MdVQ17 negatively regulates apple resistance to Glomerella leaf spot by promoting MdWRKY17-mediated salicylic acid degradation and pectin lyase activity.

Glomerella leaf spot (GLS) is a fungal disease caused by Colletotrichum fructicola, which severely restricts the yield and quality of apples. Valine-glutamine (VQ) proteins are transcriptional regulators involved in the regulation of plant growth and stress responses. However, little is known about the role of VQ proteins in the biotic stress response in apple. Here, a VQ gene, MdVQ17, that was highly induced by C. fructicola infection was identified. Overexpression of MdVQ17 in apple increased susceptibility to C. fructicola and significantly reduced the salicylic acid content and ß-1,3-glucanase and chitinase activities. Based on yeast two-hybrid screening, MdWRKY17, which promotes susceptibility to C. fructicola, was identified as an MdVQ17-interacting protein. Co-expression of MdVQ17 can promote the binding and transcriptional activation activity of MdWRKY17 on the promoter of Downy Mildew Resistant 6 (MdDMR6), thereby promoting MdWRKY17-mediated salicylic acid degradation. Based on DNA affinity purification sequencing, the pectin lyase-encoding gene MdPL-like was identified as a direct target of MdWRKY17. MdWRKY17 can directly bind to the promoter of MdPL-like and activate its transcription, and the binding and activation of MdWRKY17 on the MdPL-like promoter were significantly enhanced by MdVQ17 co-expression. Functional identification showed that MdPL-like promoted pectin lyase activity and susceptibility to C. fructicola. In sum, these results demonstrate that the MdVQ17-MdWRKY17 module mediates the response to C. fructicola infection by regulating salicylic acid accumulation and pectin lyase activity. Our findings provide novel insights into the mechanisms by which the VQ-WRKY complex modulates plant pathogen defense responses.

Study on the mechanism of hepatotoxicity of Aucklandiae radix through liver metabolomics and network pharmacology.

Background: Aucklandiae Radix (CAR) and its roasted processed products (PAR) are extensively used in various Chinese patent medicines due to their diverse pharmacological activities. However, numerous side effects of CAR have been reported and the hepatotoxicity and the corresponding mechanisms have not been thoroughly investigated. Our study aims to explore the underlying mechanism of the hepatotoxic impacts of CAR. Methods: In this study, metabolomic analysis was performed using liver tissue from the mice administered with different dosages of CAR/PAR extracts to examine the hepatotoxic impacts of CAR and elucidate the underlying mechanism. Network pharmacology was employed to predict the potential molecular targets and associated signaling pathways based on the distinctive compounds between CAR and PAR. A composition-target-GO-Bio process-metabolic pathway network was constructed by integrating the hepatotoxicity-related metabolic pathways. Finally, the target proteins related with the hepatotoxic effect of CAR were identified and validated in vivo. Results: The metabolomics analysis revealed that 33 related metabolic pathways were significantly altered in the high-dose CAR group, four of which were associated with the hepatotoxicity and could be alleviated by PAR. The network identified NQO1 as the primary target of the hepatotoxic effect induced by CAR exposure, which was subsequently verified by Western Blotting. Further evidence in vivo demonstrated that Nrf2 and HO-1, closely related to NQO1, were also the main targets through which CAR induced the liver injury, and that oxidative stress should be the primary mechanism for the CAR-induced hepatotoxicity. Conclusions: This preliminary study on the hepatic toxic injury of CAR provides a theoretical basis for the rational and safe use of CAR rationally and safely in clinical settings.

A review of 10 patients treated with the masquelet technique and microsurgical technique combined for Gustilo type III open tibial fractures.

BACKGROUND: Open tibial fractures often include severe bone loss and soft tissue defects and requires complex reconstructive operations. However, the optimal treatment is unclear. METHODS: This retrospective study enrolled patients with Gustilo type III open tibial fractures from January 2018 to January 2021 to assess the clinical utility of Masquelet technique together with microsurgical technique as a combined strategy for the treatment of open tibial fractures. The demographics and clinical outcomes including bone union time, infection, nonunion and other complications were recorded for analysis. The bone recovery quality was evaluated by the AOFAS Ankle-Hindfoot Scale score and the Paley criteria. RESULTS: We enrolled 10 patients, the mean age of the patients and length of bone defects were 31.7 years (range, 23-45 years) and 7.5 cm (range, 4.5-10 cm) respectively. Bone union was achieved for all patients, with an average healing time of 12.2 months (range, 11-16 months). Seven patients exhibited a bone healing time of less than 12 months, whereas 3 patients exhibited a bone healing time exceeding 12 months. No significant correlation was found between the length of bone loss and healing time. In addition, no deep infection or nonunion was observed, although 2 patients experienced wound fat liquefaction with exudates and 1 patient presented with a bloated skin flap. The average AOFAS Ankle-Hindfoot Scale score was 80.5 (range, 74-85), and all patients were evaluated as good or exellent based on the Paley criteria. CONCLUSIONS: Our study indicated that the use of the Masquelet technique and the microsurgical technique as a combined strategy is safe and effective for the treatment of Gustilo type III open tibial fractures.

Circulating microRNA expression and nonalcoholic fatty liver disease in adolescents with severe obesity.

BACKGROUND: Nonalcoholic fatty liver disease (NAFLD) is one of the most common chronic liver diseases in children and adolescents. NAFLD ranges in severity from isolated hepatic steatosis to nonalcoholic steatohepatitis (NASH), wherein hepatocellular inflammation and/or fibrosis coexist with steatosis. Circulating microRNA (miRNA) levels have been suggested to be altered in NAFLD, but the extent to which miRNA are related to NAFLD features remains unknown. This analysis tested the hypothesis that plasma miRNAs are significantly associated with histological features of NAFLD in adolescents. AIM: To investigate the relationship between plasma miRNA expression and NAFLD features among adolescents with NAFLD. METHODS: This study included 81 adolescents diagnosed with NAFLD and 54 adolescents without NAFLD from the Teen-Longitudinal Assessment of Bariatric Surgery study. Intra-operative core liver biopsies were collected from participants and used to characterize histological features of NAFLD. Plasma samples were collected during surgery for miRNA profiling. A total of 843 plasma miRNAs were profiled using the HTG EdgeSeq platform. We examined associations of plasma miRNAs and NAFLD features using logistic regression after adjusting for age, sex, race, and other key covariates. Ingenuity Pathways Analysis was used to identify biological functions of miRNAs that were associated with multiple histological features of NAFLD. RESULTS: We identified 16 upregulated plasma miRNAs, including miR-193a-5p and miR-193b-5p, and 22 downregulated plasma miRNAs, including miR-1282 and miR-6734-5p, in adolescents with NAFLD. Moreover, 52, 16, 15, and 9 plasma miRNAs were associated with NASH, fibrosis, ballooning degeneration, and lobular inflammation, respectively. Collectively, 16 miRNAs were associated with two or more histological features of NAFLD. Among those miRNAs, miR-411-5p was downregulated in NASH, ballooning, and fibrosis, while miR-122-5p, miR-1343-5p, miR-193a-5p, miR-193b-5p, and miR-7845-5p were consistently and positively associated with all histological features of NAFLD. Pathway analysis revealed that most common pathways of miRNAs associated with multiple NAFLD features have been associated with tumor progression, while we also identified linkages between miR-122-5p and hepatitis C virus and between miR-199b-5p and chronic hepatitis B. CONCLUSION: Plasma miRNAs were associated with NAFLD features in adolescent with severe obesity. Larger studies with more heterogeneous NAFLD phenotypes are needed to evaluate miRNAs as potential biomarkers of NAFLD.

Ternary CdS@MoS2-Co3O4 Multiheterojunction Photocatalyst for Boosting Photocatalytic H2 Evolution.

Turning the carrier dynamics in heterojunction photocatalysts is a direct and effective strategy for improving the solar energy conversion efficiency of photocatalysts. Herein, we report a ternary CdS@MoS2-Co3O4 multiheterojunction photocatalyst consisting of the p-n junction of MoS2-Co3O4 and the type-I junction of CdS@MoS2, wherein MoS2 located at the frontier between CdS and Co3O4 acts as an intermediate bridge. The type-I junction allows the directional transfer of photoinduced charge from CdS to MoS2, suppressing the photocorrosion of CdS. Notably, the single-particle photoluminescence technique demonstrates the sequential one-direction hole transfer from MoS2 to Co3O4 aroused by the p-n junction, resulting in a long-lifetime charge separation in the carrier lifetime (54-58 ns). Compared to the bare CdS and type-I CdS@MoS2, the CdS@MoS2-Co3O4 photocatalyst affords a 347-fold and 3.5-fold enhancement of the H2 evolution rate, a quantum efficiency of 28.6% at 450 nm, and a 20 h of long-term stability. This work provides a new understanding of the rational regulation of the charge-transfer mechanism of type-I systems by constructing multiheterojunction photocatalysts.

Development of a prognostic nomogram for patients with malignant mesothelioma with bone metastasis.

Malignant mesothelioma (MM) is a rare aggressive tumor, and bone metastasis often occurs in later stages of this disease. This study aimed to establish a nomogram to predict the prognosis of bone metastasis of patients with MM. Data from the Surveillance, Epidemiology, and End Results database were screened and retrieved. This study included 311 patients with MM with bone metastases. Prognostic factors were analyzed using the Kaplan-Meier method and Cox proportional hazards model. A nomogram for overall survival (OS) was established and evaluated using statistically significant prognostic factors, and cancer-specific survival (CSS) analysis was performed to investigate its prognostic factors. In addition, the metastasis patterns of patients with MM were investigated, and the effects of different sites of metastasis on survival were compared using the Kaplan-Meier method. Age, sex, histological type, and chemotherapy were identified as the independent risk factors for OS. The 1-, 2-, and 3-year areas under the curve of the nomogram were 0.792, 0.774, and 0.928, and 0.742, 0.733, and 0.733 in the training and validation sets, respectively. Compared to OS, histological type, radiotherapy, and chemotherapy were independent risk factors for CSS. Different metastatic sites in MM have significantly different effects on prognosis.

Metabolic Signatures of Youth Exposure to Mixtures of Per- and Polyfluoroalkyl Substances: A Multi-Cohort Study.

BACKGROUND: Exposure to per- and polyfluoroalkyl substances (PFAS) is ubiquitous and has been associated with an increased risk of several cardiometabolic diseases. However, the metabolic pathways linking PFAS exposure and human disease are unclear. OBJECTIVE: We examined associations of PFAS mixtures with alterations in metabolic pathways in independent cohorts of adolescents and young adults. METHODS: Three hundred twelve overweight/obese adolescents from the Study of Latino Adolescents at Risk (SOLAR) and 137 young adults from the Southern California Children's Health Study (CHS) were included in the analysis. Plasma PFAS and the metabolome were determined using liquid-chromatography/high-resolution mass spectrometry. A metabolome-wide association study was performed on log-transformed metabolites using Bayesian regression with a g-prior specification and g-computation for modeling exposure mixtures to estimate the impact of exposure to a mixture of six ubiquitous PFAS (PFOS, PFHxS, PFHpS, PFOA, PFNA, and PFDA). Pathway enrichment analysis was performed using Mummichog and Gene Set Enrichment Analysis. Significance across cohorts was determined using weighted Z-tests. RESULTS: In the SOLAR and CHS cohorts, PFAS exposure was associated with alterations in tyrosine metabolism (meta-analysis p=0.00002) and de novo fatty acid biosynthesis (p=0.03), among others. For example, when increasing all PFAS in the mixture from low (∼30th percentile) to high (∼70th percentile), thyroxine (T4), a thyroid hormone related to tyrosine metabolism, increased by 0.72 standard deviations (SDs; equivalent to a standardized mean difference) in the SOLAR cohort (95% Bayesian credible interval (BCI): 0.00, 1.20) and 1.60 SD in the CHS cohort (95% BCI: 0.39, 2.80). Similarly, when going from low to high PFAS exposure, arachidonic acid increased by 0.81 SD in the SOLAR cohort (95% BCI: 0.37, 1.30) and 0.67 SD in the CHS cohort (95% BCI: 0.00, 1.50). In general, no individual PFAS appeared to drive the observed associations. DISCUSSION: Exposure to PFAS is associated with alterations in amino acid metabolism and lipid metabolism in adolescents and young adults. https://doi.org/10.1289/EHP11372.

Transforaminal Endoscopic Lumbar Discectomy versus Open Decompression Discectomy for Lumbar Disc Herniation.

OBJECTIVE: To compare the efficacy of transforaminal endoscopic lumbar discectomy (TELD) and open decompression discectomy (ODD) in the treatment of single-segment L4/5disc herniation. STUDY DESIGN: Descriptive study. PLACE AND DURATION OF STUDY: Department of Orthopedics, General Hospital of the Yangtze River Shipping, Jiang'an District, Wuhan, Hubei, China, from January to December 2021. METHODOLOGY: Using random number table allocation, 94 patients with lumbar disc herniation (LDH, at level L4/5) received were divided into two groups of 47 patients each in the control group treated with ODD and 47 patients in the observation group treated with TELD. The perioperative-related indexes, VAS score, ODI index, modified Macnab evaluation criteria, and the incidence of complications were compared between the two groups. RESULTS: The incision length in the observation group was shorter than that in the control group, the amount of intraoperative blood loss was less than that in the control group, and bedtime and hospital stay were shorter than that in the control group, but the operation time was longer than that in the control group, with statistically significant differences (p <0.01). The VAS score, ODI index, and incidence of postoperative complications in the observation group were lower than those in the control group at 7d, 1 month, 3 months, and 6 months after surgery, while modified Macnab evaluation criteria of lumbar function recovery were higher than that in the control group, the difference was statistically significant (p <0.01). CONCLUSION: TELD is safe and effective for patients with LDH, which can relieve postoperative pain and help restore lumbar function. KEY WORDS: Lumbar disc herniation (LDH), Transforaminal endoscopic lumbar discectomy (TELD), Open decompression discectomy (ODD), Visual analogue score (VAS), Oswestry disability index (ODI), Modified Macnab evaluation criteria.

Genome-wide association study of Klebsiella pneumoniae identifies variations linked to carbapenems resistance.

Klebsiella pneumoniae (KP) is one of the microorganisms that can acquire carbapenem-resistance (CR), and few antimicrobial therapy options exist for infections caused by Carbapenem-Resistant KP (CRKP). In recent years, with the increase of carbapenem resistance rates, treating CRKP has become a serious public health threat in clinical practice. We have collected 2,035 clinical KP isolates from a tertiary hospital in China. Whole genome sequencing data coupled with their binary antimicrobial susceptibility testing data were obtained to conduct the genome-wide association study using a bayesian-based method, including single nucleotide polymorphisms (SNPs) and genes. We identified 28 and 37 potential maker genes associated with imipenem and meropenem resistance, respectively. Among which 19 of them were selected in both drugs by genome-wide association study (GWAS), 11 genes among them were simultaneously validated in independent datasets. These genes were likely related to biofilm formation, efflux pump, and DNA repairing. Moreover, we identified 13 significant CR related SNPs in imipenem or meropenem, with one SNP located in the non-coding region and validated in the independent datasets. Our study indicates complex mechanisms of carbapenems resistance and further investigation of CRKP-related factors are warranted to better understand their contributions to carbapenems resistance. These identified biomarkers may provide targets for future drug interventions or treatments.

Effects of Mind Mapping Combined with Microvideo Explanation on Disease Perception Control and Nursing Cooperation during Membrane Induction Therapy in Patients with Infectious Nonunion after Tibial Trauma.

Objective: To study the effects of mind mapping combined with microvideo explanation on disease perception control and nursing cooperation during membrane induction therapy in patients with infectious nonunion after tibial trauma. Methods: 30 patients with infectious nonunion after tibial trauma treated in the hospital between March 2018 and March 2022 were selected as the research subjects, and the patients were divided into a control group (n = 15) and an observation group (n = 15) by the random number table method. During membrane induction therapy, the control group adopted a routine nursing method while the observation group was given a nursing method of mind mapping combined with microvideo explanation. The disease perception control, nursing cooperation, and self-care ability of the two groups of patients were compared. Results: After nursing, the scores of aspects of "life impact (3.87 ± 0.92)," "duration (3.20 ± 1.01)," and "emotions (3.93 ± 0.59)" of the Brief Illness Perception Questionnaire (B-IPQ) in the observation group were lower than those in the control group (5.27 ± 0.88, 4.67 ± 1.05, and 4.93 ± 0.80, respectively) (P < 0.05) while the scores of "self-control (6.80 ± 1.21)" and "disease awareness (7.27 ± 0.70)" were higher than those in the control group (5.00 ± 1.07 and 5.93 ± 0.70, respectively) (P < 0.05). There was no significant difference in the total compliance rate between the two groups (P > 0.05). After nursing, the scores of dimensions and the total score of the Exercise of Self-Care Agency Scale (ESCA) were increased in the two groups, and the scores were higher in the observation group than those in the control group (P < 0.05). Conclusion: Mind mapping combined with microvideo explanation can improve the disease perception control during membrane induction therapy in patients with infectious nonunion after tibial trauma, improve the self-care ability, and facilitate the smooth progress of treatment.

Large-Scale Genomic Epidemiology of Klebsiella pneumoniae Identified Clone Divergence with Hypervirulent Plus Antimicrobial-Resistant Characteristics Causing Within-Ward Strain Transmissions.

Global dissemination of K. pneumoniae clones poses health hazards to the public. Genomic epidemiology studies with comprehensive data set further revealed clone divergence, showing a high complexity in evolution. Moreover, clones carrying both acquired virulent and antimicrobial-resistant genes emerged and might replace the carbapenem-resistant clones. Co-occurrence of virulence and resistance is emerging. An unbiased collection of 3,061 clinical K. pneumoniae isolates (January 5, 2013 to July 24, 2018) underwent whole-genome sequencing. Pairwise core-genome single-nucleotide polymorphism (cgSNP) distances identified clone divergence and transmission events. A sum of 2,193 nonduplicated genomes clustered into four phenotypically indistinguishable species complexes. 93% (n = 2,035) were KpI with its largest clonal group (CG) being CG11 (n = 406). Three hundred ninety-three were ST11 and three hundred seventy-four carried blaKPC-2. Noticeably, CG11 is divided into two main subclones based on the capsule synthesis K loci (KL). CG11-KL64 showed a clear hypervirulent plus antimicrobial-resistant (hv+AMR) characteristic. Besides, the phylogenetic structure revealed the clone divergence of CG25, and this is the first report with sufficient CG25 genomes to identify the divergence. The outcomes of the hv+AMR CG25 cluster 1 affected patients were poorer (P < 0.05). Moreover, two episodes of strain transmissions were associated with CG25 cluster 1. Other transmissions were associated with ST20 and ST307. Genomic epidemiology identified clone divergence of CG11 and CG25. The hv+AMR subclones pose greater threats on a global scale. Nosocomial transmissions of the high-risk clones raised our concerns about the evolution and transmission of emerging clones among newborns and critically ill patients. IMPORTANCE The convergence of AMR and acquired virulence posing higher risks to the public is a focusing point. With sufficient genomes and genotypes, we successfully identify the convergence in two subclones, the previously reported CG11-KL64, and the newly reported CG25 cluster 1. The novel finding of the CG25 divergence was not only revealed by the phylogenetic tree but also confirmed by the clinical outcome data and the accessory genome patterns. Moreover, the transmission subclones circulated in two clinically important wards highlights the deficiency of infection control program using conventional methods. Without the assistance of whole-genome sequencing, the transmissions of high-risk clones could not be identified.

Multienzyme interactions of the de novo purine biosynthetic protein PAICS facilitate purinosome formation and metabolic channeling.

There is growing evidence that mammalian cells deploy a mitochondria-associated metabolon called the purinosome to perform channeled de novo purine biosynthesis (DNPB). However, the molecular mechanisms of this substrate-channeling pathway are not well defined. Here, we present molecular evidence of protein-protein interactions (PPIs) between the human bifunctional phosphoribosylaminoimidazole carboxylase/succinocarboxamide synthetase (PAICS) and other known DNPB enzymes. We employed two orthogonal approaches: bimolecular fluorescence complementation, to probe PPIs inside live, intact cells, and co-immunoprecipitation using StrepTag-labeled PAICS that was reintegrated into the genome of PAICS-knockout HeLa cells (crPAICS). With the exception of amidophosphoribosyltransferase, the first enzyme of the DNPB pathway, we discovered PAICS interacts with all other known DNPB enzymes and with MTHFD1, an enzyme which supplies the 10-formyltetrahydrofolate cofactor essential for DNPB. We show these interactions are present in cells grown in both purine-depleted and purine-rich conditions, suggesting at least a partial assembly of these enzymes may be present regardless of the activity of the DNPB pathway. We also demonstrate that tagging of PAICS on its C terminus disrupts these interactions and that this disruption is correlated with disturbed DNPB activity. Finally, we show that crPAICS cells with reintegrated N-terminally tagged PAICS regained effective DNPB with metabolic signatures of channeled synthesis, whereas crPAICS cells that reintegrated C-terminally tagged PAICS exhibit reduced DNPB intermediate pools and a perturbed partitioning of inosine monophosphate into AMP and GMP. Our results provide molecular evidence in support of purinosomes and suggest perturbing PPIs between DNPB enzymes negatively impact metabolite flux through this important pathway.

Nickel-Based Selenides with a Fractal Structure as an Excellent Bifunctional Electrocatalyst for Water Splitting.

Nickel-based selenides are believed to be promising non-precious metal electrocatalysts, and have been widely used for both oxygen evolution reactions (OER) and hydrogen evolution reactions (HER). Here, we control the aging time to prepare NixSey with different fractal structures as a bifunctional catalyst. An obtained sample with an aging time of 80 min shows outstanding electrocatalytic performance for hydrogen evolution reactions (HER) with an overpotential of 225 mV (η@10 mA/cm2) and for oxygen evolution reactions (OER) with an overpotential of 309 mV (η@50 mA/cm2). Moreover, to further improve catalytic activity, we doped Fe in NixSey to obtain the ternary nickel-based selenide, Fe0.2Ni0.8Se (FNSs). The HER activity of FNS increased two-fold at 10 mA/cm2, and the overpotential of OER decreased to 255 mV at 50 mA/cm2. The synthetic strategy and research results of this work have a certain reference value for other low-cost and high-efficiency transition metal catalysts for electrocatalytic water splitting.

Promoting the Oxygen Evolution Activity of Perovskite Nickelates through Phase Engineering.

Perovskite oxides have emerged as promising candidates for the oxygen evolution reaction (OER) electrocatalyst due to their flexible lattice structure, tunable electronic structure, superior stability, and cost-effectiveness. Recent research studies have mostly focused on the traditional methods to tune the OER performance, such as cation/anion doping, A-/B-site ordering, epitaxial strain, oxygen vacancy, and so forth, leading to reasonable yet still limited activity enhancement. Here, we report a novel strategy for promoting the OER activity for perovskite LaNiO3 by crystal phase engineering, which is realized by breaking long-range chemical bonding through amorphization. We provide the first and direct evidence that perovskite oxides with an amorphous structure can induce the self-adaptive process, which helps enhance the OER performance. This is evidenced by the fact that an amorphous LaNiO3 film on glassy carbon shows a 9-fold increase in the current density compared to that of an epitaxial LaNiO3 single crystalline film. The obtained current density of 1038 µΑ cm-2 (@ 1.6 vs RHE) is the largest value among the literature reported values. Our work thus offers a new protocol to boost the OER performance for perovskite oxides for future clean energy applications.

Metabolic channeling: predictions, deductions, and evidence.

With the elucidation of myriad anabolic and catabolic enzyme-catalyzed cellular pathways crisscrossing each other, an obvious question arose: how could these networks operate with maximal catalytic efficiency and minimal interference? A logical answer was the postulate of metabolic channeling, which in its simplest embodiment assumes that the product generated by one enzyme passes directly to a second without diffusion into the surrounding medium. This tight coupling of activities might increase a pathway's metabolic flux and/or serve to sequester unstable/toxic/reactive intermediates as well as prevent their access to other networks. Here, we present evidence for this concept, commencing with enzymes that feature a physical molecular tunnel, to multi-enzyme complexes that retain pathway substrates through electrostatics or enclosures, and finally to metabolons that feature collections of enzymes assembled into clusters with variable stoichiometric composition. Lastly, we discuss the advantages of reversibly assembled metabolons in the context of the purinosome, the purine biosynthesis metabolon.

Longitudinal Study of the Drug Resistance in Klebsiella pneumoniae of a Tertiary Hospital, China: Phenotypic Epidemiology Analysis (2013-2018).

PURPOSE: Multi-drug resistant Klebsiella pneumoniae (MDR KP) is spreading worldwide and has posed a huge medical burden to public health. However, studies on drug resistance surveillance of KP, especially MDR KP, with a large longitudinal sample size in a tertiary hospital are rare. This study aims to investigate phenotypic epidemiology characteristics of 4128 KP isolates in a Chinese tertiary hospital covering a period of 5 years. METHODS: All the KP clinical isolates were retrospectively collected from a tertiary hospital in Hunan province of China from Jan 5, 2013 to Jul 24, 2018. All the isolates were identified by MALDI-TOF MS analysis. Twenty-four antimicrobial agents were tested by antimicrobial susceptibility testing. Fisher exact test and logistic regression were used to analyze the association between clinical factors and antimicrobial non-susceptibility for seven second-choice antimicrobials. RESULTS: A total of 4128 KP isolates were collected in our study. The non-susceptible rates (NSRs) to ertapenem, imipenem and tigecycline increased considerably from 2013 to 2018 (13.6% to 28.6%, 10.1% to 28.9%, 10.8% to 46.5%, respectively). Amikacin presents the lowest NSR among 3 aminoglycosides (3.8-22.8%). The multi-drug NSRs among KP isolates to second-choice antimicrobials (88.6-100%) were higher than to all drugs (68.0%). The NSRs varied significantly among departments and sample sources. Higher ETP/IPM/AK NSRs (39.8/39.7/30.6%) were observed in Intensive Care Unit, and ETP/IPM non-susceptible isolates tended to distribute in cerebrospinal fluid. From 2015 to 2017, the NSRs of ETP, IPM, and AK showed an opposite trend of seasonal fluctuations to SXT. CONCLUSION: Higher multi-drug resistance (MDR) rates were observed in KP isolates to second-choice antimicrobials than to others, among which MDR rates to carbapenems or AK are the highest. A unique pattern of MIC and time distributions of MDR were observed. Clinical factors including gender were correlated with MDR rates of KP. Isolates in ICU and CSF showed higher NSRs in carbapenems which should be paid more attention to, and temporal distribution of NSRs was observed.

Circ_0010220-mediated miR-503-5p/CDCA4 axis contributes to osteosarcoma progression tumorigenesis.

CircRNAs are reported to exert a significant role in modulating genes in cancers, including osteosarcoma progression. Up to now, the function of circ_0010220 in osteosarcoma is still poorly known. The aim of our work was to figure out the potential mechanism of circ_0010220/miR-503-5p/CDCA4 axis in osteosarcoma progression. Firstly, quantitative RT-qPCR was utilized to measure the expression of circ_0010220 in osteosarcoma cells. Then, osteosarcoma cell proliferation, apoptosis, cell cycle, migration and invasion after loss of circ_0010220 were evaluated using CCK-8, flow cytometry, transwell migration, invasion and tumorigenesis experiments respectively. Circ_0010220 expression was markedly increased in osteosarcoma cells. Additionally, knockdown of circ_0010220 significantly depressed tumor growth. CCK-8 analysis indicated that down-regulation of circ_0010220 inhibited osteosarcoma cells proliferation. Flow cytometry assay showed that knockdown of circ_0010220 induced cell apoptosis and blocked cell cycle in the G1 phase. Meanwhile, cell migration an invasion was reduced by circ_0010220. Furthermore, miR-503-5p was predicted as the target for circ_0010220 and miR-503-5p inhibitors reversed cell growth suppressed through silencing circ_0010220. Then, our study demonstrated that Cell Division Cycle-Associated protein 4 (CDCA4) could be a downstream target of miR-503-5p. Additionally, circ_0010220 down-regulation reduced CDCA4 expression level and the inhibitors of miR-503-5p reversed that. In conclusion, we indicated circ_0010220 can be an important biomarker for osteosarcoma via regulating miR-503-5p and CDCA4.

Mapping Post-Translational Modifications of de Novo Purine Biosynthetic Enzymes: Implications for Pathway Regulation.

Purines represent a class of essential metabolites produced by the cell to maintain cellular homeostasis and facilitate cell proliferation. In times of high purine demand, the de novo purine biosynthetic pathway is activated; however, the mechanisms that facilitate this process are largely unknown. One plausible mechanism is through intracellular signaling, which results in enzymes within the pathway becoming post-translationally modified to enhance their individual enzyme activities and the overall pathway metabolic flux. Here, we employ a proteomic strategy to investigate the extent to which de novo purine biosynthetic pathway enzymes are post-translationally modified in 293T cells. We identified 7 post-translational modifications on 135 residues across the 6 human pathway enzymes. We further asked whether there were differences in the post-translational modification state of each pathway enzyme isolated from cells cultured in the presence or absence of purines. Of the 174 assigned modifications, 67% of them were only detected in one experimental growth condition in which a significant number of serine and threonine phosphorylations were noted. A survey of the most-probable kinases responsible for these phosphorylation events uncovered a likely AKT phosphorylation site at residue Thr397 of PPAT, which was only detected in cells under purine-supplemented growth conditions. These data suggest that this modification might alter enzyme activity or modulate its interaction(s) with downstream pathway enzymes. Together, these findings propose a role for post-translational modifications in pathway regulation and activation to meet intracellular purine demand.

Generation and characterization of functional phosphoserine-incorporated neuronal nitric oxide synthase holoenzyme.

Phosphorylation is an important pathway for the regulation of nitric oxide synthase (NOS) at the posttranslational level. However, the molecular underpinnings of NOS regulation by phosphorylations remain unclear to date, mainly because of the problems in making a good amount of active phospho-NOS proteins. Herein, we have established a system in which recombinant rat nNOS holoprotein can be produced with site-specific incorporation of phosphoserine (pSer) at residue 1412, using a specialized bacterial host strain for pSer incorporation. The pSer1412 nNOS protein demonstrates UV-Vis, far-UV CD and fluorescence spectral properties that are identical to those of nNOS overexpressed in other bacterial strains. The protein is also functional, possessing normal NO production and NADPH oxidation activities in the presence of abundant substrate L-Arg. Conversely, the rate of FMN-heme interdomain electron transfer (IET) in pSer1412 nNOS is considerably lower than that of wild-type (wt) nNOS, while the phosphomimetic S1142E mutant possesses similar electron transfer kinetics to that of wt. The successful incorporation and high yield of pSer1412 into rat nNOS and the significant change in the IET kinetics upon the phosphorylation demonstrate a highly useful method for incorporating native phosphorylation sites as a substantial improvement to commonly used phosphomimetics.

An Empirical Approach Leveraging Tumorgrafts to Dissect the Tumor Microenvironment in Renal Cell Carcinoma Identifies Missing Link to Prognostic Inflammatory Factors.

By leveraging tumorgraft (patient-derived xenograft) RNA-sequencing data, we developed an empirical approach, DisHet, to dissect the tumor microenvironment (eTME). We found that 65% of previously defined immune signature genes are not abundantly expressed in renal cell carcinoma (RCC) and identified 610 novel immune/stromal transcripts. Using eTME, genomics, pathology, and medical record data involving >1,000 patients, we established an inflamed pan-RCC subtype (IS) enriched for regulatory T cells, natural killer cells, TH1 cells, neutrophils, macrophages, B cells, and CD8+ T cells. IS is enriched for aggressive RCCs, including BAP1-deficient clear-cell and type 2 papillary tumors. The IS subtype correlated with systemic manifestations of inflammation such as thrombocytosis and anemia, which are enigmatic predictors of poor prognosis. Furthermore, IS was a strong predictor of poor survival. Our analyses suggest that tumor cells drive the stromal immune response. These data provide a missing link between tumor cells, the TME, and systemic factors.Significance: We undertook a novel empirical approach to dissect the renal cell carcinoma TME by leveraging tumorgrafts. The dissection and downstream analyses uncovered missing links between tumor cells, the TME, systemic manifestations of inflammation, and poor prognosis. Cancer Discov; 8(9); 1142-55. ©2018 AACR.This article is highlighted in the In This Issue feature, p. 1047.