentil.
Lentil (Lens culinaris Medik.) is a self-pollinating, diploid, annual, cool-season, food legume crop that is cultivated throughout the world. Ascochyta blight (AB), caused by Ascochyta lentis Vassilievsky, is an economically important and widespread disease of lentil. Development of cultivars with high levels of durable resistance provides an environmentally acceptable and economically feasible method for AB control. A detailed understanding of the genetic basis of AB resistance is hence highly desirable, in order to obtain insight into the number and influence of resistance genes. Genetic linkage maps based on single nucleotide polymorphisms (SNP) and simple sequence repeat (SSR) markers have been developed from three recombinant inbred line (RIL) populations. The IH × NF map contained 460 loci across 1461.6 cM, while the IH × DIG map contained 329 loci across 1302.5 cM and the third map, NF × DIG contained 330 loci across 1914.1 cM. Data from these maps were combined with a map from a previously published study through use of bridging markers to generate a consensus linkage map containing 689 loci distributed across seven linkage groups (LGs), with a cumulative length of 2429.61 cM at an average density of one marker per 3.5 cM. Trait dissection of AB resistance was performed for the RIL populations, identifying totals of two and three quantitative trait loci (QTLs) explaining 52 and 69% of phenotypic variation for resistance to infection in the IH × DIG and IH × NF populations, respectively. Presence of common markers in the vicinity of the AB_IH1- and AB_IH2.1/AB_IH2.2-containing regions on both maps supports the inference that a common genomic region is responsible for conferring resistance and is associated with the resistant parent, Indianhead. The third QTL was derived from Northfield. Evaluation of markers associated with AB resistance across a diverse lentil germplasm panel revealed that the identity of alleles associated with AB_IH1 predicted the phenotypic responses with high levels of accuracy (~86%), and therefore have the potential to be widely adopted in lentil breeding programs. The availability of RIL-based maps, a consensus map, and validated markers linked to AB resistance provide important resources for lentil improvement.


Skeletal muscle strength and endurance are maintained during moderate dehydration.
This study investigated the effects of moderate dehydration (~2.5% body weight) on muscle strength and endurance using percutaneous electrical stimulation to quantify central and peripheral fatigue, and isolate the combined effects of exercise-heat stress and dehydration, vs. the effect of dehydration alone. Force production and voluntary activation were calculated in 10 males during 1 brief and 15 repeated maximal voluntary isometric contractions performed prior to (control) walking in the heat (35°C), immediately following exercise, and the next morning (dehydration). The protocol was also performed in a euhydrated state. During the brief contractions, force production and voluntary activation were maintained in all trials. In contrast, force production decreased throughout the repeated contractions, regardless of hydration status (P<0.001). The decline in force was greater immediately following exercise-heat stress dehydration compared with control and euhydration (P<0.001). When dehydration was isolated from acute post-exercise dehydration, force production was maintained similarly to control and euhydration. Despite the progressive decline in force production and the increased fatigability observed during the repeated contractions, voluntary activation remained elevated throughout each muscle function test. Therefore, moderate dehydration, isolated from acute exercise-heat stress, does not appear to influence strength during a single contraction or enhance fatigability.


Osteochondritis Dissecans in the Knee of Skeletally Immature Patients: Rates of Persistent Pain, Osteoarthritis, and Arthroplasty at Mean 14-Years' Follow-Up.
Osteochondritis dissecans (OCD) is a knee disorder of predominately pediatric populations. Because of low incidence, it has traditionally been difficult to study OCD. The purpose of this study was to report long-term outcomes of skeletally immature OCD lesions and determine risk factors for persistent knee pain at final follow-up. A geographic database of more than 500,000 patients was reviewed to identify patients with knee OCD. Clinical course including operative management, persistent knee pain, and total knee arthroplasty (TKA) were analyzed through review of radiographs, magnetic resonance images, and physician notes. A total of 95 skeletally immature patients (70 male, 25 female, mean age 12.5 ± 2.0 years) were followed for a mean of 14 years (range, 2-40 years). Fifty-three patients were treated operatively and 42 were treated nonoperatively. At final follow-up, 13 patients noted persistent knee pain, 8 treated operatively versus 5 treated nonoperatively. Risk factors for knee pain were female gender, patellar lesions, and unstable lesions. Four patients (8%) treated operatively and 2 patients (5%) treated nonoperatively developed symptomatic osteoarthritis at a mean of 28.6 years following diagnosis. Three patients underwent TKA at a mean age of 52 years, significantly younger than that observed for primary TKA at our institution (P = 0.004). Skeletally immature OCD patients have promising histories, with an estimated 14% risk of persistent knee pain, 6% symptomatic osteoarthritis, and 3% conversion to TKA at 14 years' mean follow-up. Females, patellar lesions, and unstable lesions demonstrated increased persistent knee pain risk. Patients with OCD undergo TKA at a significantly younger age than the general population.


Short-Term biomechanical adaptation of the rat carotid to acute hypertension: contribution of smooth muscle.
The biomechanical adaptation of the arterial wall to hypertension has been studied extensively in recent years; however, the exact biomechanical contribution of vascular smooth muscle cells (VSMCs) during the adaptation process in conduit vessels is not known. We induced hypertension in 8 wk old Wistar rats by total ligation of the aorta between the two kidneys. Mean blood pressure increased from 92 +/- 2 (mean +/- SE) mm Hg to approximately 150 mmHg. Rats were sacrificed 2, 4, and 8 d after surgery and the left common carotid artery was excised for analysis. Wall thickness increased by 18% in 8 d and the opening angle by 32% in 4 d. The elastic properties were measured under normal VSMC tone (i.e., the amount of VSMC tone under normal conditions also called basal VSMC tone or normal resting VSMC tone), under maximally contracted VSMC (NE, 5 x 10(-7) mol/L) and under totally relaxed VSMC conditions (papaverine, 10(-4) mol/L). The most pronounced modifications were the changes in elastic properties related to normal VSMC tone. The functional contraction ratio at 100 mm Hg, defined as the relative contraction under normal conditions (normal VSMC tone), increased by 439% 4 d after the induction of hypertension. The total contraction capacity of the VSMC increased by 38% within 8 d. The changes in normal VSMC tone led to important changes in the mechanical properties of the arterial wall. Under normal VSMC conditions, compliance at mean pressure (148 mm Hg) increased by 159% within 8 d, whereas in the absence of VSMC tone, compliance did not increase significantly. We conclude that in conduit vessels, the VSMC, which is the sensing and effecting element of the adaptation process, is subjected to large-scale changes during the early phase of arterial adaptation to acute hypertension.


Molecular organizations of conical mesogenic fullerenes.
We have studied liquid crystal phases formed by fullerenes functionalized with mesogenic groups yielding a cone-shaped molecular structure. We have modelled these shuttlecock-like molecules with a set of Gay-Berne particles grafted with flexible springs to a spherical core and we have studied, using Monte Carlo simulations, their phase organization, also with a view to examining their possible use as candidate organic photovoltaic materials. We have found that, upon cooling from the isotropic phase, the system forms a columnar phase, like in the experimental work of Kato and coworkers [T. Kato et al., Nature, 2002, 419, 702]. However the phase is made of polar stacks extending not more than about ten molecules, which could limit their usefulness in enhancing and directing charge transport for possible photovoltaic applications.


Predictive scoring model of mortality in Gram-negative bloodstream infection.
Mortality is a well-recognized complication of Gram-negative bloodstream infection (BSI). The aim of this study was to develop a model to predict mortality in patients with Gram-negative BSI by using the Pitt bacteraemia score (PBS) and other clinical and laboratory variables. A cohort of 683 unique adult patients who were followed for at least 28 days after admission to Mayo Clinic Hospitals with Gram-negative BSI from 1 January 2001 to 31 October 2006 and who received clinically predefined appropriate empirical antimicrobial therapy was retrospectively identified. Multivariable logistic regression was used to identify independent risk factors for 28-day all-cause mortality. Regression coefficients from a multivariable model were used to develop a risk score to predict mortality following Gram-negative BSI. Malignancy (OR 3.48, 95% CI 1.94-6.22), liver cirrhosis (OR 5.42, 95% CI 2.52-11.65), source of BSI other than urinary tract or central venous catheter infection (OR 5.54, 95% CI 2.42-12.69), and PBS (OR 1.98, 95% CI 0.92-4.25 for PBS of 2-3 and OR 6.42, 95% CI 3.11-13.24 for PBS ≥4) were identified as independent risk factors for 28-day mortality in patients with Gram-negative BSI. A risk-score model was created by adding points for each independent risk factor, and had a c-statistic of 0.84. Patients with risk scores of 0, 4, 8, 12 and 16 had estimated 28-day mortality rates of approximately 0%, 3%, 14%, 45%, and 81%, respectively. The Gram-negative BSI risk score described herein estimated mortality risk with high discrimination in patients with Gram-negative BSI who received clinically adequate empirical antimicrobial therapy.


Screening of Pleural Mesotheliomas for DNA-damage Repair Players by Digital Gene Expression Analysis Can Enhance Clinical Management of Patients Receiving Platin-Based Chemotherapy.
Malignant pleural mesothelioma (MPM) is a rare, predominantly asbestos-related and biologically highly aggressive tumour leading to a dismal prognosis. Multimodality therapy consisting of platinum-based chemotherapy is the treatment of choice. The reasons for the rather poor efficacy of platinum compounds remain largely unknown. For this exploratory mRNA study, 24 FFPE tumour specimens were screened by digital gene expression analysis. Based on data from preliminary experiments and recent literature, a total of 366 mRNAs were investigated using a Custom CodeSet from NanoString. All statistical analyses were calculated with the R i386 statistical programming environment. CDC25A and PARP1 gene expression were correlated with lymph node spread, BRCA1 and TP73 expression levels with higher IMIG stage. NTHL1 and XRCC3 expression was associated with TNM stage. CHECK1 as well as XRCC2 expression levels were correlated with tumour progression in the overall cohort of patients. CDKN2A and MLH1 gene expression influenced overall survival in this collective. In the adjuvant treated cohort only, CDKN2A, CHEK1 as well as ERCC1 were significantly associated with overall survival. Furthermore, TP73 expression was associated with progression in this subgroup. DNA-damage response plays a crucial role in response to platin-based chemotherapeutic regimes. In particular, CHEK1, XRCC2 and TP73 are strongly associated with tumour progression. ERCC1, MLH1, CDKN2A and most promising CHEK1 are prognostic markers for OS in MPM. TP73, CDKN2A, CHEK1 and ERCC1 seem to be also predictive markers in adjuvant treated MPMs. After a prospective validation, these markers may improve clinical and pathological practice, finally leading to a patients' benefit by an enhanced clinical management.


Controlled generation of single photons in a coupled atom-cavity system at a fast repetition-rate.
We have demonstrated high-speed controlled generation of single photons in a coupled atom-cavity system. A single 85Rb atom, pumped with a nanosecond-pulse laser, generates a single photon into the cavity mode, and the photon is then emitted out the cavity rapidly. By employing cavity parameters for a moderate coupling regime, the single-photon emission process was optimized for both high efficiency and fast bit rates up to 10 MHz. The temporal single-photon wave packet was studied by means of the photon-arrival-time distribution relative to the pump pulse and the efficiency of the single-photon generation was investigated as the pump power. The single-photon nature of the emission was confirmed by the second-order correlation of emitted photons.


Rosiglitazone affects nitric oxide synthases and improves renal outcome in a rat model of severe ischemia/reperfusion injury.
Background. Nitric oxide (NO)-signal transduction plays an important role in renal ischemia/reperfusion (I/R) injury. NO produced by endothelial NO-synthase (eNOS) has protective functions whereas NO from inducible NO-synthase (iNOS) induces impairment. Rosiglitazone (RGZ), a peroxisome proliferator-activated receptor (PPAR)-γ agonist exerted beneficial effects after renal I/R injury, so we investigated whether this might be causally linked with NOS imbalance. Methods. RGZ (5 mg/kg) was administered i.p. to SD-rats (f) subjected to bilateral renal ischemia (60 min). Following 24 h of reperfusion, inulin- and PAH-clearance as well as PAH-net secretion were determined. Morphological alterations were graded by histopathological scoring. Plasma NO(x)-production was measured. eNOS and iNOS expression was analyzed by qPCR. Cleaved caspase 3 (CC3) was determined as an apoptosis indicator and ED1 as a marker of macrophage infiltration in renal tissue. Results. RGZ improves renal function after renal I/R injury (PAH-/inulin-clearance, PAH-net secretion) and reduces histomorphological injury. Additionally, RGZ reduces NO(x) plasma levels, ED-1 positive cell infiltration and CC3 expression. iNOS-mRNA is reduced whereas eNOS-mRNA is increased by RGZ. Conclusion. RGZ has protective properties after severe renal I/R injury. Alterations of the NO pathway regarding eNOS and iNOS could be an explanation of the underlying mechanism of RGZ protection in renal I/R injury.


Inositol trisphosphate and calcium oscillations.
InsP3 has two important functions in generating Ca2+ oscillations. It releases Ca2+ from the internal store and it can contribute to Ca2+ entry. A hypothesis has been developed to describe a mechanism for Ca2+ oscillations with particular emphasis on the way agonist concentration regulates oscillator frequency. The main idea is that the InsP3 receptors are sensitized to release Ca2+ periodically by cyclical fluctuations of Ca2+ within the lumen of the endoplasmic reticulum. Each time a pulse of Ca2+ is released, the luminal level of Ca2+ declines and has to be replenished before the InsP3 receptors are resensitized to deliver the next pulse of Ca2+. It is this loading of the internal store that explains why frequency is sensitive to external Ca2+ and may also account for how variations in agonist concentration are translated into changes in oscillation frequency. Variations in agonist-induced entry of external Ca2+, which can occur through different mechanisms, determine the variable rates of store loading responsible for adjusting the sensitivity of the InsP3 receptors to produce the periodic pulses of Ca2+. The Ca2+ oscillator is an effective analogue-to-digital converter in that variations in the concentration of the external stimulus are translated into a change in oscillator frequency.


Experimental chemotherapy for Chagas disease: 15 years of research contributions from in vivo and in vitro studies.
Chagas disease, which is caused by the intracellular parasite Trypanosoma cruzi, is a neglected illness with 12-14 million reported cases in endemic geographic regions of Latin America. While the disease still represents an important public health problem in these affected areas, the available therapy, which was introduced more than four decades ago, is far from ideal due to its substantial toxicity, its limited effects on different parasite stocks, and its poor activity during the chronic phase of the disease. For the past 15 years, our group, in collaboration with research groups focused on medicinal chemistry, has been working on experimental chemotherapies for Chagas disease, investigating the biological activity, toxicity, selectivity and cellular targets of different classes of compounds on T. cruzi. In this report, we present an overview of these in vitro and in vivo studies, focusing on the most promising classes of compounds with the aim of contributing to the current knowledge of the treatment of Chagas disease and aiding in the development of a new arsenal of candidates with anti-T. cruzi efficacy.


Revealing the assembly of filamentous proteins with scanning transmission electron microscopy.
Filamentous proteins are responsible for the superior mechanical strength of our cells and tissues. The remarkable mechanical properties of protein filaments are tied to their complex molecular packing structure. However, since these filaments have widths of several to tens of nanometers, it has remained challenging to quantitatively probe their molecular mass density and three-dimensional packing order. Scanning transmission electron microscopy (STEM) is a powerful tool to perform simultaneous mass and morphology measurements on filamentous proteins at high resolution, but its applicability has been greatly limited by the lack of automated image processing methods. Here, we demonstrate a semi-automated tracking algorithm that is capable of analyzing the molecular packing density of intra- and extracellular protein filaments over a broad mass range from STEM images. We prove the wide applicability of the technique by analyzing the mass densities of two cytoskeletal proteins (actin and microtubules) and of the main protein in the extracellular matrix, collagen. The high-throughput and spatial resolution of our approach allow us to quantify the internal packing of these filaments and their polymorphism by correlating mass and morphology information. Moreover, we are able to identify periodic mass variations in collagen fibrils that reveal details of their axially ordered longitudinal self-assembly. STEM-based mass mapping coupled with our tracking algorithm is therefore a powerful technique in the characterization of a wide range of biological and synthetic filaments.


Impact of serum triglyceride and high density lipoprotein cholesterol levels on early-phase insulin secretion in normoglycemic and prediabetic subjects.
Increased triglycerides (TGs) and decreased high density lipoprotein cholesterol (HDL-C) levels are established as diabetic risks for nondiabetic subjects. The aim of this study was to investigate the relationship among TG, HDL-C, TG/HDL-C ratio, and early-phase insulin secretion in normoglycemic and prediabetic subjects. We evaluated 663 Japanese subjects who underwent the 75-g oral glucose tolerance test. On the basis of these results, the subjects were divided into four groups: those with normal glucose tolerance (NGT; n=341), isolated impaired fasting glucose (i-IFG; n=211), isolated impaired glucose tolerance (i-IGT; n=71), and combined IFG and IGT (IFG+IGT; n=40). Insulin secretion was estimated by the insulinogenic index (IGI) (Δinsulin/Δglucose [30 to 0 minutes]) and disposition index (DI) (IGI/homeostasis model assessment of insulin resistance). In prediabetic subjects (i-IFG, i-IGT, and IFG+IGT), linear regression analyses revealed that IGI and DI were positively correlated with HDL-C levels. Moreover, in subjects with i-IGT and (IFG+IGT), but not with i-IFG, the indices of insulin secretion were negatively correlated with the log-transformed TG and TG/HDL-C ratio. In both the subjects with i-IGT, multivariate linear regression analyses revealed that DI was positively correlated with HDL-C and negatively with log-transformed TG and TG/HDL-C ratio. On the other hand, in subjects with NGT, there was no association between insulin secretion and lipid profiles. These results revealed that serum TG and HDL-C levels have different impacts on early-phase insulin secretion on the basis of their glucose tolerance status.


Laparoscopic approach is safe and effective in the management of Mirizzi syndrome.
Mirizzi syndrome (MS), an unusual complication of gallstone disease is due to mechanical obstruction of the common hepatic duct and is associated with clinical presentation of obstructive jaundice. Pre-operative identification of this entity is difficult and surgical management constitutes a formidable challenge to the operating surgeon. To analyse the clinical presentation, pre-operative diagnostic strategies, operative management and outcome of patients operated for MS in a tertiary care centre. This retrospective study identified patients operated for MS between January 2006 and August 2013 and recorded and analysed their pre-operative demographics, pre-operative diagnostic strategies, operative management, and outcome. A total of 20 patients was identified out of 1530 cholecystectomies performed during the study period giving an incidence of 1.4%. There were 11 males and 9 females with a mean age of 55.6 years. Abdomen pain and jaundice were predominant symptoms and alteration of liver function test was seen in 14 patients. Endoscopic retrograde cholangiopancreatography (ERCP) the mainstay of diagnosis was diagnostic of MS in 72% of patients, while the rest were identified intra-operatively. The most common type of MS was Type II with an incidence of 40%. Cholecystectomy was completed by laparoscopy in 14 patients with a conversion rate of 30%. A choledochoplasty was sufficed in most of the patients and none required a hepaticojejunostomy. The laparoscopic cohort had a shorter length of hospital stay when compared to the entire group. MS, a rare complication of cholelithiasis is a formidable diagnostic and therapeutic challenge and pre-operative ERCP as a main diagnostic strategy enables the surgeon to identify and minimize bile duct injury. A choledochoplasty might be sufficient in the majority of the types of MS, while a laparoscopic approach is feasible and safe in most cases as well.


Convalescent plasma therapy for the treatment of patients with COVID-19: Assessment of methods available for antibody detection and their correlation with neutralising antibody levels.
The lack of approved specific therapeutic agents to treat coronavirus disease (COVID-19) associated with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has led to the rapid implementation of convalescent plasma therapy (CPT) trials in many countries, including the United Kingdom. Effective CPT is likely to require high titres of neutralising antibody (nAb) in convalescent donations. Understanding the relationship between functional neutralising antibodies and antibody levels to specific SARS-CoV-2 proteins in scalable assays will be crucial for the success of a large-scale collection. We assessed whether neutralising antibody titres correlated with reactivity in a range of enzyme-linked immunosorbent assays (ELISA) targeting the spike (S) protein, the main target for human immune response. Blood samples were collected from 52 individuals with a previous laboratory-confirmed SARS-CoV-2 infection. These were assayed for SARS-CoV-2 nAbs by microneutralisation and pseudo-type assays and for antibodies by four different ELISAs. Receiver operating characteristic (ROC) analysis was used to further identify sensitivity and specificity of selected assays to identify samples containing high nAb levels. All samples contained SARS-CoV-2 antibodies, whereas neutralising antibody titres of greater than 1:20 were detected in 43 samples (83% of those tested) and >1:100 in 22 samples (42%). The best correlations were observed with EUROimmun immunoglobulin G (IgG) reactivity (Spearman Rho correlation coefficient 0.88; p < 0.001). Based on ROC analysis, EUROimmun would detect 60% of samples with titres of >1:100 with 100% specificity using a reactivity index of 9.1 (13/22). Robust associations between nAb titres and reactivity in several ELISA-based antibody tests demonstrate their possible utility for scaled-up production of convalescent plasma containing potentially therapeutic levels of anti-SARS-CoV-2 nAbs.


The potent and novel thiosemicarbazone chelators di-2-pyridylketone-4,4-dimethyl-3-thiosemicarbazone and 2-benzoylpyridine-4,4-dimethyl-3-thiosemicarbazone affect crucial thiol systems required for ribonucleotide reductase activity.
Di-2-pyridylketone-4,4-dimethyl-3-thiosemicarbazone possesses potent and selective antitumor activity. Its cytotoxicity has been attributed to iron chelation leading to inhibition of the iron-containing enzyme ribonucleotide reductase (RR). Thiosemicarbazone iron complexes have been shown to be redox-active, although their effect on cellular antioxidant systems is unclear. Using a variety of antioxidants, we found that only N-acetylcysteine significantly inhibited thiosemicarbazone-induced antiproliferative activity. Thus, we examined the effects of thiosemicarbazones on major thiol-containing systems considering their key involvement in providing reducing equivalents for RR. Thiosemicarbazones significantly (p < 0.001) elevated oxidized trimeric thioredoxin levels to 213 ± 5% (n = 3) of the control. This was most likely due to a significant (p < 0.01) decrease in thioredoxin reductase activity to 65 ± 6% (n = 4) of the control. We were surprised to find that the non-redox-active chelator desferrioxamine increased thioredoxin oxidation to a lower extent (152 ± 9%; n = 3) and inhibited thioredoxin reductase activity (62 ± 5%; n = 4), but at a 10-fold higher concentration than thiosemicarbazones. In contrast, only the thiosemicarbazones significantly (p < 0.05) reduced the glutathione/oxidized-glutathione ratio and the activity of glutaredoxin that requires glutathione as a reductant. All chelators significantly decreased RR activity, whereas the NADPH/NADP(total) ratio was not reduced. This was important to consider because NADPH is required for thiol reduction. Thus, thiosemicarbazones could have an additional mechanism of RR inhibition via their effects on major thiol-containing systems.


The potential of GPNMB as novel neuroprotective factor in amyotrophic lateral sclerosis.
Amyotrophic lateral sclerosis (ALS) is an incurable and fatal neurodegenerative disease characterized by the loss of motor neurons. Despite substantial research, the causes of ALS remain unclear. Glycoprotein nonmetastatic melanoma protein B (GPNMB) was identified as an ALS-related factor using DNA microarray analysis with mutant superoxide dismutase (SOD1(G93A)) mice. GPNMB was greatly induced in the spinal cords of ALS patients and a mouse model as the disease progressed. It was especially expressed in motor neurons and astrocytes. In an NSC34 cell line, glycosylation of GPNMB was inhibited by interaction with SOD1(G93A), increasing motor neuron vulnerability, whereas extracellular fragments of GPNMB secreted from activated astrocytes attenuated the neurotoxicity of SOD1(G93A) in neural cells. Furthermore, GPNMB expression was substantial in the sera of sporadic ALS patients than that of other diseased patients. This study suggests that GPNMB can be a target for therapeutic intervention for suppressing motor neuron degeneration in ALS.


Estimating peer effects in networks with peer encouragement designs.
Peer effects, in which the behavior of an individual is affected by the behavior of their peers, are central to social science. Because peer effects are often confounded with homophily and common external causes, recent work has used randomized experiments to estimate effects of specific peer behaviors. These experiments have often relied on the experimenter being able to randomly modulate mechanisms by which peer behavior is transmitted to a focal individual. We describe experimental designs that instead randomly assign individuals' peers to encouragements to behaviors that directly affect those individuals. We illustrate this method with a large peer encouragement design on Facebook for estimating the effects of receiving feedback from peers on posts shared by focal individuals. We find evidence for substantial effects of receiving marginal feedback on multiple behaviors, including giving feedback to others and continued posting. These findings provide experimental evidence for the role of behaviors directed at specific individuals in the adoption and continued use of communication technologies. In comparison, observational estimates differ substantially, both underestimating and overestimating effects, suggesting that researchers and policy makers should be cautious in relying on them.


Emergency department tobacco cessation program: staff participation and intervention success among patients.
The emergency department (ED) is often the primary source of healthcare for uninsured and underinsured patients. To evaluate ED staff attitudes toward and participation in referring patients to a tobacco cessation program, and to assess the program's effectiveness. A nonvalidated survey on smoking cessation and preventative services for ED patients was mailed to ED staff at a suburban hospital. After survey completion, ED staff was encouraged to refer smokers with diagnoses substantially worsened by tobacco use to a brief intervention delivered in the ED. An incentive was provided to staff beginning in the second month of the 3-month period. Referred patients were briefly counseled by a hospital social worker or an ED physician or nurse. Follow-up telephone interviews with patients occurred 1 to 3 months postintervention. Of the 70 ED staff contacted, 63 (90%) responded to the survey. Most staff members (81%) agreed that they should facilitate clinical prevention. Fewer staff (60%) were comfortable advising patients to quit tobacco use (P<.03), and fewer still (51%) agreed that ED staff should assist patients in tobacco cessation (P<.001). Tobacco users were more likely to favor implementing patient education in the ED (P=.01) and were less comfortable advising patients to quit (P=.06). Staff referrals increased with program incentives (P=.008), with a total of 150 interventions occurring in the 3-month span. Of the 36 patients (24%) reached for follow-up, 13 (36%) attempted to quit and 6 (17%) succeeded. Overall, 45% of the patients reached for follow-up either cut down or quit tobacco use. Staff members' attitudes toward tobacco cessation are not a firm barrier to the successful implementation of an ED tobacco cessation program. In addition, the ED provides an important opportunity to encourage patients to quit or cut down tobacco use.


Electrical Lysis and RNA Extraction from Single Cells Fixed by Dithiobis(succinimidyl propionate).
We present a microfluidic method for electrical lysis and RNA extraction from single fixed cells leveraging reversible cross-linker dithiobis(succinimidyl propionate) (DSP). Our microfluidic system captures a single DSP-fixed cell at a hydrodynamic trap, reverse-cross-links the DSP molecules on a chip with dithiothreitol, lyses the plasma membrane via electrical field, and extracts cytoplasmic RNA with isotachophoresis-aided nucleic acids extraction. All of the on-chip processes complete in less than 5 min. We demonstrated the method using K562 leukemia cells and benchmarked the performance of RNA extraction with reverse transcription quantitative polymerase chain reaction. We also demonstrated the integration of our method with single-cell RNA sequencing.


Somatic mutation screening: identification of individuals harboring K-ras mutations with the use of plasma DNA.
Many cancers are attributed to somatic mutation of DNA. We investigated whether it is feasible to detect cancer-associated somatic mutations in patients with neoplasms by using plasma DNA. Plasma samples were prospectively collected from 240 patients undergoing colonoscopy. Colorectal biopsies were performed as clinically indicated in 135 patients, and risk factor information was available from 232 patients. DNA was extracted from plasma and colorectal tissue and was amplified by use of a polymerase chain reaction method that enriches for mutations in codon 12 of the K-ras oncogene. Molecular, histologic, and clinical data were compared by use of two-sided Fisher's exact test. Mutations in the K-ras gene detected in the plasma of 64 (28%) of 232 patients were statistically significantly associated with colorectal cancer risk factors (P =.0002). Of those patients having tissue available for comparison (n = 135), mutations in the K-ras gene were found in the tissues of 35 patients, and 29 (83%) of these 35 showed mutations in plasma samples. In contrast, the plasma assay was negative in 93 of the 100 patients whose tissue K-ras was wild-type. Among patients without biopsies (n = 105), 28 had mutated K-ras in their plasma DNA, despite the absence of remarkable colonoscopy findings; 24 of these 28 patients had risk factors for colorectal cancer. Overall, 25 (39%) of 64 patients showing mutations in plasma DNA had colorectal neoplasms with K-ras mutations compared with five (3%) of 176 patients without K-ras mutations in plasma DNA. Plasma DNA assays for the detection of mutations in K-ras codon 12 may provide a feasible method to screen populations for somatic mutations frequently found in neoplasms. The clinical utility of using this test in screening populations requires further study.


Does being a retired or employed caregiver affect the association between behaviours in Alzheimer's disease and caregivers' health-related quality-of-life?
We examined whether caregivers' employment status (i.e., retired or employed) might modify the association between the behaviours of persons with Alzheimer's disease (PwAD) and caregivers' health-related quality-of-life (HRQoL). Data came from a cross-sectional study of the primary informal caregivers of 200 persons with mild or moderate Alzheimer's disease. Caregivers completed the EQ-5D-3L to rate their HRQoL and generate health utility scores, and the Dementia Behaviour Disturbance Scale (DBDS) to assess the degree to which PwAD exhibited each of 28 behaviours. Caregivers' health utility scores were regressed on overall DBDS scores, with caregiver employment status (retired, employed) treated as an effect modifier and confounder in separate regression models. We also controlled for age, sex, income, education, caregivers' relationship to the PwAD, and whether caregivers gave up paid employment/cut down working hours to care for PwAD. Effect modification by caregiver employment status is possible, with the inverse association between DBDS score and health utility score largely existing for retired versus employed caregivers. Research using larger samples and longitudinal data would further inform this area of inquiry.


Neonatal hyperbilirubinemia due to ABO incompatibility: does blood group matter?
Newborn infants with maternal-fetal ABO incompatibility are at a greater risk for developing subsequent significant hyperbilirubinemia, and therefore, prediction of probable risk factors, such as the degree of hemolysis, gains importance. In this study, we aimed to evaluate the effect of fetal-neonatal blood group on the severity of hemolysis and jaundice due to maternal-fetal ABO incompatibility. In a retrospective analysis of 166 cases with ABO hemolytic disease of the newborn, risk factors for the severity of jaundice were compared in infants with blood group A or B. Both groups had similar demographic parameters such as birth weight, gender and day of admission. Similarly, there were no statistically significant differences in hematological parameters, such as initial hemoglobin levels, initial and final indirect bilirubin levels, frequency of positive direct Coombs test and hemolytic findings on peripheral blood smear, duration of phototherapy, number of exchange transfusions, and intravenous immunoglobulin (IVIG) therapy (p>0.05). We conclude that blood type has no effect on the severity of the hemolytic jaundice in ABO incompatibility.


Breast cancer patients on endocrine therapy reveal more symptoms when self-reporting than in pivotal trials: an outcome research study.
The purpose of this investigation was firstly to assess the overall frequency of subjectively experienced symptoms self-reported by patients receiving endocrine therapy and secondly to compare these symptoms with side effects assessed by clinicians in pivotal trials. Unselected patients with early and advanced breast cancer receiving endocrine therapy were approached consecutively during a routine outpatient visit. They received a questionnaire called Checklist for Patients with Endocrine Therapy (C-PET), a validated self-assessment tool to determine prespecified symptoms associated with endocrine therapy. Data on toxicity were also obtained from previously published trials. 405 patients were approached and 373 agreed to participate in this study. Some symptoms were significantly more often recorded by the women in the adjuvant setting completing the C-PET than by physicians' reports in pivotal trials: hot flushes/sweats (C-PET 70%, ATAC 40% and BIG1-98 38%), low energy (C-PET 45%, ATAC 15% and BIG1-98 9%), fluid retention (C-PET 22% and BIG1-98 7%) and vaginal dryness (C-PET 30% and BIG1-98 3%). Similar differences were observed in the metastatic and adjuvant setting. A simple tool like the C-PET questionnaire is able to reflect the treatment burden of endocrine therapies and may be helpful to improve communication between patients and care providers. Some symptoms were significantly more often reported by the women in the C-PET than by physicians in pivotal trials.


SCOPE: a web server for practical de novo motif discovery.
SCOPE is a novel parameter-free method for the de novo identification of potential regulatory motifs in sets of coordinately regulated genes. The SCOPE algorithm combines the output of three component algorithms, each designed to identify a particular class of motifs. Using an ensemble learning approach, SCOPE identifies the best candidate motifs from its component algorithms. In tests on experimentally determined datasets, SCOPE identified motifs with a significantly higher level of accuracy than a number of other web-based motif finders run with their default parameters. Because SCOPE has no adjustable parameters, the web server has an intuitive interface, requiring only a set of gene names or FASTA sequences and a choice of species. The most significant motifs found by SCOPE are displayed graphically on the main results page with a table containing summary statistics for each motif. Detailed motif information, including the sequence logo, PWM, consensus sequence and specific matching sites can be viewed through a single click on a motif. SCOPE's efficient, parameter-free search strategy has enabled the development of a web server that is readily accessible to the practising biologist while providing results that compare favorably with those of other motif finders. The SCOPE web server is at <http://genie.dartmouth.edu/scope>.


Tonsillar homing of Epstein-Barr virus-specific CD8+ T cells and the virus-host balance.
Patients with infectious mononucleosis (IM) undergoing primary EBV infection show large expansions of EBV-specific CD8+ T cells in the blood. While latent infection of the B cell pool is quickly controlled, virus shedding from lytically infected cells in the oropharynx remains high for several months. We therefore studied how responses localize to the tonsil, a major target site for EBV, during primary infection and persistence. In acute IM, EBV-specific effectors were poorly represented among CD8+ T cells in tonsil compared with blood, coincident with absence of the CCR7 lymphoid homing marker on these highly activated cells. In patients who had recently recovered from IM, latent epitope reactivities were quicker than lytic reactivities both to acquire CCR7 and to accumulate in the tonsil, with some of these cells now expressing the CD103 integrin, which mediates retention at mucosal sites. By contrast, in long-term virus carriers in whom both lytic and latent infections had been controlled, there was 2- to 5-fold enrichment of lytic epitope reactivities and 10- to 20-fold enrichment of latent epitope reactivities in tonsil compared with blood; up to 20% of tonsillar CD8+ T cells were EBV specific, and many now expressed CD103. We suggest that efficient control of EBV infection requires appropriate CD8+ T cell homing to oropharyngeal sites.


Broadly Neutralizing Antibody Responses in a Large Longitudinal Sub-Saharan HIV Primary Infection Cohort.
Broadly neutralizing antibodies (bnAbs) are thought to be a critical component of a protective HIV vaccine. However, designing vaccines immunogens able to elicit bnAbs has proven unsuccessful to date. Understanding the correlates and immunological mechanisms leading to the development of bnAb responses during natural HIV infection is thus critical to the design of a protective vaccine. The IAVI Protocol C program investigates a large longitudinal cohort of primary HIV-1 infection in Eastern and South Africa. Development of neutralization was evaluated in 439 donors using a 6 cross-clade pseudo-virus panel predictive of neutralization breadth on larger panels. About 15% of individuals developed bnAb responses, essentially between year 2 and year 4 of infection. Statistical analyses revealed no influence of gender, age or geographical origin on the development of neutralization breadth. However, cross-clade neutralization strongly correlated with high viral load as well as with low CD4 T cell counts, subtype-C infection and HLA-A*03(-) genotype. A correlation with high overall plasma IgG levels and anti-Env IgG binding titers was also found. The latter appeared not associated with higher affinity, suggesting a greater diversity of the anti-Env responses in broad neutralizers. Broadly neutralizing activity targeting glycan-dependent epitopes, largely the N332-glycan epitope region, was detected in nearly half of the broad neutralizers while CD4bs and gp41-MPER bnAb responses were only detected in very few individuals. Together the findings suggest that both viral and host factors are critical for the development of bnAbs and that the HIV Env N332-glycan supersite may be a favorable target for vaccine design.


Recombineering, transfection, Western, IP and ChIP methods for protein tagging via gene targeting or BAC transgenesis.
Protein tagging offers many advantages for proteomic and regulomic research. Ideally, protein tagging is equivalent to having a high affinity antibody for every chosen protein. However, these advantages are compromised if the tagged protein is overexpressed, which is usually the case from cDNA expression vectors. Physiological expression of tagged proteins can be achieved by gene targeting to knock-in the protein tag or by BAC transgenesis. BAC transgenes usually retain the native gene architecture including all cis-regulatory elements as well as the exon-intron configurations. Consequently most BAC transgenes are authentically regulated (e.g. by transcription factors, cell cycle, miRNA) and can be alternatively spliced. Recombineering has become the method of choice for generating targeting constructs or modifying BACs. Here we present methods with detailed protocols for protein tagging by recombineering for BAC transgenesis and/or gene targeting, including the evaluation of tagged protein expression, the retrieval of associated protein complexes for mass spectrometry and the use of the tags in ChIP (chromatin immunoprecipitation).


Awareness and attitude to the law banning smoking in public places in Osun State, Nigeria.
This study determined the awareness and attitude towards the Osun state prohibition of smoking in public places law. Descriptive cross-sectional study design. 520 consenting respondents recruited using a convenience sampling method were interviewed using a semi-structured questionnaire covering their smoking pattern, awareness and attitude towards the law of prohibition of smoking in public places in Osun State. Data analyzed using descriptive and chi-square statistics. Only 38% were aware of the law while none had seen the document. Fifty six percent felt cigarette smoking is a problem that required the law to be implemented, while only 20% agreed that the law will stop tobacco use. The radio (58%), bill boards (45%) and newspapers (44%) were the major sources of awareness of the law. The perception of risk posed to the public and family health by cigarette smoking was poor among the participants. There is poor awareness and attitude to the law of prohibition of smoking in public places in Osun State. It is necessary to increase sensitization of the general public and enforcement of the law.


Results of balloon dilatation of stenotic homografts in pulmonary position in children and young adults.
To evaluate the results of balloon dilatation of stenotic homografts in children, adolescents, and young adults and to identify factors that might influence or predict the effect of the dilatation. Homografts are widely used in congenital cardiac surgery; however, the longevity remains a problem mostly because of stenosis in the homograft. The effect of treatment by balloon dilatation is unclear. In a retrospective study, the effect of balloon dilatation was determined by the percentage of reduction of the peak systolic pressure gradient over the homograft during catheterisation and the postponement of re-intervention or replacement of the homograft in months. Successful dilatations - defined in this study as a reduction of more than 33% and postponement of more than 18 months - were compared with unsuccessful dilatations in search of factors influencing or predicting the results. The mean reduction of the peak systolic pressure gradient was 30% in 40 procedures. Re-intervention or replacement of the homograft was postponed by a mean of 19 months. In all, 14 balloon dilatations (35%) were successful; the mean reduction was 49% and the mean postponement was 34 months. The time since homograft implantation, the presence of calcification, the homograft/balloon ratio, and the pressure applied during dilatation all tended to correlate with outcome, but were not statistically significant. Balloon dilatation is able to reduce the peak systolic pressure gradient over homografts in a subgroup of patients and can be of clinical significance to postpone re-intervention or pulmonary valve replacement.


Microplastic litter composition of the Turkish territorial waters of the Mediterranean Sea, and its occurrence in the gastrointestinal tract of fish.
Microplastic pollution of marine environment is receiving increased publicity over the last few years. The present survey is, according to our knowledge, the survey with the largest sample size analyzed, to date. In total, 1337 specimens of fish were examined for the presence of plastic microlitter representing 28 species and 14 families. In addition, samples of seawater and sediment were also analyzed for the quantification of microplastic in the same region. Samples of water/sediment were collected from 18 locations along the Mediterranean coast of Turkey. 94% of all collected plastic microlitter from the sea was in the size range between 0.1 and 2.5 mm, while the occurrence of other sizes was rare. The quantity of microplastic particles in surface water samples ranged from 16 339 to 520 213 per km2. Fish were collected from 10 locations from which 8 were either shared with or situated in the proximity of water/sediment sampling locations. A total of 1822 microplastic particles were extracted from stomach and intestines of fish. Majority of ingested particles were represented by fibers (70%) and hard plastic (20.8%), while the share of other groups: nylon (2.7%), rubber (0.8%) and miscellaneous plastic (5.5%) were low. The blue color of plastic was the most dominant color. 34% of all examined fish had microplastic in the stomach. On average, fish which had microplastic contained 1.80 particles per stomach. 41% of all fish had microplastic in the intestines with an average of 1.81 particles per fish. 771 specimens contained microplastic in either stomach and/or intestines representing 58% of the total sample with an average of 2.36 particles per fish. Microplastic was found in all species/families that had sample size of at least 2 individuals. The number of particles present in either stomach or intestines ranged between 1 and 35. Ingested microplastic had an average diameter ±SD of 656 ± 803 μm, however particles as small as 9 μm were detected. The trophic level of fish species had no influence whatsoever on the amount of ingested microplastic. Pelagic fish ingested more microplastic than demersal species. In general, fish that ingested higher number of microplastic particles originated from the sites that also had a higher particle count in the seawater and sediment.


Amyloid fibril formation by bovine α1-acid glycoprotein in a reducing environment: The role of disulfide bridges on the observed aggregation kinetics.
Bovine α1-acid glycoprotein (bAGP), a thermostable counterpart of its human homologue, is a positive acute phase protein involved in binding and transportation of a large number of bio-active molecules and drugs across the body. We have investigated the effect of low pH and reducing conditions on the structure of the protein and found that it aggregates at high temperatures. The aggregates show a fibrillar structure when observed with electron microscopy. Aggregation assays using the amyloid-specific dye Thioflavin T show the presence of a lag phase which was neither abolished nor shortened when seeds were added. A priori reduction of the two disulfide bridges of bAGP, on the other hand, abolished the lag phase and reveals a connection between the kinetics of reduction and aggregation. We provide a kinetic interpretation and the corresponding rate laws allowing to model the process of fibril formation by bAGP under reducing conditions. Our interpretation allows to assess the role of disulfide bridges on the fibrillation kinetics of bAGP and can provide a more accurate interpretation of the fibrillation kinetics of other amyloidogenic proteins containing disulfide bridges.


Characterization of hepatocellular carcinomas with triphasic CT and correlation with histopathologic findings.
To determine the utility of triphasic CT in the characterization of hepatocellular carcinomas (HCCs) and correlations with histopathologic findings. Thirty patients with hepatocellular carcinomas were included in the study and triphasic CT examinations were performed. The CT protocol included hepatic arterial, portal venous and late phases. A histopathologic examination was carried out in all but 3 patients, and the diagnosis and degrees of differentiation were determined. Hepatocellular carcinomas were hyperattenuated in 17 (57%) and hypoattenuated in 13 (43%) of the 30 patients in arterial phase images. The lesions were hypoattenuated in 26 (87%) and hyperattenuated in 4 (13%) patients in portal venous phase images. These hyperattenuated tumors were well-differentiated in the histopathologic examinations (P < or = 0.05). Portal vein invasion was seen in 50% of the patients and this relationship was significant in patients whose lesions was greater than 10 cm (P < 0.05). Capsule formation, abnormal internal vessels and necrosis were detected in 57%, 53% and 40% of the patients, respectively. Triphasic CT can aid in the histopathologic differentiation of HCCs, in addition to their characterization. Hyperattenuation in PVP images was found to be associated with well-differentiated HCCs and portal vein invasion was more frequent in tumors larger than 10 cm.


Genetic polymorphisms of the renin-angiotensin system and complications of insulin-dependent diabetes mellitus.
Patients with insulin-dependent diabetes mellitus (IDDM) have a high risk of developing diabetic nephropathy, retinopathy and cardiovascular diseases. The contribution of gene polymorphisms of the renin angiotensin system to these complications is controversial and may differ among populations. In 257 Dutch IDDM patients (188 with urinary albumin excretion (UAE) <30 mg/24 h), logistic regression analysis was used to study the relationships among, on the one hand, the insertion/deletion gene polymorphism of the angiotensin-converting enzyme gene (ACE-ID), the M235T gene polymorphism of the angiotensinogen gene (AGT-M235T), and the A1166C gene polymorphism of the angiotensin type 1 receptor gene (AT1-A1166C), and, on the other hand, UAE, retinopathy, hypertension, and coronary heart disease. The T-allele of the AGT-M235T polymorphism was associated with an increased risk of an elevated UAE (odds ratio (OR) 3.03; 95% confidence interval (CI) 1.06-8.61), but only when interaction with the D-allele of the ACE-ID polymorphism was considered. A previously described positive interaction between the T-allele of the AGT-M235T polymorphism and the D-allele of the ACE-ID polymorphism could not be confirmed. The T-allele was also associated with an increased risk of retinopathy (OR 3.89, 95% CI 1.79-8.47). The CC-genotype of the AT1-A1166C polymorphism was associated with hypertension (OR 3.58; 95% CI 1. 23-10.37). In a Dutch IDDM population, including 69 patients with (incipient) diabetic nephropathy, the T-allele of the AGT-M235T polymorphism is associated with an elevated UAE and diabetic retinopathy and the CC-genotype of the AT1-A1166C polymorphism is associated with hypertension. A previously described interaction between the AGT-M235T and the ACE-ID polymorphisms could not be confirmed. Since the number of nephropathic patients in this study is small, these conclusions must be interpreted with caution.


Novel Nested Peptide Epitopes Recognized by CD4+ T Cells Induced by HIV-1 Conserved-Region Vaccines.
CD4+ T-cell responses play an important role in the immune control of the human immunodeficiency virus type 1 (HIV-1) infection and as such should be efficiently induced by vaccination. It follows that definition of HIV-1-derived peptides recognized by CD4+ T cells in association with HLA class II molecules will guide vaccine development. Here, we have characterized the fine specificity of CD4+ T cells elicited in human recipients of a candidate vaccine delivering conserved regions of HIV-1 proteins designated HIVconsv. The majority of these 19 most immunogenic regions contained novel epitopes, that is, epitopes not listed in the Los Alamos National Laboratory HIV Sequence Database, which were able in vitro to stimulate vaccinees' CD4+ T cells to proliferate and produce interferon-γ and tumor necrosis factor-α. Accumulation of HLA class II epitopes will eventually accelerate development of HIV-1 prophylactic and therapeutic vaccines.


Escherichia coli expression and substrate specificities of canine cytochrome P450 3A12 and rabbit cytochrome P450 3A6.
High level Escherichia coli expression of cytochromes P450 3A12 and 3A6 has facilitated the characterization of proteins which exhibit limited activity as purified hepatic enzymes in reconstituted systems. Three 3A12 and two 3A6 constructs modified at the 5'-end to encode the bovine 17 alpha-sequence (Barnes et al., Proc. Natl. Acad. Sci. U.S.A. 88: 5597-5601, 1991), or related sequences, exhibited expression levels ranging from 2 to 89 nmol of cytochrome P450 liter-1. Recombinant canine 3A12 catalyzed steroid 6 beta-hydroxylation and erythromycin demethylation at rates comparable to those obtained in phenobarbital-induced canine liver microsomes. In contrast, 3A12 troleandomycin demethylase activity (2.5 nmol/min/nmol) was significantly lower than that of canine phenobarbital-induced liver microsomes (6.6 nmol/min/nmol). This difference in activity suggests that at least two 3A forms, which may differ functionally, are present within the canine liver. Purification of recombinant rabbit 3A6 revealed that homogeneous and E. coli-solubilized membrane preparations of 3A6 exhibit similar metabolic rates and identical substrate specificities; 3A activity was modulated by 25 microM alpha-naphthoflavone, which stimulated an unidentified progesterone metabolite 9-fold in 3A6 reconstituted systems in contrast to the 4-fold stimulation of 3A12. Furthermore, 25 microM alpha-naphthoflavone inhibited erythromycin demethylation 64 and 33% by purified recombinant 3A6- or 3A6-solubilized membrane fractions, respectively; 3A12-mediated erythromycin demethylation in solubilized membrane fractions was resistant to flavonoid inhibition. These results indicate that, although 3A substrate specificities are highly conserved between species, functional differences exist between canine 3A12 and rabbit 3A6, which may be utilized to better understand 3A structure-function relationships.


A different approach to patients and loved ones who request "futile" treatments.
The author describes an alternative approach that care-providers may want to consider when caring for patients who request interventions that careproviders see as futile. This approach is based, in part, on findings of recent neuroimaging research. The author also provides several examples of seemingly justifiable "paternalistic omissions," taken from articles in this issue of The Journal of Clinical Ethics (JCE). The author suggests that while careproviders should always give patients and their loved ones all potentially relevant information regarding "futile" decisions, care-providers may wish to consider, paradoxically, not giving advice in these situations, when the advice is based mostly or wholly on their own moral views, based on this same, ethical rationale.


Extracellular retention of a cyclopamine nanoformulation leveraging larger size and more negative charge for improved breast cancer treatment.
Compared with intracellular drug delivery, drugs with extracellular targeting sites are rarely considered. As one of these drugs, cyclopamine (CYC) is a promising anticancer drug that functions by targeting the cell membrane receptor. For improving therapeutic effect, an albumin-based nano-system (ABN) with the capacity for extracellular retention was developed. The ABN was formulated by incorporating bovine serum albumin (BSA) into nanoparticles at the denaturing temperature of BSA, with CYC acting as a hydrophobic nucleation site, followed by stabilization upon heat-induced disulfide cross-linking. The resultant ABNs are negatively charged with a nanoparticle size that can be delicately regulated by varying the reaction time. In MDA-MB-231 cells, the size and charge of ABNs significantly affected the extracellular retention capacity, with ABN-300 nm exhibiting an enhanced cytotoxic effect. In vivo fluorescence imaging revealed obvious and persistent tumor accumulation of ABNs. A therapeutic study in an orthotopic mammary fat pad tumor model shows that ABN-300 nm possesses the most remarkable antitumor effect compared with the control groups. These results provide a new strategy for improving the efficacy of drug targeting at extracellular sites.


A "PROPP" for the Bronx: preterm birth prevention education in the inner city.
As one component of the multifaceted community-wide preterm prevention program known as "A PROPP for the Bronx" (Program to Reduce Obstetric Problems and Prematurity), a prenatal education videotape in both English and Spanish was developed. Its impact was evaluated in 615 high-risk patients at the Bronx Municipal Hospital Center. The 12-minute videotape focused on the implications of preterm birth, the signs and symptoms of preterm labor, and behavioral modification to reduce life-style risk factors. An instrument was constructed to examine baseline knowledge, information transfer, and knowledge retention, and was validated and tested for reliability (r = 0.95). The results demonstrated a statistically significant knowledge transfer for patients viewing the videotape (P less than .0001) and significant knowledge retention through the postpartum period (P less than .0001) for Spanish-speaking as well as English-speaking parturients. These data emphasize the importance of the educational component of a preterm prevention program in an impoverished population at risk for early delivery.


Removal of arsenic from high ionic strength solutions: effects of ionic strength, pH, and preformed versus in situ formed HFO.
Arsenic sorption to hydrous ferric oxide (HFO) is an effective treatment method for removing dissolved arsenic from fresh drinking water sources. However, detailed information is limited regarding arsenic removal from solutions of high ionic strength such as brackish groundwater, seawater, or high-pressure membrane process residuals. Bench-scale treatment experiments were conducted exploring arsenic removal from simple solutions with ionic strengths ranging from 0.008 to 1.5 M by addition of ferric chloride followed by solid/liquid separation (microfiltration or ultrafiltration). Arsenic removal from these solutions during in situ iron precipitation was approximately 90% at Fe:As molar ratios of 10 to 15 and > 95% for Fe:As molar ratios greater than 20. Arsenic removal at iron doses of 10(-6) to 10(-4) mol-Fe/L improved when pH was lowered from 8 to less than 6.5 at ionic strength 0.2 M; this improvement was not as significant at ionic strength 0.7 M. Arsenic removal diminished when alkalinity was increased from 400 to 1,400 mg/L as calcium carbonate; however, arsenic removal at the higher alkalinity improved when pH was lowered from approximately 8 to less than 7. Arsenic removal with preformed HFO solids and subsequent microfiltration was significantly less than that observed with in situ HFO precipitation. Increased removal by in situ precipitation compared to that of preformed solids is explained by an increased number of adsorption sites due to uptake during iron oxy-hydroxide polymerization as well as an increase in surface area resulting in diminished surface charge effects. Model simulations of arsenic uptake by in situ precipitation adequately captured these effect by changing the model parameters used to model arsenic uptake by preformed HFO, specificallythe total number of surface sites and surface area.


Avirulence of rough mutants of Shigella flexneri: requirement of O antigen for correct unipolar localization of IcsA in the bacterial outer membrane.
Mutations in the lipopolysaccharide (LPS) of Shigella spp. result in attenuation of the bacteria in both in vitro and in vivo models of virulence, although the precise block in pathogenesis is not known. We isolated defined mutations in two genes, galU and rfe, which directly affect synthesis of the LPS of S. flexneri 2a, in order to determine more precisely the step in virulence at which LPS mutants are blocked. The galU and rfe mutants invaded HeLa cells but failed to generate the membrane protrusions (fireworks) characteristic of intracellular motility displayed by wild-type shigellae. Furthermore, the galU mutant was unable to form plaques on a confluent monolayer of eucaryotic cells and the rfe mutant generated only tiny plaques. These observations indicated that the mutants were blocked in their ability to spread from cell to cell. Western immunoblot analysis of expression of IcsA, the protein essential for intracellular motility and intercellular spread, demonstrated that both mutants synthesized IcsA, although they secreted less of the protein to the extracellular medium than did the wild-type parent. More strikingly, the LPS mutants showed aberrant surface localization of IcsA. Unlike the unipolar localization of IcsA seen in the wild-type parent, the galU mutant expressed the protein in a circumferential fashion. The rfe mutant had an intermediate phenotype in that it displayed some localization of IcsA at one pole while also showing diffuse localization around the bacterium. Given the known structures of the LPS of wild-type S. flexneri 2a, the rfe mutant, and the galU mutant, we hypothesize that the core and O-antigen components of LPS are critical elements in the correct unipolar localization of IcsA. These observations indicate a more precise role for LPS in Shigella pathogenesis.


Charcot-Marie-Tooth disease-linked protein SIMPLE functions with the ESCRT machinery in endosomal trafficking.
Mutations in small integral membrane protein of lysosome/late endosome (SIMPLE) cause autosomal dominant, Charcot-Marie-Tooth disease (CMT) type 1C. The cellular function of SIMPLE is unknown and the pathogenic mechanism of SIMPLE mutations remains elusive. Here, we report that SIMPLE interacted and colocalized with endosomal sorting complex required for transport (ESCRT) components STAM1, Hrs, and TSG101 on early endosomes and functioned with the ESCRT machinery in the control of endosome-to-lysosome trafficking. Our analyses revealed that SIMPLE was required for efficient recruitment of ESCRT components to endosomal membranes and for regulating endosomal trafficking and signaling attenuation of ErbB receptors. We found that the ability of SIMPLE to regulate ErbB trafficking and signaling was impaired by CMT-linked SIMPLE mutations via a loss-of-function, dominant-negative mechanism, resulting in prolonged activation of ERK1/2 signaling. Our findings indicate a function of SIMPLE as a regulator of endosomal trafficking and provide evidence linking dysregulated endosomal trafficking to CMT pathogenesis.


Antiretroviral treatment for children with peripartum nevirapine exposure.
Single-dose nevirapine is the cornerstone of the regimen for prevention of mother-to-child transmission of human immunodeficiency virus (HIV) in resource-limited settings, but nevirapine frequently selects for resistant virus in mothers and children who become infected despite prophylaxis. The optimal antiretroviral treatment strategy for children who have had prior exposure to single-dose nevirapine is unknown. We conducted a randomized trial of initial therapy with zidovudine and lamivudine plus either nevirapine or ritonavir-boosted lopinavir in HIV-infected children 6 to 36 months of age, in six African countries, who qualified for treatment according to World Health Organization (WHO) criteria. Results are reported for the cohort that included children exposed to single-dose nevirapine prophylaxis. The primary end point was virologic failure or discontinuation of treatment by study week 24. Enrollment in this cohort was terminated early on the recommendation of the data and safety monitoring board. A total of 164 children were enrolled. The median percentage of CD4+ lymphocytes was 19%; a total of 56% of the children had WHO stage 3 or 4 disease. More children in the nevirapine group than in the ritonavir-boosted lopinavir group reached a primary end point (39.6% vs. 21.7%; weighted difference, 18.6 percentage-points; 95% confidence interval, 3.7 to 33.6; nominal P=0.02). Baseline resistance to nevirapine was detected in 18 of 148 children (12%) and was predictive of treatment failure. No significant between-group differences were seen in the rate of adverse events. Among children with prior exposure to single-dose nevirapine for perinatal prevention of HIV transmission, antiretroviral treatment consisting of zidovudine and lamivudine plus ritonavir-boosted lopinavir resulted in better outcomes than did treatment with zidovudine and lamivudine plus nevirapine. Since nevirapine is used for both treatment and perinatal prevention of HIV infection in resource-limited settings, alternative strategies for the prevention of HIV transmission from mother to child, as well as for the treatment of HIV infection, are urgently required. (Funded by the National Institutes of Health; ClinicalTrials.gov number, NCT00307151.).


Influence of thyroid state on mechanical restitution of rat myocardium.
The purpose of this study was to determine whether thyroid state affects the beat-to-beat regulation of contractile strength in cardiac muscle. Transmembrane action potential and isometric force were simultaneously recorded in right ventricular papillary muscles from euthyroid, hypothyroid, and hyperthyroid rats. Large thyroid state-dependent alterations in the contractile response of the muscles were not accompanied by any significant difference in the action potential. During steady-state stimulation, single test stimuli were interpolated at varying intervals. Action potential duration and peak force of the test responses were plotted against the test stimulus interval to produce electrical and mechanical restitution curves. In all muscles studied, electrical and mechanical restitution followed different time courses; over a wide range of test intervals, action potential duration and peak force of the test responses changed in opposite directions. Thyroid state profoundly affected the recovery of contractile strength, while only minor differences were found among the electrical restitution curves of the three groups of preparations. Mechanical recovery was much faster in hyperthyroid and slower in hypothyroid than in euthyroid muscles. We conclude that electrical and mechanical restitutions occur through separate processes and that the thyroid state affects only the mechanisms responsible for the contractile recovery of rat myocardium. The modifications induced by thyroid dysfunction on contractile recovery might be accounted for by an effect of thyroid state on a time-dependent recycling of calcium by the sarcoplasmic reticulum.


Redox-active star molecules incorporating the 4-benzoylpyridinium cation: implications for the charge transfer efficiency along branches vs across the perimeter in dendrimers.
We report the redox properties of four star systems incorporating the 4-benzoyl-N-alkylpyridinium cation; the redox potential varies along the branches but remains constant at fixed radii. Bulk electrolysis shows that at a semi-infinite time scale all redox centers are electrochemically accessible. However, voltammetric analysis (cyclic voltammetry and differential pulse voltammetry) shows that only two of the three redox-active centers in the perimeter are electrochemically accessible during potential sweeps as slow as 20 mV s-1 and as fast as 10 V s-1. On the contrary, both redox centers along branches are accessible electrochemically within the same time frame. These results are explained in terms of slow through-space charge transfer and the globular 3-D folding of the molecules and are discussed in terms of their implications on the design of efficient redox functional dendrimers.


An integrated hierarchical Bayesian model for multivariate eQTL mapping.
Recently, expression quantitative loci (eQTL) mapping studies, where expression levels of thousands of genes are viewed as quantitative traits, have been used to provide greater insight into the biology of gene regulation. Originally, eQTLs were detected by applying standard QTL detection tools (using a "one gene at-a-time" approach), but this method ignores many possible interactions between genes. Several other methods have proposed to overcome these limitations, but each of them has some specific disadvantages. In this paper, we present an integrated hierarchical Bayesian model that jointly models all genes and SNPs to detect eQTLs. We propose a model (named iBMQ) that is specifically designed to handle a large number G of gene expressions, a large number S of regressors (genetic markers) and a small number n of individuals in what we call a ``large G, large S, small n'' paradigm. This method incorporates genotypic and gene expression data into a single model while 1) specifically coping with the high dimensionality of eQTL data (large number of genes), 2) borrowing strength from all gene expression data for the mapping procedures, and 3) controlling the number of false positives to a desirable level. To validate our model, we have performed simulation studies and showed that it outperforms other popular methods for eQTL detection, including QTLBIM, R-QTL, remMap and M-SPLS. Finally, we used our model to analyze a real expression dataset obtained in a panel of mice BXD Recombinant Inbred (RI) strains. Analysis of these data with iBMQ revealed the presence of multiple hotspots showing significant enrichment in genes belonging to one or more annotation categories.


Crystal structure of the pyrazinamidase of Mycobacterium tuberculosis: insights into natural and acquired resistance to pyrazinamide.
Pyrazinamidase (PncA) activates the first-line antituberculous drug pyrazinamide into pyrazinoic acid. The crystal structure of the Mycobacterium tuberculosis PncA protein has been determined, showing significant differences in the substrate binding cavity when compared to the pyrazinamidases from Pyrococcus horikoshii and Acinetobacter baumanii. In M. tuberculosis, this region was found to hold a Fe(2+) ion coordinated by one aspartate and three histidines, one of them corresponding to His57 which is replaced by Asp in Mycobacterium bovis, a species naturally resistant to pyrazinamide. The binding cavity also contains a Cys138-Asp8-Lys96 motif evocating a cysteine-based catalytic mechanism. Mutants have been constructed and investigated by kinetic and thermal shift assays, highlighting the importance of protein folding and thermal stability in the pyrazinamidase activity.


Identification and angiogenic role of the novel tumor endothelial marker CLEC14A.
Tumor endothelial markers (TEMs) that are highly expressed in human tumor vasculature compared with vasculature in normal tissue hold clear therapeutic potential. We report that the C-type lectin CLEC14A is a novel TEM. Immunohistochemical and immunofluorescence staining of tissue arrays has shown that CLEC14A is strongly expressed in tumor vasculature when compared with vessels in normal tissue. CLEC14A overexpression in tumor vessels was seen in a wide range of solid tumor types. Functional studies showed that CLEC14A induces filopodia and facilitates endothelial migration, tube formation and vascular development in zebrafish that is, CLEC14A regulates pro-angiogenic phenotypes. CLEC14A antisera inhibited cell migration and tube formation, suggesting that anti-CLEC14A antibodies may have anti-angiogenic activity. Finally, in endothelial cultures, expression of CLEC14A increased at low shear stress, and we hypothesize that low shear stress due to poor blood flow in the disorganized tumor vasculature induces expression of CLEC14A on tumor vessels and pro-angiogenic phenotypes.


[Quantitative analysis and pharmaco-toxicity of cannabinoids in commercially available cannabis seeds].
delta 9-Tetrahydrocannabinol (delta 9-THC), cannabinol, cannabidiol and cannabichromene were detected in commercially available cannabis seeds by silica gel TLC and gas chromatography. These cannabinoids existed in rather high content (0.10-2.02 mg/100 g of seeds) in the feed for birds especially bracts (82.3-441 mg/100 g). When the suspension prepared from the benzene washing solution of cannabis seeds, BenW, was administered at a dose of 3 mg/kg corresponding to delta 9-THC into a mouse, i.v., BenW caused hypothermia, catalepsy, pentobarbital-induced sleep prolongation and suppression of locomotor activity. These pharmacological activities of BenW were significantly higher than those of delta 9-THC (3 mg/kg, i.v.). These results may indicate the necessity to reconsider the present regulations on marihuana.


Neurog3 gene dosage regulates allocation of endocrine and exocrine cell fates in the developing mouse pancreas.
The basic helix-loop-helix transcription factor Neurog3 (Neurogenin3 or Ngn3) actively drives endodermal progenitor cells towards endocrine islet cell differentiation during embryogenesis. Here, we manipulate Neurog3 expression levels in endocrine progenitor cells without altering its expression pattern using heterozygosity and a hypomorph. Lowered Neurog3 gene dosage in the developing pancreatic epithelium reduces the overall production of endocrine islet cells without significantly affecting the proportions of various islet cell types that do form. A reduced Neurog3 production level in the endocrine-directed pancreatic progenitor population activates the expression of Neurog3 in an increased number of epithelial progenitors. Yet a significant number of these Neurog3+ cells detected in heterozygous and hypomorphic pancreata, possibly those that express low levels of Neurog3, move on to adopt pancreatic ductal or acinar fates. These data directly demonstrate that achieving high levels of Neurog3 expression is a critical step for endocrine commitment from multipotent pancreatic progenitors. These findings also suggest that a high level of Neurog3 expression could mediate lateral inhibition or other unknown feedback mechanisms to regulate the number of cells that initiate Neurog3 transcription and protein production. The control of Neurog3+ cell number and the Neurog3 threshold-dependent endocrine differentiation mechanism combine to select a specific proportion of pancreatic progenitor cells to adopt the islet cell fate.


Sub-epidermal moisture versus traditional and visual skin assessments to assess pressure ulcer risk in surgery patients.
To compare the predictive ability of subepidermal moisture (SEM) measurement versus traditional risk assessment and visual skin assessment (VSA) as means of detecting early pressure ulcer (PU) damage development among adults undergoing surgery. A non-experimental, comparative, descriptive cohort study design was used. Following ethical approval, participants who had given their informed written consent had their skin assessed over the areas that were weight-bearing during surgery, using VSA and the SEM measurement. Visual PUs were graded according to the European Pressure Ulcer Advisory Panel and National Pressure Ulcer Advisory Panel ulcer grading system. Assessments took place preoperatively, then daily on the ward, beginning on day one postoperatively and continuing for three days or until discharge. Of the 231 participants, who had a mean age of 57.50 years, 55.8% (n=129) were male. The most common comorbidity was cardiology/vascular (n=42; 18.2%). Just over half (52.4%; n=121) underwent orthopaedic surgery and 47.6% (n=110) underwent non-orthopaedic surgery; 70% (n=163) received a general anaesthetic and 43% (n=100) were in the supine decubitus position during surgery. PU incidence was 51% (n=116), according to SEM measurement, and 3% (n=7) according to VSA. Among the seven participants who developed a visual PU, 10 PUs at stage 1 developed (31%); some patients developed more than one PU. Of the participants who had assessments for three days postoperatively, 94% (n=61) had a persistently high SEM delta on day three. The variables that emerged as statistically significantly related to abnormal SEM measurement deltas among these participants were: surgery duration (p=0.038); having orthopaedic surgery (p=0.020); supine surgical position (p=0.003); spinal anaesthetic type (p=0.0001); and Waterlow and Braden mobility subscale day one postoperatively (p=0.0001). None of the variables had a statistically significant influence on abnormal VSA. Surgical patients, because of immobility, are vulnerable to the action of compression and shear forces. These forces cause changes at a cellular level that trigger inflammation, which is a precursor to early tissue damage. SEM measurement can detect this tissue damage from the increase in the underlying tissue water content that results from inflammation. From the findings of this study, SEM measurement is very promising in the detection of early tissue damage in those at risk of PU development among the surgical population.


Suicide screening in schools, primary care and emergency departments.
Every year, suicide claims the lives of tens of thousands of young people worldwide. Despite its high prevalence and known risk factors, suicidality is often undetected. Early identification of suicide risk may be an important method of mitigating this public health crisis. Screening youth for suicide may be a critical step in suicide prevention. This paper reviews suicide screening in three different settings: schools, primary care clinics and emergency departments (EDs). Unrecognized and thus untreated suicidality leads to substantial morbidity and mortality. With the onus of detection falling on nonmental health professionals, brief screening tools can be used to initiate more in-depth evaluations. Nonetheless, there are serious complexities and implications of screening all children and adolescents for suicide. Recent studies show that managing positive screens is a monumental challenge, including the problem of false positives and the burden subsequently posed on systems of care. Furthermore, nearly 60% of youth in need of mental health services do not receive the care they need, even after suicide attempt. Schools, primary care clinics and EDs are logical settings where screening that leads to intervention can be initiated. Valid, brief and easy-to-administer screening tools can be utilized to detect risk of suicide in children and adolescents. Targeted suicide screening in schools, and universal suicide screening in primary care clinics and EDs may be the most effective way to recognize and prevent self-harm. These settings must be equipped to manage youth who screen positive with effective and timely interventions. Most importantly, the impact of suicide screening in various settings needs to be further assessed.


In vitro inhibition of Giardia lamblia and Trichomonas vaginalis growth by bithionol, dichlorophene, and hexachlorophene.
Bithionol, dichlorophene, and hexachlorophene, which are used in treating some helminthic infections, killed trophozoites of Giardia lamblia and Trichomonas vaginalis in modified BI-S-33 and Asami media, respectively. Virtually all G. lamblia and T. vaginalis cells were killed within 24 h with a 0.42 mM concentration of these compounds, except that 0.93 mM dichlorophene was required for sterilizing T. vaginalis in the same period. In modified BI-S-33 and Asami media from which bovine and human sera were omitted, respectively, the inhibitory actions of the compounds against in vitro growth of these protozoa were significantly enhanced. Trophozoites of G. lamblia and T. vaginalis could be killed in shorter than 10 min with 0.074 mM dichlorophene and 0.0025 mM hexachlorophene, respectively, in serum-free media. G. lamblia, which was incubated in the complete medium containing dichlorophene, showed a characteristic swelling of the ventral side which led to disruption of the parasite, whereas bithionol caused a thin crack in the cytoplasm of T. vaginalis incubated in Asami medium. The crack appeared to enlarge and result in vacuolization of T. vaginalis. These observations suggest that bithionol, dichlorophene, and hexachlorophene merit further evaluation to ascertain whether they are useful for treatment of giardiasis and trichomoniasis.


Management of Osteoarthritis with Avocado/Soybean Unsaponifiables.
Osteoarthritis (OA) is a painful and life-altering disease that severely limits the daily activity of millions of Americans, and is one of the most common causes of disability in the world. With obesity on the rise and the world's population living longer, the prevalence of OA is expected to increase dramatically in the coming decades, generating burdensome socioeconomic costs. This review summarizes current pharmaceutical, non-pharmaceutical, and prospective new treatments for OA, with primary focus on the dietary supplement Avocado/Soybean Unsaponifiables (ASU). ASU modulates OA pathogenesis by inhibiting a number of molecules and pathways implicated in OA. Anticatabolic properties prevent cartilage degradation by inhibiting the release and activity of matrix metalloproteinases (MMP-2,3,13) and increasing tissue inhibitors of these catabolic enzymes (TIMP-1). ASU also inhibits fibrinolysis by stimulating the expression of plasminogen activator inhibitor (PAI-1). Anabolic properties promote cartilage repair by stimulating collagen and aggrecan synthesis via inhibition of inflammatory cytokines such as IL1, IL6, IL8, TNF, ERK, and PGE2. Chondroprotective effects are mediated by correcting growth factor abnormalities, increasing TGFβ while decreasing vascular endothelial growth factor (VEGF) in synovial fluid. ASU also inhibits cholesterol absorption and endogenous cholesterol biosynthesis, which mediate reactive oxygen species pathology in chondrocytes. At the clinical level, ASU reduces pain and stiffness while improving joint function, resulting in decreased dependence on analgesics.


Wolbachia modulates Chikungunya replication in Aedes albopictus.
The Aedes albopictus mosquito has been involved as the principal vector of recent major outbreaks due to the chikungunya virus (CHIKV). The species is naturally infected by two strains of Wolbachia (wAlbA and wAlbB). Wolbachia infections are thought to have spread by manipulating the reproduction of their hosts; cytoplasmic incompatibility is the mechanism used by Wolbachia to invade natural populations of many insects including Ae. albopictus. Here, we report a study on the effects of removing Wolbachia from Ae. albopictus on CHIKV replication and examine the consequences of CHIKV infection on some life-history traits (survival and reproduction) of Wolbachia-free Ae. albopictus. We found that Wolbachia-free mosquitoes maintained a highly heterogeneous CHIKV replication compared to Wolbachia-infected individuals. In Wolbachia-infected Ae. albopictus, the regular increase of CHIKV followed by a steady viral load from day 4 post-infection onwards was concomitant with a decline in Wolbachia density. This profile was also detected when examining the two key organs for viral transmission, the midgut and the salivary glands. Moreover, Wolbachia-free Ae. albopictus was not altered in life-history traits such as survival, oviposition and hatching characteristics whether infected or not with CHIKV. We found that Wolbachia is not essential for viral replication, its presence could lead to optimize replication from day 4 post-infection onwards, coinciding with a decrease in Wolbachia density. Wolbachia may regulate viral replication in Ae. albopictus, with consequences on survival and reproduction.