Protocol for neonatal respiratory syncytial virus infection in mice and immune analysis of infected lungs and bronchoalveolar lavage fluid.

Respiratory syncytial virus (RSV) infection in infants and toddlers is a major public health problem. Here, we provide a protocol for neonatal RSV infection in mice and immune analysis of infected lungs and bronchoalveolar lavage (BAL) fluid. We describe steps for anesthesia and intranasal inoculation, weight monitoring, and whole lung collection. We then detail BAL fluid immune and whole lung analyses. This protocol can be used for neonatal pulmonary infection with other viruses or bacteria.

Expansion and mechanistic insights into de novo DEAF1 variants in DEAF1-associated neurodevelopmental disorders.

De novo deleterious and heritable biallelic mutations in the DNA binding domain (DBD) of the transcription factor deformed epidermal autoregulatory factor 1 (DEAF1) result in a phenotypic spectrum of disorders termed DEAF1-associated neurodevelopmental disorders (DAND). RNA-sequencing using hippocampal RNA from mice with conditional deletion of Deaf1 in the central nervous system indicate that loss of Deaf1 activity results in the altered expression of genes involved in neuronal function, dendritic spine maintenance, development, and activity, with reduced dendritic spines in hippocampal regions. Since DEAF1 is not a dosage-sensitive gene, we assessed the dominant negative activity of previously identified de novo variants and a heritable recessive DEAF1 variant on selected DEAF1-regulated genes in 2 different cell models. While no altered gene expression was observed in cells over-expressing the recessive heritable variant, the gene expression profiles of cells over-expressing de novo variants resulted in similar gene expression changes as observed in CRISPR-Cas9-mediated DEAF1-deleted cells. Altered expression of DEAF1-regulated genes was rescued by exogenous expression of WT-DEAF1 but not by de novo variants in cells lacking endogenous DEAF1. De novo heterozygous variants within the DBD of DEAF1 were identified in 10 individuals with a phenotypic spectrum including autism spectrum disorder, developmental delays, sleep disturbance, high pain tolerance, and mild dysmorphic features. Functional assays demonstrate these variants alter DEAF1 transcriptional activity. Taken together, this study expands the clinical phenotypic spectrum of individuals with DAND, furthers our understanding of potential roles of DEAF1 on neuronal function, and demonstrates dominant negative activity of identified de novo variants.

Enhanced preferential cytotoxicity through surface modification: synthesis, characterization and comparative in vitro evaluation of TritonX-100 modified and unmodified zinc oxide nanoparticles in human breast cancer cell (MDA-MB-231).

BACKGROUND: Nanoparticles (NPs) are receiving increasing interest in biomedical research owing to their comparable size with biomolecules, novel properties and easy surface engineering for targeted therapy, drug delivery and selective treatment making them a better substituent against traditional therapeutic agents. ZnO NPs, despite other applications, also show selective anticancer property which makes it good option over other metal oxide NPs. ZnO NPs were synthesized by chemical precipitation technique, and then surface modified using Triton X-100. Comparative study of cytotoxicity of these modified and unmodified NPs on breast cancer cell line (MDA-MB-231) and normal cell line (NIH 3T3) were carried out. RESULTS: ZnO NPsof average size 18.67 ± 2.2 nm and Triton-X modified ZnO NPs of size 13.45 ± 1.42 nm were synthesized and successful characterization of synthesized NPs was done by Fourier transform infrared spectroscopy (FT-IR), X-Ray diffraction (XRD), transmission electron microscopy (TEM) analysis. Surface modification of NPs was proved by FT-IR analysis whereas structure and size by XRD analysis. Morphological analysis was done by TEM. Cell viability assay showed concentration dependent cytotoxicity of ZnO NPs in breast cancer cell line (MDA-MB-231) whereas no positive correlation was found between cytotoxicity and increasing concentration of stress in normal cell line (NIH 3T3) within given concentration range. Half maximum effective concentration (EC50) value for ZnO NPs was found to be 38.44 µg/ml and that of modified ZnO NPs to be 55.24 µg/ml for MDA-MB-231. Crystal violet (CV) staining image showed reduction in number of viable cells in NPs treated cell lines further supporting this result. DNA fragmentation assay showed fragmented bands indicating that the mechanism of cytotoxicity is through apoptosis. CONCLUSIONS: Although use of surfactant decreases particle size, toxicity of modified ZnO NPs were still less than unmodified NPs on MDA-MB-231 contributed by biocompatible surface coating. Both samples show significantly less toxicity towards NIH 3T3 in concentration independent manner. But use of Triton-X, a biocompatible polymer, enhances this preferentiality effect. Since therapeutic significance should be analyzed through its comparative effect on both normal and cancer cells, possible application of biocompatible polymer modified nanoparticles as therapeutic agent holds better promise.Graphical abstractSurface coating, characterization and comparative in vitro cytotoxicity study on MDA-MB 231 and NIH 3T3 of ZnO NPs showing enhanced preferentiality by biocompatible surface modification.

Subject preferences of first- and second-year medical students for their future specialization at Chitwan Medical College and Teaching Hospital, Chitwan, Nepal - a questionnaire-based study.

INTRODUCTION: The selection of a discipline for future specialization may be an important factor for the medical students' future career, and it is influenced by multiple factors. The interest of students in the early stages can be improved in subjects related to public health or of academic importance, as per need. METHODS: A questionnaire-based study was conducted among 265 first- and second-year medical students of Chitwan Medical College, Nepal to find out their subject of preference for postgraduation and the factors affecting their selection along with their interesting basic science subject. Only the responses from 232 completely filled questionnaires were analyzed. RESULTS: The preference of the students for clinical surgical (50.9%), clinical medical (45.3%), and basic medical (3.9%) sciences for postgraduation were in descending order. The most preferred specialty among male students was clinical surgical sciences (56.3%), and among female students, it was clinical medical sciences (53.6%). Although all the students responded to their preferred specialty, only 178 students specified the subject of their interest. General surgery (23.4%), pediatrics (23.4%), and anatomy (2.4%) were the most favored subjects for postgraduation among clinical surgical, clinical medical, and basic medical sciences specialties, respectively. More common reasons for selection of specific subject for future career were found to be: personal interests, good income, intellectual challenge, and others. CONCLUSION: Many students preferred clinical surgical sciences for their future specialization. Among the reasons for the selection of the specialty for postgraduation, no significant reason could be elicited from the present study.