Seed and Soil: Consensus Molecular Subgroups (CMS) and Tumor Microenvironment Features Between Primary Lesions and Metastases of Different Organ Sites in Colorectal Cancer.

Purpose: Consensus molecular subtypes (CMS) are mainly used for biological interpretability and clinical stratification of colorectal cancer (CRC) in primary tumors (PT) but few in metastases. The heterogeneity of CMS distribution in metastases and the concordance of CMS between PT and metastases still lack sufficient study. We used CMS to classify CRC metastases and combine it with histopathological analysis to explore differences between PT and distant metastases. Patients and Methods: We obtained gene expression profiles for 942 PT samples from TCGA database (n=376) and GEO database (n=566), as well as 442 metastasis samples from GEO database. Among these, 765 PT samples and 442 metastasis samples were confidently identified with CMS using the "CMS classifier" and enrolled for analysis. Clinicopathological manifestation and CMS classification of CRC metastases were assessed with data from GEO, TCGA, and cBioPortal. Overall, 105 PT-metastasis pairs were extracted from 10 GEO datasets to assess CMS concordance. Tumor microenvironment (TME) features between PT and metastases were analyzed by immune-stromal infiltration with ESTIMATE and xCell algorithms. Finally, TME features were validated with multiplex immunohistochemistry in 27 PT-metastasis pairs we retrospectively collected. Results: Up to 64% of CRC metastases exhibited concordant CMS groups with matched PT, and the TME of metastases was similar to that of PT. For most common distant metastases, liver metastases were predominantly CMS2 and lung and peritoneal metastases were mainly CMS4, highlighting "seed" of tumor cells of different CMS groups had a preference for metastasis to "soil" of specific organs. Compared with PT, cancer-associated fibroblasts (CAF) reduced in liver metastases, CD4+T cells and M2-like macrophages increased in lung metastases, and M2-like macrophages and CAF increased in peritoneal metastases. Conclusion: Our findings underscore the importance of CMS-guided specific organ monitoring and treatment post-primary tumor surgery for patients. Differences in immune-stromal infiltration among different metastases provide targeted therapeutic opportunities for metastatic CRC.

Development and Validation of a Deep Learning and Radiomics Combined Model for Differentiating Complicated From Uncomplicated Acute Appendicitis.

RATIONALE AND OBJECTIVES: This study aimed to develop and validate a deep learning and radiomics combined model for differentiating complicated from uncomplicated acute appendicitis (AA). MATERIALS AND METHODS: This retrospective multicenter study included 1165 adult AA patients (training cohort, 700 patients; validation cohort, 465 patients) with available abdominal pelvic computed tomography (CT) images. The reference standard for complicated/uncomplicated AA was the surgery and pathology records. We developed our combined model with CatBoost based on the selected clinical characteristics, CT visual features, deep learning features, and radiomics features. We externally validated our combined model and compared its performance with that of the conventional combined model, the deep learning radiomics (DLR) model, and the radiologist's visual diagnosis using receiver operating characteristic (ROC) curve analysis. RESULTS: In the training cohort, the area under the ROC curve (AUC) of our combined model in distinguishing complicated from uncomplicated AA was 0.816 (95% confidence interval [CI]: 0.785-0.844). In the validation cohort, our combined model showed robust performance across the data from three centers, with AUCs of 0.836 (95% CI: 0.785-0.879), 0.793 (95% CI: 0.695-0.872), and 0.723 (95% CI: 0.632-0.802). In the total validation cohort, our combined model (AUC = 0.799) performed better than the conventional combined model, DLR model, and radiologist's visual diagnosis (AUC = 0.723, 0.755, and 0.679, respectively; all P < 0.05). Decision curve analysis showed that our combined model provided greater net benefit in predicting complicated AA than the other three models. CONCLUSION: Our combined model allows the accurate differentiation of complicated and uncomplicated AA.

Quantitative composite testing model based on measurement uncertainty and its application for the detection of phthalate esters.

To improve the quantitative detection efficiency of chemical analysis and reduce the detection cost, the sample pass rate was estimated and mathematical statistics were used to calculate the optimal group size (K opt) of the composite testing to save on the maximum workload. A quantitative composite testing model was developed based on chemical analysis measurement uncertainty. Using this model, the maximum allowable number of composited samples (K max) is first calculated using parameters of regulated limits (L), limit of quantification (LOQ), and method measured uncertainty (U rel) to ensure that the sensitivity of the composite testing can meet the limit requirements. Finally, the appropriate composite group size (K a) can be obtained by creating a balance between K opt, K max, and the practical information used for that particular test. Furthermore, based on a constructed model, a practical quantitative composite testing method of 3-10 samples was established for the routine detection of toy phthalates (PAEs). The experimental results showed that the quantitative limits of 7 PAEs were 9.1-41.8 mg/kg, the relative expansion uncertainties were 16.6%-23.2%, and the recovery rates were 91.0%-112.3%, with a relative deviation of less than 10%. All these meet international PAEs standards. Compared with the traditional individual and qualitative composite testing, this model will not decrease the detection sensitivity, but can save up to 17.9%-80.4% of the workload when it is employed in toy PAEs testing with the pass rate of 80%-99%. This quantitative composite testing method will be implemented in the coming revision of ISO 8124-6 toy PAEs standards.

Analysis of abdominal adhesion using the ileostomy model.

Abdominal adhesion occurs commonly in clinical practice, causing unfavorable symptoms and readmission. The ileostomy operation is a common surgical procedure and we utilized this model to evaluate abdominal adhesion. Adhesion grade score was calculated in 35 patients (Cohort 1) and subjected to correlation and receiver operating characteristic analysis. Then 98 consecutive patients (Cohort 2) who underwent ileostomy and ileostomy closure were included into a retrospective study. Logistic regression analysis was performed, and the risk of small bowel obstruction was also assessed. The time of ileostomy closure correlated with adhesion grade score in Cohort 1, justifying its use as an indicator of abdominal adhesion. All patients in Cohort 2 were then divided into the high- and low-adhesion group. A multi-variable logistic regression analysis indicated that type of surgery and peritoneum suture during ileostomy were significant factors affecting the risk of abdominal adhesion. Abdominal adhesion had the trend to prolong the length of stay postoperatively without increasing the risk of bowel obstruction. Nine patients suffered bowel obstruction, and age older than 65 significantly increased the risk. We proposed the ileostomy procedure to be a model of abdominal adhesion, and the operative time of ileostomy closure could be used as an alternative of adhesion score. Type of surgery and peritoneum suture may be risk factors of abdominal adhesion. Older age increased the risk of small bowel obstruction after ileostomy surgery.

Simple transanal total mesorectal resection versus laparoscopic transabdominal total mesorectal resection for the treatment of low rectal cancer: a single-center retrospective case-control study.

Aim: To evaluate the efficacy and safety of simple TaTNE in the treatment of low rectal cancer compared with laparoscopic transabdominal TME. Methods: We collected patients with low rectal cancer admitted to our hospital between January 2019 and November 2021 who received simple TaTME or laparoscopic transabdominal TME. The main outcome was the integrity of the TME specimen. Secondary outcomes were the number of lymph nodes dissected, intraoperative blood loss, operative time, surgical conversion rate, Specimen resection length, circumferential margin (CRM), and distal resection margin (DRM), complication rate. In addition, the Wexner score and LARS score of fecal incontinence were performed in postoperative follow-up. Results: Pathological tissues were successfully resected in all patients. all circumferential margins of the specimen were negative. Specimen resection length was not statistically significant (9.94 ± 2.85 vs. 8.90 ± 2.49, P > 0.05). The incidence of postoperative complications in group A (n = 0) was significantly lower than that in group B (n = 3) (P > 0.05). There was no significant difference in operation time between group A and group B (296 ± 60.36 vs. 305 ± 58.28, P > 0.05). Among the patients with follow-up time less than 1 year, there was no significant difference in Wexner score and LARS score between group A and group B (P > 0.05). However, in patients who were followed up for more than 1 year, the Wexner score in group A (9.25 ± 2.73) was significantly lower than that in group B (17.36 ± 10.95) and was statistically significant (P < 0.05). Conclusion: For radical resection of low rectal cancer, Simple TaTME resection may be as safe and effective as laparoscopic transabdominal TME, and the long-term prognosis may be better.

Copy number variation of gasdermin D gene is associated with atrial fibrillation-related thromboembolic stroke.

AIMS: Atrial fibrillation (AF) is one of the major causes of ischaemic stroke. In addition to clinical risk evaluated by the CHA2DS2-VASC score, the impact of genetic factors on the risk of AF-related thromboembolic stroke has been largely unknown. We found several copy number variations (CNVs) in novel genes that were associated with thromboembolic stroke risk in our AF patients by genome-wide approach. Among them, the gasdermin D (GSDMD) gene was related to inflammation. We aimed to test whether GSDMD deletion was associated with AF-related stroke. METHODS AND RESULTS: A total of 400 patients with documented non-familial AF were selected, of which 100 patients were diagnosed with ischaemic stroke. The baseline characteristics of age, sex, valvular heart disease, coronary artery disease, heart failure, and CHA2DS2-VASc scores were not statistically different between cases and controls. We found that individuals who carried GSDMD homozygous deletion genotype had a higher risk for ischaemic stroke (odds ratio 2.195; 95% confidence interval, 1.24-3.90; P = 0.007), even adjusted by CHA2DS2-VASc scores. We also validated the association of GSDMD with AF stroke in a large Caucasian population (UK Biobank). CONCLUSION: We found a link between the homozygous deletion of the GSDMD gene and an increased risk of stroke in patients with AF. This may implicate the use of therapy targeting GSDMD in the prevention of ischaemic stroke for AF patients.

Clinical Efficacy of Intersphincteric Resection for Low Rectal Cancer Compared With Abdominoperineal Resection: A Single-Center Retrospective Study.

BACKGROUND: In recent years, intersphincteric resection (ISR) has been increasingly used to replace abdominoperineal resection (APR) in the surgical treatment of ultra-low rectal cancer. AIM: This study was to compare the clinical efficacy of ISR and APR. METHODS: Between 2012 and 2018, 74 consecutive patients with ultra-low rectal cancer underwent ISR or APR in our medical center. A retrospective comparison of these 2 procedures was performed. RESULTS: A total of 43 patients underwent ISR and 31 underwent APR were included in the study. No significant differences were found between 2 groups in gender, age, BMI, and ASA score. Intersphincteric resection group showed shorter operative time (P = .02) and less blood loss (P = .001). Hospital stays, time to soft diet, and postoperative 30-day complications were not significantly different between the 2 groups. R0 resection achieved 100% in both the groups. As for the long-term outcomes, the survival and recurrence rate were similar between 2 groups. Moreover, the LARS and Wexner score showed that the postoperative anal function after ISR were satisfactory. CONCLUSION: This study suggested that ISR was feasible and safe for selected patients with ultra-low rectal cancer, with clinically superior outcomes in select patients (small tumors/further from the anal verge) and similar oncological outcomes to APR, and the anal functional outcomes after ISR were acceptable.

The Effect of Mesenchymal Stem Cells, Adipose Tissue Derived Stem Cells, and Cellular Stromal Vascular Fraction on the Repair of Acute Anal Sphincter Injury in Rats.

BACKGROUND: Anal sphincter incontinence (ASI) can cause a serious decline in the quality of life and can cause a socioeconomic burden. Studies have shown that bone marrow mesenchymal stem cells (MSC) have significant therapeutic effects on ASI, but the cost and risk of MSC harvest limit their further application. In contrast, adipose tissue derived stem cells (ADSC) and cellular stromal vascular fraction (CSVF) as stem cell sources have multipotency and the advantage of easy harvest. OBJECTIVE: Here we aim to investigate the effects of ADSC and CSVF on treating ASI and compare them to that of bone marrow MSC. METHODS: Bone marrow MSC, ADSC, and CSVF were obtained and labeled with green fluorescent protein (GFP), and CSVF was labeled with DIL. Sprague Dawley (SD) rats were divided into 5 groups. Four groups were injected with 0.2 mL phosphate buffer saline (PBS), 1 × 107/0.2 mL of MSC, ADSC, or CSVF, respectively, after model establishment. The control group received no treatment. The repair was assessed by anal functional tests and immunostaining on day 5 and day 10 after injection. RESULTS: MSC, ADSC, and CSVF significantly promoted tissue repair and the recovery of muscle contraction and electromyographic activity in ASI. The generation of myosatellite cells by injected MSC, ADSC, and CSVF was found in the wounded area. On day 5, CSVF showed highest therapeutic effect, while on day 10, MSC and ADSC showed higher therapeutic effects than CSVF. When comparing the effects of MSC and ADSC, ADSC was slightly better than MSC in the indexes of anal pressure, etc. Conclusion: ADSC and CVSF are alternative stem cell sources for ASI repair.

The Effect of Tissue Stromal Vascular Fraction as Compared to Cellular Stromal Vascular Fraction to Treat Anal Sphincter Incontinence.

BACKGROUND: The long-term prognosis of current treatments for anal sphincter incontinence (ASI) is poor. Here, we explored the efficacy of tissue adipose stromal vascular fraction SVF (tSVF) on ASI and compared it to that of cellular SVF (cSVF). We then investigated possible mechanisms. METHODS: Rat cSVF and tSVF were isolated and labeled with DIL. One day after modeling, three groups received phosphate-buffered saline (PBS), cSVF, tSVF, respectively. The control group received nil modeling nor any treatments. The effect was assessed by function test for anal pressure and electromyography, and staining for fiber content, proliferation and differentiation at day 5 and day 10. RESULTS: cSVF injection resulted in faster healing than tSVF. The cSVF group showed significant improvement on anal pressure on day 10. For the electromyography test, cSVF showed significant improvement for the frequencies on day 10, and for the peak values on both time points, while tSVF showed significant improvement for the peak values on day 10. The two SVF both alleviated fibrosis. Immunofluorescence tracing identified differentiation of some injected cells towards myosatellite cells and smooth muscle cells in both SVF groups. For all the tests, the tSVF group tends to have similar or lower effects than the cSVF group with no significant difference. CONCLUSION: cSVF and tSVF are both safe and effective in treating ASI, while the effect of cSVF is slighter higher than tSVF.

Mitoquinone treatment for the prevention of surgical adhesions via regulation of the NRF2/HO-1 signaling pathway in mice.

BACKGROUND: Postoperative adhesion is a common cause of long-term morbidity after abdominal or pelvic surgery. The development of postoperative adhesion involves oxidative stress, inflammatory response, and collagen deposition mechanisms. Here, we demonstrate that mitoquinone could be useful for the treatment of postoperative adhesion. METHODS: A murine adhesion model was established by induction of peritoneal ischemic buttons. Mice received different doses of mitoquinone via the tail vein. All the ischemic buttons were dissected at 1 day and 7 days after surgery to investigate the effect of mitoquinone in the early and late stage of the adhesion process, respectively. Human peritoneal mesothelial cells were treated with H2O2 to examine the potential mechanisms of mitoquinone in oxidative insult. RESULTS: Postoperative adhesion scores were markedly decreased in mitoquinone-treated mice compared with the control mice. The degree of oxidative stress, inflammatory injury, and collagen deposition were also significantly reduced in the mitoquinone-treated mice. The expression of plasminogen-activating inhibitor, interleukin-1, interleukin-6, tumor necrosis factor-α, vascular endothelial growth factor, malondialdehyde, and nitric oxide was decreased, while the expression of tissue-type plasminogen activator, glutathione, superoxide dismutase, and Nrf2 was increased in the peritoneal ischemic buttons after mitoquinone treatment. Cellular reactive oxygen species and the canonical inflammatory pathway were inhibited in mitoquinone-treated human peritoneal mesothelial cells after H2O2 challenge. Mechanistically, mitoquinone was found to enhance the activity of Nrf2 and heme oxygenase-1 and to induce nuclear translocation of Nrf2 in human peritoneal mesothelial cells. CONCLUSION: The mitochondria-targeting antioxidant molecule mitoquinone attenuates postoperative adhesion formation by inhibiting oxidative stress, inflammation, and collagen accumulation, and therefore provides a therapeutic agent for the management of surgical adhesion.

Highly efficient generation of sheep with a defined FecBB mutation via adenine base editing.

Base editing has the potential to improve important economic traits in agriculture and can precisely convert single nucleotides in DNA or RNA sequences into minimal double-strand DNA breaks (DSB). Adenine base editors (ABE) have recently emerged as a base editing tool for the conversion of targeted A:T to G:C, but have not yet been used in sheep. ABEmax is one of the latest versions of ABE, which consists of a catalytically-impaired nuclease and a laboratory-evolved DNA-adenosine deaminase. The Booroola fecundity (FecBB) mutation (g.A746G, p.Q249R) in the bone morphogenetic protein receptor 1B (BMPR1B) gene influences fecundity in many sheep breeds. In this study, by using ABEmax we successfully obtained lambs with defined point mutations that result in an amino acid substitution (p.Gln249Arg). The efficiency of the defined point mutations was 75% in newborn lambs, since six lambs were heterozygous at the FecBB mutation site (g.A746G, p.Q249R), and two lambs were wild-type. We did not detect off-target mutations in the eight edited lambs. Here, we report the validation of the first gene-edited sheep generated by ABE and highlight its potential to improve economically important traits in livestock.

Intersphincteric Resection Versus Abdominoperineal Resection for Low Rectal Cancer: A Meta-Analysis.

Background. Abdominoperineal resection (APR) has been the standard surgery for ultra-low rectal cancer for a century. In recent years, intersphincteric resection (ISR) has been increasingly used to avoid the permanent colostomy. Up to now, there is no relevant meta-analysis comparing the clinical efficacy of ISR and APR. This meta-analysis aimed to compare the outcomes of these 2 procedures. Methods. A comprehensive search of online databases was performed on PubMed, EMBASE, and the Cochrane Library to obtain comparative studies of ISR and APR. Then the data from studies that met the inclusion criteria were extracted and analyzed. Results. A total of 12 studies covering 2438 patients were included. No significant differences were found between ISR and APR in gender, body mass index, distance from tumor to anal edge, operative time, and blood loss. In addition, hospital stay (weighted mean differences = -2.98 days; 95% confidence interval [CI] = -3.54 to -2.43; P < .00001) and postoperative morbidity (odds ratio [OR] = 0.76; 95% CI = 0.59 to 0.99; P = .04) were significantly lower in ISR group compared with APR group. However, patients who underwent ISR showed lower pathological T-stage (T3T4%, OR = 0.49; 95% CI = 0.28 to 0.86; P = .01) and lymph node metastasis rate (OR = 0.77; 95% CI = 0.59 to 1.01; P = .06) compared with those who underwent APR. Moreover, oncological outcomes were similar between the 2 groups. Conclusion. ISR may provide a safe alternative to APR, with shorter hospital stays, lower postoperative morbidity, and similar oncological outcomes. Well-designed randomized controlled trials are needed to confirm and update the findings of this analysis.

Follistatin like protein-1 modulates macrophage polarization and aggravates dextran sodium sulfate-induced colitis.

Follistatin-like protein 1 (FSTL1) is a pleiotropic cytokine involved in multiple processes including organ development, carcinogenesis, metastasis and so on. Some recent studies have suggested a possible role of FSTL1 in the inflammatory diseases. We for the first time tried to unravel its effect on the colitis, and explore the possible mechanisms. Here we found that FSTL1 was upregulated in active human and murine colitis. It facilitated proinflammatory M1 polarization of macrophages and inhibited the M2 anti-inflammatory phenotype, leading to excessive production of multiple inflammatory cytokines in vitro and in vivo. Haplodeletion of FSTL1 in mice significantly reduced the clinical and histological activity of colitis. Most importantly, macrophage depletion diminished the difference between DSS-treated WT and FSTL1+/- mice. Altogether, our results suggested that FSTL1 may also serve as an important contributor in the colonic inflammation. The possible mechanism may be related to its modulation on macrophage polarization.

Glove single-port laparoscopy-assisted transanal total mesorectal excision for low rectal cancer: a preliminary report.

PURPOSE: Glove single-port laparoscopy-assisted transanal total mesorectal excision (TaTME) has been successfully carried out in our medical center. The purpose of this study is to evaluate the feasibility of this emerging operation. METHODS: This technique was performed by self-made glove single-port laparoscopic platform to radically resect low rectal cancer. Short-term postoperative results, including complications, length of hospital stay, and follow-up results were collected and analyzed statistically. RESULTS: There are five consecutive patients (three males, two females) who underwent this surgery and included in this study. The mean distance from the tumor to the anal verge was 4.8 cm (range 4.0-6.0). The surgery was completed in all cases, and the rectal tumor was removed successfully without conversion; circumferential margins of all the excised specimens were negative. The mean time of operation was 338.00 min (range 280-400). The average number of lymph node dissection was 12.20. The average postoperative hospital stay was 8.60 days. During the follow-up (14.80 ± 1.92 months), all preventive ileostomies were successfully closed in about 3 months after the surgery, all patients had satisfactory anal function, and no tumor recurrence was found. CONCLUSION: Glove single-port laparoscopy-assisted TaTME has a significant effect in specific patients with low rectal cancer, with rapid recovery and high safety. Prospective randomized studies involving more case counts and long-term follow-up results, especially oncologic outcomes, are needed to validate this technique.

Base pair editing in goat: nonsense codon introgression into FGF5 results in longer hair.

The ability to alter single bases without homology directed repair (HDR) of double-strand breaks provides a potential solution for editing livestock genomes for economic traits, which are often multigenic. Progress toward multiplex editing in large animals has been hampered by the costly inefficiencies of HDR via microinjection of in vitro manipulated embryos. Here, we designed sgRNAs to induce nonsense codons (C-to-T transitions) at four target sites in caprine FGF5, which is a crucial regulator of hair length in mammals. Initial transfections of the third generation Base Editor (BE3) plasmid and four different sgRNAs into caprine fibroblasts were ineffective in altering FGF5. In contrast, all five progenies produced from microinjected single-cell embryos had alleles with a targeted nonsense mutation. The effectiveness of BE3 to make single base changes varied considerably based on sgRNA design. In addition, the rate of mosaicism differed between animals, target sites, and tissue type. The phenotypic effects on hair fiber were characterized by hematoxylin and eosin, immunofluorescence staining, and western blotting. Differences in morphology were detectable, even though mosaicism was probably affecting the levels of FGF5 expression. PCR amplicon and whole-genome resequencing analyses for off-target changes caused by BE3 were low at a genome-wide scale. This study provided the first evidence of base editing in large mammals produced from microinjected single-cell embryos. Our results support further optimization of BEs for introgressing complex human disease alleles into large animal models, to evaluate potential genetic improvement of complex health and production traits in a single generation.

Programmable Base Editing of the Sheep Genome Revealed No Genome-Wide Off-Target Mutations.

Since its emergence, CRISPR/Cas9-mediated base editors (BEs) with cytosine deaminase activity have been used to precisely and efficiently introduce single-base mutations in genomes, including those of human cells, mice, and crop species. Most production traits in livestock are induced by point mutations, and genome editing using BEs without homology-directed repair of double-strand breaks can directly alter single nucleotides. The p.96R > C variant of Suppressor cytokine signaling 2 (SOCS2) has profound effects on body weight, body size, and milk production in sheep. In the present study, we successfully obtained lambs with defined point mutations resulting in a p.96R > C substitution in SOCS2 by the co-injection of BE3 mRNA and a single guide RNA (sgRNA) into sheep zygotes. The observed efficiency of the single nucleotide exchange in newborn animals was as high as 25%. Observations of body size and body weight in the edited group showed that gene modification contributes to enhanced growth traits in sheep. Moreover, targeted deep sequencing and unbiased family trio-based whole genome sequencing revealed undetectable off-target mutations in the edited animals. This study demonstrates the potential for the application of BE-mediated point mutations in large animals for the improvement of production traits in livestock species.

STING-mediated intestinal barrier dysfunction contributes to lethal sepsis.

BACKGROUND: Gut integrity is compromised in abdominal sepsis with increased cellular apoptosis and altered barrier permeability. Intestinal epithelial cells (IEC) form a physiochemical barrier that separates the intestinal lumen from the host's internal milieu and is strongly involved in the mucosal inflammatory response and immune response. Recent research indicates the involvement of the stimulator of interferons genes (STING) pathway in uncontrolled inflammation and gut mucosal immune response. METHODS: We investigated the role of STING signaling in sepsis and intestinal barrier function using intestinal biopsies from human patients with abdominal sepsis and with an established model of abdominal sepsis in mice. FINDINGS: In human abdominal sepsis, STING expression was elevated in peripheral blood mononuclear cells and intestinal biopsies compared with healthy controls, and the degree of STING expression in the human intestinal lamina propria correlated with the intestinal inflammation in septic patients. Moreover, elevated STING expression was associated with high levels of serum intestinal fatty acid binding protein that served as a marker of enterocyte damage. In mice, the intestinal STING signaling pathway was markedly activated following the induction of sepsis induced by cecal ligation perforation (CLP). STING knockout mice showed an alleviated inflammatory response, attenuated gut permeability, and decreased bacterial translocation. Whereas mice treated with a STING agonist (DMXAA) following CLP developed greater intestinal apoptosis and a more severe systemic inflammatory response. We demonstrated that mitochondrial DNA (mtDNA) was released during sepsis, inducing the intestinal inflammatory response through activating the STING pathway. We finally investigated DNase I administration at 5 hours post CLP surgery, showing that it reduced systemic mtDNA and inflammatory cytokines levels, organ damage, and bacterial translocation, suggesting that inhibition of mtDNA-STING signaling pathway protects against CLP-induced intestinal barrier dysfunction. INTERPRETATION: Our results indicate that the STING signaling pathway can contribute to lethal sepsis by promoting IEC apoptosis and through disrupting the intestinal barrier. Our findings suggest that regulation of the mtDNA-STING pathway may be a promising therapeutic strategy to promote mucosal healing and protect the intestinal barrier in septic patients. FUND: National Natural Science Foundation of China.

Trio-Based Deep Sequencing Reveals a Low Incidence of Off-Target Mutations in the Offspring of Genetically Edited Goats.

Unintended off-target mutations induced by CRISPR/Cas9 nucleases may result in unwanted consequences, which will impede the efficient applicability of this technology for genetic improvement. We have recently edited the goat genome through CRISPR/Cas9 by targeting MSTN and FGF5, which increased muscle fiber diameter and hair fiber length, respectively. Using family trio-based sequencing that allow better discrimination of variant origins, we herein generated offspring from edited goats, and sequenced the members of four family trios (gene-edited goats and their offspring) to an average of ∼36.8× coverage. This data was to systematically examined for mutation profiles using a stringent pipeline that comprehensively analyzed the sequence data for de novo single nucleotide variants, indels, and structural variants from the genome. Our results revealed that the incidence of de novo mutations in the offspring was equivalent to normal populations. We further conducted RNA sequencing using muscle and skin tissues from the offspring and control animals, the differentially expressed genes (DEGs) were related to muscle fiber development in muscles, skin development, and immune responses in skin tissues. Furthermore, in contrast to recently reports of Cas9 triggered p53 expression alterations in cultured cells, we provide primary evidence to show that Cas9-mediated genetic modification does not induce apparent p53 expression changes in animal tissues. This work provides adequate molecular evidence to support the reliability of conducting Cas9-mediated genome editing in large animal models for biomedicine and agriculture.