Mechanistic insights into transposon cleavage and integration by TnsB of ShCAST system.

The type V-K CRISPR-associated transposons (CASTs) allow RNA-guided DNA integration and have great potential as a programmable site-specific gene insertion tool. Although all core components have been independently characterized structurally, the mechanism of how the transposase TnsB associates with AAA+ ATPase TnsC and catalyzes donor DNA cleavage and integration remains ambiguous. In this study, we demonstrate that TniQ-dCas9 fusion can direct site-specific transposition by TnsB/TnsC in ShCAST. TnsB is a 3'-5' exonuclease that specifically cleaves donor DNA at the end of the terminal repeats and integrates the left end prior to the right end. The nucleotide preference and the cleavage site of TnsB are markedly different from those of the well-documented MuA. We also find that TnsB/TnsC association is enhanced in a half-integration state. Overall, our results provide valuable insights into the mechanism and application expansion of CRISPR-mediated site-specific transposition by TnsB/TnsC.

Highly stable Ru-complex-based metal-covalent organic frameworks as novel type of electrochemiluminescence emitters for ultrasensitive biosensing.

Developing novel types of high-performance electrochemiluminescence (ECL) emitters is of great significance for constructing ultrasensitive ECL sensors. Herein, a highly stable metal-covalent organic framework (MCOF), termed Ru-MCOF, has been devised and synthesized by employing a classic ECL luminophore, tris(4,4'-dicarboxylicacid-2,2'-bipyridyl)ruthenium(II) (Ru(dcbpy)32+), as building unit and applied as a novel ECL probe to construct an ultrasensitive ECL sensor for the first time. Impressively, the topologically ordered and porous architectures of the Ru-MCOF not only allow Ru(bpy)32+ units to precisely locate and homogeneously distribute in the skeleton via strong covalent bonds but also facilitate the transport of co-reactants and electrons/ions in channels to promote the electrochemical activation of both external and internal Ru(bpy)32+ units. All these features endow the Ru-MCOF with excellent ECL emission, high ECL efficiency, and outstanding chemical stability. As expected, the constructed ECL biosensor based on the Ru-MCOF as a high-efficiency ECL probe accomplishes the ultrasensitive detection of microRNA-155. Overall, the synthesized Ru-MCOF not only enriches the MCOF family but also displays excellent ECL performance and thus expands the application of MCOFs in bioassays. Considering the structural diversity and tailorability of MCOFs, this work opens a new horizon to design and synthesize high-performance ECL emitters, therefore paving a new way to develop highly stable and ultrasensitive ECL sensors and motivating further research on MCOFs.

Ruthenium(II) complex-grafted conductive metal-organic frameworks with conductivity- and confinement-enhanced electrochemiluminescence for ultrasensitive biosensing application.

Improving the electrochemiluminescence (ECL) performance of luminophores is an ongoing research hotspot in the ECL realm. Herein, a high-performance metal-organic framework (MOF)-based ECL material (Ru@Ni3(HITP)2, HITP = 2,3,6,7,10,11-hexaiminotriphenylene) with conductivity- and confinement-enhanced ECL was successfully constructed by using conductive MOF Ni3(HITP)2 as the carrier to graft Ru(bpydc)34- (H2bpydc = 2,2'-bipyridine-4,4'-dicarboxylic acid) into the channels of Ni3(HITP)2. Compared to Ru@Cu3(HITP)2 and Ru@Co3(HITP)2 with relatively low conductivity, the ECL intensity of Ru@Ni3(HITP)2 was prominently increased about 6.76 times and 18.8 times, respectively, which demonstrated that the increase in conductivity induced the ECL enhancement of the MOF-based ECL materials. What's more, the hydrophobic and porous Ni3(HITP)2 can not only effectively enrich the lipophilic tripropylamine (TPrA) coreactants in its channels to enhance the electrochemical oxidation efficiency of TPrA, but also provide a conductive reaction micro-environment to boost the ECL reaction between Ru(bpydc)33- intermediates and TPrA• in confined spaces, thus realizing a remarkable confinement-enhanced ECL. Considering the excellent ECL performance of Ru@Ni3(HITP)2, an ultrasensitive ECL biosensor was prepared based on the Ru@Ni3(HITP)2 ECL indicator combining an exonuclease I-aided target cycling amplification strategy for thrombin determination. The constructed ECL biosensor showcased a wide linear range from 1 fM to 1 nM with a low detection limit of 0.62 fM. Overall, the conductivity- and confinement-enhanced ECL based on Ru@Ni3(HITP)2 provided effective and feasible strategies to enhance ECL performance, which paved a promising avenue for exploring high-efficient MOF-based ECL materials and thus broadened the application scope of conductive MOFs.

Pyrene-Based Hydrogen-Bonded Organic Frameworks as New Emitters with Porosity- and Aggregation-Induced Enhanced Electrochemiluminescence for Ultrasensitive MicroRNA Assay.

Exploring new electrochemiluminescence (ECL) luminophores with strong ECL emission is highly desirable for developing ultrasensitive ECL sensors. Herein, a pyrene-based hydrogen-bonded organic framework (Py-HOF) featuring prominent ECL performance was prepared by utilizing 1,3,6,8-tetrakis(p-benzoic acid) pyrene (H4TBAPy) with an aggregation-induced enhanced emission (AIEE) property as a building block, exhibiting a stronger ECL emission than those of H4TBAPy monomers, H4TBAPy aggregates, the low-porosity Py-HOF-210 °C and Py-HOF-180 °C. We have coined the term "the porosity- and aggregation-induced enhanced ECL (PAIE-ECL)" for this intriguing phenomenon. The Py-HOF displayed superb and stable ECL intensity, not only because the luminophore H4TBAPy was assembled into the Py-HOF via four pairs of O-H···O hydrogen bonds, which constrained the intramolecular movements to reduce nonradiative transition, but also because the H4TBAPy in Py-HOF was stacked in a slipped face-to-face mode to form J-aggregates that benefited the ECL enhancement. Furthermore, the high porosity of Py-HOF allowed the enrichment of coreactants and facilitated the migration of ions, electrons, and coreactants, which made it possible for the inner and outer H4TBAPy to be electrochemically excited. Considering the remarkable ECL performance, Py-HOF was first employed as an ECL probe combined with a 3D DNA nanomachine amplification strategy to assemble a hypersensitive "on-off" ECL sensor for the microRNA-141 assay, presenting a satisfactory linear range (100 aM to 1 nM) with a detection limit of 14.4 aM. The PAIE-ECL manifested by Py-HOF provided a bright avenue for the design and synthesis of outstanding HOF-based ECL materials and offered new opportunities for the development of ECL biosensors with excellent sensitivity.

Ruthenium(II) Complex-Grafted Hollow Hierarchical Metal-Organic Frameworks with Superior Electrochemiluminescence Performance for Sensitive Assay of Thrombin.

Metal-organic frameworks (MOFs) with porous structures exhibit favorable promise in synthesizing high-performance electrochemiluminescence (ECL) materials, yet their micropores and narrow channels not only restrict the loading capacity of ECL luminophores but also constrain the diffusion of coreactants, ions, and electrons. Hence, we developed a new and simple hydrothermal etching strategy for the fabrication of a hollow hierarchical MOF (HH-UiO-66-NH2) with a hierarchical-pore shell, which was employed as a carrier to graft Ru(bpy)2(mcpbpy)2+ (bpy = 2,2'-bipyridine, mcpbpy = 4-(4'-methyl-[2,2'-bipyridin]-4-yl) butanoic acid) onto the coordinatively unsaturated Zr6 nodes of HH-UiO-66-NH2, creating the Ru-complex-grafted HH-UiO-66-NH2 (abbreviated as HH-Ru-UiO-66-NH2). Impressively, the HH-Ru-UiO-66-NH2 presented brilliant ECL emission. On the one hand, the HH-UiO-66-NH2 with a hierarchical-pore shell and hollow cavity was conducive to immobilize the Ru(bpy)2(mcpbpy)2+ of large steric hindrance into the interior of the MOF, markedly improving the load number of luminophores. On the other hand, the hierarchical-pore shell of HH-UiO-66-NH2 permitted fast diffusion of coreactants, ions, and electrons that facilitated the excitation of more grafted luminophores and greatly enhanced the utilization ratio of ECL luminophores. Inspired by the superior ECL performance of HH-Ru-UiO-66-NH2, an ECL sensing platform was constructed on the basis of HH-Ru-UiO-66-NH2 as an ECL beacon combining catalytic hairpin assembly as a signal amplification strategy, showing excellent selectivity and high sensitivity for thrombin determination. This proof-of-concept work proposed a simple and feasible hydrothermal etching strategy to construct hollow hierarchical MOFs that served as carrier materials to immobilize ECL luminophores, providing significant inspiration to develop highly efficient ECL materials and endowing hollow hierarchical MOFs with ECL sensing applications for the first time.

Lithium promotes proliferation and suppresses migration of Schwann cells.

Schwann cell proliferation, migration and remyelination of regenerating axons contribute to regeneration after peripheral nervous system injury. Lithium promotes remyelination by Schwann cells and improves peripheral nerve regeneration. However, whether lithium modulates other phenotypes of Schwann cells, especially their proliferation and migration remains elusive. In the current study, primary Schwann cells from rat sciatic nerve stumps were cultured and exposed to 0, 5, 10, 15, or 30 mM lithium chloride (LiCl) for 24 hours. The effects of LiCl on Schwann cell proliferation and migration were examined using the Cell Counting Kit-8, 5-ethynyl-2'-deoxyuridine, Transwell and wound healing assays. Cell Counting Kit-8 and 5-ethynyl-2'-deoxyuridine assays showed that 5, 10, 15, and 30 mM LiCl significantly increased the viability and proliferation rate of Schwann cells. Transwell-based migration assays and wound healing assays showed that 10, 15, and 30 mM LiCl suppressed the migratory ability of Schwann cells. Furthermore, the effects of LiCl on the proliferation and migration phenotypes of Schwann cells were mostly dose-dependent. These data indicate that lithium treatment significantly promotes the proliferation and inhibits the migratory ability of Schwann cells. This conclusion will inform strategies to promote the repair and regeneration of peripheral nerves. All of the animal experiments in this study were ethically approved by the Administration Committee of Experimental Animal Center of Nantong University, China (approval No. 20170320-017) on March 2, 2017.

Screening of 485 Pesticide Residues in Fruits and Vegetables by Liquid Chromatography-Quadrupole-Time-of-Flight Mass Spectrometry Based on TOF Accurate Mass Database and QTOF Spectrum Library.

This paper uses the LC-quadrupole-time-of-flight MS technique to evaluate the behavioral characteristics of MS of 485 pesticides under different conditions and has developed an accurate mass database and spectra library. A high-throughput screening and confirmation method has been developed for the 485 pesticides in fruits and vegetables. Through the optimization of parameters such as accurate mass number, time of retention window, ionization forms, etc., the method has improved the accuracy of pesticide screening, thus avoiding the occurrence of false-positive and false-negative results. The method features a full scan of fragments, with 80% of pesticide qualitative points over 10, which helps increase pesticide qualitative accuracy. The abundant differences of fragment categories help realize the effective separation and qualitative identification of isomer pesticides. Four different fruits and vegetables-apples, grapes, celery, and tomatoes-were chosen to evaluate the efficiency of the method at three fortification levels of 5, 10, and 20 µg/kg, and satisfactory results were obtained. With this method, a national survey of pesticide residues was conducted between 2012 and 2015 for 12 551 samples of 146 different fruits and vegetables collected from 638 sampling points in 284 counties across 31 provincial capitals/cities directly under the central government, which provided scientific data backup for ensuring pesticide residue safety of the fruits and vegetables consumed daily by the public. Meanwhile, the big data statistical analysis of the new technique also further proves it to be of high speed, high throughput, high accuracy, high reliability, and high informatization.

Mycophenolate Mofetil Protects Septic Mice via the Dual Inhibition of Inflammatory Cytokines and PD-1.

Due to the imbalance between hyper-inflammation and hypo-inflammation, which are characterized by excessive cytokine productions and programmed death 1 (PD-1) upregulation, respectively, sepsis remains a highly lethal inflammatory syndrome with limited effective therapies. Mycophenolate mofetil (MMF), an immunosuppressant, has been reported to attenuate various inflammatory diseases. However, the role of MMF in sepsis therapy remains to be elucidated. C57BL-6J mice underwent cecal ligation and puncture (CLP) and were treated either with or without MMF. Survival rate and organ injuries were compared. Cytokine levels, bacteria clearance, apoptosis of spleen and peritoneal macrophages, and PD-1 expression were assessed. At the beginning of CLP, 60 mg/kg MMF administered by gavage significantly protected septic mice, which was evidenced by improved survival and attenuated organ injuries, decreased cytokines, lower bacterial loads, and alleviated immune cell apoptosis. In addition, immune cells in the MMF mice showed lower PD-1 expression and improved immune response to pathogeny stimuli. MMF protects septic mice via the dual inhibition of cytokine releasing and PD-1 expression.

Biliary tract external drainage protects against multiple organs injuries of severe acute pancreatitis rats via heme oxygenase-1 upregulation.

OBJECTIVE: To investigate the effect of biliary tract external drainage (BTED) on severe acute pancreatitis (SAP) in rats and the relationship with heme oxygenase-1 (HO-1) pathway. METHODS: Thirty SD rats weighing 250-300 g were randomly assigned into five groups (n = 6): sham surgery (SS) group, SAP group, SAP + BTED group, SAP + zinc protoporphyrin IX (ZnPP) group, SAP + BTED + ZnPP group. The SAP model was induced via retrograde injection of 4% sodium taurocholate (1 mL/kg) into biliopancreatic duct through duodenal wall. BTED was performed by inserting a cannula into the bile duct of SAP rats. Tissue and blood samples were collected 24 h after surgery. Pathological changes in organs were scored. The level of amylase, alanine transaminase (ALT), aspartate aminotransferase (AST), diamine oxidase (DAO), lipopolysaccharide (LPS), myeloperoxidase (MPO) and ability to inhibit hydroxyl radical(·OH) in serum were measured. The expression of hemeoxygenase-1 (HO-1), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) in tissues were analyzed by RT- PCR and western-blot. RESULTS: Organs damage in SAP rats was significantly alleviated by BTED (p < 0.05). Compared to the SAP group, the serum level of amylase, ALT, AST, DAO, MPO, and LPS were significantly lower in the SAP + BTED group, and the ability to inhibit ·OH was significantly higher (p < 0.05). The BETD treatment led to a significant reduction of TNF-α, IL-6 level and a significant increase of HO-1 level in tissues than in SAP rats (p < 0.05). ZnPP significantly inhibited all above mentioned changes. CONCLUSIONS: BTED protected multiple organs against SAP related injuries via HO-1 upregulation.

The skeletal effects of thiazolidinedione and metformin on insulin-resistant mice.

To explore the skeletal effects and the potential underlying mechanisms of treatment with two thiazolidinediones (rosiglitazone and pioglitazone) or metformin in insulin-resistant mice, 24 female, 12-week-old C57BL6J ob/ob mice were evaluated according to the following treatment groups for 6 weeks: placebo group, pioglitazone group (Pio), rosiglitazone group (Rosi), and metformin group (Met). Bone mineral density (BMD), bone microarchitecture, bone histomorphometry, and expression of three phenotype-specific gene markers, including bone morphogenetic protein 2 (Bmp2), runt-related transcription factor 2 (Runx2), and fatty acid-binding protein 4 (Fabp4), were compared across the four groups. At the femur, the Met group had the highest BMD (0.084 ± 0.001 g/cm(2)) and trabecular bone volume/total volume (0.644 ± 0.018 %) and the lowest trabecular spacing (Tb.Sp.) (0.143 ± 0.008 µm), whereas the Rosi group had lower BMD (0.076 ± 0.003 g/cm(2)) and a relatively higher degree of Tb.Sp. (0.173 ± 0.024 µm). A histomorphometric analysis revealed that in the Rosi group the number of adipocytes was fourfold higher than in the placebo group and fivefold higher than in the Met group, whereas the highest osteoid width and mineral apposition rate were found in the Met group (49.88 ± 48.53 µm and 4.46 ± 1.72 µm/day). Furthermore, the Rosi group displayed the highest level of Fabp4 gene expression, which was accompanied by normal expression levels of Bmp2 and Runx2. Seemingly, metformin is a bone-friendly antidiabetic drug. Rosiglitazone had adverse effects on the skeleton at the trabecular bone even in insulin-resistant mice, whereas no evidence of adverse effects was found for pioglitazone.

Partial scapulectomy for congenital elevation of the scapula.

We treated 26 patients with congenital elevation of the scapula by excising the superomedial part of the scapula. We also simultaneously resected the omovertebral bone when present. A modified inverted L-shape incision was used to release the contracted tissue around the medial edge of the scapula. We evaluated 28 shoulders (26 patients) at a minimum of 10 months followup (mean, 3.9 years; range, 10 months-7 years). The improvement rate for range of shoulder abduction was 59.11%. Eighteen shoulders with preoperative abduction less than 120 degrees achieved an average improvement of 52 degrees, whereas 10 shoulders with a preoperative abduction range greater than 120 degrees achieved an average improvement of 19 degrees. However, there was no difference in the improvement rate of the range of shoulder abduction between the two groups (60.94% versus 56%). Twenty-three shoulders (82.24%) attained various degrees of cosmetic improvement after the operation. There were no neurologic complications during or after surgery. No patients complained of scar problems. Excising the superomedial part of the scapula is a safe, simple operation for treating congenital elevation of the scapula.

Interactions of lipid metabolism and intestinal physiology with Tremella fuciformis Berk edible mushroom in rats fed a high-cholesterol diet with or without Nebacitin.

Male adult Wistar rats were randomly divided into six groups in a 2 x 3 factorial design and fed diets containing different levels of Tremella fuciformis Berk (TFB) dietary fiber (0, 5, or 10%) and 1 g of cholesterol/100 g of diet with or without 0.7% Nebacitin for 4 weeks. TFB contained 6.2% soluble dietary fiber and 57.3% insoluble dietary fiber. The results showed that the serum LDL-cholesterol, hepatic total cholesterol, and triglyceride levels were significantly decreased (P < 0.05) in the rats fed diets with TFB content with or without Nebacitin. However, the serum total cholesterol, VLDL-cholesterol, and triglyceride levels were significantly decreased (P < 0.05) by Nebacitin. In feces, the presence of TFB (T5, T10, AT5, and AT10) in the diet significantly increased the total neutral steroids and bile acid excretions and undigested fiber concentrations as compared to T0 or AT0. In the small intestine, the Nebacitin diets increased the weights of both cecum and colon-rectum contents and lowered short-chain fatty acid (SCFA) concentrations of serum and cecal contents more than no Nebacitin diets did. It was suggested that the hypocholesterolemic effect of TFB dietary fiber may be mediated by the increase in fecal neutral steroids and total bile acids excretion and the increase in SCFA productions. The TFB edible mushroom dietary supplement altered the intestinal physiology of the rats.