LINC-PINT plays an anti-tumor role in nasopharyngeal carcinoma by binding to XRCC6 and affecting its function.

BACKGROUND: LINC-PINT was downregulated in nasopharyngeal carcinoma (NPC) and correlated with treatment efficiency of NPC. However, the underlying mechanism of LINC-PINT in NPC has not yet been fully explored. METHOD: We used CellTiter luminescent assay, clone formation assay, Hoechst staining, and SYTO-9/PI staining to examine cell viability and cell apoptosis regulated by LINC-PINT in NPC cells. Xenograft tumor model, HE staining, Ki67 staining, and TUNEL assay were conducted to assess the role of LINC-PINT in vivo. Bioinformatics and RNA immunoprecipitation assay was performed to identify the binding protein of LINC-PINT. Fluorescence in situ hybridization and immunofluorescence were utilized to measure the colocalization of XRCC6 with LINC-PINT and DNA-PKcs. Mito-Tracker red CMXRos staining was used to label mitochondria in cells specifically. RESULT: We found LINC-PINT was downregulated in many tumors (including NPC) and associated with poor prognosis. The cell viability was significantly inhibited and cell apoptosis was remarkably promoted in LINC-PINT overexpressed cells in contrast to control cells. The growth of tumor xenografts was significantly suppressed and the tumor weight was significantly decreased in LINC-PINT overexpression group compared to the control group. Correspondingly, the positive Ki67 foci was decreased while TUNEL foci was increased in LINC-PINT overexpression group. Mechanically, we verified XRCC6 as a new binding protein of LINC-PINT through RNA binding domains prediction, RIP and colocalization of LINC-PINT and XRCC6. By binding to XRCC6, LINC-PINT interfered the formation of DNA-PK complex, regulated mitochondria accumulation status and affected the modification of apoptosis proteins, leading to more cell apoptosis. CONCLUSION: Our study provided the first evidence that LINC-PINT promotes cell apoptosis in NPC by binding to XRCC6 and affecting its function.

Statistical Insight into China's Indigenous Diagnosis-Related-Group System Evolution.

The use of Diagnosis-Related Groups (DRG) is a prevalent payment system employed to control hospitalization costs and improve medical efficiency. China has developed an indigenized DRG payment system including Single Disease Payment (SDP), DRGs, and Big Data Diagnosis-Intervention Packet (DIP). In this study, we took cholecystitis as an example, drawing on both primary and secondary data to verify the effectiveness of China's indigenized DRG system and to introduce the evolution of DRGs in China. Primary data were gathered from Qilu Hospital in 2019-2021. Secondary data were collected from published literature from 2004-2016. Only studies with both pre-SDP/DRG and post-SDP/DRG groups were included. Among the studies included, 92.9% (13/14) reported a significant reduction in hospitalization costs after the implementation of SDP while other studies identified length of stay (LOS) and age as the most significant influential factors in SDP. Furthermore, we elaborated the efficiency of DRGs using data from 2738 inpatients in Qilu hospital. Moreover, 60% (6/10) of the studies from the databases also showed the efficiency of DRGs in different regions. SDP is efficient in saving hospitalization costs, but its implementation is limited. DRGs have a broader scope of application, but their effectiveness remains to be validated. DIP is a brand new concept in China, and more data are needed to assess its efficiency.

Endoscopic construction of an antireflux mucosal barrier for the treatment of GERD: a pilot study (with video).

BACKGROUND AND AIMS: Based on the porcine natural antireflux mechanism, we developed a novel endoscopic procedure to build an antireflux mucosal flap to block acid reflux and treat GERD. METHODS: The antireflux mucosal valvuloplasty (ARMV) procedure is performed by releasing and reconstructing three-fourths of the circumference of cardiac mucosa at the lesser curvature side into a double-layer mucosal flap. The mucosal flap works together with cardiac scarring to block reflux. We retrospectively reviewed 30 patients who underwent ARMV from 2019 to 2021. Subjective and objective data evaluating GERD were collected before and after ARMV. RESULTS: All 30 ARMV procedures were performed successfully, with a mean operation time of 72.6 ± 20.3 minutes. One patient had postoperative bleeding that required endoscopic hemostasis. The mean follow-up time was 28.9 ± 13.9 months. Twenty-five of 30 patients (83.3%) and 23 of 26 patients (88.5%) reported discontinuation or reduction in proton pump inhibitor therapy 3 months and 1 year after ARMV, respectively. GERD questionnaire and GERD Health-Related Quality of Life questionnaire scores improved significantly from 14.0 ± 2.6 and 48.7 ± 15.0, respectively, before ARMV to 7.7 ± 2.5 and 10.2 ± 5.9, respectively, 12 months after ARMV (P < .0001 in both comparisons). Eleven patients received 24-hour esophageal pH monitoring before and after ARMV. The mean acid exposure time and DeMeester score dropped from 56.9% ± 23.7% and 167.1 ± 80.1, respectively, before ARMV to 5.5% ± 3.0% and 18.6 ± 11.9, respectively, after ARMV (P < .0001 in both comparisons). CONCLUSIONS: This pilot study showed that ARMV is a safe, feasible, and effective procedure for GERD patients. Further prospective and comparative trials are needed to confirm its role among endoscopic antireflux therapies.

CRISPR/Cas9-Mediated Gene Knockout in Cells and Tissues Using Lentivirus.

CRISPR-Cas9 has become a powerful and popular gene editing tool. However, successful application of this tool in the lab can still be quite daunting to many newcomers to molecular biology, mostly because it is a relatively lengthy process involving multiple steps with variations of each step. Here, we provide a reliable, stepwise, and newcomer-friendly protocol to knock out a target gene in wild-type human fibroblasts. This protocol involves sgRNA design using CRISPOR, construction of an "all-in-one" vector expressing both sgRNA and Cas9 using Golden Gate cloning, streamlined production of high-titer lentiviruses in 1 week after molecular cloning, and transduction of cells to generate a knockout cell pool. We further introduce a protocol for lentiviral transduction of ex vivo mouse embryonic salivary epithelial explants. In summary, our protocol is useful for new researchers to apply CRISPR-Cas9 to generate stable gene knockout cells and tissue explants using lentivirus. Published 2023. This article is a U.S. Government work and is in the public domain in the USA. Basic Protocol 1: sgRNA design Basic Protocol 2: Cloning sgRNA in plasmid vector containing Cas9 encoding sequence using golden gate cloning Basic Protocol 3: Lentivirus packaging Basic Protocol 4: Lentivirus transduction of cells Basic Protocol 5: Lentivirus transduction of salivary gland epithelial buds.

Benefits of precise endoscopic incision on post-dilation mucosal scars to treat refractory esophageal stricture after endoscopic submucosal dissection.

Endoscopic dilation (ED) is the mainstream treatment for esophageal stricture after endoscopic submucosal dissection (ESD). However, some complex esophageal strictures do not respond well to dilation. Endoscopic radial incision (ERI) has proved to be effective in treating anastomotic strictures, but it is rarely used to treat post-ESD esophageal strictures due to technical difficulties and risks, not to mention the optimal method and timing to perform ERI. Here, we developed an integrated procedure in which ED was performed first, followed by ERI on the stiff scars that remained intact after dilation. The ED + ERI procedure resulted in complete, uniform expansion of the esophageal lumen. Between 2019 and 2022, 5 post-ESD patients who received a median number of 11 sessions of ED (range, 4-28) of ED over a period of 322 days (range, 246-584) but still had moderate to severe dysphagia were admitted. 2 or 3 sessions of ED + ERI were performed for each patient interspersed with ED. After a median number of 4 treatments (range, 2-9), all patients were symptom-free or had minimal symptoms. No serious complications occurred in any patients who underwent ED + ERI. Therefore, ED + ERI is safe, feasible, and may serve as a useful therapeutic method for refractory esophageal stricture after ESD.

A Neural Network Based Approach to Inverse Kinematics Problem for General Six-Axis Robots.

Inverse kinematics problems (IKP) are ubiquitous in robotics for improved robot control in widespread applications. However, the high non-linearity, complexity, and equation coupling of a general six-axis robotic manipulator pose substantial challenges in solving the IKP precisely and efficiently. To address this issue, we propose a novel approach based on neural network (NN) with numerical error minimization in this paper. Within our framework, the complexity of IKP is first simplified by a strategy called joint space segmentation, with respective training data generated by forward kinematics. Afterwards, a set of multilayer perception networks (MLP) are established to learn from the foregoing data in order to fit the goal function piecewise. To reduce the computational cost of the inference process, a set of classification models is trained to determine the appropriate forgoing MLPs for predictions given a specific input. After the initial solution is sought, being improved with a prediction error minimized, the refined solution is finally achieved. The proposed methodology is validated via simulations on Xarm6-a general 6 degrees of freedom manipulator. Results further verify the feasibility of NN for IKP in general cases, even with a high-precision requirement. The proposed algorithm has showcased enhanced efficiency and accuracy compared to NN-based approaches reported in the literature.

HOXA11-AS facilitates the proliferation, cell cycle process and migration of keloid fibroblasts through sponging miR-188-5p to regulate VEGFA.

BACKGROUND: Abnormal expression of long non-coding RNA (lncRNA) has been proved to be related to the formation of keloid. Homeobox A11 antisense (HOXA11-AS) has been found to be a significantly upregulated lncRNA in keloid tissues. OBJECTIVE: To explore the mechanism of HOXA11-AS regulates keloid formation. METHODS: Primary fibroblasts were isolated from keloid tissues and normal skin tissues. The expression of HOXA11-AS, microRNA (miR)-188-5p and vascular endothelial growth factor A (VEGFA) was determined using quantitative real-time PCR (qRT-PCR). Cell counting kit 8 (CCK8) assay, EdU staining, flow cytometry and wound healing assay were performed to assess the proliferation, cell cycle process, apoptosis and migration of keloid fibroblasts. Importantly, some marker protein levels and VEGFA protein level were examined by western blot (WB) analysis. The interaction between miR-188-5p and HOXA11-AS or VEGFA was confirmed using dual-luciferase reporter assay, RNA pull-down assay and RIP assay. Animal experiments were performed to further confirm the role of HOXA11-AS in keloid growth. RESULTS: HOXA11-AS was markedly upregulated in keloid tissues and fibroblasts. Knockdown of HOXA11-AS repressed the proliferation, cell cycle process, migration and promoted the apoptosis of keloid fibroblasts. Further analysis suggested that HOXA11-AS could sponge miR-188-5p to positively regulate VEGFA. The inhibition of HOXA11-AS silencing on the biological functions of keloid fibroblasts could be reversed by miR-188-5p inhibitor. In addition, VEGFA overexpression also abolished the suppressive effect of miR-188-5p on the biological functions of keloid fibroblasts. Interferences of HOXA11-AS suppressed keloid growth in vivo. CONCLUSION: HOXA11-AS might regulate the miR-188-5p/VEGFA axis to promote keloid formation.

Cell-3D matrix interactions: recent advances and opportunities.

Tissues consist of cells and their surrounding extracellular matrix (ECM). Cell-ECM interactions play crucial roles in embryonic development, differentiation, tissue remodeling, and diseases including fibrosis and cancer. Recent research advances in characterizing cell-matrix interactions include detailed descriptions of hundreds of ECM and associated molecules, their complex intermolecular interactions in development and disease, identification of distinctive modes of cell migration in different 3D ECMs, and new insights into mechanisms of organ formation. Exploring the roles of the physical features of different ECM microenvironments and the bidirectional regulation of cell signaling and matrix organization emphasize the dynamic nature of these interactions, which can include feedback loops that exacerbate disease. Understanding mechanisms of cell-matrix interactions can potentially lead to targeted therapeutic interventions.

Genetic Polymorphisms of Long Non-coding RNA Linc00312 Are Associated With Susceptibility and Predict Poor Survival of Nasopharyngeal Carcinoma.

BACKGROUND: Linc00312 is dysregulated in nasopharyngeal carcinoma (NPC) and participates in the initiation and progression of NPC. Our previous studies suggested that linc00312 was able to enhance the sensitivity of NPC cells to irradiation and NPC patients with higher expression of linc00312 was associated with better short-term curative effect and overall survival. The single nucleotide polymorphisms (SNPs) of lncRNAs may influence the disease course and outcome by affecting the expression, secondary structure or function of lncRNAs. However, the role of SNPs in linc00312 on the occurrence and survival of NPC remains unknown. METHODS: We recruited 684 NPC patients and 823 healthy controls to evaluate the association between linc00312 SNPs and NPC susceptibility by using multivariate logistic regression analysis. Kaplan-Meier analysis and Cox proportional hazards regression were applied to assess the effect of linc00312 SNPs on the survival of NPC patients. The relative expression of linc00312 in NPC tissues was determined by real-time PCR. The interaction between linc00312 and mir-411-3p was explored by luciferase reporter assay. In silico prediction of the changes on linc00312 folding structure was conducted by RNAfold WebServer. RESULT: We demonstrated that rs12497104 (G > A) GA genotype carriers had a higher risk than others for suffering from NPC (GA vs GG, OR = 1.437, P = 0.003). Besides, patients with rs12497104 AA genotype showed a poorer overall survival in contrast to GG genotype (AA vs GG, HR = 2.117, P = 0.011). In addition, the heterozygous carriers of rs15734 (G > A) and rs164966 (A > G) were correlated with decreased risk of NPC (GA vs GG, OR = 0.778, P = 0.031; GA vs AA, OR = 0.781, P = 0.033, respectively). We found that the three SNPs might influence the expression of linc00312 in a genotype specific feature. The local centroid secondary structure as well as the minimum free energy of linc00312 were changed following the candidate SNPs alterations. Besides, we revealed that the G to A alteration at rs12497104 disrupted the binding between mir-411-3p and linc00312. CONCLUSION: Our results indicated genetic polymorphisms of linc00312 might serve as potential biomarkers for NPC carcinogenesis and prognosis.

The mutated Bacillus amyloliquefaciens strain shows high resistance to Aeromonas hydrophila and Aeromonas veronii in grass carp.

Bacillus amyloliquefaciens X030 (BaX030) has broad-spectrum antibacterial activity against the fish pathogens Aeromonas hydrophila and Aeromonas veronii. To improve its antibacterial effect, BaX030 was subjected to compound mutagenesis of atmospheric and room temperature plasma (ARTP) and nitrosoguanidine (NTG). The results showed that, compared with the original strain, the production of macrolactin A and oxydifficidin in mutated strain N-11 increased to 39 % and 268 %, respectively. The re-sequencing analysis suggested that there were SNPs and InDels in the gene clusters focused on the sucrose utilization pathway, glycolysis pathway and fatty acid synthesis pathway. Scanning electron microscopy revealed that strain N-11 became thin and long. The qRT-PCR results indicated that the expression of immune factors in the liver or kidney tissue of grass carp increased after feeding with N-11. H&E staining and protection experiments also showed that the mortality and surface symptoms of grass carp infected by the two pathogens were significantly reduced. The study identified a probiotic strain with potential application value in aquaculture production and provided a new strategy for the discovery of new strains with higher antibacterial biological activity.

Pelvic peritoneum closure reduces postoperative complications of laparoscopic abdominoperineal resection: 6-year experience in single center.

BACKGROUND: To investigate feasibility of laparoscopic abdominoperineal resection with pelvic peritoneum closure (LAPR-PPC) for lower rectal cancer. METHODS: LAPR-PPC has been used for lower rectal cancer in our institution since 2014. In this study, we retrospectively analyzed the data from 86 patients who underwent LAPR-PPC and compared with the data from 96 patients who underwent laparoscopic APR without PPC (LAPR) from January 2013 to December 2018. RESULTS: The rate of perineal surgical site infection (SSI) (18.75% (18/96) vs. 5.81% (5/86), p < 0.01), delayed (> 4 weeks) perineal healing (12.50% (12/96) vs. 3.49% (3/86), p = 0.027), ileus (7.29% (7/96) vs 1.16% (1/86), p = 0.044), and postoperative perineal hernia (PPH, 5.21% (5/96) vs. 0% (0/86), p = 0.032) were significantly lower in LAPR-PPC group than LAPR group. The patients in LAPR-PPC group had shorter hospitalization time (21.32 ± 11.95 days vs. 13.93 ± 11.51 days, p < 0.01). CONCLUSIONS: PPC procedure enabled the reduction in perineal wound complications, ileus, PPH, and consequently shortened hospitalization time. LAPR-PPC is beneficial for the patients with lower rectal cancer.

Effects of SpoIVA on the formation of spores and crystal protein in Bacillus thuringiensis.

In addition to forming spores, Bacillus thuringiensis (Bt) 4.0718 can produce toxins, insecticidal crystal protein (ICP) and vegetative insecticidal protein (Vip). The Bt spoIVA was successfully knocked out by gene recombination and was shown to inhibit sporulation. The mutant strain also exhibited significantly decreased growth and crystal formation, which inhibited spore formation and partially reduced the rate of crystal synthesis. The 50 % lethal concentrations (LC50) values of Bt 4.0718, replacement, complementation and multi-copy mutant strains against the fourth larval stage of H. armigera was determined as 5.422, 6.776, 6.223 and 5.018 µg/mL, respectively. A total of 1814 proteins were identified through isobaric tags for relative and absolute protein (iTRAQ), with 41 and 54 up and downregulated proteins observed. Gene ontology enrichment analysis showed that differentially expressed proteins were primarily involved in the biological process and molecular function. Quantitative real-time PCR analysis confirmed that 9 differential expressed genes exhibited a positive correlation between changes at transcriptional and translational levels. The results of this study provide a basis for further studies of the metabolic regulatory network of spores and crystal protein formation. Moreover, they can be used to ecologically safe insecticide of farmland production because the constructed Bt spoIVA mutants did not produce spores.Provides new ideas for the targeted improvement and application of environmentally friendly spore-free strains.

Alteration of the gut microbiome and immune factors of grass carp infected with Aeromonas veronii and screening of an antagonistic bacterial strain (Streptomyces flavotricini).

Aeromonas veronii is a widely distributed novel pathogen that can affect humans and animals, it can cause sepsis in fish with high mortality and serious economic losses to aquaculture. In the study, the gut microbiome of the infected and uninfected grass carp with Aeromonas veronii were analyzed probiotics and pathogenic bacteria by the Miseq high-throughput sequencing, the results showed that the infected fish were significantly higher in Proteobacteria, Firmicutes, Fusobacteria, and the immune factors in liver and kidney were up-regulated by qRT-PCR. In order to effectively inhibit the pathogen, we screened an actinomycete strain and had good antibacterial effect on Aeromonas veronii. The new antagonistic bacteria was named as Streptomyces flavotricini X101, the whole genome sequencing revealed that the metabolic process was most active. After grass carp was inoculated with the minimum inhibitory concentration of 900 µg/mL of the strain's fermentation supernatant, then Aeromonas veronii was injected, we found that the pathological symptoms such as body surface, anus and abdominal congestion were alleviated by H&E staining. Cellular experiments showed that it wasn't toxic to liver cells of grass carp. Overall, this is the first study of changes in intestinal flora, phenotype, and immune factors in grass crap infected with Aeromonas veronii, it had important theoretical significance and application value for immunization and prevention.

Basement Membrane Regulates Fibronectin Organization Using Sliding Focal Adhesions Driven by a Contractile Winch.

We have discovered that basement membrane and its major components can induce rapid, strikingly robust fibronectin organization. In this new matrix assembly mechanism, α5ß1 integrin-based focal adhesions slide actively on the underlying matrix toward the ventral cell center through the dynamic shortening of myosin IIA-associated actin stress fibers to drive rapid fibronectin fibrillogenesis distal to the adhesion. This mechanism contrasts with classical fibronectin assembly based on stable or fixed-position focal adhesions containing αVß3 integrins plus α5ß1 integrin translocation into proximal fibrillar adhesions. On basement membrane components, these sliding focal adhesions contain standard focal adhesion constituents but completely lack classical αVß3 integrins. Instead, peripheral α3ß1 or α2ß1 adhesions mediate initial cell attachment but over time are switched to α5ß1 integrin-based sliding focal adhesions to assemble fibronectin matrix. This basement-membrane-triggered mechanism produces rapid fibronectin fibrillogenesis, providing a mechanistic explanation for the well-known widespread accumulation of fibronectin at many organ basement membranes.

N-myc downstream-regulated gene 1 inhibits the proliferation of colorectal cancer through emulative antagonizing NEDD4-mediated ubiquitylation of p21.

BACKGROUND: N-myc downstream-regulated gene 1 (NDRG1) has been shown to play a key role in tumor metastasis. Recent studies demonstrate that NDRG1 can suppress tumor growth and is related to tumor proliferation; however, the mechanisms underlying these effects remain obscure. METHODS: Immunohistochemistry (IHC) was used to detect NDRG1 and p21 protein expression in colorectal cancer tissue, and clinical significance of NDRG1 was also analyzed. CCK-8 assay, colony formation assay, flow cytometry, and xenograft model were used to assess the effect of NDRG1 on tumor proliferation in vivo and in vitro. The mechanisms underlying the effect of NDRG1 were investigated using western blotting, immunofluorescence, immunoprecipitation, and ubiquitylation assay. RESULTS: NDRG1 was down-regulated in CRC tissues and correlated with tumor size and patient survival. NDRG1 inhibited tumor proliferation through increasing p21 expression via suppressing p21 ubiquitylation. NDRG1 and p21 had a positive correlation both in vivo and in vitro. Mechanistically, E3 ligase NEDD4 could directly interact with and target p21 for degradation. Moreover, NDRG1 could emulatively antagonize NEDD4-mediated ubiquitylation of p21, increasing p21 expression and inhibit tumor proliferation. CONCLUSION: Our study could fulfill potential mechanisms of the NDRG1 during tumorigenesis and metastasis, which may serve as a tumor suppressor and potential target for new therapies in human colorectal cancer.

Role of hsp20 in the Production of Spores and Insecticidal Crystal Proteins in Bacillus thuringiensis.

The small heat shock protein plays an important role in response to stresses. We wanted to investigate how Hsp20 affects sporulation and production of insecticidal crystal proteins (ICPs) in Bacillus thuringiensis (Bt) at the stationary growth phase when cells are starved. The hsp20 gene was knocked out in Bt4.0718 (wide type), which is a B. thuringiensis strain screened in our laboratory, using endonuclease I-SceI mediated unmarked gene replacement method. Deletion of Hsp20 resulted in a decrease in both sporulation and ICPs production. Bt4-Δhsp20 cells and its ICP did not have a significant difference in shape and size but entered the decline phase 2 h earlier than the Bt4.0718. In order to find the mechanism that underlies these phenotypes, we completed a proteomic study of differentially expressed proteins (DEPs). In Bt4-Δhsp20 cells, 11 DEPs were upregulated and 184 DEPs downregulated. These affected DEPs are involved in multiple metabolic pathways: (1) six DEPs (two upregulated and four downregulated) are directly related to the sporulation and ICPs synthesis; (2) supply of amino acids including amino acid synthesis and protein recycling; (3) the energy supplementation (the tricarboxylic acid cycle and glycolysis); (4) purine metabolism and mRNA stability. These results suggest that hsp20 may be critical in maintaining the homeostasis of B. thuringiensis during the production of spores and ICPs, and could provide new sight into the sporulation and ICPs formation in B. thuringiensis.

Extracellular matrix dynamics in cell migration, invasion and tissue morphogenesis.

This review describes how direct visualization of the dynamic interactions of cells with different extracellular matrix microenvironments can provide novel insights into complex biological processes. Recent studies have moved characterization of cell migration and invasion from classical 2D culture systems into 1D and 3D model systems, revealing multiple differences in mechanisms of cell adhesion, migration and signalling-even though cells in 3D can still display prominent focal adhesions. Myosin II restrains cell migration speed in 2D culture but is often essential for effective 3D migration. 3D cell migration modes can switch between lamellipodial, lobopodial and/or amoeboid depending on the local matrix environment. For example, "nuclear piston" migration can be switched off by local proteolysis, and proteolytic invadopodia can be induced by a high density of fibrillar matrix. Particularly, complex remodelling of both extracellular matrix and tissues occurs during morphogenesis. Extracellular matrix supports self-assembly of embryonic tissues, but it must also be locally actively remodelled. For example, surprisingly focal remodelling of the basement membrane occurs during branching morphogenesis-numerous tiny perforations generated by proteolysis and actomyosin contractility produce a microscopically porous, flexible basement membrane meshwork for tissue expansion. Cells extend highly active blebs or protrusions towards the surrounding mesenchyme through these perforations. Concurrently, the entire basement membrane undergoes translocation in a direction opposite to bud expansion. Underlying this slowly moving 2D basement membrane translocation are highly dynamic individual cell movements. We conclude this review by describing a variety of exciting research opportunities for discovering novel insights into cell-matrix interactions.