Transcriptional profiling of mucus production in rhesus macaque endocervical cells under hormonal regulation.

OBJECTIVE: Endocervical mucus production is a key regulator of fertility throughout the menstrual cycle. With cycle-dependent variability in mucus quality and quantity, cervical mucus can either facilitate or block sperm ascension into the upper female reproductive tract. This study seeks to identify genes involved in the hormonal regulation of mucus production, modification, and regulation through profiling the transcriptome of endocervical cells from the non-human primate, the rhesus macaque (Macaca mulatta). INTERVENTION: We treated differentiated primary endocervical cultures with estradiol (E2) and progesterone (P4) to mimic peri-ovulatory and luteal-phase hormonal changes. Using RNA-sequencing, we identified differential expression of gene pathways and mucus producing and modifying genes in cells treated with E2 compared to hormone-free conditions and E2 compared to E2-primed cells treated with P4. MAIN OUTCOME MEASURES: We pursued differential gene expression analysis on RNA-sequenced cells. Sequence validation was done using qPCR. RESULTS: Our study identified 158 genes that show significant differential expression in E2-only conditions compared to hormone-free control, and 250 genes that show significant differential expression in P4-treated conditions compared to E2-only conditions. From this list, we found hormone-induced changes in transcriptional profiles for genes across several classes of mucus production, including ion channels and enzymes involved in post-translational mucin modification that have not previously been described as hormonally regulated. CONCLUSION: Our study is the first to use an in vitro culture system to create an epithelial-cell specific transcriptome of the endocervix. As a result, our study identifies new genes and pathways altered by sex-steroids in cervical mucus production.

Combinatorial Inhibition of Complement Factor D and BCL2 for Early-Onset Colorectal Cancer.

BACKGROUND: The tumor immune microenvironment is distinct between early-onset and late-onset colorectal cancer, which facilitates tumor progression. We previously identified several genes, including complement factor D, as having increased expression in patients with early-onset colorectal cancer. OBJECTIVE: This study aimed to assess and validate the differential expression of immune genes in early-onset and late-onset colorectal cancer. We also aimed to test known drugs targeting genes increased in early-onset colorectal cancer in preclinical mouse models. DESIGN: A retrospective cohort study with analysis was performed using tumor RNA from formalin-fixed paraffin-embedded cell culture and immunohistochemistry to validate gene expression and function and in vivo preclinical tumor study to assess drug efficacy. SETTINGS: The Oregon Colorectal Cancer Registry was queried to identify patients with colorectal cancer. PATIENTS: The study included 67 patients with early-onset colorectal cancer and 54 patients with late-onset colorectal cancer. INTERVENTIONS: Preclinical animal models using the HCT-116 colon cancer cell line were treated with the complement factor D inhibitor danicopan and the BCL2 inhibitor venetoclax, or with vehicle controls. MAIN OUTCOME MEASURES: Elevated RNA signatures using NanoString data were evaluated by the retrospective cohort. When inhibiting these markers in the mouse preclinical model, tumor volume and weight were the main outcome measures. RESULTS: After updating our sample size from our previously published data, we found that complement factor D and BCL2, genes with known function and small molecule inhibitors, are elevated in patients with early-onset colorectal cancer. When inhibiting these markers with the drugs danicopan and venetoclax in a mouse model, we found that the combination of these drugs decreased tumor burden but also resulted in toxicity. LIMITATIONS: This study is limited by a small sample size and a subcutaneous tumor model. CONCLUSIONS: Combinatorial inhibition of early-onset associated genes complement factor D and BCL2 slows the growth of early-onset colorectal cancer in a mouse preclinical model. See Video Abstract . INHIBICIN COMBINADA DEL FACTOR DCOMPLEMENTARIO Y DEL BCL EN CASOS DE CNCER COLORRECTAL DE APARICIN TEMPRANA: ANTECEDENTES:El microambiente inmunológico del tumor es distinto entre el cáncer colorrectal de aparición temprana y el de aparición tardía, lo que facilita la progresión de dicho tumor. Anteriormente identificamos varios genes, incluidos el factor D-Complementario, con una mayor expresión en pacientes con cáncer colorrectal de aparición temprana.OBJETIVO:El presente estudio tuvo como objetivo el evaluar y validar la expresión diferenciada de genes inmunes en casos de cáncer colorrectal de aparición temprana y tardía. También nos propusimos evaluar los fármacos conocidos dirigidos sobre los genes aumentados en el cáncer colorrectal de aparición temprana en modelos pre-clínicos en ratones.DISEÑO:Estudio de cohortes con análisis retrospectivo utilizando el ARN tumoral procedente de cultivos celulares fijados con formalina e incluidos en parafina, y el analisis por inmunohistoquímica para validar la expresión y la función genética. Se realizó el estudio pre-clínico de los tumores in vivo para evaluar la eficacia de los fármacos.AJUSTES:Se consultó el Registro de Oregon de casos de Cáncer Colorrectal para encontrar los pacientes afectados.SUJETOS:67 pacientes con cáncer colorrectal de aparición temprana y 54 pacientes con cáncer colorrectal de aparición tardía.INTERVENCIONES (SI LAS HUBIESE):Los modelos animales pre-clínicos que utilizaron la línea celular de cáncer de colon HCT-116 se trataron con el inhibidor del factor D-Complementario o Danicopan y con el inhibidor de BCL-2 o Venetoclax, ambos con control del transportador.PRINCIPALES MEDIDAS DE RESULTADO:Se evaluaron las firmas de ARN elevadas utilizando los datos del NanoString a partir de la cohorte retrospectiva. Al inhibir estos marcadores del modelo pre-clínico en los ratones, el volumen y el peso del tumor fueron las principales medidas de resultado.RESULTADOS:Después de actualizar el tamaño de nuestra muestra a partir de datos publicados con anterioridad, encontramos que el factor D-Complementario y BCL-2, genes con función conocida e inhibidores de moléculas pequeñas, se encuentran elevados en aquellos pacientes con cáncer colorrectal de aparición temprana. Al inhibir estos marcadores con los medicamentos Danicopan y Venetoclax en el modelo de ratones vivos, encontramos que la combinación de estos dos farmacos disminuyó la carga tumoral pero también produjo toxicidad.LIMITACIONES:Estudio limitado por un tamaño de muestra pequeño y el modelo de tumor subcutáneo.CONCLUSIONES:La inhibición combinada de genes asociados de aparición temprana, el factor D-Complementario y el BCL-2, enlentecen el crecimiento del cáncer colorrectal de aparición temprana del modelo preclínico en ratones. (Traducción-Dr. Xavier Delgadillo ).

The Mechanism of Hepatic Encephalopathy Induced by Thioacetamide Based on Metabolomics and Proteomics: A Preliminary Study.

Hepatic encephalopathy (HE) is a central nervous system dysfunction syndrome caused by acute and chronic liver failure or various portal systemic shunt disorders. HE arises from metabolic disorder and excludes other known types of encephalopathy. HE is a major cause of death in people with liver disease. Early diagnosis and timely treatment are key to improving HE prognosis. Herein, we established a model of HE and performed metabolomics to identify 50 significantly differential metabolites between the HE group and control group. The main metabolic pathways associated with these differential metabolites were the purine metabolism, pyrimidine metabolism, aminoacyl tRNA biosynthesis, and glucose metabolism. Through proteomics analysis, we identified 226 significantly differential proteins (52 up-regulated and 174 down-regulated). The main (Kyoto Encyclopedia of Genes and Genomes) enrichment pathways were the Staphylococcus aureus infection, vitamin digestion and absorption, and complement and coagulation cascades. Through the conjoint analysis of proteomics and metabolomics, the differentially present proteins and metabolites were found to be involved in vitamin digestion and absorption, and ferroptosis pathways. In HE, malondialdehyde was significantly elevated, but glutathione was significantly diminished, and the redox balance was destroyed, thus leading to changes in proteins' levels associated with the ferroptosis pathway. In conclusion, this study preliminarily explored the molecular and metabolic mechanisms underlying HE.

A rare incidence of acute ischaemic stroke with reversible middle cerebral artery occlusion in a methamphetamine addict: Case report.

Background: Methamphetamine is an illegal drug that poses serious public health concerns worldwide. Previous studies have demonstrated a strong association between methamphetamine abuse and non-lethal haemorrhagic stroke. Ischaemic stroke after methamphetamine intake is less common than haemorrhagic stroke. The present study investigated the clinical features and potential pathogenesis in a young methylamphetamine addict that presented with acute ischaemic stroke and reversible middle cerebral artery (MCA) occlusion. Methods: A retrospective data analysis was performed for the young methylamphetamine addict admitted to a hospital for acute ischaemic stroke followed by a literature review to explore the possible pathogenesis. Results: The patient had been receiving methamphetamine for past 2 years. His recurrent headache occurred half an hour after each consumption and was relieved within a few hours. The patient was admitted for acute ischaemic stroke. Urine toxicology screening was positive for methamphetamine. Magnetic resonance angiography revealed occlusion of the right MCA. After discontinuing medication and routine treatment, digital subtraction angiography revealed normal blood flow in the right MCA, indicating reversible MCA occlusion. Conclusion: For young patients with a stroke, a thorough investigation of the history of illicit drug use and toxicological screening of urine and serum samples should be performed. Young methamphetamine users need to be aware of the elevated risk of stroke as well as early signs and symptoms. Transient headaches in young methamphetamine users may be caused by cerebral vasospasms, suggesting the possibility of future catastrophic stroke events.

Using a k-means clustering to identify novel phenotypes of acute ischemic stroke and development of its Clinlabomics models.

Objective: Acute ischemic stroke (AIS) is a heterogeneous condition. To stratify the heterogeneity, identify novel phenotypes, and develop Clinlabomics models of phenotypes that can conduct more personalized treatments for AIS. Methods: In a retrospective analysis, consecutive AIS and non-AIS inpatients were enrolled. An unsupervised k-means clustering algorithm was used to classify AIS patients into distinct novel phenotypes. Besides, the intergroup comparisons across the phenotypes were performed in clinical and laboratory data. Next, the least absolute shrinkage and selection operator (LASSO) algorithm was used to select essential variables. In addition, Clinlabomics predictive models of phenotypes were established by a support vector machines (SVM) classifier. We used the area under curve (AUC), accuracy, sensitivity, and specificity to evaluate the performance of the models. Results: Of the three derived phenotypes in 909 AIS patients [median age 64 (IQR: 17) years, 69% male], in phenotype 1 (N = 401), patients were relatively young and obese and had significantly elevated levels of lipids. Phenotype 2 (N = 463) was associated with abnormal ion levels. Phenotype 3 (N = 45) was characterized by the highest level of inflammation, accompanied by mild multiple-organ dysfunction. The external validation cohort prospectively collected 507 AIS patients [median age 60 (IQR: 18) years, 70% male]. Phenotype characteristics were similar in the validation cohort. After LASSO analysis, Clinlabomics models of phenotype 1 and 2 were constructed by the SVM algorithm, yielding high AUC (0.977, 95% CI: 0.961-0.993 and 0.984, 95% CI: 0.971-0.997), accuracy (0.936, 95% CI: 0.922-0.956 and 0.952, 95% CI: 0.938-0.972), sensitivity (0.984, 95% CI: 0.968-0.998 and 0.958, 95% CI: 0.939-0.984), and specificity (0.892, 95% CI: 0.874-0.926 and 0.945, 95% CI: 0.923-0.969). Conclusion: In this study, three novel phenotypes that reflected the abnormal variables of AIS patients were identified, and the Clinlabomics models of phenotypes were established, which are conducive to individualized treatments.

The potential contribution of aberrant cathepsin K expression to gastric cancer pathogenesis.

The role of cathepsin K (CTSK) expression in the pathogenesis and progression of gastric cancer (GC) remains unclear. Hence, the primary objective of this study is to elucidate the precise expression and biological role of CTSK in GC by employing a combination of bioinformatics analysis and in vitro experiments. Our findings indicated a significant upregulation of CTSK in GC. The bioinformatics analysis revealed that GC patients with a high level of CTSK expression exhibited enrichment of hallmark gene sets associated with angiogenesis, epithelial-mesenchymal transition (EMT), inflammatory response, KRAS signaling up, TNFα signaling via KFκB, IL2-STAT5 signaling, and IL6-JAK-STAT3 signaling. Additionally, these patients demonstrated elevated levels of M2-macrophage infiltration, which was also correlated with a poorer prognosis. The results of in vitro experiments provided confirmation that the over-expression of CTSK leads to an increase in the proliferative and invasive abilities of GC cells. However, further evaluation was necessary to determine the impact of CTSK on the migration capability of these cells. Our findings suggested that CTSK has the potential to facilitate the initiation and progression of GC by augmenting the invasive capacity of GC cells, engaging in tumor-associated EMT, and fostering the establishment of an immunosuppressive tumor microenvironment (TME).

What sperm parameters effect blastocyst formation and quality during ICSI with severe male infertility.

Sperm-derived genetic material contributes half of the genome to the embryo, hence it's crucial to investigate which sperm parameter influences blastocyst formation in the intracytoplasmic sperm injection (ICSI) cycles with severe male infertility. The retrospective study analyzed 296 ICSI cycles with severe oligoasthenoteratozoospermia (OAT) and 99 ICSI cycles with preimplantation genetic testing for aneuploidy (PGT-A). Following the correlation analysis, data stratifications were performed in the OAT ICSI subgroup. The results showed that the matching blastocyst in the OAT ICSI cycles had inferior sperm parameters. DFI and sperm morphology had an influence on the blastocyst formation rate and the high-quality blastocysts formation rate on Day6, but no significant effect on the blastocyst development on Day 5. The high-quality blastocysts formation rate and ratio of high-quality blastocyst on Day 6 were demonstrably better in the subgroup of the teratozoospermic morphology when DFI was within the normal range. In the case of the normal sperm morphology, no statistically significant difference was found in blastocyst development, although there were numerical differences within different DFI subgroups. It was concluded that the blastocyst quality and development declined with the decreased sperm qualities.

Ion Dynamics at the Intermediate Charging State of the Sodium Vanadium Fluorophosphate Cathode.

Na super ionic conductor (NASICON)-type polyanionic vanadium fluorophosphate Na3V2O2(PO4)2F (NVOPF) is a promising cathode material for high-energy sodium-ion batteries. The dynamic diffusion and exchange of sodium ions in the lattice of NVOPF are crucial for its electrochemical performance. However, standard characterizations are mostly focused on the as-synthesized material without cycling, which is different from the actual battery operation conditions. In this work, we investigated the hopping processes of sodium in NVOPF at the intermediate charging state with 23Na solid-state nuclear magnetic resonance (ssNMR) and density functional theory (DFT) calculations. Our experimental characterizations revealed six distinct sodium coordination sites in the intermediate structure and determined the exchange rates among these sites at variable temperatures. The theoretical calculations showed that these dynamic processes correspond to different ion transport pathways in the crystalline lattice. Our combined experimental and theoretical study uncovered the underlying mechanisms of the ion transport in cycled NVOPF and these understandings may help the optimization of cathode materials for sodium-ion batteries.

Structural Evolution of Lithium-Exchanged Na3(VO)2(PO4)2F Cathode under Operation in Sodium Ion Batteries.

Na superionic conductor (NASICON)-type Na3(VO)2(PO4)2F (NVOPF) exhibits excellent cycling stability for high-voltage sodium ion batteries. Various strategies have been developed to form ion-exchanged NVOPF which can enhance the ionic and electronic conductivity. However, the underlying ion transport mechanism and complex structural transitions during battery operation remained uninvestigated. In this work, we prepared lithium-exchanged NVOPF (namely NLVOPF) which shows improved ionic conductivity and increased capacity at high discharging rates. Solid-state nuclear magnetic resonance (SSNMR) revealed the distinctive presence of two kinds of Li-exchanged sites in the NLVOPF, which are attributed to the occupied lithium ions at the Na1 and Na2 sites (namely Li1 and Li2, respectively). The Li1 site was metastably replaced in the first cycle, yet the Li2 site participated in ion insertion/extraction in the subsequent cycles. Our characterizations show that the dynamic doping of lithium in NLVOPF could contribute to the improved cycling stability and capacity retention.

Process Optimization of SiC-Reinforced Aluminum Matrix Composites Prepared Using Laser Powder Bed Fusion and the Effect of Particle Morphology on Performance.

Process parameters and powder spreading quality are important factors for aluminum matrix composites (AMCs) prepared using laser powder bed fusion (LPBF). In this study, a Box-Behnken Design (BBD) was used to optimize the process parameters, and near-spherical ß-SiC was selected to improve the quality of powder spreading. The rationality of parameter optimization was verified by testing the density of samples prepared using different laser power levels. Al4C3 diffraction peaks were found in XRD patterns, which indicated that interface reactions occurred to form good interface bonding between the Al matrix and the SiC particles. The tensile strength and plasticity of LPBF α-SiC/AlSi10Mg were lower than that of LPBF AlSi10Mg, which was mainly due to the poor fluidity of the powder mixtures and powder spreading quality. For LPBF ß-SiC/AlSi10Mg, the tensile strength increased and elongation decreased slightly compared to LPBF α-SiC/AlSi10Mg. The data in this study were compared with the data in other studies. In this study, LPBF AlSi10Mg and LPBF ß-SiC/AlSi10Mg not only showed the inherent high strength of their LPBF parts, but also had relatively high plasticity. Matching between strength and plasticity was mainly dependent on the scanning strategy. Most studies use uni-directional or bi-directional scanning strategies with a certain rotation angle between layers. A chessboard scanning strategy was used in this study to form a coarse remelted connected skeleton inside the material and significantly improve plasticity. This study lays a theoretical and experimental foundation for the controllable preparation of SiC-reinforced AMCs using LPBF.

Investigating the antiviral activity of Erigeron annuus (L.) pers extract against RSV and examining its active components.

ETHNOPHARMACOLOGICAL RELEVANCE: The plants in the genus Erigeron are known to exhibit antiviral activities, including those against the respiratory syncytial virus (RSV). In traditional medicine Erigeron annuus (L.) Pers (EA) has been used in the treatment of pulmonary diseases and acute infectious hepatitis. AIM OF THIS STUDY: The aim of this study is to determine the optimum extraction method to produce the most potent anti-RSV extract, elucidate its mode and mechanisms of antiviral activity in both in vitro and in vivo models, and identify the chemical structures of the bioactive compounds. MATERIALS AND METHODS: The whole plant of EA was extracted with ethyl acetate, methanol, ethanol, water, aqueous methanol (60, 80% and 100%) and aqueous ethanol (50, 75% and 95%) using maceration, reflux, and ultrasound-assisted extraction methods. The antiviral activities of the extracts were determined in vitro. The in vitro antiviral activities of the extracts were determined using Hep-2 cells. Four in vitro experiments were performed to determine the mode of antiviral activity of the most active extract, ethyl acetate fraction (EAE) of Erigeron annuus whole plant extract prepared by refluxing with 50% ethanol, by examining its ability to inactivate the virus directly, inhibit viral adsorption and penetration, inhibit viral replication and preventive effect. The effect of temperature and duration of treatment on these modes of action was also determined. The antiviral activity of the EAE was also assessed in vivo in a mouse model. The lung index, viral load, and lung tissue histology were measured. qRT-PCR and ELISA studies were performed to determine the expression of key genes (TLR-3 and TLR-4) and proteins (IL-2, IFN-γ, and TNF-α) related to RSV infection. The most active antiviral compound was isolated using chromatography techniques, and its chemical structure was identified through electrospray triple quadrupole mass spectroscopy and nuclear magnetic resonance spectroscopy. RESULTS: The EAE was the most active on RSV. In vitro experiments showed that the antiviral activity of EAE is via direct inactivation, inhibition of entry, and inhibition of the proliferation of the virus. In vivo experiments showed that the EAE effectively inhibited the proliferation of RSV in the lungs and alleviated the lung tissue lesions in RSV-infected mice. The antiviral activity of the EAE is mediated by downregulating the expression of TLR3 and TLR4 in the lung, upregulating the expression of IL-2 and IFN-γ, and downregulating the expression of TNF-α. Apigenin 7-O-methylglucuronide was found to be a major bioactive compound in EAE. CONCLUSIONS: The results of this study confirmed the antiviral activity of EA by inactivating, inhibiting the entry, and inhibiting the proliferation of RSV. The activity is mediated by regulating the immunity and inflammatory mediators. Apigenin 7-O-methylglucuronide is the bioactive compound present in EA.

Improvements in lung function following vitamin C supplementation to pregnant smokers are associated with buccal DNA methylation at 5 years of age.

BACKGROUND: We previously reported in the "Vitamin C to Decrease the Effects of Smoking in Pregnancy on Infant Lung Function" randomized clinical trial (RCT) that vitamin C (500 mg/day) supplementation to pregnant smokers is associated with improved respiratory outcomes that persist through 5 years of age. The objective of this study was to assess whether buccal cell DNA methylation (DNAm), as a surrogate for airway epithelium, is associated with vitamin C supplementation, improved lung function, and decreased occurrence of wheeze. METHODS: We conducted epigenome-wide association studies (EWAS) using Infinium MethylationEPIC arrays and buccal DNAm from 158 subjects (80 placebo; 78 vitamin C) with pulmonary function testing (PFT) performed at the 5-year visit. EWAS were performed on (1) vitamin C treatment, (2) forced expiratory flow between 25 and 75% of expired volume (FEF25-75), and (3) offspring wheeze. Models were adjusted for sex, race, study site, gestational age at randomization (≤ OR > 18 weeks), proportion of epithelial cells, and latent covariates in addition to child length at PFT in EWAS for FEF25-75. We considered FDR p < 0.05 as genome-wide significant and nominal p < 0.001 as candidates for downstream analyses. Buccal DNAm measured in a subset of subjects at birth and near 1 year of age was used to determine whether DNAm signatures originated in utero, or emerged with age. RESULTS: Vitamin C treatment was associated with 457 FDR significant (q < 0.05) differentially methylated CpGs (DMCs; 236 hypermethylated; 221 hypomethylated) and 53 differentially methylated regions (DMRs; 26 hyper; 27 hypo) at 5 years of age. FEF25-75 was associated with one FDR significant DMC (cg05814800), 1,468 candidate DMCs (p < 0.001), and 44 DMRs. Current wheeze was associated with 0 FDR-DMCs, 782 candidate DMCs, and 19 DMRs (p < 0.001). In 365/457 vitamin C FDR significant DMCs at 5 years of age, there was no significant interaction between time and treatment. CONCLUSIONS: Vitamin C supplementation to pregnant smokers is associated with buccal DNA methylation in offspring at 5 years of age, and most methylation signatures appear to be persistent from the prenatal period. Buccal methylation at 5 years was also associated with current lung function and occurrence of wheeze, and these functionally associated loci are enriched for vitamin C associated loci. Clinical trial registration ClinicalTrials.gov, NCT01723696 and NCT03203603.

The TET-Sall4-BMP regulatory axis controls craniofacial cartilage development.

Craniofacial microsomia (CFM) is a congenital defect that usually results from aberrant development of embryonic pharyngeal arches. However, the molecular basis of CFM pathogenesis is largely unknown. Here, we employ the zebrafish model to investigate mechanisms of CFM pathogenesis. In early embryos, tet2 and tet3 are essential for pharyngeal cartilage development. Single-cell RNA sequencing reveals that loss of Tet2/3 impairs chondrocyte differentiation due to insufficient BMP signaling. Moreover, biochemical and genetic evidence reveals that the sequence-specific 5mC/5hmC-binding protein, Sall4, binds the promoter of bmp4 to activate bmp4 expression and control pharyngeal cartilage development. Mechanistically, Sall4 directs co-phase separation of Tet2/3 with Sall4 to form condensates that mediate 5mC oxidation on the bmp4 promoter, thereby promoting bmp4 expression and enabling sufficient BMP signaling. These findings suggest the TET-BMP-Sall4 regulatory axis is critical for pharyngeal cartilage development. Collectively, our study provides insights into understanding craniofacial development and CFM pathogenesis.

Effects of repeated alcohol abstinence on within-subject prefrontal cortical gene expression in rhesus macaques.

Male rhesus monkeys (n = 24) had a biopsy of prefrontal cortical area 46 prior to chronic ethanol self-administration (n = 17) or caloric control (n = 7). Fourteen months of daily self-administration (water vs. 4% alcohol, 22 h access/day termed "open-access") was followed by two cycles of prolonged abstinence (5 weeks) each followed by 3 months of open-access alcohol and a final abstinence followed by necropsy. At necropsy, a biopsy of Area 46, contralateral to the original biopsy, was obtained. Gene expression data (RNA-Seq) were collected comparing biopsy/necropsy samples. Monkeys were categorized by drinking status during the final post-abstinent drinking phase as light (LD), binge (BD), heavy (HD) and very heavy (VHD drinkers). Comparing pre-ethanol to post-abstinent biopsies, four animals that converted from HD to VHD status had significant ontology enrichments in downregulated genes (necropsy minus biopsy n = 286) that included immune response (FDR < 9 × 10-7) and plasma membrane changes (FDR < 1 × 10-7). Genes in the immune response category included IL16 and 18, CCR1, B2M, TLR3, 6 and 7, SP2 and CX3CR1. Upregulated genes (N = 388) were particularly enriched in genes associated with the negative regulation of MAP kinase activity (FDR < 3 × 10-5), including DUSP 1, 4, 5, 6 and 18, SPRY 2, 3, and 4, SPRED2, BMP4 and RGS2. Overall, these data illustrate the power of the NHP model and the within-subject design of genomic changes due to alcohol and suggest new targets for treating severe escalated drinking following repeated alcohol abstinence attempts.

Development and Validation of a Novel Placental DNA Methylation Biomarker of Maternal Smoking during Pregnancy in the ECHO Program.

BACKGROUND: Maternal cigarette smoking during pregnancy (MSDP) is associated with numerous adverse health outcomes in infants and children with potential lifelong consequences. Negative effects of MSDP on placental DNA methylation (DNAm), placental structure, and function are well established. OBJECTIVE: Our aim was to develop biomarkers of MSDP using DNAm measured in placentas (N=96), collected as part of the Vitamin C to Decrease the Effects of Smoking in Pregnancy on Infant Lung Function double-blind, placebo-controlled randomized clinical trial conducted between 2012 and 2016. We also aimed to develop a digital polymerase chain reaction (PCR) assay for the top ranking cytosine-guanine dinucleotide (CpG) so that large numbers of samples can be screened for exposure at low cost. METHODS: We compared the ability of four machine learning methods [logistic least absolute shrinkage and selection operator (LASSO) regression, logistic elastic net regression, random forest, and gradient boosting machine] to classify MSDP based on placental DNAm signatures. We developed separate models using the complete EPIC array dataset and on the subset of probes also found on the 450K array so that models exist for both platforms. For comparison, we developed a model using CpGs previously associated with MSDP in placenta. For each final model, we used model coefficients and normalized beta values to calculate placental smoking index (PSI) scores for each sample. Final models were validated in two external datasets: the Extremely Low Gestational Age Newborn observational study, N=426; and the Rhode Island Children's Health Study, N=237. RESULTS: Logistic LASSO regression demonstrated the highest performance in cross-validation testing with the lowest number of input CpGs. Accuracy was greatest in external datasets when using models developed for the same platform. PSI scores in smokers only (n=72) were moderately correlated with maternal plasma cotinine levels. One CpG (cg27402634), with the largest coefficient in two models, was measured accurately by digital PCR compared with measurement by EPIC array (R2=0.98). DISCUSSION: To our knowledge, we have developed the first placental DNAm-based biomarkers of MSDP with broad utility to studies of prenatal disease origins. https://doi.org/10.1289/EHP13838.

Effect of metabolic status on response to SIV infection and antiretroviral therapy in nonhuman primates.

Current antiretroviral therapy (ART) regimens efficiently limit HIV replication, thereby improving life expectancy of people living with HIV, but also cause metabolic side effects. The ongoing obesity epidemic has resulted in more people with metabolic comorbidities at the time of HIV infection, yet the impact of pre-existing metabolic dysregulation on infection sequelae and response to ART is unclear. Here, to investigate the impact of preexisting obesity and insulin resistance on acute infection and subsequent long-term ART, we infected a cohort of lean and obese adult male macaques with SIV and administered ART. The responses of lean and obese macaques to SIV and ART were similar with respect to plasma and cell-associated viral loads, ART drug levels in plasma and tissues, SIV-specific immune responses, adipose tissue and islet morphology, and colon inflammation, with baseline differences between lean and obese groups largely maintained. Both groups exhibited a striking depletion of CD4+ T cells from adipose tissue that did not recover with ART. However, differential responses to SIV and ART were observed for body weight, omental adipocyte size, and the adiponectin/leptin ratio, a marker of cardiometabolic risk. Thus, obesity and insulin resistance had limited effects on multiple responses to acute SIV infection and ART, while several factors that underlie long-term metabolic comorbidities were influenced by prior obesity and insulin resistance. These studies provide the foundation for future investigations into the efficacy of adjunct therapies such as metformin and glucagon-like peptide-1 receptor agonists in the prevention of metabolic comorbidities in people living with HIV.

Cytomegalovirus vaccine vector-induced effector memory CD4 + T cells protect cynomolgus macaques from lethal aerosolized heterologous avian influenza challenge.

An influenza vaccine approach that overcomes the problem of viral sequence diversity and provides long-lived heterosubtypic protection is urgently needed to protect against pandemic influenza viruses. Here, to determine if lung-resident effector memory T cells induced by cytomegalovirus (CMV)-vectored vaccines expressing conserved internal influenza antigens could protect against lethal influenza challenge, we immunize Mauritian cynomolgus macaques (MCM) with cynomolgus CMV (CyCMV) vaccines expressing H1N1 1918 influenza M1, NP, and PB1 antigens (CyCMV/Flu), and challenge with heterologous, aerosolized avian H5N1 influenza. All six unvaccinated MCM died by seven days post infection with acute respiratory distress, while 54.5% (6/11) CyCMV/Flu-vaccinated MCM survived. Survival correlates with the magnitude of lung-resident influenza-specific CD4 + T cells prior to challenge. These data demonstrate that CD4 + T cells targeting conserved internal influenza proteins can protect against highly pathogenic heterologous influenza challenge and support further exploration of effector memory T cell-based vaccines for universal influenza vaccine development.