Expression of EBV Encoded viral RNA 1, 2 and anti-inflammatory Cytokine (interleukin-10) in FFPE lymphoma specimens: a preliminary study for diagnostic implication in Pakistan

<p>Abstract</p> <p>Background</p> <p>Epstein Barr Virus (EBV) plays a significant role as a cofactor in the process of tumorigenesis and has consistently been associated with a variety of malignancies. EBV encoded RNAs (EBER1 and EBER2) are the most abundant viral transcripts in latently EBV-infected cells and their role in viral infection is still unclear. Formalin Fixed Paraffin Embedded (FFPE) tissues of surgically removed carcinoma biopsies are widely available form but have never been exploited for expressional studies previously in Pakistan. Immunohistochemistry (IHC) and <it>in situ </it>hybridization (ISH) in FFPE biopsy tissues remains the gold standard for proving EBV relationship in a histopathological lesion but their reagents associated limitations confines their reliability in some applications. Recently introduced targeted drug delivery systems induce viral lytic gene expression and therefore require more sensitive method to quantify viral as well as cellular gene expression.</p> <p>Methods</p> <p>Eight (8) lymphoma samples were screened to detect the EBV genome. Qualitative and quantitative expression of EBV Encoded RNAs (EBER1, EBER2) and anti-inflammatory cytokine (interleukin-10) in FFPE EBV positive lymphoma tissue samples were then analysed by using Reverse transcriptase Polymerase Chain Reaction (RT-PCR) and Real Time Polymerase Chain Reaction (qRT-PCR), respectively.</p> <p>Results</p> <p>In this study we have successfully quantified elevated expressional levels of both cellular and viral transcripts, namely EBER1, EBER2 and anti-inflammatory cytokine (IL-10) in the FFPE Burkitt's lymphoma (BL) specimens of Pakistani origin.</p> <p>Conclusions</p> <p>These results indicate that FFPE samples may retain viral as well as cellular RNA expression information at detectable level. To our knowledge, this is first study which represents elevated expressional levels of EBER1, EBER2 and IL-10 in FFPE tissue samples of Burkitt's lymphoma in Pakistan. These observations will potentially improve current lacunas in clinical as well as diagnostic practices in Pakistan and can be further exploited to develop new strategies for studying cellular and/or viral gene expression.</p

Microfluidic Perfusion for Regulating Diffusible Signaling in Stem Cells

Background Autocrine & paracrine signaling are widespread both in vivo and in vitro, and are particularly important in embryonic stem cell (ESC) pluripotency and lineage commitment. Although autocrine signaling via fibroblast growth factor-4 (FGF4) is known to be required in mouse ESC (mESC) neuroectodermal specification, the question of whether FGF4 autocrine signaling is sufficient, or whether other soluble ligands are also involved in fate specification, is unknown. The spatially confined and closed-loop nature of diffusible signaling makes its experimental control challenging; current experimental approaches typically require prior knowledge of the factor/receptor in order to modulate the loop. A new approach explored in this work is to leverage transport phenomena at cellular resolution to downregulate overall diffusible signaling through the physical removal of cell-secreted ligands. Methodology/Principal Findings We develop a multiplex microfluidic platform to continuously remove cell-secreted (autocrine\paracrine) factors to downregulate diffusible signaling. By comparing cell growth and differentiation in side-by-side chambers with or without added cell-secreted factors, we isolate the effects of diffusible signaling from artifacts such as shear, nutrient depletion, and microsystem effects, and find that cell-secreted growth factor(s) are required during neuroectodermal specification. Then we induce FGF4 signaling in minimal chemically defined medium (N2B27) and inhibit FGF signaling in fully supplemented differentiation medium with cell-secreted factors to determine that the non-FGF cell-secreted factors are required to promote growth of differentiating mESCs. Conclusions/Significance Our results demonstrate for the first time that flow can downregulate autocrine\paracrine signaling and examine sufficiency of extracellular factors. We show that autocrine\paracrine signaling drives neuroectodermal commitment of mESCs through both FGF4-dependent and -independent pathways. Overall, by uncovering autocrine\paracrine processes previously hidden in conventional culture systems, our results establish microfluidic perfusion as a technique to study and manipulate diffusible signaling in cell systems.National Institutes of Health (U.S.) (NIH grant No. EB007278)Swiss National Science FoundationSwiss National Science Foundatio

A La Autoantigen Homologue Is Required for the Internal Ribosome Entry Site Mediated Translation of Giardiavirus

Translation of Giardiavirus (GLV) mRNA is initiated at an internal ribosome entry site (IRES) in the viral transcript. The IRES localizes to a downstream portion of 5′ untranslated region (UTR) and a part of the early downstream coding region of the transcript. Recent studies indicated that the IRES does not require a pre-initiation complex to initiate translation but may directly recruit the small ribosome subunit with the help of a number of trans-activating protein factors. A La autoantigen homologue in the viral host Giardia lamblia, GlLa, was proposed as one of the potential trans-activating factors based on its specific binding to GLV-IRES in vitro. In this study, we further elucidated the functional role of GlLa in GLV-IRES mediated translation in Giardia by knocking down GlLa with antisense morpholino oligo, which resulted in a reduction of GLV-IRES activity by 40%. An over-expression of GlLa in Giardia moderately stimulated GLV-IRES activity by 20%. A yeast inhibitory RNA (IRNA), known to bind mammalian and yeast La autoantigen and inhibit Poliovirus and Hepatitis C virus IRES activities in vitro and in vivo, was also found to bind to GlLa protein in vitro and inhibited GLV-IRES function in vivo. The C-terminal domain of La autoantigen interferes with the dimerization of La and inhibits its function. An over-expression of the C-terminal domain (200–348aa) of GlLa in Giardia showed a dominant-negative effect on GLV-IRES activity, suggesting a potential inhibition of GlLa dimerization. HA tagged GlLa protein was detected mainly in the cytoplasm of Giardia, thus supporting a primary role of GlLa in translation initiation in Giardiavirus

An exploration of the knowledge and attitudes towards breastfeeding among a sample of Chinese mothers in Ireland

<p>Abstract</p> <p>Background</p> <p>Psychological factors are important in influencing breastfeeding practices. This retrospective study explored knowledge and attitudes related to breastfeeding of Chinese mothers living in Ireland.</p> <p>Methods</p> <p>A cross-sectional self-administrated survey written in Chinese was distributed to a convenience sample of 322 immigrant Chinese mothers mainly <it>via </it>Chinese supermarkets and Chinese language schools in Dublin, with the involvement of the snowball method to increase sample size. Maternal breastfeeding knowledge and attitudes were described, their associations with socio-demographic variables were explored by Chi-square analysis, and their independent associations with breastfeeding behaviours were estimated by binary logistic regression analyses.</p> <p>Results</p> <p>In spite of considerable awareness of the advantages of breastfeeding (mean score = 4.03 ± 0.73), some misconceptions (<it>e</it>.<it>g</it>. 'mother should stop breastfeeding if she catches a cold') and negative attitudes (<it>e</it>.<it>g</it>. breastfeeding inconvenient, embarrassing, and adverse to mothers' figure) existed, especially among the less educated mothers. Cultural beliefs concerning the traditional Chinese postpartum diet were prevalent, particularly among those who had lived in Ireland for a shorter duration (P = 0.004). Psychological parameters had strong independent associations with breastfeeding practices in this study. Those who had lower awareness score (OR = 2.98, 95% CI: 1.87-4.73), more misconceptions and negative attitudes (P < 0.05), and weaker cultural beliefs (P < 0.05) were less likely to breastfeed.</p> <p>Conclusions</p> <p>Findings highlight a need to focus resources and education on correcting the misconceptions identified and reversing the negative attitudes towards breastfeeding among Chinese mothers in Ireland, in particular those with primary/secondary level of education. Mothers' cultural beliefs should also be acknowledged and understood by healthcare providers.</p

Reproducible, Ultra High-Throughput Formation of Multicellular Organization from Single Cell Suspension-Derived Human Embryonic Stem Cell Aggregates

Background: Human embryonic stem cells (hESC) should enable novel insights into early human development and provide a renewable source of cells for regenerative medicine. However, because the three-dimensional hESC aggregates [embryoid bodies (hEB)] typically employed to reveal hESC developmental potential are heterogeneous and exhibit disorganized differentiation, progress in hESC technology development has been hindered. Methodology/Principal Findings: Using a centrifugal forced-aggregation strategy in combination with a novel centrifugalextraction approach as a foundation, we demonstrated that hESC input composition and inductive environment could be manipulated to form large numbers of well-defined aggregates exhibiting multi-lineage differentiation and substantially improved self-organization from single-cell suspensions. These aggregates exhibited coordinated bi-domain structures including contiguous regions of extraembryonic endoderm- and epiblast-like tissue. A silicon wafer-based microfabrication technology was used to generate surfaces that permit the production of hundreds to thousands of hEB per cm 2. Conclusions/Significance: The mechanisms of early human embryogenesis are poorly understood. We report an ultra high throughput (UHTP) approach for generating spatially and temporally synchronised hEB. Aggregates generated in this manner exhibited aspects of peri-implantation tissue-level morphogenesis. These results should advance fundamental studies into early human developmental processes, enable high-throughput screening strategies to identify conditions tha

Macrosomia and large for gestational age in Asia:One size does not fit all

Macrosomia, usually defined as infant birth weight of >= 4000 g, does not consider gestational age, sex, or country/region-specific differences in mean birth weight and maternal body weight. This issue is particularly relevant for Asia, where 60% of the world's population lives, due to variations in maternal size and birth weights across populations. Large for gestational age (LGA), defined as birth weight > 90th centile, is a more sensitive measure as it considers gestational age and sex, though it is dependent on the choice of growth charts. We aimed to review reporting of macrosomia and LGA in Asia. We reviewed the literature on prevalence and risk of macrosomia and LGA in Asia over the last 29 years. Prevalence of macrosomia ranged from 0.5% (India) to 13.9% (China) while prevalence of LGA ranged from 4.3% (Korea) to 22.1% (China), indicating substantial variation in prevalence within and between Asian countries. High pre-pregnancy body mass index, excessive gestational weight gain, and impaired glucose tolerance conferred risk of macrosomia/LGA. Incidence of macrosomia and LGA varies substantially within and between Asian countries, as do the growth charts and definitions. The latter makes it impossible to make comparisons but suggests differences in intrauterine growth between populations. Reporting LGA, using standardized country/regional growth charts, would better capture the incidence of high birth weight and allow for comparison and identification of contributing factors. Better understanding of local drivers of excessive intrauterine growth could enable development of improved strategies for prevention and management of LGA

Pooled analysis of WHO Surgical Safety Checklist use and mortality after emergency laparotomy

Background The World Health Organization (WHO) Surgical Safety Checklist has fostered safe practice for 10 years, yet its place in emergency surgery has not been assessed on a global scale. The aim of this study was to evaluate reported checklist use in emergency settings and examine the relationship with perioperative mortality in patients who had emergency laparotomy. Methods In two multinational cohort studies, adults undergoing emergency laparotomy were compared with those having elective gastrointestinal surgery. Relationships between reported checklist use and mortality were determined using multivariable logistic regression and bootstrapped simulation. Results Of 12 296 patients included from 76 countries, 4843 underwent emergency laparotomy. After adjusting for patient and disease factors, checklist use before emergency laparotomy was more common in countries with a high Human Development Index (HDI) (2455 of 2741, 89.6 per cent) compared with that in countries with a middle (753 of 1242, 60.6 per cent; odds ratio (OR) 0.17, 95 per cent c.i. 0.14 to 0.21, P <0001) or low (363 of 860, 422 per cent; OR 008, 007 to 010, P <0.001) HDI. Checklist use was less common in elective surgery than for emergency laparotomy in high-HDI countries (risk difference -94 (95 per cent c.i. -11.9 to -6.9) per cent; P <0001), but the relationship was reversed in low-HDI countries (+121 (+7.0 to +173) per cent; P <0001). In multivariable models, checklist use was associated with a lower 30-day perioperative mortality (OR 0.60, 0.50 to 073; P <0.001). The greatest absolute benefit was seen for emergency surgery in low- and middle-HDI countries. Conclusion Checklist use in emergency laparotomy was associated with a significantly lower perioperative mortality rate. Checklist use in low-HDI countries was half that in high-HDI countries.Peer reviewe

Single-dose administration and the influence of the timing of the booster dose on immunogenicity and efficacy of ChAdOx1 nCoV-19 (AZD1222) vaccine: a pooled analysis of four randomised trials.

BACKGROUND: The ChAdOx1 nCoV-19 (AZD1222) vaccine has been approved for emergency use by the UK regulatory authority, Medicines and Healthcare products Regulatory Agency, with a regimen of two standard doses given with an interval of 4-12 weeks. The planned roll-out in the UK will involve vaccinating people in high-risk categories with their first dose immediately, and delivering the second dose 12 weeks later. Here, we provide both a further prespecified pooled analysis of trials of ChAdOx1 nCoV-19 and exploratory analyses of the impact on immunogenicity and efficacy of extending the interval between priming and booster doses. In addition, we show the immunogenicity and protection afforded by the first dose, before a booster dose has been offered. METHODS: We present data from three single-blind randomised controlled trials-one phase 1/2 study in the UK (COV001), one phase 2/3 study in the UK (COV002), and a phase 3 study in Brazil (COV003)-and one double-blind phase 1/2 study in South Africa (COV005). As previously described, individuals 18 years and older were randomly assigned 1:1 to receive two standard doses of ChAdOx1 nCoV-19 (5 × 1010 viral particles) or a control vaccine or saline placebo. In the UK trial, a subset of participants received a lower dose (2·2 × 1010 viral particles) of the ChAdOx1 nCoV-19 for the first dose. The primary outcome was virologically confirmed symptomatic COVID-19 disease, defined as a nucleic acid amplification test (NAAT)-positive swab combined with at least one qualifying symptom (fever ≥37·8°C, cough, shortness of breath, or anosmia or ageusia) more than 14 days after the second dose. Secondary efficacy analyses included cases occuring at least 22 days after the first dose. Antibody responses measured by immunoassay and by pseudovirus neutralisation were exploratory outcomes. All cases of COVID-19 with a NAAT-positive swab were adjudicated for inclusion in the analysis by a masked independent endpoint review committee. The primary analysis included all participants who were SARS-CoV-2 N protein seronegative at baseline, had had at least 14 days of follow-up after the second dose, and had no evidence of previous SARS-CoV-2 infection from NAAT swabs. Safety was assessed in all participants who received at least one dose. The four trials are registered at ISRCTN89951424 (COV003) and ClinicalTrials.gov, NCT04324606 (COV001), NCT04400838 (COV002), and NCT04444674 (COV005). FINDINGS: Between April 23 and Dec 6, 2020, 24 422 participants were recruited and vaccinated across the four studies, of whom 17 178 were included in the primary analysis (8597 receiving ChAdOx1 nCoV-19 and 8581 receiving control vaccine). The data cutoff for these analyses was Dec 7, 2020. 332 NAAT-positive infections met the primary endpoint of symptomatic infection more than 14 days after the second dose. Overall vaccine efficacy more than 14 days after the second dose was 66·7% (95% CI 57·4-74·0), with 84 (1·0%) cases in the 8597 participants in the ChAdOx1 nCoV-19 group and 248 (2·9%) in the 8581 participants in the control group. There were no hospital admissions for COVID-19 in the ChAdOx1 nCoV-19 group after the initial 21-day exclusion period, and 15 in the control group. 108 (0·9%) of 12 282 participants in the ChAdOx1 nCoV-19 group and 127 (1·1%) of 11 962 participants in the control group had serious adverse events. There were seven deaths considered unrelated to vaccination (two in the ChAdOx1 nCov-19 group and five in the control group), including one COVID-19-related death in one participant in the control group. Exploratory analyses showed that vaccine efficacy after a single standard dose of vaccine from day 22 to day 90 after vaccination was 76·0% (59·3-85·9). Our modelling analysis indicated that protection did not wane during this initial 3-month period. Similarly, antibody levels were maintained during this period with minimal waning by day 90 (geometric mean ratio [GMR] 0·66 [95% CI 0·59-0·74]). In the participants who received two standard doses, after the second dose, efficacy was higher in those with a longer prime-boost interval (vaccine efficacy 81·3% [95% CI 60·3-91·2] at ≥12 weeks) than in those with a short interval (vaccine efficacy 55·1% [33·0-69·9] at <6 weeks). These observations are supported by immunogenicity data that showed binding antibody responses more than two-fold higher after an interval of 12 or more weeks compared with an interval of less than 6 weeks in those who were aged 18-55 years (GMR 2·32 [2·01-2·68]). INTERPRETATION: The results of this primary analysis of two doses of ChAdOx1 nCoV-19 were consistent with those seen in the interim analysis of the trials and confirm that the vaccine is efficacious, with results varying by dose interval in exploratory analyses. A 3-month dose interval might have advantages over a programme with a short dose interval for roll-out of a pandemic vaccine to protect the largest number of individuals in the population as early as possible when supplies are scarce, while also improving protection after receiving a second dose. FUNDING: UK Research and Innovation, National Institutes of Health Research (NIHR), The Coalition for Epidemic Preparedness Innovations, the Bill & Melinda Gates Foundation, the Lemann Foundation, Rede D'Or, the Brava and Telles Foundation, NIHR Oxford Biomedical Research Centre, Thames Valley and South Midland's NIHR Clinical Research Network, and AstraZeneca