Identification of a novel LFNG variant in a Chinese fetus with spondylocostal dysostosis and a systematic review.

Spondylocostal dysostosis (SCDO) encompasses a group of skeletal disorders characterized by multiple segmentation defects in the vertebrae and ribs. SCDO has a complex genetic etiology. This study aimed to analyze and identify pathogenic variants in a fetus with SCDO. Copy number variant sequencing and whole exome sequencing were performed on a Chinese fetus with SCDO, followed by bioinformatics analyses, in vitro functional assays and a systematic review on the reported SCDO cases with LFNG pathogenic variants. Ultrasound examinations in utero exhibited that the fetus had vertebral malformation, scoliosis and tethered cord, but rib malformation was not evident. We found a novel homozygous variant (c.1078 C > T, p.R360C) within the last exon of LFNG. The variant was predicted to cause loss of function of LFNG by in silico prediction tools, which was confirmed by an in vitro assay of LFNG enzyme activity. The systematic review listed a total of 20 variants of LFNG in SCDO. The mutational spectrum spans across all exons of LFNG except the last one. This study reported the first Chinese case of LFNG-related SCDO, revealing the prenatal phenotypes and expanding the mutational spectrum of the disorder.

Targeting LINC070974 inhibits lung adenocarcinoma cell proliferation and progression by interacting with Y-box binding protein 1.

Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related mortality worldwide. Increasing evidence suggests that long noncoding RNAs play crucial roles in lung cancer pathogenesis. We previously identified a novel lncRNA, LINC070974, which is associated with tumor cell proliferation. In the present study, we find that knockdown of LINC070974 inhibits cell proliferation, migration and invasion as well as tumor formation both in vitro and in nude mice. LINC070974 silencing also improves cisplatin efficacy in A549/DDP cells. The function of LINC070974 may depend on its interaction with YBX1. Knockdown of LINC070974 reduces the recruitment of YBX1 to the CCND1 promoter and delays tumor progression through its coregulatory genes, which are mainly involved in the p53 signaling pathway. We utilize nebulized inhalation to deliver siRNAs targeting LINC070974 and find that LINC070974 significantly prevents tumor metastasis and growth in lung tissues. These findings reveal the role of LINC070974 in lung cancer and suggest a promising therapeutic approach involving siRNA inhalation.

Well-defined N3 C1 -anchored Single-Metal-Sites for Oxygen Reduction Reaction.

N-, C-, O-, S-coordinated single-metal-sites (SMSs) have garnered significant attention due to the potential for significantly enhanced catalytic capabilities resulting from charge redistribution. However, significant challenges persist in the precise design of well-defined such SMSs, and the fundamental comprehension has long been impeded in case-by-case reports using carbon materials as investigation targets. In this work, the well-defined molecular catalysts with N3 C1 -anchored SMSs, i.e., N-confused metalloporphyrins (NCPor-Ms), are calculated for their catalytic oxygen reduction activity. Then, NCPor-Ms with corresponding N4 -anchored SMSs (metalloporphyrins, Por-Ms), are synthesized for catalytic activity evaluation. Among all, NCPor-Co reaches the top in established volcano plots. NCPor-Co also shows the highest half-wave potential of 0.83 V vs. RHE, which is much better than that of Por-Co (0.77 V vs. RHE). Electron-rich, low band gap and regulated d-band center contribute to the high activity of NCPor-Co. This study delves into the examination of well-defined asymmetric SMS molecular catalysts, encompassing both theoretical and experimental facets. It serves as a pioneering step towards enhancing the fundamental comprehension and facilitating the development of high-performance asymmetric SMS catalysts.

Isomeric Dual-Pore Two-Dimensional Covalent Organic Frameworks.

Two-dimensional (2D) covalent organic frameworks (COFs) with hierarchical porosity have been increasingly recognized as promising materials in various fields. Besides, the 2D COFs with kagome (kgm) topology can exhibit unique optoelectronic features and have extensive applications. However, rational synthesis of the COFs with kgm topology remains challenging because of competition with a square-lattice topology. Herein, we report two isomeric dual-pore 2D COFs with kgm topology using a novel geometric strategy to reduce the symmetry of their building blocks, which are four-armed naphthalene-based and azulene-based isomeric monomers. Owing to the large dipole moment of azulene, as-prepared azulene-based COF (COF-Az) possesses a considerably narrow band gap of down to 1.37 eV, which is much narrower than the naphthalene-based 2D COF (COF-Nap: 2.28 eV) and is the lowest band gap among reported imine-linked dual-pore 2D COFs. Moreover, COF-Az was used as electrode material in a gas sensor and exhibits high selectivity for NO2, including a high response rate (58.7%) to NO2 (10 ppm), fast recovery (72 s), up to 10 weeks of stability, and resistance to 80% relative humidity, which are superior to those of reported COF-based NO2 gas sensors. The calculation and in situ experimental results indicate that the large dipole moment of azulene boosts the sensitivity of the imine linkages. The usage of isomeric building blocks not only enables the synthesis of 2D COFs with isometric kgm topology but also provides an azulene-based 2D platform for studying the structure-property correlations of COFs.

Regulation of the integrin αVß3- actin filaments axis in early osteogenic differentiation of human mesenchymal stem cells under cyclic tensile stress.

BACKGROUND: Integrins are closely related to mechanical conduction and play a crucial role in the osteogenesis of human mesenchymal stem cells. Here we wondered whether tensile stress could influence cell differentiation through integrin αVß3. METHODS: We inhibited the function of integrin αVß3 of human mesenchymal stem cells by treating with c(RGDyk). Using cytochalasin D and verteporfin to inhibit polymerization of microfilament and function of nuclear Yes-associated protein (YAP), respectively. For each application, mesenchymal stem cells were loaded by cyclic tensile stress of 10% at 0.5 Hz for 2 h daily. Mesenchymal stem cells were harvested on day 7 post-treatment. Western blotting and quantitative RT-PCR were used to detect the expression of alkaline phosphatase (ALP), RUNX2, ß-actin, integrin αVß3, talin-1, vinculin, FAK, and nuclear YAP. Immunofluorescence staining detected vinculin, actin filaments, and YAP nuclear localization. RESULTS: Cyclic tensile stress could increase the expression of ALP and RUNX2. Inhibition of integrin αVß3 activation led to rearrangement of actin filaments and downregulated the expression of ALP, RUNX2 and promoted YAP nuclear localization. When microfilament polymerization was inhibited, ALP, RUNX2, and nuclear YAP nuclear localization decreased. Inhibition of YAP nuclear localization could reduce the expression of ALP and RUNX2. CONCLUSIONS: Cyclic tensile stress promotes early osteogenesis of human mesenchymal stem cells via the integrin αVß3-actin filaments axis. YAP nuclear localization participates in this process of human mesenchymal stem cells. Video Abstract.

Fully Automated CRISPR-LAMP Platform for SARS-CoV-2 Delta and Omicron Variants.

Integrated clustered regularly interspaced short palindromic repeat (CRISPR)-loop-mediated amplification (LAMP) technology is of great importance in CRISPR-based diagnostic systems, which urgently needs to be developed to improve diagnostic accuracy. A labor-free, contamination-free, and fully automated droplet manipulation platform for the CRISPR-LAMP technology has not been developed before. Herein, we propose a fully automated CRISPR-LAMP platform, which can precisely manipulate the CRISPR-LAMP droplet and perform combined reactions with high sensitivity and specificity. SARS-CoV-2 Spike T478K, D614G, P681R, and P681H mutations, typical point mutations of B.1.617.2 (Delta) and Omicron variants, are monitored with this platform with a detection limit of 102 copies/µL. Allele discrimination between the mutants and wild type is significant with the designed one/two-mismatch CRISPR RNA (crRNA) at a limit of 102 copies/µL. Chemically synthesized and modified crRNAs greatly increase the CRISPR-LAMP signal, which advance the wide application. Combined with the previously developed RdRp CRISPR-LAMP assay, clinical results showed that Spike T478K and P681H can discriminate the mutant type form the wild type with 70% (49.66-85.50%, 95% confidence interval) and 78% (57.27-90.62%, 95% confidence interval) sensitivity, respectively, and 100% specificity (51.68-100%, 95% confidence interval), and the RdRp target can detect SARS-CoV-2 strains with 85% sensitivity (65.39-95.14%, 95% confidence interval) and 100% specificity (51.68-100%, 95% confidence interval). We believe that this automatic digital microfluid (DMF) system can advance the integrated CRISPR-LAMP technology with higher stability, sensitivity, and practicability, also for other CRISPR-associated diagnostic platforms.

B/N-Enriched Semi-Conductive Polymer Film for Micro-Supercapacitors with AC Line-Filtering Performance.

Microsupercapacitors (MSCs) have drawn great attention for use as miniaturized electrochemical energy storage devices in portable, wearable, as well as implantable electronics. Many materials have been developed as electrodes for MSCs. However, the thin-film fabrication for most of these materials involves multistep operations, including filtration, spray coating, and sputtering. Most importantly, these methods present challenges for the preparation of thin films at the atomic or molecular scale. Therefore, the understanding of performance of ultrathin-film-based MSCs remains challenge. Herein, a B/N-enriched polymer film is successfully prepared using the photoassisted interfacial approach. The as-synthesized polymer film exhibits typical semiconductive characteristics and can be easily scaled up to a large area of up to tens of square centimeters. This ultrathin polymer film can be directly transferred to silicon wafers to fabricate MSC through laser scribing. The prepared MSC exhibits specific volumetric capacitance as high as 20.9 F cm-3, corresponding to volumetric energy density of 2.9 mWh cm-3 (at 0.1 V s-1). Moreover, the volumetric power density can reach 1461 W cm-3, surpassing most existing semiconductive polymer film-based MSC devices. In addition, the prepared MSC exhibits typical AC line-filtering ability (-67° at 120 Hz). This study offers a facile interfacial approach to preparing semiconductive polymer films with aromatic moieties for microsized energy storage devices.

Current and potential biomarkers in gastric cancer: a critical review of the literature.

Gastric cancer is the fourth most common type of cancer worldwide and the second most lethal. Gastric cancer biomarkers can be used for diagnosis, prediction of sensitivity to treatment, and prognosis. The following search terms were applied to PubMed as of December 2020: 'gastric cancer classification', 'gastric cancer epidemiology', 'cancer metastasis' and 'gastric cancer biomarker'. Only experimental studies were reported in the 'biomarkers' section. Some biomarkers can serve as therapeutic targets for antitumoral drugs. The genes analyzed include E-cadherin, RPRM, XAF1, MINT25, TFF1, p16 and p53. The miRNAs analyzed include miR-18a, miR185-5p, miR-125b and miR-21. Some molecules were associated with metastasis of gastric cancer, specifically those involved with EMT process and tissue degradation.

Lay abstract Gastric cancer is the fourth most common type of cancer worldwide and the second most lethal. Gastric cancer biomarkers are molecules that have different expressions in tumor cells than in normal body cells, and can be used for diagnosis, prediction of sensitivity to treatment, and prognosis. Biomarkers in gastric cancer can include genes that suppress tumor progression, genes that increase tumor progression by binding to growth molecules, molecules related to the body's immune response to the tumor, and non-coding RNA molecules (RNA molecules that do not produce proteins but regulate the cell's genetic material). Some biomarkers can serve as therapeutic targets for anti-tumoral drugs.

[Effects of Human Immunodeficiency Virus-positive Mothers Receiving Antiretroviral Therapy to Prevent Mother-to-child Transmission on the Growth and Development of 18-month-old Children in Lingshan County of Guangxi].

Objective To evaluate the effects of antiretroviral therapy(ART)for the prevention of mother-to-child transmission(PMTCT)of acquired immune deficiency syndrome(AIDS)on the growth and development of 18-month-old children born by human immunodeficiency virus(HIV)-positive pregnant women in Lingshan County,Guangxi Zhuang Autonomous Region,and provide scientific evidence for improving the ART medication plan for PMTCT.Methods Lingshan County,ranking the first in the HIV-epidemic counties of Guangxi,was selected as the research site.According to the design of retrospective case-control study,we assigned all the subjects into the case group and the control group:(1)The case group included the HIV-positive pregnant women who had received ART for PMTCT and their HIV-negative infants in Lingshan County from 2010 to 2017.The historical cards and PMTCT data of them were collected from the national PMTCT database.(2)The control group included the healthy pregnant women and their healthy babies born in the Lingshan Maternity and Infant Hospital in 2017,and the children's growth and development data were collected.The stunted growth in children was defined as at least one of the three main indicators of body height,body weight,and head circumference below the normal range.Results The number of HIV-positive mothers and their infants in the case group was 391 and 368,respectively,and 87.21%(341/391)and 95.38%(351/368)of mothers and infants respectively received ART medication.The HIV positive rate,mortality rate,and mother-to-child transmission rate of 18-month-old children were 1.36%(5/368),4.35%(16/368),and 2.01%(5/249),respectively.The incidence of stunted growth of 18-month-old children in the case group and the control group was 42.12%(155/368)and 23.06%(101/438),respectively,with significant difference(χ2=33.520,P<0.001).Conclusion After HIV-positive mothers in Lingshan County of Guangxi received ART for PMTCT,the incidence of growth stunting in 18-month-old children increased.

Ionic Polyimide Derived Porous Carbon Nanosheets as High-Efficiency Oxygen Reduction Catalysts for Zn-Air Batteries.

Two-dimensional (2D) porous carbon nanosheets (2DPCs) have attracted great attention for their good porosity and long-distance conductivity. Factors such as templates, precursors, and carbonization-activation methods, directly determine their performance. However, rational design and preparation of porous carbon materials with controlled 2D morphology and heteroatom dopants remains a challenge. Therefore, an ionic polyimide with both sp2 - and sp3 -hybridized nitrogen atoms was prepared as a precursor for fabricating N-doped hexagonal porous carbon nanosheets through a hard-template approach. Because of the large surface area and efficient charge-mass transport, the resulting activated 2D porous carbon nanosheets (2DPCs-a) displayed promising electrocatalytic properties for oxygen reduction reaction (ORR) in alkaline and acidic media, such as ultralow half-wave potential (0.83 vs. 0.84 V of Pt/C) and superior limiting current density (5.42 vs. 5.14 mA cm-2 of Pt/C). As air cathodes in Zn-air batteries, the as-developed 2DPCs-a exhibited long stability and high capacity (up to 614 mA h g-1 ), which are both higher than those of commercial Pt/C. This work provides a convenient method for controllable and scalable 2DPCs fabrication as well as new opportunities to develop high-efficiency electrocatalysts for ORR and Zn-air batteries.

A charge-sensing region in the stromal interaction molecule 1 luminal domain confers stabilization-mediated inhibition of SOCE in response to S-nitrosylation.

Store-operated Ca2+ entry (SOCE) is a major Ca2+ signaling pathway facilitating extracellular Ca2+ influx in response to the initial release of intracellular endo/sarcoplasmic reticulum (ER/SR) Ca2+ stores. Stromal interaction molecule 1 (STIM1) is the Ca2+ sensor that activates SOCE following ER/SR Ca2+ depletion. The EF-hand and the adjacent sterile α-motif (EFSAM) domains of STIM1 are essential for detecting changes in luminal Ca2+ concentrations. Low ER Ca2+ levels trigger STIM1 destabilization and oligomerization, culminating in the opening of Orai1-composed Ca2+ channels on the plasma membrane. NO-mediated S-nitrosylation of cysteine thiols regulates myriad protein functions, but its effects on the structural mechanisms that regulate SOCE are unclear. Here, we demonstrate that S-nitrosylation of Cys49 and Cys56 in STIM1 enhances the thermodynamic stability of its luminal domain, resulting in suppressed hydrophobic exposure and diminished Ca2+ depletion-dependent oligomerization. Using solution NMR spectroscopy, we pinpointed a structural mechanism for STIM1 stabilization driven by complementary charge interactions between an electropositive patch on the core EFSAM domain and the S-nitrosylated nonconserved region of STIM1. Finally, using live cells, we found that the enhanced luminal domain stability conferred by either Cys49 and Cys56S-nitrosylation or incorporation of negatively charged residues into the EFSAM electropositive patch in the full-length STIM1 context significantly suppresses SOCE. Collectively, our results suggest that S-nitrosylation of STIM1 inhibits SOCE by interacting with an electropositive patch on the EFSAM core, which modulates the thermodynamic stability of the STIM1 luminal domain.

Two-Dimensional Porous Polymers: From Sandwich-like Structure to Layered Skeleton.

Inorganic porous materials have long dominated the field of porous materials due to their stable structure and wide applications. In the past decade, porous polymers have generated considerable interest among researchers because of their easily tunable porosity, carbon-rich backbones, and prominent physical properties. These attributes enable porous polymers to be used in various applications such as sensing, gas separation and storage, catalysis, and energy storage. However, poor dispersibility has long hindered the development of porous polymers. A majority of the reported porous polymers can only be synthesized with amorphous structure through direct precipitation from solutions during reactions. The rational design and synthesis of porous polymers with controllable morphology, such as two-dimensional (2D) morphology, remains great challenge. Two-dimensional nanomaterials have attracted considerable interest because of their unique properties, which originate from the intrinsic chemical structures and 2D dimensionality. Among 2D nanomaterials, 2D porous polymers, which possess the advanced features of polymers, porous materials, and 2D nanomaterials, have been a rising star. Conventionally, polymerization strategies for generating 2D porous polymers mainly include the cross-linking of multiarmed monomers in 2D-space-confined environments, such as crystalline solid surfaces, liquid-liquid interfaces, and liquid-air interfaces. However, these methods always involve complicate operations, e.g., under vacuum, sophisticated equipment, film transfer technology, exfoliation, and so on and, most importantly, are difficult to scale up. To overcome this synthesis obstacle, 2D nanomaterials, such as graphene, can be used as 2D templates for synthesis of sandwich-like 2D porous polymers and porous carbon nanosheets. p-Bromobenzene-, p-cyanobenzene-, polyacrylonitrile-, and amino-functionalized graphene are used as templates for direct surface polymerization through reactions such as Sonogashira-Hagihara coupling reaction, condensation reaction, ionothermal reaction, reversible addition-fragmentation chain transfer polymerization, Friedel-Crafts reaction, and oxidation reaction. Therefore, sandwich-like 2D conjugated microporous polymers, Schiff-base type porous polymers, covalent triazine frameworks, hyper-cross-linked porous polymers, and mesoporous conducting polymers can be easily prepared. Beyond graphene, other excellent 2D nanomaterials, e.g., MoS2, can also act 2D templates to construct 2D porous polymers and corresponding hybrid materials. In addition, 2D morphology for porous polymer can be achieved without 2D templates in a few cases. For instance, olefin-linkage-linked covalent organic frameworks can be synthesized through Knoevenagel condensation reaction. As is known, porous polymers can serve as carbon-rich precursors to generate heteroatom doped porous carbons for energy storage and catalysis. Thus, one benefit of 2D porous polymers is new access toward porous carbon nanosheets through direct pyrolysis without using inorganic porous templates. In this Account, we summarize recent research on 2D porous polymers and corresponding porous carbon nanosheets.

Gemcitabine induces apoptosis and autophagy via the AMPK/mTOR signaling pathway in pancreatic cancer cells.

Gemcitabine (GEM)-based chemotherapy is a commonly used treatment for pancreatic cancer. However, acquired drug resistance, a major problem in pancreatic cancer treatment, causes a reduction in the survival rate of patients with cancer. In this study, we attempted to reveal the molecular mechanism of GEM resistance. Our data showed that GEM treatment inhibits cell growth, induces apoptosis, and activates autophagy via the AMP-activated protein kinase (AMPK) pathway. The combination of GEM treatment and AMPK knockdown resulted in a dramatic increase of apoptosis and inhibition of autophagy. Additionally, inhibition of mammalian target of Rapamycin induced autophagy. Our findings show the potential therapeutic implications of the combined treatment with GEM and AMPK inhibitors for pancreatic cancer.

The STIM-Orai Pathway: STIM-Orai Structures: Isolated and in Complex.

Considerable progress has been made elucidating the molecular mechanisms of calcium (Ca2+) sensing by stromal interaction molecules (STIMs) and the basis for Orai channel activity. This chapter focuses on the available high-resolution structural details of STIM and Orai proteins with respect to the regulation of store-operated Ca2+ entry (SOCE). Solution structures of the Ca2+-sensing domains of STIM1 and STIM2 are reviewed in detail, crystal structures of cytosolic coiled-coil STIM fragments are discussed, and an overview of the closed Drosophila melanogaster Orai hexameric structure is provided. Additionally, we highlight structures of human Orai1 N-terminal and C-terminal domains in complex with calmodulin and human STIM1, respectively. Ultimately, the accessible structural data are discussed in terms of potential mechanisms of action and cohesiveness with functional observations.

Combined open and laparoscopic technique for extraperitoneal mesh repair of large sac inguinal hernias.

BACKGROUND: Laparoscopic total extraperitoneal (TEP) hernia repair has been confirmed as an effective procedure in several studies but is considered technically demanding. Separating the hernial sac and spermatic cord is difficult when a large sac inguinal hernia is encountered. This study aimed to investigate the feasibility and effectiveness of a combined open and laparoscopic TEP repair of large sac inguinal hernias. METHODS: From June 2012 to May 2015, laparoscopic TEP (112 cases) and combined open and laparoscopic TEP (COL-TEP) (44 cases) were performed in patients with large sac hernia. There was no clear definition of large sac inguinal hernia; therefore, we defined a large sac as one with the sac base cranial to or over outer ring that could not be easily resected laparoscopically. Using this definition, the laparoscopic TEP group was divided into a small sac TEP (SS-TEP) group (68 cases) and a large sac TEP (LS-TEP) group (44 cases). Direct hernias were included in the SS-TEP group because the hernial sac was easily dissected laparoscopically. The patient demographics, perioperative parameters, complications, and recurrence were compared between the three groups. RESULTS: No significant differences were found between the groups in mean age, gender, body mass index, comorbidities, number of previous laparotomies, or recurrence rate. Compared with the LS-TEP group, both the SS-TEP and COL-TEP groups had a significantly lower surgical duration (51.4 ± 10.9 vs. 32.8 ± 13.1 and 36.2 ± 11.2 min, respectively), conversion rate (13.6 vs. 0 and 0 %, respectively), and total complication rate (27.3 vs. 13.2 and 11.3 %, respectively). CONCLUSION: The combined technique was safe and effective for repair of large sac inguinal hernias. The combined technique was associated with decreased technical difficulty, surgical duration, and conversion and total complication rates.

Laparoscopic Choledocholithotomy With a Novel Articulating Forceps.

PURPOSE: The aim of this study was to assess the safety and effectiveness of laparoscopic common bile duct (CBD) exploration with a novel articulating forceps. METHODS: A retrospective analysis was carried out of 90 patients who underwent laparoscopic transcholedochal CBD exploration for choledocholithiasis between May 2006 and June 2014. Forty-five patients underwent laparoscopic choledocholithotomy using the routine instruments (group A). Forty-five patients underwent laparoscopic choledocholithotomy using the routine instruments plus the novel articulating forceps (group B). The 2 group populations were similar with regard to demographic data and clinical presentations. RESULTS: Laparoscopic transcholedochal CBD exploration was successful in all 90 patients. The mean diameter of the CBD was 14.42 mm in group A and 14.73 mm in group B (P > .05). The average number of stones extracted per patient was 4.22 in group A and 4.67 in group B (P > .05). The patients in group A had a significantly longer operative time than the patients in group B (109.38 vs 80.49 minutes; P < .01). The intraoperative blood loss was minimal in both groups, and no major complications were observed in either group. The mean hospitalization stay was 6.60 days in group A and 5.58 days in group B (P < .01). CONCLUSION: Laparoscopic transcholedochal CBD exploration with the novel articulating forceps is a safe and effective approach to the management of choledocholithiasis that offers a short operating time and short postoperative hospital stay.

Inhibition of protein phosphatase 5 (PP5) suppresses survival and growth of colorectal cancer cells.

Protein phosphatase 5 (PP5) is a unique member of the protein phosphatases family that functions in multiple signaling pathways involved in DNA damage, cell cycle control, cell growth, and apoptosis. Recent evidence indicated that PP5 may play a role in cancer progression. In this study, we aimed to examine the biological effect of PP5 on cell growth and apoptosis in human colorectal cancer (CRC). We first knocked down PP5 expression in RKO cells via a short hairpin RNA containing lentivirus system. Then, methylthiazoletetrazolium assay, colony formation assay, and flow cytometry analysis were performed. The proliferation and colony formation ability of RKO cells were remarkably suppressed in PP5-silenced groups, as compared with control groups. Moreover, downregulation of PP5 resulted in a significant G0/G1 phase cell cycle arrest and an induction of apoptosis. In all, these results demonstrated the importance of PP5 in CRC cell growth, and it might be used as a potential therapeutic target for the treatment of CRC.

A sensitive and high-throughput LC-MS/MS method for inhibition assay of seven major cytochrome P450s in human liver microsomes using an in vitro cocktail of probe substrates.

A sensitive and high-throughput LC-MS/MS method was established and validated for the simultaneous quantification of seven probe substrate-derived metabolites (cocktail assay) for assessing the in vitro inhibition of cytochrome P450 (CYP) enzymes in pooled human liver microsomes. The metabolites acetaminophen (CYP1A2), hydroxy-bupropion (CYP2B6), n-desethyl-amodiaquine (CYP2C8), 4'-hydroxy-diclofenac (CYP2C9), 4'-hydroxy-mephenytoin (CYP2C19), dextrorphan (CYP2D6) and 1'-hydroxy-midazolam (CYP3A4/5), together with the internal standard verapamil, were eluted on an Agilent 1200 series liquid chromatograph in <7 min. All metabolites were detected by an Agilent 6410B tandem mass spectrometer. The concentration of each probe substrate was selected by substrate inhibition assay that reduced potential substrate interactions. CYP inhibition of seven well-known inhibitors was confirmed by comparing a single probe substrate assay with cocktail assay. The IC50 values of these inhibitors determined on this cocktail assay were highly correlated (R(2) > 0.99 for each individual probe substrate) with those on single assay. The method was selective and showed good accuracy (85.89-113.35%) and between-day (RSD <13.95%) and within-day (RSD <9.90%) precision. The sample incubation extracts were stable at 25 °C for 48 h and after three freeze-thaw cycles. This seven-CYP inhibition cocktail assay significantly increased the efficiency of accurately assessing compounds' potential inhibition of the seven major CYPs in drug development settings.

Wound healing after mesh inner embedding and outer binding pancreaticojejunostomy in piglets.

BACKGROUND: The aim of this study was to introduce a novel technique of pancreaticojejunostomy, namely, mesh inner embedding and outer binding pancreaticojejunostomy, and to evaluate wound healing after this operation in piglets. METHODS: Thirty-six domestic piglets were randomly divided into 2 groups after pancreaticoduodenectomy: the mesh inner embedding and outer binding pancreaticojejunostomy group (n = 18) and the conventional double-deck invaginated pancreaticojejunostomy group (n = 18). Bursting pressure and breaking strength were assessed on the operative day and on days 7 and 14 postoperatively. The pathologic findings and collagen content of the anastomotic site were evaluated on days 7 and 14 postoperatively. RESULTS: Both the bursting pressure and breaking strength were significantly higher in the mesh inner embedding and outer binding pancreaticojejunostomy group than in the double-deck invaginated pancreaticojejunostomy group on days 0, 7, and 14 (P < .01). The collagen content of the anastomotic site was significantly higher in the mesh inner embedding and outer binding pancreaticojejunostomy group than in the double-deck invaginated pancreaticojejunostomy group on days 7 and 14 postoperatively (P < .01). The anastomotic site was more completely repaired by connective and granulation tissue in the mesh inner embedding and outer binding pancreaticojejunostomy group on day 7 than in the double-deck invaginated pancreaticojejunostomy group. CONCLUSION: Mesh inner embedding and outer binding pancreaticojejunostomy significantly enhanced the anastomotic firmness and sped up the wound healing process compared with conventional mesh inner embedding and outer binding pancreaticojejunostomy. Therefore, it may decrease the risk of pancreatic fistulas after pancreaticoduodenectomy.

A phosphorylated guanidine chitosan and UiO-66-NH2 modified magnetic nanoparticle platform for enrichment and detection of multiple bacteria.

Wastewater-based epidemiology (WBE) is a powerful tool for early warning of infectious disease outbreaks. Hence, a rapid and portable pathogen monitoring system is urgent needed for on-site detection. In this work, we first reported synthesis of an artificial modulated wide-spectrum bacteria capture nanoparticle (Arg-CSP@UiO@Fe3O4). Arginine-modified phosphorylated chitosan (Arg-CSP) coating could provide strongly positive charged guanidinium group for pathogen interaction by electrostatic attraction, and UiO-66-NH2 layer could help Arg-CSP graft onto Fe3O4 magnetic particles. The capture efficiency of Arg-CSP@UiO@Fe3O4 reached 92.2 % and 97.3 % for Escherichia coli (E.coli) and Staphylococcus epidermidis (S.epidermidis)within 40 min, in 10 mL sample. To prevent pathogen degradation in sewage, a portable nucleic acid extraction-free method was also developed. UiO-66-NH2 could disintegrate in buffer with high concentration of PO43- for bacterium desorption, and then nucleic acid of the bacteria was released by heating. The DNA template concentration obtained by this method was 779.28 times higher than that of the direct thermal lysis product and 8.63 times higher than that of the commercial kit. Afterwards, multiple detection of bacteria was realized by loop-mediated isothermal amplification (LAMP). Artificial regulated pathogen desorption could prevent non-specific adsorption of nucleic acid by nanoparticles. The detection limit of Arg-CSP@UiO@Fe3O4-LAMP method was 80 cfu/mL for E.coli and 300 cfu/mL for S.epidermidis. The accuracy and reliability of the method was validated by spiked sewage samples. In conclusion, this bio-monitoring system was able to detect multiple bacteria in environment conveniently and have good potential to become an alternative solution for rapid on-site pathogen detection.