Correlation between serum 25-OH vitamin D expression and non-alcoholic fatty liver disease.

This study investigated changes in the level of serum 25-OH vitamin D [25-hydroxyvitamin D, 25(OH)D] in patients with non-alcoholic fatty liver disease (NAFLD) and the correlation between the severity of NAFLD and 25(OH)D. A retrospective analysis was performed on 385 NAFLD patients (NAFLD group) admitted to the Zhongshan Hospital Affiliated to Xiamen University from January 2015 to December 2017 and 347 healthy people with physical examination (control group). The height and weight of all subjects were measured, and BMI was calculated. Fasting venous blood was extracted for the determination of blood glucose, blood lipid and 25(OH)D. The indicator levels of patients in the two groups were compared and analyzed. Spearman's correlation analysis was used to investigate the correlation between the severity of NAFLD and the level of 25(OH)D. The levels of BMI, FPG, FPI, HbA1c, TG, TC and LDL-C of patients in the NAFLD group were significantly higher than those in control group (P<0.05). The level of 25(OH)D in the NAFLD group was lower than that in control group (P<0.05). There was a significant negative correlation between 25(OH)D and the severity of patients in the NAFLD group (r=-0.868, P<0.001). BMI, FPG, FPI, HbA1c, TG, TC and LDL-C were independent risk factors for the low level of 25(OH)D (P<0.05). Lowly expressed in the serum of NAFLD patients, 25(OH)D has a significant negative correlation with the severity of NAFLD, which is of guiding significance for the prevention and treatment. 25(OH)D is a novel biomarker for NAFLD diagnosis and a potential drug target.

Characterization of AEBSF-antibody modifications for a protease inhibitor supplementation strategy.

Application of a protease inhibitor, 4-(2-aminoethyl) benzenesulfonyl fluoride (AEBSF), during the cell culture process was demonstrated to effectively reduce proteolytic activity at a specific amino acid site during the production of an HIV-1 broadly neutralizing antibody (bNAb). However, the addition of AEBSF could potentially introduce some modifications to the bNAb protein. Experimental design from sample preparation to LC-MS characterization was performed using middle-up and bottom-up approaches to identify AEBSF-modified species for the bNAb using an AEBSF supplementation in the cell culture media. Modified species along with the unmodified control sample were also subjected to binding activity assessment. The results showed that two amino acids (Tyr177 and Lys250) were susceptible to AEBSF modification in the bNAb test articles but at a negligible level and not in the CDR regions, which therefore did not reduce the in vitro binding activity of the bNAb.

Cardiac RNA induces inflammatory responses in cardiomyocytes and immune cells via Toll-like receptor 7 signaling.

We have recently reported that extracellular RNA (exRNA) released from necrotic cells induces cytokine production in cardiomyocytes and immune cells and contributes to myocardial ischemia/reperfusion injury. However, the signaling mechanism by which exRNA exhibits its pro-inflammatory effect is unknown. Here we hypothesize that exRNA directly induces inflammation through specific Toll-like receptors (TLRs). To test the hypothesis, we treated rat neonatal cardiomyocytes, mouse bone marrow-derived macrophages (BMDM), or mouse neutrophils with RNA (2.5-10 µg/ml) isolated from rat cardiomyocytes or the hearts from mouse, rat, and human. We found that cellular RNA induced production of several cytokines such as macrophage inflammatory protein-2 (MIP-2), ILs, TNFα, and the effect was completely diminished by RNase, but not DNase. The RNA-induced cytokine production was partially inhibited in cells treated with TLR7 antagonist or genetically deficient in TLR7. Deletion of myeloid differentiation primary response protein 88 (MyD88), a downstream adapter of TLRs including TLR7, abolished the RNA-induced MIP-2 production. Surprisingly, genetic deletion of TLR3 had no impact on the RNA-induced MIP-2 response. Importantly, extracellular RNA released from damaged cardiomyocytes also induced cytokine production. Finally, mice treated with 50 µg of RNA intraperitoneal injection exhibited acute peritonitis as evidenced by marked neutrophil and monocyte migration into the peritoneal space. Together, these data demonstrate that exRNA of cardiac origin exhibits a potent pro-inflammatory property in vitro and in vivo and that exRNA induces cytokine production through TLR7-MyD88 signaling.

Importance of Toll-like receptor 2 in mitochondrial dysfunction during polymicrobial sepsis.

BACKGROUND: Toll-like receptor 2 (TLR2) contributes to sepsis pathogenesis such as deleterious systemic inflammation, cardiac dysfunction, and high mortality in animal studies. Mitochondrial dysfunction is a key molecular event that is associated with organ injury in sepsis. The role of TLR2 in sepsis-induced mitochondrial dysfunction remains unclear. METHODS: Intracellular hydrogen peroxide (H2O2), mitochondrial superoxide (O2), mitochondrial membrane potential (ΔΨm), and intracellular adenosine triphosphate (ATP) were measured in peritoneal leukocytes. A mouse model of polymicrobial sepsis was generated by cecum ligation and puncture (CLP). Wild-type and TLR2-deficient (TLR2) mice were subjected to sham or CLP. Mitochondrial functions including reactive oxygen species (ROS), ΔΨm, intracellular ATP, and complex III activity were measured. RESULTS: TLR2/1 activation by Pam3Cys enhanced intracellular H2O2 and mitochondrial O2 production in leukocytes, but had no effect on mitochondrial ΔΨm and ATP production. The effect was specific for TLR2/1 as TLR3 or TLR9 ligands did not induce ROS production. Polymicrobial sepsis induced mitochondrial dysfunction in leukocytes, as demonstrated by increased H2O2 and mitochondrial O2- production (CLP vs. sham; H2O2: 3,173±498, n=5 vs. 557±38, n=4; O2-: 707±66, n=35 vs. 485±35, n=17, mean fluorescence intensity, mean±SEM), attenuated complex III activity (13±2, n=16 vs. 30±3, n=7, millioptical densities/min), loss of mitochondrial ΔΨm, and depletion of intracellular ATP (33±6, n=11 vs. 296±29, n=4, nmol/mg protein). In comparison, there was significant improvement in mitochondrial function in septic TLR2-/- mice as evidenced by attenuated mitochondrial ROS production, better-maintained mitochondrial ΔΨm, and higher cellular ATP production. CONCLUSIONS: TLR2 signaling plays a critical role in mediating mitochondrial dysfunction in peritoneal leukocytes during polymicrobial sepsis.

Role of extracellular RNA and TLR3-Trif signaling in myocardial ischemia-reperfusion injury.

BACKGROUND: Toll-like receptor 3 (TLR3) was originally identified as the receptor for viral RNA and represents a major host antiviral defense mechanism. TLR3 may also recognize extracellular RNA (exRNA) released from injured tissues under certain stress conditions. However, a role for exRNA and TLR3 in the pathogenesis of myocardial ischemic injury has not been tested. This study examined the role of exRNA and TLR3 signaling in myocardial infarction (MI), apoptosis, inflammation, and cardiac dysfunction during ischemia-reperfusion (I/R) injury. METHODS AND RESULTS: Wild-type (WT), TLR3(-/-), Trif(-/-), and interferon (IFN) α/ß receptor-1 deficient (IFNAR1(-/-)) mice were subjected to 45 minutes of coronary artery occlusion and 24 hours of reperfusion. Compared with WT, TLR3(-/-) or Trif(-/-) mice had smaller MI and better preserved cardiac function. Surprisingly, unlike TLR(2/4)-MyD88 signaling, lack of TLR3-Trif signaling had no impact on myocardial cytokines or neutrophil recruitment after I/R, but myocardial apoptosis was significantly attenuated in Trif(-/-) mice. Deletion of the downstream IFNAR1 had no effect on infarct size. Importantly, hypoxia and I/R led to release of RNA including microRNA from injured cardiomyocytes and ischemic heart, respectively. Necrotic cardiomyocytes induced a robust and dose-dependent cytokine response in cultured cardiomyocytes, which was markedly reduced by RNase but not DNase, and partially blocked in TLR3-deficient cardiomyocytes. In vivo, RNase administration reduced serum RNA level, attenuated myocardial cytokine production, leukocytes infiltration and apoptosis, and conferred cardiac protection against I/R injury. CONCLUSION: TLR3-Trif signaling represents an injurious pathway during I/R. Extracellular RNA released during I/R may contribute to myocardial inflammation and infarction.

Complement factor B is the downstream effector of TLRs and plays an important role in a mouse model of severe sepsis.

Severe sepsis involves massive activation of the innate immune system and leads to high mortality. Previous studies have demonstrated that various types of TLRs mediate a systemic inflammatory response and contribute to organ injury and mortality in animal models of severe sepsis. However, the downstream mechanisms responsible for TLR-mediated septic injury are poorly understood. In this article, we show that activation of TLR2, TLR3, and TLR4 markedly enhanced complement factor B (cfB) synthesis and release by macrophages and cardiac cells. Polymicrobial sepsis, created by cecal ligation and puncture in a mouse model, augmented cfB levels in the serum, peritoneal cavity, and major organs including the kidney and heart. Cecal ligation and puncture also led to the alternative pathway activation, C3 fragment deposition in the kidney and heart, and cfB-dependent C3dg elevation. Bacteria isolated from septic mice activated the serum alternative pathway via a factor D-dependent manner. MyD88 deletion attenuated cfB/C3 upregulation as well as cleavage induced by polymicrobial infection. Importantly, during sepsis, absence of cfB conferred a protective effect with improved survival and cardiac function and markedly attenuated acute kidney injury. cfB deletion also led to increased neutrophil migratory function during the early phase of sepsis, decreased local and systemic bacterial load, attenuated cytokine production, and reduced neutrophil reactive oxygen species production. Together, our data indicate that cfB acts as a downstream effector of TLR signaling and plays a critical role in the pathogenesis of severe bacterial sepsis.

Aldolase A-HBsAg interaction and its effect on ultraviolet radiation induced apoptosis in 293FT cells.

BACKGROUND AND AIM: Hepatitis B virus (HBV) infection poses great challenges to humans, claiming one million lives annually worldwide. Solid data have related HBV to hepatocellular carcinoma. METHODS: In the present research, we verified the interaction between surface protein (HBs) encoded by HBV and aldolase A (ALDA) using yeast two-hybrid, mammalian two-hybrid, co-immunoprecipitation, GST pull-down and laser scanning confocal. RESULTS: Anti-ALDA antibody precipitated Gal4-HBs fusion protein in the presence of HBs. Anti-HBs antibody precipitated p65ΔN-ALDA only in the presence of ALDA. Small HBs could be pulled down by GST-ALDA. Cells transfected with pCMV-AD-ALDA showed a protection from ultraviolet radiation-induced apoptosis (21.3% ± 1.3% for ALDA, 35.4% ± 2.1% for control, P < 0.05). CONCLUSIONS: An interaction does exist between ALDA and HBs. The S region within HBs is sufficient for binding ALDA. In addition, ALDA conferred protection to ultraviolet radiation-induced apoptosis, and this effect was enhanced by the interaction between HBs and ALDA.

Competitive priming PCR: a versatile method to generate cohesive terminus.

In the post-genomic era, providing a detailed description of protein functions poses a formidable challenge. To gain functional insights, we have to construct many kinds of expression vectors. DNA recombination based on polymerase chain reaction (PCR) and digestion followed by ligation is the preferred method for vector construction. However, this existing pattern is intrinsically limited by the selection of restriction endonuclease, prompting researchers to use commercial suppliers rather than amplifying genes themselves. Moreover, this method would introduce additional bases into the PCR products, which may be undesired for the construction of epitope expressing plasmid. A PCR-based gene synthesis method, referred to as competitive priming PCR (CP-PCR), is described here to efficiently assemble the plasmid expressing fusion protein of fibrinogen alpha chain and binding domain of galactose transcription factor. A pair of competitive sense primers were designed for the same target sequence. With the presence of antisense primer, PCR amplification of target sequence was performed in the same one system. The PCR product was underwent single digestion by using PstI, followed by ligation with the vector pCMV-BD linearized with EcoRIand PstI. The reconstructed plasmid was validated by sequencing and the fusion protein was affirmed by western blot. CP-PCR combines the superior convenience and precision of PCR. Moreover, it is perfectly capable of generating nearly all kinds of cohesive terminuses, which are ready to recombination in the presence of single digestion or even in the absence of digestion. We demonstrate, by using CP-PCR, the feasibility of directed cloning interested sequence only in the requirement of single digestion or even in the absence of digestion. Competitive priming PCR is demonstrated with convenience and precision equivalent to the traditional method. More than that, "seamless" DNA recombination may be achieved by this novel strategy.

Interacting with HBsAg compromises resistance of jumping translocation breakpoint protein to ultraviolet radiation-induced apoptosis in 293FT cells.

Jumping translocation breakpoint protein (JTB) is suppressed in many cancers, implying it plays a role in the neoplastic transformation of cells. In order to explore the role of JTB in the carcinogenesis of liver, we used mammalian two-hybrid, co-immunoprecipitation, GST pull-down and laser scanning confocal to verify the interaction between HBs and JTB. According to the results, HBs interacts with JTB. In addition, we further determined that S region within HBs is sufficient for binding JTB. Overexpression of JTB conferred resistance to apoptosis induced by ultraviolet radiation, whereas this effect was compromised by the co-overexpression of HBs.

Integrated application of uniform design and least-squares support vector machines to transfection optimization.

BACKGROUND: Transfection in mammalian cells based on liposome presents great challenge for biological professionals. To protect themselves from exogenous insults, mammalian cells tend to manifest poor transfection efficiency. In order to gain high efficiency, we have to optimize several conditions of transfection, such as amount of liposome, amount of plasmid, and cell density at transfection. However, this process may be time-consuming and energy-consuming. Fortunately, several mathematical methods, developed in the past decades, may facilitate the resolution of this issue. This study investigates the possibility of optimizing transfection efficiency by using a method referred to as least-squares support vector machine, which requires only a few experiments and maintains fairly high accuracy. RESULTS: A protocol consists of 15 experiments was performed according to the principle of uniform design. In this protocol, amount of liposome, amount of plasmid, and the number of seeded cells 24 h before transfection were set as independent variables and transfection efficiency was set as dependent variable. A model was deduced from independent variables and their respective dependent variable. Another protocol made up by 10 experiments was performed to test the accuracy of the model. The model manifested a high accuracy. Compared to traditional method, the integrated application of uniform design and least-squares support vector machine greatly reduced the number of required experiments. What's more, higher transfection efficiency was achieved. CONCLUSION: The integrated application of uniform design and least-squares support vector machine is a simple technique for obtaining high transfection efficiency. Using this novel method, the number of required experiments would be greatly cut down while higher efficiency would be gained. Least-squares support vector machine may be applicable to many other problems that need to be optimized.

Identification of two AFLP markers linked to bacterial wilt resistance in tomato and conversion to SCAR markers.

Tomato bacterial wilt (BW) incited by Ralstonia solanacearum is a constraint on tomato production in tropical, subtropical and humid regions of the world. In this paper, we present the results of a research aimed at the identification of PCR-based markers amplified fragment length polymorphism (AFLP) linked to the genes that confer resistance to tomato BW. To this purpose, bulked segregant analysis was applied to an F(2) population segregating for the BW resistant gene and derived from the pair-cross between a BW resistant cultivar T51A and the susceptible cultivar T9230. Genetic analysis indicated that tomato BW was conferred by two incomplete dominant genes. A CTAB method for total DNA extraction, developed by Murray and Thompson with some modifications was used to isolation the infected tomato leaves. Thirteen differential fragments were detected using 256 primer combinations, and two AFLP markers were linked to the BW resistance. Subsequently, the AFLP markers were converted to co-dominant SCAR markers, named TSCAR(AAT/CGA) and TSCAR(AAG/CAT). Linkage analysis showed that the two markers are on the contralateral side of TRSR-1. Genetic distance between TSCAR(AAT/CGA) and TRS-1 was estimated to 4.6 cM, while 8.4 cM between TSCAR(AAG/CAT) and TRS-1.