A universal all-in-one RPA-Cas12a strategy with de novo autodesigner and its application in on-site ultrasensitive detection of DNA and RNA viruses.

Revolutionary all-in-one RPA-CRISPR assays are rapidly becoming the most sought-after tools for point-of-care testing (POCT) due to their high sensitivity and ease of use. Despite the availability of one-pot methods for specific targets, the development of more efficient methods for new targets remains a significant challenge. In this study, we present a rapid and universal approach to establishing an all-in-one RPA-Cas12a method CORDSv2 based on rational balancing amplification and Cas12a cleavage, which achieves ultrasensitive detection of several targets, including SARS-CoV-2, ASFV, HPV16, and HPV18. CORDSv2 demonstrates a limit of detection (LOD) of 0.6 cp/µL and 100% sensitivity for SARS-CoV-2, comparable to qPCR. Combining with our portable device(hippo-CORDS), it has a visual detection LOD of 6 cp/µL and a sensitivity up to 100% for SARS-CoV-2 and 97% for Ct<35 ASFV samples, surpassing most one-pot visual methods. To simplify and accelerate the process for new targets, we also develop a de novo autodesigner by which the optimal couples of primers and crRNA can be selected rapidly. As a universal all-in-one RPA-CRISPR method for on-site testing, CORDSv2 becomes an attractive choice for rapid and accurate diagnosis in resource-limited settings.

miR-130b duplex (miR-130b-3p/miR-130b-5p) negatively regulates goat intramuscular preadipocyte lipid droplets accumulation by inhibiting Krüppel-like factor 3 expression.

Intramuscular lipid deposition is important for meat quality improvement. microRNAs and their target mRNAs provide a new approach for studying the mechanism of fat deposition. The present study aimed to investigate the effect of miR-130b duplex (miR-130b-5p, miR-130b-3p) and its target gene KLF3 in regulating goat intramuscular adipocyte differentiation. Goat intramuscular preadipocytes were isolated from 7-d-old male Jianzhou big-ear goats and identified by Oil red O staining after differentiation induction. miR-130b-5p and miR-130b-3p mimics or inhibitors and their corresponding controls were transfected into goat intramuscular preadipocytes, respectively, and differentiation was induced by 50µM oleic acid for 48 h. Oil red O and Bodipy staining indicated that both miR-130b-5p and miR-130b-3p can reduce lipid droplets accumulation and triglyceride (TG) content (P < 0.01). Differentiation markers C/EBPα, C/EBPß, PPARγ, pref1, fatty acids synthesis markers ACC, FASN, DGAT1, DGAT2, AGPAT6, TIP47, GPAM, ADRP, AP2, SREBP1, and TG markers LPL, ATGL, HSL were assessed by qPCR. All the markers measured were downregulated by miR-130b-5p and miR-130b-3p analog (P < 0.01), suggesting that miR-130b inhibits goat intramuscular adipocyte adipogenic differentiation, fatty acids synthesis, and lipid lipolysis. To examine the mechanism of miR-130b duplex inhibition of lipid deposition, TargetScan, miRDB, and starBase were used to predict the potential targets, KLF3 was found to be the only one intersection. Furthermore, the 3'UTR of KLF3 was cloned, qPCR analysis and dual luciferase activity assay showed that both miR-130b-5p and miR-130b-3p could directly regulate KLF3 expression (P < 0.01). In addition, overexpression and interference of KLF3 were conducted, it was found that KLF3 positively regulated lipid droplets accumulation by Oil red O, Bodipy staining, and TG content detection (P < 0.01). Quantitative PCR result indicated that KLF3 overexpression promoted lipid droplets accumulation relative genes C/EBPß, PPARγ, pref1, ACC, FASN, DGAT1, DGAT2, AGPAT6, TIP47, GPAM, ADRP, SREBP1, LPL, and ATGL expression (P < 0.01). Downregulation of KLF3 inhibited the expression of genes such as C/EBPα, C/EBPß, PPARγ, pref1, TIP47, GPAM, ADRP, AP2, LPL, and ATGL expression (P < 0.01). Taken together, these results indicate that miR-130b duplex could directly inhibit KLF3 expression, then attenuated adipogenic and TG synthesis genes expression, thus leading to its anti-adipogenic effect.

microRNAs (miRNAs) are small (19 to 24 nucleotides), single-stranded, noncoding RNAs that are evolutionarily conserved and can be complimentary bound to the 3ʹ-untranslated region (3ʹUTR) of their target mRNA for cleavage or translation inhibition to participate in almost all biological processes. We demonstrated miR-130b duplex (miR-130b-3p/miR-130b-5p) negatively regulates goat intramuscular preadipocyte lipid droplets accumulation by targeting Krüppel-like factor 3 (KLF3) expression. This research opens new visions to study and understand the functions and mechanisms of goat miRNAs in lipid deposition.

Knockout of ABC transporters by CRISPR/Cas9 contributes to reliable and accurate transporter substrate identification for drug discovery.

It is essential to explore the relationship between drugs and transporters in the process of drug development. Strong background signals in nonhuman MDCK or LLC-PK1 cells and overlapping interference of inhibitors or RNAi in human Caco-2 cells mean that an ideal alternative could be to knock out specific transporter genes in Caco-2 cells. However, the application of gene knockout (KO) to Caco-2 cells is challenging because it is still inefficient to obtain rapidly growing Caco-2 subclones with double-allele KO through long-term monoclonal cultivation. Herein, CRISPR/Cas9, a low cost but more efficient and precise gene editing technology, was utilized to singly or doubly knockout the P-gp, BCRP, and MRP2 genes in Caco-2 cells. By combining this with single cell expansion, rapidly growing transporter-deficient subclones were successfully screened and established. Bidirectional transport assays with probe substrates and three protease inhibitors indicated that more reliable and detailed data could be drawn easily with these KO Caco-2 models. The six robust KO Caco-2 subclones could contribute to efficient in vitro drug transport research.

Validation Study to Determine the Accuracy of Widespread Promoterless EGFP Reporter at Assessing CRISPR/Cas9-Mediated Homology Directed Repair.

An accurate visual reporter system to assess homology-directed repair (HDR) is a key prerequisite for evaluating the efficiency of Cas9-mediated precise gene editing. Herein, we tested the utility of the widespread promoterless EGFP reporter to assess the efficiency of CRISPR/Cas9-mediated homologous recombination by fluorescence expression. We firstly established a promoterless EGFP reporter donor targeting the porcine GAPDH locus to study CRISPR/Cas9-mediated homologous recombination in porcine cells. Curiously, EGFP was expressed at unexpectedly high levels from the promoterless donor in porcine cells, with or without Cas9/sgRNA. Even higher EGFP expression was detected in human cells and those of other species when the porcine donor was transfected alone. Therefore, EGFP could be expressed at certain level in various cells transfected with the promoterless EGFP reporter alone, making it a low-resolution reporter for measuring Cas9-mediated HDR events. In summary, the widespread promoterless EGFP reporter could not be an ideal measurement for HDR screening and there is an urgent need to develop a more reliable, high-resolution HDR screening system to better explore strategies of increasing the efficiency of Cas9-mediated HDR in mammalian cells.

Efficient generation of a CYP3A4-T2A-luciferase knock-in HepaRG subclone and its optimized differentiation.

CRISPR/Cas9 has allowed development of better and easier-to-use ADME models than traditional methods by complete knockout or knock-in of genes. However, gene editing in HepaRG cells remains challenging because long-term monoclonal cultivation may alter their differentiation capacity to a large extent. Here, CRISPR/Cas9 was used to generate a CYP3A4-T2A-luciferase knock-in HepaRG subclone by Cas9-mediated homologous recombination and monoclonal cultivation. The knock-in HepaRG-#9 subclone retained a similar differentiation potential to wildtype HepaRG cells (HepaRG-WT). To further improve differentiation and expand the applications of knock-in HepaRG cells, two optimized differentiation procedures were evaluated by comparison with the standard differentiation procedure using the knock-in HepaRG-#9 subclone and HepaRG-WT. The results indicated that addition of forskolin (an adenylate cyclase activator) and SB431542 (a TGF-ß pathway inhibitor) to the first optimized differentiation procedure led to better differentiation consequence in terms of not only the initiation time for differentiation and morphological characterization, but also the mRNA levels of hepatocyte-specific genes. These data may contribute to more extensive applications of genetically modified HepaRG cells in ADME studies.

Integrative Analysis of lncRNA-miRNA-mRNA Regulatory Network Reveals the Key lncRNAs Implicated Potentially in the Differentiation of Adipocyte in Goats.

Goats are popular in China because of their superior meat quality, delicate flesh, and unique flavor. Long noncoding RNAs (lncRNAs) play important roles in transcriptional and post-transcriptional regulation of gene expression. However, the effects of lncRNAs on adipocyte differentiation in goat has not been fully elucidated yet. In this investigation, we performed RNA-Seq analysis of intramuscular and subcutaneous adipocytes from Jianzhou Daer goat before and after differentiation, including both intramuscular preadipocytes (IMPA) vs. intramuscular adipocytes (IMA) and subcutaneous preadipocytes (SPA) vs. subcutaneous adipocytes (SA). A total of 289.49 G clean reads and 12,519 lncRNAs were obtained from 20 samples. In total, 3,733 differentially expressed RNAs (182 lncRNAs and 3,551 mRNAs) were identified by pairwise comparison. There were 135 differentially expressed lncRNAs (DELs) specific to intramuscular adipocytes, 39 DELs specific to subcutaneous adipocytes, and 8 DELs common to both adipocytes in these 182 DELs. Some well-known and novel pathways associated with preadipocyte differentiation were identified: fat acid metabolism, TGF-beta signaling pathway and PI3K-Akt signaling pathway. By integrating miRNA-seq data from another study, we also identified hub miRNAs in both types of fat cells. Our analysis revealed the unique and common lncRNA-miRNA-mRNA networks of two kinds of adipocytes. Several lncRNAs that regulate potentially goat preadipocyte differentiation were identified, such as XR_001918 647.1, XR_001917728.1, XR_001297263.2 and LNC_004191. Furthermore, our findings from the present study may contribute to a better understanding of the molecular mechanisms underlying in goat meat quality and provide a theoretical basis for further goat molecular breeding.

Polymer-Metal Interfacial Friction Characteristics under Ultrasonic Plasticizing Conditions: A United-Atom Molecular Dynamics Study.

Understanding the properties of polymer-metal interfacial friction is critical for accurate prototype design and process control in polymer-based advanced manufacturing. The transient polymer-metal interfacial friction characteristics are investigated using united-atom molecular dynamics in this study, which is under the boundary conditions of single sliding friction (SSF) and reciprocating sliding friction (RSF). It reflects the polymer-metal interaction under the conditions of initial compaction and ultrasonic vibration, so that the heat generation mechanism of ultrasonic plasticization microinjection molding (UPMIM) is explored. The contact mechanics, polymer segment rearrangement, and frictional energy transfer features of polymer-metal interface friction are investigated. The results reveal that, in both SSF and RSF modes, the sliding rate has a considerable impact on the dynamic response of the interfacial friction force, where the amplitude has a response time of about 0.6 ns to the friction. The high frequency movement of the polymer segment caused by dynamic interfacial friction may result in the formation of a new coupled interface. Frictional energy transfer is mainly characterized by dihedral and kinetic energy transitions in polymer chains. Our findings also show that the ultrasonic amplitude has a greater impact on polymer-metal interfacial friction heating than the frequency, as much as it does under ultrasonic plasticizing circumstances on the homogeneous polymer-polymer interface. Even if there are differences in thermophysical properties at the heterointerface, transient heating will still cause heat accumulation at the interface with a temperature difference of around 35 K.

RNA-seq analysis reveals the positive role of KLF5 in the differentiation of subcutaneous adipocyte in goats.

As the largest energy storage reservoir, subcutaneous adipose tissue (SAT) stores excess lipids by adipocytes enlargement and/or recruitment of new precursor cells. Energy overload can cause ectopic fat deposition and metabolic diseases. In this study, 6814 differentially expressed genes (DEGs) were screened in goat subcutaneous preadipocytes and mature adipocytes by RNA-seq technique. The relative expression of the DEGs were verified by qPCR, such as PLIN2, MECR, ADCY7, PEBP1 and KLF5, and their expression level was found to be consistent with the trend of RNA-seq analysis. The KLF5 was selected for further functional verification. Overexpression of KLF5 promoted both the adipogenesis and the differentiation of preadipocytes, while the expression of preadipocyte marker gene: preadipocyte factor 1(Pref1) was decreased, as well as, peroxisome proliferator activation Receptor γ(PPARγ), CCAAT enhancer binding protein ß(C/EBPß) and Sterol regulatory element binding protein isoform 1(SREBP1) were increased. On the contrary, the interference of KLF5 could reduce adipogenesis, enhance the expression of Pref1, and reduce the expression of C/EBPß and SREBP1. Our research provides a basic reference for revealing the mechanism of subcutaneous adipocyte differentiation in goats.

Rapid and accurate detection of SARS-CoV-2 mutations using a Cas12a-based sensing platform.

The increasing prevalence of SARS-CoV-2 variants with spike mutations has raised concerns owing to higher transmission rates, disease severity, and escape from neutralizing antibodies. Rapid and accurate detection of SARS-CoV-2 variants provides crucial information concerning the outbreaks of SARS-CoV-2 variants and possible lines of transmission. This information is vital for infection prevention and control. We used a Cas12a-based RT-PCR combined with CRISPR on-site rapid detection system (RT-CORDS) platform to detect the key mutations in SARS-CoV-2 variants, such as 69/70 deletion, N501Y, and D614G. We used type-specific CRISPR RNAs (crRNAs) to identify wild-type (crRNA-W) and mutant (crRNA-M) sequences of SARS-CoV-2. We successfully differentiated mutant variants from wild-type SARS-CoV-2 with a sensitivity of 10-17 M (approximately 6 copies/µL). The assay took just 10 min with the Cas12a/crRNA reaction after a simple RT-PCR using a fluorescence reporting system. In addition, a sensitivity of 10-16 M could be achieved when lateral flow strips were used as readouts. The accuracy of RT-CORDS for SARS-CoV-2 variant detection was 100% consistent with the sequencing data. In conclusion, using the RT-CORDS platform, we accurately, sensitively, specifically, and rapidly detected SARS-CoV-2 variants. This method may be used in clinical diagnosis.

Overexpression of Krueppel like factor 3 promotes subcutaneous adipocytes differentiation in goat Capra hircus.

Previous research reported that KLF3 plays different roles in the regulation of adipose deposition across species. However, the exact function of KLF3 in goat subcutaneous adipocyte remains unknown. Here, the goat KLF3 gene was firstly cloned and showed that the mRNA sequence of the goat KLF3 gene was 1,264 bp (GenBank accession number: KU041753.1) and its coding sequence was 1,037 bp, encoding 345 amino acids with three classic zinc finger domains of KLFs family at its C-terminus. The alignment of the amino acid sequence of KLF3 among various species demonstrated that goat had the highest homology to that of sheep, presenting 99.4% similarity, while the homology similarity to that of mice presented only 93.62% in contrast. Furthermore, KLF3 had highest mRNA level in fat tissue and lowest level in the heart in comparison. Additionally, the mRNA level of KLF3 gradually tended to increase during adipogenesis. Interestingly, overexpression of KLF3 increased lipid accumulation. In line with this, the gain-of-function of KLF3 dramatically elevated the mRNA levels of TG synthetic genes and adipogenic maker genes (p < .01) . Moreover, overexpression of KLF3 upregulated all the potential target genes, except for C/EBPα. These results suggested that KLF3 is a positive regulator for subcutaneous adipocyte differentiation in goats.

PSTPIP2 inhibits cisplatin-induced acute kidney injury by suppressing apoptosis of renal tubular epithelial cells.

Cisplatin (CP) is an effective chemotherapeutic agent widely used in the treatment of various solid tumours. However, CP nephrotoxicity is an important limitation for CP use; currently, there is no method to ameliorate cisplatin-induced acute kidney injury (AKI). Recently, we identified a specific role of proline-serine-threonine phosphatase-interacting protein 2 (PSTPIP2) in cisplatin-induced AKI. PSTPIP2 was reported to play an important role in a variety of diseases. However, the functions of PSTPIP2 in experimental models of cisplatin-induced AKI have not been extensively studied. The present study demonstrated that cisplatin downregulated the expression of PSTPIP2 in the kidney tissue. Administration of AAV-PSTPIP2 or epithelial cell-specific overexpression of PSTPIP2 reduced cisplatin-induced kidney dysfunction and inhibited apoptosis of renal tubular epithelial cells. Small interfering RNA-based knockdown of PSTPIP2 expression abolished PSTPIP2 regulation of epithelial cell apoptosis in vitro. Histone acetylation may impact gene expression at the epigenetic level, and histone deacetylase (HDAC) inhibitors were reported to prevent cisplatin-induced nephrotoxicity. The UCSC database was used to predict that acetylation of histone H3 at lysine 27 (H3K27ac) induces binding to the PSTPIP2 promoter, and this prediction was validated by a ChIP assay. Interestingly, an HDAC-specific inhibitor (TSA) was sufficient to potently upregulate PSTPIP2 in epithelial cells. Histone acetylation-mediated silencing of PSTPIP2 may contribute to cisplatin nephrotoxicity. PSTPIP2 may serve as a potential therapeutic target in the prevention of cisplatin nephrotoxicity.

Sennoside A prevents liver fibrosis by binding DNMT1 and suppressing DNMT1-mediated PTEN hypermethylation in HSC activation and proliferation.

Hepatic stellate cell (HSC) activation is an essential event during liver fibrogenesis. Phosphatase and tension homolog deleted on chromosome 10 (PTEN) is a negative regulator of this process. DNA methyltransferase 1 (DNMT1), which catalyzes DNA methylation and subsequently leads to the transcriptional repression of PTEN, is selectively induced in myofibroblasts from diseased livers. Sennoside A (SA), a major purgative constituent of senna and the Chinese herb rhubarb, is widely used in China and other Asian countries as an irritant laxative. SA is reported to improve hepatic steatosis. However, the effect and mechanism of SA on liver fibrosis remain largely unknown. We recently identified a novel strategy for protecting liver fibrosis via epigenetic modification by targeting DNMT1. A Surface Plasmon Resonance (SPR) assay first reported that SA could directly bind DNMT1 and inhibit its activity. Administration of SA significantly prevented liver fibrosis, as evidenced by the dramatic downregulation of α-smooth muscle actin (α-SMA) and type I collagen alpha-1 (Col1α1) protein levels in a CCl4 -induced mouse hepatic fibrosis model and in TGF-ß1-activated HSC-T6 cells, in vivo and in vitro. SA decreased the expression of Cyclin D1, CDK, and C-myc, indicating that SA may inhibit the activation and proliferation of TGF-ß1-induced HSC-T6. Moreover, SA significantly promoted the expression of PTEN and remarkably inhibited the expression of p-AKT and p-ERK in vitro. Blocking PTEN or overexpressing DNMT1 could reduce the effect of SA on liver fibrosis. These data suggest that SA directly binds and inhibits the activity and that attenuated DNMT1-mediated PTEN hypermethylation caused the loss of PTEN expression, followed by the inhibition of the AKT and ERK pathways and prevented the development of liver fibrosis. Hence, SA might be employed as a promising natural supplement for liver fibrosis drug therapy.

Coinfection of parvovirus and astrovirus in gout-affected goslings.

Outbreaks of gosling gout have occurred in China since 2017 and caused a considerable economic impact on the poultry industry. While gosling astrovirus (GoAstV) is believed to be the main causal pathogen of gout, the full-blown disease of gout cannot be well reproduced by infecting the goslings with GoAstV, suggesting the possibility of other infectious agents being involved with the development of gosling gout. To assess other possible infectious agents, we collected tissues from gout-affected goslings in 12 goose farms in China, followed by PCR detection of GoAstV, goose reovirus (GRV), goose parvovirus (GPV), fowl adenovirus (FAdV), goose circovirus (GcoV), Tembusu virus (TMUV) and goose haemorrhagic polyomavirus (GHPV). Our data showed that all gout-affected goslings carried both of GoAstV and GPV determined by PCRs, and this was further confirmed by fluorescence multiplex immunohistochemical staining, and phylogenetic analysis of ORF2 gene of GoAstV and VP3 gene of GPV. In addition to the haemorrhage in the kidney, liver, spleen and lung of the gout-affected goslings, histological examinations showed also extensive infiltration of heterophil myelocytes in the kidney, liver, spleen, bursa of Fabricius, thymus, lungs and pancreas. Our findings strongly suggest that coinfection of GoAstV and GPV increases the severity of gout. While this is the first study to report GPV in gout-affected goslings, further studies including infection model are warranted to investigate the role of GPV and its coinfection with GoAstV in the development of gosling gout.

Inhibition of Histone H3K27 Acetylation Orchestrates Interleukin-9-Mediated and Plays an Anti-Inflammatory Role in Cisplatin-Induced Acute Kidney Injury.

Nephrotoxicity is a major side effect of cisplatin (CP)- and platinum-related chemotherapy, and inflammation contributes to disease pathogenesis. Interleukin-9 (IL-9) is a pleiotropic cytokine associated with inflammation. Here, we investigated the key role of IL-9 as a regulator of protective mechanisms in CP-induced acute kidney injury (AKI). We observed that IL-9 was decreased not only in a CP-induced AKI mouse model but also in THP-1 and RAW264.7 cell lines. Seventy-two hours post-CP injection, renal dysfunction and tubule injury were significantly attenuated in IL-9 overexpression adeno-associated virus 9 (AAV9)-treated mice. The levels of serum urea, serum creatinine, kidney injury molecule-1 (KIM-1), and histological damage were partially diminished following treatment with IL-9. The renoprotective effects of IL-9 may be attributed to the regulation of cytokines, and we found that IL-9 acted on macrophages in a regulatory manner, promoting an anti-inflammatory phenotype. Furthermore, IL-9 enhanced the suppression of macrophage-driven renal inflammation. Inhibition of H3K27 acetylation orchestrated IL-9-mediated renoprotection in CP-induced AKI. Thus, our findings indicate novel and potent anti-inflammatory properties of IL-9 that confer preservation of kidney function and structure in CP-induced AKI, which may counteract kidney disease procession.

Wogonin attenuates liver fibrosis via regulating hepatic stellate cell activation and apoptosis.

Liver fibrosis is the representative features of liver chronic inflammation and the characteristic of early cirrhosis. To date, effective therapy for liver fibrosis is lacking. Recently, Traditional Chinese Medicine (TCM) has attracted increasing attention due to its wide pharmacological effects and more uses in clinical. Wogonin, as one major active constituent of Scutellaria radix, has been reported it plays an important role in anti-inflammatory, anti-cancer, anti-viral, anti-angiogenesis, anti-oxidant and neuro-protective effects. However, the anti-fibrotic effect of wogonin is never covered in liver. In this study, we evaluated the protect effect of wogonin in liver fibrosis. Wogonin significantly attenuated liver fibrosis both in CCl4-induced mice and TGF-ß1 activated HSCs. Meanwhile, wogonin can enhances apoptosis of TGF-ß1 activated HSC-T6 cell from rat and LX-2 cell from human detected by flow cytometry. Additionally, wogonin can largely enhances cle-caspase3, cle-caspase9 expression and the ratio of Bax/Bcl-2 in T6 cells. Pro-apoptosis effect of wogonin in vivo was further verified in situ. In conclusion, wogonin can attenuate liver fibrosis via regulating the activation and apoptosis of hepatic stellate cells, and may be an effective drug to treat and prevent liver fibrosis.

Improved defined approaches for predicting skin sensitization hazard and potency in humans.

Since the EU banned animal testing for cosmetic products and ingredients in 2013, many defined approaches (DA) for skin sensitization assessment have been developed. Machine learning models were shown to be effective in DAs, but the predictivity might be affected by data imbalance (i.e. more numbers of sensitizers than non-sensitizers) and limited information in the databases. To improve the predictivity of DAs, here we attempted to apply data-rebalancing ensemble learning (bagging with support vector machine (SVM)) and a novel and comprehensive Cosmetics Europe database. For predicting human hazard and three-class potency, 12 models were built for each using a training set of 96 substances and a test set of 32 substances from the database. The model with the highest accuracy for predicting hazard (90.63% for the test set and 88.54% for the training set, named hazard-DA) used the SVM-bagging with combinations of all variables (V6), while the model with the highest accuracy for predicting potency (68.75% for the test set and 82.29% for the training set, named potency-DA) used SVM alone. Both DAs showed higher performance than LLNA and other machine-learning-based DAs, and the potency-DA could provide more in-depth assessment. Those findings indicated that SVM-bagging-based DAs provided enhanced predictivity for hazard assessment by further data rebalancing. Meanwhile, the effect of imbalanced data might be offset by more detailed categorization of sensitizers for potency assessment, thus SVM-based DA without bagging could provide sufficient predictivity. The improved DAs in this study could be promising tools for skin sensitization assessment without animal testing.

Advancing the predictivity of skin sensitization by applying a novel HMOX1 reporter system.

Reporter cell lines are a particularly useful tool to screen for the skin sensitization potential of chemicals. Current cell models based on Keap1-Nrf2 mimic induction by conducting antioxidant response element-luciferase plasmids. However, plasmid-based reporters may ignore comprehensive aspects of induction, thus affecting the accuracy of hazard identification. Herein, we developed a novel HaCaT-based reporter system, EndoSens, whereby luciferase was specifically inserted into the cassette for heme oxygenase (decycling) 1 (HMOX1, the most consistent marker induced by skin sensitizers) by CRISPR/Cas9. Testing data from 20 coded substances showed an accuracy of 90%, sensitivity of 91.7%, and specificity of 87.5%, which exceeded the OECD requirement. Among the 35 chemicals examined, predictivity was better than reported for the validated KeratinoSens™. These results indicate that the EndoSens assay could advance the predictivity of skin sensitization, thus making it a promising tool for in vitro skin sensitization testing.

Postoperative Catheter-Directed Thrombolysis Versus Systemic Anticoagulation for Acute Superior Mesenteric Venous Thrombosis.

BACKGROUND: Little data evaluate catheter-directed thrombolysis (CDT) therapy as a sequential treatment of emergent surgery for patients with acute superior mesenteric venous thrombosis (ASMVT). We compared the outcomes of ASMVT patients receiving CDT via superior mesenteric artery (SMA) with those who had systemic anticoagulation after emergent laparotomy. METHODS: A single-center retrospective study of ASMVT patients receiving emergent laparotomy from May 2012 to April 2014 was performed. Patients in group I had postoperative systemic anticoagulation and patients in group II underwent postoperative CDT. The demography, etiology, imaging features, clinical outcomes, and complications were compared. Moreover, univariate analysis was performed to identify confounding variables of 30-day mortality. RESULTS: Thirty-two patients (20 males, mean age of 44.9 ± 10.6 years) were included, 17 in group I and 15 in group II. No significant differences of demographic data, etiology, baseline value, and perioperative comorbidity were found. The rate of complete thrombus removal was significantly higher in group II than group I (29.4% vs. 80.0%, P = 0.001). The second-look laparotomy and repeat bowel resection (58.8% vs. 13.3%, P = 0.002) were required in fewer patients in group II (20.0% vs. 70.6%, P = 0.001). The incidence of short-bowel syndrome (SBS; 41.2% vs. 6.7%, P = 0.001) and 30-day mortality (41.2% vs. 6.7%, P = 0.001) were lower in group II. The 1-year survival was also better in group II (52.9% vs. 93.3%, P = 0.014). The incidence of massive abdominal hemorrhage requiring blood transfusion and surgical intervention was 11.8% in group I and 20.0% in group II (P = 0.645). The age, serum D-dimer level, SBS, and postoperative CDT were significant risk factors of 30-day mortality in this study. CONCLUSIONS: For ASMVT patients receiving emergent surgery and intraoperative thrombectomy, the algorithm with postoperative CDT via SMA is associated with more favorable clinical outcome compared with systemic anticoagulation.

Systemic and Splanchnic Lipopolysaccharide and Endothelin-1 Plasma Levels in Liver Cirrhosis before and after Transjugular Intrahepatic Portosystemic Shunt.

Lipopolysaccharide (LPS) and endothelin- (ET-) 1 may aggravate portal hypertension by increasing intrahepatic resistance and splanchnic blood flow. In the portal vein, after TIPS shunting, LPS and ET-1 were significantly decreased. Our study suggests that TIPS can benefit cirrhotic patients not only in high hemodynamics related variceal bleeding but also in intestinal bacterial translocation associated complications such as endotoxemia.

Biliary Obstruction following Transjugular Intrahepatic Portosystemic Shunt Creation in Patients with Variceal Bleeding.

This report describes an unusual complication after creation of a transjugular intrahepatic portosystemic shunt (TIPS). Biliary obstruction developed in two patients with portal hypertension accompanied by portal vein thrombosis, one patient with and the other without portal cavernous transformation. The biliary obstruction was thought to be secondary to compression of the bile duct by the stent graft placed in the TIPS. Awareness of this possible complication is important for its early diagnosis.