12/28/2023 0 Comments Premier biosoft beacon designer 8.02![]() The immature brain is particularly susceptible to HI injury. Therefore, more in-depth research into neuronal cell death and the mechanisms of brain injury after HI is warranted in order to develop more effective therapies for preventing and treating neonatal brain injury. Although erythropoietin treatment has demonstrated remarkable neuroprotection in infants, the window of opportunity and optimal dosage is still controversial. Therapeutic hypothermia within 6 h of hypoxia–ischemia (HI) onset has been clinically shown to be a promising therapeutic intervention, but it only reduces the risk of death and disability by about 11%, meaning that up to 40% of the treated infants still develop neurological deficits. HIE is a global problem with an estimated incidence ranging from 1 to live births in developed countries to live births in underdeveloped countries. It is a major cause of mortality in neonates and can result in profound and devastating lifelong mental and physical disabilities, including cerebral palsy, seizures, and cognitive impairments in both term and preterm neonates. Hypoxic–ischemic encephalopathy (HIE) is a severe central nervous system injury caused by oxygen deprivation and limited blood flow in the neonatal brain. Altogether, these findings corroborate earlier studies and further demonstrate that AIF is involved in oxidative stress, which contributes to the sex-specific differences observed in neonatal HI brain injury. We also found that AIF stimulated carbohydrate metabolism in young males. Under physiological conditions (without HI), the doublecortin-positive area in the dentate gyrus of females was 1.15 times larger than in males, indicating that AIF upregulation effectively promoted neurogenesis in females in the long term. As compared to females, male mice exhibited more severe brain injury, correlating with reduced antioxidant capacities, more pronounced protein carbonylation and nitration, and increased neuronal cell death. We found that the male sex significantly aggravated AIF-driven brain damage, as indicated by the injury volume in the gray matter (2.25 times greater in males) and by the lost volume of subcortical white matter (1.71 greater in males) after HI. Based on previous findings that AIF overexpression aggravates neonatal HI brain injury, we further investigated potential sex differences in the severity and molecular mechanisms underlying the injury using mice that overexpress AIF from homozygous transgenes. Gene expression analysis of individual cells enables characterization of heterogeneous and rare cell populations, yet widespread implementation of existing single-cell gene analysis techniques has been hindered due to limitations in scale, ease, and cost.There are sex differences in the severity, mechanisms, and outcomes of neonatal hypoxia–ischemia (HI) brain injury, and apoptosis-inducing factor (AIF) may play a critical role in this discrepancy. Here, we present a novel microdroplet-based, one-step reverse-transcriptase polymerase chain reaction (RT-PCR) platform and demonstrate the detection of three targets simultaneously in over 100,000 single cells in a single experiment with a rapid read-out. Our customized reagent cocktail incorporates the bacteriophage T7 gene 2.5 protein to overcome cell lysate-mediated inhibition and allows for one-step RT-PCR of single cells encapsulated in nanoliter droplets. Fluorescent signals indicative of gene expressions are analyzed using a probabilistic deconvolution method to account for ambient RNA and cell doublets and produce single-cell gene signature profiles, as well as predict cell frequencies within heterogeneous samples. ![]() We also developed a simulation model to guide experimental design and optimize the accuracy and precision of the assay. ![]() ![]() Using mixtures of in vitro transcripts and murine cell lines, we demonstrated the detection of single RNA molecules and rare cell populations at a frequency of 0.1%. This low cost, sensitive, and adaptable technique will provide an accessible platform for high throughput single-cell analysis and enable a wide range of research and clinical applications.
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