Growth hormone secretagogue receptor (GHSR) and its ligand, ghrelin, are important modulators in weight control and energy homeostasis. Recently, ghrelin is also involved in experimental colitis, but the role of GHSR in the development of colitis is unclear. The aim was to examine the underlying mechanism of GHSR in IBD development.

The Krüppel-associated box (KRAB) domain is a transcription repression module from the largest family of transcriptional regulators encoded by higher vertebrates. We developed a drug-controllable regulation system based on an artificial KRAB-containing repressor (tTS) that targets the endogenous Hprt gene to explore the regulatory mechanism and molecular basis of KRAB-containing regulators within the context of an endogenous gene in vivo. We show that KRAB can mediate irreversible and reversible regulation of endogenous genes in mouse that is dependent on embryonic developmental stage. KRAB-induced stable DNA methylation within the KRAB binding region during the early embryonic stage, resulting in irreversible gene repression. In later stages, KRAB mainly induced de-acetylation and methylation of histone, resulting in reversible gene repression. Thus, we have characterized the KRAB-mediated regulation system within the context of an endogenous gene and multiple spatiotemporal ranges, thereby providing a basis for identifying the function of KRAB-containing regulators and aiding development of novel KRAB-based gene regulation tools in vivo.

Methods for generating loss-of-function mutations, such as conventional or conditional gene knockout, are widely used in deciphering gene function in vivo. By contrast, inducible and reversible regulation of endogenous gene expression has not been well established. Using a mouse model, we demonstrate that a chimeric transcriptional repressor molecule (tTS) can reversibly inhibit the expression of an endogenous gene, Nmyc. In this system, a tetracycline response element (TRE) artificially inserted near the target gene's promoter region turns the gene on and off in a tetracycline-inducible manner. Nmyc(TRE) mice were generated by inserting a TRE into the first intron of Nmyc by the knockin technique. Nmyc(TRE) mice were crossed to tTS transgenic mice to produce Nmyc(TRE/TRE): tTS embryos. In these embryos, tTS blocked Nmyc expression, and embryonic lethality was observed at E11.5d. When the dam was exposed to drinking water containing doxycycline (dox), normal endogenous Nmyc expression was rescued, and the embryo survived to birth. This novel genetic modification strategy based on the tTS-dox system for inducible and reversible regulation of endogenous mouse genes will be a powerful tool to investigate target genes that cause embryonic lethality or other defects where reversible regulation or temporary shutdown of the target gene is needed.

c-Rel plays important roles in many inflammatory diseases. Revealing the dynamic expression of c-Rel in disease processes in vivo is critical for understanding c-Rel functions and for developing anti-inflammatory drugs. In this paper, a transgenic mouse line, B6-Tg(c-Rel-luc)(Mlit), which incorporated the transgene firefly luciferase driven by a 14.5-kb fragment containing mouse c-Rel gene Rel promoter, was generated to monitor Rel expression in vivo. Luciferase expression could be tracked in living mice by the method of bioluminescence imaging in a variety of inflammatory processes, including LPS induced sepsis and EAE disease model. The luciferase expression in transgenic mice was comparable to the endogenous Rel expression and could be suppressed by administration of anti-inflammatory drug dexamethasone or aspirin. These results indicate that the B6-Tg(c-Rel-luc)(Mlit) mouse is a valuable animal model to study Rel expression in physiological and pathological processes, and the effects of various drug treatments in vivo.

Gastrointestinal mucositis is a major side effect of chemotherapy, leading to life quality reduction in patients and interrupting the therapy of cancer. Chimonanthus nitens var. salicifolius (CS) is a traditional Chinese herb for enteral disease. Considering the protective effect of CS on intestine, we hypothesize that the aqueous extract of CS could be benefcial to gastrointestinal mucositis. To verify this, a mouse mucositis model was induced by 5-Fluorouracil (5-Fu). Male Balb/C mice were treated with CS aqueous extract (5, 10, and 20 g/kg) or loperamide (0.2 mg/kg) intragastrically for 11 days, and the severity of mucositis was evaluated. Furthermore, the chemical compounds of CS aqueous extract were also analysed by high-performance liquid chromatography (HPLC). Our results demonstrated that CS aqueous extract improved mice body weight, diarrhoea, and faecal blood, maintained the liver function and intestinal length, alleviated villus shortening, and suppressed the apoptosis and inflammation in small intestine. We concluded that CS could protect mice against 5-Fu induced mucositis by inhibiting apoptosis and inflammation, and this protective effect might be associated with the 3 flavonoids (rutin, quercetin, and kaempferol) identified in CS aqueous extract.

Liuwei Dihuang (LWDH), a famous traditional Chinese medicine, is widely used in the clinical treatment of aging-related diseases in China. However, its pharmacological mechanisms are not clear. In the present study, we evaluated the lifespan extension effect of LWDH in C. elegans and mice and revealed its underlying mechanisms. The results showed that LWDH significantly extended the lifespan of C. elegans in a dose-dependent manner. LWDH also conferred protection to nematodes against oxidative stress and reduced their fat storage. Genetics analysis and microarray data showed that the longevity effect of LWDH was attributed to the regulation of the innate immune response, proteolysis, lipid metabolism, and the oxidation-reduction process and was dependent on daf-16. Among the six herbs in the formula, Radix Rehmanniae Preparata and Fructus Macrocarpii contributed most to the longevity effect of this medicine, while the other four components had a synergistic effect on the longevity effect of the prescription. The lack of any single herb reduced the efficacy of the complete formula. LWDH also extended the lifespan and reduced both the weight and oxidant stress status in aged mice. Taken together, these results suggested that LWDH might function in a multi-target manner to extend the lifespan in both C. elegans and aged mice, and the best effect was achieved with the complete formula.

Testis-specific genes are prone to affect spermatogenesis or sperm fertility, and thus may play pivotal roles in male reproduction. However, whether a gene really affects male reproduction in vivo needs to be confirmed using a gene knock-out (KO) model, a 'gold standard' method. Increasing studies have found that some of the evolutionarily conserved testis-enriched genes are not essential for male fertility. In this study, we report that 1700121C10Rik, a previously uncharacterized gene, is specifically expressed in the testis and produces two long noncoding RNAs (lncRNAs) in mouse: Transcript 1 and Transcript 2. qRT-PCR, northern blotting, and in situ hybridization revealed that expression of both the lncRNAs commenced at the onset of sexual maturity and was predominant in round and elongating spermatids during spermiogenesis. Moreover, we found different subcellular localization of Transcript 1 and Transcript 2 that was predominant in the cytoplasm and nucleus, respectively. 1700121C10Rik-KO mouse model disrupting Transcript 1 and Transcript 2 expression was generated by CRISPR/Cas9 to determine their role in male reproduction. Results showed that 1700121C10Rik-KO male mice were fully fertile with approximately standard testis size, testicular histology, sperm production, sperm morphology, sperm motility, and induction of acrosome reaction. Thus, we conclude that both the testis-specific 1700121C10Rik-produced lncRNAs are dispensable for male fertility in mice under standard laboratory conditions.

To investigate the risk factors of cervical lymph node (LN) metastasis in papillary thyroid microcarcinoma (PTMC) patients.

While genome-wide studies have identified genomic loci in hosts associated with life-threatening Covid-19 (critical Covid-19), the challenge of resolving these loci hinders further identification of clinically actionable targets and drugs. Building upon our previous success, we here present a priority index solution designed to address this challenge, generating the target and drug resource that consists of two indexes: the target index and the drug index. The primary purpose of the target index is to identify clinically actionable targets by prioritising genes associated with Covid-19. We illustrate the validity of the target index by demonstrating its ability to identify pre-existing Covid-19 phase-III drug targets, with the majority of these targets being found at the leading prioritisation (leading targets). These leading targets have their evolutionary origins in Amniota ('four-leg vertebrates') and are predominantly involved in cytokine-cytokine receptor interactions and JAK-STAT signaling. The drug index highlights opportunities for repurposing clinically approved JAK-STAT inhibitors, either individually or in combination. This proposed strategic focus on the JAK-STAT pathway is supported by the active pursuit of therapeutic agents targeting this pathway in ongoing phase-II/III clinical trials for Covid-19.

Degeneration of the dopaminergic neurons in the substantia nigra and the resultant dopamine depletion from the striatum are the hallmarks of Parkinson's disease (PD) and are responsible for the disease's cardinal motor symptoms. The transcriptional repressor Neuron-Restrictive Silencer Factor (NRSF), also known as RE1-Silencing Transcription Factor (REST), was originally identified as a negative regulator of neuron-specific genes in non-neuronal cells. Our previous study showed that mice deficient in neuronal NRSF/REST expression were more vulnerable to the noxious effects of the dopaminergic neurotoxin MPTP. Here, we found that brain-specific deletion of NRSF/REST led to more severe damages to the nigrostriatal pathway and long-lasting behavioral impairments in mice challenged with MPTP. Moreover, compared to wild-type controls, these mice showed increased neurogenesis shortly after MPTP exposure, but reduced neurogenesis later on. These results suggest that NRSF/REST acts as a negative modulator of neurogenesis and a pro-survival factor of neural stem cells under both normal conditions and during the course of PD.

The aim of the present study was to establish the effect of silencing L1 cell adhesion molecule (L1CAM) on the proliferation, invasion, cell cycle progression and apoptosis of pancreatic cancer cells, and to determine the potential molecular mechanisms that are involved. The human Capan-2 pancreatic cancer cell line was infected with lentivirus-mediated short hairpin RNA (shRNA) to target L1CAM. Cell proliferation and invasion were analyzed using cell counting kit-8 and Transwell assays, respectively, and cell cycle progression and apoptosis were analyzed using flow cytometry. L1CAM protein expression in Capan-2 cells decreased following shRNA-L1CAM infection. Furthermore, knockdown of L1CAM significantly inhibited cell proliferation and reduced the number of invasive cells, while increasing the percentage of cells in the G0/G1 phase (P<0.05). However, the effect on apoptosis was not identified to be statistically significant. In addition, L1CAM silencing may induce activation of p38/extracellular signal regulated kinase 1/2. Downregulation of L1CAM may inhibit proliferation, invasion and arrests cell cycle progression in pancreatic cancer via p38/ERK1/2 signal pathway, and therefore, L1CAM may serve as a potential target for gene therapy in pancreatic cancer.

As a traditional herbal medicine, the clinical efficacy of Ganoderma lucidum (G. lucidum, also known as Lingzhi in China) has been proved by clinical research and a large number of animal experiments. However, its pharmacological mechanism is not clear. Here, we used the Caenorhabditis elegans as an animal model to study the anti-oxidative stress and anti-aging effects of G. lucidum water extract. Our results showed that G. lucidum effectively promoted the nematodes to resist the oxidative stress of paraquat and heavy metal Cr6+, and significantly prolonged the lifespan of the nematodes. The underlining mechanisms were further investigated by focusing on the signaling pathways that regulate the stress responses and the lifespan. We found that G. lucidum protected the nematode against the insults of paraquat and heavy metals through the diet restriction pathway and the mTOR/S6K signaling pathway, respectively. Whereas, the effect of G. lucidum on the longevity of the nematode mainly depended on the germline signaling pathway. Microarray assays were conducted to reveal the gene expression profiles. The expression levels of 2746 genes were significantly changed during the aging process, of which 34 genes were reversed in their expression by the treatment of G. lucidum in aged nematodes. These results suggest that G. lucidum regulates the biophysiological processes in the nematodes through multiple signaling pathways.

Hepatocytes perform most of the functions of the liver and are considered terminally differentiated cells. Recently, it has been suggested that hepatocytes might have the potential to transdifferentiate or dedifferentiate under physiological or pathological conditions in vivo. Epithelial-mesenchymal transition of hepatocytes in liver fibrosis has also been proposed. However, these findings have not been fully confirmed. In this study, hepatocytes were genetically labelled for cell fate tracing using lacZ via the tamoxifen-induced CreERT/loxP system. After induction with tamoxifen, alb + cells were permanently marked by lacZ expression, and all progeny lacZ + cells were derived from a single source with no interference. We did not observe transdifferentiation or dedifferentiation of hepatocytes into cholangiocytes or hepatic progenitor cells under conditions of liver homeostasis or following a 2/3 partial hepatectomy. Meanwhile, lacZ/OPN-positive cells were observed in livers of 3,5-diethoxycarbonyl-1,4-dihydrocollidine-fed mice, and lacZ/alpha-smooth muscle actin-positive cells were detected in carbon tetrachloride-induced chronic liver injury models. These results suggested that some existing differentiated alb + cells might have the potential of transdifferentiation/dedifferentiation or epithelial-to-mesenchymal transition in vivo in some liver injury models, but the proportion of these alb + cells in liver was very low, and their significance and actual function during the pathological process remains to be elucidated.

G protein-coupled receptor 116 (GPR116) is a novel member of the G protein-coupled receptors and its function is largely unknown. To investigate the physiological function of GPR116 in vivo, we generated adipose tissue specific conditional Gpr116 knockout mice (CKO) and fed them on standard chow or high fat diets. Selective deletion of Gpr116 in adipose tissue caused a pronounced glucose intolerance and insulin resistance in mice, especially when challenged with a high fat diet. Biochemical analysis revealed a more severe hepatosteatosis in CKO mice. Additionally, we found that CKO mice showed a lowered concentration of circulating adiponectin and an increased level of serum resistin. Our study suggests that GPR116 may play a critical role in controlling adipocyte biology and systemic energy homeostasis.

Mutations in the parkin gene are currently thought to be the most common cause of recessive familial Parkinsonism. Parkin functions as an E3 ligase to regulate protein turnover, and its function in mitochondrial quality control has been reported recently. Overexpression of parkin has been found to prevent neuronal degeneration under various conditions both in vivo and in vitro. Here, we generated a transgenic mouse model in which expression of wild type parkin was driven by neuron-specific enolase (NSE) promoter. We reported that both young and old parkin transgenic mice exhibited less reduction of striatal TH protein and number of TH positive neurons in the substantia nigra induced by 1-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine (MPTP), compared to wild type littermates. MPTP-induced mitochondrial impairment in the substantia nigra was improved in young parkin transgenic mice. Decreased striatal α-synuclein was demonstrated in old parkin transgenic mice. These results provide reliable evidence from the transgenic mouse model for parkin that overexpression of parkin may attenuate dopaminergic neurodegeneration induced by MPTP through protection of mitochondria and reduction of α-synuclein in the nigrostriatal pathway.

The inflammatory response in acute pancreatitis (AP) is associated with acinar-to-dendritic cell transition. The CD4+ T-cell-mediated adaptive immune response is necessary for pancreatic inflammatory damage. However, the effect of acinar-to-dendritic cell transition on the CD4+ T-cell response and the regulatory mechanism remain undefined. A mouse animal model of AP was established by repeated intraperitoneal injection of CAE. The mTOR inhibitor rapamycin was administered before AP induction. Primary acinar cells were isolated and co-incubated with subsets of differentiated CD4+ T cells. The expression of DC-SIGN was also assessed in pancreatic tissues from human AP patients. We found acinar cells expressed DC-SIGN and displayed the phenotype of dendritic cells (DCs), which promoted the differentiation of naive CD4+ T cells into CD4+/IFN-γ+ Th1 and CD4+/IL-17A+ Th17 cells in pancreatic tissues during AP. DC-SIGN was the target gene of Myc. The mTOR inhibitor rapamycin inhibited AP-induced DC-SIGN expression, CD4+ Th1/Th17 cell differentiation and the pro-inflammatory response via Myc. Acinar cells expressed DC-SIGN in pancreatic tissues of human patients with AP. In conclusion, acinar-to-dendritic cell transition is implicated in the CD4+ T-cell immune response via mTOR-Myc-DC-SIGN axis, which might be an effective target for the prevention of local pancreatic inflammation in AP.

Prime editing was used to insert and correct various pathogenic mutations except for beta-thalassemia variants, which disrupt functional beta-globin and prevent hemoglobin assembly in erythrocytes. This study investigated the effect of gene correction using prime editor version 3 (PE3) in a mouse model with the human beta-thalassemia IVS-II-654 mutation (C > T). The T conversion generates a 5′ donor site at intron 2 of the beta-globin gene resulting in aberrant splicing of pre-mRNA, which affects beta-globin expression. We microinjected PE3 components (pegRNA, nick sgRNA, and PE2 mRNA) into the zygotes from IVS-II-654 mice to generate mutation-edited mice. Genome sequencing of the IVS-II-654 site showed that PE3 installed the correction (T > C), with an editing efficiency of 14.29%. Reverse transcription-PCR analysis showed that the PE3-induced conversion restored normal splicing of beta-globin mRNA. Subsequent comprehensive phenotypic analysis of thalassemia symptoms, including anemic hematological parameters, anisocytosis, splenomegaly, cardiac hypertrophy, extramedullary hematopoiesis, and iron overload, showed that the corrected IVS-II-654 mice had a normal phenotype identical to the wild type mice. Off-target analysis of pegRNA and nick sgRNA additionally showed the genomic safety of PE3. These results suggest that correction of beta-thalassemia mutation by PE3 may be a straightforward therapeutic strategy for this disease.

Retinol dehydrogenase 13 (RDH13) is a mitochondrion-localized member of the short-chain dehydrogenases/reductases (SDRs) superfamily that participates in metabolism of some compounds. Rdh13 mRNA is most highly expressed in mouse liver. Rdh13 deficiency reduces the extent of liver injury and fibrosis, reduces hepatic stellate cell (HSC) activation, attenuates collagen I (II), tissue inhibitor of metalloproteinase 1 (TIMP-1) and transforming growth factor beta 1 (Tgf-β1) expression. The results indicate an important role of Rdh13 and suggest RDH13 as a possible new therapeutic target for CCl4-induced fibrosis.

Neuroinflammation, as an important pathological characteristic of Parkinson's disease (PD), is primarily mediated by activated astrocytes and microglia. Neuron-restrictive silencer factor/repressor element 1 (RE1)-silencing transcription factor (NRSF/REST) regulates many genes and signal pathways involved in the inflammatory process in astrocytes. In the present study, we established the GFAP-Cre:NRSFflox/flox conditional knockout (cKO) mice. The expression of inflammation-associated molecules were measured in primary astrocytes from wild type (WT) and cKO mice after stimulation by 1-Methyl-4-phenylpyridine (MPP+), LPS, and conditioned medium (CM) of LPS-treated BV-2 microglial cells. The inflammatory molecule expression in BV-2 microglial cells exposed to conditioned medium of MPP+-treated primary astrocytes were also analyzed. Moreover, a subacute regimen of 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine hydrochloride (MPTP) was used to establish mouse PD model and the damages to the nigrostriatal pathway were comprehensively evaluated in WT and cKO mice. We found that MPP+ induced a remarkable increase of NRSF expression in cultured astrocytes. Compared to WT astrocytes, the expression of inflammatory molecules IL-1β, IL-6, COX-2, and iNOS increased dramatically in NRSF deficient astrocytes challenged with CM of LPS-treated BV-2 cells. COX-2 and IL-1β transcripts were significantly elevated in BV-2 microglial cells exposed to CM of MPP+-treated NRSF deficient astrocytes compared to WT astrocytes. In cKO mice, the activation of astrocytes and microglial cells was more obvious, and the nigrostriatal dopaminergic system was more heavily injured compared to their WT counterparts after MPTP administration. Our results suggest that reactive NRSF deficient astrocytes orchestrated with microglial cells aggravate the pathophysiological progress in PD.

CD8+ T cells play a critical role during adaptive immune response, which often change locations and expand or contract in numbers under different states. In the past, many attempts to develop CD8+T cells that express luciferase in vivo have involved the use of viral transduction, which has drawbacks of hardly tracked via detection of luciferase signal in untouched natural states. Here, we generate a transgenic mouse model via CRISPR-mediated genome editing, C57BL/6-CD8aem(IRES-AkaLuci-2A-EGFP) knock-in mice(CD8a-Aka mice), as a novel tool for non-invasive imaging of CD8+ T cells, which expressed a highly sensitive luciferase-Akaluciferase. Our study offers a convenient and robust tool for understanding fundamental CD8+ T cell biology in experimental applications and preclinical translational studies.