Substrate and functional characterization of the lysine acetyltransferase MsKat and deacetylase MsCobB in Mycobacterium smegmatis.

Tuberculosis (TB) is a serious cause of infectious death worldwide. Recent studies have reported that about 30% of the Mtb proteome was modified post-translationally, indicating that their functions are essential for drug resistance, mycobacterial survival, and pathogenicity. Among them, lysine acetylation, reversibly regulated by acetyltransferase and deacetylase, has important roles involved in energy metabolism, cellular adaptation, and protein interactions. However, the substrate and biological functions of these two important regulatory enzymes remain unclear. Herein, we utilized the non-pathogenic M. smegmatis strain as a model and systematically investigated the dynamic proteome changes in response to the overexpressing of MsKat/MsCobB in mycobacteria. A total of 4179 proteins and 1236 acetylated sites were identified in our data. Further analysis of the dynamic changes involved in proteome and acetylome showed that MsKat/MsCobB played a regulatory role in various metabolic pathways and nucleic acid processes. After that, the quantitative mass spectrometric method was utilized and proved that the AMP-dependent synthetase, Citrate synthase, ATP-dependent specificity component of the Clp protease, and ATP-dependent DNA/RNA helicases were identified to be the substrates of MsKat. Overall, our study provided an important resource underlying the substrates and functions of the acetylation regulatory enzymes in mycobacteria. SIGNIFICANCE: In this study, we systematically analyzed the dynamic molecular changes in response to the MsKat/MsCobB overexpression in mycobacteria at proteome and lysine acetylation level by using a TMT-based quantitative proteomic approach. Pathways related with glycolysis, degradation of branched chain amino acids, phosphotransferase system were affected after disturbance of the two regulates enzymes involved in lysine acetylation. We also proved that AMP-dependent synthetase Clp protease, ATP-dependent DNA/RNA helicases and citrate synthase was the substrate of MsKat according to our proteomic data and biological validation. Together, our study underlined the substrates and functions of the acetylation regulatory enzymes in mycobacteria.

Characterization of diverse lysine acylations in Bacillus thuringiensis: Substrate profiling and functional exploration.

Lysine acylation has been extensively investigated due to its regulatory role in a diverse range of biological functions across prokaryotic and eukaryotic species. In-depth acylomic profiles have the potential to enhance comprehension of the biological implications of organisms. However, the extent of research on global acylation profiles in microorganisms is limited. Here, four lysine acylomes were conducted in Bacillus thuringiensis by using the LC-MS/MS based proteomics combined with antibody-enrichment strategies, and a total of 3438 acetylated sites, 5797 propionylated sites, 1705 succinylated sites, and 925 malonylated sites were identified. The motif analysis of these modified proteins revealed a high conservation of glutamate in acetylation and propionylation, whereas such conservation was not observed in succinylation and malonylation modifications. Besides, conservation analysis showed that homologous acylated proteins in Bacillus subtilis and Escherichia coli were connected with ribosome and aminoacyl-tRNA biosynthesis. Further biological experiments showed that lysine acylation lowered the RNA binding ability of CodY and impaired the in vivo protein activity of MetK. In conclusion, our study expanded the current understanding of the global acylation in Bacillus, and the comparative analysis demonstrated that shared acylation proteins could play important roles in regulating both metabolism and RNA transcription progression.

A clinical medicine level test at Jinan University School of Medicine reveals the importance of training medical students in clinical history-taking.

Backgrounds: Training in the basic interview skills of clinical history-taking has always been a significant component of medical education. Purpose: This study was designed to identify the factors influence medical students' history-taking skills learning and develop a way to improve these skills. Methods: We firstly analysed the academic performance of medical students at Jinan University School of Medicine in different disciplines of the Clinical Medicine Level Test (CMLT), to ensure the students have obtained comprehensive medical education prior to beginning their clinical internships. Next, we conducted a survey among the CMLT participants to seek the underlying causes and corresponding measures to improve history-taking in the future. Before these medical students entered their fifth-year clinical practice, we finally provide them with pre-internship training, including the history-taking workshops with standard patients (SP). Results: The analysis of the clinical skill sections of the CMLT revealed that the students performed significantly better on clinical operations from multiple disciplines than on medical history-taking. Principal component analysis of the survey questionnaire indicated that the capability of history-taking, course assessments, and awareness of the value of medical history-taking emerged as the key factors forming a cohesive clue for sustaining history-taking implementation. The intervention workshops of employing SP had a positive impact, as evidenced by the students' feedback and suggestions for improving their ability of history-taking. Conclusions: This study suggests that strengthening of medical history-taking training is indispensable for training qualified medical students. Workshops with SP is a successful teaching strategy for practicing history-taking, allowing students to spot minute errors and training communication skills.

Global landscape of lysine acylomes in Bacillus subtilis.

Lysine acetylation is a common posttranslational modification that regulates numerous biochemical functions in both eukaryotic and prokaryotic species. In addition, several new non-acetyl acylations are structurally different from lysine acetylation and participate in diverse physiological functions. Here, a comprehensive analysis of several lysine acylomes was performed by combining the high-affinity antibody enrichment with high-resolution LC-MS/MS. In total, we identified 2536 lysine acetylated sites, 4723 propionylated sites, 2150 succinylated sites and 3001 malonylated sites in Bacillus subtilis, respectively. These acylated proteins account for 35.8% of total protein in this bacterium. The four lysine acylomes showed a motif preference for glutamate surrounding the modified lysine residues, and a functional preference for several metabolic pathways, such as carbon metabolism, fatty acid metabolism, and ribosome. In addition, more protein-protein interaction clusters were identified in the propionylated substrates than other three lysine acylomes. In summary, our study presents a global landscape of acylation in the Gram-positive model organism Bacillus and their potential functions in metabolism and physiology.

Study on the Tribological Behaviour of Nanolubricants during Micro Rolling of Copper Foils.

Water-based lubricants with different fractions of TiO2 nanoparticles ranging from 1.0 to 9.0 wt.% were utilized to study the lubrication mechanisms during micro rolling tests and the tribological behaviour of nanolubricants during the micro rolling of copper foils. The results indicate that the application of TiO2 nanolubricants remarkably improves the surface quality of rolled copper foils during rolling processes. For lubricants with inadequate TiO2 nanoparticles, it is found that few TiO2 nanoparticles enter the contact regions between the rolls and foils, causing insufficient lubrication during rolling processes. Instead, for lubricants with excessive TiO2 nanoparticles, obvious agglomeration occurs at the contact regions and promotes the generation of voids on the surface of the rolled foils, thereby deteriorating the surface quality of the rolled copper foils. In addition, it is found that the surface quality of rolled foils is improved by utilizing a large reduction ratio. Overall, the fraction of 3.0 wt.% TiO2 nanolubricants is optimal to improve the lubrication conditions at the contact regions, thereby improving the surface quality of the rolled copper foils.

An Integrative Proteome-Based Pharmacologic Characterization and Therapeutic Strategy Exploration of SAHA in Solid Malignancies.

Targeting histone epigenetic modification is an important strategy for anticancer therapy. Histone deacetylase inhibitors (HDACis) have been clinically approved in the treatment of diverse hematological cancers, but mechanisms of drug resistance and poor therapeutic efficacy in solid malignancies remain largely unknown. In this study, we applied a mass spectrometry-based quantitative proteomic strategy to investigate the molecular differences in HDACi vorinostat (SAHA) sensitive and resistant cell lines. The proteomic results revealed that the glycolysis pathway was highly enriched after vorinostat treatment in the resistant cell line, leading to the prediction of a new drug combination, SAHA and hexokinase inhibitor (2-deoxyglucose). The efficacy of this combination was further verified in several solid tumor cell lines. Quantitative proteomics revealed that alterations in the transcription process and protein homeostasis could play roles in the synergetic utilization of these two compounds. Our study showed the application of proteomics in elucidating the drug mechanism and predicting drug combination and the potential of expanding the utilization of HDACi.

ß-elemonic acid inhibits growth and triggers apoptosis in human castration-resistant prostate cancer cells through the suppression of JAK2/STAT3/MCL-1 and NF-ĸB signal pathways.

Castration-resistant prostate cancer (CRPC) has become a significant problem in the current treatment of prostate cancer (PCa) with the characteristics of high metastatic potential, resistance and easy recurrence. The abnormal activation of JAK2/STAT3/MCL-1 and NF-κB has been confirmed as the main reason for the development of CRPC. We previously found that ß-elemonic acid (ß-EA) as a natural triterpene has potential anti-inflammatory and anti-osteosarcoma effects with lower toxicity. But it remains unknown whether it had effects on CRPC. The present research in vitro and in vivo systematically investigates anti-cancer effects and mechanisms of ß-EA on human CRPC. ß-EA treatment resulted in apoptotic cell death in human PCa cells by mitochondrial apoptotic pathways (including up-regulation of cleaved caspase-3, cleaved PARP, and Bax or down-regulation of Bcl-2). Besides, ß-EA at relatively lower levels inhibited colony-forming, the migration and invasion potential of PCa cells, indicating its anti-proliferation and anti-metastasis activities. After exploring the potential mechanism, our results suggested that it subsequently inhibited the activation of JAK2/STAT3/MCL-1 and NF-κB signaling pathway by the administration of ß-EA. The silencing of NF-κB/p65, JAK2 and STAT3, respectively, increased the sensitivity of the PCa cells to ß-EA induced apoptosis. Moreover, ß-EA exhibited a strong affinity with its essential proteins JAK2, RELA/p65, NF-κBIα/IκBα by molecular docking analysis. Importantly, ß-EA retards tumor growth in a murine xenograft model, consistent with our study in vitro. Taken together, findings from this study reveal for the first time the potential role and mechanisms of ß-EA on CRPC.

Aucubin, a natural iridoid glucoside, attenuates oxidative stress-induced testis injury by inhibiting JNK and CHOP activation via Nrf2 up-regulation.

BACKGROUND: Eucommia ulmoides has been used for many years as a successful strategy to treat male infertility. Aucubin (AU) is the active ingredient extracted from Eucommia ulmoides. However, its protective action and exact mechanism on testicular injury is not yet known. PURPOSE: Here, the protective effect and the mechanism of action of AU on testis damage under oxidative stress was investigated in vivo and in vitro. METHODS: As regard the in vivo experiment, male mice were divided into five groups and testicular injury model was established by Triptolide (TP) (120 µg/kg) intraperitoneal injection for two weeks. Animals in the treatment group were pretreated with an intraperitoneal injection of AU at different doses (5, 10 and 20 mg/kg) for 1 h and subsequently treated with TP (120 µg/kg). At the end of the experimental period, the testis was collected for biochemical and histological examination. As regard the in vitro experiment, Sertoli cells (SCs) were used to investigate the protective effect and mechanism of action of AU against disruption of the blood-testis-barrier (BTB) and apoptosis induced by TP via apoptosis detection, western blot, immunofluorescence analysis, and siRNA transient transfection. RESULTS: TP-treated animals showed testicular atrophy, BTB disruption, increased ROS levels and spermatogenic dysfunction. Pre-administration of AU resulted in a significant protection on keeping a normal testicular weight, sperm morphology, BTB integrity, and a normal level of oxidative stress markers and antioxidants. Furthermore, AU prevented apoptosis through an effective inhibition of PERK/CHOP and JNK dependent apoptosis pathway, as well as protected the integrity of BTB by up-regulating the expression of tight junction proteins (ZO-1, Occludin, Claudin-11) and gap junction protein (Cx43). The mechanistic study revealed that AU significantly triggered Nrf2 translocation, thus increasing nuclear Nrf2 accumulation and then induced antioxidant enzymes expression in the testis and SCs. Furthermore, Nrf2 silencing unsuccessfully reversed the increased CHOP and p-JNK expression induced by TP, abolishing the protective effect of AU. CONCLUSION: These results indicate that AU might be considered as a potential protective agent against testicular injury.

MitoQ ameliorates testis injury from oxidative attack by repairing mitochondria and promoting the Keap1-Nrf2 pathway.

Mitochondrial dysfunctions induced by oxidative stress could play a pivotal role in the development of testicular damage and degeneration, leading to impaired fertility in adulthood. MitoQ as mitochondria-targeted antioxidant has been used in many diseases for a long time, but its therapeutic effects on testicular injury 'have not been reported yet. Here, we examined the protective action mechanism of MitoQ on testicular injury from oxidative stress induced by triptolide (TP). Mice were orally administrated with MitoQ (1.3, 2.6 and 5 .2mg/kg, respectively) in a TP-induced model of testicular damage for 14 days. And then testis injuries were comprehensively evaluated in terms of morphological changes, spermatogenesis assessment, blood-testis barrier (BTB) integrity, and apoptosis. The results demonstrated MitoQ effectively increased testicular weight, maintained the integrity of BTB, protected microstructure of testicular tissue and sperm morphology by inhibition of oxidative stress. Further mechanism studies revealed that MitoQ markedly activates the Keap1-Nrf2 antioxidative defense system characterized by increasing the expression of Nrf2 and its target genes HO-1 and NQO1. Meanwhile, MitoQ upregulated the expression of mitochondrial dynamics proteins Mfn2 and Drp-1and exerted a protective effect on mitochondria. On this basis, the results from pharmacokinetic study indicated that the MitoQ could enter into testis tissues after oral administration in despite of the low absolute bioavailability, which provided the material basis for MitoQ in the treatment of testicular damage. More importantly, MitoQ reached mitochondria quickly and had an outstanding feature of mitochondria targeting in Sertoli cells. Therefore, these results provide information for the application of MitoQ against testicular injury diseases.

Facile fabrication of amphiphobic surfaces on copper substrates with a mixed modified solution.

Fabricating amphiphobic surfaces is often complex, difficult to control, and time-consuming, making the fabrication process very difficult. Herein, a facile and time-saving modification method using a mixed modified solution of stearic acid and perfluorooctanoic acid was initially proposed, which played a key role in the achievement of the superhydrophobicity and highly oleophobicity. The effects of reaction time on surface morphology and wettability as well as the content of perfluorooctanoic acid in the mixed modified solution on wettability were investigated to determine the optimal experiment parameters that maximized the amphiphobicity of the surfaces. The as-fabricated amphiphobic surfaces displayed high oil contact angles of 133.5°, higher water contact angles of 156.8°, ultra-low water sliding angles of less than 5° and excellent self-cleaning properties. The facile, easy to control, and efficient method can provide new insights into fabricating amphiphobic surfaces and can open up a new way for the basic research and practical application of amphiphobic surfaces.

Raddeanin A inhibits growth and induces apoptosis in human colorectal cancer through downregulating the Wnt/ß-catenin and NF-κB signaling pathway.

AIMS: Colorectal cancer (CRC) remains one of the most lethal human malignancies with high incidence and lack of effective therapy. Raddeanin A (RA), an active triterpenoid saponins, has been demonstrated the ability to inhibit the growth of tumor. But the therapeutic effects and mechanisms of RA in CRC remain elusive. Here, we investigated the efficacy and mechanism of RA in CRC both in vitro and in vivo. MAIN METHODS: Cell viability was investigated to evaluate cytotoxic activity by MTT method. Apoptosis induced by RA was studied using Annexin V-FITC/PI binding and JC-1 staining by flow cytometry analysis. The xenograft mouse model of CRC was used to investigate anti-tumor effects in vivo. The key proteins involved in mitochondrial apoptotic, Wnt/ß-catenin and NF-κB pathway were detected by Western blotting, Immunofluorescence, and Immunohistochemistry. KEY FINDINGS: RA induced apoptosis and inhibited cell proliferation of SW480 and LOVO cells in a concentration-dependent manner. Moreover, RA efficiently inhibited tumor growth in xenograft mouse model. RA could down regulate the Wnt/ß-catenin signaling to display anti-tumor effects via suppression of p-LRP6, induction of AKT inactivation, removal of GSK-3ß inhibition and attenuation of ß-catenin. Meanwhile, RA also suppressed the NF-κB pathway by decreasing the phosphorylation of IKBα to induce subsequently mitochondrial apoptotic pathway. SIGNIFICANCE: In summary, RA suppressed the growth and triggered the apoptosis of CRC through discontinuing Wnt/ß-catenin signaling and inhibiting the NF-κB pathway. These findings suggested that RA may hold a promise as a novel therapeutic agent for CRC therapy.

Clinical simulation training improves the clinical performance of Chinese medical students.

BACKGROUND: Modern medical education promotes medical students' clinical operating capacity rather than the mastery of theoretical knowledge. To accomplish this objective, clinical skill training using various simulations was introduced into medical education to cultivate creativity and develop the practical ability of students. However, quantitative analysis of the efficiency of clinical skill training with simulations is lacking. METHODS: In the present study, we compared the mean scores of medical students (Jinan University) who graduated in 2013 and 2014 on 16 stations between traditional training (control) and simulative training groups. In addition, in a clinical skill competition, the objective structured clinical examination (OSCE) scores of participating medical students trained using traditional and simulative training were compared. The data were statistically analyzed and qualitatively described. RESULTS: The results revealed that simulative training could significantly enhance the graduate score of medical students compared with the control. The OSCE scores of participating medical students in the clinical skill competition, trained using simulations, were dramatically higher than those of students trained through traditional methods, and we also observed that the OSCE marks were significantly increased for the same participant after simulative training for the clinical skill competition. CONCLUSIONS: Taken together, these data indicate that clinical skill training with a variety of simulations could substantially promote the clinical performance of medical students and optimize the resources used for medical education, although a precise analysis of each specialization is needed in the future.

Steps towards preventive HIV treatment in Fujian, China: problems identified via an assessment of initial antiretroviral therapy provision.

BACKGROUND: At the end of 2009, a total of 501 AIDS patients were receiving antiretroviral therapy (ART) in Fujian Province in China, yet there were no assessments to determine treatment efficacy and HIV-1 preventive potency under the current health care delivery system. METHODS: During the period of 2005-2009, we assessed the outcomes of initial ART by following up 381 patients for 12 months in Fujian Province. CD4⁺ T-lymphocyte (CD4) count, plasma viral load (VL), and patient characteristics were analysed. The results were compared between 4 groups divided by the baseline CD4 values at the 25, 50 (median), and 75 percentiles. FINDINGS: Over three-quarters of the subjects reported heterosexual contact as the probable route of transmission. After 12 months of ART, CD4 recovery varied between the 4 groups (P < 0.001), but VL sharply declined regardless of the baseline CD4 count (P = 0.136). Although this VL decline indicates the potency of ART as an HIV-1 prevention tool, the time between positive diagnosis and ART initiation suggests serious delay in both diagnosis and treatment; the medians of periods for the lowest and highest baseline CD4 quartiles were 1.2 and 9.6 months, respectively. CONCLUSION: Current limitations in VL determination make it difficult to assess the efficacy of initial ART, and delays in diagnosis and treatment suggest that subjects contributed to HIV-1 transmission while they were not receiving ART. The current National Free ART scheme does not provide free treatment for sexually transmitted infection (STI), and there is no link between ART and the STI care delivery system. This may interfere with the HIV-1 preventive potency of ART. We highly recommend establishing a collaborating mechanism with STI care, strengthening the VL determination system, and promoting HIV tests and early ART initiation.

[Establishment and preliminary application of multiplex PCR for detecting the microsatellite markers in silkies].

In order to obtain the stable PCR combinations for silkies genetic analysis, multiplex PCR was used and its reaction condition was optimized. Five combinations of multiplex PCR with good effects were obtained from eighteen microsatellite markers. Three of them were quadruplex-PCRs, and two were triplex-PCRs. The products of multiplex PCR were further combined into three sets for the electrophoresis on ABI 3100-Avant Genetic Analyzer. The results suggested that the eighteen microsatellite markers could be successfully applied to the silkies genetic analysis.

[A linkage map of mirosatellite on chromosome 1 in Liangshan semi-wool sheep].

The 9 microsatellite markers on Chromosomes 1 were used to construct a linkage map of Liangshan semi-wool sheep with a 387 members half-sib pedigrees after paternity testing. The results were as follows: the number of alleles for 9 markers was from 5 to 15; the heterozygosity was from 0.202 to 0.831, the average heterozygosity was 0.617 and the polymorphic information content (PIC) was 0.604. Length of the linkage map of chromosome 1 we built was 311.0 cM which is in consistent with the sheep linkage map of USDA and IMF, and can be used in Quantitative Trait Loci searching in Liangshan semi-wool sheep.