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Abstract During myocardial injury, inflammatory mediators and oxidative stress significantly increase to impair cardiac mitochondria. Emerging evidence has highlighted interplays between circadian protein—period 2 (Per2) and mitochondrial metabolism. However, besides circadian rhythm regulation, the direct role of Per2 in mitochondrial performance particularly following acute stress, remains unknown. In this study, we aim to determine the importance of Per2 protein’s regulatory role in mitochondrial function following exposure to inflammatory cytokine TNFα and oxidative stressor H2O2 in human cardiomyocytes. Global warm ischemia (37 °C) significantly impaired complex I activity with concurrently reduced mitochondrial Per2 in adult mouse hearts. TNFα or H2O2 decreased Per2 protein levels and damaged mitochondrial respiratory function in adult mouse cardiomyocytes. Next, mitochondrial membrane potential ( $$\Delta \psi$$ Δ ψ M) using JC-1 fluorescence probe and mitochondrial respiration capacity via Seahorse Cell Mito Stress Test were then detected in Per2 or control siRNA transfected AC16 Human Cardiomyocytes (HCM) that were subjected to 2 h-treatment of TNFα (100 ng/ml) or H2O2 (100 μM). After 4 h-treatment, cell death was also measured using Annexin V and propidium iodide apoptosis kit through flow cytometry. We found that knockdown of Per2 enhanced TNFα-induced cell death and TNFα- or H2O2-disrupted $$\Delta \psi$$ Δ ψ M, as well as TNFα- or H2O2-impaired mitochondrial respiration function. In conclusion, Per2 knockdown increases likelihood of cell death and mitochondrial dysfunction in human cardiomyocytes exposed to either TNFα or H2O2, supporting the protective role of Per2 in HCM during stress with a focus on mitochondrial function.

Necrotizing enterocolitis (NEC) is a destructive gastrointestinal disease primarily affecting preterm babies. Despite advancements in neonatal care, NEC remains a significant cause of morbidity and mortality in neonatal intensive care units worldwide and the etiology of NEC is still unclear. Risk factors for NEC include prematurity, very low birth weight, feeding with formula, intestinal dysbiosis and bacterial infection. A review of the literature would suggest that supplementation of prebiotics and probiotics prevents NEC by altering the immune responses. Innate T cells, a highly conserved subpopulation of T cells that responds quickly to stimulation, develops differently from conventional T cells in neonates. This review aims to provide a succinct overview of innate T cells in neonates, encompassing their phenotypic characteristics, functional roles, likely involvement in the pathogenesis of NEC, and potential therapeutic implications.

Abstract Hydrogen produced from neutral seawater electrolysis faces many challenges including high energy consumption, the corrosion/side reactions caused by Cl-, and the blockage of active sites by Ca2+/Mg2+ precipitates. Herein, we design a pH-asymmetric electrolyzer with a Na+ exchange membrane for direct seawater electrolysis, which can simultaneously prevent Cl- corrosion and Ca2+/Mg2+ precipitation and harvest the chemical potentials between the different electrolytes to reduce the required voltage. In-situ Raman spectroscopy and density functional theory calculations reveal that water dissociation can be promoted with a catalyst based on atomically dispersed Pt anchored to Ni-Fe-P nanowires with a reduced energy barrier (by 0.26 eV), thus accelerating the hydrogen evolution kinetics in seawater. Consequently, the asymmetric electrolyzer exhibits current densities of 10 mA cm−2 and 100 mA cm−2 at voltages of 1.31 V and 1.46 V, respectively. It can also reach 400 mA cm−2 at a low voltage of 1.66 V at 80 °C, corresponding to the electricity cost of US$1.36 per kg of H2 ($0.031/kW h for the electricity bill), lower than the United States Department of Energy 2025 target (US$1.4 per kg of H2).

Abstract Necrotizing enterocolitis (NEC) continues to be a devastating disease in preterm neonates and has a paucity of medical management options. Chondroitin sulfate (CS) is a naturally occurring glycosaminoglycan (GAG) in human breast milk (HM) and has been shown to reduce inflammation. We hypothesized that supplementation with CS in an experimental NEC model would alter microbial diversity, favorably alter the cytokine profile, and (like other sulfur compounds) improve outcomes in experimental NEC via the eNOS pathway. NEC was induced in 5‐day‐old pups. Six groups were studied (n = 9–15/group): (1) WT breastfed and (2) Formula fed controls, (3) WT NEC, (4) WT NEC + CS, (5) eNOS KO (knockout) NEC, and (6) eNOS KO NEC + CS. Pups were monitored for clinical sickness score and weights. On postnatal day 9, the pups were killed. Stool was collected from rectum and microbiome analysis was done with 16 s rRNA sequencing. Intestinal segments were examined histologically using a well‐established injury scoring system and segments were homogenized and analyzed for cytokine profile. Data were analyzed using GraphPad Prism with p

The significant morphological differences and abundant germplasm resources of Chinese indigenous dog breeds can be attributed to the diverse geographical environment, including plateaus, mountains, and a long history of raising dogs. The combination of both natural and artificial selection during the past several thousand years has led to hundreds of dog breeds with distinct morphological traits and environmental adaptations. China is one of the earliest countries to domesticate dogs and there are more than 50 ancient indigenous dog breeds. In this study, the run of homozygosity (ROH) and proportion of the autosomal genome covered by ROHs (FROH) were calculated for 10 dog breeds that are the most representative Chinese indigenous dogs based on 170K SNP microarray. The results of FROH showed that the Chuandong hound dogs (HCSSC) have the highest level of inbreeding among the tested breeds. The inbreeding in HCSSC occurred more recently than the Liangshan dogs (SCLSQ) dogs because of more numbers of long ROHs in HCSSC dogs, and the former also have higher inbreeding degree. In addition, there are significant differences in the inbreeding degree among different subpopulations of the same breed, such as the Thin dogs from Shaanxi and Shandong province. To explore genome-wide selection signatures among different breeds, including coat color, ear shape, and altitude adaptability, we performed genome selection analyses of FST and cross population extended haplotype homozygosity (XP-EHH). For the coat color, the FST analysis between Xiasi dogs (XSGZ) and HCSSC dogs was performed and identified multiple genes involved in coat color, hair follicle, and bone development, including MC1R, KITLG, SOX5, RSPO2, and TBX15. For the plateau adaptability, we performed FST and XP-EHH analyses between dogs from Tibet (Tibetan Mastiffs and Nyingchi dogs) and plain regions (Guangxi Biwei dogs GXBWQ and Guandong Sharpei dogs). The results showed the EPAS1 gene in dogs from Tibet undergo strong selection. Multiple genes identified for selection signals based on different usage of dogs. Furthermore, the results of ear shape analyses showed that MSRB3 was likely to be the main gene causing the drop ear of domestic dogs. Our study provides new insights into further understanding of Chinese indigenous dogs.

Necrotizing enterocolitis (NEC) is a devastating condition of multi-factorial origin that affects the intestine of premature infants and results in high morbidity and mortality. Infants that survive contend with several long-term sequelae including neurodevelopmental impairment (NDI)—which encompasses cognitive and psychosocial deficits as well as motor, vision, and hearing impairment. Alterations in the gut-brain axis (GBA) homeostasis have been implicated in the pathogenesis of NEC and the development of NDI. The crosstalk along the GBA suggests that microbial dysbiosis and subsequent bowel injury can initiate systemic inflammation which is followed by pathogenic signaling cascades with multiple pathways that ultimately lead to the brain. These signals reach the brain and activate an inflammatory cascade in the brain resulting in white matter injury, impaired myelination, delayed head growth, and eventual downstream NDI. The purpose of this review is to summarize the NDI seen in NEC, discuss what is known about the GBA, explore the relationship between the GBA and perinatal brain injury in the setting of NEC, and finally, highlight the existing research into possible therapies to help prevent these deleterious outcomes.

Abstract Although differential expression of genes is apparent during the adipogenic/osteogenic differentiation of marrow mesenchymal stem cells (MSCs), it is not known whether this is associated with changes in chromosomal structure. In this study, we used ATAC-sequencing technology to observe variations in chromatin assembly during the early stages of MSC differentiation. This showed significant changes in the number and distribution of chromosome accessibility at different time points of adipogenic/osteogenic differentiation. Sequencing of differential peaks indicated alterations in transcription factor motifs involved in MSC differentiation. Gene Ontology (GO) and pathway analysis indicated that changes in biological function resulted from the alterations in chromatin accessibility. We then integrated ATAC-seq and RNA-seq and found that only a small proportion of the overlapping genes were screened out from ATAC-seq and RNA-seq overlapping. Through GO and pathway analysis of these overlapped genes, we not only observed some known biological functions related to adipogenic/osteogenic differentiation but also noticed some unusual biological clustering during MSC differentiation. In summary, our work not only presents the landscape of chromatin accessibility of MSC during differentiation but also helps to further our understanding of the underlying mechanisms of gene expression in these processes.

Gastrointestinal (GI) diseases have a high prevalence throughout the United States. Screening and diagnostic modalities are often expensive and invasive, and therefore, people do not utilize them effectively. Lack of proper screening and diagnostic assessment may lead to delays in diagnosis, more advanced disease at the time of diagnosis, and higher morbidity and mortality rates. Research on the intestinal microbiome has demonstrated that dysbiosis, or unfavorable alteration of organismal composition, precedes the onset of clinical symptoms for various GI diseases. GI disease diagnostic research has led to a shift towards non-invasive methods for GI screening, including chemical-detection tests that measure changes in volatile organic compounds (VOCs), which are the byproducts of bacterial metabolism that result in the distinct smell of stool. Many of these tools are expensive, immobile benchtop instruments that require highly trained individuals to interpret the results. These attributes make them difficult to implement in clinical settings. Alternatively, electronic noses (E-noses) are relatively cheaper, handheld devices that utilize multi-sensor arrays and pattern recognition technology to analyze VOCs. The purpose of this review is to (1) highlight how dysbiosis impacts intestinal diseases and how VOC metabolites can be utilized to detect alterations in the microbiome, (2) summarize the available VOC analytical platforms that can be used to detect aberrancies in intestinal health, (3) define the current technological advancements and limitations of E-nose technology, and finally, (4) review the literature surrounding several intestinal diseases in which headspace VOCs can be used to detect or predict disease.

Abstract Xenotransplantation (cross-species transplantation) using genetically-engineered pig organs offers a potential solution to address persistent organ shortage. Current evaluation of porcine genetic modifications is to monitor the nonhuman primate immune response and survival after pig organ xenotransplantation. This measure is an essential step before clinical xenotransplantation trials, but it is time-consuming, costly, and inefficient with many variables. We developed an efficient approach to quickly examine human-to-pig xeno-immune responses in vitro. A porcine endothelial cell was characterized and immortalized for genetic modification. Five genes including GGTA1, CMAH, β4galNT2, SLA-I α chain, and β2-microglobulin that are responsible for the production of major xenoantigens (αGal, Neu5Gc, Sda, and SLA-I) were sequentially disrupted in immortalized porcine endothelial cells using CRISPR/Cas9 technology. The elimination of αGal, Neu5Gc, Sda, and SLA-I dramatically reduced the antigenicity of the porcine cells, though the cells still retained their ability to provoke human natural killer cell activation. In summary, evaluation of human immune responses to genetically modified porcine cells in vitro provides an efficient method to identify ideal combinations of genetic modifications for improving pig-to-human compatibility, which should accelerate the application of xenotransplantation to humans.

Abstract Introduction Gut microbiota has been reported to contribute to obesity and the pathology of obesity‐related diseases but the underlying mechanisms are largely unknown. Mucosal‐associated invariant T (MAIT) cells are a unique subpopulation of T cells characterized by the expression of a semi‐invariant T cell receptor (TCR) α chain (Vα19 in mice; Vα7.2 in humans). The expansion and maturation of MAIT cells require the gut microbiota and antigen‐presenting molecule MR1, suggesting that MAIT cells may play a unique role in bridging gut microbiota, obesity, and obesity‐associated inflammation. Methods The levels of human MAIT cells from obese patients, as well as mouse MAIT cells from obese mouse models, were determined by flow cytometry. By comparing to controls, we analyzed the change of MAIT cells in obese subjects. Results We found obese patients had fewer circulating MAIT cells than healthy‐weight donors and the difference was more distinct in male patients. Consistently, male mice (but not female mice) have shown reduced MAIT cells in the liver and adipose tissue after a 10‐week Western diet compared to mice on a control diet. We also explored the possibility of utilizing high‐throughput technology (i.e., quantitative polymerase chain reaction [qPCR]), other than flow cytometry, to determine the expression levels of the invariant TCR of human MAIT cells. But a minimal correlation (R2 = 0.23, p = .11) was observed between qPCR and flow cytometry data. Conclusion Our study suggests that there is a sex discrepancy in the impact of obesity on MAIT cells: MAIT cells in male (but not female) humans and male mice are reduced by obesity.

MicroRNAs (miRNAs) regulate the functions of granulosa cells by interacting with their target mRNAs. Insulin receptor substrate 2 (IRS2) is one of the targets of miR-431 and can be regulated by ovarian hormones. However, the role of miR-431 and the associated signal transduction pathway in ovarian development has not been studied previously. In this study, we first analyzed the expression of miR-431 and IRS2 following stimulation with pregnant mare serum gonadotropin (PMSG) during the estrous cycle or different stages of ovarian development in mice. Subsequently, we investigated the role, function, and signaling pathway of miR-431 in the human granulosa cell line, COV434. The results showed that follicle stimulating hormone (FSH) gradually decreased miR-431 levels, induced IRS2, and promoted pAKT expression. Moreover, miR-431 overexpression and IRS2 knockdown attenuated AKT activation, inhibited cell proliferation, and decreased estradiol (E2) and progesterone (P4) synthesis. Further, luciferase reporter assay demonstrated that IRS2 was a direct target of miR-431. In conclusion, this study demonstrated that miR-431 regulates granulosa cell function through the IRS2/PI3K/AKT signaling pathway.

Abstract Background The process of bone repair is heavily dependent on the ability of human bone marrow mesenchymal stem cells (hMSCs) to undergo osteogenic differentiation. MicroRNAs have been shown to regulate this osteogenic process. This study aimed to investigate the role of miR-765 in the osteogenic differentiation of hMSCs. Methods We transfected hMSCs with lentiviral constructs to knock down or overexpress this miRNA, allowing us to assess its role in osteogenesis via assessing the expression of the relevant markers alkaline phosphatase (ALP), runt-related gene-2 (RUNX-2), and osteocalcin (OCN), with further functional measurements made via quantifying ALP activity and conducting Alizarin Red S staining. The targeting of the 3′-untranslated region (UTR) of BMP6 by miR-765 was examined via luciferase assay. We used hMSCs with altered miR-765 expression to assess p-Smad1/5/9 levels via Western blotting over the course of osteogenic differentiation. We also assessed the osteogenic differentiation of hMSCs in which miR-765 was knocked down and at the same time as a BMP/Smad signaling inhibitor was added to disrupt Smad1/5/9 phosphorylation. Results We found miR-765 overexpression to inhibit osteogenesis-associated gene upregulation during osteogenic differentiation of hMSCs, whereas knockdown of this miRNA was associated with increased expression of these genes. Using luciferase reporter assays, we confirmed direct miR-765 binding to the 3′-untranslated region (UTR) of BMP6. We also found that miR-765 overexpression reduced Smad1/5/9 phosphorylation, and knockdown of this miRNA enhanced this phosphorylation on BMP6/Smad1/5/9 signaling. The osteogenic differentiation of hMSCs in which miR-765 had been knocked down was further weakened upon the addition of a BMP/Smad signaling inhibitor relative to miR-765 knockdown alone. Conclusions Together, these results thus suggest that miR-765 is able to inhibit hMSC osteogenic differentiation by targeting BMP6 via regulation of the BMP6/Smad1/5/9 signaling pathway. Our findings may offer molecular insights of value for the development of novel therapeutic treatments for bone diseases including osteoporosis.

A cohort of 542 individuals in 166 families with congenital hearing loss was recruited for whole-exome sequencing analysis. Here, we report the identification of three variants in five affected individuals in two unrelated families. In family 1, a nonsense mutation (c.1516C>T, p.R506*) in the ATP6V1B2 gene, a known causal allele for dominant deafness-onychodystrophy (DDOD), was identified in the mother and son with DDOD. However, a novel heterozygous variant (c.1590T>G, p.D530E) in TJP2, a known causal gene for hearing-loss, was also detected in the patients. In family 2, the same mutation (c.1516C>T, p.R506*) of ATP6V1B2 was detected from the father and daughter with DDOD. Furthermore, a novel heterozygous variant (c.733A>G, p.M245V) in the KIF11 gene was identified from the spouse with sensorineural hearing-loss and epilepsy. Notably, genotype-phenotype analysis of KIF11-associated disorders revealed that the p.M245V and two reported hearing-loss-associated variants (p.S235C and p.H244Y) are all mapped to a single β-sheet (Ser235∼M245) in the kinesin motor domain. Together, this is the first demonstration that ATP6V1B2-caused DDOD is an autosomal dominant genetic disease, compared to previous cases with de novo mutation. Our findings expand the variant spectrum of hearing-loss-associated genes and provide new insights on understanding of hearing-loss candidate genes ATP6V1B2, TJP2, and KIF11.

Hydrogen sulfide has been recently identified as the third biological gasotransmitter, along with the more well studied nitric oxide (NO) and carbon monoxide (CO). Intensive studies on its potential as a therapeutic agent for cardiovascular, inflammatory, infectious and neuropathological diseases have been undertaken. Here we review the possible direct targets of H2S in mammals. H2S directly interacts with reactive oxygen/nitrogen species and is involved in redox signaling. H2S also reacts with hemeproteins and modulates metal-containing complexes. Once being oxidized, H2S can persulfidate proteins by adding -SSH to the amino acid cysteine. These direct modifications by H2S have significant impact on cell structure and many cellular functions, such as tight junctions, autophagy, apoptosis, vesicle trafficking, cell signaling, epigenetics and inflammasomes. Therefore, we conclude that H2S is involved in many important cellular and physiological processes. Compounds that donate H2S to biological systems can be developed as therapeutics for different diseases.

Abstract The ubiquitin-specific protease 22 (USP22) is an oncogene and its expression is upregulated in many types of cancer. In the nucleus, USP22 functions as one subunit of the SAGA to regulate gene transcription. However, the genome-wide USP22 binding sites and its direct target genes are yet clear. In this study, we characterized the potential genomic binding sites of UPS22 and GCN5 by ChIP-seq using specific antibodies in HeLa cells. There were 408 overlapping putative target genes bound by both USP22 and GCN5. Motif analysis showed that the sequences bound by USP22 and GCN5 shared two common motifs. Gene ontology (GO) and pathway analysis indicated that the genes targeted by USP22 and GCN5 were involved in different physiological processes and pathways. Further RNA-seq, GO and pathway analyses revealed that knockdown of UPS22 induced differential expression of many genes that participated in diverse physiological processes, such as metabolic process. Integration of ChIP-seq and RNA-seq data revealed that UPS22 bound to the promoters of 56 genes. These findings may provide new insights into the regulation of USP22 on gene expression during the development of cervical cancer.

Neurofibromin 1 (NF1) is a tumor suppressor gene encoding a Ras GTPase that negatively regulates Ras signaling pathways. Mutations in NF1 are linked to neurofibromatosis type 1, juvenile myelomonocytic leukemia and Watson syndrome. In terms of antitumor immunity, CD1d-dependent natural killer T (NKT) cells play an important role in the innate antitumor immune response. Generally, Type-I NKT cells protect (and Type-II NKT cells impair) host antitumor immunity. We have previously shown that CD1d-mediated antigen presentation to NKT cells is regulated by cell signaling pathways. To study whether a haploinsufficiency in NF1 would affect CD1d-dependent activation of NKT cells, we analyzed the NKT-cell population as well as the functional expression of CD1d in Nf1+/− mice. Nf1+/− mice were found to have similar levels of NKT cells as wildtype (WT) littermates. Interestingly, however, reduced CD1d expression was observed in Nf1+/− mice compared with their WT littermates. When inoculated with a T-cell lymphoma in vivo, Nf1+/− mice survived longer than their WT littermates. Furthermore, blocking CD1d in vivo significantly enhanced antitumor activity in WT, but not in Nf1+/− mice. In contrast, a deficiency in Type-I NKT cells increased antitumor activity in Nf1+/− mice, but not in WT littermates. Therefore, these data suggest that normal NF1 expression impairs CD1d-mediated NKT-cell activation and antitumor activity against a T-cell lymphoma.

CD1d molecules are structurally similar to MHC class I, but present lipid antigens as opposed to peptides. Here, we show that MHC class I molecules physically associate with (and regulate the functional expression of) mouse CD1d on the surface of cells. Low pH (3.0) acid stripping of MHC class I molecules resulted in increased surface expression of murine CD1d on antigen presenting cells as well as augmented CD1d-mediated antigen presentation to NKT cells. Consistent with the above results, TAP1-/- mice were found to have a higher percentage of type I NKT cells as compared to wild type mice. Moreover, bone marrow-derived dendritic cells from TAP1-/- mice showed increased antigen presentation by CD1d compared to wild type mice. Together, these results suggest that MHC class I molecules can regulate NKT cell function, in part, by masking CD1d.