Acute Methylmercury Exposure and the Hypoxia-Inducible Factor-1α Signaling Pathway under Normoxic Conditions in the Rat Brain and Astrocytes in Vitro.
As a ubiquitous environmental pollutant, methylmercury (MeHg) induces toxic effects in the nervous system, one of its main targets. However, the exact mechanisms of its neurotoxicity have not been fully elucidated. Hypoxia-inducible factor-1α (HIF-1α), a transcription factor, plays a crucial role in adaptive and cytoprotective responses in cells and is involved in cell survival, proliferation, apoptosis, inflammation, angiogenesis, glucose metabolism, erythropoiesis, and other physiological activities. The aim of this study was to explore the role of HIF-1α in response to acute MeHg exposure in rat brain and primary cultured astrocytes to improve understanding of the mechanisms of MeHg-induced neurotoxicity and the development of effective neuroprotective strategies. Primary rat astrocytes were treated with MeHg (0-10μM) for 0.5h. Cell proliferation and cytotoxicity were assessed with a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl diphenyltetrazolium bromide (MTT) assay and a lactate dehydrogenase (LDH) release assay, respectively. Reactive oxygen species (ROS) levels were analyzed to assess the level of oxidative stress using 2',7'-dichlorofluorescin diacetate (DCFH-DA) fluorescence. HIF-1α, and its downstream proteins, glucose transporter 1 (GLUT-1), erythropoietin (EPO), and vascular endothelial growth factor A (VEGF-A) were analyzed by means of Western blotting. Real-time PCR was used to detect the expression of HIF-1α mRNA. Pretreatment with protein synthesis inhibitor (CHX), proteasome inhibitor (MG132), or proline hydroxylase inhibitor (DHB) were applied to explore the possible mechanisms of HIF-1α inhibition by MeHg. To investigate the role of HIF-1α in MeHg-induced neurotoxicity, cobalt chloride (CoCl2), 2-methoxyestradiol (2-MeOE2), small interfering RNA (siRNA) transfection and adenovirus overexpression were used. Pretreatment with N-acetyl-L-cysteine (NAC) and vitamin E (Trolox) were used to investigate the putative role of oxidative stress in MeHg-induced alterations in HIF-1α levels. The expression of HIF-1α and related downstream proteins was detected in adult rat brain exposed to MeHg (0-10mg/kg) for 0.5h in vivo. MeHg caused lower cell proliferation and higher cytotoxicity in primary rat astrocytes in a time- and concentration-dependent manner. In comparison with the control cells, exposure to 10μM MeHg for 0.5h significantly inhibited the expression of astrocytic HIF-1α, and the downstream genes GLUT-1, EPO, and VEGF-A (p<0.05), in the absence of a significant decrease in HIF-1α mRNA levels. When protein synthesis was inhibited by CHX, MeHg promoted the degradation rate of HIF-1α. MG132 and DHB significantly blocked the MeHg-induced decrease in HIF-1α expression (p<0.05). Overexpression of HIF-1α significantly attenuated the decline in MeHg-induced cell proliferation, whereas the inhibition of HIF-1α significantly increased the decline in cell proliferation (p<0.05). NAC and Trolox, two established antioxidants, reversed the MeHg-induced decline in HIF-1α protein levels and the decrease in cell proliferation (p<0.05). MeHg suppressed the expression of HIF-1α and related downstream target proteins in adult rat brain. MeHg induced a significant reduction in HIF-1α protein by activating proline hydroxylase (PHD) and the ubiquitin proteasome system (UPS) in primary rat astrocytes. Additionally, ROS scavenging by antioxidants played a neuroprotective role via increasing HIF-1α expression in response to MeHg toxicity. Moreover, we established that up-regulation of HIF-1α might serve to mitigate the acute toxicity of MeHg in astrocytes, affording a novel therapeutic target for future exploration. https://doi.org/10.1289/EHP5139.


Can We Agree on Expectations and Assessments of Graduating Residents?: 2016 AOA Critical Issues Symposium.
Orthopaedic educators are responsible for training a prepared and competent workforce that will provide effective care for a growing number of patients with musculoskeletal conditions. Currently, there are both internal and external forces that pose substantial challenges to medical students, residents, program directors, faculty members, and chairs in achieving this goal. One area of particular concern is the education of surgeons, whose knowledge and professional behavior must be matched by their ability to acquire procedural skills. In order to address this issue, many training systems have implemented a competency-based training approach into their curricula. This article discusses the efforts that orthopaedic training bodies in Canada and Australia have taken toward competency-based education and what steps the American Board of Orthopaedic Surgery (ABOS), the Council of Orthopaedic Residency Directors (CORD), the American Orthopaedic Association (AOA), the American Academy of Orthopaedic Surgeons (AAOS), and the Accreditation Council for Graduate Medical Education (ACGME) are considering to improve residency education in the current and future environments.


Biochemical reconstitution of hemorrhagic-fever arenavirus envelope glycoprotein-mediated membrane fusion.
The membrane-anchored proteins of enveloped viruses form labile spikes on the virion surface, primed to undergo large-scale conformational changes culminating in virus-cell membrane fusion and viral entry. The prefusion form of these envelope glycoproteins thus represents an important molecular target for antiviral intervention. A critical roadblock to this endeavor has been our inability to produce the prefusion envelope glycoprotein trimer for biochemical and structural analysis. Through our studies of the GPC envelope glycoprotein of the hemorrhagic fever arenaviruses, we have shown that GPC is unique among class I viral fusion proteins in that the mature complex retains a stable signal peptide (SSP) in addition to the conventional receptor-binding and transmembrane fusion subunits. In this report we show that the recombinant GPC precursor can be produced as a discrete native-like trimer and that its proteolytic cleavage generates the mature glycoprotein. Proteoliposomes containing the cleaved GPC mediate pH-dependent membrane fusion, a characteristic feature of arenavirus entry. This reaction is inhibited by arenavirus-specific monoclonal antibodies and small-molecule fusion inhibitors. The in vitro reconstitution of GPC-mediated membrane-fusion activity offers unprecedented opportunities for biochemical and structural studies of arenavirus entry and its inhibition. To our knowledge, this report is the first to demonstrate functional reconstitution of membrane fusion by a viral envelope glycoprotein.


Simplified PCR for detection of Haemophilus ducreyi and diagnosis of chancroid.
A simplified PCR was developed for detection of Haemophilus ducreyi in samples from chancroid patients. The strategy included a straightforward chloroform extraction sample preparation method, a one-tube nested PCR to minimize contamination risks, and a colorimetric method for detection of products. Primers were designed from published nucleotide sequences of the 16S rRNA gene of H. ducreyi, with longer outer primers for annealing at a higher temperature and shorter inner primers labelled with biotin and digoxigenin for binding with avidin and colorimetric detection. The PCR technique detected all 35 strains of H. ducreyi tested, from four different geographical regions, and was negative for other, related strains of bacteria and for the common contaminating bacteria tested. Of 25 samples from H. ducreyi culture-positive chancroid patients, 24 were PCR positive and 1 produced a weak reaction. Of 83 samples from clinical cases of chancroid in the Republic of South Africa, 69 were PCR positive. The sensitivity of PCR compared with that of clinical diagnosis was 83%. All 50 negative control samples were negative. Encouraging results were also obtained with a consecutive series of 25 genital ulcer patients in Tanzania, of whom 9 were PCR positive. The adaptations of this simplified PCR strategy, at the sensitivity and specificity levels obtained, mean it will be useful for detection of H. ducreyi in areas where the organism is endemic, particularly where testing by culture is difficult or impossible.


Iron removal in highly contaminated acid mine drainage using passive biochemical reactors.
Passive biochemical reactors (PBRs) are a viable alternative to neutralization plants for the treatment of acid mine drainage (AMD) because they require lower investment costs and use residual materials. However, high iron (Fe) concentrations (≥0.5 g/L) in AMD are challenging for their long-term efficiency. Sorption and precipitation are the main Fe removal mechanisms, but the relative importance of each is mostly unknown. In this study, locally available natural materials (organic and inorganic) were characterized and tested for their performance in Fe removal from highly contaminated AMD (pH 3.5, 4 g/L of Fe, and 9 g/L of sulfate). Iron retention capacity of the materials was then evaluated and the efficiency of eight mixtures of materials was compared through 40-day laboratory batch tests. All batch-type PBRs increased the pH up to 6.5 and decreased dissolved metals concentrations, including Fe, up to 99%. Results showed that organic residual materials (manures, municipal wastewater sludge, and compost) were the best substrates for Fe removal.These findings allowed for the selection of three reactive mixtures with distinct characteristics (mixture #1 - 30% organic wastes; mixture #4 - 50% calcite; and mixture #7 - 50% sand) to be further evaluated in column type PBRs.


Reasons for increasing administrative costs in hospitals.
To evaluate trends in administrative expenditures by examining changes in administrative costs compared with other areas of the hospital budget, changes in expenditures for patient care departments compared with nonpatient care departments, and departments with a greater number of regulatory requirements compared with those with fewer regulatory requirements. Annual hospital operating budgets submitted to Blue Cross were examined for an 8-year period from 1983 to 1990. Seventy hospitals in Western Pennsylvania. Annual operating expenditures. Total administrative expenditures showed a larger growth rate (90%) than service departments (29%), ancillary services (30%), professional care (52%), miscellaneous expenses (70%), and the total hospital budget (45%). Administrative costs increased from 10.6% as a proportion of the total hospital budget in 1983 to 13.9% in 1990. These increases were seen regardless of hospital size. Departments with a greater number of regulatory obligations had a greater increase in expenditures (84%) than did departments with fewer regulatory requirements (5%). However, overall expenditures in departments with direct patient care responsibilities did not increase appreciably faster (44%) than in departments not providing clinical services (46%), possibly reflecting the fact that administrative costs may be increasing equally in both areas. Administrative costs were found to be one of the fastest growing components of hospital budgets. Future research should determine the effect of these increases on the quality of patient care.


CTGF antagonism with mAb FG-3019 enhances chemotherapy response without increasing drug delivery in murine ductal pancreas cancer.
Pancreatic ductal adenocarcinoma (PDA) is characterized by abundant desmoplasia and poor tissue perfusion. These features are proposed to limit the access of therapies to neoplastic cells and blunt treatment efficacy. Indeed, several agents that target the PDA tumor microenvironment promote concomitant chemotherapy delivery and increased antineoplastic response in murine models of PDA. Prior studies could not determine whether chemotherapy delivery or microenvironment modulation per se were the dominant features in treatment response, and such information could guide the optimal translation of these preclinical findings to patients. To distinguish between these possibilities, we used a chemical inhibitor of cytidine deaminase to stabilize and thereby artificially elevate gemcitabine levels in murine PDA tumors without disrupting the tumor microenvironment. Additionally, we used the FG-3019 monoclonal antibody (mAb) that is directed against the pleiotropic matricellular signaling protein connective tissue growth factor (CTGF/CCN2). Inhibition of cytidine deaminase raised the levels of activated gemcitabine within PDA tumors without stimulating neoplastic cell killing or decreasing the growth of tumors, whereas FG-3019 increased PDA cell killing and led to a dramatic tumor response without altering gemcitabine delivery. The response to FG-3019 correlated with the decreased expression of a previously described promoter of PDA chemotherapy resistance, the X-linked inhibitor of apoptosis protein. Therefore, alterations in survival cues following targeting of tumor microenvironmental factors may play an important role in treatment responses in animal models, and by extension in PDA patients.


Exercise Dose and Weight Loss in Adolescents with Overweight-Obesity: A Meta-Regression.
A recent meta-analysis reported that exercise interventions are effective for promoting weight loss in adolescents with overweight-obesity. However, the meta-analysis did not investigate whether there is an optimal exercise dose for promoting weight loss in overweight and obese adolescents. A common method of expressing exercise dose is the calculation of metabolic equivalents (METs), expressed as MET-h/week. The objective of this study was to determine the association between exercise dose (MET-h/week) and weight loss [body weight, body mass index (BMI)] in adolescents with overweight-obesity. Trials included in the original meta-analysis were extracted, and a subsequent search to identify studies published between May 2015 and May 2018 was conducted. The search included electronic databases (PubMed, Web of Science SPORTDiscus, Google Scholar) and the reference lists of eligible articles and relevant reviews. The inclusion criteria were as follows: (i) randomized controlled trial; (ii) structured exercise intervention, alone or combined with other intervention components; (iii) control group received no structured exercise or behavioral modification designed to increase physical activity; (iv) participants overweight or obese (BMI ≥ 85th percentile); and (v) participants aged between 10 and 19 years. Fifteen trials were extracted from the original meta-analysis. The current search identified an additional five trials (three articles). Data from 20 trials (16 articles) involving 1091 participants (54% female, 17% not reported) were included in the analysis. Effect sizes were reported as mean difference, and random effects meta-regression quantified the association between exercise dose and weight loss. Study quality was assessed using a modified Jadad's scale. Total body weight change (decrease) ranged from - 2.7 to 19.3 (median 2.5) kg, and BMI change (decrease) ranged from - 1.6 to 6.3 (median 0.9) kg/m2. MET-h/week ranged from 5.4 to 36.0 (median 6.0). Each MET-h/week was associated with a 0.13 kg/m2 (95% confidence interval [CI] 0.08-0.19) and 0.33 kg (95% CI 0.08-0.59) decrease in BMI and body weight, respectively. The prescribed exercise dose for the majority of trials was low. As such, we were unable to discern whether there was an optimal exercise dose for weight loss (i.e., if the association between dose and weight loss was non-linear). Additionally, most trials had small sample sizes (median n = 34) and 17 trials had methodological limitations. Each MET-h/week was associated with a 0.13 kg/m2 and 0.33 kg decrease in BMI and body weight, respectively. While this relationship appears to be linear, i.e., no optimal exercise dose, it should be emphasized that the exercise prescription dose for the majority of trials was low. Subsequent trials, with greater exercise dosage, are required to determine whether there is an 'optimal' dose for promoting weight loss in adolescents with overweight-obesity. However, the current findings lend support to the use of exercise prescription for promoting weight loss and improving health outcomes in this population.


Stability of nucleosomes containing homogenously ubiquitylated H2A and H2B prepared using semisynthesis.
Post-translational modifications (PTMs) of histones are an essential feature in the dynamic regulation of chromatin. One of these modifications, ubiquitylation, has been speculated to directly influence the stability of the nucleosome, which represents the basic building block of chromatin. Here we report a strategy for the semisynthesis of site-specifically ubiquitylated histone H2A (uH2A). This branched protein was generated through a three-piece expressed protein ligation approach including a traceless ligation at valine. uH2A could be efficiently incorporated into nucleosomes, thereby opening the way to detailed biochemical and biophysical studies on the function of this PTM. Accordingly, we used uH2A, as well as a previously generated ubiquitylated H2B, in chaperone-coupled nucleosome stability assays to demonstrate that the direct effect of ubiquitylated histones on nucleosomal stability is in fact modest.


Mechanisms of nuclear pore complex disassembly by the mitotic Polo-like kinase 1 (PLK-1) in C. elegans embryos.
The nuclear envelope, which protects and organizes the genome, is dismantled during mitosis. In the Caenorhabditis elegans zygote, nuclear envelope breakdown (NEBD) of the parental pronuclei is spatially and temporally regulated during mitosis to promote the unification of the maternal and paternal genomes. Nuclear pore complex (NPC) disassembly is a decisive step of NEBD, essential for nuclear permeabilization. By combining live imaging, biochemistry, and phosphoproteomics, we show that NPC disassembly is a stepwise process that involves Polo-like kinase 1 (PLK-1)-dependent and -independent steps. PLK-1 targets multiple NPC subcomplexes, including the cytoplasmic filaments, central channel, and inner ring. PLK-1 is recruited to and phosphorylates intrinsically disordered regions (IDRs) of several multivalent linker nucleoporins. Notably, although the phosphosites are not conserved between human and C. elegans nucleoporins, they are located in IDRs in both species. Our results suggest that targeting IDRs of multivalent linker nucleoporins is an evolutionarily conserved driver of NPC disassembly during mitosis.


Involvement of the IL-2 receptor gamma-chain (gammac) in the control by IL-4 of human monocyte and macrophage proinflammatory mediator production.
IL-4 has potent anti-inflammatory properties on monocytes and suppresses both IL-1beta and TNF-alpha production. Well-characterized components of the IL-4 receptor on monocytes include the 140-kDa alpha-chain and the IL-2R gamma-chain, gammac, which normally dimerize 1:1 for signaling from the receptor. However, mRNA levels for gammac were very low in 7-day-cultured monocytes. As mRNA levels for gammac declined with culture, so too did the ability of IL-4 to down-regulate LPS-induced TNF-alpha production. In contrast, IL-4 consistently down-regulated IL-1beta production by cultured monocytes. Immunoprecipitation and Western blot analyses demonstrated that 7-day-cultured monocytes do not express the functionally active 64-kDa gammac protein. This was associated with decreased STAT6 activation by IL-4. Studies with Abs to gammac and an IL-4 mutant that is unable to bind to gammac showed that IL-4 can suppress IL-1beta but not TNF-alpha production by LPS-stimulated monocytes in the presence of little or no functioning gammac. IL-4 also suppressed IL-1beta but not TNF-alpha production by Mono Mac 6 cells, which express minimal levels of gammac. For gammac-expressing LPS/PMA-activated U937 cells, IL-4 decreased both TNF-alpha and IL-1beta production. These results suggest that functional gammac is not present on in vitro-derived macrophages, and that while some anti-inflammatory responses to IL-4 are lost with this down-regulation of functional gammac, others are retained. We conclude that different functional responses to IL-4 by human monocytes and macrophages are regulated by different IL-4 receptor configurations.


Traction injury of the recurrent laryngeal nerve: Results of continuous intraoperative neuromonitoring in a swine model.
Recurrent laryngeal nerve (RLN) palsy is the most serious complication after thyroidectomy. However, little is known about the degree of traction injury that causes loss of signal. The purpose of this study was to evaluate traction injuries in the swine RLN using continuous intraoperative neuromonitoring (IONM) and determine the traction power that results in loss of signal. Thirteen swine underwent traction injury to the RLNs with continuous IONM, and stress-strain curves were determined for 8 nerves using the universal material testing machine in an ex vivo model. Traction injury at a mean power of 2.83 MPa caused loss of signal. The mean physiologic limit strain and tensile strength of the swine RLNs were found to be 15.0% and 4.9 MPa, respectively. Histological analysis showed no abnormal structural findings. Traction injury of swine RLNs causes loss of signal at a power of 2.83 MPa. However, all injured nerves recovered within 7 days with no observed structural damage.


Aggregatibacter actinomycetemcomitans leukotoxin cytotoxicity occurs through bilayer destabilization.
The Gram-negative bacterium, Aggregatibacter actinomycetemcomitans, is a common inhabitant of the human upper aerodigestive tract. The organism produces an RTX (Repeats in ToXin) toxin (LtxA) that kills human white blood cells. LtxA is believed to be a membrane-damaging toxin, but details of the cell surface interaction for this and several other RTX toxins have yet to be elucidated. Initial morphological studies suggested that LtxA was bending the target cell membrane. Because the ability of a membrane to bend is a function of its lipid composition, we assessed the proficiency of LtxA to release of a fluorescent dye from a panel of liposomes composed of various lipids. Liposomes composed of lipids that form nonlamellar phases were susceptible to LtxA-induced damage while liposomes composed of lipids that do not form non-bilayer structures were not. Differential scanning calorimetry demonstrated that the toxin decreased the temperature at which the lipid transitions from a bilayer to a nonlamellar phase, while (31) P nuclear magnetic resonance studies showed that the LtxA-induced transition from a bilayer to an inverted hexagonal phase occurs through the formation of an isotropic intermediate phase. These results indicate that LtxA cytotoxicity occurs through a process of membrane destabilization.


Intrastromal delivery of bevacizumab using microneedles to treat corneal neovascularization.
This study tested the hypothesis that highly targeted intrastromal delivery of bevacizumab using coated microneedles allows dramatic dose sparing compared with subconjunctival and topical delivery for treatment of corneal neovascularization. Stainless steel microneedles 400 μm in length were coated with bevacizumab. A silk suture was placed in the cornea approximately 1 mm from the limbus to induce corneal neovascularization in the eyes of New Zealand white rabbits that were divided into different groups: untreated, microneedle delivery, topical eye drop, and subconjunctival injection of bevacizumab. All drug treatments were initiated 4 days after suture placement and area of neovascularization was measured daily by digital photography for 18 days. Eyes treated once with 4.4 μg bevacizumab using microneedles reduced neovascularization compared with untreated eyes by 44% (day 18). Eyes treated once with 2500 μg bevacizumab using subconjunctival injection gave similar results to microneedle-treated eyes. Eyes treated once with 4.4 μg subconjunctival bevacizumab showed no significant effect compared with untreated eyes. Eyes treated with 52,500 μg bevacizumab by eye drops three times per day for 14 days reduced the neovascularization area compared with untreated eyes by 6% (day 18), which was significantly less effective than the single microneedle treatment. Visual exam and histological analysis showed no observable effect of microneedle treatment on corneal transparency or microanatomical structure. This study shows that microneedles can target drug delivery to corneal stroma in a minimally invasive way and demonstrates effective suppression of corneal neovascularization after suture-induced injury using a much lower dose compared with conventional methods.


Enhancing Transgene Expression from Recombinant AAV8 Vectors in Different Tissues Using Woodchuck Hepatitis Virus Post-Transcriptional Regulatory Element.
Adeno-associated virus (AAV) vectors have been utilized extensively in gene therapy and gene function studies, as strong transgene expression is a prerequisite for positive outcomes. AAV8 was reported as the most efficient AAV serotype for transduction of the liver, brain and muscle compared with other serotypes. However, AAV8-mediated transduction of human hepatocytes is rather poor with approximately 20-fold lower efficiency compared with that of mouse hepatocytes. Therefore, we applied the woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) to enhance AAV8-mediated transgene expression driven by a combination promoter (CAG promoter) with a CMV-IE enhancer and chicken beta-actin promoter for a more efficient viral vector. Transgene expression from recombinant AAV8 (rAAV8) vectors harboring a red fluorescent protein (RFP) reporter gene with or without WPRE were evaluated in vitro and in vivo. The results demonstrated that WPRE improved AAV8-mediated RFP expression in different cell lines with clear increases of transgene expression in the liver, brain or muscle of animals. The findings of this study will help to substantially reduce the quantity of viral particles that must be injected in order to reach a therapeutic level of transgene expression in gene therapy. Consequently, such dose reductions may lessen the potential risks associated with high doses of viral vectors.


Involvement of synaptonemal complex proteins in sex chromosome segregation during marsupial male meiosis.
Marsupial sex chromosomes break the rule that recombination during first meiotic prophase is necessary to ensure reductional segregation during first meiotic division. It is widely accepted that in marsupials X and Y chromosomes do not share homologous regions, and during male first meiotic prophase the synaptonemal complex is absent between them. Although these sex chromosomes do not recombine, they segregate reductionally in anaphase I. We have investigated the nature of sex chromosome association in spermatocytes of the marsupial Thylamys elegans, in order to discern the mechanisms involved in ensuring their proper segregation. We focused on the localization of the axial/lateral element protein SCP3 and the cohesin subunit STAG3. Our results show that X and Y chromosomes never appear as univalents in metaphase I, but they remain associated until they orientate and segregate to opposite poles. However, they must not be tied by a chiasma since their separation precedes the release of the sister chromatid cohesion. Instead, we show they are associated by the dense plate, a SCP3-rich structure that is organized during the first meiotic prophase and that is still present at metaphase I. Surprisingly, the dense plate incorporates SCP1, the main protein of the central element of the synaptonemal complex, from diplotene until telophase I. Once sex chromosomes are under spindle tension, they move to opposite poles losing contact with the dense plate and undergoing early segregation. Thus, the segregation of the achiasmatic T. elegans sex chromosomes seems to be ensured by the presence in metaphase I of a synaptonemal complex-derived structure. This feature, unique among vertebrates, indicates that synaptonemal complex elements may play a role in chromosome segregation.


Cannabidiol changes P-gp and BCRP expression in trophoblast cell lines.
Objectives. Marijuana is the most commonly used illicit drug during pregnancy. Due to high lipophilicity, cannabinoids can easily penetrate physiological barriers like the human placenta and jeopardize the developing fetus. We evaluated the impact of cannabidiol (CBD), a major non-psychoactive cannabinoid, on P-glycoprotein (P-gp) and Breast Cancer Resistance Protein (BCRP) expression, and P-gp function in a placental model, BeWo and Jar choriocarcinoma cell lines (using P-gp induced MCF7 cells (MCF7/P-gp) for comparison). Study design. Following the establishment of the basal expression of these transporters in the membrane fraction of all three cell lines, P-gp and BCRP protein and mRNA levels were determined following chronic (24-72 h) exposure to CBD, by Western Blot and qPCR. CBD impact on P-gp efflux function was examined by uptake of specific P-gp fluorescent substrates (calcein-AM, DiOC2(3) and rhodamine123(rh123)). Cyclosporine A (CsA) served as a positive control. Results. Chronic exposure to CBD resulted in significant changes in the protein and mRNA levels of both transporters. While P-gp was down-regulated, BCRP levels were up-regulated in the choriocarcinoma cell lines. CBD had a remarkably different influence on P-gp and BCRP expression in MCF7/P-gp cells, demonstrating that these are cell type specific effects. P-gp dependent efflux (of calcein, DiOC2(3) and rh123) was inhibited upon short-term exposure to CBD. Conclusions. Our study shows that CBD might alter P-gp and BCRP expression in the human placenta, and inhibit P-gp efflux function. We conclude that marijuana use during pregnancy may reduce placental protective functions and change its morphological and physiological characteristics.


The global hidden hunger indices and maps: an advocacy tool for action.
The unified global efforts to mitigate the high burden of vitamin and mineral deficiency, known as hidden hunger, in populations around the world are crucial to the achievement of most of the Millennium Development Goals (MDGs). We developed indices and maps of global hidden hunger to help prioritize program assistance, and to serve as an evidence-based global advocacy tool. Two types of hidden hunger indices and maps were created based on i) national prevalence data on stunting, anemia due to iron deficiency, and low serum retinol levels among preschool-aged children in 149 countries; and ii) estimates of Disability Adjusted Life Years (DALYs) attributed to micronutrient deficiencies in 136 countries. A number of countries in sub-Saharan Africa, as well as India and Afghanistan, had an alarmingly high level of hidden hunger, with stunting, iron deficiency anemia, and vitamin A deficiency all being highly prevalent. The total DALY rates per 100,000 population, attributed to micronutrient deficiencies, were generally the highest in sub-Saharan African countries. In 36 countries, home to 90% of the world's stunted children, deficiencies of micronutrients were responsible for 1.5-12% of the total DALYs. The pattern and magnitude of iodine deficiency did not conform to that of other micronutrients. The greatest proportions of children with iodine deficiency were in the Eastern Mediterranean (46.6%), European (44.2%), and African (40.4%) regions. The current indices and maps provide crucial data to optimize the prioritization of program assistance addressing global multiple micronutrient deficiencies. Moreover, the indices and maps serve as a useful advocacy tool in the call for increased commitments to scale up effective nutrition interventions.


Functional analysis of eve stripe 2 enhancer evolution in Drosophila: rules governing conservation and change.
Experimental investigations of eukaryotic enhancers suggest that multiple binding sites and trans-acting regulatory factors are often required for wild-type enhancer function. Genetic analysis of the stripe 2 enhancer of even-skipped (eve), an important developmental gene in Drosophila, provides support for this view. Given the importance of even-skipped expression in early Drosophila development, it might be predicted that many structural features of the stripe 2 enhancer will be evolutionarily conserved, including the DNA sequences of protein binding sites and the spacing between them. To test this hypothesis, we compared sequences of the stripe 2 enhancer between four species of Drosophila: D. melanogaster, D. yakuba, D. erecta and D. pseudoobscura. Our analysis revealed a large number of nucleotide substitutions in regulatory protein binding sites for bicoid, hunchback, Kruppel and giant, as well as a systematic change in the size of the enhancer. Some of the binding sites in D. melanogaster are either absent or modified in other species. One functionally important bicoid-binding site in D. melanogaster appears to be recently evolved. We, therefore, investigated possible functional consequences of sequence differences among these stripe 2 enhancers by P-element-mediated transformation. This analysis revealed that the eve stripe 2 enhancer from each of the four species drove reporter gene expression at the identical time and location in D. melanogaster embryos. Double staining of native eve protein and transgene mRNA in early embryos showed that the reporter gene mimicked native eve expression and, in every case, produced sharply defined stripes at the blastoderm stage that were coincident with eve stripe 2 protein. We argue that stripe 2 eve expression in Drosophila evolution can be viewed as being under constant stabilizing selection with respect to the location of the anterior and posterior borders of the stripe. We further hypothesize that the stripe 2 enhancer is functionally robust, so that its evolution may be governed by the fixation of both slightly deleterious and adaptive mutations in regulatory protein binding sites as well as in the spacing between binding sites. This view allows for a slow but continual turnover of functionally important changes in the stripe 2 enhancer.


Low risk of ischemic stroke in patients with reduced internal carotid artery lumen diameter distal to severe symptomatic carotid stenosis: cerebral protection due to low poststenotic flow? On behalf of the European Carotid Surgery Trialists' Collaborative Group.
Patients with recently symptomatic severe carotid stenosis have a high risk of ischemic stroke on medical treatment. The main mechanism of stroke appears to be plaque surface thrombus formation and distal embolism. It is unclear to what extent reduction in blood flow across the stenosis, and the consequent reduction in cerebral perfusion pressure, is also important. Angiographic indices of reduced cerebral perfusion may identify patients at a particularly high risk of stroke who require urgent endarterectomy. The most direct angiographic correlate of poststenotic perfusion pressure is the degree of narrowing of the distal internal carotid artery (ICA) lumen. We sought to develop criteria for the definition of poststenotic narrowing of the ICA and to determine the effect of this and other angiographic characteristics likely to be associated with reduced cerebral perfusion on the risk of ipsilateral ischemic stroke in patients with recently symptomatic carotid stenosis. We studied the carotid angiograms of 3007 patients in the European Carotid Surgery Trial. Poststenotic narrowing of the ICA was defined with use of the ratio of the lumen diameter of the ICA to that of the common carotid artery (CCA). The normal range of the ICA/CCA ratio was defined in 2966 symptomatic or contralateral carotid arteries with 0% to 49% stenosis. Arteries with 70% to 99% symptomatic stenosis and an ICA/CCA ratio below this range were categorized as narrowed. We related the presence of narrowing and other angiographic characteristics to the risk of ipsilateral ischemic stroke on medical treatment. An assessment of the ICA/CCA ratio had good interobserver reproducibility. Poststenotic narrowing of the ICA was defined as an ICA/CCA ratio of <0.42. The 5-year risk of ipsilateral carotid territory ischemic stroke on medical treatment was 8% in patients with 70% to 99% stenosis and narrowing of the ICA versus 25% in patients without narrowing (log rank test, P=0.02). This difference remained after correction for other clinical and angiographic variables (hazard ratio 0.40, 95% CI 0.17 to 0.94, P=0. 03). The other angiographic characteristics did not predict stroke. Poststenotic narrowing of the ICA was associated with a low risk of stroke on medical treatment. This suggests that low flow alone is not usually sufficient to cause ischemic stroke distal to symptomatic carotid stenosis. Poststenotic narrowing may be protective because blood flow distal to the stenosis is insufficient to carry emboli to the brain.


Analyzing Factors Associated with Fatal Road Crashes: A Machine Learning Approach.
Road traffic injury accounts for a substantial human and economic burden globally. Understanding risk factors contributing to fatal injuries is of paramount importance. In this study, we proposed a model that adopts a hybrid ensemble machine learning classifier structured from sequential minimal optimization and decision trees to identify risk factors contributing to fatal road injuries. The model was constructed, trained, tested, and validated using the Lebanese Road Accidents Platform (LRAP) database of 8482 road crash incidents, with fatality occurrence as the outcome variable. A sensitivity analysis was conducted to examine the influence of multiple factors on fatality occurrence. Seven out of the nine selected independent variables were significantly associated with fatality occurrence, namely, crash type, injury severity, spatial cluster-ID, and crash time (hour). Evidence gained from the model data analysis will be adopted by policymakers and key stakeholders to gain insights into major contributing factors associated with fatal road crashes and to translate knowledge into safety programs and enhanced road policies.


Endemic filariasis on a Pacific island. I. Clinical, epidemiologic, and parasitologic aspects.
The clinical and parasitologic aspects of filariasis were investigated in 459 inhabitants of a South Pacific island endemic for subperiodic Wuchereria bancrofti filariasis. Episodes of filarial fevers, usually with concomitant lymphangitis and/or lymphadenitis, were experienced by 26%. Lymphobstructive lesions, manifest in the later decades of life, were present in 12 with elephantiasis and 24 with hydroceles. Microfilaremia was detectable by membrane filtration of 1 ml of blood in 33% of persons, with a quarter of these having less than 50 microfilariae/ml. Microfilaremia and filarial fevers were more frequent in males than females and in residents of the most inland villages. Neither presence nor magnitude of lymphadenopathy correlated with other manifestations of filariasis except that markedly enlarged inguinal-femoral nodes were more common in those with elephantiasis or hydroceles. A history of filarial fevers did not correlate with presence of microfilaremia. Over 70% of adults had microfilaremia and/or historical or physical manifestations of filariasis. The absence of detectable microfilaremia in many residents with clinical evidence of filariasis and the presence of asymptomatic microfilaremia emphasize the limitations in relying only on microfilaremia rates or clinical findings in the study of the prevalence or manifestations of filarial infection.


Rheumatoid arthritis synovium contains plasmacytoid dendritic cells.
We have previously described enrichment of antigen-presenting HLA-DR+ nuclear RelB+ dendritic cells (DCs) in rheumatoid arthritis (RA) synovium. CD123+HLA-DR+ plasmacytoid DCs (pDCs) and their precursors have been identified in human peripheral blood (PB), lymphoid tissue, and some inflamed tissues. We hypothesized recruitment of pDCs into the inflamed RA synovial environment and their contribution as antigen-presenting cells (APCs) and inflammatory cells in RA. CD11c+ myeloid DCs and CD123+ pDCs were compared in normal and RA PB, synovial fluid (SF), and synovial tissue by flow cytometry, immunohistochemistry, and electron microscopy and were sorted for functional studies. Nuclear RelB-CD123+ DCs were located in perivascular regions of RA, in a similar frequency to nuclear RelB+CD123- DCs, but not normal synovial tissue sublining. Apart from higher expression of HLA-DR, the numbers and phenotypes of SF pDCs were similar to those of normal PB pDCs. While the APC function of PB pDCs was less efficient than that of PB myeloid DCs, RA SF pDCs efficiently activated resting allogeneic PB T cells, and high levels of IFN-gamma, IL-10, and tumor necrosis factor alpha were produced in response to incubation of allogeneic T cells with either type of SF DCs. Thus, pDCs are recruited to RA synovial tissue and comprise an APC population distinct from the previously described nuclear RelB+ synovial DCs. pDCs may contribute significantly to the local inflammatory environment.


Near-real-time analysis of the phenotypic responses of Escherichia coli to 1-butanol exposure using Raman Spectroscopy.
Raman spectroscopy was used to study the time course of phenotypic responses of Escherichia coli (DH5α) to 1-butanol exposure (1.2% [vol/vol]). Raman spectroscopy is of interest for bacterial phenotyping because it can be performed (i) in near real time, (ii) with minimal sample preparation (label-free), and (iii) with minimal spectral interference from water. Traditional off-line analytical methodologies were applied to both 1-butanol-treated and control cells to draw correlations with Raman data. Here, distinct sets of Raman bands are presented that characterize phenotypic traits of E. coli with maximized correlation to off-line measurements. In addition, the observed time course phenotypic responses of E. coli to 1.2% (vol/vol) 1-butanol exposure included the following: (i) decreased saturated fatty acids levels, (ii) retention of unsaturated fatty acids and low levels of cyclopropane fatty acids, (iii) increased membrane fluidity following the initial response of increased rigidity, and (iv) no changes in total protein content or protein-derived amino acid composition. For most phenotypic traits, correlation coefficients between Raman spectroscopy and traditional off-line analytical approaches exceeded 0.75, and major trends were captured. The results suggest that near-real-time Raman spectroscopy is suitable for approximating metabolic and physiological phenotyping of bacterial cells subjected to toxic environmental conditions.


Biomarkers for Predicting Malignant Pleural Mesothelioma in a Mexican Population.
Background: Diagnosis of malignant pleural mesothelioma (MPM) remains a challenge, especially when resources in pathology are limited. The study aimed to evaluate cost-effective tumor markers to predict the probability of MPM in plasma samples in order to accelerate the diagnostic workup of the tissue of potential cases. Methods: We conducted a case-control study stratified by gender, which included 75 incident cases with MPM from three Mexican hospitals and 240 controls frequency-matched by age and year of blood drawing. Plasma samples were obtained to determine mesothelin, calretinin, and thrombomodulin using enzyme-linked immunosorbent assays (ELISAs). We estimated the performance of the markers based on the area under the curve (AUC) and predicted the probability of an MPM diagnosis of a potential case based on the marker concentrations. Results: Mesothelin and calretinin, but not thrombomodulin were significant predictors of a diagnosis of MPM with AUCs of 0.90 (95% CI: 0.85-0.95), 0.88 (95% CI: 0.82-0.94), and 0.51 (95% CI: 0.41-0.61) in males, respectively. For MPM diagnosis in men we estimated a true positive rate of 0.79 and a false positive rate of 0.11 for mesothelin. The corresponding figures for calretinin were 0.81 and 0.18, and for both markers combined 0.84 and 0.11, respectively. Conclusions: We developed prediction models based on plasma concentrations of mesothelin and calretinin to estimate the probability of an MPM diagnosis. Both markers showed a good performance and could be used to accelerate the diagnostic workup of tissue samples in Mexico.


Enhancing cattle handling safety with the work crew performance model.
Kentucky farmers and University of Kentucky extension agents applied the principles of the Work Crew Performance Model (WCPM), used previously in mining and construction, to identify and prioritize critical action factors (CAFs) for safe handling of cattle. One focus group of farmers helped identify a set of 32 critical action factors in four categories: environmental conditions, animal behavior, facilities and equipment, and handling techniques. A second focus group of farmers used a Q-sort nominal group process to rate each of the factors on the basis of the cost consequences of failure to properly perform the activity. A three-stage binary sort process resulted in a five-point ordinal ranking of tasks within each of the four categories. The Q-sort procedure was performed with assumptions of both routine and emergency animal handling activities. The list and the rankings were validated and modified by extension agriculture agents and by an expert panel of animal scientists and an agricultural engineering educator. The validation process added seven CAFs to the original list. The result of the process was the development of a cattle safety handling checklist that has been used with success in Master Cattleman educational workshops conducted for approximately 1500 Kentucky farmers.


Guillain-barré syndrome: a clinical study of twenty children.
Guillain-Barre Syndrome (GBS) is an acute monophasic demyelinating neuropathy characterized by progressive motor weakness of limbs with areflexia. To study the clinical pattern and outcome of children with Guillain-Barre syndrome. It was a cross-sectional study conducted in a pediatric unit of tertiary care hospital over a period of 18 months. We assessed the clinical manifestations, results of electro-diagnostic tests, functional status, treatment instituted and outcome of 20 children diagnosed with GBS. Of the 20 (male to female ratio = 2.3:1) children studied, all had motor weakness, 5 (25%) had sensory loss, 4 (20 %) had cranial nerve palsies and 4 (20%) had autonomic disturbances. Respiratory paralysis was found in 7 (35%) children requiring assisted ventilation. Antecedent illness preceding GBS was recorded in 50% children. The GBS subtype distribution as per electrodiagnostic studies was as follows: acute motor axonal neuropathy (AMAN) in 7 (38.9%), acute motor sensory axonal neuropathy (AMSAN) in 4 (22.2%), acute inflammatory demyelinating polyradiculoneuropathy (AIDP) in 4 (22.2%) and both axonal and demyelinating neuropathy in 3 (16.7%). Intravenous immunoglobulins (IVIG) constituted the treatment given in majority of the patients. Plasmapharesis was performed in one child in view of poor response to IVIG. Complete recovery was observed in 14 children and the remaining 3 children experienced only incomplete recovery. Male preponderance and presence of antecedent illness in a majority of subjects was observed in our study. Regardless of the severity of illness at admission and electrophysiological subtypes, a majority achieved full recovery. Intravenous Immunoglobulin and supportive care form the cornerstone of management in childhood GBS.


Hemodynamic studies in acute-on-chronic liver failure.
Patients with decompensated cirrhosis and acute liver failure have circulatory dysfunctions leading to high portal pressure and cardiac output (CO) and low systemic vascular resistance (SVR). Circulatory changes in acute-on-chronic liver failure (ACLF) patients have not been studied. We studied the portal, systemic, and pulmonary hemodynamics in patients with ACLF and compared them with compensated and decompensated cirrhotics. Clinical features and hemodynamic profile were studied in patients with ACLF and compared with age- and sex-matched compensated and decompensated cirrhotics with portal hypertension. The study cohort comprised 144 patients categorized into one of three groups (ACLF, compensated cirrhosis, and decompensated cirrhosis), with 48 (33%) patients in each group. All values are given as the mean +/- standard deviation, except for frequencies (%). The mean arterial pressure (MAP) and SVR were lower in the ACLF than the compensated group and were similar to those of the decompensated group (MAP 90 +/- 16 vs. 99 +/- 15 vs. 96 +/- 16 mmHg; SVR 912 +/- 435 vs. 1350 +/- 449 vs. 891 +/- 333 dyn s/cm(5)). The mean CO of the ACLF patients was higher than that of the compensated group and similar to that of the decompensated group (CO 8.9 +/- 3.5 vs. 6.1 +/- 1.7 vs. 9.0 +/- 3.0 l/min). The pulmonary vascular resistance (PVR) and pulmonary capillary wedge pressures (PCWP) were similar in all the three groups (PVR 78 +/- 48 vs. 109 +/- 70 vs. 61 +/- 47 dyn s/cm(5); PCWP 8 +/- 4 vs. 8 +/- 4 vs. 10 +/- 5 mmHg). The mean hepatic venous pressure gradient (HVPG) in the ACLF group was 15.1 +/- 6.3 mmHg, which was significantly higher than that of the compensated group (11.7 +/- 6.3 mmHg), but lower than that of the decompensated cirrhosis group (20.2 +/- 6.0 mmHg). When patients of ACLF were categorized on the basis of their variceal size, the mean HVPG in ACLF patients with small varices was similar to that of compensated cirrhotics (13.7 +/- 5.7 vs. 11.7 +/- 6.3 mmHg; P = 0.146), while in the ACLF patients with large varices, the HVPG was comparable to that of the decompensated cirrhotics (18.7 +/- 6.6 vs. 20.2 +/- 6.0 mmHg; P = 0.442). The systemic hemodynamics in patients with ACLF is similar to that in decompensated cirrhotics. The portal pressure in these patients is higher than that in the compensated cirrhotics, and in the subgroup with large varices, it becomes similar to that of decompensated cirrhotics.


Development of a neutralizing antibody response during acute primary human immunodeficiency virus type 1 infection and the emergence of antigenic variants.
We monitored the primary humoral response to human immunodeficiency virus type 1 infection and showed that, in addition to antibodies to p24 and gp41, antigens which form the basis of most diagnostic assays, the response included a significant antibody response directed to the gp120 region of the infecting viral quasispecies. When tested in a recombinant virus neutralization assay, these antibodies were capable of inhibiting viral growth. We found the primary viral quasispecies to solely utilize the CCR-5 chemokine receptor; however, recombinant viruses differed in their cytopathology and in their sensitivity to beta-chemokine inhibition of viral growth. Sequence analysis of the gp120 open reading frames showed that amino acid changes in the C1 (D-->G at position 62) and C4 (V-->A at position 430) regions accounted for the phenotypic differences. These data demonstrate that early in infection, polymorphism exists in envelope glycoprotein coreceptor interactions and imply that therapeutic strategies targeted at this step in the viral life cycle may lead to rapid resistance.


Feasibility of 3T dynamic contrast-enhanced magnetic resonance-guided biopsy in localizing local recurrence of prostate cancer after external beam radiation therapy.
The objective of this study was to assess the feasibility of the combination of magnetic resonance (MR)-guided biopsy (MRGB) and diagnostic 3T MR imaging in the localization of local recurrence of prostate cancer (PCa) after external beam radiation therapy (EBRT). Twenty-four consecutive men with biochemical failure suspected of local recurrence after initial EBRT were enrolled prospectively in this study. All patients underwent a diagnostic 3T MR examination of the prostate. T2-weighted and dynamic contrast-enhanced MR images (DCE-MRI) were acquired. Two radiologists evaluated the MR images in consensus for tumor suspicious regions (TSRs) for local recurrence. Subsequently, these TSRs were biopsied under MR-guidance and histopathologically evaluated for the presence of recurrent PCa. Descriptive statistical analysis was applied. Tissue sampling was successful in all patients and all TSRs. The positive predictive value on a per patient basis was 75% (15/20) and on a per TSR basis 68% (26/38). The median number of biopsies taken per patient was 3, and the duration of an MRGB session was 31 minutes. No intervention-related complications occurred. The combination of MRGB and diagnostic MR imaging of the prostate was a feasible technique to localize PCa recurrence after EBRT using a low number of cores in a clinically acceptable time.


Functional mapping of single spines in cortical neurons in vivo.
The individual functional properties and spatial arrangement of afferent synaptic inputs on dendrites have a critical role in the processing of information by neurons in the mammalian brain. Although recent work has identified visually-evoked local dendritic calcium signals in the rodent visual cortex, sensory-evoked signalling on the level of dendritic spines, corresponding to individual afferent excitatory synapses, remains unexplored. Here we used a new variant of high-resolution two-photon imaging to detect sensory-evoked calcium transients in single dendritic spines of mouse cortical neurons in vivo. Calcium signals evoked by sound stimulation required the activation of NMDA (N-methyl-D-aspartate) receptors. Active spines are widely distributed on basal and apical dendrites and pure-tone stimulation at different frequencies revealed both narrowly and widely tuned spines. Notably, spines tuned for different frequencies were highly interspersed on the same dendrites: even neighbouring spines were mostly tuned to different frequencies. Thus, our results demonstrate that NMDA-receptor-dependent single-spine synaptic inputs to the same dendrite are highly heterogeneous. Furthermore, our study opens the way for in vivo mapping of functionally defined afferent sensory inputs with single-synapse resolution.


Stakeholders views on the ethical aspects of oocyte banking for third-party assisted reproduction: a qualitative interview study with donors, recipients and professionals.
What are the moral considerations held by donors, recipients and professionals towards the ethical aspects of the intake and distribution of donor bank oocytes for third-party assisted reproduction? Interviews with oocyte donors, oocyte recipients and professionals demonstrate a protective attitude towards the welfare of the donor and the future child. The scarcity of donor oocytes challenges the approach towards the many ethical aspects that arise in establishing and operating an oocyte bank for third-party assisted reproduction. Including experiences and moral considerations originating from practice provides useful insight on how to overcome these challenges. The project was set-up as a qualitative interview study and took place between October 2016 and August 2017. We conducted 25 semi-structured interviews with professionals engaged in the practice of oocyte banking (n = 10), recipients of donor oocytes (n = 7) and oocyte donors (n = 8). Key themes were formulated by means of a thematic analysis. Based on the interviews, we formulated four main themes describing stakeholders' views regarding the ethical aspects of the intake and distribution of donor bank oocytes. First, respondents articulated that when selecting donors and recipients, healthcare workers should prevent donors from making a wrong decision and safeguard the future child's well-being by minimizing health risks and selecting recipients based on their parental capabilities. Second, they proposed to provide a reasonable compensation and to increase societal awareness on the scarcity of donor oocytes to diminish barriers for donors. Third, respondents considered the prioritization of recipients in case of scarcity a difficult choice, because they are all dependent on donor oocytes to fulfil their wish for a child. They emphasized that treatment attempts should be limited, but at least include one embryo transfer. Fourth and finally, the importance of good governance of oocyte banks was mentioned, including a homogenous policy and the facilitation of exchange of experiences between oocyte banks. The possibility of selection bias exists, because we interviewed donors and recipients who were selected according to the criteria currently employed in the clinics. Respondents' moral considerations regarding the ethical aspects of the intake and distribution of donor oocytes demonstrate a protective attitude towards the welfare of the donor and the future child. At the same time, respondents also questioned whether such a (highly) protective attitude was justified. This finding may indicate there is room for reconsidering strategies for the collection and distribution of donor bank oocytes. This study was funded by ZonMw: The Dutch Organization for Health Research and Development (Grant number 70-73000-98-200). A.M.E.B. and B.C.J.M.F. are the initiators of the UMC Utrecht oocyte bank. J.J.P.M.P. is the director of the MCK Fertility Centre. IMC is working as a gynaecologist at the AMC Amsterdam oocyte bank. During the most recent 5-year period, BCJM Fauser has received fees or grant support from the following organizations (in alphabetic order): Actavis/Watson/Uteron, Controversies in Obstetrics & Gynaecologist (COGI), Dutch Heart Foundation, Dutch Medical Research Counsel (ZonMW), Euroscreen/Ogeda, Ferring, London Womens Clinic (LWC), Merck Serono (GFI), Myovant, Netherland Genomic Initiative (NGI), OvaScience, Pantharei Bioscience, PregLem/Gedeon Richter/Finox, Reproductive Biomedicine Online (RBMO), Roche, Teva and World Health Organization (WHO). The authors have no further competing interests to declare. N/A.


Adjunctive Nutraceuticals for Depression: A Systematic Review and Meta-Analyses.
There is burgeoning interest in augmentation strategies for improving inadequate response to antidepressants. The adjunctive use of standardized pharmaceutical-grade nutrients, known as nutraceuticals, has the potential to modulate several neurochemical pathways implicated in depression. While many studies have been conducted in this area, to date no specialized systematic review (or meta-analysis) has been conducted. A systematic search of PubMed, CINAHL, Cochrane Library, and Web of Science was conducted up to December 2015 for clinical trials using adjunctive nutrients for depression. Where sufficient data were available, a random-effects model analyzed the standard mean difference between treatment and placebo in the change from baseline to endpoint, combining the effect size data. Funnel plot and heterogeneity analyses were also performed. Primarily positive results were found for replicated studies testing S-adenosylmethionine (SAMe), methylfolate, omega-3 (primarily EPA or ethyl-EPA), and vitamin D, with positive isolated studies for creatine, folinic acid, and an amino acid combination. Mixed results were found for zinc, folic acid, vitamin C, and tryptophan, with nonsignificant results for inositol. No major adverse effects were noted in the studies (aside from minor digestive disturbance). A meta-analysis of adjunctive omega-3 versus placebo revealed a significant and moderate to strong effect in favor of omega-3. Conversely, a meta-analysis of folic acid revealed a nonsignificant difference from placebo. Marked study heterogeneity was found in a Higgins test for both omega-3 and folic acid studies; funnel plots also revealed asymmetry (reflecting potential study bias). Current evidence supports adjunctive use of SAMe, methylfolate, omega-3, and vitamin D with antidepressants to reduce depressive symptoms.


7-Tesla Magnetic Resonance Imaging for Brain Iron Quantification in Homozygous and Heterozygous PANK2 Mutation Carriers.
Pantothenate-kinase-associated neurodegeneration (PKAN) is an autosomal recessive disorder characterized by iron deposits in basal ganglia. The aim of this study was to quantify iron concentrations of deep gray matter structures in heterozygous PANK2 mutation carriers and in PKAN patients using quantitative susceptibility mapping MRI. By determining iron concentration, we intended to find mutation-specific brain parenchymal stigmata in heterozygous PANK2 mutation carriers in comparison to age-matched healthy volunteers. We studied 11 heterozygous PANK2 gene mutation carriers (mean age: 43.4 years; standard deviation [SD]: 10.5), who were found to be clinically asymptomatic by neurological examination. These carriers were compared to 2 clinically affected PKAN patients 21 and 32 years of age and to 13 age-matched, healthy controls (mean age: 39.7; SD, 13.6). Scanning was performed on a 7.0-Tesla whole-body scanner applying three-dimensional susceptibility-weighted gradient echo acquisitions. Susceptibility maps were calculated by threshold-based k-space division with single-orientation acquisition. Magnetic susceptibility values, relative to the occipital white matter, were determined for the following regions of interest (ROI): globus pallidus (GP), thalamus, putamen, internal capsule (IC), caudate nucleus, substantia nigra (SN), and red nucleus. Heterozygous PANK2 mutation carriers did not show increased brain iron concentrations, compared to healthy controls (P > 0.05), in any of the examined ROIs. In PKAN patients, more than 3 times higher concentrations of iron were found in the GP, SN, and IC. Our results suggest that heterozygous mutations in PANK2 gene do not cause brain iron accumulation nor do they cause movement disorders.


Solution structure of the granular starch binding domain of Aspergillus niger glucoamylase bound to beta-cyclodextrin.
Carbohydrate-binding domains are usually small and physically separate from the catalytic domains of hydrolytic enzymes. Glucoamylase 1 (G1) from Aspergillus niger, an enzyme used widely in the food and brewing industries, contains a granular starch binding domain (SBD) which is separated from the catalytic domain by a semi-rigid linker. The aim of this study was to determine how the SBD binds to starch, and thereby more generally to throw light on the role of carbohydrate-binding domains in the hydrolysis of insoluble polysaccharides. The solution structure of the SBD of A. niger G1 bound to beta-cyclodextrin (betaCD), a cyclic starch analogue, shows that the well-defined beta-sheet structure seen in the free SBD is maintained in the SBD-betaCD complex. The main differences between the free and bound states of the SBD are observed in loop regions, in or near the two starch-binding sites. The two binding sites, each of which binds one molecule of betaCD, are structurally different. Binding site 1 is small and accessible, and its structure changes very little upon ligand binding. Site 2 is longer and undergoes a significant structural change on binding. Part of this site comprises a flexible loop, which appears to allow the SBD to bind to starch strands in a range of orientations. The two starch-binding sites of the SBD probably differ functionally as well as structurally; site 1 probably acts as the initial starch recognition site, whereas site 2 is involved in specific recognition of appropriate regions of starch. The two starch strands are bound at approximately 90 degrees to each other. This may be functionally important, as it may force starch strands apart thus increasing the hydrolyzable surface, or alternatively it may localize the enzyme to noncrystalline (more hydrolyzable) areas of starch. The region of the SBD where the linker to the catalytic domain is attached is flexible, allowing the catalytic site to access a large surface area of the starch granules.


Meningiomas of the tuberculum and diaphragma sellae.
Introduction Although tuberculum sellae (TS) and diaphragma sellae (DS) meningiomas have different anatomical origins, they are frequently discussed as a single entity. Here we review the radiologic and intraoperative findings of TS and DS meningiomas and propose a radiologic classification. Methods We retrospectively reviewed 10 consecutive TS and DS meningiomas. Data regarding clinical presentation, preoperative imaging, and intraoperative findings were analyzed. Three sellar dimensions were measured on magnetic resonance imaging (MRI): the tuberculum-sellar floor interval (TSFI), the planum-tuberculum interval (PTI), and the total height. Results Three distinct anatomical patterns were recognized: exclusively tubercular meningiomas (type A) were accompanied by elongation of the TSFI and, more significantly, of the PTI; combined TS and DS meningiomas (type B) were associated with relative elongation of both the PTI and TSFI; and the sole exclusively DS meningioma (type C) was associated with elongation of neither PTI nor TSFI. Conclusion Suprasellar meningiomas can be classified as tubercular, combined, or diaphragmatic based on preoperative MRI. Exclusively tubercular meningiomas (type A) require only a supradiaphragmatic approach. Tumor involvement of the sellar diaphragm (type B or C) requires resection of the diaphragm and thus a combined infra- and supradiaphragmatic approach.


Force spectroscopy predicts thermal stability of immobilized proteins by measuring microbead mechanics.
Optimal immobilization of enzymes on porous microbeads enables the fabrication of highly active and stable heterogeneous biocatalysts to implement biocatalysis in synthetic and analytical chemistry. However, empirical procedures for enzyme immobilization still prevail over rational ones because there is an unmet need for more comprehensive characterization techniques that aid to understand and trace the immobilization process. Here, we present the use of atomic force spectroscopy (AFS) as an innovative solution to indirectly characterize immobilized proteins on porous materials and monitor the immobilization process in real time. We investigate the mechanical properties of porous agarose microbeads immobilizing proteins by indenting a colloidal probe (silica microparticle) into a single bead. AFS demonstrates that the binding of proteins to the solid matrix of an agarose microbead alters its stiffness. Interestingly, we discovered that irreversible and multivalent immobilizations that make microbeads stiffer also stabilize the immobilized proteins against the temperature. Hence, we propose atomic force spectroscopy as a useful technique to indirectly unravel the stability of the immobilized enzymes investigating the mechanics of the heterogenous biocatalysts as a solid biomaterial beyond the intrinsic mechanics of the proteins.


Projected Uptake of New Antiretroviral (ARV) Medicines in Adults in Low- and Middle-Income Countries: A Forecast Analysis 2015-2025.
With anti-retroviral treatment (ART) scale-up set to continue over the next few years it is of key importance that manufacturers and planners in low- and middle-income countries (LMICs) hardest hit by the HIV/AIDS pandemic are able to anticipate and respond to future changes to treatment regimens, generics pipeline and demand, in order to secure continued access to all ARV medicines required. We did a forecast analysis, using secondary WHO and UNAIDS data sources, to estimate the number of people living with HIV (PLHIV) and the market share and demand for a range of new and existing ARV drugs in LMICs up to 2025. UNAIDS estimates 24.7 million person-years of ART in 2020 and 28.5 million person-years of ART in 2025 (24.3 million on first-line treatment, 3.5 million on second-line treatment, and 0.6 million on third-line treatment). Our analysis showed that TAF and DTG will be major players in the ART regimen by 2025, with 8 million and 15 million patients using these ARVs respectively. However, as safety and efficacy of dolutegravir (DTG) and tenofovir alafenamide (TAF) during pregnancy and among TB/HIV co-infected patients using rifampicin is still under debate, and ART scale-up is predicted to increase considerably, there also remains a clear need for continuous supplies of existing ARVs including TDF and EFV, which 16 million and 10 million patients-respectively-are predicted to be using in 2025. It will be important to ensure that the existing capacities of generics manufacturers, which are geared towards ARVs of higher doses (such as TDF 300mg and EFV 600mg), will not be adversely impacted due to the introduction of lower dose ARVs such as TAF 25mg and DTG 50mg. With increased access to viral load testing, more patients would be using protease inhibitors containing regimens in second-line, with 1 million patients on LPV/r and 2.3 million on ATV/r by 2025. However, it will remain important to continue monitoring the evolution of ARV market in LMICs to guarantee the availability of these medicines.


Human papillomavirus vaccination 2020 guideline update: American Cancer Society guideline adaptation.
The American Cancer Society (ACS) presents an adaptation of the current Advisory Committee on Immunization Practices recommendations for human papillomavirus (HPV) vaccination. The ACS recommends routine HPV vaccination between ages 9 and 12 years to achieve higher on-time vaccination rates, which will lead to increased numbers of cancers prevented. Health care providers are encouraged to start offering the HPV vaccine series at age 9 or 10 years. Catch-up HPV vaccination is recommended for all persons through age 26 years who are not adequately vaccinated. Providers should inform individuals aged 22 to 26 years who have not been previously vaccinated or who have not completed the series that vaccination at older ages is less effective in lowering cancer risk. Catch-up HPV vaccination is not recommended for adults aged older than 26 years. The ACS does not endorse the 2019 Advisory Committee on Immunization Practices recommendation for shared clinical decision making for some adults aged 27 through 45 years who are not adequately vaccinated because of the low effectiveness and low cancer prevention potential of vaccination in this age group, the burden of decision making on patients and clinicians, and the lack of sufficient guidance on the selection of individuals who might benefit.


Ballistocardiogram correction in simultaneous EEG/ fMRI recordings: a comparison of average artifact subtraction and optimal basis set methods using two popular software tools.
Electroencephalography data recorded during functional magnetic resonance imaging acquisition are subject to large cardiac-related artifacts that must be corrected during postprocessing. This study compared two widely used ballistocardiogram (BCG) correction algorithms as implemented in two software programs. Reduction of BCG amplitude, correlation of corrected data with electrocardiogram traces, correlation of independent components with electrocardiogram traces, and event-related potential signal-to-noise ratio from each algorithm were compared. Both algorithms effectively reduced the BCG artifact, with a slight advantage of average artifact subtraction over the optimal basis set method (0.1-2.2%) when the quality of the correction was examined at the individual subject level. This study provides users of these software tools with an important, practical, and previously unavailable comparison of the performance of these two methods.


The importance of patient expectations in predicting functional outcomes after total joint arthroplasty.
To evaluate the relationship between patient expectations of total joint arthroplasty (TJA) and health related quality of life plus satisfaction 6 months after surgery. Methods. This prospective cohort study included patients undergoing primary total hip (THA) and knee arthroplasty (TKA). Patients were evaluated with self-report questionnaires prior to surgery and 6 months post-surgery. Medical Outcomes Study Short Form 36 (SF-36), the Western Ontario McMaster Universities Osteoarthritis Index (WOMAC), and a satisfaction scale were used to evaluate outcomes at final followup. Multivariate regression models were used to evaluate the impact of expectations on outcomes. There were 102 patients with THA and 89 with TKA. Mean age was 66 years. All patients achieved significant improvements in their WOMAC and SF-36 scores following surgery. Patient expectations regarding surgery were not associated with their age, gender, index joint of surgery, marital status, or race. Expectations were not correlated with pre-operative functional health status. Expectation of complete pain relief after surgery was an independent predictor of better physical function and improvement in level of pain at 6 months post-surgery. Expectation of low risk of complications from TJA was an independent predictor of greater satisfaction. Patient expectations were important independent predictors of improved functional outcomes and satisfaction following TJA. Greater understanding of the relationship between expectations and outcomes may improve the process of care and outcomes of TJA.


Heterostructures of germanium nanowires and germanium-silicon oxide nanotubes and growth mechanisms.
We report on a method to fabricate one-dimensional heterostructures of germanium nanowires (GeNWs) and germanium-silicon oxide nanotubes (GeSiO(x)NTs). The synthesis of the wire-tube heterostructures is carried out using a simple furnace set-up with germanium tetraiodide and germanium powders as growth precursors, gold-dotted silicon wafers as substrates and by controlling the temperature ramp rate/sequence of the growth precursors. Two types of wire-tube heterostructures resulting from distinct growth mechanisms are obtained. The type-1 heterostructure consists of a GeNW, grown via a gold-catalyzed vapour-liquid-solid process, at the lower end and a GeSiO(x)NT at the upper end. In contrast, the type-2 heterostructure is made up of a solid wire at the upper end and a hollow tube at the lower end. The solid wire portion of the type-2 heterostructure is formed through an oxide-assisted growth process.


The Ecology of Social Learning in Animals and its Link with Intelligence.
Classical ethology and behavioral ecology did not pay much attention to learning. However, studies of social learning in nature reviewed here reveal the near-ubiquity of reliance on social information for skill acquisition by developing birds and mammals. This conclusion strengthens the plausibility of the cultural intelligence hypothesis for the evolution of intelligence, which assumes that selection on social learning abilities automatically improves individual learning ability. Thus, intelligent species will generally be cultural species. Direct tests of the cultural intelligence hypothesis require good estimates of the amount and kind of social learning taking place in nature in a broad variety of species. These estimates are lacking so far. Here, we start the process of developing a functional classification of social learning, in the form of the social learning spectrum, which should help to predict the mechanisms of social learning involved. Once validated, the categories can be used to estimate the cognitive demands of social learning in the wild.


Theoretical prediction of spectral and optical properties of bacteriochlorophylls in thermally disordered LH2 antenna complexes.
A general approach for calculating spectral and optical properties of pigment-protein complexes of known atomic structure is presented. The method, that combines molecular dynamics simulations, quantum chemistry calculations, and statistical mechanical modeling, is demonstrated by calculating the absorption and circular dichroism spectra of the B800-B850 bacteriochlorophylls of the LH2 antenna complex from Rs. molischianum at room temperature. The calculated spectra are found to be in good agreement with the available experimental results. The calculations reveal that the broadening of the B800 band is mainly caused by the interactions with the polar protein environment, while the broadening of the B850 band is due to the excitonic interactions. Since it contains no fitting parameters, in principle, the proposed method can be used to predict optical spectra of arbitrary pigment-protein complexes of known structure.


Effect of drought stress on the genetic architecture of photosynthate allocation and remobilization in pods of common bean (Phaseolus vulgaris L.), a key species for food security.
Common bean is the most important staple grain legume for direct human consumption and nutrition. It complements major sources of carbohydrates, including cereals, root crop, or plantain, as a source of dietary proteins. It is also a significant source of vitamins and minerals like iron and zinc. To fully play its nutritional role, however, its robustness against stresses needs to be strengthened. Foremost among these is drought, which commonly affects its productivity and seed quality. Previous studies have shown that photosynthate remobilization and partitioning is one of the main mechanisms of drought tolerance and overall productivity in common bean. In this study, we sought to determine the inheritance of pod harvest index (PHI), a measure of the partitioning of pod biomass to seed biomass, relative to that of grain yield. We evaluated a recombinant inbred population of the cross of ICA Bunsi and SXB405, both from the Mesoamerican gene pool, to determine the effects of intermittent and terminal drought stresses on the genetic architecture of photosynthate allocation and remobilization in pods of common bean. The population was grown for two seasons, under well-watered conditions and terminal and intermittent drought stress in one year, and well-watered conditions and terminal drought stress in the second year. There was a significant effect of the water regime and year on all the traits, at both the phenotypic and QTL levels. We found nine QTLs for pod harvest index, including a major (17% of variation explained), stable QTL on linkage group Pv07. We also found eight QTLs for yield, three of which clustered with PHI QTLs, underscoring the importance of photosynthate remobilization in productivity. We also found evidence for substantial epistasis, explaining a considerable part of the variation for yield and PHI. Our results highlight the genetic relationship between PHI and yield and confirm the role of PHI in selection of both additive and epistatic effects controlling drought tolerance. These results are a key component to strengthen the robustness of common bean against drought stresses.


Should biomarker estimates of HIV incidence be adjusted?
To evaluate adjustment procedures that have been proposed to correct HIV incidence rates derived from cross-sectional surveys of biomarkers (e.g. BED capture enzyme immunoassay). These procedures were motivated by some reports that the biomarker BED approach overestimates incidence when compared to cohort studies. Consideration of the Hargrove and McDougal adjustment procedures that adjust biomarker estimates of HIV incidence rates for misclassification with respect to the timing of infections. : Performed mathematical and statistical analysis of the adjustment formulas. Evaluated sources of error in cohort studies of incidence that could also explain discrepancies between cohort and biomarker estimates. The McDougal adjustment has no net effect on the estimate of HIV incidence because false positives exactly counterbalance false negatives. The Hargrove adjustment has a mathematical error that can cause significant underestimation of HIV incidence rates, especially if there is a large pool of prevalent long-standing infections. The two adjustment procedures of biomarker incidence estimates evaluated here that purport to correct for misclassification do not increase accuracy and in some situations can introduce significant bias. Instead, the accuracy of biomarker estimates can be increased through improvements in the estimates of the mean window period of the populations under study and the representativeness of the cross-sectional samples. Cohort estimates of incidence are also subject to important sources of error and should not blindly be considered the gold standard for assessing the validity of biomarker estimates.


Attenuation of vascular permeability by methylnaltrexone: role of mOP-R and S1P3 transactivation.
Endothelial cell (EC) barrier dysfunction (i.e., increased vascular permeability) is observed in inflammatory states, tumor angiogenesis, atherosclerosis, and both sepsis and acute lung injury. Therefore, agents that preserve vascular integrity have important clinical therapeutic implications. We examined the effects of methylnaltrexone (MNTX), a mu opioid receptor (mOP-R) antagonist, on human pulmonary EC barrier disruption produced by edemagenic agents including morphine, the endogenous mOP-R agonist DAMGO, thrombin, and LPS. Pretreatment of EC with MNTX (0.1 muM, 1 h) or the uncharged mOP-R antagonist naloxone attenuated morphine- and DAMGO-induced barrier disruption in vitro. However, MNTX, but not naloxone, pretreatment of EC inhibited thrombin- and LPS-induced barrier disruption, indicating potential mOP-R-independent effects of MNTX. In addition, intravenously delivered MNTX attenuated LPS-induced vascular hyperpermeability in the murine lung. We next examined the mechanistic basis for this MNTX barrier protection and observed that silencing of mOP-R attenuated the morphine- and DAMGO-induced EC barrier disruption, but not the permeability response to either thrombin or LPS. Because activation of the sphingosine 1-phosphate receptor, S1P(3), is key to a number of barrier-disruptive responses, we examined the role of this receptor in the permeability response to mOP-R ligation. Morphine, DAMGO, thrombin, and LPS induced RhoA/ROCK-mediated threonine phosphorylation of S1P(3), which was blocked by MNTX, suggesting S1P(3) transactivation. In addition, silencing of S1P(3) receptor expression (siRNA) abolished the permeability response to each edemagenic agonist. These results indicate that MNTX provides barrier protection against edemagenic agonists via inhibition of S1P(3) receptor activation and represents a potentially useful therapeutic agent for syndromes of increased vascular permeability.


Do acute psychological and psychobiological responses to trauma predict subsequent symptom severities of PTSD and depression?
The study investigated the relationship between the acute psychological and psychobiological trauma response and the subsequent development of posttraumatic stress disorder (PTSD) and depressive symptoms in 53 accident survivors attending an emergency department. Lower levels of salivary cortisol measured in the emergency room predicted greater symptom levels of PTSD and depression 6 months later, and lower diastolic blood pressure, past emotional problems, greater dissociation and data-driven processing predicted greater PTSD symptoms. Heart rate was not predictive. Low cortisol levels correlated with data-driven processing during the accident, and, in female participants only, with prior trauma and prior emotional problems. Higher evening cortisol 6 months after the accident correlated with PTSD and depressive symptoms at 6 months, but this relationship was no longer significant when levels of pain were controlled. The results support the role of the acute response to trauma in the development and maintenance of PTSD and provide promising preliminary evidence for a meaningful relationship between psychobiological and psychological factors in the acute trauma phase.


Midkine (MK), a heparin-binding growth/differentiation factor, is regulated by retinoic acid and epithelial-mesenchymal interactions in the developing mouse tooth, and affects cell proliferation and morphogenesis.
Midkine (MK) is the first cloned gene in a new family of heparin-binding growth/differentiation factors involved in the regulation of growth and differentiation. We have analyzed the expression of MK mRNA and protein during tooth development in mouse embryos and studied the regulation of MK expression and the biological effects of MK protein in organ cultures. MK expression was restricted and preferential in the tooth area as compared to the rest of the developing maxillary and mandibular processes suggesting specific functions for MK during tooth morphogenesis. MK mRNA and protein were expressed during all stages of tooth formation (initiation, morphogenesis, and cell differentiation), and shifts of expression were observed between the epithelial and mesenchymal tissue components. However, the expression of mRNA and protein showed marked differences at some stages suggesting paracrine functions for MK. Tissue recombination experiments showed that MK gene and protein expression are regulated by epithelial-mesenchymal interactions, and, moreover, that dental tissue induces the ectopic expression of MK protein in non-dental tissue. The expression of MK gene and protein in the mandibular arch mesenchyme from the tooth region were stimulated by local application of retinoic acid in beads. Cell proliferation was inhibited in dental mesenchyme around the beads releasing MK, but this effect was modulated by simultaneous application of FGF-2. Morphogenesis and cell differentiation were inhibited in tooth germs cultured in the presence of neutralizing antibodies for MK, whereas the development of other organs (e.g., salivary gland, kidney) was unaffected. These results suggest important roles for MK in the molecular cascade that regulates tooth development.


How graphene crosses a grain boundary on the catalyst surface during chemical vapour deposition growth.
The chemical vapour deposition (CVD) growth of graphene is normally an epitaxial process, where the atomic structure of the adlayer should copy the texture of the substrate. However, it has been widely observed that single crystalline graphene grown on metal foil may cross a grain boundary (GB) of the substrate without forming any line defect, a necessary condition to change its crystalline orientation and maintain the structure registry with the substrate on the other side of the GB. Here, we present a comprehensive theoretical study on graphene growth behavior on polycrystalline metal substrates. Our density functional theory (DFT) calculations reveal that for graphene growth on most metal surfaces, the binding energy difference between the epitaxial and non-epitaxial graphene on the substrate is not large enough to compensate for the formation energy of a GB in graphene and therefore, during the CVD process, the growing graphene can pass through a GB on the metal surface without changing its crystalline orientation. Hence, graphene CVD growth cannot be strictly regarded as an epitaxial process; this conclusion is further verified by atomic simulations. The present study shows that the growth of graphene on a metal catalyst surface should be regarded rather as a quasi-epitaxial process, where a graphene domain is aligned only on the single crystalline metal facet on which it nucleates, but this structural registry with the metal substrate may be lost when the graphene crosses a GB on the metal surface.


Sequence variation and structural conservation allows development of novel function and immune evasion in parasite surface protein families.
Trypanosoma and Plasmodium species are unicellular, eukaryotic pathogens that have evolved the capacity to survive and proliferate within a human host, causing sleeping sickness and malaria, respectively. They have very different survival strategies. African trypanosomes divide in blood and extracellular spaces, whereas Plasmodium species invade and proliferate within host cells. Interaction with host macromolecules is central to establishment and maintenance of an infection by both parasites. Proteins that mediate these interactions are under selection pressure to bind host ligands without compromising immune avoidance strategies. In both parasites, the expansion of genes encoding a small number of protein folds has established large protein families. This has permitted both diversification to form novel ligand binding sites and variation in sequence that contributes to avoidance of immune recognition. In this review we consider two such parasite surface protein families, one from each species. In each case, known structures demonstrate how extensive sequence variation around a conserved molecular architecture provides an adaptable protein scaffold that the parasites can mobilise to mediate interactions with their hosts.


Sequence homology and microhomology dominate chromosomal double-strand break repair in African trypanosomes.
Genetic diversity in fungi and mammals is generated through mitotic double-strand break-repair (DSBR), typically involving homologous recombination (HR) or non-homologous end joining (NHEJ). Microhomology-mediated joining appears to serve a subsidiary function. The African trypanosome, a divergent protozoan parasite, relies upon rearrangement of subtelomeric variant surface glycoprotein (VSG) genes to achieve antigenic variation. Evidence suggests an absence of NHEJ but chromosomal repair remains largely unexplored. We used a system based on I-SceI meganuclease and monitored temporally constrained DSBR at a specific chromosomal site in bloodstream form Trypanosoma brucei. In response to the lesion, adjacent single-stranded DNA was generated; the homologous strand-exchange factor, Rad51, accumulated into foci; a G(2)M checkpoint was activated and >50% of cells displayed successful repair. Quantitative analysis of DSBR pathways employed indicated that inter-chromosomal HR dominated. HR displayed a strong preference for the allelic template but also the capacity to interact with homologous sequence on heterologous chromosomes. Intra-chromosomal joining was predominantly, and possibly exclusively, microhomology mediated, a situation unique among organisms examined to date. These DSBR pathways available to T. brucei likely underlie patterns of antigenic variation and the evolution of the vast VSG gene family.


Molecular Variation of Sporisorium scitamineum in Mainland China Revealed by RAPD and SRAP Markers.
Sugarcane smut caused by Sporisorium scitamineum occurs worldwide, causing serious losses in sugar yield and quality. To study the molecular variation of S. scitamineum, 23 S. scitamineum isolates collected from the six primary sugarcane production areas in mainland China (Guangxi, Yunnan, Guangdong, Hainan, Fujian, and Jiangxi provinces) were assessed by random amplified polymorphic DNA (RAPD) and sequence-related amplified polymorphism (SRAP) markers. The results of RAPD, SRAP, and RAPD-SRAP combined analysis showed that, whereas the molecular variation of S. scitamineum was associated with geographic origin, there was no evidence of co-evolution between sugarcane and the pathogen. The results of RAPD, SRAP, or RAPD-SRAP combined analysis also did not provide any information about race differentiation of S. scitamineum. This suggests that the mixture of spores from sori collected from different areas should be used in artificial inoculations for resistance breeding and selection.


A meta-analysis of the neuropsychological sequelae of HIV infection.
This meta-analysis summarizes the broad spectrum of neuropsychological research on HIV disease across a sample of 41 primary studies and an aggregate of 8,616 participants for 10 major neuropsychological ability areas. Analyses of the course of cognitive decline within and across Centers for Disease Control classifications reveals statistically significant cognitive deficits from asymptomatic HIV to AIDS. Effect sizes (Cohen, 1988) were calculated to reflect between-group (asymptomatic, symptomatic, AIDS) differences in each neuropsychological domain. Relatively small effect sizes were obtained for the asymptomatic (0.05-0.21) patients, and generally small to moderate effect sizes were obtained for symptomatic (0.18-0.65) HIV+ patients, with motor functioning exhibiting the greatest effects in this later disease stage. The most notable deficits in cognitive functioning were found in the AIDS group with moderate (attention and concentration) to large (motor functioning) effect sizes with values ranging from 0.42-0.82. Comparison of cognitive functioning as a function of disease progression revealed that motor functioning, executive skills, and information processing speed were among the cognitive domains showing the greatest decline from early to later stages of HIV. These findings indicate that cognitive deficits in the early stages of HIV are small and increase in the later phases of the illness, and that specific patterns of cognitive deficits can be detected with disease progression. These results and their clinical utility are further discussed.


Exercise-induced bronchial hyperresponsiveness and parental ISAAC questionnaire responses.
The predictive value of parental questionnaire responses for exercise-induced bronchoconstriction in childhood asthma has not been fully clarified. The aim of this study was to compare exercise-induced bronchial hyperresponsiveness in 7 year old children with parental responses to core questions in the International Study of Asthma and Allergies in Childhood (ISAAC) study. A cross-sectional study was conducted on 191 (91% of eligible) children from seven randomly selected schools in Southern Tasmania. Study measurements included a parental questionnaire and exercise challenge testing, using a recently validated 6 min free-running protocol. The response to exercise was assessed using forced expiratory volume in one second (FEV1) measurement. The median percentage fall in FEV1 was significantly higher in children whose parents responded positively to ISAAC questions on a history of wheeze (p = 0.0031) or asthma (p = 0.0005), recent wheeze (p = 0.0005), sleep disturbance due to wheeze (p = 0.0005), or exercise-induced wheeze (p = 0.0015). Receiver operating characteristic (ROC) curve analysis showed exercise-induced bronchial hyperresponsiveness to be a good indicator of current asthma status. Using a 12% or greater fall in FEV1 postexercise as a positive test response, the exercise challenge had sensitivity and specificity estimates for current asthma and exercise-induced wheeze of (0.58 and 0.77) and (0.60 and 0.77), respectively. In conclusion, the respiratory response to exercise was consistent with parental responses to the ISAAC questionnaire in a population-based sample of 7 year old children. These findings will assist interpretation of large ISAAC studies in terms of asthma prevalence.


Isokinetic Dynamometry as a Tool to Predict Shoulder Injury in an Overhead Athlete Population: A Systematic Review.
Prospective and cross-sectional studies have used pre-season isokinetic dynamometry strength and endurance measurements of shoulder internal rotation (IR) and external rotation (ER) to determine if they can be correlated to injury. However, to date, no review has provided a synthesis of all available literature on this topic. The aim of this systematic review was to identify isokinetic dynamometry studies that assess shoulder IR and ER strength and endurance in the overhead athletic population in relation to shoulder injury. Electronic databases (PubMed, CINAHL, and SportDiscus) were searched through September 2019 using pre-determined search terms. Both prospective and cross-sectional studies were included in this review. Studies were assessed for quality using either Appraisal Tool for Cross-sectional Studies (AXIS) or Critical Appraisal Skills Programme (CASP). Data on outcome measures of strength and endurance peak torque (PT) and ratios (ER:IR) were extracted and further analysed using a best evidence synthesis approach. A total of 13 articles met the inclusion criteria. Conflicting evidence was found when reviewing all studies without differentiating by study type. Prospective study designs revealed strong evidence that reduced IR endurance and reduced strength ratios are predictive of shoulder injury. Cross-sectional literature showed only conflicting and limited evidence for all outcome measures. At this stage, more research is needed in individual sporting populations using prospective cohort designs.


Bioactivity of five components of Chinese herbal formula Jiangzhi granules against hepatocellular steatosis.
This study aims to evaluate the bioactivity of five components of the traditional Chinese medicine complex prescription Jiangzhi granules against hepatocellular steatosis. The five major components, including protopanaxadiol, tanshinone IIA, emodin, chlorogenic acid, and nuciferine, were extracted from Jiangzhi granules. Their cytotoxicity was assessed to determine the safe dose of each component for HepG2 cells. HepG2 cellular steatosis was induced using 1 mmol/L of free fatty acids (FFAs) for 24 h, and then treated with each component at high, intermediate, and low doses (500, 50, and 5 μmol/L), respectively for another 24 h. The effects on HepG2 steatosis were observed directly under optical phase microscopy, or through oil red O staining and Nile red assays. In addition, the levels of reactive oxygen species (ROS) in the steatotic HepG2 cells with and without high-dose protopanaxadiol treatment were measured using fluorescent dye 2',7'-dichlorodihydrofluorescein diacetate staining. No obvious cytotoxicity was observed in the HepG2 cells incubated with each of the five components at up to 500 μmol/L. At 24 h after incubation with FFAs, the HepG2 cells swelled and many lipid droplets accumulated. The lipid content was attenuated after 24 h of incubation with protopanaxadiol, tanshinone IIA, and emodin at 500 or 50 μmol/L (P < 0.05), especially with 500 μmol/L protopanaxadiol (P < 0.01). In addition, the ROS level was elevated in steatotic cells, but decreased after intervention with 500 μmol/L protopanaxadiol (P < 0.05). Protopanaxadiol, tanshinone IIA, and emodin alleviate hepatocellular steatosis in a dose-dependent manner, and oxidative stress regulation may partially contribute to the effects of protopanaxadiol.