SENP1 protects cisplatin-induced AKI by attenuating apoptosis through regulation of HIF-1α.

BACKGROUND: Acute kidney injury is a clinical syndrome with both high morbidity and mortality. However, the underlying molecular mechanism of AKI is still largely unknown. The role of SENP1 in AKI is unclear, while one of its substrates, HIF-1α possesses nephroprotective effect in AKI. Herein, this study aimed to reveal the role of SENP1/HIF-1α axis in AKI by using both cell and animal models. METHODS: We investigated the effects of AKI on SENP1 expression using clinical samples, and cisplatin-induced AKI model based on mice or HK-2 cells. The influence of SENP1 knockdown or over-expression on cisplatin-induced AKI was studied in vitro and in vivo. Following the exploration of the change in HIF-1α expression brought by AKI, the synergistic effects of SENP1 knockdown and HIF-1α over-expression on AKI were examined. RESULTS: The results showed the up-regulation of SENP1 in clinical specimens, as well as cell and animal models. The knockdown or over-expression of SENP1 in HK-2 cells could promote or inhibit AKI through regulating cell apoptosis, respectively. Moreover, SENP1+/- mice suffered from much more serious AKI compared with mice in wild type group. Furthermore, we found that HIF-1α over-expression could attenuate the promoted cell apoptosis as well as AKI induced by SENP1 knockdown. CONCLUSIONS: we showed that SENP1 provided protection for kidney in AKI via regulating cell apoptosis and through the regulation of HIF-1α. This study could benefit for the understanding of the pathogenesis of AKI and provide potential therapeutic target for AKI treatment.

PKD2 gene variants in Chinese patients with autosomal dominant polycystic kidney disease.

PKD2 gene variants account for 4.5% to 20% of patients with autosomal dominant polycystic kidney disease (ADPKD). Little is known about the clinical characteristics of PKD2 variants in Chinese patients with ADPKD. Herein, we performed a comprehensive search for variants of PKD2 gene in 44 Chinese ADPKD pedigrees and a total of 37 variants were identified. Of these 37 variants, 18 were nonsense variants, 10 frameshift variants, 4 missense variants, and 5 splice site variants. 11/37 variants were detected for the first time. The median age at diagnosis was 30.5 years, and positive family history was detected in 77.27% patients, liver cysts in 68.18%, hypertension in 45.45%, nephrolithiasis in 31.82%, macro-hematuria in 22.73%, and proteinuria in 13.63%. The level of estimated glomerular filtration rate in 8/39 patients were blow 60 ml/min/1.73m2 . 11/17 patients were classified as rapid progression by Mayo Clinic classification. The end stage renal disease (ESRD) events were reported in 9/22 pedigrees, and the presence of nephrolithiasis and macro-hematuria were significantly associated with ESRD in the pedigrees with PKD2 variants. The identified variants and clinical features will facilitate the early diagnosis and prognosis prediction in Chinese ADPKD patients with PKD2 variants.

Functional characterization of a PROTAC directed against BRAF mutant V600E.

The RAF family kinases function in the RAS-ERK pathway to transmit signals from activated RAS to the downstream kinases MEK and ERK. This pathway regulates cell proliferation, differentiation and survival, enabling mutations in RAS and RAF to act as potent drivers of human cancers. Drugs targeting the prevalent oncogenic mutant BRAF(V600E) have shown great efficacy in the clinic, but long-term effectiveness is limited by resistance mechanisms that often exploit the dimerization-dependent process by which RAF kinases are activated. Here, we investigated a proteolysis-targeting chimera (PROTAC) approach to BRAF inhibition. The most effective PROTAC, termed P4B, displayed superior specificity and inhibitory properties relative to non-PROTAC controls in BRAF(V600E) cell lines. In addition, P4B displayed utility in cell lines harboring alternative BRAF mutations that impart resistance to conventional BRAF inhibitors. This work provides a proof of concept for a substitute to conventional chemical inhibition to therapeutically constrain oncogenic BRAF.

[A retrospective study on risk factors for prognosis of children with sepsis].

OBJECTIVE: To explore the risk factors that may affect the prognosis of sepsis in children. METHODS: A retrospective study was conducted. Septic children who aged 28 days to 18 years old admitted to the department of critical care medicine of Xi'an Children's Hospital from January 2018 to May 2019 were enrolled. The general demographic data and clinical indicators within 8 hours after admission were collected, and the 28-day mortality was the end point. The differences of the indexes between the survival and the dead children were compared, and the risk factors of prognosis by binary Logistic regression analysis were analyzed. The predictive value of related risk factors on the prognosis was verified by receiver operating characteristic (ROC) curve. RESULTS: A total of 165 children with sepsis were selected, 75 died in 28-days with a 28-day mortality of 45.45%. Compared with the survival group, the alanine aminotransferase (ALT), aspertate aminotransferase (AST), serum creatinine (SCr), creatine kinase isoenzyme (CK-MB), lactate (Lac) and procalcitonin (PCT) in the dead group significantly increased [ALT (U/L): 404.99±88.26 vs. 181.64±35.17, AST (U/L): 453.37±35.37 vs. 210.69±92.50, SCr (µmol/L): 156.46±105.65 vs. 54.32±25.46, CK-MB (U/L): 244.86±53.68 vs. 97.29±19.11, Lac (mmol/L): 5.55±1.66 vs. 2.49±1.29, PCT (ng/L): 35.55±15.87 vs. 18.66±4.91, all P < 0.01], while platelet count (PLT), serum ionized calcium concentration (iCa2+) significantly decreased [PLT (×109/L): 81.49±29.53 vs. 165.43±97.81, iCa2+ (mmol/L): 0.89±0.16 vs. 1.84±0.14, both P < 0.01]. There was no significant difference in gender, age, prothrombin time (PT), activated partial thromboplastin time (APTT), albumin (ALB) and blood urea nitrogen (BUN) between the two groups. Binary Logistic regression analysis showed that Lac, SCr and PCT were the risk factors for the prognosis of children with sepsis [odds ratio (OR) and 95% confidence interval (95%CI) were 2.18 (1.22-3.09), 2.01 (1.00-2.07), 1.14 (1.07-1.21), respectively, all P < 0.01], while PLT and iCa2+ were protective factors [OR and 95%CI were 0.95 (0.93-0.98), 0.32 (0.12-0.42), respectively, both P < 0.01]. Further ROC curve analysis showed that Lac, PCT and SCr were all of predictive value for the prognosis of children with sepsis, and the area under ROC curve (AUC) was 0.881, 0.864, 0.711, respectively (all P < 0.01); the combined predicted AUC of Lac, SCr, PCT, PLT and iCa2+ influencing factors was as high as 0.981, with the sensitivity of 97.6%, and the specificity of 98.7%. CONCLUSIONS: Lac, PCT and SCr are independent risk factors for the prognosis of children with sepsis.

SOCS3 protects against neonatal necrotizing enterocolitis via suppressing NLRP3 and AIM2 inflammasome activation and p65 nuclear translocation.

BACKGROUND: Necrotizing enterocolitis (NEC) is an acquired disorder of mucosal damage characterized by the diffuse or local necrosis of the intestine. The suppressor of cytokine signaling 3 (SOCS3) has been demonstrated to possess anti-inflammatory action in gastritis, ulcerative colitis and other inflammatory diseases. The present study aims to explore the effects of SOCS3 on LPS-induced colonic cell model of NEC, and investigate the underlying mechanisms. METHODS: Expression of SOCS3 in tissue samples of NEC and LPS-induced enterocytes were evaluated by real-time quantitative PCR (RT-qPCR). Western blotting and enzyme-linked immunosorbent assay (ELISA) were applied to examine the effect of SOCS3 on inflammatory molecules. Co-immunoprecipitation assay were devoted to explore the relation between SOCS3 and TLR4. RESULTS: We proved that SOCS3 was expressed at a low level in tissue samples of NEC and LPS-induced enterocytes, and LPS inhibited SOCS3 expression via JAK2/STAT3 pathway. Overexpression of SOCS3 weaken the LPS-induced inflammatory response in FHC and CACO2 cells. Moreover, SOCS3 downregulates proinflammatory cytokines by targeting TLR4, thus mediating the p65 nuclear translocation, and the activation of NLR family pyrin domain containing 3/absent in melanoma-2 (NLRP3/AIM2) inflammasome, ultimately reveals its anti-inflammatory effects. CONCLUSIONS: Taken together, our data revealed that LPS inhibited SOCS3 expression via JAK2/STAT3 pathway, and SOCS3 protects enterocytes against NEC through mediating p65 nuclear translocation and NLRP3/AIM2 inflammasome activation in a TLR4 dependent manner.

Acute kidney injury burden in different clinical units: Data from nationwide survey in China.

BACKGROUND: The inpatient morbidity and mortality of acute kidney injury (AKI) vary considerably in different clinical units, yet studies to compare the difference remain limited. METHODS: We compared the clinical characteristics of AKI in Intensive Care Unit (ICU), medical and surgical departments by using the data derived from the 2013 nationwide cross-sectional survey of AKI in China to capture variations among different clinical departments in recognition, management, and outcomes of AKI. Suspected AKI patients were identified based on changes in serum creatinine during hospitalization, and confirmed by reviewing medical records. RESULTS: The detection rate of AKI was the highest in ICU (22.46%), followed by the rates in medical (1.96%) and surgical departments (0.96%). However, the absolute number of cases was the largest in medical departments, which contributed to 50% of the cases. In medical departments, 78% of AKI cases were extensively distributed in cardiac, nephrology, oncology, gastroenterology, pneumology and neurology departments. In contrast, 87% of AKI cases in surgical departments were mainly from urology, general surgery and cardiothoracic departments. The in-time recognition rates were extremely low in all departments except nephrology. Only 10.5~15.0% AKI patients from non-nephrology departments received renal referral. Among all the death cases, 50% and 39% came from ICU and medical departments while only 11% from surgical departments. Older age, higher AKI stage and renal replacement therapy indication were identified as risk factors for high mortality in all departments. Delayed recognition and no renal referral were significantly associated with increased mortality in medical and ICU patients. CONCLUSIONS: These findings suggest that ICU and medical departments are major affected departments in China with a large number of AKI cases and subsequent high mortality. The reality is more alarming considering the low awareness of AKI and the paucity of effective interventions in the high-risk patients in these departments.

Methylation of the Constitutive Androstane Receptor Is Involved in the Suppression of CYP2C19 in Hepatitis B Virus-Associated Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC), one of the most dangerous malignancies with an increasing incidence and a high mortality rate, represents a major international health problem. HCC progression is known to involve genome-wide alteration of epigenetic modifications, leading to aberrant gene expression patterns. The activity of CYP2C19, an important member of the cytochrome P450 superfamily, was reported to be compromised in HCC, but the underlying mechanism remains unclear. To understand whether epigenetic modification in HCC is associated with a change in CYP2C19 activity, we evaluated the expression levels of CYP2C19 and its transcription factors by quantitative real-time polymerase chain reaction using mRNA extracted from both primary hepatocytes and paired tumor versus nontumor liver tissues of patients infected with hepatitis B virus (HBV). DNA methylation was examined by bisulfite sequencing and methylation-specific polymerase chain reaction. Our results indicated that CYP2C19 could be regulated by e-box methylation of the constitutive androstane receptor (CAR). Decreased CYP2C19 expression in tumorous tissues of HBV-infected patients with HCC was highly correlated with suppressed expression and promoter hypermethylation of CAR. Our study demonstrates that aberrant CAR methylation is involved in CYP2C19 regulation in HBV-related HCC and may play a role in liver tumorigenesis.

A Tumor-Specific Neo-Antigen Caused by a Frameshift Mutation in BAP1 Is a Potential Personalized Biomarker in Malignant Peritoneal Mesothelioma.

Malignant peritoneal mesothelioma (MPM) is an aggressive rare malignancy associated with asbestos exposure. A better understanding of the molecular pathogenesis of MPM will help develop a targeted therapy strategy. Oncogene targeted depth sequencing was performed on a tumor sample and paired peripheral blood DNA from a patient with malignant mesothelioma of the peritoneum. Four somatic base-substitutions in NOTCH2, NSD1, PDE4DIP, and ATP10B and 1 insert frameshift mutation in BAP1 were validated by the Sanger method at the transcriptional level. A 13-amino acids neo-peptide of the truncated Bap1 protein, which was produced as a result of this novel frameshift mutation, was predicted to be presented by this patient's HLA-B protein. The polyclonal antibody of the synthesized 13-mer neo-peptide was produced in rabbits. Western blotting results showed a good antibody-neoantigen specificity, and Immunohistochemistry (IHC) staining with the antibody of the neo-peptide clearly differentiated neoplastic cells from normal cells. A search of the Catalogue of Somatic Mutations in Cancer (COSMIC) database also revealed that 53.2% of mutations in BAP1 were frameshift indels with neo-peptide formation. An identified tumor-specific neo-antigen could be the potential molecular biomarker for personalized diagnosis to precisely subtype rare malignancies such as MPM.

Fugitive methane emissions from leak-prone natural gas distribution infrastructure in urban environments.

Fugitive emissions from natural gas systems are the largest anthropogenic source of the greenhouse gas methane (CH4) in the U.S. and contribute to the risk of explosions in urban environments. Here, we report on a survey of CH4 emissions from 100 natural gas leaks in cast iron distribution mains in Metro Boston, MA. Direct measures of CH4 flux from individual leaks ranged from 4.0 - 2.3 × 10(4) g CH4•day(-1). The distribution of leak size is positively skewed, with 7% of leaks contributing 50% of total CH4 emissions measured. We identify parallels in the skewed distribution of leak size found in downstream systems with midstream and upstream stages of the gas process chain. Fixing 'superemitter' leaks will disproportionately stem greenhouse gas emissions. Fifteen percent of leaks surveyed qualified as potentially explosive (Grade 1), and we found no difference in CH4 flux between Grade 1 leaks and all remaining leaks surveyed (p = 0.24). All leaks must be addressed, as even small leaks cannot be disregarded as 'safely leaking.' Key methodological impediments to quantifying and addressing the impacts of leaking natural gas distribution infrastructure involve inconsistencies in the manner in which gas leaks are defined, detected, and classified. To address this need, we propose a two-part leak classification system that reflects both the safety and climatic impacts of natural gas leaks.

Inhibition of SCF ubiquitin ligases by engineered ubiquitin variants that target the Cul1 binding site on the Skp1-F-box interface.

Skp1-Cul1-F-box (SCF) E3 ligases play key roles in multiple cellular processes through ubiquitination and subsequent degradation of substrate proteins. Although Skp1 and Cul1 are invariant components of all SCF complexes, the 69 different human F-box proteins are variable substrate binding modules that determine specificity. SCF E3 ligases are activated in many cancers and inhibitors could have therapeutic potential. Here, we used phage display to develop specific ubiquitin-based inhibitors against two F-box proteins, Fbw7 and Fbw11. Unexpectedly, the ubiquitin variants bind at the interface of Skp1 and F-box proteins and inhibit ligase activity by preventing Cul1 binding to the same surface. Using structure-based design and phage display, we modified the initial inhibitors to generate broad-spectrum inhibitors that targeted many SCF ligases, or conversely, a highly specific inhibitor that discriminated between even the close homologs Fbw11 and Fbw1. We propose that most F-box proteins can be targeted by this approach for basic research and for potential cancer therapies.

Epigenetic Regulation of Cytochrome P450 Enzymes and Clinical Implication.

CYPs are a large and diverse group of drug-metabolizing enzymes, which govern the metabolism of the majority of xenobiotic substances as well as endogenous components. The high inter-subject variability of CYP bioactivity has been largely attributed to gene polymorphism until the rapid development in epigenetics in the last decade that revealed another aspect of regulatory mechanism of drug-related genes. Epigenetics is the study of changes in gene expression or cellular phenotype that are not caused by changes in the underlying DNA sequence. The modification of histone proteins, together with DNA methylation and miRNAs, is the most extensively studied epigenetic mechanism in mammals. Recently, it has been demonstrated that alterations in epigenetic regulation occur during multiple pathological processes, especially carcinogenesis. As CYPs play an important role in carcinogen and anti-cancer drug biotransformation, epigenetic changes in CYP genes would lead to interindividual differences in drug responses. In this review, we provide an up-to-date summary of epigenetic studies on human CYPs, and discuss how such information could be integrated with clinical application.

Distinguishing characteristics of idiopathic calcium oxalate kidney stone formers with low amounts of Randall's plaque.

BACKGROUND: Overgrowth of calcium oxalate on Randall's plaque is a mechanism of stone formation among idiopathic calcium oxalate stone-formers (ICSFs). It is less clear how stones form when there is little or no plaque. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: Participants were a consecutive cohort of ICSFs who underwent percutaneous nephroscopic papillary mapping in the kidney or kidneys containing symptomatic stones and a papillary tip biopsy from a representative calyx during a stone removal procedure between 2009 and 2013. The distribution of Randall's plaque coverage was analyzed and used to divide ICSFs into those with a high (≥5%; mean, 10.5%; n=10) versus low (<5%; mean, 1.5%; n=32) amount of plaque coverage per papilla. Demographic and laboratory features were compared between these two groups. RESULTS: Low-plaque stone formers tended to be obese (50% versus 10%; P=0.03) and have a history of urinary tract infection (34% versus 0%; P=0.04). They were less likely to have multiple prior stone events (22% versus 80%; P=0.002) and had a lower mean 24-hour urine calcium excretion (187±86 mg versus 291±99 mg; P<0.01). Morphologically, stones from patients with low amounts of plaque lacked a calcium phosphate core by microcomputed tomography. Papillary biopsies from low plaque stone-formers revealed less interstitial and basement membrane punctate crystallization. CONCLUSIONS: These findings suggest that other pathways independent of Randall's plaque may contribute to stone pathogenesis among a subgroup of ICSFs who harbor low amounts of plaque.

[Water extract from Codonopsis thalictrifolia wall affects the reproductive system of male infant rats].

OBJECTIVE: To study the impact of the water extract from Codonopsis thalictrifolia Wall (CTW) on the reproductive METHODS: We divided 32 male SD infant rats into four groups of equal number to be treated intragastrical-system of male infant rats. ly with distilled water (control) and CTW at 10 g/kg (low dose) , 20 g/kg (medium dose), and 40 g/kg (high dose), respectively, twice a day for 2 weeks. Then we killed the rats, measured the levels of testosterone (T), luteinizing hormone (LH) and follicle-stimulating hormone (FSH) in the serum, obtained the testis weight, body weight, testis visceral coefficient and sperm concentration, and detected sperm viability, sperm motility and the level of cyclic adenosine monophosphate (cAMP) in the Leydig cells, followed by RESULTS: Compared with the control group, the low-dose, me-analysis of differences among different groups using the SPSS software. Medium-dose and high-dose CTW groups showed significant decreases in the serum T level ([3.09 +/-0.42] vs [1.22 +/-0. 32] , [1.06 +/- 0.29] and [0.57 +/-0.18] nmol/L, P<0.01), testis weight ([1.40 +/-0.16] vs [0.96 +/-0.09], [0.92 +/-0.11] and [0.91 +/- 0.08] g, P <0.01), and sperm concentration ([1.03 +/-0.16] vs [0.19 +/-0.07], [0.17 +/-0.08] and [0.16 +/-0.07] x 10(6)/ml, P <0.01), but a dramatic elevation in the testis visceral coefficient ([42.22 +/- 3.02] vs [51.39 +/- 3.09], [52.28 +/- 4.86] and [54.13 +/-6.06] mg/10 g, P <0.01); the medium- and high-dose CTW groups exhibited remarkable increases in the levels of serum LH ([13.62+/-0.89] vs [14.69 +/-0.12] and [14.93 +/-0.28] ng/L, P<0.01) and FSH ([4.32 +/-0.18] vs [4.77 +/-0.23] and [4.89 +/-0. 38] IU/L, P <0.05); all the three CTW groups showed markedly inhibited serum T secretion ([1.85 +/- 0.18] vs [1.42 +/-0.15], [1.12+/-0.18] and [0.88 +/-0.21] nmol/L, P<0.01) and intracellular cAMP ([5.51 +/-0.12] vs [4.39+/-0.06], [4.28 +/-0.07] and [4.11 +/- 0.10] nmol/L, P <0.01) in the Leydig cells. CONCLUSION: The water extract from CTW may reduce the synthesis of testosterone in the serum of male infant rats through the PKA pathway and consequently inhibit their testicular development and sperm production and affect the development of their reproductive system.

[Detection of somatic mutations in deteriorated cell of peritoneal mesothelioma by whole genome sequencing].

OBJECTIVE: To detect the somatic mutations in peritoneal mesothelioma with whole genome sequencing technique. METHODS: Surgically resected cancer and pericancerous tissue samples from one patient with peritoneal mesothelioma were obtained. The whole genome sequences of tumor tissue and pericancerous tissue were examined by the second generation sequencing technique and compared with reference sequences from human genome database. RESULTS: There were 639 717 single nucleotide variations (Single Nucleotide Variation SNV) found in both tumor and pericancerous tissue cells; while 20 302 SNVs were unique for tumor cells and 2 185 SNVs unique for pericancerous tissue, but still 223 SNVs found in cancer and pericancerous tissue were differed from those in human genome database. CONCLUSION: The preliminary results indicate that merely comparing the gene sequences of cancer and pericancerous tissue samples in an individual with the human genome reference sequence can not accurately locate all somatic mutations in pathological cells. For those individualized diseases caused by random somatic mutations, it is suggested to sequence the whole genome at birth or at least to reserve a DNA sample at early age for both research and clinical needs.

Novel conjugation of tumor-necrosis-factor-related apoptosis-inducing ligand (TRAIL) with monomethyl auristatin E for efficient antitumor drug delivery.

Monomethyl auristatin E (MMAE) is conjugated with TNF-related apoptosis-inducing ligand (TRAIL) via a linker that is stable in extracellular fluid, while it is cleaved by cathepsin once the conjugate has entered a tumor cell, thus activating the antimitotic mechanism of MMAE. The TRAIL-MMAE conjugate is a conceptually viable therapeutic strategy with improved in vitro antitumor activity, cell circle arrest and specific accumulation in tumor to treat TRAIL-resistant tumors.

An allosteric inhibitor of the human Cdc34 ubiquitin-conjugating enzyme.

In the ubiquitin-proteasome system (UPS), E2 enzymes mediate the conjugation of ubiquitin to substrates and thereby control protein stability and interactions. The E2 enzyme hCdc34 catalyzes the ubiquitination of hundreds of proteins in conjunction with the cullin-RING (CRL) superfamily of E3 enzymes. We identified a small molecule termed CC0651 that selectively inhibits hCdc34. Structure determination revealed that CC0651 inserts into a cryptic binding pocket on hCdc34 distant from the catalytic site, causing subtle but wholesale displacement of E2 secondary structural elements. CC0651 analogs inhibited proliferation of human cancer cell lines and caused accumulation of the SCF(Skp2) substrate p27(Kip1). CC0651 does not affect hCdc34 interactions with E1 or E3 enzymes or the formation of the ubiquitin thioester but instead interferes with the discharge of ubiquitin to acceptor lysine residues. E2 enzymes are thus susceptible to noncatalytic site inhibition and may represent a viable class of drug target in the UPS.

CDK activity antagonizes Whi5, an inhibitor of G1/S transcription in yeast.

Cyclin-dependent kinase (CDK) activity initiates the eukaryotic cell division cycle by turning on a suite of gene expression in late G1 phase. In metazoans, CDK-dependent phosphorylation of the retinoblastoma tumor suppressor protein (Rb) alleviates repression of E2F and thereby activates G1/S transcription. However, in yeast, an analogous G1 phase target of CDK activity has remained elusive. Here we show that the cell size regulator Whi5 inhibits G1/S transcription and that this inhibition is relieved by CDK-mediated phosphorylation. Deletion of WHI5 bypasses the requirement for upstream activators of the G1/S transcription factors SBF/MBF and thereby accelerates the G1/S transition. Whi5 is recruited to G1/S promoter elements via its interaction with SBF/MBF in vivo and in vitro. In late G1 phase, CDK-dependent phosphorylation dissociates Whi5 from SBF and drives Whi5 out of the nucleus. Elimination of CDK activity at the end of mitosis allows Whi5 to reenter the nucleus to again repress G1/S transcription. These findings harmonize G1/S control in eukaryotes.