Public Interest in Acetyl Hexapeptide-8: Longitudinal Analysis.

BACKGROUND: Acetyl hexapeptide-8, also known as Argireline, is a topical, short-acting, synthetic peptide that has recently gained popularity for its antiwrinkle effects. This agent has emerged as a more accessible alternative to botulinum neurotoxin. OBJECTIVE: This study evaluates the public interest in acetyl hexapeptide-8 in the United States from 2013 to 2023, as described by search volume on Google, the most-used search engine. METHODS: We analyzed the longitudinal relative monthly search volume from January 1, 2013, to January 1, 2023, for acetyl hexapeptide-related terms. We compared the internet search trends for "Botox" during this period to "Argireline." RESULTS: The terms "Argireline" and "Botox in a Bottle" both had substantial increases in search volume in 2022. Although its search volume is drastically increasing, "Argireline" was less searched than "Botox," which had a stable, up-trending search volume over the past decade. CONCLUSIONS: The increasing interest in acetyl hexapeptide-8 may be due to its cost-effectiveness and use as a botulinum neurotoxin alternative. Affordability, over-the-counter availability, and ease of self-application of the agent suggest its potential to enhance accessibility to cosmetic dermatologic care.

Integrative epigenome profiling of 47XXY provides insights into whole genomic DNA hypermethylation and active chromatin accessibility.

Klinefelter syndrome (KS, 47XXY) is a disorder characterized by sex chromosomal aneuploidy, which may lead to changes in epigenetic regulations of gene expression. To define epigenetic architectures in 47XXY, we annotated DNA methylation in euploid males (46XY) and females (46XX), and 47XXY individuals using whole genome bisulfite sequencing (WGBS) and integrated chromatin accessbilty, and detected abnormal hypermethylation in 47XXY. Furthermore, we detected altered chromatin accessibility in 47XXY, in particular in chromosome X, using Assay for Transposase-Accessible Chromatin sequencing (ATAC-seq) in cultured amniotic cells. Our results construct the whole genome-wide DNA methylation map in 47XXY, and provide new insights into the early epigenomic dysregulation resulting from an extra chromosome X in 47XXY.

A Knock-In Mouse Model of Thymoma With the GTF2I L424H Mutation.

INTRODUCTION: The pathogenesis of thymic epithelial tumors remains largely unknown. We previously identified GTF2I L424H as the most frequently recurrent mutation in thymic epithelial tumors. Nevertheless, the precise role of this mutation in tumorigenesis of thymic epithelial cells is unclear. METHODS: To investigate the role of GTF2I L424H mutation in thymic epithelial cells in vivo, we generated and characterized a mouse model in which the Gtf2i L424H mutation was conditionally knocked-in in the Foxn1+ thymic epithelial cells. Digital spatial profiling was performed on thymomas and normal thymic tissues with GeoMx-mouse whole transcriptome atlas. Immunohistochemistry staining was performed using both mouse tissues and human thymic epithelial tumors. RESULTS: We observed that the Gtf2i mutation impairs development of the thymic medulla and maturation of medullary thymic epithelial cells in young mice and causes tumor formation in the thymus of aged mice. Cell cycle-related pathways, such as E2F targets and MYC targets, are enriched in the tumor epithelial cells. Results of gene set variation assay analysis revealed that gene signatures of cortical thymic epithelial cells and thymic epithelial progenitor cells are also enriched in the thymomas of the knock-in mice, which mirrors the human counterparts in The Cancer Genome Atlas database. Immunohistochemistry results revealed similar expression pattern of epithelial cell markers between mouse and human thymomas. CONCLUSIONS: We have developed and characterized a novel thymoma mouse model. This study improves knowledge of the molecular drivers in thymic epithelial cells and provides a tool for further study of the biology of thymic epithelial tumors and for development of novel therapies.

The regulation of CD73 in non-small cell lung cancer.

BACKGROUND: Lung cancer is the leading cause of global cancer-related mortality. Although immune checkpoint therapy has achieved remarkable results in lung cancer, EGFR-mutant or ALK-positive non-smallcell lung cancer patients show limited benefit. Besides the low tumor mutational burden, PD-L1 expression and CD8+ tumor-infiltrating T cells, upregulation of CD73/adenosine pathway also contributes to the immune-inert microenvironment of EGFR-mutant NSCLC. However, the detailed mechanism underlying the regulation of CD73 is unclear. METHODS: TCGA data was used to analyze the CD73 expression in cancer patients. Western blotting, qPCR, and ChIP-PCR were performed in multiple NSCLC cancer cell lines and patient derived organoids were used to explore the regulation of CD73 expression using western blotting. RESULTS: CD73 expression was highly expressed in multiple cancer types. Pharmacological or genetic inhibition of EGFR, MEK, KRAS, or ALK dramatically reduced the CD73 mRNA and protein expression in NSCLC cancer cells and patient-derived organoids with EGFR mutation, KRAS mutation or ALK-rearrangement. C-Jun overexpression-induced CD73 mRNA and protein expression. ChIP assay showed that c-Jun bind to CD73 genomic regions. CONCLUSIONS: Higher CD73 expression in NSCLC cancer cells and patient-derived organoids with EGFR mutation, KRAS mutation or ALK-rearrangement. Mechanistically, CD73 is regulated by ERK-Jun pathway, wherein c-Jun regulates CD73 expression via binding to CD73 genomic regions.

Single-Cell Transcriptomics of Cultured Amniotic Fluid Cells Reveals Complex Gene Expression Alterations in Human Fetuses With Trisomy 18.

Trisomy 18, commonly known as Edwards syndrome, is the second most common autosomal trisomy among live born neonates. Multiple tissues including cardiac, abdominal, and nervous systems are affected by an extra chromosome 18. To delineate the complexity of anomalies of trisomy 18, we analyzed cultured amniotic fluid cells from two euploid and three trisomy 18 samples using single-cell transcriptomics. We identified 6 cell groups, which function in development of major tissues such as kidney, vasculature and smooth muscle, and display significant alterations in gene expression as detected by single-cell RNA-sequencing. Moreover, we demonstrated significant gene expression changes in previously proposed trisomy 18 critical regions, and identified three new regions such as 18p11.32, 18q11 and 18q21.32, which are likely associated with trisomy 18 phenotypes. Our results indicate complexity of trisomy 18 at the gene expression level and reveal genetic reasoning of diverse phenotypes in trisomy 18 patients.

Rapid Overlap Initiation Protocol Using Low Dose Buprenorphine for Opioid Use Disorder Treatment in an Outpatient Setting: A Case Series.

OBJECTIVES: Fear and risk of precipitated withdrawal are barriers for initiating buprenorphine in individuals with opioid use disorder, particularly among those using fentanyl. A buprenorphine rapid overlap initiation (ROI) protocol (also knownas "rapidmicro-dosing") utilizing small, escalating doses of buprenorphine can overcome this barrier, reaching therapeutic doses in 3 to 4 days. We sought to demonstrate the feasibility of implementing a buprenorphine ROI protocol for buprenorphine initiation in the outpatient setting. METHODS: We conducted a retrospective chart review of patients prescribed an outpatient ROI protocol at the Office-based Buprenorphine Induction Clinic from October to December 2020. The ROI protocol utilizes divided doses of sublingual buprenorphine tablets and blister packaging for easier dosing. Patients were not required to stop other opioid use and were advised to follow up on day 4 of initiation. RESULTS: Twelve patients were included, of whom eleven (92%) were using fentanyl at intake. Eleven patients picked up their prescription. Seven patients returned for follow-up (58%), and all 7 completed the ROI protocol. One patient reported any withdrawal symptoms, which were mild. At 30 days, 7 patients (58%) were retained in care, and 5 (42%) were still receiving buprenorphine treatment, 4 (33%) of whom had been abstinent from nonprescribed opioid use for ≥2 weeks. CONCLUSIONS: The ROI protocol was successful in initiating buprenorphine treatment for patients in our outpatient clinic, many of whom were using fentanyl. The ROI protocol may offer a safe alternative to traditional buprenorphine initiation and warrants further study.

Exploiting mesothelin in thymic carcinoma as a drug delivery target for anetumab ravtansine.

BACKGROUND: Thymic epithelial tumours (TETs) are rare tumours comprised of thymomas and thymic carcinoma. Novel therapies are needed, especially in thymic carcinoma where the 5-year survival rate hovers at 30%. Mesothelin (MSLN), a surface glycoprotein that is cleaved to produce mature MSLN (mMSLN) and megakaryocyte potentiating factor (MPF), is expressed in limited tissues. However, its expression is present in various cancers, including thymic carcinoma, where it is expressed in 79% of cases. METHODS: We utilised flow cytometry, in vitro cytotoxicity assays, and an in vivo xenograft model in order to demonstrate the ability of the MSLN targeting antibody-drug conjugate (ADC) anetumab ravtansine (ARav) in inhibiting the growth of thymic carcinoma. RESULTS: Thymoma and thymic carcinoma cell lines express MSLN, and anetumab, the antibody moiety of ARav, was capable of binding MSLN expressing thymic carcinoma cells and internalising. ARav was effective at inhibiting the growth of thymic carcinoma cells stably transfected with mMSLN in vitro. In vivo, 15 mg/kg ARav inhibited T1889 xenograft tumour growth, while combining 7.5 mg/kg ARav with 4 mg/kg cisplatin yielded an additive effect on inhibiting tumour growth. CONCLUSIONS: These data demonstrate that anetumab ravtansine inhibits the growth of MSLN positive thymic carcinoma cells in vitro and in vivo.

Distinct roles of Fto and Mettl3 in controlling development of the cerebral cortex through transcriptional and translational regulations.

Proper development of the mammalian cerebral cortex relies on precise gene expression regulation, which is controlled by genetic, epigenetic, and epitranscriptomic factors. Here we generate RNA demethylase Fto and methyltransferase Mettl3 cortical-specific conditional knockout mice, and detect severe brain defects caused by Mettl3 deletion but not Fto knockout. Transcriptomic profiles using RNA sequencing indicate that knockout of Mettl3 causes a more dramatic alteration on gene transcription than that of Fto. Interestingly, we conduct ribosome profiling sequencing, and find that knockout of Mettl3 leads to a more severe disruption of translational regulation of mRNAs than deletion of Fto and results in altered translation of crucial genes in cortical radial glial cells and intermediate progenitors. Moreover, Mettl3 deletion causes elevated translation of a significant number of mRNAs, in particular major components in m6A methylation. Our findings indicate distinct functions of Mettl3 and Fto in brain development, and uncover a profound role of Mettl3 in regulating translation of major mRNAs that control proper cortical development.

Prenatal stress leads to deficits in brain development, mood related behaviors and gut microbiota in offspring.

Early exposure to stressful and adverse life events at fetal and neonatal stages is one of crucial risk factors for mood disorders such as anxiety and depressive disorder in adulthood. Intergenerational effects of prenatal stress on offspring are still not fully understood. We here uncover a significant negative impact of prenatal stress on brain development in embryos and newborns, and on mood-related behaviors and gut microbiota in adult offspring. Prenatal stress leads to reduced numbers in neural progenitors and newborn neurons, and altered gene expression profiles in the mouse embryonic cerebral cortex. Adult mouse offspring exposed to prenatal stress displays altered gene expression in the cortex and elevated responses in anxiety- and depression-like behaviors. Interestingly, prenatal stress has an enduring effect on gut microbiota, as specific microbial community structure is altered in adult F1 offspring treated with prenatal stress, compared to that of the control. Our results highlight the essential impact of prenatal stress on cortical neurogenesis, gene expression patterns, mood-related behaviors, and even gut microbiota in the next generation.

Identifying cortical specific long noncoding RNAs modified by m6A RNA methylation in mouse brains.

Proper development of the mammalian cerebral cortex relies on precise gene expression regulation. Increasing evidence shows that cortical development is regulated by both mRNAs and long noncoding RNAs (lncRNAs), which also are modified by N6-methyladenosine (m6A). Patterns of m6A-methylation in lncRNAs in the developing cortex have not been uncovered. Here we reveal differentially expressed lncRNAs and report stage-specific m6A-methylation patterns in lncRNAs expressed in mouse embryonic (E) and postnatal (P) cortices using RNA sequencing (RNA-seq) and methylated RNA immunoprecipitation (MeRIP) sequencing. Many lncRNAs show temporal differential expression, and display genic distribution in the genome. Interestingly, we detect temporal-specific m6A-methylation with consensus m6A motif GGACU in the last exon in most lncRNAs. And m6A methylation levels of lncRNAs are positively correlated with the transcript abundance of lncRNAs that have no significantly differential expression in E- and P-stages. Furthermore, the transcript abundance has a positive correlation between the m6A genic lncRNAs and their nearest m6A methylated mRNAs. Our work reveals a fundamental expression reference of lncRNAs and their nearest mRNAs, and highlights an importance of m6A-mediated epitranscriptomic modifications in lncRNAs that are temporally expressed in the developing cortex.

Unique and Specific m6A RNA Methylation in Mouse Embryonic and Postnatal Cerebral Cortices.

N6-methyladenosine (m6A)-mediated epitranscriptomic regulation is critical for various physiological processes. Genetic studies demonstrate that proper m6A-methylation is required for mouse brain development and function. Revealing landscapes of m6A-methylation in the cerebral cortex at different developmental stages will help to understand the biological meaning of epitranscriptomic regulation. Here, we depict the temporal-specific m6A-methylation status in mouse embryonic and postnatal cortices using methylated RNA immunoprecipitation (MeRIP) sequencing. We identified unique m6A binding motifs in stage-specific RNAs and found that more RNA transcripts are temporally methylated in embryonic cortices than in postnatal ones. Moreover, we found that cortical transcription factors and genes associated with neurological disorders are broadly as well specifically methylated at m6A sites. Our study highlights the importance of epitranscriptomic regulation in the developing cortex and provides a fundamental reference for future mechanistic examinations of m6A methylation-mediated gene expression regulation in normal brain development and neurological disorders.

Single-nucleotide editing: From principle, optimization to application.

Cytosine base editors (CBEs) and adenine base editors (ABEs), which are generally composed of an engineered deaminase and a catalytically impaired CRISPR-Cas9 variant, are new favorite tools for single base substitution in cells and organisms. In this review, we summarize the principle of base-editing systems and elaborate on the evolution of different platforms of CBEs and ABEs, including their deaminase, Cas9 variants, and editing outcomes. Moreover, we highlight their applications in mouse and human cells and discuss the challenges and prospects of base editors. The ABE- and CBE systems have been used in gene silencing, pathogenic gene correction, and functional genetic screening. Single base editing is becoming a new promising genetic tool in biomedical research and gene therapy.

m6A RNA Methylation Controls Neural Development and Is Involved in Human Diseases.

RNA modifications are involved in many aspects of biological functions. N6-methyladenosine (m6A) is one of the most important forms of RNA methylation and plays a vital role in regulating gene expression, protein translation, cell behaviors, and physiological conditions in many species, including humans. The dynamic and reversible modification of m6A is conducted by three elements: methyltransferases ("writers"), such as methyltransferase-like protein 3 (METTL3) and METTL14; m6A-binding proteins ("readers"), such as the YTH domain family proteins (YTHDFs) and YTH domain-containing protein 1 (YTHDC1); and demethylases ("erasers"), such as fat mass and obesity-associated protein (FTO) and AlkB homolog 5 (ALKBH5). In this review, we summarize the current knowledge on mapping mRNA positions of m6A modification and revealing molecular processes of m6A. We further highlight the biological significance of m6A modification in neural cells during development of the nervous system and its association with human diseases. m6A RNA methylation is becoming a new frontier in neuroscience and should help us better understand neural development and neurological diseases from a novel point of view.

Counter-Balance Between Gli3 and miR-7 Is Required for Proper Morphogenesis and Size Control of the Mouse Brain.

Brain morphogenesis requires precise regulation of multiple genes to control specification of distinct neural progenitors (NPs) and neuronal production. Dysregulation of these genes results in severe brain malformation such as macrocephaly and microcephaly. Despite studies of the effect of individual pathogenic genes, the counter-balance between multiple factors in controlling brain size remains unclear. Here we show that cortical deletion of Gli3 results in enlarged brain and folding structures in the cortical midline at the postnatal stage, which is mainly caused by the increased percentage of intermediate progenitors (IPs) and newborn neurons. In addition, dysregulation of neuronal migration also contributes to the folding defects in the cortical midline region. Knockdown of microRNA (miRNA) miR-7 can rescue abnormal brain morphology in Gli3 knockout mice by recovering progenitor specification, neuronal production and migration through a counter-balance of the Gli3 activity. Moreover, miR-7 likely exerts its function through silencing target gene Pax6. Our results indicate that proper brain morphogenesis is an outcome of interactive regulations of multiple molecules such as Gli3 and miR-7. Because miRNAs are easy to synthesize and deliver, miR-7 could be a potential therapeutic means to macrocephaly caused by Gli3-deficiency.

Opposite Roles of Wnt7a and Sfrp1 in Modulating Proper Development of Neural Progenitors in the Mouse Cerebral Cortex.

The Wingless (Wnt)-mediated signals are involved in many important aspects of development of the mammalian cerebral cortex. How Wnts interact with their modulators in cortical development is still unclear. Here, we show that Wnt7a and secreted frizzled-related protein 1 (Sfrp1), a soluble modulator of Wnts, are co-expressed in mouse embryonic cortical neural progenitors (NPs). Knockout of Wnt7a in mice causes microcephaly due to reduced NP population and neurogenesis, and Sfrp1 has an opposing effect compared to Wnt7a. Similar to Dkk1, Sfrp1 decreases the Wnt1 and Wnt7a activity in vitro. Our results suggest that Wnt7a and Sfrp1 play opposite roles to ensure proper NP progeny in the developing cortex.

Perception and reality of particulate matter exposure in New York City taxi drivers.

Exposure to fine particulate matter (PM2.5) and black carbon (BC) have been linked to negative health risks, but exposure among professional taxi drivers is understudied. This pilot study measured drivers' knowledge, attitudes, and beliefs (KAB) about air pollution compared with direct measures of exposures. Roadside and in-vehicle levels of PM2.5 and BC were continuously measured over a single shift on each subject, and exposures compared with central site monitoring. One hundred drivers completed an air pollution KAB questionnaire, and seven taxicabs participated in preliminary in-cab air sampling. Taxicab PM2.5 and BC concentrations were elevated compared with nearby central monitoring. Average PM2.5 concentrations per 15-min interval were 4-49 µg/m3. BC levels were also elevated; reaching>10 µg/m3. Fifty-six of the 100 drivers surveyed believed they were more exposed than non-drivers; 81 believed air pollution causes health problems. Air pollution exposures recorded suggest that driver exposures would likely exceed EPA recommendations if experienced for 24 h. Surveys indicated that driver awareness of this was limited. Future studies should focus on reducing exposures and increasing awareness among taxi drivers.

Impact of Reperfusion Calcium and pH on the Resuscitation of Hearts Donated After Circulatory Death.

BACKGROUND: Hearts donated after circulatory death may represent an additional donor source. The influx of sodium and calcium ions across the sarcolemma play a central role in the pathogenesis of ischemia-reperfusion injury; however, this process may be inhibited if the initial reperfusion solution is rendered hypocalcemic and acidic. We sought to determine the calcium concentration and pH of the initial reperfusion solution that yielded optimal functional recovery of hearts donated after circulatory death during ex vivo heart perfusion. METHODS: Pigs were anesthetized, mechanical ventilation was discontinued, and a 15-minute standoff period was observed after circulatory arrest. Hearts were reperfused with a normothermic cardioplegia of varying calcium concentrations (part 1 [50 µmol/L, n = 4; 220 µmol/L, n = 9; 500 µmol/L, n = 4; and 1,250 µmol/L, n = 5]) and pH (part 2 [7.9, n = 5; 7.4, n = 9; 6.9, n = 8; and 6.4, n = 6]). Myocardial function was then assessed in a physiologic working model 1 hour after initiation of normothermic ex vivo heart perfusion. RESULTS: The calcium concentration and pH of the cardioplegic solution affected the development of myocardial edema (part 1: 50 µmol/L = 5.8% ± 0.9%; 220 µmol/L = 4.3% ± 0.4%; 500 µmol/L = 7.0% ± 0.6%; and 1,250 µmol/L = 6.6% ± 0.8% weight gain, p = 0.015; part 2: 7.9 = 3.6% ± 0.4%, 7.4 = 4.3% ± 0.4%, 6.9 = 3.7% ± 0.6%, and 6.4 = 6.4% ± 1.3% weight gain, p = 0.056) and the recovery of myocardial function (cardiac index part 1: 50 µmol/L = 2.6 ± 0.6; 220 µmol/L = 6.0 ± 0.8; 500 µmol/L = 2.3 ± 0.5; and 1,250 µmol/L = 1.9 ± 0.6 mL · m-1 · g-1, p < 0.001; part 2: 7.9 = 1.5 ± 0.7; 7.4 = 6.0 ± 0.8; 6.9 = 8.4 ± 1.8; and 6.4 = 3.1 ± 0.8 mL · m-1 · g-1, p = 0.003) during ex vivo heart perfusion. CONCLUSIONS: Initial reperfusion of hearts donated after circulatory death with a hypocalcemic and moderately acidic cardioplegia minimizes edema and optimizes functional recovery during subsequent ex vivo heart perfusion.

High Spinal Anesthesia Enhances Anti-Inflammatory Responses in Patients Undergoing Coronary Artery Bypass Graft Surgery and Aortic Valve Replacement: Randomized Pilot Study.

BACKGROUND: Cardiac surgery induces many physiologic changes including major inflammatory and sympathetic nervous system responses. Here, we conducted a single-centre pilot study to generate hypotheses on the potential immune impact of adding high spinal anaesthesia to general anaesthesia during cardiac surgery in adults. We hypothesized that this strategy, previously shown to blunt the sympathetic response and improve pain management, could reduce the undesirable systemic inflammatory responses caused by cardiac surgery. METHODS: This prospective randomized unblinded pilot study was conducted on 14 patients undergoing cardiac surgery for coronary artery bypass grafting and/or aortic valve replacement secondary to severe aortic stenosis. The primary outcome measures examined longitudinally were serum pro-inflammatory (IL-6, IL-1b, CCL2), anti-inflammatory (IL-10, TNF-RII, IL-1Ra), acute phase protein (CRP, PTX3) and cardiovascular risk (sST2) biomarkers. RESULTS: The kinetics of pro- and anti-inflammatory biomarker was determined following surgery. All pro-inflammatory and acute phase reactant biomarker responses induced by surgical stress were indistinguishable in intensity and duration between control groups and those who also received high spinal anaesthesia. Conversely, IL-10 levels were markedly elevated in both intensity and duration in the group receiving high spinal anesthesia (p = 0.005). CONCLUSIONS: This hypothesis generating pilot study suggests that high spinal anesthesia can alter the net inflammatory response that results from cardiac surgery. In appropriately selected populations, this may add incremental benefit by dampening the net systemic inflammatory response during the week following surgery. Larger population studies, powered to assess immune, physiologic and clinical outcomes in both acute and longer term settings, will be required to better assess potential benefits of incorporating high spinal anesthesia. TRIAL REGISTRATION: ClinicalTrials.gov NCT00348920.

Assessment of donor heart viability during ex vivo heart perfusion.

Ex vivo heart perfusion (EVHP) may facilitate resuscitation of discarded donor hearts and expand the donor pool; however, a reliable means of demonstrating organ viability prior to transplantation is required. Therefore, we sought to identify metabolic and functional parameters that predict myocardial performance during EVHP. To evaluate the parameters over a broad spectrum of organ function, we obtained hearts from 9 normal pigs and 37 donation after circulatory death pigs and perfused them ex vivo. Functional parameters obtained from a left ventricular conductance catheter, oxygen consumption, coronary vascular resistance, and lactate concentration were measured, and linear regression analyses were performed to identify which parameters best correlated with myocardial performance (cardiac index: mL·min(-1)·g(-1)). Functional parameters exhibited excellent correlation with myocardial performance and demonstrated high sensitivity and specificity for identifying hearts at risk of poor post-transplant function (ejection fraction: R(2) = 0.80, sensitivity = 1.00, specificity = 0.85; stroke work: R(2) = 0.76, sensitivity = 1.00, specificity = 0.77; minimum dP/dt: R(2) = 0.74, sensitivity = 1.00, specificity = 0.54; tau: R(2) = 0.51, sensitivity = 1.00, specificity = 0.92), whereas metabolic parameters were limited in their ability to predict myocardial performance (oxygen consumption: R(2) = 0.28; coronary vascular resistance: R(2) = 0.20; lactate concentration: R(2) = 0.02). We concluded that evaluation of functional parameters provides the best assessment of myocardial performance during EVHP, which highlights the need for an EVHP device capable of assessing the donor heart in a physiologic working mode.