Satb2 acts as a gatekeeper for major developmental transitions during early vertebrate embryogenesis

Zygotic genome activation (ZGA) initiates regionalized transcription underlying distinct cellular identities. ZGA is dependent upon dynamic chromatin architecture sculpted by conserved DNA-binding proteins. However, the direct mechanistic link between the onset of ZGA and the tissue-specific transcription remains unclear. Here, we have addressed the involvement of chromatin organizer Satb2 in orchestrating both processes during zebrafish embryogenesis. Integrative analysis of transcriptome, genome-wide occupancy and chromatin accessibility reveals contrasting molecular activities of maternally deposited and zygotically synthesized Satb2. Maternal Satb2 prevents premature transcription of zygotic genes by influencing the interplay between the pluripotency factors. By contrast, zygotic Satb2 activates transcription of the same group of genes during neural crest development and organogenesis. Thus, our comparative analysis of maternal versus zygotic function of Satb2 underscores how these antithetical activities are temporally coordinated and functionally implemented highlighting the evolutionary implications of the biphasic and bimodal regulation of landmark developmental transitions by a single determinant

Optimization and Stability Testing of Four Commercially Available Dried Blood Spot Devices for Estimating Measles and Rubella IgG Antibodies.

Blood collection using dried blood spots (DBS) provides an easier alternative to venipuncture for sample collection, transport, and storage but requires additional processing that can cause variability in results. Whole-blood samples spotted on four DBS devices and respective paired serum samples were tested for antimeasles and antirubella IgG antibody concentrations by enzyme immunoassay. Elution protocols for DBS devices were optimized for comparability relative to serum samples using 12 adult volunteers. Stability of DBS collected on HemaSpot HF was assessed under various temperature conditions (+4, 22 to 25, and 45°C) at six time points (0, 7, 15, 30, 60, and 90 days) in a controlled laboratory setting using six adult volunteers. Devices were shipped and stored for 30 days at four settings with variable temperature and humidity conditions to assess the impact on antibody concentrations. Three DBS devices demonstrated comparable antibody concentrations with paired sera following optimization. Antibodies recovered from DBS were stable for at least 90 days at 4°C and for 30 days at ambient temperature (22 to 25°C) using the HemaSpot HF device. A drastic decline in antibody concentrations was observed at 45°C, resulting in quantitative and qualitative discrepancies by day 7. HemaSpot HF devices shipped to field sites and stored at ambient temperature and humidity resulted in quantitative, but not qualitative, variability. Measurement of antimeasles and antirubella IgG antibodies with DBS devices is an accurate alternative to testing serum, provided elution protocols are optimized. Stability of HemaSpot HF devices at ambient temperature enables broader use in surveys when serum processing and cold storage are not feasible. IMPORTANCE Dried blood spot (DBS) collection offers various advantages over conventional methods of blood collection, especially when collecting and transporting samples for a serosurvey. Yet use of DBS requires additional processing steps in the laboratory that can add to variability in results. We optimized a protocol to elute IgG antibodies against measles and rubella viruses in four DBS devices, demonstrating high concordance with paired venous sera for most devices. Extensive stability studies with various temperature and storage conditions in the laboratory and in the field were conducted using HemaSpot HF DBS devices prior to its use in one of the largest community-based measles and rubella serological surveys in the world

Implementing Serosurveys in India: Experiences, Lessons Learned, and Recommendations.

Serological surveillance for vaccine-preventable diseases, such as measles and rubella, can provide direct measures of population immunity across age groups, identify gaps in immunity, and document changes in immunity over time. Rigorously conducted, representative household serosurveys provide high-quality estimates with minimal bias. However, they can be logistically challenging, expensive, and have higher refusal rates than vaccine coverage surveys. This article shares lessons learned through implementing nine measles and rubella household serosurveys in five districts in India-the challenges faced, the potential impact on results, and recommendations to facilitate the conduct of serosurveys. Specific lessons learned arose from challenges related to community mobilization owing to lack of cooperation in certain settings and populations, limitations of outdated census information, nonresponse due to refusal or unavailability during survey enumeration and enrollment, data collection issues, and specimen collection and handling issues. Although some experiences are specific to serosurveys in India, these lessons are generalizable to other household surveys, particularly vaccination coverage and serosurveys conducted in low- and middle-income settings

Diagnostic Accuracy of Dried Blood Spots Collected on HemaSpot HF Devices Compared to Venous Blood Specimens To Estimate Measles and Rubella Seroprevalence.

Fingerprick blood spotted onto filter paper offers an alternative to venous blood for use in population-based surveillance because it is comparatively inexpensive, acceptable, and easy to manage in the field. Prior studies have shown excellent agreement for immunoglobulin G (IgG) antibody detection from dried blood spots (DBS) and venous blood samples. However, much of this evidence is from high-income settings or laboratories where the samples were unlikely to be exposed to extreme temperatures and humidity, factors known to degrade DBS. We report the diagnostic accuracy of DBS collected using HemaSpot HF devices against venous sera in measuring measles- and rubella-specific IgG antibodies in a household serosurvey conducted in two districts in India. Paired serum and DBS samples collected by fingerprick were collected from women aged 15 to 50 years enrolled in a serosurvey in Palghar District of Maharashtra and Kanpur Nagar District of Uttar Pradesh in India. Specimen quality and volume were assessed in the laboratory. Samples were tested for antimeasles and antirubella IgG antibodies by an enzyme-linked immunosorbent assay (ELISA) (Euroimmun). Sensitivity of antibody detection by DBS was greater than 98%, and specificity was 90% and 98%, for measles and rubella IgG, respectively. Antibody concentrations were strongly correlated between paired specimens with adequate volume (measles R2 = 0.94; rubella R2 = 0.89). Although correlation was poor if DBS specimens had lower volumes, impact on qualitative results was minimal. This study showed DBS collected with HemaSpot HF devices can generate highly accurate results of measles- and rubella-specific IgG compared to sera in community-based surveys when protocols are optimized for DBS specimens. IMPORTANCE Dried blood spot (DBS) collection provides an easy, practical, and acceptable alternative to venous blood collection, especially for community-based studies, provided that results from DBS are accurate. We demonstrated high sensitivity and specificity for measles- and rubella-specific immunoglobulin G (IgG) with DBS collected via HemaSpot HF devices compared to serum samples. This is one of the largest community-based diagnostic accuracy studies of measles and rubella antibody testing with DBS and the first application we are aware of using HemaSpot HF device for measles and rubella serology. Results support the use of DBS in community-based serosurveillance

ZNF423 patient variants, truncations, and in-frame deletions in mice define an allele-dependent range of midline brain abnormalities.

Interpreting rare variants remains a challenge in personal genomics, especially for disorders with several causal genes and for genes that cause multiple disorders. ZNF423 encodes a transcriptional regulatory protein that intersects several developmental pathways. ZNF423 has been implicated in rare neurodevelopmental disorders, consistent with midline brain defects in Zfp423-mutant mice, but pathogenic potential of most patient variants remains uncertain. We engineered ~50 patient-derived and small deletion variants into the highly-conserved mouse ortholog and examined neuroanatomical measures for 791 littermate pairs. Three substitutions previously asserted pathogenic appeared benign, while a fourth was effectively null. Heterozygous premature termination codon (PTC) variants showed mild haploabnormality, consistent with loss-of-function intolerance inferred from human population data. In-frame deletions of specific zinc fingers showed mild to moderate abnormalities, as did low-expression variants. These results affirm the need for functional validation of rare variants in biological context and demonstrate cost-effective modeling of neuroanatomical abnormalities in mice

A Prospective Observational Study Assessing the Impacts of Health Literacy and Psychosocial Determinants of Health on 30-day Readmission Risk

Our objective was to assess the utility of an assessment battery capturing health literacy (HL) and biopsychosocial determinants of health in predicting 30-day readmission in comparison to a currently well-adopted readmission risk calculator. We also sought to capture the distribution of inpatient HL, with emphasis on inadequate and marginal HL (an intermediate HL level). A prospective observational study was conducted to obtain HL and biopsychosocial data on general medicine inpatients admitted to the UCLA health system. Five hundred thirty-seven subjects were tracked prospectively for 30-day readmission after index hospitalization. HL was significantly better at predicting readmission compared to LACE + (Length, admission acuity, comorbidities, emergency room visits) alone (P = .013). A multivariate model including education, insurance, and language comfort was a strong predictor of adequate HL (P < .001). In conclusion, HL offered significant improvement in risk stratification in comparison to LACE + alone. Patients with marginal HL were high-risk, albeit difficult to characterize. Incorporating robust HL and biopsychosocial determinant assessments may allow hospital systems to allocate educational resources towards at-risk patients, thereby mitigating readmission risk

Myosin Vb Mediated Plasma Membrane Homeostasis Regulates Peridermal Cell Size and Maintains Tissue Homeostasis in the Zebrafish Epidermis

<div><p>The epidermis is a stratified epithelium, which forms a barrier to maintain the internal milieu in metazoans. Being the outermost tissue, growth of the epidermis has to be strictly coordinated with the growth of the embryo. The key parameters that determine tissue growth are cell number and cell size. So far, it has remained unclear how the size of epidermal cells is maintained and whether it contributes towards epidermal homeostasis. We have used genetic analysis in combination with cellular imaging to show that zebrafish <i>goosepimples/myosin Vb</i> regulates plasma membrane homeostasis and is involved in maintenance of cell size in the periderm, the outermost epidermal layer. The decrease in peridermal cell size in Myosin Vb deficient embryos is compensated by an increase in cell number whereas decrease in cell number results in the expansion of peridermal cells, which requires <i>myosin Vb (myoVb)</i> function. Inhibition of cell proliferation as well as cell size expansion results in increased lethality in larval stages suggesting that this two-way compensatory mechanism is essential for growing larvae. Our analyses unravel the importance of Myosin Vb dependent cell size regulation in epidermal homeostasis and demonstrate that the epidermis has the ability to maintain a dynamic balance between cell size and cell number.</p></div

Schematic representation of <i>myosin Vb</i> function in the epidermis and its link with tissue homeostasis.

<p>In wild-type peridermal cells the endocytosis and recycling is balanced maintaining the plasma membrane homeostasis (A). In Myosin Vb deficient embryos, there is increased endocytosis and reduction in recycling leading to accumulation of endosomes, which are targeted for degradation in lysosomes (A). As a consequence the plasma membrane homeostasis is perturbed leading to shrinkage of cell surface area, which results in loss of membrane projections and in cell-bulging (A). Reduction in cell surface area in <i>gsp/myoVb</i> is compensated by increase in peridermal cell number. In contrast decrease in cell number results in bigger cells in the periderm (B). Epidermis maintains the dynamic balance between cell size and cell number.</p

Reduction in endocytosis mitigates the cellular and morphological phenotype in Myosin Vb deficient embryos.

<p>Endocytosis of tracers WGA (A,B,E,F) and Dextran (C,D,G,H) in wild type (A,E, C,G) and <i>myoVb</i> morphant embryos (B,F,D,H) treated with DMSO (A,B,C,D) and dynasore (E,F,G,H) in the LynEGFP background. The dynasore treatment decreases endocytosis of both WGA and Dextran. Note that in wild type larvae there is hardly any dextran uptake indicating low rate of fluid phase endocytosis. As compared to wild type, WGA uptake appears less in the morphant. The reason being that the mucous layer is reduced in the mutants. The LynEGFP staining (I,J,M,N) in wild type (I,M) and <i>myosin Vb</i> morphants (J,N) treated with DMSO (I,J) and dynasore (M,N). Cell proliferation analysis using BrdU incorporation (K,L,O,P) in wild type (K,O) and <i>myoVb</i> morphant (L,P) treated with DMSO (K,L) and dynasore (O,P) in LynEGFP background. Note the decrease in proliferation in the morphant periderm upon dynasore treatment. Quantification of WGA and Dextran (Q) uptake by peridermal cells in different genetic backgrounds and under different treatments mentioned along the X axis. The first graph shows the number of cells per field showing uptake of the tracers. The second graph represents the number of vesicles per cell. For this analysis only the cells showing tracer uptake were considered. Quantification of area and cell proliferation for genetic backgrounds and various treatments shown along the X-axis (R). Quantification of embryos showing various degrees of phenotypic strengths (S) at 40 hpf (first graph) and 48 hpf (second graph). The morphant embryos were categorised in three phenotypic classes- strong, mild and no phenotype- based on the head periderm phenotype. The embryos classified as “no phenotypes” exhibited the decreased fin expansion and occasionally a few rounded up cells in the finfold. Bright field images showing strong (T) and mild (U) phenotypes. Scale bar in F, H corresponds to 10 µ in A,B,E,F and C,D,G,H, respectively whereas scale bars in N,P corresponds to 20 µ in I,J,M,N and K,L,O,P, respectively. Square brakets and associated aseterisks in Q and R represent the comparison by T-test and significant difference (P≤0.05), respectively.</p