Investigating the functionality of an OCT4-short response element in human induced pluripotent stem cells.

Pluripotent stem cells offer great therapeutic promise for personalized treatment platforms for numerous injuries, disorders, and diseases. Octamer-binding transcription factor 4 (OCT4) is a key regulatory gene maintaining pluripotency and self-renewal of mammalian cells. With site-specific integration for gene correction in cellular therapeutics, use of the OCT4 promoter may have advantages when expressing a suicide gene if pluripotency remains. However, the human OCT4 promoter region is 4 kb in size, limiting the capacity of therapeutic genes and other regulatory components for viral vectors, and decreasing the efficiency of homologous recombination. The purpose of this investigation was to characterize the functionality of a novel 967bp OCT4-short response element during pluripotency and to examine the OCT4 titer-dependent response during differentiation to human derivatives not expressing OCT4. Our findings demonstrate that the OCT4-short response element is active in pluripotency and this activity is in high correlation with transgene expression in vitro, and the OCT4-short response element is inactivated when pluripotent cells differentiate. These studies demonstrate that this shortened OCT4 regulatory element is functional and may be useful as part of an optimized safety component in a site-specific gene transferring system that could be used as an efficient and clinically applicable safety platform for gene transfer in cellular therapeutics

Lateral Connectivity in the Olfactory Bulb is Sparse and Segregated

Lateral connections in the olfactory bulb were previously thought to be organized for center–surround inhibition. However, recent anatomical and physiological studies showed sparse and distributed interactions of inhibitory granule cells (GCs) which tended to be organized in columnar clusters. Little is known about how these distributed clusters are interconnected. In this study, we use transsynaptic tracing viruses bearing green or red fluorescent proteins to further elucidate mitral- and tufted-to-GC connectivity. Separate sites in the glomerular layer were injected with each virus. Columns with labeling from both viruses after transsynaptic spread show sparse red or green GCs which tended to be segregated. However, there was a higher incidence of co-labeled cells than chance would predict. Similar segregation of labeling is observed from dual injections into olfactory cortex. Collectively, these results suggest that neighboring mitral and tufted cells receive inhibitory inputs from segregated subsets of GCs, enabling inhibition of a center by specific and discontinuous lateral elements

Trichostatin A accentuates doxorubicin-induced hypertrophy in cardiac myocytes

Histone deacetylase inhibitors represent a new class of anticancer therapeutics and the expectation is that they will be most effective when used in combination with conventional cancer therapies, such as the anthracycline, doxorubicin. The dose-limiting side effect of doxorubicin is severe cardiotoxicity and evaluation of the effects of combinations of the anthracycline with histone deacetylase inhibitors in relevant models is important. We used a well-established in vitro model of doxorubicin-induced hypertrophy to examine the effects of the prototypical histone deacetylase inhibitor, Trichostatin A. Our findings indicate that doxorubicin modulates the expression of the hypertrophy-associated genes, ventricular myosin light chain-2, the alpha isoform of myosin heavy chain and atrial natriuretic peptide, an effect which is augmented by Trichostatin A. Furthermore, we show that Trichostatin A amplifies doxorubicin-induced DNA double strand breaks, as assessed by γH2AX formation. More generally, our findings highlight the importance of investigating potential side effects that may be associated with emerging combination therapies for cancer

Restoring Ureagenesis in Hepatocytes by CRISPR/Cas9-mediated Genomic Addition to Arginase-deficient Induced Pluripotent Stem Cells.

Urea cycle disorders are incurable enzymopathies that affect nitrogen metabolism and typically lead to hyperammonemia. Arginase deficiency results from a mutation in Arg1, the enzyme regulating the final step of ureagenesis and typically results in developmental disabilities, seizures, spastic diplegia, and sometimes death. Current medical treatments for urea cycle disorders are only marginally effective, and for proximal disorders, liver transplantation is effective but limited by graft availability. Advances in human induced pluripotent stem cell research has allowed for the genetic modification of stem cells for potential cellular replacement therapies. In this study, we demonstrate a universally-applicable CRISPR/Cas9-based strategy utilizing exon 1 of the hypoxanthine-guanine phosphoribosyltransferase locus to genetically modify and restore arginase activity, and thus ureagenesis, in genetically distinct patient-specific human induced pluripotent stem cells and hepatocyte-like derivatives. Successful strategies restoring gene function in patient-specific human induced pluripotent stem cells may advance applications of genetically modified cell therapy to treat urea cycle and other inborn errors of metabolism

Speech Processing Research Program

Contains an introduction and reports on five research projects.National Science Foundation FellowshipNational Science Foundation Grant MIP 87-14969U.S. Navy - Office of Naval Research Contract N00014-89-J-1489U.S. Air Force - Electronic Systems Division Contract F19628-89-K-0041National Science Foundation Fellowshi

Durations of asymptomatic, symptomatic, and care-seeking phases of tuberculosis disease with a Bayesian analysis of prevalence survey and notification data

This work was supported by the Wellcome Trust (206575/Z/17/Z to PM, 200901/Z/16/Z to ELC) and the UK MRC (MR/P022081/1 to PJD). This UK funded award (PJD) is part of the EDCTP2 programme supported by the European Union. KCH is supported by the European Research Council (757699) and UK FCDO (“Leaving no-one behind: transforming gendered pathways to health for TB”). This research has been partially funded by UK aid from the UK government (to KCH).Background Ratios of bacteriologically positive tuberculosis (TB) prevalence to notification rates are used to characterise typical durations of TB disease. However, this ignores the clinical spectrum of tuberculosis disease and potentially long infectious periods with minimal or no symptoms prior to care-seeking. Methods We developed novel statistical models to estimate progression from initial bacteriological positivity including smear conversion, symptom onset and initial care-seeking. Case-detection ratios, TB incidence, durations, and other parameters were estimated by fitting the model to tuberculosis prevalence survey and notification data (one subnational and 11 national datasets) within a Bayesian framework using Markov chain Monte Carlo methods. Results Analysis across 11 national datasets found asymptomatic tuberculosis durations in the range 4–8 months for African countries; three countries in Asia (Cambodia, Lao PDR, and Philippines) showed longer durations of > 1 year. For the six countries with relevant data, care-seeking typically began half-way between symptom onset and notification. For Kenya and Blantyre, Malawi, individual-level data were available. The sex-specific durations of asymptomatic bacteriologically-positive tuberculosis were 9.0 months (95% credible interval [CrI]: 7.2–11.2) for men and 8.1 months (95% CrI: 6.2–10.3) for women in Kenya, and 4.9 months (95% CrI: 2.6–7.9) for men and 3.5 months (95% CrI: 1.3–6.2) for women in Blantyre. Age-stratified analysis of data for Kenya showed no strong age-dependence in durations. For Blantyre, HIV-stratified analysis estimated an asymptomatic duration of 1.3 months (95% CrI: 0.3–3.0) for HIV-positive people, shorter than the 8.5 months (95% CrI: 5.0–12.7) for HIV-negative people. Additionally, case-detection ratios were higher for people living with HIV than HIV-negative people (93% vs 71%). Conclusion Asymptomatic TB disease typically lasts around 6 months. We found no evidence of age-dependence, but much shorter durations among people living with HIV, and longer durations in some Asian settings. To eradicate TB transmission, greater gains may be achieved by proactively screening people without symptoms through active case finding interventions.Peer reviewe

A CANDELS - 3D-HST Synergy: Resolved Star Formation Patterns at 0.7 < z < 1.5

We analyze the resolved stellar populations of 473 massive star-forming galaxies at 0.7 < z < 1.5, with multi-wavelength broad-band imaging from CANDELS and Halpha surface brightness profiles at the same kiloparsec resolution from 3D-HST. Together, this unique data set sheds light on how the assembled stellar mass is distributed within galaxies, and where new stars are being formed. We find the Halpha morphologies to resemble more closely those observed in the ACS I band than in the WFC3 H band, especially for the larger systems. We next derive a novel prescription for Halpha dust corrections, which accounts for extra extinction towards HII regions. The prescription leads to consistent SFR estimates and reproduces the observed relation between the Halpha/UV luminosity ratio and visual extinction, both on a pixel-by-pixel and on a galaxy-integrated level. We find the surface density of star formation to correlate with the surface density of assembled stellar mass for spatially resolved regions within galaxies, akin to the so-called 'main sequence of star formation' established on a galaxy-integrated level. Deviations from this relation towards lower equivalent widths are found in the inner regions of galaxies. Clumps and spiral features, on the other hand, are associated with enhanced Halpha equivalent widths, bluer colors, and higher specific star formation rates compared to the underlying disk. Their Halpha/UV luminosity ratio is lower than that of the underlying disk, suggesting the ACS clump selection preferentially picks up those regions of elevated star formation activity that are the least obscured by dust. Our analysis emphasizes that monochromatic studies of galaxy structure can be severely limited by mass-to-light ratio variations due to dust and spatially inhomogeneous star formation histories.Comment: Accepted by The Astrophysical Journal, 18 pages, 1 table, 10 figure

Client satisfaction and experience of telemedicine and home use of mifepristone and misoprostol for abortion up to 10 weeks' gestation at British Pregnancy Advisory Service: A cross-sectional evaluation.

OBJECTIVE: Evaluate satisfaction and experience with telemedicine consultation and home use of mifepristone and misoprostol for abortion to 10 weeks' gestation. STUDY DESIGN: Cross-sectional evaluation of British Pregnancy Advisory Service (BPAS) clients who used mifepristone and misoprostol at home from 11 May to 10 July 2020. We sent a text message with a link to a web-survey 2 to 3 weeks postabortion. Questions assessed satisfaction and experiences with a service model including telephone consultation and provision of medicines by mail or collection from the clinic. We used bivariate and multivariate regression to explore associations between client characteristics and outcomes. Our primary outcomes were overall satisfaction (5-point Likert scale) and reported contact with a health care provider. RESULTS: A total of 1,333 clients participated. Respondents described home use of medications as "straightforward" (75.8%) and most were "very satisfied" (78.3%) or "satisfied" (18.6%) overall. Being "very satisfied" was associated with parity (aOR 1.53, 95% CI 1.09-2.14) and pain control satisfaction (aOR 2.22, 95% CI 1.44-3.44). Health care provider contact was reported by 14.7%; mainly to BPAS' telephone aftercare service (76.8%). Dissatisfaction with pain control (aOR 3.62, 95% CI 1.79-7.29) and waiting >1 week to use mifepristone (aOR3.71, 95% CI 1.48-9.28) were associated with health care provider contact. If needed in the future, most would prefer consultation by phone (74.3%) and home use of mifepristone and misoprostol (77.8%). CONCLUSIONS: Satisfaction with telemedicine and home use of mifepristone and misoprostol is high. Most clients do not need health care provider support when administering medicines at home or post abortion

Blockade ofthe negative co-stimulatory molecules PD-1 and CTLA-4 improves survival in primary and secondary fungal sepsis

INTRODUCTION: Fungal sepsis is an increasingly common problem in intensive care unit patients.Mortality from fungal sepsis remains high despite antimicrobial therapy that is highly active against most fungal pathogens, a finding consistent with defective host immunity that is present in many patients with disseminated fungemia.One recently recognized immunologic defect that occurs in patients with sepsis is T cell "exhaustion" due to increased expression of programmed cell death -1 (PD-1).This study tested the ability of anti-PD-1 and anti-programmed cell death ligand -1 (anti-PD-L1) antagonistic antibodies to improve survival and reverse sepsis-induced immunosuppression in two mouse models of fungal sepsis. METHODS: Fungal sepsis was induced in mice using two different models of infection, that is, primary fungal sepsis and secondary fungal sepsis occurring after sub-lethal cecal ligation and puncture (CLP).Anti-PD-1 and anti-PD-L1 were administered 24 to 48 h after fungal infection and effects on survival, interferon gamma production, and MHC II expression were examined. RESULTS: Anti-PD-1 and anti-PD-L1 antibodies were highly effective at improving survival in primary and secondary fungal sepsis.Both antibodies reversed sepsis-induced suppression of interferon gamma and increased expression of MHC II on antigen presenting cells.Blockade of cytotoxic T-lymphocyte antigen-4 (CTLA-4), a second negative co-stimulatory molecule that is up-regulated in sepsis and acts like PD-1 to suppress T cell function, also improved survival in fungal sepsis. CONCLUSIONS: Immuno-adjuvant therapy with anti-PD-1, anti-PD-L1 and anti-CTLA-4 antibodies reverse sepsis-induced immunosuppression and improve survival in fungal sepsis.The present results are consistent with previous studies showing that blockade of PD-1 and CTLA-4 improves survival in bacterial sepsis.Thus, immuno-adjuvant therapy represents a novel approach to sepsis and may have broad applicability in the disorder.Given the relative safety of anti-PD-1 antibody in cancer clinical trials to date, therapy with anti-PD-1 in patients with life-threatening sepsis who have demonstrable immunosuppression should be strongly considered