Miracle-dz/UniFAR-FTUs huggingface dataset:
Amino Acid Transport Mechanisms in Mouse Oocytes During Growth and Meiotic Maturation1

Amino acids are transported into cells by a number of different transport systems, each with their own specific range of substrates. The amino acid transport systems active in preimplantation embryos and the amino acids required by embryos for optimal development have been extensively investigated. Much less is known about amino acid transport systems active in growing and meiotically maturing oocytes or about developmental changes in their activity. As a first step in determining the array of amino acid transporters active in oocytes, the transport characteristics of nine amino acids were measured in small, medium, and large growing oocytes; in fully grown germinal vesicle (GV)-stage oocytes; in metaphase I oocytes; and in metaphase II eggs. Whether each of 11 classically defined amino acid transport systems was likely active in oocytes at each stage was determined using assays based on measuring the transport of radiolabeled amino acids into oocytes and the effect of a limited set of potential competitive inhibitors. Six amino acid transport systems were found to be active during oocyte growth or maturation. L, b0,+, and ASC/asc were active throughout oocyte growth and maturation, increasing during growth. In contrast, GLY, beta, and xc− had little or no activity during growth but became activated during meiotic maturation. Surprisingly, the presence of follicular cells surrounding medium growing oocytes or cumulus cells surrounding GV oocytes did not confer amino acid transport by additional transport systems not present in the oocyte. In some cases, however, follicular cells coupled to the oocyte enhanced uptake of amino acids by the same systems present in the oocyte.
SPAM1 Isoforms from Two Tissue Origins Are Differentially Localized Within Ejaculated Bull Sperm Membranes and Have Different Roles During Fertilization1

The deduced amino acid sequence of bull sperm, SPAM1, suggests that it possesses a transmembrane domain between the hyaluronidase and the putative zona pellucida (ZP) binding domains. The objective of this study was to determine the orientation and localization of SPAM1 in order to understand how it could fulfill these two roles. We report that two isoforms of SPAM1 are present on ejaculated bull spermatozoa: one localized on the anterior portion of the sperm head, and the other on the postacrosomal portion of the head. The first isoform is expressed intracellularly, while the second one is detected at the sperm surface with its hyaluronidase domain facing the extracellular environment. Two-dimensional electrophoresis revealed that the two isoforms have different masses (80 and 70 kDa, respectively), and LC/MS/MS analyses confirmed our previously published deduced amino acid sequence of bovine SPAM1. In addition, this approach showed that the 70-kDa isoform differs from the 80-kDa isoform in its C terminus. Our results suggest that the shorter SPAM1 form originates from the epididymis, while the longer one is produced during spermatogenesis. These results clearly demonstrate that ejaculated bull sperm possess two forms of SPAM1: one (epididymal) expressed at the surface, and one (testicular) that interacts with the ZP after the acrosome exocytosis.
Regulation of Piezo2 Mechanotransduction by Static Plasma Membrane Tension in Primary Afferent Neurons*

The Piezo2 channel is a newly identified mammalian mechanical transducer that confers rapidly adapting mechanically activated (RA-MA) currents in primary afferent neurons. The Piezo2 channels sense rapid membrane displacement, but it is not clear whether they are sensitive to osmotic swelling, which slowly increases static plasma membrane tension (SPMT). Here, we show that SPMT exerts a profound impact on the mechanical sensitivity of RA-MA channels in primary afferent neurons. RA-MA currents are greatly enhanced, and the mechanical threshold was reduced in both primary afferent neurons of rat dorsal root ganglia (DRG) and HEK293 cells heterologously expressing Piezo2 when these cells undergo osmotic swelling to increase SPMT. Osmotic swelling switches the kinetics of RA-MA currents to the slowly adapting type in both cultured DRG neurons and HEK293 cells heterologously expressing Piezo2. The potentiation of RA-MA currents is abolished when cultured DRG neurons are treated with cytochalasin D, an actin filament disruptor that prevents SPMT of cultured DRG neurons from an increase by osmotic swelling. Osmotic swelling significantly increases DRG neuron mechano-excitability such that a subthreshold mechanical stimulus can result in action potential firing. Behaviorally, the mechanical hind paw withdrawal threshold in rats is reduced following the injection of a hypotonic solution, but this osmotic effect is abolished when cytochalasin D or Gd3+ is co-administered with the hypo-osmotic solution. Taken together, our findings suggest that Piezo2-mediated mechanotransduction is regulated by SPMT in primary afferent neurons. Because SPMT can be changed by multiple biological factors, our findings may have broad implications in mechanical sensitivity under physiological and pathological conditions.
Hepatitis E virus enters liver cells through receptor‐dependent clathrin‐mediated endocytosis

Summary. We investigated the virus–host interaction for hepatitis E virus (HEV) by performing competitive binding assays using in vitro assembled virus‐like particles (VLPs). We used Escherichia coli expressed native capsid protein (pORF2) and its mutants with an attached Gly(5)‐Ala (linker) reporter [enhanced green fluorescent protein (EGFP)/firefly luciferase (Fluc)]. Transmission electron microscopy and nanoparticle tracking showed near uniform particles of approximately 30–35 nm in diameter for pORF2 VLPs and 60–100 nm for reporter‐linked VLPs. Binding of reporter‐linked full‐length (1–660aa) and N‐terminal truncated (Δ1–112aa) pORF2 VLPs to Huh7 cell surfaces was found to be specific with 1.92 ± 0.065 × 105 sites per cell. Saturation binding indicated an equilibrium dissociation constant (Kd) of 121.1 ± 23.83 and 123.8 ± 16.15 nm for pORF2‐linker‐EGFP and pORF2‐linker‐Fluc VLPs respectively. A similar binding pattern was observed for Δ1–112aa pORF2‐linker‐EGFP and Δ1–112aa pORF2‐linker‐Fluc VLPs with Kd values of 123.6 ± 10.60 and 135.6 ± 16.19 nm respectively. The affinity (log Ki) of pORF2 binding on Huh7 cells in the presence of EGFP‐tagged and Fluc‐tagged pORF2 VLPs was found to be approximately 2.0. However, no VLP formation or binding was observed with refolded C‐terminal truncated (Δ458–660aa) pORF2. We investigated HEV internalization using fluorescent VLPs (EGFP‐VLPs), which showed vesicle‐mediated uptake starting at 5 min post‐incubation. The uptake of VLPs could be stopped by inhibitors for clathrin‐dependent endocytosis, but not by caveosome inhibitors. No binding and uptake of EGFP‐VLPs were observed on non‐hepatic cell lines (HeLa and SiHa). These findings suggest that HEV attaches to the host cell via a specific high affinity receptor and enters the cytoplasm by clathrin‐mediated endocytosis.
SCCA‐IC serum levels are predictive of clinical response in HCV chronic hepatitis to antiviral therapy: a multicentric prospective study

Summary. The combination of pegylated interferon (Peg‐IFN) and ribavirin is currently the gold standard therapy in patients with HCV chronic infection. The duration of therapy, as well as the therapeutic dosage, depend on the genotype. Identification of the genotype and rapid virological response (RVR) are widely accepted as the most important predictors of clinical outcome during antiviral therapy but to optimize cost‐benefits and to reduce possible side effects, further prognostic factors are needed. Squamous cell carcinoma antigens immunocomplex (SCCA‐IC) has been reported to be increased in the serum of patients with liver cancer. In this multicentric prospective study, we investigated the serum levels of SCCA‐IC in 103 patients with HCV chronic infection. Serum HCV‐RNA was detected before the beginning of treatment, after 4, 12, 24 or 48 weeks, and at week 24 during follow‐up. RVR, early virological response and sustained virological response (SVR) were assessed following the international guidelines. SCCA‐IC levels were higher in responders (238 AU, interquartile difference 130–556 AU) and decreased significantly to 125 AU (70–290 AU). The mean baseline value in nonresponders was 149 AU (86.5–306.5 AU), but after 4 weeks of treatment the serum levels decreased to 115 AU (80–280 AU): the profile of reduction was different between patients with or without a positive SVR. Logistic regression with SVR as dependent variable identified as significant independent variables: the reduction in SCCA‐IC after 1 month (OR = 4.82; 95% CI 1.39–16.67; P = 0.131) and a genotype other than 1 (OR = 0.094; 95% CI 0.21–0.42; P = 0.002); sex and age were also significant factors influencing SVR. SCCA‐IC seems to be a reliable independent prognostic marker of therapeutic effectiveness in anti‐HCV positive patients undergoing antiviral therapy.
Mice Lacking Two Sperm Serine Proteases, ACR and PRSS21, Are Subfertile, but the Mutant Sperm Are Infertile In Vitro1

Although sperm serine protease and proteasome have long been believed to play an important role in the fertilization process, the molecular mechanism is still controversial. In this study, we have produced double-knockout mice lacking two sperm serine proteases, ACR and PRSS21, to uncover the functional role of the trypsinlike activity in fertilization. The double-knockout male mice were subfertile, likely owing to the incompleteness of fertilization in the oviductal ampulla. Despite male subfertility, the mutant epididymal sperm exhibited the inability to undergo acrosomal exocytosis on the zona pellucida (ZP) surface and to traverse the ZP, thus resulting in the failure of fertilization in vitro. The double-knockout epididymal sperm were also defective in penetration through the cumulus matrix to reach the ZP. When epididymal sperm were artificially injected into the uterus of wild-type mice, the 2-cell embryos, which had previously been fertilized by double-knockout sperm, were recovered at a low but significant level. The mutant epididymal sperm were also capable of fertilizing the oocytes in the presence of uterine fluids in vitro. These data demonstrate that the trypsinlike protease activity of ACR and PRSS21 is essential for the process of sperm penetration through the cumulus matrix and ZP in vitro, and suggest that the female reproductive tract partially compensates for the loss of the sperm function. We therefore conclude that the sperm trypsinlike activity is still important but not essential for fertilization in vivo in the mouse.
Learning collectively to collectively learn training for Emergency Duty Teams

Abstract This article begins by outlining some of the difficulties that can exist in providing training for a small and closely defined staff group in this case Emergency Duty Teams (EDTs). It describes how an idea was formed, nurtured and supported in such a way that led to the establishment of a training collective. This collective went on to set up a training day which was attended by 40 representatives from ten London Boroughs/Counties. A collaborative approach was adopted. Roles and responsibilities were shared so that no one person or team was asked to do too much. The views of the whole collective were given prominence as much as possible. Theory and practice are brought together to demonstrate how a foundation has been laid on which several years of future learning opportunities may be built if required. The article concludes by showing how, following an initial inspiration, the support and participation of others can help bring into being training which is cost effective, meaningful and relevant.
Impact of hepatitis C on survival in dialysis patients: a link with cardiovascular mortality?

Summary. Recent evidence has been accumulated showing that anti‐HCV‐positive serologic status is significantly associated with lower survival in dialysis populations, but the mechanisms underlying this negative relationship are still unclear. The aim of this study was to conduct a systematic review of the published medical literature concerning the impact of hepatitis C virus (HCV) infection on all‐cause and disease‐specific mortality of patients on regular dialysis. The relative risk of all‐cause, cardiovascular and liver disease‐related mortality was regarded as the most reliable outcome end‐point. Study‐specific relative risks were weighted by the inverse of their variance to obtain fixed‐ and random effect pooled estimates for mortality with HCV across the published studies. We identified fourteen observational studies involving 145 608 unique patients on long‐term dialysis. Pooling of study results demonstrated that anti‐HCV antibody was an independent and significant risk factor for death in patients on maintenance dialysis. The summary estimate for adjusted relative risk (all‐cause mortality) was 1.35 with a 95% confidence interval (CI) of 1.25–1.47. Stratified analysis showed that the adjusted RR for liver disease‐related death was 3.82 (95% CI, 1.92; 7.61); heterogeneity statistics, Ri = 0.58 (P‐value by Q‐test = 0.087). The adjusted RR for cardiovascular mortality was 1.26 (95% CI, 1.10; 1.45); no heterogeneity was found (NS). This meta‐analysis of observational studies indicates that anti‐HCV‐positive patients on dialysis have an increased risk of either liver or cardiovascular disease‐related mortality compared with anti‐HCV‐negative patients. Further studies are in progress to understand better the link between HCV and cardiovascular risk among patients on maintenance dialysis.
Motivation of Parents Towards Reading Multilingual eBooks To Pre-School Children

<span class="TextRun SCXW153678227" lang="EN-US" xml:lang="EN-US">The use of mobile devices for language learning, under the Mobile Assisted Language Learning (MALL) has been found to motivate children to read digital print. However, parents need to be convinced of the benefits of this new technology-assisted learning method within a multilingual learning context. A case study was conducted to propose a MALL framework in which educators and parents created a shared meaningful learning environment to develop the children’s emergent multilingual literacy skills, using mobile devices installed with interactive multilingual </span><span class="TextRun SCXW153678227" lang="EN-US" xml:lang="EN-US">eB</span><span class="TextRun SCXW153678227" lang="EN-US" xml:lang="EN-US">ooks. This paper reports the findings, specifically on the parents’ attitude and motivation towards reading multilingual </span><span class="TextRun SCXW153678227" lang="EN-US" xml:lang="EN-US">eBooks</span><span class="TextRun SCXW153678227" lang="EN-US" xml:lang="EN-US"> with their children. A group of 25 multi-ethnic families, together with their 5-year-old children participated in this study. A mixed-method explanatory research design obtained parent's attitude and motivation from a questionnaire, interview, and feedback form. The results show a high level of parents’ acceptance toward reading eBook with their children. Parents attributed their positive attitude and motivation towards MALL to the use of the multimedia, multilingual, and mobile platform. The MALL framework was also found to encourage parents and other adults to scaffold the children’s multilingual skills development. However, there were also setbacks toward the use of eBook as some parents expressed a stronger preference for printed books and were concerned with health issues associated with the children’s eBook use. Areas for improvements and future research are also made in this paper. </span>
Anti-inflammatory Effects of Traditional Chinese Medicines on Preclinical in vivo Models of Brain Ischemia-Reperfusion-Injury: Prospects for Neuroprotective Drug Discovery and Therapy

Acquired brain ischemia-and reperfusion-injury (IRI), including both Ischemic stroke (IS) and Traumatic Brain injury (TBI), is one of the most common causes of disability and death in adults and represents a major burden in both western and developing countries worldwide. China’s clinical neurological therapeutic experience in the use of traditional Chinese medicines (TCMs), including TCM-derived active compounds, Chinese herbs, TCM formulations and decoction, in brain IRI diseases indicated a trend of significant improvement in patients’ neurological deficits, calling for blind, placebo-controlled and randomized clinical trials with careful meta-analysis evaluation. There are many TCMs in use for brain IRI therapy in China with significant therapeutic effects in preclinical studies using different brain IRI-animal. The basic hypothesis in this field claims that in order to avoid the toxicity and side effects of the complex TCM formulas, individual isolated and identified compounds that exhibited neuroprotective properties could be used as lead compounds for the development of novel drugs. China’s efforts in promoting TCMs have contributed to an explosive growth of the preclinical research dedicated to the isolation and identification of TCM-derived neuroprotective lead compounds. Tanshinone, is a typical example of TCM-derived lead compounds conferring neuroprotection toward IRI in animals with brain middle cerebral artery occlusion (MCAO) or TBI models. Recent reports show the significance of the inflammatory response accompanying brain IRI. This response appears to contribute to both primary and secondary ischemic pathology, and therefore anti-inflammatory strategies have become popular by targeting pro-inflammatory and anti-inflammatory cytokines, other inflammatory mediators, reactive oxygen species, nitric oxide, and several transcriptional factors. Here, we review recent selected studies and discuss further considerations for critical reevaluation of the neuroprotection hypothesis of TCMs in IRI therapy. Moreover, we will emphasize several TCM’s mechanisms of action and attempt to address the most promising compounds and the obstacles to be overcome before they will enter the clinic for IRI therapy. We hope that this review will further help in investigations of neuroprotective effects of novel molecular entities isolated from Chinese herbal medicines and will stimulate performance of clinical trials of Chinese herbal medicine-derived drugs in IRI patients.
Cytokines Regulate β-Cell Thioredoxin-interacting Protein (TXNIP) via Distinct Mechanisms and Pathways*

Thioredoxin-interacting protein (TXNIP) is a key regulator of diabetic β-cell apoptosis and dysfunction, and TXNIP inhibition prevents diabetes in mouse models of type 1 and type 2 diabetes. Although we have previously shown that TXNIP is strongly induced by glucose, any regulation by the proinflammatory cytokines tumor necrosis factor α (TNFα), interleukin-1β (IL-1β), and interferon γ (IFNγ) has remained largely unexplored. Moreover, even though this three-cytokine mixture is widely used to mimic type 1 diabetes in vitro, the mechanisms involved are not fully understood. Interestingly, we have now found that this cytokine mixture increases β-cell TXNIP expression; however, although TNFα had no effect, IL-1β surprisingly down-regulated TXNIP transcription, whereas IFNγ increased TXNIP levels in INS-1 β-cells and primary islets. Human TXNIP promoter analyses and chromatin immunoprecipitation studies revealed that the IL-1β effect was mediated by inhibition of carbohydrate response element binding protein activity. In contrast, IFNγ increased pro-apoptotic TXNIP post-transcriptionally via induction of endoplasmic reticulum stress, activation of inositol-requiring enzyme 1α (IRE1α), and suppression of miR-17, a microRNA that targets and down-regulates TXNIP. In fact, miR-17 knockdown was able to mimic the IFNγ effects on TXNIP, whereas miR-17 overexpression blunted the cytokine effect. Thus, our results demonstrate for the first time that the proinflammatory cytokines TNFα, IL-1β, and IFNγ each have distinct and in part opposing effects on β-cell TXNIP expression. These findings thereby provide new mechanistic insight into the regulation of TXNIP and β-cell biology and reveal novel links between proinflammatory cytokines, carbohydrate response element binding protein-mediated transcription, and microRNA signaling.
Beta-Oxidation Is Essential for Mouse Oocyte Developmental Competence and Early Embryo Development1

Oocyte and embryo metabolism are closely linked with their subsequent developmental capacity. Lipids are a potent source of cellular energy, yet little is known about lipid metabolism during oocyte maturation and early embryo development. Generation of ATP from lipids occurs within mitochondria via beta-oxidation of fatty acids, with the rate-limiting step catalyzed by carnitine palmitoyl transferase I (CPT1B), a process also requiring carnitine. We sought to investigate the regulation and role of beta-oxidation during oocyte maturation and preimplantation development. Expression of Cpt1b mRNA, assessed by real-time RT-PCR in murine cumulus-oocyte complexes (COCs), increased following hormonal induction of oocyte maturation and ovulation in vivo with human chorionic gonadotropin (5 IU) and in embryos reaching the blastocyst stage. Beta-oxidation, measured by the production of 3H2O from [3H]palmitic acid, was significantly increased over that in immature COCs following induction of maturation in vitro with epidermal growth factor (3 ng/ml) and follicle-stimulating hormone (50 mIU/ml). The importance of lipid metabolism for oocyte developmental competence and early embryo development was demonstrated by assessing the rate of embryo development following inhibition or upregulation of beta-oxidation with etomoxir (an inhibitor of CPT1B) or l-carnitine, respectively. Inhibition of beta-oxidation during oocyte maturation or zygote cleavage impaired subsequent blastocyst development. In contrast, l-carnitine supplementation during oocyte maturation significantly increased beta-oxidation, improved developmental competence, and in the absence of a carbohydrate energy supply, significantly increased 2-cell cleavage. Thus, carnitine is an important cofactor for developing oocytes, and fatty acids are an important energy source for oocyte and embryo development.
Distribution and molecular detection of Theileria and Babesia in questing ticks from northern Spain

Abstract A total of 562 questing adult ixodid ticks, collected during 2003−05 in 10 recreational mountain areas in northern Spain, were analysed for piroplasm infection. Reverse line blot (RLB) analysis using a panel of probes for 23 piroplasm species identified 16 different piroplasms, with an overall prevalence of 9.3%. Most were Theileria spp. ‐positive (7.7%), 3.0% were positive for Babesia spp. and 1.4% of ticks harboured both genera. Ixodes ricinus (Linnaeus, 1758), the most abundant tick in the vegetation, ranked third with regard to piroplasm infection prevalence (11.4%) after Rhipicephalus bursa (Canestrini & Fanzago, 1878) (16.0%) and Haemaphysalis punctata (Canestrini & Fanzago, 1878) (13.5%). Infection was detected in 6.2% of Dermacentor reticulatus (Fabricius, 1794) and in 1.1% of Haemaphysalis inermis (Birula, 1895), but was absent from Haemaphysalis concinna (Koch, 1844). Ixodes ricinus carried more piroplasm species (13), followed by H. punctata (10), D. reticulatus (8), R. bursa (3) and H. inermis (1). Although most of the positive ticks harboured a single infection (76.9%), mixed infections with two or three different piroplasm species were also detected (23.1%). The various tick−pathogen associations found are discussed and prevalences of infection in ticks are compared with previous results on piroplasms infecting animals in the same region.