MitoVisualize: a resource for analysis of variants in human mitochondrial RNAs and DNA.

SUMMARY: We present MitoVisualize, a new tool for analysis of the human mitochondrial DNA (mtDNA). MitoVisualize enables visualization of: (i) the position and effect of variants in mitochondrial transfer RNA and ribosomal RNA secondary structures alongside curated variant annotations, (ii) data across RNA structures, such as to show all positions with disease-associated variants or with post-transcriptional modifications and (iii) the position of a base, gene or region in the circular mtDNA map, such as to show the location of a large deletion. All visualizations can be easily downloaded as figures for reuse. MitoVisualize can be useful for anyone interested in exploring mtDNA variation, though is designed to facilitate mtDNA variant interpretation in particular. AVAILABILITY AND IMPLEMENTATION: MitoVisualize can be accessed via https://www.mitovisualize.org/. The source code is available at https://github.com/leklab/mito_visualize/. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Development of a text message-based intervention for follow-up colposcopy among predominately underserved Black and Hispanic/Latinx women.

PURPOSE: Underserved Black and Hispanic/Latinx women show low rates of follow-up care after an abnormal Pap test, despite the fact that cervical cancer is one of the few preventable cancers if detected early. However, extant literature falls short on efficacious interventions to increase follow-up for this population. A concurrent mixed methods study was completed to evaluate the acceptability of a text message-based intervention and identify perceived barriers and facilitators to follow-up after an abnormal Pap test among underserved predominantly Black and Hispanic/Latinx women. METHODS: Patients who completed follow-up for an abnormal Pap test were recruited to complete a cross-sectional survey, qualitative interview assessing barriers and facilitators to follow-up, and text message content evaluation (N = 28). Descriptive statistics were performed to describe background variables and to evaluate the acceptability of text messages. A directed content analysis was completed for the qualitative interviews. RESULTS: Participants expressed interest in a text message-based intervention to increase abnormal Pap test follow-up. In the qualitative interviews, low knowledge about cervical risk and negative affect toward colposcopy/test results were identified as barriers to follow-up. Facilitators of follow-up included feeling relieved after the colposcopy and adequate social support. Participants rated the text messages as understandable, personally relevant, and culturally appropriate. CONCLUSION: The findings suggest that underserved Black and Hispanic/Latinx women experience cognitive and emotional barriers that undermine their ability to obtain follow-up care and a text message-based intervention may help women overcome these barriers. Future research should develop and evaluate text message-based interventions to enhance follow-up after an abnormal Pap test.

The mechanism of the ultrafast crystal growth of pure metals from their melts.

Pure metals can have ultrafast growth rates from their melts, such as a crystal of pure nickel that grows at a rate reaching 70 m s-1. These extraordinary growth rates suggest that metallic crystals might provide the next generation of phase-change materials. The huge crystal growth rates of metals are the consequence of kinetics without activated control, in sharp contrast to the prediction of the 'classic' theory of crystal growth. While the existence of barrierless growth kinetics is now well established in atomic melts, the physical explanation for the absence of an activation barrier to ordering remains unclear. It is something of a paradox that diffusion in the liquid metal is governed by thermal activation while the movement of the same atoms organizing into a crystal is not. Here we use computer simulations of crystallization in pure metals to explicitly resolve the origin of the barrierless growth kinetics.

Baseline characteristics and hospitalizations in patients with schizophrenia receiving olanzapine long-acting injection: an interim analysis from a non-interventional, prospective observational safety study.

BACKGROUND: Depot antipsychotics are a treatment option for medication nonadherence in patients with schizophrenia. Nonadherence can lead to increased relapse and hospitalization rates. This article reports hospitalization data before and after initiation of olanzapine long-acting injection (LAI), a depot antipsychotic. METHODS: Data were assessed from an ongoing, multinational, prospective, observational post-authorisation safety study being conducted to evaluate post-injection delirium/sedation syndrome (PDSS), an adverse reaction that can occur following injection of olanzapine LAI. Eligible patients were aged ≥18 years, diagnosed with schizophrenia, were prescribed olanzapine LAI, and lived outside the United States. Psychiatric hospitalization and medication data were collected retrospectively for the 6-month period before study entry and prospectively throughout the study. Paired t-tests and McNemar's tests were used to assess changes in hospitalization incidence and duration. Stepwise Cox proportional hazards models assessed factors associated with hospitalizations. Analyses were based on data from the first 3 years of the continuously enrolling study (N = 668). RESULTS: The average duration of olanzapine LAI exposure for all patients was 0.768 years. Of the 529 patients who received at least 1 injection of olanzapine LAI and were not hospitalized at study entry, 8.1% had at least 1 subsequent psychiatric hospitalization with a mean duration of 2.0 days. Of the 288 patients who had a >6-month follow-up, 8.3% had at least 1 post-baseline psychiatric hospitalization with a mean duration of 2.3 days. The incidence of hospitalizations in the 6-month period after treatment was significantly lower than that in the 6-month period prior to treatment (8.3 vs 32.6%, respectively; P < 0.001). Furthermore, mean hospitalization duration decreased from 11.5 days in the 6-month period before treatment to 2.3 days in the 6-month period after treatment (P < 0.001). Psychiatric hospitalization in the prior 12 months (P < 0.0001) and recreational drug use within 24 h of baseline visit (P = 0.015) were identified as potential predictors of time to first psychiatric hospitalization after beginning to take olanzapine LAI. At the time of interim analysis, 5 PDSS events had occurred, which was too few for a full analysis of those events. CONCLUSIONS: Results indicate a significant reduction in the incidence and days of hospitalization from the 6-month period before to the 6-month period after olanzapine LAI initiation, which suggests reduced relapse and hospitalization during treatment. Results should be interpreted with caution due to the observational nature of the study and use of retrospective baseline data.

Dynamic, sex- and diet-specific pleiotropism in the PAC1 receptor-mediated regulation of arcuate proopiomelanocortin and Neuropeptide Y/Agouti related peptide neuronal excitability by anorexigenic ventromedial nucleus PACAP neurons.

This study furthers the investigation of how pituitary adenylate cyclase activating polypeptide (PACAP) and the PAC1 receptor (PAC1R) regulate the homeostatic energy balance circuitry. We hypothesized that apoptotic ablation of PACAP neurones in the hypothalamic ventromedial nucleus (VMN) would affect both energy intake and energy expenditure. We also hypothesized that selective PAC1R knockdown would impair the PACAP-induced excitation in anorexigenic proopiomelanocortin (POMC) neurones and inhibition of orexigenic neuropeptide Y (NPY)/agouti-related peptide (AgRP) neurones in the hypothalamic arcuate nucleus (ARC). The results show CASPASE-3-induced ablation of VMN PACAP neurones leads to increased energy intake and meal frequency as well as decreased energy expenditure in lean animals. The effects were more robust in obese males, whereas we saw the opposite effects in obese females. We then utilized visualized whole-cell patch clamp recordings in hypothalamic slices. PAC1R knockdown in POMC neurones diminishes the PACAP-induced depolarization, increase in firing, decreases in energy intake and meal size, as well as increases in CO2 production and O2 consumption. Similarly, the lack of expression of the PAC1R in NPY/AgRP neurones greatly attenuates the PACAP-induced hyperpolarization, suppression of firing, decreases in energy intake and meal frequency, as well as increases in energy expenditure. The PACAP response in NPY/AgRP neurones switched from predominantly inhibitory to excitatory in fasted animals. Finally, the anorexigenic effect of PACAP was potentiated when oestradiol was injected into the ARC in ovariectomized females. This study demonstrates the critical role of anorexigenic VMN PACAP neurones and the PAC1R in exciting POMC and inhibiting NPY/AgRP neurons to control homeostatic feeding.

Development and qualification of cell-based relative potency assay for a human respiratory syncytial virus (RSV) mRNA vaccine.

Respiratory syncytial virus (RSV) is a leading cause of severe lower respiratory tract infections worldwide. A safe and effective RSV vaccine has been an elusive goal but recent advances in vaccine technology have improved the likelihood that a vaccine for the prevention of RSV could be licensed in near future. We have developed an RSV vaccine V171 consisting of four lipids and messenger ribonucleic acid (mRNA) encoding an engineered form of the RSV F protein stabilized in its prefusion conformation. The lipids form lipid nanoparticles (LNP) with mRNA encapsulated during process, which protects the mRNA from degradation and enables the mRNA to be delivered into mammalian cells. Once inside the cells, the mRNA then can be translated into RSV F protein and elicit both humoral and cellular immune responses. Preclinical results and Phase I clinical trial results indicate that this mRNA vaccine targeting RSV F protein is a promising RSV vaccine approach and should be further evaluated in clinical trials. We have developed a cell-based relative potency assay to support the Phase II development of this vaccine. Test articles and a reference standard are tested with serial dilutions in a 96-well plate pre-seeded with Hep G2 cells. Cells were incubated for 16-18 h after transfection and then permeabilized and stained with a human monoclonal antibody specific to RSV F protein, followed by a fluorophore-conjugated secondary antibody. The plate is then analyzed for percentage of transfected cells and relative potency of the test article is calculated by comparing its EC50 to that of a reference standard. This assay takes advantage of the fact that due to the inherent variability in biological test systems an absolute measure of potency is more variable than a measure of activity relative to a standard. Targeting testing relative potency range 25-250 %, our assay showed an R2 close to 1 for linearity, relative bias of 1.05-5.41 %, and intermediate precision of 11.0 %. The assay has been used for testing of process development samples, formulation development samples, as well as drug product intermediate (DPI) and drug product (DP) in support of Phase II development of our RSV mRNA vaccine.

Deep Mutational Scanning in Disease-related Genes with Saturation Mutagenesis-Reinforced Functional Assays (SMuRF).

Interpretation of disease-causing genetic variants remains a challenge in human genetics. Current costs and complexity of deep mutational scanning methods hamper crowd-sourcing approaches toward genome-wide resolution of variants in disease-related genes. Our framework, Saturation Mutagenesis-Reinforced Functional assays (SMuRF), addresses these issues by offering simple and cost-effective saturation mutagenesis, as well as streamlining functional assays to enhance the interpretation of unresolved variants. Applying SMuRF to neuromuscular disease genes FKRP and LARGE1, we generated functional scores for over 99.8% of all possible coding single nucleotide variants and resolved 310 clinically reported variants of uncertain significance with high confidence, enhancing clinical variant interpretation in dystroglycanopathies. SMuRF also demonstrates utility in predicting disease severity, resolving critical structural regions, and providing training datasets for the development of computational predictors. Our approach opens new directions for enabling variant-to-function insights for disease genes in a manner that is broadly useful for crowd-sourcing implementation across standard research laboratories.

The Association between Patient Characteristics and the Efficacy and Safety of Selinexor in Diffuse Large B-Cell Lymphoma in the SADAL Study.

Selinexor, an oral selective inhibitor of nuclear export, was evaluated in the Phase 2b SADAL study in patients with diffuse large B-cell lymphoma (DLBCL) who previously received two to five prior systemic regimens. In post hoc analyses, we analyzed several categories of patient characteristics (age, renal function, DLBCL subtype, absolute lymphocyte count, transplant status, number of prior lines of therapy, refractory status, Ann Arbor disease stage, and lactate dehydrogenase) at baseline, i.e., during screening procedures, to determine their potential contributions to the efficacy (overall response rate [ORR], duration of response [DOR], overall survival [OS]) and tolerability of selinexor. Across most categories of characteristics, no significant difference was observed in ORR or DOR. OS was significantly longer for patients < 65 vs. ≥ 65 years, and for those with lymphocyte counts ≥ 1000/µL vs. < 1000/µL or lactate dehydrogenase ≤ ULN vs. > ULN. The most common adverse events (AEs) across the characteristics were thrombocytopenia and nausea, and similar rates of grade 3 AEs and serious AEs were observed. With its oral administration, novel mechanism of action, and consistency in responses in heavily pretreated patients, selinexor may help to address an important unmet clinical need in the treatment of DLBCL.

Social and medical risk factors associated with supportive needs in the first year following localized prostate cancer treatment.

PURPOSE: Individuals who completed treatment for prostate cancer (PCa) often report poor coping and practical concerns when adapting to new roles in their lives-and strong patient-provider communication is critical for this period. However, there is limited research identifying factors associated with supportive needs after the completion of PCa treatment. This study aimed to identify the social and medical risk factors associated with supportive needs for adapting among individuals who completed treatment for localized PCa. METHODS: Using baseline data from a study evaluating a web-based support system for patients in the first year following treatment for localized PCa, self-efficacy for re-entry (e.g., maintaining relationships, symptom management), medical interactions, and practical concerns (e.g., insurance, exercise) were assessed. Multivariable regression analyses were completed to identify risk factors for low readiness. RESULTS: Participants (N = 431) with lower health literacy or income or with depressive symptoms had lower self-efficacy for re-entry, more negative interactions with medical providers, and more practical concerns (ps < .05). Lastly, non-Hispanic White participants reported greater readiness compared with all other races (ps < .05). CONCLUSIONS: Multiple social and medical risk factors are associated with greater supportive needs when adapting to new roles after PCa treatment. Understanding the risk factors for supportive needs in this period is critical. Future research is needed to help providers identify and support individuals at risk for poorer coping and greater practical concerns after treatment completion. IMPLICATIONS FOR CANCER SURVIVORS: Identifying individuals with greater supportive needs following treatment for localized PCa treatment will help ensure successful adaptation to new roles.

Duckweed hosts a taxonomically similar bacterial assemblage as the terrestrial leaf microbiome.

Culture-independent characterization of microbial communities associated with popular plant model systems have increased our understanding of the plant microbiome. However, the integration of other model systems, such as duckweed, could facilitate our understanding of plant microbiota assembly and evolution. Duckweeds are floating aquatic plants with many characteristics, including small size and reduced plant architecture, that suggest their use as a facile model system for plant microbiome studies. Here, we investigated the structure and assembly of the duckweed bacterial microbiome. First, a culture-independent survey of the duckweed bacterial microbiome from different locations in New Jersey revealed similar phylogenetic profiles. These studies showed that Proteobacteria is a dominant phylum in the duckweed bacterial microbiome. To observe the assembly dynamics of the duckweed bacterial community, we inoculated quasi-gnotobiotic duckweed with wastewater effluent from a municipal wastewater treatment plant. Our results revealed that duckweed strongly shapes its bacterial microbiome and forms distinct associations with bacterial community members from the initial inoculum. Additionally, these inoculation studies showed the bacterial communities of different duckweed species were similar in taxa composition and abundance. Analysis across the different duckweed bacterial communities collected in this study identified a set of "core" bacterial taxa consistently present on duckweed irrespective of the locale and context. Furthermore, comparison of the duckweed bacterial community to that of rice and Arabidopsis revealed a conserved taxonomic structure between the duckweed microbiome and the terrestrial leaf microbiome. Our results suggest that duckweeds utilize similar bacterial community assembly principles as those found in terrestrial plants and indicate a highly conserved structuring effect of leaf tissue on the plant microbiome.

Bacterial Production of Indole Related Compounds Reveals Their Role in Association Between Duckweeds and Endophytes.

Duckweed farming can be a sustainable practice for biofuel production, animal feed supplement, and wastewater treatment, although large scale production remains a challenge. Plant growth promoting bacteria (PGPB) have been shown to improve plant health by producing phytohormones such as auxin. While some of the mechanisms for plant growth promotion have been characterized in soil epiphytes, more work is necessary to understand how plants may select for bacterial endophytes that have the ability to provide an exogenous source of phytohormones such as auxin. We have isolated and characterized forty-seven potentially endophytic bacteria from surface-sterilized duckweed tissues and screened these bacterial strains for production of indole related compounds using the Salkowski colorimetric assay. Indole-3-acetic acid (IAA), indole-3-lactic acid (ILA), and indole produced by various bacterial isolates were verified by mass spectrometry. Using the Salkowski reagent, we found that 79% of the isolated bacterial strains from our collection may be capable of producing indole related compounds to various extents during in vitro growth. Of these bacteria that are producing indole related compounds, 19% are additionally producing indole. There is an apparent correlation between the type of indole related compound produced by a particular bacteria and the duckweed genus from which the bacterial strain is derived. These results suggest the possible association between different duckweed genera and endophytes that are producing distinct types of secondary metabolites. Understanding the role of indole related compounds during interaction between endophytes and the plant host may be useful to help design synthetic bacterial communities that could target specific or multiple species of duckweed in the future to sustainably enhance plant growth.

The capacity to form H-DNA cannot substitute for GAGA factor binding to a (CT)n*(GA)n regulatory site.

Previous studies of the Drosophila melanogaster hsp26 gene promoter have demonstrated the importance of a homopurine*homopyrimidine segment [primarily (CT)n*(GA)n] for chromatin structure formation and gene activation. (CT)n regions are known to bind GAGA factor, a dominant enhancer of PEV thought to play a role in generating an accessible chromatin structure. The (CT)n region can also form an H-DNA structure in vitro under acidic pH and negative supercoiling; a detailed map of that structure is reported here. To test whether the (CT)n sequence can function through H-DNA in vivo, we have analyzed a series of hsp26-lacZ transgenes with altered sequences in this region. The results indicate that a 25 bp mirror repeat within the homopurine.homopyrimidine region, while adequate for H-DNA formation, is neither necessary nor sufficient for positive regulation of hsp26 when GAGA factor-binding sites have been eliminated. The ability to form H-DNA cannot substitute for GAGA factor binding to the (CT)n sequence.

Using QPCR to assign infectious potencies to adenovirus based vaccines and vectors for gene therapy: toward a universal method for the facile quantitation of virus and vector potency.

The assignment of infectious potency to test articles of adenovirus has been conducted mainly using classical end-point dilution methods, which rely on virus induced cytopathology to reveal the presence of infectious virus. These assays suffer the disadvantages of labor intensity, duration, throughput restriction and variability. In the course of our development of an Ad5 based HIV vaccine for clinical evaluation, we sought a facile method for the assignment of potency to the numerous test articles generated during the development of bioprocesses for bulk manufacture, downstream purification and formulation. In this paper we describe a quantitative PCR based potency assay (QPA) which uses QPCR to quantitate adenovirus genomes replicated 24h after the inoculation of a test article on 293 cell monolayers, and then relates that mass to potency by interpolation to a standard curve of replicated adenovirus genomes constructed with a reference adenovirus standard to which infectious potency has been previously assigned in the classical end-point dilution assay. The QPA assay for adenovirus is simple and rapid, with a throughput capacity adequate to the potency assay demands of bioprocess development, and with a precision expressed as a root variability of 16.8% R.S.D., allowing for close discriminations of the products of alternative process configurations. The adenovirus QPA principle can be applied to the quantitation of infectious potency of both RNA and DNA viruses and we report briefly on the development of QPA assays for measles and mumps. QPA assays owing to their simplicity and easy automation, rapidity, capacity and precision hold promise to become widely practiced methods for the quantitation of the potency of live virus vaccines and other recombinant virus vectors.