Protein S-Nitrosylation Controls Glycogen Synthase Kinase 3ß Function Independent of Its Phosphorylation State.

RATIONALE: GSK-3ß (glycogen synthase kinase 3ß) is a multifunctional and constitutively active kinase known to regulate a myriad of cellular processes. The primary mechanism to regulate its function is through phosphorylation-dependent inhibition at serine-9 residue. Emerging evidence indicates that there may be alternative mechanisms that control GSK-3ß for certain functions. OBJECTIVES: Here, we sought to understand the role of protein S-nitrosylation (SNO) on the function of GSK-3ß. SNO-dependent modulation of the localization of GSK-3ß and its ability to phosphorylate downstream targets was investigated in vitro, and the network of proteins differentially impacted by phospho- or SNO-dependent GSK-3ß regulation and in vivo SNO modification of key signaling kinases during the development of heart failure was also studied. METHODS AND RESULTS: We found that GSK-3ß undergoes site-specific SNO both in vitro, in HEK293 cells, H9C2 myoblasts, and primary neonatal rat ventricular myocytes, as well as in vivo, in hearts from an animal model of heart failure and sudden cardiac death. S-nitrosylation of GSK-3ß significantly inhibits its kinase activity independent of the canonical phospho-inhibition pathway. S-nitrosylation of GSK-3ß promotes its nuclear translocation and access to novel downstream phosphosubstrates which are enriched for a novel amino acid consensus sequence motif. Quantitative phosphoproteomics pathway analysis reveals that nuclear GSK-3ß plays a central role in cell cycle control, RNA splicing, and DNA damage response. CONCLUSIONS: The results indicate that SNO has a differential effect on the location and activity of GSK-3ß in the cytoplasm versus the nucleus. SNO modification of GSK-3ß occurs in vivo and could contribute to the pathobiology of heart failure and sudden cardiac death.

The current status and future of cardiac stem/progenitor cell therapy for congenital heart defects from diabetic pregnancy.

Pregestational maternal diabetes induces congenital heart defects (CHDs). Cardiac dysfunction after palliative surgical procedures contributes to the high mortality of CHD patients. Autologous or allogeneic stem cell therapies are effective for improving cardiac function in animal models and clinical trials. c-kit+ cardiac progenitor cells (CPCs), the most recognized CPCs, have the following basic properties of stem cells: self-renewal, multicellular clone formation, and differentiation into multiple cardiac lineages. However, there is ongoing debate regarding whether c-kit+ CPCs can give rise to sufficient cardiomyocytes. A new hypothesis to address the beneficial effect of c-kit+ CPCs is that these cells stimulate endogenous cardiac cells through a paracrine function in producing a robust secretome and exosomes. The values of other cardiac CPCs, including Sca1+ CPCs and cardiosphere-derived cells, are beginning to be revealed. These cells may be better choices than c-kit+ CPCs for generating cardiomyocytes. Adult mesenchymal stem cells are considered immune-incompetent and effective for improving cardiac function. Autologous CPC therapy may be limited by the observation that maternal diabetes adversely affects the biological function of embryonic stem cells and CPCs. Future studies should focus on determining the mechanistic action of these cells, identifying new CPC markers, selecting highly effective CPCs, and engineering cell-free products.

Cysteine oxidative posttranslational modifications: emerging regulation in the cardiovascular system.

In the cardiovascular system, changes in oxidative balance can affect many aspects of cellular physiology through redox-signaling. Depending on the magnitude, fluctuations in the cell's production of reactive oxygen and nitrogen species can regulate normal metabolic processes, activate protective mechanisms, or be cytotoxic. Reactive oxygen and nitrogen species can have many effects including the posttranslational modification of proteins at critical cysteine thiols. A subset can act as redox-switches, which elicit functional effects in response to changes in oxidative state. Although the general concepts of redox-signaling have been established, the identity and function of many regulatory switches remains unclear. Characterizing the effects of individual modifications is the key to understand how the cell interprets oxidative signals under physiological and pathological conditions. Here, we review the various cysteine oxidative posttranslational modifications and their ability to function as redox-switches that regulate the cell's response to oxidative stimuli. In addition, we discuss how these modifications have the potential to influence other posttranslational modifications' signaling pathways though cross-talk. Finally, we review the increasing number of tools being developed to identify and quantify the various cysteine oxidative posttranslational modifications and how this will advance our understanding of redox-regulation.

The clinical-psychological features of functional dyspepsia patients with weight loss: a multi-center study from China.

AIMS: To investigate the clinical features, appetite, quality of life (QOL), and their associated psychological factors of functional dyspepsia (FD) patients with weight loss. METHODS: For a multicenter study, FD patients were recruited and divided into two groups according to the degree of weight changes during the previous 12 months or less with the onset of dyspepsia symptoms: Group A (≥5%) and Group B (<5%). Patients were evaluated based on the Nepean dyspepsia index (NDI), appetite questionnaire, Hamilton Rating Scale of Anxiety/Depression (HAMA/HAMD). RESULTS: The body mass index in Group A was lower than in Group B, while, the frequency of physician visits in Group A was higher than in Group B. There were no differences in the total scores of NDI-symptom checklist or the items of intensity and bothersomeness between them (p > 0.05), but the frequency item for Group A was significantly higher than Group B (p = 0.035). The incidence of anxiety or depression, the proportion of poor or very poor appetite for Group A was higher than those for Group B (p < 0.05). Subscale scores of the NDI-QOL for Group A were significantly lower than those for Group B (p < 0.05). CONCLUSIONS: FD patients with weight loss have lower BMI, more frequent physician visits, higher psychological disorders, poorer appetite and QoL.

The Ser/Thr protein kinase AfsK regulates polar growth and hyphal branching in the filamentous bacteria Streptomyces.

In cells that exhibit apical growth, mechanisms that regulate cell polarity are crucial for determination of cellular shape and for the adaptation of growth to intrinsic and extrinsic cues. Broadly conserved pathways control cell polarity in eukaryotes, but less is known about polarly growing prokaryotes. An evolutionarily ancient form of apical growth is found in the filamentous bacteria Streptomyces, and is directed by a polarisome-like complex involving the essential protein DivIVA. We report here that this bacterial polarization machinery is regulated by a eukaryotic-type Ser/Thr protein kinase, AfsK, which localizes to hyphal tips and phosphorylates DivIVA. During normal growth, AfsK regulates hyphal branching by modulating branch-site selection and some aspect of the underlying polarisome-splitting mechanism that controls branching of Streptomyces hyphae. Further, AfsK is activated by signals generated by the arrest of cell wall synthesis and directly communicates this to the polarisome by hyperphosphorylating DivIVA. Induction of high levels of DivIVA phosphorylation by using a constitutively active mutant AfsK causes disassembly of apical polarisomes, followed by establishment of multiple hyphal branches elsewhere in the cell, revealing a profound impact of this kinase on growth polarity. The function of AfsK is reminiscent of the phoshorylation of polarity proteins and polarisome components by Ser/Thr protein kinases in eukaryotes.

Effective separation of maslinic acid and oleanolic acid from olive pomace using high-speed shear off-line coupled with high-speed countercurrent chromatography and their antibacterial activity test.

In this work, a high-speed shear extraction off-line coupling high-speed countercurrent chromatography method was developed to separate maslinic acid and oleanolic acid from olive pomace. To improve extraction efficiency, the polar disparity between maslinic acid and oleanolic acid necessitated the concurrent utilization of both polar and non-polar solvents during high-speed shear extraction. Then, the high-speed shear extraction was directly feed to high-speed countercurrent chromatography for subsequently separation. A total of 250 min were needed to complete the extraction and separation process. This yielded two molecules from 3.3 g of defatted olive pomace: 7.2 mg of 93.8 % pure maslinic acid and 2.3 mg of 90.1 % pure oleanolic acid, both determined by HPLC at 210 nm. Furthermore, the compounds exhibited inhibitory activity against Escherichia coli and Staphylococcus aureus. At a concentration of 100 µg/mL, its efficacy in inhibiting hyaluronidase was comparable to that of the standard drug indomethacin. Compared with the conventional separation method, this coupled technique reduced the whole time due to the direct injection of sample extraction solution. This technique provides a useful approach for the separation of natural products with significant polarity differences.

Redox regulation of mitochondrial ATP synthase: implications for cardiac resynchronization therapy.

RATIONALE: Cardiac resynchronization therapy (CRT) is an effective clinical treatment for heart failure patients with conduction delay, impaired contraction, and energetics. Our recent studies have revealed that mitochondrial posttranslational modifications (PTM) may contribute to its benefits, motivating the present study of the oxidative regulation of mitochondrial ATP synthase. OBJECTIVES: We tested whether CRT alteration of ATP synthase function is linked to cysteine (Cys) oxidative PTM (Ox-PTM) of specific ATP synthase subunits. METHODS AND RESULTS: Canine left ventricular myocardium was collected under conditions to preserve Ox-PTM from control, dyssynchronous heart failure (DHF), or hearts that had undergone CRT. In-gel ATPase activity showed that CRT increased ATPase activity by approximately 2-fold (P<0.05) over DHF, approaching control levels, and this effect was recapitulated with a reducing agent. ATP synthase function and 3 Ox-PTM: disulfide bond, S-glutathionylation and S-nitrosation were assessed. ATP synthase from DHF hearts contained 2 novel disulfide bonds, between ATP synthase α subunits themselves and between α and γ subunits, both of which were decreased in CRT hearts (4.38 ± 0.13- and 4.23 ± 0.36-fold, respectively, P<0.01). S-glutathionylation of ATP synthase α subunit occurred in DHF hearts and was decreased by CRT (1.56 ± 0.16-fold, P<0.04). In contrast, S-nitrosation of ATP synthase α subunit in DHF hearts was lower than in CRT hearts (1.53 ± 0.19-fold, P<0.05). All modifications occurred at ATP synthase α subunit Cys294 and Cys to Ser mutation indicated that this residue is critical for ATP synthase function. CONCLUSIONS: A selective Cys in ATP synthase α subunit is targeted by multiple Ox-PTM suggesting that this Cys residue may act as a redox sensor modulating ATP synthase function.

Site-mapping of in vitro S-nitrosation in cardiac mitochondria: implications for cardioprotection.

S-nitrosation (SNO) of mitochondrial protein cysteines can be cardioprotective. Several targets have been implicated, yet the scope and identification of specific residues has not been fully assessed. To address this, a comprehensive assessment of mitochondrial SNO-modifiable cysteines was performed to determine nitric oxide (NO) susceptible pathways and identify novel mechanisms of oxidative cardioprotection. The biotin switch assay and mass spectrometry were used on rat cardiac mitochondrial lysates treated with the nitric oxide donor, S-nitrosoglutathione, and controls (n=3) to map 83 SNO-modified cysteine residues on 60 proteins. Of these, three sites have been reported, 30 sites are new to 21 proteins previously known to be S-nitrosated but which lacked site-specific information and 50 sites were found on 39 proteins not previously implicated in SNO pathways. The SNO-modifications occurred in only a subset of available cysteines, indicating a specific targeted effect. Functional annotation and site-specificity analysis revealed a twofold greater nitric oxide-susceptibility for proteins involved in transport; including regulators of mitochondrial permeability transition suggesting SNO-regulation and a possible protective mechanism. Additionally, we identified many novel SNO-modified proteins with cardioprotective potential involved in the electron transport chain, tricarboxylic acid cycle, oxidative stress defense, fatty acid and amino acid metabolism. These findings suggest that SNO-modification may represent a novel mechanism for the regulation of oxidative phosphorylation and/or cell death. S-nitrosation of mitochondrial permeability transition-associated proteins represents an intriguing potential link to cardioprotection.

Aldehyde dehydrogenase 2 gene rs671 G>A polymorphism is associated with an increased risk of digestive tract cancer.

OBJECTIVE: Acetaldehyde can accumulate in cells and form acetaldehyde-DNA adducts that result in digestive tract cancer development. Acetaldehyde dehydrogenase 2 (ALDH2) enzymatic activity is involved in this process. Here, we aimed to analyze the relationship between an ALDH2 gene polymorphism and the digestive tract cancer risk in the Hakka population in China. METHODS: This was a retrospective study, with the ALDH2 rs671 genotype and medical record information collected from all subjects. The relationships between these factors, including various blood cell parameters, and digestive tract cancer susceptibility were analyzed. RESULTS: Overall, 307 cancer patients and 317 controls were included. The cancer patients had significantly higher percentages with a history of smoking and drinking alcohol, as well as an increased platelet to lymphocyte ratio and lower lymphocyte to monocyte ratio, compared with the controls. The ALDH2 rs671 genotype and allele distributions were significantly different between the cancer patients and controls. Logistic regression analysis showed that the ALDH2 G/A genotype (G/A vs. G/G) and A/A genotype (A/A vs. G/G) in the co-dominant mode were risk factors for digestive tract cancer susceptibility. CONCLUSIONS: ALDH2 rs671 G/A or A/A genotype carriers may have an increased risk of developing digestive tract cancers among the Hakka people.

Proteomic analysis of early-responsive redox-sensitive proteins in Arabidopsis.

Regulation of protein function through oxidative modification has emerged as an important molecular mechanism modulating various biological processes. Here, we report a proteomic study of redox-sensitive proteins in Arabidopsis cells subjected to H(2)O(2) treatment. Four gel-based approaches were employed, leading to the identification of four partially overlapping sets of proteins whose thiols underwent oxidative modification in the H(2)O(2)-treated cells. Using a method based on differential labeling of thiols followed by immunoprecipitation and Western blotting, five of the six selected putative redox-sensitive proteins were confirmed to undergo oxidative modification following the oxidant treatment in Arabidopsis leaves. Another method, which is based on differential labeling of thiols coupled with protein electrophoretic mobility shift assay, was adopted to reveal that one of the H(2)O(2)-sensitive proteins, a homologue of cytokine-induced apoptosis inhibitor 1 (AtCIAPIN1), also underwent oxidative modification in Arabidopsis leaves after treatments with salicylic acid or the peptide elicitor flg22, two inducers of defense signaling. The redox-sensitive proteins identified from the proteomic study are involved in various biological processes such as metabolism, the antioxidant system, protein biosynthesis and processing, and cytoskeleton organization. The identification of novel redox-sensitive proteins will be helpful toward understanding of cellular components or pathways previously unknown to be redox-regulated.

Study on the Effect of Different Endoscopic Auxiliary Treatment of Gastric Mucosal Microtumor.

Objective: To explore the effect of endoscopy in the treatment of gastric mucosal microtumors. Methods: A total of 229 patients with gastric mucosal microtumors were treated in our hospital from January 2016 to December 2021. All patients were divided into three groups group A, group B, and group C. Group A was treated with a transparent cap combined with circle-assisted endoscopic resection, group B with ligator combined with circle-assisted endoscopic resection, and group C with endoscopic mucosal tumor resection. The effects of the three groups were observed. Results: There were 47 patients in group A, 17 males, and 30 females, aged 36-69 years, with an average age of 55.6 ± 9.2 years. There were 54 patients in group B, 18 males, and 36 females, aged 38-72 years, with an average age of 57.6 ± 7.7 years. There were 128 patients in group C, 29 males, and 99 females, aged 33-78 years, with an average age of 55.6 ± 8.4 years. There is no significant difference in age and sex between group A, group B, and group C (P > 0.05). The incidence of postoperative complications in group B (66.7%) was significantly higher than that in group A (57.4%) and group C (53.9%) (all P < 0.05). The incidence of postoperative complications in group A (57.4%) was higher than that in group C (53.9%), and the difference was statistically significant (P < 0.05). Conclusion: Endoscopic mucosal resection and ligation combined with circle-assisted endoscopic resection are effective and safe in the treatment of gastric mucosal microtumors, but it needs to be combined with targeted nursing measures. The transparent cap combined with ring-assisted endoscopic resection has a significant effect on the treatment of gastric mucosal micromasses, reducing operative complications.

Risk factors for rebleeding in patients with obscure gastrointestinal bleeding from southern China.

BACKGROUND: To identify the risk factors associated with rebleeding in obscure gastrointestinal bleeding (OGIB) patients from southern China. METHODS: This retrospective study involved 229 patients who underwent small bowel endoscopy in our hospital between 1 January 2018 and 1 December 2020. The clinical characteristics and risk factors related to rebleeding were retrospectively evaluated. RESULTS: Rebleeding patients were significantly older than non-rebleeding patients (53.0 ± 15.9 vs. 46.2 ± 17.8 years), had lower hemoglobin concentrations (89.2 ± 28.1 vs. 126.2 ± 25.1 g/L), and higher blood urea nitrogen concentrations (5.4 ± 2.6 vs. 4.5 ± 2.2 µmol/L), respectively. A higher percentage of rebleeding patients had diabetes mellitus (13.9% vs. 2.9%) and overt bleeding (70.4% vs. 38.6%), and required blood transfusions (43.1% vs. 8.0%), compared with non-rebleeding patients, respectively. Multivariate logistic analysis indicated that drinking alcohol (odds ratio (OR): 9.27; 95% confidence interval (CI) = 1.35-63.78), anemia (OR: 17.38; 95% CI = 5.48-55.10), and blood transfusion (OR: 3.76; 95% CI = 1.04-13.56) increased the risk of rebleeding in OGIB patients. CONCLUSION: Our data suggested that OGIB patients who drink alcohol, have anemia, and require blood transfusion have an increased risk of rebleeding.

Patients with Gastric Polyps need Colonoscopy Screening at Younger Age: A Large Prospective Cross-Sectional Study in China.

Background To date, it is not clarified whether patients with gastric polyps without any alarming symptoms for colorectal neoplasia need colonoscopy screening. The objective of this study is to prospectively determine the association between gastric polyps and colorectal neoplasia. Methods A multicenter prospective cross-sectional study was performed from July 2012 to December 2014. We compared patients with and without gastric polyps for prevalence of colorectal adenomas. The odds ratios (OR) were computed by logistic regression analysis after multivariable adjustments. Results Totally 1546 patients were included, with 770 patients in the gastric polyp group and 776 in the age- and sex- matched control group. Patients with gastric polyps had greater odds of having any colorectal adenoma (adjusted OR=2.34, 95% confidence interval [CI]: 1.79 to 3.06, p<0.001) and advanced colorectal adenomas (adjusted OR=2.71, 95% CI: 1.74 to 4.23, p<0.001) than those without. The positive association between gastric polyps and colorectal adenomas remained significant in both women (OR=2.34, 95% CI: 1.66 to 3.29, p<0.001) and men (OR=1.87, 95% CI: 1.31 to 2.66, p=0.001). Patients over the age of 40 with gastric polyps had a higher prevalence of colorectal adenomas than those without (40-49yr: OR=1.81, 95% CI=1.02-3.21, p=0.04; 50-59yr: OR=1.88, 95% CI=1.26-2.81, p<0.001; 60-74yr: OR=2.62, 95% CI=1.73-3.98, p<0.001). Conclusions The presence of gastric polyps is significantly associated with a higher prevalence of colorectal adenomas, especially advanced colorectal adenomas. Colonoscopy might be considered in patients with gastric polyps, of any gender, and over the age of 40.

Assemblies of DivIVA mark sites for hyphal branching and can establish new zones of cell wall growth in Streptomyces coelicolor.

Time-lapse imaging of Streptomyces hyphae revealed foci of the essential protein DivIVA at sites where lateral branches will emerge. Overexpression experiments showed that DivIVA foci can trigger establishment of new zones of cell wall assembly, suggesting a key role of DivIVA in directing peptidoglycan synthesis and cell shape in Streptomyces.