p53-driven replication stress in nucleoli of malignant epithelial ovarian cancer.

Malignancies including ovarian cancer (OvCa) are genetically unstable. Genomic integrity is maintained by tumor suppressor p53 and DNA damage response network, which crosstalk to each other via not well characterized mechanisms. In this work, we characterize features of damage-related signals in cultured epithelial OvCa cells and tumor biopsies. We found that endogenous burden of DNA damage in OvCa tissues were ubiquitously accumulated in high-grade malignancies than lower grade of cancer that cannot be obviously explained by disturbed function of in DNA damage response (DDR). In contrast, CHK1 phosphorylation (CHK1-pS345) marking the checkpoint activation in nucleolar compartments are prevalent in high-grade OvCa, coincident to the elevated DNA damage in nucleoplasm. Generation of CHK1-pS345 requires the presence of p53 protein in addition to the well-known activities of ATM/ATR kinases. Apparently, mutant forms of p53 possess higher activity in triggering CHK1 phosphorylation than wild type, implying a potential role of p53 in maintaining rDNA integrity. Loss of p53 function would cause replication stress in nucleoli. Altogether, our study reveals endogenous nucleoli stress in OvCa that is coupled to perturbed function of p53 in DNA repair.

Implication of Ataxia-Telangiectasia-mutated kinase in epithelium-mesenchyme transition.

Impairment of genome instability drives the development of cancer by disrupting anti-cancer barriers. Upon genotoxic insults, DNA damage responsive factors, notably ATM kinase, is crucial to protect genomic integrity while promoting cell death. Meanwhile, cytotoxic therapy-inducing DNA lesions is double-edged sword by causing cancer metastasis based on animal models and clinical observations. The underlying mechanisms for the procancer effect of cytotoxic therapies are poorly understood. Here, we report that cancer cells subjected to cytotoxic treatments elicit dramatic alteration of gene expression controlling the potential of epithelium-mesenchyme transition (EMT). Resultantly, EMT-dependent cell mobility is potently induced upon DNA damage. This stimulation of EMT is mainly Ataxia-Telangiectasia-mutated (ATM)-dependent, as the chemical inhibitor specifically inhibiting ATM kinase activity can suppress the EMT gene expression and thus cell mobility. At last, we show that cancer cells with ATM activation display increased metastatic potential in ovarian cancer tissues. Taken together, we reveal a novel role of ATM in promoting metastatic potential of cancer cells by favoring EMT gene expression.

The preparation of mass producible, highly-cycling stable Si/C anode materials with nano-sized silicon crystals embedded in highly amorphous silicon matrix.

The commercial applications of silicon nanomaterials as anode in lithium-ion batteries must solve two important problems, namely low expansion and long-term cycle stability. The former is related to nano-silicon structure, while the latter depends on silicon/carbon composite structure and preparation process. In order to suppress volume expansion appeared during lithiation, this paper selects a kind of silicon nanoparticles (SiNPs) with a high degree of amorphization (81.9%), and designs a stable silicon/carbon composite material structure. Inside this structure, graphite nanoflakes (GNFs) with high specific surface are used as the skeleton, which can provide enough surface area for SiNPs to adhere and avoid the local accumulation of SiNPs. Outside this structure is uniformly coated with a layer of amorphous carbon. Raman and x-ray diffraction results show that after the high-temperature carbonization, the nano-silicon in the composite material still maintains a high degree of amorphization (67.1%) and the average crystallite size of Si has only increased from 3.7 to 9.5 nm. The initial Coulombic efficiency and reversible specific capacity of the composite material are 86.7% and 1374.8 mAh g-1, respectively. After mixing with commercial graphite, the initial Coulombic efficiency and reversible specific capacity are 93.7% and 426.4 mAh g-1, respectively. LiNi0.8Co0.1Mn0.1O2(NCM811) is used as the cathode to produce a soft-pack battery. After 900 cycles at room temperature, the capacity remains 86.2%. The silicon/carbon anode material reported in this paper is of great potential for commercialization.

Active DNA end processing in micronuclei of ovarian cancer cells.

BACKGROUND: Ovarian cancer is one of the most deadly gynecological malignancies and inclined to recurrence and drug resistance. Previous studies showed that the tumorigenesis of ovarian cancers and their major histotypes are associated with genomic instability caused by defined sets of pathogenic mutations. In contrast, the mechanism that influences the development of drug resistance and disease recurrence is not well elucidated. Solid tumors are prone to chromosomal instability (CIN) and micronuclei formation (MN). Although MN is traditionally regarded as the outcome of genomic instability, recent investigation on its origin and final consequences reveal that the abnormal DNA metabolism in MN is a driver force for some types of catastrophic genomic rearrangements, accelerating dramatic genetic variation of cancer cells. METHODS: We used Indirect Immunofluorescent staining to visualize micronuclei and activation of DNA repair factors in ovarian cancer cell lines and biopsies. RESULTS: We show that ovarian cancer cells are disposed to form micronuclei upon genotoxic insults. Double strand DNA breaks (DSBs)-triggered insurgence of micronuclei is associated with unrepaired chromosomes passing through mitosis. According to their morphology and DNA staining, micronuclei compartments are divided into early and late stages that can be further characterized by differential staining of γH2AX and 53BP1. We also show that MN compartments do not halt controlled DNA metabolism as sequestered nuclear repair factors are enriched at DNA breaks in MN compartments and efficiently process DNA ends to generate single-stranded DNA (ssDNA) structures. Interestingly, unknown factors are required for DNA end processing in MN in addition to the nuclear resection machinery. Finally, these hallmarks of micronuclei evolution depicted in cell culture were recapitulated in different stages of ovarian cancer biopsies. CONCLUSIONS: In aggregate, our findings demonstrate that ovarian cancer cells are inclined to form micronuclei that undergo robust DNA metabolism and generate ssDNA structures, potentially destabilizing genomic structures and triggering genetic variation.

[Changes in expression of PYCARD gene and its transcript variant mRNA in peripheral blood mononuclear cells of patients with primary gout].

OBJECTIVE: To determine the expression level and role of PYCARD [PYRIN-PAAD-DAPIN domain (PYD) and a C-terminal caspase recruitment domain (CARD), PYCARD] gene and its transcript variant mRNA in peripheral blood mononuclear cells (PBMCs) of patients with primary gout (PG). METHODS: PYCARD gene and its transcript variant mRNA were measured using reverse transcription-polymerase chain reaction (RT-PCR) in PBMCs. The expression of PYCARD gene and PYCARD-1,-2 mRNA in PBMCs was compared between the patients with acute phase PG (APPG) (n=44), non-acute phase PG (NAPPG) (n= 51) and healthy controls (HC) (n=87). PYCARD and NF-kappaB (p105/p50) protein expressions were measured using Western blot in the PBMCs of participants in the PG and HC groups. Routine blood tests and blood uric acid test were undertaken in all participants. Differences in the indicators were examined among the three groups. Correlations between the expression of PYCARD gene and PYCARD-1,-2 mRNA and other indicators were analyzed. RESULTS: The expression level of PYCARD gene, PYCARD-1,-2 mRNA was significantly higher in the APPG and NAPPG group than in the HC group (P<0.01). The NAPPG group had significantly higher levels of PYCARD gene transcript variant 2x mRNA and 2y mRNA in the HC and APPG groups (P<0.05). The expression of PYCARD and NF-kappaB (p105/p50) protein was significantly higher in the PG group compared with the HC group [(4.900 +/- 1.324) vs. (3.975 +/- 0.210) and (0.263 +/- 0.106) vs. (0.127 +/- 0.008), respectively P<0.05]. The expression level of PYCARD-2 mRNA and granulocyte were positively correlated in the NAPPG group. CONCLUSION: Abnormal expression of PYCARD gene and its transcript variant and PYCARD protein in PG patients suggests that PYCARD gene and its transcript variant may play an important role in regulating the inflammatory response of PG patients.

The ABCG2 gene Q141K polymorphism contributes to an increased risk of gout: a meta-analysis of 2185 cases.

OBJECTIVES: Individual genetic association studies examining the relationship between the ABCG2 gene polymorphisms and gout have yielded inconsistent results. This study aims to evaluate the association between the ABCG2 gene variants and gout using meta-analysis. MATERIALS AND METHODS: Relevant studies were identified by searching databases extensively. The odds ratio (OR) was calculated using a random-effect or fixed-effect model. A Q statistic was used to evaluate homogeneity, and Egger's test and funnel plot were used to assess publication bias. Subgroup analyses on ethnicities and sex were also performed. RESULTS: A total of 7 studies, including 2185 gout patients and 8028 controls from 5 countries or regions, were included and identified for the current meta-analysis. It was found that the A allele or AA genotype of the ABCG2 Q141K polymorphism (rs2231142) had an increased risk of gout in the general population (A allele, p < 0.00001 and AA genotype, p < 0.00001, respectively). On the contrary, CC homozygote played a protective role against the risk of gout (p < 0.00001). Similar results were found in subgroup analyses. However, there was a significant heterogeneity among studies. CONCLUSIONS: Existing evidence indicates that the Q141K polymorphism (rs2231142, the A allele and AA genotype) is associated with an increased risk of gout.

Unveiling Challenges and Opportunities in Silicon-Based All-Solid-State Batteries: Thin-Film Bonding with Mismatch Strain.

Li-metal and silicon are potential anode materials in all-solid-state Li-ion batteries (ASSBs) due to high specific capacity. However, both materials form gaps at the interface with solid electrolytes (SEs) during charging/discharging, resulting in increased impedance and uneven current density distribution. In this perspective, the different mechanisms of formation of these gaps are elaborated in detail. For Li-metal anodes, Li-ions are repeatedly stripped and unevenly deposited on the surface, leading to gaps and Li dendrite formation, which is an unavoidable electrochemical behavior. For Si-based anodes, Li-ions inserting/extracting within the Si-based electrode causes volume changes and a local separation from the SE, which is a mechanical behavior and avoidable by mitigating the strain mismatch of thin-film bonding between anode and SE. Si electro-chemical-mechanical behaviors are also described and strategies recommended to synergistically decrease Si-based electrode strain, including Si materials, Si-based composites, and electrodes. Last, it is suggested to choose a composite polymer-inorganic SE with favorable elastic properties and high ionic conductivity and form it directly on the Si-based electrode, beneficial for increasing SE strain to accommodate stack pressure and the stability of the interface. Thus, this perspective sheds light on the development and application of Si-based ASSBs.

Human papillomavirus E7 protein induces homologous recombination defects and PARPi sensitivity.

PURPOSE: Cervical cancer is a common gynecological malignancy, pathologically associated with persistent infection of high-risk types of human papillomavirus (HPV). Previous studies revealed that HPV-positive cervical cancer displays genomic instability; however, the underlying mechanism is not fully understood. METHODS: To investigate if DNA damage responses are aggravated in precancerous lesions of HPV-positive cervical epithelium, cervical tissues were biopsied and cryosectioned, and subjected to immunofluorescent staining. Cloned HA-tagged E6 and E7 genes of HPV16 subtype were transfected into HEK293T or C33A cells, and indirect immunofluorescent staining was applied to reveal the competency of double strand break (DSB) repair. To test the synthetic lethality of E7-indued HRD and PARP inhibitor (PARPi), we expressed E7 in C33A cells in the presence or absence of olaparib, and evaluated cell viability by colony formation. RESULTS: In precancerous lesions, endogenous DNA lesions were elevated along with the severity of CIN grade. Expressing high-risk viral factor (E7) in HPV-negative cervical cells did not impair checkpoint activation upon genotoxic insults, but affected the potential of DSB repair, leading to homologous recombination deficiency (HRD). Based on this HPV-induced genomic instability, the viability of E7-expressing cells was reduced upon exposure to PARPi in comparison with control cells. CONCLUSION: In aggregate, our findings demonstrate that HPV-E7 is a potential driver for genome instability and provides a new angle to understand its role in cancer development. The viral HRD could be employed to target HPV-positive cervical cancer via synthetic lethality.

[Effects of danshen injection on the proliferation of rheumatoid arthritis fibroblast-like synoviocytes cultured with human serum].

OBJECTIVE: To investigate the effects of Danshen Injection (DSI) on the proliferation of rheumatoid arthritis fibroblast-like synoviocytes (RA FLSs) cultured in RA patients' serum. METHODS: The RA FLSs harvested from RA patients' synovial fluid were primarily cultured by routines. The cells were cultured with 10% inactivated human serum (the healthy human serum and the RA patients' serum) for 24 h. Then DSI at the final concentration of 0. 4 mg/mL was added in the cells for further 24 h culture. By taking 10% fetal calf serum as the control, the morphological changes were observed under optical microscope. The proliferation was analyzed by MTT. The apoptosis was detected by flow cytometry. The total RNA was extracted and reverse transcription was performed. The Bax mRNA expression was detected by fluorescent quantitative PCR. RESULTS: (1) After human serum was added in the healthy human serum and RA patients' serum, cells could grow adhering to the wall. Compared with the fetal calf serum group (FCS), the cell density was higher in the healthy human serum group than in the fetal calf serum group, with no obvious morphological changes. (2) MTT results showed that, compared with the fetal calf serum group, the absorbance value (OD) obviously increased in the healthy human serum group and the RA patients' serum group, showing statistical difference (P <0.01). After adding DSI at the final concentration of 0.4 mg/mL, cells from different serums were inhibited to various degrees (with OD significantly decreased, P <0.05). The OD value significantly increased more in the healthy human serum group and the RA patients' serum group than in the fetal calf serum group, showing statistical difference (P <0.01). There was statistical difference between the healthy human serum group and the RA patients' serum group (P <0.01). (3) The apoptosis rate in the RA patients' serum group obviously decreased with statistical difference, when compared with the Salvia miltiorrhiza free fetal calf serum group (P >0. 01). The apoptosis rate in the fetal calf serum group and the RA patients' serum group significantly increased after adding 0.4 mg/mL Salvia miltiorrhiza, showing statistical difference when compared with the Salvia miltiorrhiza free fetal calf serum group and the Salvia miltiorrhiza free RA patients' serum group (P <0.05). The FLSs were effected by 0.4 mg/mL Salvia miltiorrhiza, the apoptosis rate significantly decreased in the healthy human serum group and the RA patients' serum group, showing statistical difference when compared with the fetal calf serum group (P <0. 05, P <0.01). (4) The expression of Bax gene significantly increased in the RA patients' serum group and the fetal calf serum group after action of 0.4 mg/mL Salvia miltiorrhiza, showing statistical difference (P <0. 01). When 0.4 mg/mL Salvia miltiorrhiza was added, the expression of Bax mRNA obviously increased in the healthy human serum group and the RA patients' serum group, showing statistical difference when compared with the fetal calf serum group (P <0.01). CONCLUSIONS: (1) Although healthy human serum can be favorable to the growth of RA FLSs, the fetal calf serum could reflect the actual results better in the cyto biological research on specific diseases (if there is no serum from patients with corresponding disease). (2) DSI could inhibit the proliferation of RA FLSs through promoting their apoptosis.

Altered expression of TPP1 in fibroblast-like synovial cells might be involved in the pathogenesis of rheumatoid arthritis.

We undertook this study to determine whether the altered expression of telomeric proteins TPP1 and POT1 in fibroblast-like synovial cells (FLS) could provide insights into the pathogenesis of rheumatoid arthritis (RA). FLS were isolated from patients with RA, osteoarthritis (OA) and traumatic joint disease, and cultured in vitro. TPP1 and POT1 mRNA level of FLS were measured using real-time quantitative polymerase chain reaction (RT-qPCR) in 42 RA, 23 OA and 13 healthy cases. Immunofluorescence staining and Western blot were used to detect the expression of TPP1 and POT1 protein. Expression of TPP1 and POT1 mRNA was significantly reduced in RA cases (P < 0.001, respectively), and no significant difference was observed between OA and healthy cases (P > 0.05, respectively). Confocal microscopy images showed TPP1 and POT1 proteins mainly located in nucleus of FLS. Western blot demonstrated that TPP1 protein level was significantly reduced in RA cases (P < 0.001), and POT1 protein expression was not statistical significance among RA, OA patients and healthy cases (P > 0.05). Significant negative correlation was observed between level of TPP1 mRNA and titers of anti-CCP antibody (P < 0.001), RF (P < 0.01). Altered expression of TPP1 might contribute to persistent proliferation of FLS in RA, further study on functions of telomeric proteins in RA would be needed.

Increased body mass index linked to decreased neutralizing antibody titers of inactivated SARS-CoV-2 vaccine in healthcare workers.

Objective: Obesity is an important risk factor for COVID-19. However, whether obesity affects SARS-CoV-2 antibody production is unclear. This study aimed to identify the influence of obesity on neutralizing antibody production of an inactivated SARS-CoV-2 vaccine to better guide vaccination strategies. Methods: This cross-sectional study recruited a total of 239 healthcare workers (age, 21-50 years) from Suining Central Hospital during 22-23 April 2021. An electronic questionnaire on basic characteristics was completed by all participants. A general physical exam and fasting blood sampling by venipuncture were performed. Peripheral leukocyte counts and the ratios of leukocyte subsets, hepatorenal function, and the neutralizing antibody titers against SARS-CoV-2 were measured. Results: Among 239 healthcare workers, the participants with underweight, normal weight, overweight, and obesity accounted for 10.88%, 64.44%, 23.01%, and 1.67%, respectively. The highest peripheral monocyte counts were observed in the group with obesity, whereas the lowest were observed in the group with normal weight. Similar results were obtained with respect to percentage of peripheral monocytes. Participants with obesity had higher peripheral eosinophil counts and percentages than the other three groups. The median neutralizing antibody titer was 12.70 AU/mL, with 85.36% (n = 204) of participants were sufficiently protected against SARS-CoV-2. The lowest neutralizing antibody titers were observed in the group with obesity, whereas the highest were observed in the group that was underweight. Additionally, high BMI was significantly associated with high peripheral monocyte counts [B (95% CI) = 0.008 (0.002, 0.013)] and low neutralizing antibody titers [B (95% CI) = -1.934 (-3.663, -0.206)]. Conclusions: Obesity could induce chronic inflammation, and associated with lower neutralizing antibody titers against SARS-CoV-2 after inactivated SARS-CoV-2 vaccination.

The associations between air pollutant exposure and neutralizing antibody titers of an inactivated SARS-CoV-2 vaccine.

Air pollution is a critical risk factor for the prevalence of COVID-19. However, few studies have focused on whether air pollution affects the efficacy of the SARS-CoV-2 vaccine. To better guide the knowledge surrounding this vaccination, we conducted a cross-section study to identify the relationships between air pollutant exposure and plasma neutralizing antibody (NAb) titers of an inactivated SARS-CoV-2 vaccine (Vero cell, CoronaVac, SINOVΛC, China). We recruited 239 healthcare workers aged 21-50 years who worked at Suining Central Hospital. Of these, 207 were included in this study, depending on vaccination date. The data regarding air pollutants were collected to calculate individual daily exposure dose (DED). The geometric mean of all six pollutant DEDs was applied to estimate the combined toxic effects (DEDcomplex). Then, the participants were divided into two groups based on the mean value of DEDcomplex. The median plasma NAb titer was 12.81 AU/mL, with 85.99% vaccine efficacy in healthcare workers against SARS-CoV-2. In exposure group, observations included lower plasma NAb titers (median: 11.13 AU/mL vs. 14.56 AU/mL), more peripheral counts of white blood cells and monocytes (mean: 6.71 × 109/L vs. 6.29 × 109/L and 0.49 × 109/L vs. 0.40 × 109/L, respectively), and a higher peripheral monocyte ratio (7.38% vs. 6.50%) as compared to the reference group. In addition, elevated air pollutant DEDs were associated with decreased plasma NAb titers. To our knowledge, this study is the first to report the relationship between air pollutant exposure and plasma NAb titers of the SARS-CoV-2 vaccine. This suggests that long-term exposure to air pollutants may inhibit plasma NAb expression by inducing chronic inflammation. Therefore, to achieve early herd immunity and hopefully curb the COVID-19 epidemic, vaccinations should be administered promptly to those eligible, and environmental factors should be considered as well.

Lipoxin A4 attenuates MSU-crystal-induced NLRP3 inflammasome activation through suppressing Nrf2 thereby increasing TXNRD2.

Gout is a common inflammatory disease. The activation of NLRP3 inflammasome induced by monosodium urate (MSU) crystals has a critical role in gout, and its prevention is beneficial for patients. Lipoxin A4 (LXA4) is an endogenous lipoxygenase-derived eicosanoid mediator with powerful anti-inflammatory properties. However, whether LXA4 can suppress NLRP3 inflammasome activation induced by MSU crystals remains unclear. This study aimed to investigate the protective effect of LXA4 on MSU-crystal-induced NLRP3 inflammasome activation and its underlying molecular mechanisms. We found that LXA4 inhibited MSU-crystal-induced NLRP3 inflammasome activation, interleukin (IL)-1ß maturation, and pyroptosis. More specifically, LXA4 suppressed the assembly of the NLRP3 inflammasome, including oligomerization and speck formation of ASC, and ASC-NLRP3 interaction. Furthermore, LXA4 suppressed oxidative stress, the upstream events for NLRP3 inflammasome activation, as evidenced by the fact that LXA4 eliminated total reactive oxygen species (ROS) generation and alleviated nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation and mitochondrial dysfunction. However, LXA4 also depressed the Nrf2 activation, a critical molecule in the antioxidant pathway, and then exerted an inhibitory impact on Klf9 expression and promotional impact on TXNRD2 expression, two molecules located downstream of Nrf2 in sequence. Knockdown of TXNRD2 reversed the LXA4-induced depression of ROS and NLRP3 inflammasome. Moreover, LXA4 alleviated joint inflammation and decreased the production of cleaved caspase-1 and matured IL-1ß in gouty arthritis rats. Taken together, our findings demonstrate that LXA4 can attenuate MSU-crystal-induced NLRP3 inflammasome activation, probably through suppressing Nrf2 activation to increase TXNRD2 expression. The present study highlights the potential of LXA4 as an attractive new gout treatment candidate.

High Effective Preparation of Amorphous-Like Si Nanoparticles Using Spark Erosion Followed by Bead Milling.

This work aims to prepare the silicon nanoparticles with the nanocrystal-embedded amorphous structure through spark erosion followed by bead milling. Spark erosion breaks up monocrystal silicon ingots into micro/nanoparticles, refines the crystal grains, makes the crystals randomly disordered, and increases isotropic character. Bead milling further refines the crystal grains to a few nanometers and increases the amorphous portion in the structure, eventually forming an amorphous structure with the nanocrystals embedded. Spark erosion saves much time and energy for bead milling. The crystallite size and the amount of amorphous phase could be controlled through varying pulse durations of spark discharge and bead milling time. The final particles could contain the nanocrystals as small as 4 nm and the content of amorphous phase as high as 84% and could be considered as amorphous-like Si nanoparticles. This processing route for Si nanoparticles greatly reduced the production time and the energy consumption and, more importantly, is structure-controllable and scalable for mass production of the products with higher purity.

Lipoxin A4 attenuates uric acid-activated, NADPH oxidase-dependent oxidative stress by interfering with translocation of p47phox in human umbilical vein endothelial cells.

LipoxinA4 (LXA4) is a well-known key mediator of endogenous anti-inflammation and of the resolution of inflammation. Considerable oxidative stress occurs during inflammation due to the generation of reactive oxidative species (ROS). Moreover, high levels of uric acid (UA) contribute to endothelial cell dysfunction, which can promote disease-related morbidity, and NADPH oxidase-derived ROS are crucial regulatory factors in these responses. However, LXA4 also has the potential to reduce oxidative stress. The aim of the present study was to examine whether LXA4 could suppress UA-induced oxidative stress in human umbilical vein endothelial cells (HUVECs) and to investigate its mechanisms of action in vitro. HUVECs were incubated with or without LXA4, followed by the addition of UA. ROS levels were then measured using 2,7-dichlorodihydrofluorescein diacetate and lucigenin-enhanced chemiluminescence was used to evaluate NADPH oxidase activity. p47phox or p22phox small interfering (si)RNA were transfected into HUVECs and protein levels of p47phox were detected using western blot analysis. LXA4 significantly inhibited UA-induced generation of ROS to the same extent as the NADPH oxidase inhibitor, diphenyleneiodonium chloride. Notably, transfection of p47phox siRNA attenuated the generation of ROS and the activation of NADPH oxidase. Cells transfected with p22phox siRNA demonstrated a significant reduction in the expression of p47phox on the membrane. Further experiments demonstrated that LXA4 interfered with the transfer of p47phox from the cytoplasm to the cell membrane. These findings suggested that LXA4 inhibited the release of NADPH oxidase derived ROS in HUVECs stimulated by UA. A potential mechanism of action underlying this effect could be LXA4-mediated suppression of NADPH oxidase activity, leading to inhibition of p47phox translocation from the cytoplasm to the cell membrane.

MicroRNA-30b participates in the pathological process of hyperuricemia by regulating interleukin-6 receptor.

The present study aimed to examine the expression of hyperuricemia (HUA)-related factors in the body fluids of HUA patients and in renal tissues and body fluids of HUA mice to elucidate the underlying mechanism of HUA and provide theoretical basis for the diagnosis, prevention and treatment of this disease. A total of 51 HUA patients (HUA group), and 36 healthy subjects (control group) were included in the present study. The peripheral blood and urine were collected from all patients and healthy subjects. A total of 20 male Kunming mice were used to construct HUA model, and another 20 mice were used as controls. The kidney tissues, peripheral blood and urine were collected from all mice. ELISA was performed to determine the levels of interleukin-6 receptor (IL-6R) proteins in the serum and urine of human or mice, while western blotting was employed to determine the protein expression in the kidney tissues of mice. Quantitative real-time polymerase chain reaction was used to measure the expression of mRNA and miR-30b in all sample types. Dual luciferase reporter assay was performed to identify the direct interaction between 3'-untranslated region of IL-6R mRNA and miR-30b. The expression of IL-6R mRNA and protein was increased in serum and urine of HUA patients, while the expression of miR-30b was reduced in HUA patients when compared with healthy subjects. The contents of uric acid, urea nitrogen and creatinine in the blood of HUA mice model were significantly elevated. Similarly, the expression of IL-6R mRNA and protein was increased in kidney, serum and urine of HUA mice model, while the expression of miR-30b was reduced in kidney tissues, serum and urine of HUA mice model. Dual luciferase reporter assay showed that miR-30b was able to bind with 3'-UTR seed region of IL-6R mRNA to regulate its expression. These findings demonstrated that the expression of IL-6R in patients and mouse with HUA is elevated, which is related with the down-regulation of miR-30b. Therefore, miR-30b might participate in the pathological process of HUA by regulating IL-6R.

Relationship between oxidative stress and inflammation in hyperuricemia: Analysis based on asymptomatic young patients with primary hyperuricemia.

The average age of hyperuricemia patients has gradually decreased, but young patients with primary hyperuricemia often do not exhibit clinical symptoms and have not received sufficient attention. However, a lack of symptoms with primary hyperuricemia does not mean that high serum uric acid (UA) levels cannot lead to pathological effects, such as oxidative stress and inflammation, and the specific damage is still unclear. We aimed to determine the relationship between oxidative stress and inflammation to explore the possible role of pathological damage in asymptomatic young patients with primary hyperuricemia.A total of 333 participants were enrolled in our study: 158 asymptomatic young patients with primary hyperuricemia and 175 healthy persons from a health check-up population. Malondialdehyde (MDA), superoxide dismutase (SOD), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and general biochemical markers were measured.We found no differences in biochemical markers (fasting glucose, TG, TC, LDL-C, HDL-C, SCr, BUN, AST, and ALT) between the patients and healthy persons. Subsequent analyses of oxidative stress and inflammation revealed that the serum levels of MDA, IL-6, and TNF-α in the patients were significantly higher than those in the healthy control group (P < .001), and the SOD activity was significantly lower (P < .001). As the UA levels increased, MDA increased significantly and SOD decreased significantly; likewise, IL-6 and TNF-α increased significantly as the UA level increased. MDA showed a significant positive correlation with IL-6 (r = 0.367, P < .001) and TNF-α (r = 0.319, P < .001), and SOD was negatively correlated with IL-6 (r = -0.241, P < .01) and TNF-α (r = -0.308, P < .001). Multivariable logistic regression analysis showed that UA (OR: 2.379, 95% CI: 1.698-3.286, P < .001; OR: 3.261, 95% CI: 1.729-3.857, P < .001; for IL-6 and TNF-α, respectively) and MDA (OR: 1.836, 95% CI: 1.283-2.517, P < .01; OR: 2.532, 95% CI: 1.693-3.102, P < .001; for IL-6 and TNF-α, respectively) were risk factors for high IL-6 and TNF-α and that SOD (OR: 0.517, 95% CI: 0.428-0.763, P < .01; OR: 0.603, 95% CI: 0.415-0.699, P < .001; for IL-6 and TNF-α, respectively) was a protective factor.In our study, some abnormal pathological effects were found in asymptomatic young patients with hyperuricemia, suggesting that in young hyperuricemia patients, oxidative stress, inflammation and the inflammatory response may be related to the oxidative stress induced by UA. Therefore, we should pay more attention to the pathological damage caused by these alterations.

Characterization of a novel PKA phosphorylation site, serine-2030, reveals no PKA hyperphosphorylation of the cardiac ryanodine receptor in canine heart failure.

Hyperphosphorylation of the cardiac Ca2+ release channel (ryanodine receptor, RyR2) by protein kinase A (PKA) at serine-2808 has been proposed to be a key mechanism responsible for cardiac dysfunction in heart failure (HF). However, the sites of PKA phosphorylation in RyR2 and their phosphorylation status in HF are not well defined. Here we used various approaches to investigate the phosphorylation of RyR2 by PKA. Mutating serine-2808, which was thought to be the only PKA phosphorylation site in RyR2, did not abolish the phosphorylation of RyR2 by PKA. Two-dimensional phosphopeptide mapping revealed two major PKA phosphopeptides, one of which corresponded to the known serine-2808 site. Another, novel, PKA phosphorylation site, serine 2030, was identified by Edman sequencing. Using phospho-specific antibodies, we showed that the novel serine-2030 site was phosphorylated in rat cardiac myocytes stimulated with isoproterenol, but not in unstimulated cells, whereas serine-2808 was considerably phosphorylated before and after isoproterenol treatment. We further showed that serine-2030 was stoichiometrically phosphorylated by PKA, but not by CaMKII, and that mutations of serine-2030 altered neither the FKBP12.6-RyR2 interaction nor the Ca2+ dependence of [3H]ryanodine binding. Moreover, the levels of phosphorylation of RyR2 at serine-2030 and serine-2808 in both failing and non-failing canine hearts were similar. Together, our data indicate that serine-2030 is a major PKA phosphorylation site in RyR2 responding to acute beta-adrenergic stimulation, and that RyR2 is not hyperphosphorylated by PKA in canine HF.

[Construction of recombinant E. coli-L. interrogans shuttle plasmid pGKINVA and screening recombinant leptospira strains].

OBJECTIVE: To further investigate pathogenic function of invA in Leptospira, recombinant L. biflexa-Escherichia coli shuttle vector pGKINVA was constructed, and electropored into L. biflexa strain Patoc I . METHODS: The invA gene was cloned into shuttle vector pGKble24 to construct a recombinant L. biflexa-Escherichia coli shuttle vector pGKINVA. The recombinant vectors were electropored into L. biflexa serovar Patoc strain Patoc I . RESULTS: A 558 bp invA was amplified from L. interrogans serovars Lai strain 017 and ligated with Aat II and Xma I digested pGKble24. After screening by kanamycin and blue/white color, the recombinant pGKINVA was obtained and sequenced. Subsequently, the recombinant pGKINVA was electropored into L. biflexa strain Patoc I. The selected mutants were identified by PCR. CONCLUSION: Recombinant L. biflexa-Escherichia coli shuttle vector with invA insert was constructed and two strains of pGKINVA transformed Leptospira were obtained. These pGKINVA transformed Leptospira strains would provid an experimental model to investigate potential pathogenic functions of invA in vivo.

[Prokaryotic expression and characterization of the Rv2994 gene from Mycobacterium tuberculosis].

OBJECTIVE: To construct a prokaryotic expression vector bearing Rv2994 gene from Mycobacterium tuberculosis and provide materials for investigating the function of the gene. METHODS: The Rv2994 gene was amplified by Polymerase Chain Reaction from Mycobacterium tuberculosis H37Rv strain and cloned into prokaryotic expression vector pGEX-1lamdaXT. The recombinant plasmid pGEX-2994 was sequenced and transformed into E. coli JM109 to be induced with IPTG and expressed the 73kDa fusion protein GST-Rv2994. It's antigenicity was confirmed by Western blotting. The expression product was purified and immunized the new Zealand rabbits. RESULTS: The Rv2994 gene was amplified accurately from the genome DNA of H37Rv. A recombinant fused expression vector pGEX-Rv2994 was constructed and GST-Rv2994 protein was purifiel to immunize New Zealand rabbit. CONCLUSION: The prokaryotic expression vector pGEX-2994 was constructed, and the 73kDa fusion protein GST-Rv2994 was expressed and purified successfully. It provided the basis for the further study of the Rv2994 gene.