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.

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.

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.

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.

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.

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.

Early prophylactic anticoagulation via transjugular intrahepatic route for portal vein thrombosis after splenectomy in cirrhotic portal hypertension.

PURPOSE: To evaluate early transcatheter anticoagulation via the transjugular intrahepatic route to prevent portal vein thrombosis (PVT) after splenectomy in cirrhotic patients with portal hypertension. MATERIALS AND METHODS: This retrospective study included 98 cirrhotic patients with portal hypertension who underwent open splenectomy (48 men and 50 women; age, 45.4 y ± 13.6). Systemic anticoagulation was given to 52 patients in group I, and transcatheter anticoagulation was performed in 46 patients in group II. RESULTS: The technical success rate of catheterization by the transjugular intrahepatic route was 93.5% in group II. The 30-day (6.52% vs 23.1%, P < .05) and 6-month (8.70% vs 26.9%, P < .05) incidences of PVT were significantly lower in group II than in group I. The postoperative bleeding rate was 6.52% in group II and 25% in group I (P < .05). There was no significant difference between groups in 30-day (5.77% vs 2.17%) and 6-month (1.92% vs 6.52%) mortality. After splenectomy, the portal trunk vessel diameter was 16.0 mm ± 3.5 in group I and 14.5 mm ± 2.5 in group II (P < .05). The portal flow velocity was 25.9 cm/s ± 7.1 in group I and 28.2 cm/s ± 5.3 in group II (P > .05). During the first week after splenectomy, notable hypercoagulability was detected within the portal vein compared with peripheral blood. Decreased portal flow velocity was considered an independent risk factor for PVT by univariate and multivariate analysis. CONCLUSIONS: Transcatheter anticoagulation via the transjugular intrahepatic route can decrease the incidence of PVT and postoperative bleeding after open splenectomy in cirrhotic patients with portal hypertension.

Continuous renal replacement therapy reduces the systemic and pulmonary inflammation induced by venovenous extracorporeal membrane oxygenation in a porcine model.

Pulmonary changes in veno-venous extracorporeal membrane oxygenation (VV-ECMO) are rarely determined. We compared the contribution of VV-ECMO and cannulation based on the observation of pulmonary inflammatory reaction and parenchymal construction in a porcine model of low tidal volume (VT ) ventilation. We also evaluated the effect of adding continuous renal replacement therapy (CRRT) to the ECMO circuit, because CRRT is known to reduce systemic cytokine release induced by VV-ECMO. A total of 18 pigs undergoing low-VT ventilation were randomly divided into three groups (group 1, cannulation; group 2, VV-ECMO; group 3, VV-ECMO + CRRT) and studied for 24 h. Hemodynamic and ventilation parameters were recorded. We assessed plasma and alveolar cytokines, expression of pulmonary inflammatory genes, histopathological grading, and ultrastructural changes of the lungs. During the process, inspiratory volume increased and PaO2 decreased in group 1. Systemic tumor necrosis factor-α (TNF-α) and interleukin 6 (IL-6) levels increased at 2 h in group 2 and partly decreased in group 3. At 24 h, the levels of bronchoalveolar lavage fluid, TNF-α, and IL-6 in group 2 were remarkably higher than those in groups 1 and 3. Pulmonary mRNA expression of cytokines did not differ between the groups. We observed an increased score of pulmonary pathological findings in pro-inflammatory cell infiltration and interstitial thickening of the lungs in group 2. The epithelium of the blood-air barrier after VV-ECMO was swollen. In group 3, the pulmonary parenchyma and blood-air barrier were well preserved. We concluded that in a porcine model of low-VT ventilation, both VV-ECMO and VV-ECMO in combination with CRRT provided adequate oxygenation and carbon dioxide removal. Compared with VV-ECMO alone, VV-ECMO in combination with CRRT better preserved the lung parenchyma by eliminating systemic cytokines.

Hemodynamic effects of continuous versus bolus infusion of terlipressin for portal hypertension: a randomized comparison.

BACKGROUND AND AIM: The hemodynamics of patients with portal hypertension within 4 h after a single injection of terlipressin has been studied. However, the hemodynamics in a longer phase under different infusion styles is unknown. This study aims to compare the effects of bolus and continuous infusion of terlipressin on systemic and hepatic hemodynamics in patients with portal hypertension. METHODS: Twenty patients who underwent transjugular intrahepatic portosystemic shunt procedure were randomly assigned to be treated with either intravenous bolus infusion of terlipressin (1 mg) followed by a continuous infusion (4 mg/24 h, n = 10), or intravenous bolus injection of terlipressin (2 mg) followed by intermittent injections (1 mg/6 h, n = 10). The mean arterial pressure, heart rate, and portal venous pressure (PVP) were monitored and recorded at baseline, 1 min, 5 min, 10 min, 30 min, and then once an hour. Serum renin activity, serum angiotensin II, and aldosterone levels were measured prior to and 24 h after the administration of terlipressin. RESULTS: PVP dropped rapidly in both groups, and reduced 16.46% and 28.22%, respectively, at the 1-h time point. Thereafter, PVP remained stable in continuous group while rebounded obviously in intermittent group. One hour after the start of drug administration, heart rate decreased significantly in both groups (84.1 ± 12.8 vs 73.8 ± 12.6 in intermittent group and 86.7 ± 11.5 vs 77.1 ± 13.6 in continuous group, P < 0.005), and mean arterial pressure increased in both groups, although no statistical differences were found. CONCLUSION: Continuous infusion of terlipressin reduces PVP stably and may become an alternative to traditional bolus injection.

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.

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.

[Sleep disturbance induced by cocaine abstinence involving in A2A receptor over-expression in rat hypothalamus].

Adult rats were implanted with sleep-wake recording electrodes in our experiments. Polygraphic signs of undisturbed sleep-wake activities were recorded for 24 h before cocaine administration, cocaine withdrawal day 1 (acute), day 8 (subacute), and day 14 (subchronic). Western blot method was performed to examine the expression levels of adenosine receptor subtypes in hypothalamus and cerebellum. Non rapid eye movement (NREM) sleep was significantly increased during nighttime (P < 0.01) and daytime (P < 0.05) on withdrawal day 8. The increase of NREM sleep was significant during nighttime (P < 0.01) and slight during daytime on withdrawal day 14, whereas both daytime and nighttime rapid eye movement (REM) sleeps were reduced markedly (P < 0.01) on withdrawal day 8 and 14. In addition, A2A receptor level was significantly enhanced on cocaine withdrawal day 8 and day 14 (P < 0.05), whereas A1 receptor level reduced markedly on withdrawal day 14 (P < 0.05). However, compared with that in the control group, no significant changes existed among adenosine A1, A2A and A2B receptors in rat cerebellum on cocaine withdrawal day 1, day 8 and day 14. Our findings suggest that sleep disorder caused by subacute and subchronic cocaine abstinence may be associated with over-expression of adenosine A2A receptor in rat hypothalamus to some extent.

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.

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.

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.

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.

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.

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.

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.

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.