KLHL21 suppresses gastric tumourigenesis via maintaining STAT3 signalling equilibrium in stomach homoeostasis.

OBJECTIVE: Precancerous metaplasia transition to dysplasia poses a risk for subsequent intestinal-type gastric adenocarcinoma. However, the molecular basis underlying the transformation from metaplastic to cancerous cells remains poorly understood. DESIGN: An integrated analysis of genes associated with metaplasia, dysplasia was conducted, verified and characterised in the gastric tissues of patients by single-cell RNA sequencing and immunostaining. Multiple mouse models, including homozygous conditional knockout Klhl21-floxed mice, were generated to investigate the role of Klhl21 deletion in stemness, DNA damage and tumour formation. Mass-spectrometry-based proteomics and ribosome sequencing were used to elucidate the underlying molecular mechanisms. RESULTS: Kelch-like protein 21 (KLHL21) expression progressively decreased in metaplasia, dysplasia and cancer. Genetic deletion of Klhl21 enhances the rapid proliferation of Mist1+ cells and their descendant cells. Klhl21 loss during metaplasia facilitates the recruitment of damaged cells into the cell cycle via STAT3 signalling. Increased STAT3 activity was confirmed in cancer cells lacking KLHL21, boosting self-renewal and tumourigenicity. Mechanistically, the loss of KLHL21 promotes PIK3CB mRNA translation by stabilising the PABPC1-eIF4G complex, subsequently causing STAT3 activation. Pharmacological STAT3 inhibition by TTI-101 elicited anticancer effects, effectively impeding the transition from metaplasia to dysplasia. In patients with gastric cancer, low levels of KLHL21 had a shorter survival rate and a worse response to adjuvant chemotherapy. CONCLUSIONS: Our findings highlighted that KLHL21 loss triggers STAT3 reactivation through PABPC1-mediated PIK3CB translational activation, and targeting STAT3 can reverse preneoplastic metaplasia in KLHL21-deficient stomachs.

CircUBA2 promotes the cancer stem cell-like properties of gastric cancer through upregulating STC1 via sponging miR-144-5p.

BACKGROUND: Cancer stem cells (CSCs) are critical factors that limit the effectiveness of gastric cancer (GC) therapy. Circular RNAs (circRNAs) are confirmed as important regulators of many cancers. However, their role in regulating CSC-like properties of GC remains largely unknown. Our study aimed to investigate the role of circUBA2 in CSC maintenance and the underlying mechanisms. METHODS: We identified circUBA2 as an upregulated gene using circRNA microarray analysis. qRT-PCR was used to examine the circUBA2 levels in normal and GC tissues. In vitro and in vivo functional assays were performed to validate the role of circUBA2 in proliferation, migration, metastasis and CSC-like properties of GC cell. The relationship between circUBA2, miR-144-5p and STC1 was characterised using bioinformatics analysis, a dual fluorescence reporter system, FISH, and RIP assays. RESULTS: CircUBA2 expression was significantly increased in GC tissues, and patients with GC with high circUBA2 expression had a poor prognosis. CircUBA2 enhances CSC-like properties of GC, thereby promoting cell proliferation, migration, and metastasis. Mechanistically, circUBA2 promoted GC malignancy and CSC-like properties by acting as a sponge for miR-144-5p to upregulate STC1 expression and further activate the IL-6/JAK2/STAT3 signaling pathway. More importantly, the ability of circUBA2 to enhance CSC-like properties was inhibited by tocilizumab, a humanised Interleukin-6 receptor (IL-6R) antibody. Thus, circUBA2 knockdown and tocilizumab synergistically inhibited CSC-like properties. CONCLUSIONS: Our study demonstrated the critical role of circUBA2 in regulating CSC-like properties in GC. CircUBA2 may be a promising prognostic biomarker for GC.

Production of Four-Gene (GTKO/hCD55/hTBM/hCD39)-Edited Donor Pigs and Kidney Xenotransplantation.

BACKGROUND: The number of multigene-modified donor pigs for xenotransplantation is increasing with the advent of gene-editing technologies. However, it remains unclear which gene combination is suitable for specific organ transplantation. METHODS: In this study, we utilized CRISPR/Cas9 gene editing technology, piggyBac transposon system, and somatic cell cloning to construct GTKO/hCD55/hTBM/hCD39 four-gene-edited cloned (GEC) pigs and performed kidney transplantation from pig to rhesus monkey to evaluate the effectiveness of these GEC pigs. RESULTS: First, 107 cell colonies were obtained through drug selection, of which seven were 4-GE colonies. Two colonies were selected for somatic cell nuclear transfer (SCNT), resulting in seven fetuses, of which four were GGTA1 biallelic knockout. Out of these four, two fetuses had higher expression of hCD55, hTBM, and hCD39. Therefore, these two fetuses were selected for two consecutive rounds of cloning, resulting in 97 live piglets. After phenotype identification, the GGTA1 gene of these pigs was inactivated, and hCD55, hTBM, and hCD39 were expressed in cells and multiple tissues. Furthermore, the numbers of monkey IgM and IgG binding to the peripheral blood mononuclear cells (PBMCs) of the 4-GEC pigs were markedly reduced. Moreover, 4-GEC porcine PBMCs had greater survival rates than those from wild-type pigs through complement-mediated cytolysis assays. In pig-to-monkey kidney xenotransplantation, the kidney xenograft successfully survived for 11 days. All physiological and biochemical indicators were normal, and no hyperacute rejection or coagulation abnormalities were found after transplantation. CONCLUSION: These results indicate that the GTKO/hCD55/hTBM/hCD39 four-gene modification effectively alleviates immune rejection, and the pig kidney can functionally support the recipient monkey's life.

Unraveling the impact of hyperleptinemia on female reproduction: insights from transgenic pig model.

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.

In vitro and in vivo development of interspecies Asian elephant embryos reconstructed with pig enucleated oocytes.

Interspecies somatic cell nuclear transfer (iSCNT) has an immense potential to rescue endangered animals and extinct species like mammoths. In this study, we successfully established an Asian elephant's fibroblast cell lines from ear tissues, performed iSCNT with porcine oocytes and evaluated the in vitro and in vivo development of reconstructed embryos. A total of 7780 elephant-pig iSCNT embryos were successfully reconstructed and showed in vitro development with cleavage rate, 4-cell, 8-cell and blastocyst rate of 73.01, 30.48, 5.64, and 4.73%, respectively. The total number of elephant-pig blastocyte cells and diameter of hatched blastocyte was 38.67 and 252.75 µm, respectively. Next, we designed species-specific markers targeting EDNRB, AGRP and TYR genes to verify the genome of reconstructed embryos with donor nucleus/species. The results indicated that 53.2, 60.8, and 60.8% of reconstructed embryos (n = 235) contained elephant genome at 1-cell, 2-cell and 4-cell stages, respectively. However, the percentages decreased to 32.3 and 32.7% at 8-cell and blastocyst stages, respectively. Furthermore, we also evaluated the in vivo development of elephant-pig iSCNT cloned embryos and transferred 2260 reconstructed embryos into two surrogate gilts that successfully became pregnant and a total of 11 (1 and 10) fetuses were surgically recovered after 17 and 19 days of gestation, respectively. The crown-rump length and width of elephant-pig cloned fetuses were smaller than the control group. Unfortunately, none of these fetuses contained elephant genomes, which suggested that elephant embryos failed to develop in vivo. In conclusion, we successfully obtained elephant-pig reconstructed embryos for the first time and these embryos are able to develop to blastocyst, but the in vivo developmental failure needs further investigated.

Divergent Total Syntheses of (+)-Vulgarisins†A-E.

The asymmetric total syntheses of (+)-vulgarisins A-E, which share a rare and highly oxygenated [5-6-4-5] tetracyclic core structure that were isolated from P. vulgaris Linn., have been described for the first time in a divergent manner. Key transformations include: 1) a catalytic asymmetric intramolecular cyclopropanation to forge the A ring bearing desired stereochemistry at C14; 2) a one-pot borylation/conjugate addition process for creation of the C1-C11 bond; 3) a Wolff ring contraction to assemble the bicyclo[3.2.0]heptane subunit (CD rings); and 4) a stereocontrolled pinacol cyclization for construction of the central B ring of the natural products.

Delayed body development with reduced triglycerides levels in leptin transgenic pigs.

Leptin is a well-known adipokine that plays critical role in adiposity. To further investigate the role of leptin in adiposity, we utilized leptin overexpressing transgenic pigs and evaluated the effect of leptin on growth and development, fat deposition, and lipid metabolism at tissue and cell level. Leptin transgenic pigs were produced and divided into two groups: elevated leptin expression (leptin ( +)) and normal leptin expression group (control). Results indicated that leptin ( +) pigs had elevated leptin protein and mRNA expression levels and exhibited sluggish growth and development followed by decreased subcutaneous fat thickness, low serum triglycerides, saturated, unsaturated fatty acids and high cholesterol esters (p < 0.05). There were differences in the lipid metabolism related genes at different fat depots, including upregulation of PPARγ, AGPAT6, PLIN2, HSL and ATGL in subcutaneous, PPARγ in perirenal, and FAT/CD36 and PLIN2 in mesenteric adipose tissues and downregulation of AGPAT6 and ATGL in perirenal and AGPAT6 in mesenteric adipose tissues (p < 0.05). Additionally, in-vitro cultured leptin ( +) preadipocytes exhibited upregulation of PPARγ, FAT/CD36, ACACA, AGPAT, PLIN2, ATGL and HSL as compared to control (p < 0.05). These findings suggested that homeostasis imbalance in lipolysis and lipogenesis at adipose tissue and adipocytes levels led to low subcutaneous fat depots in leptin overexpression pigs. These pigs can act as model for obesity and related metabolic disorder.

Total Synthesis of (-)-Rhodomollanol A.

An asymmetric approach for the first total synthesis of (-)-rhodomollanol A, a highly oxidized diterpenoid, is described. The efficient synthetic strategy features three key transformations: (1) an oxidative dearomatization-induced (5 + 2) cycloaddition/pinacol-type 1,2-acyl migration cascade to build up the bicyclo[3.2.1]octane skeleton; (2) a retro-Dieckmann fragmentation/vinylogous Dieckmann cyclization cascade to assemble the bicyclo[3.3.0]octane subunit; and (3) a photo-Nazarov cyclization/intramolecular cycloetherification cascade to forge the 7-oxabicyclo[4.2.1]nonane core structure of the natural product.

Improved production of GTKO/hCD55/hCD59 triple-gene-modified Diannan miniature pigs for xenotransplantation by recloning.

Multiple genetic modification is necessary for successful xenotransplantation from pigs. However, multiple-genetically modified cells usually suffer from various drug selections and long-term in vitro culture, which have a poor performance for somatic cell nuclear transfer (SCNT) to produce genetically modified pigs. We used to generate GTKO/hCD55/hCD59 triple-gene modified pigs by using drug-selective cell lines for SCNT, but the majority of cloned pigs were transgenic-negative individuals. In this study, to improve the production efficiency of multiple genetically modified pigs, we performed the recloning process by using transgenic porcine fetal fibroblast cells. As a result, two fetuses expressing hCD55 and hCD59 were obtained from 12 live-cloned fetuses, and one carrying high transgene expression was selected as a source of donor cells for recloning. Then we obtained 12 cloned piglets, all GTKO and carrying hCD55 and hCD59. Both hCD55 and hCD59 were expressed in fibroblast cells, but the expression levels of hCD55 and hCD59 were different among these piglets. Furthermore, piglet P5# had the highest expression of hCD55 and hCD59 in fibroblast cells than other piglets. Correspondingly, fibroblast cells of piglet P5# had significantly higher resistance against human serum-mediated cytolysis than those of piglet P11#. In conclusion, our results firstly provide support for improving efficiency of generating multiple genetically modified pig by recloning.

Generation of GHR-modified pigs as Laron syndrome models via a dual-sgRNAs/Cas9 system and somatic cell nuclear transfer.

BACKGROUND: Laron syndrome is an autosomal disease resulting from mutations in the growth hormone receptor (GHR) gene. The only therapeutic treatment for Laron syndrome is recombinant insulin-like growth factor I (IGF-I), which has been shown to have various side effects. The improved Laron syndrome models are important for better understanding the pathogenesis of the disease and developing corresponding therapeutics. Pigs have become attractive biomedical models for human condition due to similarities in anatomy, physiology, and metabolism relative to humans, which could serve as an appropriate model for Laron syndrome. METHODS: To further improve the GHR knockout (GHRKO) efficiency and explore the feasibility of precise DNA deletion at targeted sites, the dual-sgRNAs/Cas9 system was designed to target GHR exon 3 in pig fetal fibroblasts (PFFs). The vectors encoding sgRNAs and Cas9 were co-transfected into PFFs by electroporation and GHRKO cell lines were established by single cell cloning culture. Two biallelic knockout cell lines were selected as the donor cell line for somatic cell nuclear transfer for the generation of GHRKO pigs. The genotype of colonies, cloned fetuses and piglets were identified by T7 endonuclease I (T7ENI) assay and sequencing. The GHR expression in the fibroblasts and piglets was analyzed by confocal microscopy, quantitative polymerase chain reaction (q-PCR), western blotting (WB) and immunohistochemical (IHC) staining. The phenotype of GHRKO pigs was recapitulated through level detection of IGF-I and glucose, and measurement of body weight and body size. GHRKO F1 generation were generated by crossing with wild-type pigs, and their genotype was detected by T7ENI assay and sequencing. GHRKO F2 generation was obtained via self-cross of GHRKO F1 pigs. Their genotypes of GHRKO F2 generation was also detected by Sanger sequencing. RESULTS: In total, 19 of 20 single-cell colonies exhibited biallelic modified GHR (95%), and the efficiency of DNA deletion mediated by dual-sgRNAs/Cas9 was as high as 90% in 40 GHR alleles of 20 single-cell colonies. Two types of GHR allelic single-cell colonies (GHR-47/-1, GHR-47/-46) were selected as donor cells for the generation of GHRKO pigs. The reconstructed embryos were transferred into 15 recipient gilts, resulting in 15 GHRKO newborn piglets and 2 fetuses. The GHRKO pigs exhibited slow growth rates and small body sizes. From birth to 13 months old, the average body weight of wild-type pigs varied from 0.6 to 89.5 kg, but that of GHRKO pigs varied from only 0.9 to 37.0 kg. Biochemically, the knockout pigs exhibited decreased serum levels of IGF-I and glucose. Furthermore, the GHRKO pigs had normal reproduction ability, as eighteen GHRKO F1 piglets were obtained via mating a GHRKO pig with wild-type pigs and five GHRKO F2 piglets were obtained by self-cross of F1 generation, indicating that modified GHR alleles can pass to the next generation via germline transmission. CONCLUSION: The dual-sgRNAs/Cas9 is a reliable system for DNA deletion and that GHRKO pigs conform to typical phenotypes of those observed in Laron patients, suggesting that these pigs could serve as an appropriate model for Laron syndrome.

Efficient generation of P53 biallelic knockout Diannan miniature pigs via TALENs and somatic cell nuclear transfer.

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.

Enlightenment of robotic gastrectomy from 527 patients with gastric cancer in the minimally invasive era: 5 years of optimizing surgical performance in a high-volume center - a retrospective cohort study.

BACKGROUND: Learning curves have been used in the field of robotic gastrectomy (RG). However, it should be noted that the previous study did not comprehensively investigate all changes related to the learning curve. This study aims to establish a learning curve for radical RG and evaluate its effect on the short-term outcomes of patients with gastric cancer. METHODS: The clinicopathological data of 527 patients who underwent RG between August 2016 and June 2021 were retrospectively analyzed. Learning curves related to the operation time and postoperative hospital stay were determined separately using cumulative sum (CUSUM) analysis. Then, the impact of the learning curve on surgical efficacy was analyzed. RESULTS: Combining the CUSUM curve break points and technical optimization time points, the entire cohort was divided into three phases (patients 1-100, 101-250, and 251-527). The postoperative complication rate and postoperative recovery time tended to decrease significantly with phase advancement ( P <0.05). More extraperigastric examined lymph nodes (LN) were retrieved in phase III than in phase I (I vs. III, 15.12±6.90 vs. 17.40±7.05, P =0.005). The rate of LN noncompliance decreased with phase advancement. Textbook outcome (TO) analysis showed that the learning phase was an independent factor in TO attainment ( P <0.05). CONCLUSION: With learning phase advancement, the short-term outcomes were significantly improved. It is possible that our optimization of surgical procedures could have contributed to this improvement. The findings of this study facilitate the safe dissemination of RG in the minimally invasive era.

Dysfunction of Complementarity Determining Region 1 Encoded by T Cell Receptor Beta Variable Gene Is Potentially Associated with African Swine Fever Virus Infection in Pigs.

The beta T-cell receptor (TRB) expressed by beta T cells is essential for foreign antigen recognition. The TRB locus contains a TRBV family that encodes three complementarity determining regions (CDRs). CDR1 is associated with antigen recognition and interactions with MHC molecules. In contrast to domestic pigs, African suids lack a 284-bp segment spanning exons 1 and 2 of the TRBV27 gene that contains a sequence encoding CDR1. In this study, we used the African swine fever virus (ASFV) as an example to investigate the effect of deleting the TRBV27-encoded CDR1 on the resistance of domestic pigs to exotic pathogens. We first successfully generated TRBV27-edited fibroblasts with disruption of the CDR1 sequence using CRISPR/Cas9 technology and used them as donor cells to generate gene-edited pigs via somatic cell nuclear transfer. The TRBV-edited and wild-type pigs were selected for synchronous ASFV infection. White blood cells were significantly reduced in the genetically modified pigs before ASFV infection. The genetically modified and wild-type pigs were susceptible to ASFV and exhibited typical fevers (>40 °C). However, the TRBV27-edited pigs had a higher viral load than the wild-type pigs. Consistent with this, the gene-edited pigs showed more clinical signs than the wild-type pigs. In addition, both groups of pigs died within 10 days and showed similar severe lesions in organs and tissues. Future studies using lower virulence ASFV isolates are needed to determine the relationship between the TRBV27 gene and ASFV infection in pigs over a relatively long period.

PGC-based cryobanking, regeneration through germline chimera mating, and CRISPR/Cas9-mediated TYRP1 modification in indigenous Chinese chickens.

Primordial germ cells (PGCs) are vital for producing sperm and eggs and are crucial for conserving chicken germplasm and creating genetically modified chickens. However, efforts to use PGCs for preserving native chicken germplasm and genetic modification via CRISPR/Cas9 are limited. Here we show that we established 289 PGC lines from eight Chinese chicken populations with an 81.6% success rate. We regenerated Piao chickens by repropagating cryopreserved PGCs and transplanting them into recipient chickens, achieving a 12.7% efficiency rate. These regenerated chickens carried mitochondrial DNA from female donor PGC and the rumplessness mutation from both male and female donors. Additionally, we created the TYRP1 (tyrosinase-related protein 1) knockout (KO) PGC lines via CRISPR/Cas9. Transplanting KO cells into male recipients and mating them with wild-type hens produced four TYRP1 KO chickens with brown plumage due to reduced eumelanin production. Our work demonstrates efficient PGC culture, cryopreservation, regeneration, and gene editing in chickens.

Circ-0075305 hinders gastric cancer stem cells by indirectly disrupting TCF4-ß-catenin complex and downregulation of SOX9.

CircRNAs are covalently closed, single-stranded RNA that form continuous loops and play a crucial role in the initiation and progression of tumors. Cancer stem cells (CSCs) are indispensable for cancer development; however, the regulation of cancer stem cell-like properties in gastric cancer (GC) and its specific mechanism remain poorly understood. We elucidate the specific role of Circ-0075305 in GC stem cell properties. Circ-0075305 associated with chemotherapy resistance was identified by sequencing GC cells. Subsequent confirmation in both GC tissues and cell lines revealed that patients with high expression of Circ-0075305 had significantly better overall survival (OS) rates than those with low expression, particularly when treated with postoperative adjuvant chemotherapy for GC. In vitro and in vivo experiments confirmed that overexpression of Circ-0075305 can effectively reduce stem cell-like properties and enhance the sensitivity of GC cells to Oxaliplatin compared with the control group. Circ-0075305 promotes RPRD1A expression by acting as a sponge for corresponding miRNAs. The addition of LF3 (a ß-catenin/TCF4 interaction antagonist) confirmed that RPRD1A inhibited the formation of the TCF4-ß-catenin transcription complex through competitive to ß-catenin and suppressed the transcriptional activity of stem cell markers such as SOX9 via the Wnt/ß-catenin signaling pathway. This leads to the downregulation of stem cell-like property-related markers in GC. This study revealed the underlying mechanisms that regulate Circ-0075305 in GCSCs and suggests that its role in reducing ß-catenin signaling may serve as a potential therapeutic candidate.

AND-Gated Nanosensor for Imaging of Glutathione and Apyrimidinic Endonuclease 1 in Cells, Animals, and Organoids.

The development of a strategy for imaging of glutathione (GSH) and apurinic/apyrimidinic endonuclease 1 (APE1) in an organism remains challenging despite their significance in elaborating the correlated pathophysiological processes. Therefore, in this study, we propose a DNA-based AND-gated nanosensor for fluorescence imaging of the GSH as well as APE1 in living cells, animals, and organoids. The DNA probe is composed of a G-strand and A-strand. The disulfide bond in the G-strand is cleaved through a GSH redox reaction, and the hybridization stability between the G-strand and A-strand is decreased, leading to a conformational change of the A-strand. In the presence of APE1, the apurinic/apyrimidinic (AP) site in the A-strand is digested, producing a fluorescence signal for the correlated imaging of GSH and APE1. This nanosensor enables monitoring of the expression level change of GSH and APE1 in cells. Additionally, we illustrate the capability of this "dual-keys-and-locked" conceptual methodology in achieving specific tumor imaging when GSH and APE1 are present simultaneously (overexpressed GSH and APE1 in tumor cells) with improving tumor-to-normal tissue ratio in vivo. Furthermore, using this nanosensor, the GSH and APE1 also are visualized in organoids that recapitulate the phenotypic and functional traits of the original biological specimens. Overall, this study demonstrates the potential of our proposed biosensing technology in investigating the roles of various biological molecules involved in specific diseases.

Weighted Gene Co-Expression Network Analysis Reveals Hub Genes for Fuzz Development in Gossypium hirsutum.

Fuzzless Gossypium hirsutum mutants are ideal materials for investigating cotton fiber initiation and development. In this study, we used the fuzzless G. hirsutum mutant Xinluzao 50 FLM as the research material and combined it with other fuzzless materials for verification by RNA sequencing to explore the gene expression patterns and differences between genes in upland cotton during the fuzz period. A gene ontology (GO) enrichment analysis showed that differentially expressed genes (DEGs) were mainly enriched in the metabolic process, microtubule binding, and other pathways. A weighted gene co-expression network analysis (WGCNA) showed that two modules of Xinluzao 50 and Xinluzao 50 FLM and four modules of CSS386 and Sicala V-2 were highly correlated with fuzz. We selected the hub gene with the highest KME value among the six modules and constructed an interaction network. In addition, we selected some genes with high KME values from the six modules that were highly associated with fuzz in the four materials and found 19 common differential genes produced by the four materials. These 19 genes are likely involved in the formation of fuzz in upland cotton. Several hub genes belong to the arabinogalactan protein and GDSL lipase, which play important roles in fiber development. According to the differences in expression level, 4 genes were selected from the 19 genes and tested for their expression level in some fuzzless materials. The modules, hub genes, and common genes identified in this study can provide new insights into the formation of fiber and fuzz, and provide a reference for molecular design breeding for the genetic improvement of cotton fiber.

Generation of stable integration-free pig induced pluripotent stem cells under chemically defined culture condition.

Genome integration-free pig induced pluripotent stem cells (iPSCs) bring tremendous value in pre-clinical testing of regenerative medicine, as well as conservation and exploitation of endangered or rare local pig idioplasmatic resources. However, due to a lack of appropriate culture medium, efficient induction and stable maintenance of pig iPSCs with practical value remains challenging. Here, we established an efficient induction system for exogenous gene-independent iPSCs under chemically defined culture condition previously used for generation of stable pig pre-gastrulation epiblast stem cells (pgEpiSCs). WNT suppression was found to play an essential role in establishment of exogenous gene-independent iPSCs. Strikingly, stable integration-free pig iPSCs could be established from pig somatic cells using episomal vectors in this culture condition. The iPSCs had pluripotency features and transcriptome characteristics approximating pgEpiSCs. More importantly, this induction system may be used to generate integration-free iPSCs from elderly disabled rare local pig somatic cells and the iPSCs could be gene-edited and used as donor cells for nuclear transfer. Our results provide novel insights into potential applications for genetic breeding of livestock species and pre-clinical evaluation of regenerative medicine.

Indocyanine green fluorescence imaging-guided versus conventional laparoscopic lymphadenectomy for gastric cancer: long-term outcomes of a phase 3 randomised clinical trial.

Indocyanine green (ICG) fluorescence imaging-guided lymphadenectomy has been demonstrated to be effective in increasing the number of lymph nodes (LNs) retrieved in laparoscopic gastrectomy for gastric cancer (GC). Previously, we reported the primary outcomes and short-term secondary outcomes of a phase 3, open-label, randomized clinical trial (NCT03050879) investigating the use of ICG for image-guided lymphadenectomy in patients with potentially resectable GC. Patients were randomly (1:1 ratio) assigned to either the ICG or non-ICG group. The primary outcome was the number of LNs retrieved and has been reported. Here, we report the primary outcome and long-term secondary outcomes including three-year overall survival (OS), three-year disease-free survival (DFS), and recurrence patterns. The per-protocol analysis set population is used for all analyses (258 patients, ICG [n = 129] vs. non-ICG group [n = 129]). The mean total LNs retrieved in the ICG group significantly exceeds that in the non-ICG group (50.5 ± 15.9 vs 42.0 ± 10.3, P < 0.001). Both OS and DFS in the ICG group are significantly better than that in the non-ICG group (log-rank P = 0.015; log-rank P = 0.012, respectively). There is a difference in the overall recurrence rates between the ICG and non-ICG groups (17.8% vs 31.0%). Compared with conventional lymphadenectomy, ICG guided laparoscopic lymphadenectomy is safe and effective in prolonging survival among patients with resectable GC.

Loss of ATOH1 in Pit Cell Drives Stemness and Progression of Gastric Adenocarcinoma by Activating AKT/mTOR Signaling through GAS1.

Gastric cancer stem cells (GCSCs) are self-renewing tumor cells that govern chemoresistance in gastric adenocarcinoma (GAC), whereas their regulatory mechanisms remain elusive. Here, the study aims to elucidate the role of ATOH1 in the maintenance of GCSCs. The preclinical model and GAC sample analysis indicate that ATOH1 deficiency is correlated with poor GAC prognosis and chemoresistance. ScRNA-seq reveals that ATOH1 is downregulated in the pit cells of GAC compared with those in paracarcinoma samples. Lineage tracing reveals that Atoh1 deletion strongly confers pit cell stemness. ATOH1 depletion significantly accelerates cancer stemness and chemoresistance in Tff1-CreERT2; Rosa26Tdtomato and Tff1-CreERT2; Apcfl/fl ; p53fl/fl (TcPP) mouse models and organoids. ATOH1 deficiency downregulates growth arrest-specific protein 1 (GAS1) by suppressing GAS1 promoter transcription. GAS1 forms a complex with RET, which inhibits Tyr1062 phosphorylation, and consequently activates the RET/AKT/mTOR signaling pathway by ATOH1 deficiency. Combining chemotherapy with drugs targeting AKT/mTOR signaling can overcome ATOH1 deficiency-induced chemoresistance. Moreover, it is confirmed that abnormal DNA hypermethylation induces ATOH1 deficiency. Taken together, the results demonstrate that ATOH1 loss promotes cancer stemness through the ATOH1/GAS1/RET/AKT/mTOR signaling pathway in GAC, thus providing a potential therapeutic strategy for AKT/mTOR inhibitors in GAC patients with ATOH1 deficiency.