Modular click chemistry libraries for functional screens using a diazotizing reagent.

Click chemistry is a concept in which modular synthesis is used to rapidly find new molecules with desirable properties1. Copper(I)-catalysed azide-alkyne cycloaddition (CuAAC) triazole annulation and sulfur(VI) fluoride exchange (SuFEx) catalysis are widely regarded as click reactions2-4, providing rapid access to their products in yields approaching 100% while being largely orthogonal to other reactions. However, in the case of CuAAC reactions, the availability of azide reagents is limited owing to their potential toxicity and the risk of explosion involved in their preparation. Here we report another reaction to add to the click reaction family: the formation of azides from primary amines, one of the most abundant functional groups5. The reaction uses just one equivalent of a simple diazotizing species, fluorosulfuryl azide6-11 (FSO2N3), and enables the preparation of over 1,200 azides on 96-well plates in a safe and practical manner. This reliable transformation is a powerful tool for the CuAAC triazole annulation, the most widely used click reaction at present. This method greatly expands the number of accessible azides and 1,2,3-triazoles and, given the ubiquity of the CuAAC reaction, it should find application in organic synthesis, medicinal chemistry, chemical biology and materials science.

Sodium butyrate alleviates experimental autoimmune prostatitis by inhibiting oxidative stress and NLRP3 inflammasome activation via the Nrf2/HO-1 pathway.

BACKGROUND: Chronic prostatitis and chronic pelvic pain syndrome (CP/CPPS) leads to severe discomfort in males and loss of sperm quality. Current therapeutic options have failed to achieve satisfactory results. Sodium butyrate (NaB) plays a beneficial role in reducing inflammation, increasing antioxidant capacities, and improving organ dysfunction; additionally NaB has good safety prospects and great potential for clinical application. The purpose of the current research was to study the effect of NaB on CP/CPPS and the underlying mechanisms using a mouse model of experimental autoimmune prostatitis (EAP) mice. METHODS: The EAP mouse model was successfully established by subcutaneously injecting a mixture of prostate antigen and complete Freund's adjuvant. Then, EAP mice received daily intraperitoneal injections of NaB (100, 200, or 400 mg/kg/day) for 16 days, from Days 26 to 42. We then explored anti-inflammatory potential mechanisms of NaB by studying the effects of Nrf2 inhibitor ML385 and HO-1 inhibitor zinc protoporphyrin on prostate inflammation and pelvic pain using this model. On Day 42, hematoxylin-eosin staining and dihydroethidium staining were used to evaluate the histological changes and oxidative stress levels of prostate tissues. Chronic pelvic pain was assessed by applying Von Frey filaments to the lower abdomen. The levels of inflammation-related cytokines, such as interleukin (IL)-1ß, IL-6, and tumor necrosis factor were detected by enzyme-linked immunosorbent assay. The regulation of Nrf2/HO-1 signaling pathway and the expression of NLRP3 inflammasome-related protein in EAP mice were detected by western blot analysis assay. RESULTS: Compared with the EAP group, chronic pain development, histological manifestations, and cytokine levels showed that NaB reduced the severity of EAP. NaB treatment could inhibit NLRP3 inflammasome activation. Mechanism studies showed that NaB intervention could alleviate oxidative stress in EAP mice through Nrf2/HO-1 signal pathway. Nrf2/HO-1 pathway inhibitors can inhibit NaB -mediated oxidative stress. The inhibitory effect of NaB on the activation of NLRP3 inflammasome and anti-inflammatory effect can also be blocked by Nrf2/HO-1 pathway. CONCLUSIONS: NaB treatment can alleviates prostatic inflammation and pelvic pain associated with EAP by inhibiting oxidative stress and NLRP3 inflammasome activation via the Nrf2/HO-1 pathway. NaB has the potential as an effective agent in the treatment of EAP.

Modular preparation of biphenyl triazoles via click chemistry as non-competitive hyaluronidase inhibitors.

Hyaluronidase is a promising target in drug discovery, given its overexpression in a range of physiological and pathological processes, including tumor migration, skin aging, sagging, and wrinkling, as well as inflammation and bacterial infections. In this study, to identify novel hyaluronidase inhibitors, we applied click chemistry for the modular synthesis of 370 triazoles in 96-well plates, starting with biphenyl azide. Utilizing an optimized turbidimetric screening assay in microplates, we identified Fmoc-containing triazoles 5 and 6, as well as quinoline-containing triazoles 15 and 16, as highly effective hyaluronidase inhibitors. Subsequent research indicated that these triazoles potentially interact with a novel binding site of hyaluronidase. Notably, these inhibitors displayed minimal cytotoxicity and showed promising anti-inflammatory effects in LPS-stimulated macrophages. Remarkably, compound 6 significantly reduced NO release by 74 % at a concentration of 20 µM.

Urethral-sparing laparoscopic simple prostatectomy for the treatment of benign prostatic hyperplasia with asymptomatic urethral stricture after urethral stricture surgery.

OBJECTIVE: To evaluate the efficacy of urethral-sparing laparoscopic simple prostatectomy (US-LSP) for the treatment of large-volume (>80 ml) benign prostatic hyperplasia (BPH) with asymptomatic urethral stricture (urethral lumen > 16 Fr) after urethral stricture surgery. METHODS: We retrospectively analyzed clinical data of 39 large-volume BPH patients with asymptomatic urethral stricture after urethral stricture surgery who underwent US-LSP from January 2016 to October 2021. Postoperative follow-ups were scheduled at 1, 3, and 6 months. RESULTS: All patients affected by significant BPH-related lower urinary tract symptoms (LUTS) including 22 cases with asymptomatic anterior urethral stricture and 17 cases with asymptomatic posterior urethral stricture. Median operative time was 118 min (interquartile range [IQR]100-145). Median estimated blood loss was 224 ml (IQR: 190-255). 33 patients(84.6%) avoided continuous bladder irrigation. Postoperative complications occurred in 5 patients (12.8%), including 4 cases with Clavien-Dindo grade 1 and grade 2 and 1 case with grade 3a. During follow-up, US-LSP presented statistically significant improvements in LUTS compared to baseline (P < 0.05). A total of 25 patients had normal ejaculation preoperatively and 3 patients (12%) complained retrograde ejaculation postoperatively. Two patients (5.1%) reported stress urinary incontinence (SUI) and no patient reported aggravated urethral stricture during follow-up. CONCLUSIONS: US-LSP was safe and effective in treating large-volume BPH with asymptomatic urethral stricture after urethral stricture surgery. Meanwhile, US-LSP could reduce the risk of SUI in patients with asymptomatic posterior urethral stricture and maintain ejaculatory function in a high percentage of patients.

The distribution characteristics of strabismus surgery types in a tertiary hospital in the Central Plains region during the COVID-19 epidemic.

OBJECTIVE: This study aimed to analyze the distribution of different types of strabismus surgery in a tertiary hospital in Central China during the three-year period of the COVID-19 pandemic. METHODS: A retrospective analysis was conducted on the clinical data of strabismus patients who underwent surgery and were admitted to the Department of Strabismus and Pediatric Ophthalmology at the First Affiliated Hospital of Zhengzhou University between January 2020 and December 2022. RESULTS: A total of 3939 strabismus surgery patients were collected, including 1357 in 2020, 1451 in 2021, and 1131 in 2022. The number of surgeries decreased significantly in February 2020, August 2021, and November and December 2022. Patients aged 0-6 years accounted for 37% of the total number of strabismus surgery patientsr. The majority (60%) of all strabismus surgery patients were diagnosed with exotropia, with intermittent exotropia accounting for the highest proportion (53%). There was no statistically significant difference in the proportion of intermittent exotropia and constant exotropia during the three-year period (χ2 = 2.642, P = 0.267 and χ2 = 3.012, P = 0.221, respectively). Among patients with intermittent exotropia, insufficient convergence type was the most common form of strabismus (accounting for over 70%). Non-accommodative esotropia accounted for more than 50% of all internal strabismus cases. CONCLUSION: During the period from 2020 to 2022, the total number of strabismus surgeries in our hospital did not show significant fluctuations, but there was a noticeable decrease in the number of surgeries during months affected by the pandemic. Exotropia accounted for the highest proportion among strabismus surgery patients. Intermittent exotropia was the most common type among patients undergoing surgery for exotropia, and the most prevalent subtype was the insufficient convergence type. The age distribution of patients varied in different months, with a concentration of surgeries for strabismus patients in the 7-12 years old age group during the months of July and August each year.

Identification of simple arylfluorosulfates as potent agents against resistant bacteria.

Sulfur fluoride exchange (SuFEx), a next generation of click chemistry, opens an avenue for drug discovery. We report here the discovery and structure-activity relationship studies of a series of arylfluorosulfates, synthesized via SuFEx, as antibacterial agents. Arylfluorosulfates 3, 81, and 101 showed potency to overcome multidrug resistance and were not susceptible to the generation of resistance. They exhibited rapid bactericidal potency and selectively killed gram-positive bacterial strains. These compounds also exhibited the ability to disrupt established bacterial biofilm and kill persisters derived from biofilm. Furthermore, arylfluorosulfate 3 had a synergistic effect with streptomycin and gentamicin. In addition, their anti-MRSA potency was evaluated and determined by the Caenorhabditis elegans model.

Si-B Functional Group Exchange Reaction Enabled by a Catalytic Amount of BH3: Scope, Mechanism, and Application.

Functional group exchanges based on single-bond transformation are rare and challenging. In this regard, functional group exchange reactions of hydrosilanes proved to be more problematic. This is because this exchange requires the cleavage of the C-Si bond, while the Si-H bond is relatively easily activated for hydrosilanes. Herein, we report the first Si-B functional group exchange reactions of hydrosilanes with hydroboranes simply enabled by BH3 as a catalyst. Our methodology works for various aryl and alkyl hydrosilanes and different hydroboranes with the tolerance of general functional groups (up to 115 examples). Control experiments and density functional theory (DFT) studies reveal a distinct reaction pathway that involves consecutive C-Si/B-H and C-B/B-H σ-bond metathesis. Further investigations of using more readily available chlorosilanes, siloxane, fluorosilane, and silylborane for Si-B functional group exchanges, Ge-B functional group exchanges, and depolymerizative Si-B exchanges of polysilanes are also demonstrated. Moreover, the regeneration of MeSiH3 from polymethylhydrosiloxane (PMHS) is achieved. Notably, the formal hydrosilylation of a wide range of alkenes with SiH4 and MeSiH3 to selectively produce (chiral)trihydrosilanes and (methyl)dihydrosilanes is realized using inexpensive and readily available PhSiH3 and PhSiH2Me as gaseous SiH4 and MeSiH3 surrogates.

Basic sensory properties of essential oils from aromatic plants and their applications: a critical review.

With higher standards in terms of diet and leisure enjoyment, spices and essential oils of aromatic plants (APEOs) are no longer confined to the food industry. The essential oils (EOs) produced from them are the active ingredients that contribute to different flavors. The multiple odor sensory properties and their taste characteristics of APEOs are responsible for their widespread use. The research on the flavor of APEOs is an evolving process attracting the attention among scientists in the past decades. For APEOs, which are used for a long time in the catering and leisure industries, it is necessary to analyze the components associated with the aromas and the tastes. It is important to identify the volatile components and assure quality of APEOs in order to expand their application. It is worth celebrating the different means by which the loss of flavor of APEOs can be retarded in practice. Unfortunately, relatively little research has been done on the structure and flavor mechanisms of APEOs. This also points the way to future research on APEOs.Therefore, this paper reviews the principles of flavor, identification of components and sensory pathways in humans for APEOs. Moreover, the article outlines the means of increasing the efficiency of using of APEOs. Finally, with respect to the sensory applications of APEOs, the review focuses on the practical application of APEOs in food sector and in aromatherapy.

The role of glomerular lesions in the prognosis of patients with acute kidney injury during hemorrhagic fever with renal syndrome.

OBJECTIVE: This study aimed to explore the role of glomerular lesions in patients who suffered from acute kidney injury (AKI) during hemorrhagic fever with renal syndrome (HFRS). METHODS: The study comprised 66 patients with AKI during HFRS treated at the National Clinical Research Center of Kidney Diseases of China, Jinling Hospital, from January 2014 to December 2018. According to the kidney pathological findings, the 66 patients were divided into two groups: the tubulointerstitial injury group (HFRS-TI group, n = 43) and the tubulointerstitial injury with glomerular lesions group (HFRS-GL group, n = 23). The clinical and pathological characteristics of the 66 patients were analyzed. RESULTS: There were 9 cases of IgA nephropathy, 1 case of membranous nephropathy, 2 cases of diabetic nephropathy, and 11 cases of mesangial proliferative glomerulonephritis in the HFRS-GL group. There were more males in the HFRS-GL group than in the HFRS-TI group (92.3% vs. 69.8%, p < .05). A higher proportion of interstitial fibrosis (56.5% vs. 27.9%, p < .05) and more immunoglobulin and complement depositions (p < .001) were observed in the HFRS-GL group than in the HFRS-TI group. Rates of remission of AKI were lower in the HFRS-GL group than in the HFRS-TI group (73.9% vs. 95.3%, p < .05). The presence of glomerular lesions (HR = 5.636, 95% CI = 1.121-28.329, p = .036) and moderate tubulointerstitial injury (HR = 3.598, 95% CI = 1.278-10.125, p = .015) were found to be independent risk factors for kidney prognosis. CONCLUSIONS: Patients with AKI during HFRS can have glomerular lesions or glomerulonephritis. Patients with AKI during HFRS who have glomerular lesions or moderate renal tubulointerstitial injury proven by kidney biopsy have a relatively poor kidney prognosis. A kidney biopsy can help determine long-term prognosis in patients with AKI during HFRS.

Phosphoproteome Profiling of uEVs Reveals p-AQP2 and p-GSK3ß as Potential Markers for Diabetic Nephropathy.

Diabetic nephropathy (DN) contributes to increased morbidity and mortality among patients with diabetes and presents a considerable global health challenge. However, reliable biomarkers of DN have not yet been established. Phosphorylated proteins are crucial for disease progression. However, their diagnostic potential remains unexplored. In this study, we used ultra-high-sensitivity quantitative phosphoproteomics to identify phosphoproteins in urinary extracellular vesicles (uEVs) as potential biomarkers of DN. We detected 233 phosphopeptides within the uEVs, with 47 phosphoproteins exhibiting significant alterations in patients with DN compared to those in patients with diabetes. From these phosphoproteins, we selected phosphorylated aquaporin-2 (p-AQP2[S256]) and phosphorylated glycogen synthase kinase-3ß (p-GSK3ß[Y216]) for validation, as they were significantly overrepresented in pathway analyses and previously implicated in DN pathogenesis. Both phosphoproteins were successfully confirmed through Phos-tag western blotting in uEVs and immunohistochemistry staining in kidney sections, suggesting that phosphoprotein alterations in uEVs reflect corresponding changes within the kidney and their potential as candidate biomarkers for DN. Our research proposes the utilization of phosphoproteins in uEVs as a liquid biopsy, presenting a highly feasible diagnostic tool for kidney disease.

CKAMP44 controls synaptic function and strength of relay neurons during early development of the dorsal lateral geniculate nucleus.

Relay neurons of the dorsal lateral geniculate nucleus (dLGN) receive inputs from retinal ganglion cells via retinogeniculate synapses. These connections undergo pruning in the first 2 weeks after eye opening. The remaining connections are strengthened several-fold by the insertion of AMPA receptors (AMPARs) into weak or silent synapses. In this study, we found that the AMPAR auxiliary subunit CKAMP44 is required for receptor insertion and function of retinogeniculate synapses during development. Genetic deletion of CKAMP44 resulted in decreased synaptic strength and a higher number of silent synapses in young (P9-11) mice. Recovery from desensitisation of AMPARs was faster in CKAMP44 knockout (CKAMP44-/- ) than in wild-type mice. Moreover, loss of CKAMP44 increased the probability of inducing plateau potentials, which are known to be important for eye-specific input segregation and retinogeniculate synapse maturation. The anatomy of relay neurons in the dLGN was changed in young CKAMP44-/- mice showing a transient increase in dendritic branching that normalised during later development (P26-33). Interestingly, input segregation in young CKAMP44-/- mice was not affected when compared to wild-type mice. These results demonstrate that CKAMP44 promotes maturation and modulates function of retinogeniculate synapses during early development of the visual system without affecting input segregation. KEY POINTS: Expression of CKAMP44 starts early during development of the dorsal lateral geniculate nucleus (dLGN) and remains stable in relay neurons and interneurons. Genetic deletion of CKAMP44 decreases synaptic strength and increases silent synapse number in dLGN relay neurons; increases the rate of recovery from desensitisation of AMPA receptors in dLGN relay neurons; and reduces synaptic short-term depression in retinogeniculate synapses. The probability of inducing plateau potentials is elevated in relay neurons of CKAMP44-/- mice. Eye-specific input segregation is unaffected in the dLGN of CKAMP44-/- mice. Deletion of CKAMP44 mildly affects dendritic arborisation of relay neurons in the dLGN.

CXCR3 antagonist AMG487 ameliorates experimental autoimmune prostatitis by diminishing Th1 cell differentiation and inhibiting macrophage M1 phenotypic activation.

BACKGROUND: Chronic prostatitis and chronic pelvic pain syndrome (CP/CPPS) is an inflammatory immune disease that is characterized by infiltrating inflammatory cells in the prostate and pelvic or by perineal pain. Receptor CXCR3modulates immune and inflammatory responses; however, the effects of CXCR3 antagonist AMG487 in the context of CP/CPPS are unknown. Therefore, we investigated the effect of AMG487 in experimental autoimmune prostatitis (EAP) mice and explored the potential functional mechanisms. METHODS: The EAP model was induced by intradermally injecting a mixture of prostate antigens and complete Freund's adjuvant on Days 0 and 28. To evaluate the effect of AMG487 on EAP mice, treatment with AMG487 and vehicle solution was conducted for the indicated period. Then, procedures were performed, including behavioral test, to evaluate the pain response to stimulation before the mice were killed and a histological assessment to evaluate the inflammation after the mice were killed. Immunofluorescence, flow cytometry, and Western blot assay were used to analyze the functional phenotype and regulation mechanism of AMG487 on T helper type 1 (Th1) cells and macrophages. RESULTS: We found high expression of CXCR3 in human benign prostate tissues with inflammation and EAP mice. The elevated CXCR3 in prostate tissues correlates with the severity of inflammation. CXCR3 antagonist AMG487 treatment ameliorated the inflammatory changes and the pelvic pain of EAP mice. AMG487 inhibits Th1 cell differentiation through the IL-12/STAT4pathway and inhibits pro-inflammatory M1 macrophages through the lipopolysaccharide/NF-κB p65signaling. AMG487 could inhibit the secretion of inflammatory mediators in EAP mice. CONCLUSION: CXCR3 antagonist AMG487 could ameliorate the inflammatory changes and the pelvic pain of EAP mice by diminishing Th1 cell differentiation and inhibiting macrophage M1 phenotypic activation. Thus, the results imply that AMG487 has the potential as an effective therapeutic agent in the prevention and treatment of EAP.

Identification and validation of an E2F-related gene signature for predicting recurrence-free survival in human prostate cancer.

BACKGROUND: It is well-established that biochemical recurrence is detrimental to prostate cancer (PCa). In the present study, we explored the mechanisms underlying PCa progression. METHODS: Five cohorts from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus databases were used to perform gene set variation analysis (GSVA) between nonrecurrent and recurrent PCa patients. We obtained the intersection of pathway enrichment results and extracted the corresponding gene list. LASSO Cox regression analysis was used to identify recurrence-free survival (RFS)-related significant genes and establish an RFS prediction gene signature and nomogram. MTT and colony formation assays were conducted to validate our findings. RESULTS: The E2F signaling pathway was activated in recurrent PCa patients compared to nonrecurrent patients. We established an E2F-related gene signature for RFS prediction based on the four identified E2F-related genes (CDKN2C, CDKN3, RACGAP1, and RRM2) using LASSO Cox regression in the Memorial Sloan Kettering Cancer Center (MSKCC) cohort. The risk score of each patient in MSKCC was calculated based on the expression levels of CDKN2C, CDKN3, RACGAP1, and RRM2. PCa patients with low-risk scores exhibited higher RFS than those with high-risk scores. Receiver operating characteristic (ROC) curve analysis validated the good performance and prognostic accuracy of the E2F-related gene signature, which was validated in the TCGA-prostate adenocarcinoma (TCGA-PRAD) cohort. Compared to patients with low Gleason scores and early T stages, PCa patients with high Gleason scores and advanced T stages had high-risk scores. Moreover, the E2F-related gene signature-based nomogram yielded good performance in RFS prediction. Functional experiments further confirmed these results. CONCLUSIONS: The E2F signaling pathway is associated with biochemical recurrence in PCa. Our established E2F-related gene signature and nomogram yielded good accuracy in predicting the biochemical recurrence in PCa.

Advances in efficient extraction of essential oils from spices and its application in food industry: A critical review.

With the increase of people's awareness of food safety, it is crucial to find natural and green antimicrobial agents to replace traditional antimicrobial agents. Essential oils of spices (SEOs) are low toxicity or nontoxic, which exhibited antioxidants and antimicrobial activity according to many in vitro and in situ experiments. Spices are widely available and low cost as a plant raw material for the extraction of SEOs. This review summarized highly efficient extraction techniques for SEOs, such as physical field assisted extraction technology, supercritical fluid extraction, and biological-based techniques. Furthermore, purification of SEOs and components were also recapitulated. Purification techniques of SEOs improve their utilization value due to the increased content of bioactive components. Finally, the review concentrated on the applications of SEOs in food industry, including food preservation, food active packaging by means of films or coatings, antioxidant properties. In addition, addressing the problem of unstability of SEOs and its role to inhibit the pathogenic bacteria, the encapsulation of SEOs for use in the food industrial sectors reduces the safety risk to human health.

Erythropoietin and iron for anemia in HIV-infected patients undergoing maintenance hemodialysis in China: a cross-sectional study.

BACKGROUND: Anemia is a common complication of chronic kidney disease (CKD) and HIV infection. The number of people living with HIV on hemodialysis (HD) is increasing. However, there is no data about anemia and related therapies in this kind of patients in China. We aim to assess the difference in hemoglobin (Hgb) and treatments like erythropoietin and iron between HIV-HD patients and HD patients in Chengdu, China. METHODS: This cross-sectional study was conducted with data collection from January 2020 to June 2020. Thirty-four HIV-infected HD patients and thirty-five non-HIV-infected HD patients were included. Age, gender, dialysis vintage, single-pool (sp) Kt/V, Hgb, the dose of erythropoietin, ferritin, use of iron preparations, and serum albumin were collected in all patients. Time since HIV diagnosis, counts of CD4 + T cells, HIV RNA, and antiretroviral therapy for HIV infection were collected in HIV-infected patients. T-test, Mann-Whitney U test, and chi-square statistics were applied in SPSS. RESULTS: The Hgb of HIV-HD and HD groups were 105.70 (95.93-112.08) g/L and 112.00 (93.00-126.00) g/L respectively (P = 0.064). There was a statistically significant higher erythropoietin dosage used in the HIV-HD population (222.55 ± 115.47 U/kg/week) compared to the HIV-negative HD group (161.86 ± 110.31 U/kg/week) (P = 0.029). 16/34 (47.06%) HIV-HD patients and 5/35 (14.29%) HD patients were treated with iron preparations (P = 0.003). The ferritin levels were 316.50 (117.38-589.75) ng/ml and 272.70 (205.00-434.00) ng/ml in HIV-HD and HD groups respectively. CONCLUSIONS: A higher erythropoietin dosage and a higher probability of iron preparations may be required to maintain Hgb in HIV-HD patients compared with HD patients.

Identification of sitagliptin binding proteins by affinity purification mass spectrometry.

Type 2 diabetes mellitus (T2DM) is recognized as a serious public health concern with increasing incidence. The dipeptidyl peptidase-4 (DPP-4) inhibitor sitagliptin has been used for the treatment of T2DM worldwide. Although sitagliptin has excellent therapeutic outcome, adverse effects are observed. In addition, previous studies have suggested that sitagliptin may have pleiotropic effects other than treating T2DM. These pieces of evidence point to the importance of further investigation of the molecular mechanisms of sitagliptin, starting from the identification of sitagliptin-binding proteins. In this study, by combining affinity purification mass spectrometry (AP-MS) and stable isotope labeling by amino acids in cell culture (SILAC), we discover seven high-confidence targets that can interact with sitagliptin. Surface plasmon resonance (SPR) assay confirms the binding of sitagliptin to three proteins, i. e., LYPLAL1, TCP1, and CCAR2, with binding affinities (K D) ranging from 50.1 µM to 1490 µM. Molecular docking followed by molecular dynamic (MD) simulation reveals hydrogen binding between sitagliptin and the catalytic triad of LYPLAL1, and also between sitagliptin and the P-loop of ATP-binding pocket of TCP1. Molecular mechanics Poisson-Boltzmann Surface Area (MMPBSA) analysis indicates that sitagliptin can stably bind to LYPLAL1 and TCP1 in active sites, which may have an impact on the functions of these proteins. SPR analysis validates the binding affinity of sitagliptin to TCP1 mutant D88A is ~10 times lower than that to the wild-type TCP1. Our findings provide insights into the sitagliptin-targets interplay and demonstrate the potential of sitagliptin in regulating gluconeogenesis and in anti-tumor drug development.

Phase Separation of MAGI2-Mediated Complex Underlies Formation of Slit Diaphragm Complex in Glomerular Filtration Barrier.

BACKGROUND: Slit diaphragm is a specialized adhesion junction between the opposing podocytes, establishing the final filtration barrier to urinary protein loss. At the cytoplasmic insertion site of each slit diaphragm there is an electron-dense and protein-rich cellular compartment that is essential for slit diaphragm integrity and signal transduction. Mutations in genes that encode components of this membrane-less compartment have been associated with glomerular diseases. However, the molecular mechanism governing formation of compartmentalized slit diaphragm assembly remains elusive. METHODS: We systematically investigated the interactions between key components at slit diaphragm, such as MAGI2, Dendrin, and CD2AP, through a combination of biochemical, biophysical, and cell biologic approaches. RESULTS: We demonstrated that MAGI2, a unique MAGUK family scaffold protein at slit diaphragm, can autonomously undergo liquid-liquid phase separation. Multivalent interactions among the MAGI2-Dendrin-CD2AP complex drive the formation of the highly dense slit diaphragm condensates at physiologic conditions. The reconstituted slit diaphragm condensates can effectively recruit Nephrin. A nephrotic syndrome-associated mutation of MAGI2 interfered with formation of the slit diaphragm condensates, thus leading to impaired enrichment of Nephrin. CONCLUSIONS: Key components at slit diaphragm (e.g., MAGI2 and its complex) can spontaneously undergo phase separation. The reconstituted slit diaphragm condensates can be enriched in adhesion molecules and cytoskeletal adaptor proteins. Therefore, the electron-dense slit diaphragm assembly might form via phase separation of core components of the slit diaphragm in podocytes.

Effects of miR-599 targeting YAP1 on proliferation, invasion and apoptosis of bladder urothelial carcinoma cells.

The aim of this study was to investigate the effects of microRNA (miR)-599 targeting Yes-associated protein 1 (YAP1) on the proliferation, invasion and apoptosis of bladder urothelial carcinoma cell line J82. J82 cells cultured in vitro were divided into miR-nc group (transfected with analog negative control miR-NC), miR-599 group (transfected with miR-599 analog), miR-599 + pcDNA3.1 group (co transfected with miR-599 analog and pcDNA3.1 empty vector), miR-599 + pcDNA3.1-YAP1 group (co transfected with miR-599 analog and pcDNA3.1-YAP1 over expression vector), the targeting relationship between miR-599 and YAP1 was detected by double luciferase reporter gene assay, the expression level of miR-599 was detected by real-time fluorescence quantitative PCR, the expression of YAP1 was detected by Western blot, cell proliferation was detected by MTT method, cell invasion was detected by Transwell's cell experiment, and apoptosis was detected by flow cytometry. The miR-599 was able to bind to YAP1 targetingly; compared with those in miR-NC group, miR-599 expression level and apoptosis rate in miR-599 group were significantly higher, however, the expression level of YAP1 protein and the abilities of cell proliferation and invasion were significantly lower (P < 0.05); there was no significant difference in the above indexes between miR-599 group and miR-599 + pcDNA3.1 group (P > 0.05); compared with those in miR-599 + pcDNA3.1 group, the expression level of YAP1 protein and the abilities of cell proliferation and invasion in miR-599 + pcDNA3.1-YAP1 group were significantly higher, while the apoptotic rate was significantly lower (P < 0.05). MiR-599 may inhibit the proliferation, invasion and promote apoptosis of bladder urothelial carcinoma J82 cells by targetingly regulating YAP1.

Radiation-free digital cholangioscopy-guided laser lithotripsy for large common bile duct stones: feasibility and technical notes.

AIMS: Although endoscopic retrograde cholangiopancreatography (ERCP) for large common bile duct (CBD) stones is facilitated by digital cholangioscopy-guided lithotripsy, it is performed by fluoroscopy guidance. Here, we report our experience of non-radiation ERCP for large CBD stones using digital cholangioscopy-guided laser lithotripsy. METHODS: Sixteen patients with large CBD stones underwent non-radiation digital cholangioscopy-guided laser lithotripsy and lithotomy. Data relevant to procedure details, adverse events, and short-term follow-up were analyzed. RESULTS: Biliary access was achieved in all patients using standard guidewire-assisted cannulation, double-guidewire technique, and transpancreatic precut in twelve, two, and two patients, respectively. Balloons of 10 mm, 8 mm, and 6 mm in diameter were applied for EPBD in 8, 2, and 6 patients, respectively. Complete stone removal in one session was achieved in all patients. One round of laser lithotripsy was needed for stone ≤ 25 mm, and three-to-five rounds were needed for stones > 25 mm or multiple stones. One or two clips were used for endoscopic clipping. The time lengths of biliary access, digital cholangioscopy-assisted laser lithotripsy and stone extraction, and whole procedure were 3.5 ± 3.2 (0.5-12) minutes, 52.5 ± 30.6 (45-97) minutes, and 76 ± 23.3 (58-106) minutes, respectively. Asymptomatic hyperleukocytose, hyperamylasemia, and mild pancreatitis were present in 1, 2, and 1 patient(s), respectively. No other complications occurred. No cholangitis or recurrent CBD stones were observed. CONCLUSION: Non-radiation digital cholangioscopy-guided laser lithotripsy is technically feasible and can be safely performed for endoscopic management of large CBD stones.

Isoliquiritigenin attenuates septic acute kidney injury by regulating ferritinophagy-mediated ferroptosis.

Defined differently from apoptosis, necrosis, and autophagy, ferroptosis has been implicated in acute kidney injury (AKI) such as ischemia-reperfusion injury induced AKI, folic acid caused AKI and cisplatin induced AKI. However, whether ferroptosis is involved in LPS induced AKI could be remaining unclear and there is still a lack of therapies associated with ferroptosis in LPS induced AKI without side effects. This study aimed to elucidate the role of isoliquiritigenin (ISL) in ferroptosis of LPS-induced AKI. We used LPS to induce renal tubular injury, followed by treatment with ISL both in vitro and in vivo. Human renal tubular HK2 cells were pretreated with 50 µM or 100 µM ISL for 5 h before stimulation with 2 µg/mL LPS. Mice were administered a single dose of either 50 mg/kg ISL orally or 5 mg/kg ferroptosis inhibitor ferrostatin-1 intraperitoneally before 10 mg/kg LPS injection. We found that LPS could induce mitochondria injury of renal tubular presented as the shape of mitochondria appeared smaller than normal with increased membrane density and are faction or destruction of mitochondrial crista through scanning electron microscope. Ferrostatin-1 significantly protected mice against renal dysfunction and renal tubular damage in LPS-induced AKI. ISL inhibited Fe2+ and lipid peroxidation accumulation in LPS-stimulated HK2 cells. It also increased the expression of GPX4 and xCT, reduced the expression of HMGB1 and NCOA4 then attenuated mitochondria injury in renal tubular following LPS stimulation. These results indicated the potential role of ISL against ferritinophagy-mediated ferroptosis in renal tubular following LPS stimulation.