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Background: Antiviral drugs show significant efficacy in non-severe COVID-19 cases, yet there remains a subset of moderate COVID-19 patients whose pneumonia continues to progress post a complete course of treatment. Plasma-activated water (PAW) possesses anti-SARS-CoV-2 properties. To explore the potential of PAW in improving pneumonia in COVID-19 patients following antiviral treatment failure, we conducted this study. Methods: This was a randomized, controlled trial. Moderate COVID-19 patients with antiviral treatment failure were randomly assigned to the experimental group or the control group. They inhaled nebulized PAW or saline respectively. This was done twice daily for four consecutive days. We assessed improvement in chest CT on day 5, the rate of symptom resolution within 10 days, and safety. Results: A total of 23 participants were included, with 11 receiving PAW and 12 receiving saline. The baseline characteristics of both groups were comparable. The experimental group showed a higher improvement rate in chest CT on day 5 (81.8% vs. 33.3%, p = 0.036). The cumulative disappearance rate of cough within 10 days was higher in the experimental group. Within 28 days, 4 patients in each group progressed to severe illness, and no patients died. No adverse reactions were reported from inhaling nebulized PAW. Conclusion: This pilot trial preliminarily confirmed that nebulized inhalation of PAW can alleviate pneumonia in moderate COVID-19 patients with antiviral treatment failure, with no adverse reactions observed. This still needs to be verified by large-scale studies. Trial registration: Chinese Clinical Trial Registry; No.: ChiCTR2300078706 (retrospectively registered, 12/15/2023); URL: www.chictr.org.cn. [ABSTRACT FROM AUTHOR]

Background: Infertility is a growing global health concern affecting millions of couples worldwide. Among several factors, an extreme body weight adversely affects reproductive functions. Leptin is a well-known adipokine that serves as an endocrine signal between adiposity and fertility. However, the exact mechanisms underlying the effects of high leptin level on female reproduction remain unclear. Methods: Transgenic pigs overexpressing leptin (♀) were produced by backcrossing and screened for leptin overexpression. The growth curve, fat deposition, reproductive performance, apoptosis, serum hormones and cholesterol production, RNA sequencing, and single-nucleus RNA sequencing (snRNA-seq) of the leptin-overexpressing pigs and wild-type group were evaluated. Results: Transgenic pigs overexpressing leptin (♀) were obtained, which exhibited significantly reduced body weight, body size, and back fat thickness. These pigs manifested a late onset of puberty (330 ± 54.3 vs. 155 ± 14.7 days), irregular estrous behavior characterized by increased inter-estrous interval (29.2 ± 0 vs. 21.3 ± 0.7 days), and more number of matings until pregnancy (at least 3 times). This reproductive impairment in leptin pigs was related to hormonal imbalances characterized by increased levels of FSH, LH, prolactin, E2, P4, and TSH, altered steroidogenesis such as increased levels of serum cholesterol esters along with steroidogenic markers (StAR, CYP19A), and ovarian dysfunctions manifested by neutrophilic infiltration and low expression of caspase-3 positive cells in the ovaries. Moreover, bulk RNA sequencing of the ovaries also revealed neutrophilic infiltration followed by upregulation of inflammation-related genes. Furthermore, snRNA-seq reflected that leptin overexpression triggered immune response, suppressed follicle development and luteinization, resulting in metabolic dysfunction and hormone imbalance in the ovary. Conclusions: Low body weight in leptin overexpressing pigs adversely affects the reproductive performance, causing delayed puberty, irregular estrous cycles, and reduced breeding efficiency. This is linked to metabolic imbalances, an increased immune response, and altered ovarian functions. This study provides a theoretical basis for the complex mechanisms underlying leptin, and infertility by employing leptin-overexpressing female pigs. [ABSTRACT FROM AUTHOR]

Background: Neurodevelopmental disorders (NDD), such as autism spectrum disorders (ASD) and intellectual disorders (ID), are highly debilitating childhood psychiatric conditions. Genetic factors are recognized as playing a major role in NDD, with a multitude of genes and genomic regions implicated. While the functional validation of NDD-associated genes has predominantly been carried out using mouse models, the significant differences in brain structure and gene function between mice and humans have limited the effectiveness of mouse models in exploring the underlying mechanisms of NDD. Therefore, it is important to establish alternative animal models that are more evolutionarily aligned with humans. Results: In this study, we employed CRISPR/Cas9 and somatic cell nuclear transplantation technologies to successfully generate a knockout miniature pig model of the MIR137 gene, which encodes the neuropsychiatric disorder-associated microRNA miR-137. The homozygous knockout of MIR137 (MIR137–/–) effectively suppressed the expression of mature miR-137 and led to the birth of stillborn or short-lived piglets. Transcriptomic analysis revealed significant changes in genes associated with neurodevelopment and synaptic signaling in the brains of MIR137–/– miniature pig, mirroring findings from human ASD transcriptomic data. In comparison to miR-137-deficient mouse and human induced pluripotent stem cell (hiPSC)-derived neuron models, the miniature pig model exhibited more consistent changes in critical neuronal genes relevant to humans following the loss of miR-137. Furthermore, a comparative analysis identified differentially expressed genes associated with ASD and ID risk genes in both miniature pig and hiPSC-derived neurons. Notably, human-specific miR-137 targets, such as CAMK2A, known to be linked to cognitive impairments and NDD, exhibited dysregulation in MIR137–/– miniature pigs. These findings suggest that the loss of miR-137 in miniature pigs affects genes crucial for neurodevelopment, potentially contributing to the development of NDD. Conclusions: Our study highlights the impact of miR-137 loss on critical genes involved in neurodevelopment and related disorders in MIR137–/– miniature pigs. It establishes the miniature pig model as a valuable tool for investigating neurodevelopmental disorders, providing valuable insights for potential applications in human research. [ABSTRACT FROM AUTHOR]

Background: Acute ischemic stroke (AIS) complicating an acute myocardial infarction (AMI) is not uncommon, but can severely worsen the clinical prognosis. This study aimed to investigate whether remote ischemic conditioning (RIC) could provide clinical benefits to patients with AIS complicating AMI. Methods: Subjects with AIS complicating AMI were recruited in this double-blind, randomized, controlled trial; assigned to the RIC and sham groups; and respectively underwent twice daily RIC and sham RIC for 2 weeks. All subjects received standard medical therapy. The primary endpoint was the rate of major adverse cardiac and cerebrovascular events (MACCEs) within 3 months after enrollment. MACCEs comprise of death from all causes, unstable anginas, AMI, acute ischemic strokes, and transient ischemic attacks. Results: Eighty subjects were randomly assigned; 37 patients in the RIC group and 40 patients in the sham-RIC group completed the 3-month follow-up and were included in the final analysis. Both RIC and sham RIC procedures were well tolerated. At 3-month follow-up, 11 subjects (29.7%) in the RIC group experienced MACCEs compared to 21 (52.5%) in the sham group (hazard ratio [HR], 0.396; 95% confidence interval, 0.187–0.838; adjusted p < 0.05). Six subjects (16.2%) in the RIC group had died at the 3-month follow up, significantly lower than the 15 (37.5%) deaths in the sham group (adjusted HR 0.333; 95% CI 0.126–0.881; p = 0.027). Seventeen subjects (45.9%) in the RIC group and 6 subjects (15.0%) in the sham group achieved functional independence (mRS score ≤ 2) at 3-month follow-up (adjusted OR 12.75; 95% CI 2.104–77.21; p = 0.006). Conclusions: Among patients with acute ischemic stroke complicating acute myocardial infarction, treatment with remote ischemic conditioning decreased the major adverse cardiac and cerebrovascular events and improved functional outcomes at 90 days. Trial registration: URL: www.clinicaltrials.gov. Unique identifier: NCT03868007. Registered 8 March 2019. [ABSTRACT FROM AUTHOR]

Background: Pigs have many features that make them attractive as biomedical models for various diseases, including cancer. P53 is an important tumor suppressor gene that exerts a central role in protecting cells from oncogenic transformation and is mutated in a large number of human cancers. P53 mutations occur in almost every type of tumor and in over 50% of all tumors. In a recent publication, pigs with a mutated P53 gene were generated that resulted in lymphoma and renal and osteogenic tumors. However, approximately 80% of human tumors have dysfunctional P53. A P53-deficient pig model is still required to elucidate.Methods: Transcription activator-like effector nucleases (TALENs) were designed to target porcine P53 exon 4. The targeting activity was evaluated using a luciferase SSA recombination assay. P53 biallelic knockout (KO) cell lines were established from single-cell colonies of fetal fibroblasts derived from Diannan miniature pigs followed by electroporation with TALENs plasmids. One cell line was selected as the donor cell line for somatic cell nuclear transfer (SCNT) for the generation of P53 KO pigs. P53 KO stillborn fetuses and living piglets were obtained. Gene typing of the collected cloned individuals was performed by T7EI assay and sequencing. Fibroblast cells from Diannan miniature piglets with a P53 biallelic knockout or wild type were analyzed for the P53 response to doxorubicin treatment by confocal microscopy and western blotting.Results: The luciferase SSA recombination assay revealed that the targeting activities of the designed TALENs were 55.35-fold higher than those of the control. Eight cell lines (8/19) were mutated for P53, and five of them were biallelic knockouts. One of the biallelic knockout cell lines was selected as nuclear donor cells for SCNT. The cloned embryos were transferred into five recipient gilts, three of them becoming pregnant. Five live fetuses were obtained from one surrogate by caesarean section after 38 days of gestation for genotyping. Finally, six live piglets and one stillborn piglet were collected from two recipients by caesarean section. Sequencing analyses of the target site confirmed the P53 biallelic knockout in all fetuses and piglets, consistent with the genotype of the donor cells. The qPCR analysis showed that the expression of the P53 mRNA had significant reduction in various tissues of the knockout piglets. Furthermore, confocal microscopy and western blotting analyses demonstrated that the fibroblast cells of Diannan miniature piglets with a P53 biallelic knockout were defective in mediating DNA damage when incubated with doxorubicin.Conclusion: TALENs combined with SCNT was successfully used to generate P53 KO Diannan miniature pigs. Although these genetically engineered Diannan miniature pigs had no tumorigenic signs, the P53 gene was dysfunctional. We believe that these pigs will provide powerful new resources for preclinical oncology and basic cancer research. [ABSTRACT FROM AUTHOR]

Background: Glycyrrhizin (Gly) protects against brain injury induced by stroke. We studied whether Gly achieves its protection by inhibiting T cell activity and high-mobility group box 1 (HMGB1) release in the ischemic brain.Methods: Stroke was induced by transient middle cerebral artery occlusion in rats and mice. Gly was injected intraperitoneally before or after stroke. We measured infarction, neuroinflammatory cells, gene expressions of interferon-γ (IFNγ), IL-4, and IL-10 in CD4 T cells, HMGB1 release, and T cell proliferation in cultured splenocytes.Results: Gly treatment reduced infarctions and neuroinflammation characterized by the infiltration of CD68-positive macrophages and myeloperoxidase-positive neutrophils, which corresponds to a reduction in the number of T cells and their subsets, CD4 and CD8 T cells, in the ischemic brain, as measured by flow cytometry. Unlike in wild-type animals, Gly did not offer protection in nude rats and severe combined immunodeficient (SCID) mice who had no T cells, while Gly reduced infarction in both nude rats and SCID mice whose T cells were reconstituted, suggesting that T cells should be the target of Gly. In addition, Gly administration inhibited T cell proliferation stimulated by ConA in in vitro assays and inhibited HMGB1 release from the ischemic brain. Furthermore, Gly attenuated gene expression of IFNγ, but not IL-4 and IL-10 in CD4 T cells. Lastly, HMGB1 promoted T cell proliferation stimulated by ConA, which was inhibited by the addition of Gly.Conclusions: Gly blocks infarction by inhibiting IFNγ-mediated T cell activity, which is at least partly modulated by HMGB1 activity. [ABSTRACT FROM AUTHOR]

We read with great interest the article by Yin et al. (Parasit Vectors 14:238, 2021). The authors found that Cryptosporidium infection induced significantly aberrant expression of circular RNA profiles in HCT-8 cells, a finding which has far-reaching implications. However, due to the high number of false positives caused by multiple comparisons, statistical methods for microarray analysis should be carefully selected. Accurate analysis results will provide a convincing basis for subsequent experiments. In addition, we recommend several more appropriate methods in this article. [ABSTRACT FROM AUTHOR]

Background: Vitamin D (VD) insufficiency or deficiency is a frequent comorbidity in Chinese women with postmenopausal osteoporosis (PMO). The present study aimed to investigate 25-hydroxyvitamin D [25(OH) D] improvement and calcium-phosphate metabolism in Chinese PMO patients treated with 70 mg of alendronate sodium and 5600 IU of vitamin D3 (ALN/D5600).Methods: Chinese PMO women (n = 219) were treated with 12-month ALN/D5600 (n = 111) or calcitriol (n = 108). Changes in 25(OH) D at month 12 were post hoc analyzed by the baseline 25 (OH) D status using the longitudinal analysis. The main safety outcome measures included serum calcium and phosphate and 24-h urine calcium, and the repeated measures mixed model was used to assess the frequencies of the calcium-phosphate metabolic disorders.Results: Absolute change in mean serum 25(OH) D level was the greatest in VD-deficient patients and least in VD-sufficient patients at months six and 12 (both, P < 0.01). Serum calcium level remained significantly lower in the ALN/D5600 treatment group than in the calcitriol treatment group throughout the 12 months. Mean 24-h urine calcium slightly increased in the ALN/D5600 treatment group and significantly increased in the calcitriol treatment group (+ 1.1 and + 0.9 mmol/L at months six and 12; both, P < 0.05). Calcitriol treatment was associated with more frequent hypercalciuria at month six (9.4% vs. 18.5%, P = 0.05), but not at month 12 (12.3% vs. 13.0%).Conclusion: Baseline VD status predicted 25(OH) D improvement in PMO patients on 12-month ALN/D5600 treatment. The daily use of 0.25 μg of calcitriol was associated with more frequent hypercalciuria at month six, compared to ALN/5600 treatment, necessitating the safety re-evaluation of calcitriol at a higher dosage. [ABSTRACT FROM AUTHOR]

Background: Pigs have many features that make them attractive as biomedical models for various diseases, including cancer. P53 is an important tumor suppressor gene that exerts a central role in protecting cells from oncogenic transformation and is mutated in a large number of human cancers. P53 mutations occur in almost every type of tumor and in over 50% of all tumors. In a recent publication, pigs with a mutated P53 gene were generated that resulted in lymphoma and renal and osteogenic tumors. However, approximately 80% of human tumors have dysfunctional P53. A P53-deficient pig model is still required to elucidate., Methods: Transcription activator-like effector nucleases (TALENs) were designed to target porcine P53 exon 4. The targeting activity was evaluated using a luciferase SSA recombination assay. P53 biallelic knockout (KO) cell lines were established from single-cell colonies of fetal fibroblasts derived from Diannan miniature pigs followed by electroporation with TALENs plasmids. One cell line was selected as the donor cell line for somatic cell nuclear transfer (SCNT) for the generation of P53 KO pigs. P53 KO stillborn fetuses and living piglets were obtained. Gene typing of the collected cloned individuals was performed by T7EI assay and sequencing. Fibroblast cells from Diannan miniature piglets with a P53 biallelic knockout or wild type were analyzed for the P53 response to doxorubicin treatment by confocal microscopy and western blotting., Results: The luciferase SSA recombination assay revealed that the targeting activities of the designed TALENs were 55.35-fold higher than those of the control. Eight cell lines (8/19) were mutated for P53, and five of them were biallelic knockouts. One of the biallelic knockout cell lines was selected as nuclear donor cells for SCNT. The cloned embryos were transferred into five recipient gilts, three of them becoming pregnant. Five live fetuses were obtained from one surrogate by caesarean section after 38 days of gestation for genotyping. Finally, six live piglets and one stillborn piglet were collected from two recipients by caesarean section. Sequencing analyses of the target site confirmed the P53 biallelic knockout in all fetuses and piglets, consistent with the genotype of the donor cells. The qPCR analysis showed that the expression of the P53 mRNA had significant reduction in various tissues of the knockout piglets. Furthermore, confocal microscopy and western blotting analyses demonstrated that the fibroblast cells of Diannan miniature piglets with a P53 biallelic knockout were defective in mediating DNA damage when incubated with doxorubicin., Conclusion: TALENs combined with SCNT was successfully used to generate P53 KO Diannan miniature pigs. Although these genetically engineered Diannan miniature pigs had no tumorigenic signs, the P53 gene was dysfunctional. We believe that these pigs will provide powerful new resources for preclinical oncology and basic cancer research.

Background: α1,3-Galactosyltransferase (GGTA1) is essential for the biosynthesis of glycoproteins and therefore a simple and effective target for disrupting the expression of galactose α-1,3-galactose epitopes, which mediate hyperacute rejection (HAR) in xenotransplantation. Miniature pigs are considered to have the greatest potential as xenotransplantation donors. A GGTA1-knockout (GTKO) miniature pig might mitigate or prevent HAR in xenotransplantation., Methods: Transcription activator-like effector nucleases (TALENs) were designed to target exon 6 of porcine GGTA1 gene. The targeting activity was evaluated using a luciferase SSA recombination assay. Biallelic GTKO cell lines were established from single-cell colonies of fetal fibroblasts derived from Diannan miniature pigs following transfection by electroporation with TALEN plasmids. One cell line was selected as donor cell line for somatic cell nuclear transfer (SCNT) for the generation of GTKO pigs. GTKO aborted fetuses, stillborn fetuses and live piglets were obtained. Genotyping of the collected cloned individuals was performed. The Gal expression in the fibroblasts and one piglet was analyzed by fluorescence activated cell sorting (FACS), confocal microscopy, immunohistochemical (IHC) staining and western blotting., Results: The luciferase SSA recombination assay revealed that the targeting activities of the designed TALENs were 17.1-fold higher than those of the control. Three cell lines (3/126) showed GGTA1 biallelic knockout after modification by the TALENs. The GGTA1 biallelic modified C99# cell line enabled high-quality SCNT, as evidenced by the 22.3 % (458/2068) blastocyst developmental rate of the reconstructed embryos. The reconstructed GTKO embryos were subsequently transferred into 18 recipient gilts, of which 12 became pregnant, and six miscarried. Eight aborted fetuses were collected from the gilts that miscarried. One live fetus was obtained from one surrogate by caesarean after 33 d of gestation for genotyping. In total, 12 live and two stillborn piglets were collected from six surrogates by either caesarean or natural birth. Sequencing analyses of the target site confirmed the homozygous GGTA1-null mutation in all fetuses and piglets, consistent with the genotype of the donor cells. Furthermore, FACS, confocal microscopy, IHC and western blotting analyses demonstrated that Gal epitopes were completely absent from the fibroblasts, kidneys and pancreas of one GTKO piglet., Conclusions: TALENs combined with SCNT were successfully used to generate GTKO Diannan miniature piglets.

Background: Glycyrrhizin (Gly) protects against brain injury induced by stroke. We studied whether Gly achieves its protection by inhibiting T cell activity and high-mobility group box 1 (HMGB1) release in the ischemic brain., Methods: Stroke was induced by transient middle cerebral artery occlusion in rats and mice. Gly was injected intraperitoneally before or after stroke. We measured infarction, neuroinflammatory cells, gene expressions of interferon-γ (IFNγ), IL-4, and IL-10 in CD4 T cells, HMGB1 release, and T cell proliferation in cultured splenocytes., Results: Gly treatment reduced infarctions and neuroinflammation characterized by the infiltration of CD68-positive macrophages and myeloperoxidase-positive neutrophils, which corresponds to a reduction in the number of T cells and their subsets, CD4 and CD8 T cells, in the ischemic brain, as measured by flow cytometry. Unlike in wild-type animals, Gly did not offer protection in nude rats and severe combined immunodeficient (SCID) mice who had no T cells, while Gly reduced infarction in both nude rats and SCID mice whose T cells were reconstituted, suggesting that T cells should be the target of Gly. In addition, Gly administration inhibited T cell proliferation stimulated by ConA in in vitro assays and inhibited HMGB1 release from the ischemic brain. Furthermore, Gly attenuated gene expression of IFNγ, but not IL-4 and IL-10 in CD4 T cells. Lastly, HMGB1 promoted T cell proliferation stimulated by ConA, which was inhibited by the addition of Gly., Conclusions: Gly blocks infarction by inhibiting IFNγ-mediated T cell activity, which is at least partly modulated by HMGB1 activity.

Background: The aim of this study was to identify the candidate genes of esophageal squamous cell carcinoma (ESCC)., Methods: Gene expression profiling of 17 ESCC samples and 17 adjacent normal samples, GSE20347, was downloaded from Gene Expression Omnibus database. The raw data were preprocessed, and the differentially expressed genes (DEGs) between ESCC and normal samples were identified by using SAM software (false discovery rate <0.001). Then, the co-expression network of DEGs was constructed based on Pearson's correlation test (r-value ≥0.8). Furthermore, the topological properties of the co-expression network were analyzed through NetworkAnalyzer (default settings) of Cytoscape. The expression fold changes of DEGs and topological properties were utilized to identify the candidate genes of ESCC (Crin score >4), which were further analyzed based on DAVID functional enrichment analysis (P-value <0.05)., Results: A total of 1,063 DEGs were identified, including 490 up-regulated and 573 down-regulated DEGs. Then, the co-expression network of DEGs was constructed, containing 999 nodes and 46,323 edges. Based on the expression fold changes of DEGs and the topological properties of the co-expression network, DEGs were ranked, and the top 24 genes were candidate genes of ESCC, such as CRISP3, EREG, CXCR2, and CRNN. Furthermore, the 24 genes were significantly enriched in bio-functions regarding cell differentiation, glucan biosynthetic process and immune response., Conclusion: The present study suggested that CRISP3, EREG, CXCR2, and CRNN might be causative genes of ESCC, and play vital roles in the development of ESCC. However, further experimental studies are needed to confirm our results.