Time-dependent patterning of the mesoderm and endoderm by Nodal signals in zebrafish

BACKGROUND: The vertebrate body plan is generated during gastrulation with the formation of the three germ layers. Members of the Nodal-related subclass of the TGF-β superfamily induce and pattern the mesoderm and endoderm in all vertebrates. In zebrafish, two nodal-related genes, called squint and cyclops, are required in a dosage-dependent manner for the formation of all derivatives of the mesoderm and endoderm. These genes are expressed dynamically during the blastula stages and may have different roles at different times. This question has been difficult to address because conditions that alter the timing of nodal-related gene expression also change Nodal levels. We utilized a pharmacological approach to conditionally inactivate the ALK 4, 5 and 7 receptors during the blastula stages without disturbing earlier signaling activity. This permitted us to directly examine when Nodal signals specify cell types independently of dosage effects. RESULTS: We show that two drugs, SB-431542 and SB-505124, completely block the response to Nodal signals when added to embryos after the mid-blastula transition. By blocking Nodal receptor activity at later stages, we demonstrate that Nodal signaling is required from the mid-to-late blastula period to specify sequentially, the somites, notochord, blood, Kupffer's vesicle, hatching gland, heart, and endoderm. Blocking Nodal signaling at late times prevents specification of cell types derived from the embryo margin, but not those from more animal regions. This suggests a linkage between cell fate and length of exposure to Nodal signals. Confirming this, cells exposed to a uniform Nodal dose adopt progressively more marginal fates with increasing lengths of exposure. Finally, cell fate specification is delayed in squint mutants and accelerated when Nodal levels are elevated. CONCLUSION: We conclude that (1) Nodal signals are most active during the mid-to-late blastula stages, when nodal-related gene expression and the movement of responding cells are at their most dynamic; (2) Nodal signals specify cell fates along the animal-vegetal axis in a time-dependent manner; (3) cells respond to the total cumulative dose of Nodal signals to which they are exposed, as a function of distance from the source and duration of exposure

O-GlcNAc modifications regulate cell survival and epiboly during zebrafish development

<p>Abstract</p> <p>Background</p> <p>The post-translational addition of the monosaccharide O-linked β-<it>N</it>-acetylglucosamine (O-GlcNAc) regulates the activity of a wide variety of nuclear and cytoplasmic proteins. The enzymes O-GlcNAc Transferase (Ogt) and O-GlcNAcase (Oga) catalyze, respectively, the attachment and removal of O-GlcNAc to target proteins. In adult mice, Ogt and Oga attenuate the response to insulin by modifying several components of the signal transduction pathway. Complete loss of <it>ogt </it>function, however, is lethal to mouse embryonic stem cells, suggesting that the enzyme has additional, unstudied roles in development. We have utilized zebrafish as a model to determine role of O-GlcNAc modifications in development. Zebrafish has two <it>ogt </it>genes, encoding six different enzymatic isoforms that are expressed maternally and zygotically.</p> <p>Results</p> <p>We manipulated O-GlcNAc levels in zebrafish embryos by overexpressing zebrafish <it>ogt</it>, human <it>oga </it>or by injecting morpholinos against <it>ogt </it>transcripts. Each of these treatments results in embryos with shortened body axes and reduced brains at 24 hpf. The embryos had 23% fewer cells than controls, and displayed increased rates of cell death as early as the mid-gastrula stages. An extensive marker analysis indicates that derivatives of three germ layers are reduced to variable extents, and the embryos are severely disorganized after gastrulation. Overexpression of Ogt and Oga delayed epiboly and caused a severe disorganization of the microtubule and actin based cytoskeleton in the extra-embryonic yolk syncytial layer (YSL). The cytoskeletal defects resemble those previously reported for embryos lacking function of the Pou5f1/Oct4 transcription factor <it>spiel ohne grenzen</it>. Consistent with this, Pou5f1/Oct4 is modified by O-GlcNAc in human embryonic stem cells.</p> <p>Conclusion</p> <p>We conclude that O-GlcNAc modifications control the activity of proteins that regulate apoptosis and epiboly movements, but do not seem to regulate germ layer specification. O-GlcNAc modifies the transcription factor Spiel ohne grenzen/Pou5f1 and may regulate its activity.</p

Publisher Correction: A novel therapeutic antibody screening method using bacterial high-content imaging reveals functional antibody binding phenotypes of Escherichia coli ST131.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.</jats:p

A novel therapeutic antibody screening method using bacterial high-content imaging reveals functional antibody binding phenotypes of Escherichia coli ST131

Funder: National Institute for Health Research; doi: http://dx.doi.org/10.13039/501100000272Funder: MRC Proximity to Discovery: Industry Engagement Fund Biomedical Research Exchange ProgrammeAbstract: The increase of antimicrobial resistance (AMR), and lack of new classes of licensed antimicrobials, have made alternative treatment options for AMR pathogens increasingly attractive. Recent studies have demonstrated anti-bacterial efficacy of a humanised monoclonal antibody (mAb) targeting the O25b O-antigen of Escherichia coli ST131. To evaluate the phenotypic effects of antibody binding to diverse clinical E. coli ST131 O25b bacterial isolates in high-throughput, we designed a novel mAb screening method using high-content imaging (HCI) and image-based morphological profiling to screen a mAb targeting the O25b O-antigen. Screening the antibody against a panel of 86 clinical E. coli ST131 O25:H4 isolates revealed 4 binding phenotypes: no binding (18.60%), weak binding (4.65%), strong binding (69.77%) and strong agglutinating binding (6.98%). Impaired antibody binding could be explained by the presence of insertion sequences or mutations in O-antigen or lipopolysaccharide core biosynthesis genes, affecting the amount, structure or chain length of the O-antigen. The agglutinating binding phenotype was linked with lower O-antigen density, enhanced antibody-mediated phagocytosis and increased serum susceptibly. This study highlights the need to screen candidate mAbs against large panels of clinically relevant isolates, and that HCI can be used to evaluate mAb binding affinity and potential functional efficacy against AMR bacteria

Tensiomyography Derived Parameters Reflect Skeletal Muscle Architectural Adaptations Following 6-Weeks of Lower Body Resistance Training

Measurement of muscle specific contractile properties in response to resistance training (RT) can provide practitioners valuable information regarding physiological status of individuals. Field based measurements of such contractile properties within specific muscle groups, could be beneficial when monitoring efficacy of training or rehabilitation interventions. Tensiomyography (TMG) quantifies contractile properties of individual muscles via an electrically stimulated twitch contraction and may serve as a viable option in the aforementioned applications. Thus, aims of this study were; (i) to investigate the potential use of TMG to quantify training adaptations and differences, in response to exercise specific lower limb RT; and (ii) investigate any associations between TMG parameters and accompanying muscle architectural measures. Non-resistance trained male participants (n = 33) were randomly assigned to 1 of 3 single-exercise intervention groups (n = 11 per group); back squat (BS), deadlift (DL), or hip thrust (HT). Participants completed a 6-week linearized training program (2× per week), where the assigned exercise was the sole method of lower body training. Pre- and post-intervention testing of maximal dynamic strength was assessed by one repetition maximum (1RM) of BS, DL, and HT. Radial muscle belly displacement (Dm) and contraction time (Tc) were obtained via TMG from the rectus femoris (RF) and vastus lateralis (VL) pre- and post-intervention, alongside muscle architectural measures (pennation angle and muscle thickness). All three groups displayed significant increases all 1RM strength tests (p < 0.001; pη2 = 0.677–0.753). Strength increases were accompanied by significant overall increases in RF muscle thickness (p < 0.001, pη2 = 0.969), and pennation angle (p = 0.007, pη2 = 0.220). Additionally, an overall reduction in RF Dm (p < 0.001, pη2 = 0.427) was observed. Significant negative relationships were observed between RF Dm and pennation angle (p = 0.003, r = −0.36), and with RF Dm and muscle thickness (p < 0.001, r = −0.50). These findings indicate that TMG is able to detect improved contractile properties, alongside improvements in muscle function within an untrained population. Furthermore, the observed associations between Dm and muscle architecture suggest that TMG contractile property assessments could be used to obtain information on muscle geometry

Chemical Synergy between Ionophore PBT2 and Zinc Reverses Antibiotic Resistance.

The World Health Organization reports that antibiotic-resistant pathogens represent an imminent global health disaster for the 21st century. Gram-positive superbugs threaten to breach last-line antibiotic treatment, and the pharmaceutical industry antibiotic development pipeline is waning. Here we report the synergy between ionophore-induced physiological stress in Gram-positive bacteria and antibiotic treatment. PBT2 is a safe-for-human-use zinc ionophore that has progressed to phase 2 clinical trials for Alzheimer's and Huntington's disease treatment. In combination with zinc, PBT2 exhibits antibacterial activity and disrupts cellular homeostasis in erythromycin-resistant group A Streptococcus (GAS), methicillin-resistant Staphylococcus aureus (MRSA), and vancomycin-resistant Enterococcus (VRE). We were unable to select for mutants resistant to PBT2-zinc treatment. While ineffective alone against resistant bacteria, several clinically relevant antibiotics act synergistically with PBT2-zinc to enhance killing of these Gram-positive pathogens. These data represent a new paradigm whereby disruption of bacterial metal homeostasis reverses antibiotic-resistant phenotypes in a number of priority human bacterial pathogens.IMPORTANCE The rise of bacterial antibiotic resistance coupled with a reduction in new antibiotic development has placed significant burdens on global health care. Resistant bacterial pathogens such as methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus are leading causes of community- and hospital-acquired infection and present a significant clinical challenge. These pathogens have acquired resistance to broad classes of antimicrobials. Furthermore, Streptococcus pyogenes, a significant disease agent among Indigenous Australians, has now acquired resistance to several antibiotic classes. With a rise in antibiotic resistance and reduction in new antibiotic discovery, it is imperative to investigate alternative therapeutic regimens that complement the use of current antibiotic treatment strategies. As stated by the WHO Director-General, "On current trends, common diseases may become untreatable. Doctors facing patients will have to say, Sorry, there is nothing I can do for you.

Food Insecurity Prevalence Across Diverse Sites During COVID-19: A Year of Comprehensive Data

Key Findings NFACT includes 18 study sites in 15 states as well as a national poll, collectively representing a sample size of more than 26,000 people. Some sites have implemented multiple survey rounds, here we report results from 22 separate surveys conducted during the year since the COVID-19 pandemic began in March 2020. 18 out of 19 surveys in 14 sites with data for before and since the pandemic began found an increase in food insecurity since the start of the COVID-19 pandemic as compared to before the pandemic. In nearly all surveys (18/19) that measured food insecurity both before and during the pandemic, more Black, Indigenous, and People of Color (BIPOC) were classified as food insecure during the pandemic as compared to before it began. Prevalence of food insecurity for BIPOC respondents was higher than the overall population in the majority of surveys (19/20) sampling a general population. In almost all surveys (21/22), the prevalence of food insecurity for households with children was higher than the overall prevalence of food insecurity. Food insecurity prevalence was higher for households experiencing a negative job impact during the pandemic (i.e. job loss, furlough, reduction in hours) in nearly all surveys and study sites (21/22). Food insecurity prevalence in most sites was significantly higher before COVID-19 than estimates from that time period. Reporting a percent change between pre and during COVID-19 prevalence may provide additional information about the rate of change in food insecurity since the start of the pandemic, which absolute prevalence of food insecurity may not capture. Results highlight consistent trends in food insecurity outcomes since the start of the COVID-19 pandemic, across diverse study sites, methodological approaches, and time

Screening of healthcare workers for SARS-CoV-2 highlights the role of asymptomatic carriage in COVID-19 transmission

Significant differences exist in the availability of healthcare worker (HCW) SARS-CoV-2 testing between countries, and existing programmes focus on screening symptomatic rather than asymptomatic staff. Over a 3-week period (April 2020), 1,032 asymptomatic HCWs were screened for SARS-CoV-2 in a large UK teaching hospital. Symptomatic staff and symptomatic household contacts were additionally tested. Real-time RT-PCR was used to detect viral RNA from a throat+nose self-swab. 3% of HCWs in the asymptomatic screening group tested positive for SARS-CoV-2. 17/30 (57%) were truly asymptomatic/pauci-symptomatic. 12/30 (40%) had experienced symptoms compatible with coronavirus disease 2019 (COVID-19) &gt;7 days prior to testing, most self-isolating, returning well. Clusters of HCW infection were discovered on two independent wards. Viral genome sequencing showed that the majority of HCWs had the dominant lineage B·1. Our data demonstrates the utility of comprehensive screening of HCWs with minimal or no symptoms. This approach will be critical for protecting patients and hospital staff