Thermoanaerobacter indiensis BSB-33 has been earlier shown to reduce Fe(III) and Cr(VI) anaerobically at 60°C optimally. Further, the Gram-positive thermophilic bacterium contains Cr(VI) reduction activity in both the membrane and cytoplasm. The soluble fraction prepared from T. indiensis cells grown at 60°C was found to contain the majority of Fe(III) reduction activity of the microorganism and produced four distinct bands in nondenaturing Fe(III) reductase activity gel. Proteins from each of these bands were partially purified by chromatography and identified by mass spectrometry (MS) with the help of T. indiensis proteome sequences. Two paralogous dihydrolipoamide dehydrogenases (LPDs), thioredoxin reductase (Trx), NADP(H)-nitrite reductase (Ntr), and thioredoxin disulfide reductase (Tdr) were determined to be responsible for Fe(III) reductase activity. Amino acid sequence and three-dimensional (3D) structural similarity analyses of the T. indiensis Fe(III) reductases were carried out with Cr(VI) reducing proteins from other bacteria. The two LPDs and Tdr showed very significant sequence and structural identity, respectively, with Cr(VI) reducing dihydrolipoamide dehydrogenase from Thermus scotoductus and thioredoxin disulfide reductase from Desulfovibrio desulfuricans. It appears that in addition to their iron reducing activity T. indiensis LPDs and Tdr are possibly involved in Cr(VI) reduction as well. The involvement of iron in a number of physiological functions, such as a cofactor for enzymes or an electron acceptor in anaerobic respiration, of most organisms has been recognized . HoweverOne of the strategies bacteria have evolved for assimilation and utilization of iron is the reduction of Fe(III) to Fe(II) . Bacteri Clostridium kluyveri an NAD(P)H-dependent flavoenzyme dihydrolipoyl dehydrogenase  reduces Fe(III) complexes of citrate, ATP, and ADP . 3D structures were compared using DaliLite [http://www.ebi.ac.uk/Tools/structure/dalilite] and PDBeFold [http://www.ebi.ac.uk/msd-srv/ssm/cgi-bin/ssmserver]. For visualization and superposition of 3D structures UCSF Chimera v.1.8 downloaded from http://www.cgl.ucsf.edu/chimera was used .In order to purify each of the four activities from nonferric reductase proteins respective Q Sepharose fractions were then passed through hydroxyapatite and mono Q columns. In each step the number of background protein bands was reduced. However, none of the four activities could be completely purified from nonferric reductase protein background (data not shown). Hence, after the mono Q step the protein bands showing Fe(III) reductase activity in preparative nondenaturing gels were excised and analyzed by MS which has the capability to identify multiple proteins from a mixture [ T. indiensis with high Mascot scores  as the template (MS spectra obtained from band 1 matched against two dihydrolipoamide dehydrogenases (LPDs) int scores . The twotemplate . High setemplate . These a Clostridium kluyveri dihydrolipoyl dehydrogenase  BfmBC reduces Fe(III) complexes of citrate, ATP, and ADP in an NAD(P)H-dependent manner [ C. kluyveri NADH transfers a hydride ion to enzyme-bound flavin and FADH2 thus produced donates an electron to ferric chelates [LPDs belong to pyridine nucleotide-disulfide oxidoreductase family and are found in many species as a part of pyruvate dehydrogenase complex . In the t manner . It has  C. Kluyveri BfmBC with T. indiensis LPD1 and LPD2, respectively  as the template ( T. indiensis proteins as well as C. kluyveri BfmBC contain the most conserved FAD-binding sequence motif XhXhGXGXXGXXXhXXh(X)8hXhE(D), where X is any residue and h is a hydrophobic residue, as a part of the Rossmann fold [ C. kluyveri enzyme either of the T. indiensis LPDs would be able to reduce Fe(III) complexes which is consistent with the zymogram result  reductase activity is thioredoxin reductase (Trx). Thioredoxin reductase-like ferric reductase (FeS) as well as typical thioredoxin reductase (Trx) is also present in T. scotoductus SA-01 [ T. scotoductus enzymes can reduce Fe(III)-NTA complex although reduction by Trx is less efficient [ Magnetospirillum gryphiswaldense MSR-1 thioredoxin reductase FeR5 has also been shown to possess ferric reductase activity [ T. indiensis Trx showed 29% sequence similarity (92% query coverage) with T. scotoductus Trx and 24% sequence similarity (95% query coverage) with M. gryphiswaldense FeR5 by BLAST analysis  as the template and the 3D structure of T. scotoductus Trx by using Deinococcus radiodurans thioredoxin reductase crystal structure (PDB ID 2Q7V) as the template -binding domain. It could possibly catalyze electron transfer from NAD(P)H to FAD and the reduced flavin could, in turn, reduce Fe(III) substrate [ T. scotoductus FeS absence of CXXC has been found to be advantageous with respect to Fe(III) reduction [ T. indiensis Trx also appears to be a thioredoxin reductase-like ferric reductase.Among the high-scoring matches obtained by MS of the protein mixture from band 2 the onlyus SA-01 . Both T.fficient . Magnetoactivity . T. indianalysis . ESyPredtemplate . PDBeFolnse FeR5 . In spit T. indiensis NAD(P)H-nitrite reductase which was the only protein in the mixture likely to be an Fe(III) reductase. InterProScan identified a FAD/NAD(P)-binding domain in the protein and suggested that it belonged to pyridine nucleotide-disulfide oxidoreductase family of proteins. BLAST analysis found significant sequence similarity of T. indiensis nitrite reductase to NAD(P)H-nitrite reductases from Clostridium tetani, Bacillus cereus, and Heliobacterium modesticaldum (data not shown). Domain analysis showed that all these nitrite reductases belonged to the pyridine nucleotide-disulfide oxidoreductase family. A search in the whole PDB archive found Pyrococcus furiosus nitrite reductase to be structurally similar to T. indiensis nitrite reductase . It is not known if any of these pyridine nucleotide-disulfide oxidoreductases could reduce iron. However, as described earlier, one pyridine nucleotide-disulfide oxidoreductase, C. kluyveri dihydrolipoamide dehydrogenase BfmBC, has been shown to reduce Fe(III) complexes of citrate, ATP, and ADP in an NAD(P)H-dependent manner [ T. indiensis nitrite reductase was predicted by ESyPred#D using Novosphingobium aromaticivorans ferredoxin reductase ArR crystal structure (PDB ID 3LXD) as the template ( T. indiensis nitrite reductase with C. kluyveri BfmBC were found to be moderate ( T. indiensis NAD(P)H-nitrite reductase is responsible for the reduction of Fe(III)-nitrilotriacetic acid in the native gel as shown in MS with samples extracted from band 3 showed the presence oftemplate . Sequencmoderate . Hence,  T. indiensis protein in band 4 most likely to be a Fe(III) reductase is thioredoxin disulfide reductase (Tdr)  as the template  as evident from the zymogram  . BLAST anse FeR5 . The 3D template . 3D strut values . Besideszymogram .T. indiensis Tdr also showed 43% similarity (97% query coverage) with a thioredoxin reductase (TR) from the hyperthermophilic bacterium Thermotoga maritima [ T. maritima was predicted using Salmonella enteric AhpF crystal structure (PDB ID 1HYU) as the template (Z = 15.95 and rmsd = 1.90). Both enzymes have a typical FAD-binding motif GXGXXG near the N-terminus, a nicotinamide nucleotide-binding motif GGGXXA near the middle, and an active redox center CXXC [ T. indiensis BSB-33, T. maritima is capable of growing as a respiratory organism when Fe(III) is provided as an electron acceptor and T. maritima cell suspension reduces Fe(III) [ T. maritima TR is involved in Fe(III) reduction has not yet been shown.maritima . Three-dtemplate . PDBeFolter CXXC . Like th Fe(III) . However T. indiensis BSB-33 is capable of reducing Cr(VI) besides Fe(III) [ Shewanella oneidensis outer membrane proteins MtrC and OmcA are involved in both Fe(III) and Cr(VI) reduction [ Paracoccus denitrificans [ Pseudomonas putida and YieF from Escherichia coli [It has been reported earlier that like several other mesophilic and thermophilic microorganisms Fe(III) . In fact Fe(III) , 21. In eduction . However Thermus scotoductus SA-01 a peripheral membrane-associated protein, identified as a dihydrolipoamide dehydrogenase (LPD) by sequence homology, has been shown to couple NAD(P)H oxidation to Cr(VI) reduction [ T. indiensis LPDs were found to possess significant sequence similarity with the T. scotoductus protein  as the template ( T. indiensis LPDs with T. scotoductus LPD gave high Z-score and rmsd ( T. scotoductus LPD as well as T. indiensis LPDs FAD- and NAD(P)-binding Rossmann folds amino acid sequence motif GX1-2GXXG together with either GXXXG or GXXXA motifs where the first glycyl residue of these motifs and the third glycyl residue of the GX1-2GXXG motif are the same [ T. indiensis LPDs would also reduce Cr(VI) in an NAD(P)H-dependent manner.Ineduction . The enzeduction . Both T. protein . T. scottemplate . Structuand rmsd  and therand rmsd . Multiplthe same . It is,  D. desulfuricans a thioredoxin reductase MreD has been shown to be involved in NAD(P)H-dependent reduction of U(VI) and Cr(VI) in association with thioredoxin and possibly an oxidoreductase protein MreG [ D. desulfuricans MreD, thioredoxin, and MreG and corresponding proteins of T. indiensis were predicted by ESyPred3D using PDB templates as enlisted in Further, inein MreG . The redein MreG . Reducedein MreG . Three-dT. indiensis thioredoxin was found to contain the typical thioredoxin fold which is a distinct structural motif consisting of a four-stranded β-sheet and three flanking α-helices [ T. indiensis and D. desulfuricans thioredoxins gave Z = 9.10, rmsd = 1.597 (PDBeFold) and Z = 13.5, rmsd = 1.7 . T. indiensis also contained a protein indolepyruvate ferredoxin oxidoreductase which was structurally similar to Desulfovibrio pyruvate ferredoxin/flavodoxin oxidoreductase MreG .-helices . Clustal T. indiensis Tdr and D. desulfuricans MreE was found to be 37% with 97% query coverage. Structural identity between the two enzymes as shown by Z-score, rmsd, and superposition was very high . It is also to be noted that neither T. indiensis Tdr nor LPDs showed any significant sequence or structural match with ChrR, YieF, or FerB indicating the possibility of different chromate reduction mechanisms in this bacterium.Sequence similarity betweenery high . Besides T. indiensis contains four distinct Fe(III) reductase activities. The annotated genome sequence of the thermophilic bacterium has enabled identification of the proteins responsible for Fe(III) reductase activity by chromatography and mass spectrometry. Two of these activities are due to pyridine nucleotide-disulfide family of enzymes, namely, two paralogous dihydrolipoamide dehydrogenases and NAD(P)H-nitrite reductase. A thioredoxin reductase-like protein is responsible for the third activity whereas the fourth activity is caused by a thioredoxin disulfide reductase having all the characteristics of a true thioredoxin reductase. Besides, dihydrolipoamide dehydrogenases and thioredoxin-thioredoxin reductase system of T. indiensis are also possibly responsible for chromate reductase activity of the bacterium. The genome sequence of T. indiensis will enable cloning and overproduction of the reductases identified in this work and further analysis to elucidate the similarities and differences of iron and chromium reduction pathways.In conclusion, the soluble fraction of


Human herpesvirus-8 (HHV-8), the etiologic agent of Kaposi sarcoma (KS), establishes lifelong latent infection with periodic lytic replication (“shedding”) at mucosal sites, especially the oropharynx. Patterns of HHV-8 shedding are not well understood, and require elucidation to better predict risk of HHV-8 related malignancies in those infected. We sought to characterize patterns of HHV-8 oropharyngeal shedding among diverse cohorts that enrolled HHV-8 seropositive persons.We quantified HHV-8 oral shedding using PCR among HHV-8 seropositive persons who collected at least 14 days of oral swabs in 22 studies on 3 continents. We excluded persons taking antivirals during sampling or any prior use of antiretrovirals in those who were HIV-infected.p < 0.0001). HHV-8 was detected in ≥1 sample in 55 % of participants with a median of 7 % of days in the US and Kenya, 0 % in Uganda and Peru, and 18 % in Cameroon. Median episode duration was three days, and episodes with high median quantity lasted longer . In persons with multiple observations over time, 66 % of shedding rate variance was attributable to differences between individuals.248 participants were enrolled from the US, Peru, Cameroon, Uganda, and Kenya; 61 % were men, 58 % were HIV seropositive, and 16 % had KS. Overall, 3,123 of 10,557 samples (29.6 %) had HHV-8 detected. Quantity of virus shed was highly correlated with shedding rate, (ρ = 0.72, In HHV-8 infected individuals from diverse settings, oral mucosal shedding rate, quantity, and duration were correlated; individual shedding was highly variable. Studies are needed to determine factors accounting for between-person variation and the relationship of HHV-8 shedding to development of associated diseases. Human herpesvirus-8 (HHV-8), also known as Kaposi sarcoma-associated herpesvirus (KSHV), establishes lifelong latent infection, punctuated with periods of lytic replication at mucosal sites (“shedding”) . SheddinDespite high seroprevalence of HHV-8 in many regions, HHV-8 associated diseases are relatively uncommon, except in the endemic regions of SSA, or in persons with advanced immunosuppression. KS remains the second most prevalent cancer in men and the fifth most prevalent cancer in women in the WHO Africa region . Previou4 copies/mL in 23 (16 %), and unmeasured in 70 (49 %). Sixteen (44 %) had endemic KS, and 20 (56 %) had HIV-associated KS. All participants from Peru and Cameroon were HIV-infected, while other sites enrolled both HIV-infected and uninfected persons [Table Between 1993 and 2011, 248 subjects met inclusion criteria and contributed 298 sessions (observation periods) to the primary analysis, with median duration of 30 days (range 14–136) per session [Fig. Overall, 10,557 swabs were analyzed for HHV-8 DNA, and HHV-8 was detected in 3,123 (29.5 %). Within the entire cohort, 137 persons (55 %) had HHV-8 DNA detected at least once. The median (IQR) shedding rate per session was 7 % (0–73 %) in the US, 0 % (0–33 %) in Peru, 18 % (0–56 %) in Cameroon, 0 % 0–34 %) in Uganda, 7 % (0–43 %) in Kenya, and 4 % (0–54 %) overall [Table  % in Uga10 copies/mL [Fig. p < 0.0001) [Fig. p < 0.0001) (data not shown).The median quantity of HHV-8 DNA in samples with HHV-8 detected was 4.7 IQR: 3.7–5.6) log.7–5.6 lo10 copies/mL) had a predicted median duration of 42 days; for episodes with lower median quantity (≤4.5 log10 copies/mL), the median duration was 3 days (p < 0.0001). We investigated the duration of shedding episodes and intervals without HHV-8 shedding between episodes. “Short” episodes were defined as ≤ 3 days  and “long” episodes as > 3 days. Short episodes were preceded by a median 13-day interval without shedding whereas long episodes were preceded by a median 7-day interval (p = 0.035). A sensitivity analysis reclassifying episodes censored prior to 3 days (26 episodes) as “short” instead of “long” provided similar estimates.We defined a shedding episode as a period of detectable HHV-8 of at least one day bounded by at least two preceding and subsequent negative swabs, and could include singular missed or negative swabs . When inTo illustrate patterns of HHV-8 shedding, data from participants who collected swabs for 30–60 days and who shed at least once were included in a heatmap Fig. . We did Stability of shedding rate over time was evaluated in participants contributing multiple sessions. Thirty-nine participants, all of whom were from the United States, had at least one additional session within two years of the first: 19 persons had 2, 9 persons had 3, 8 persons had 4, and 3 persons had 5, totaling 112 sessions. The median shedding rate of first sessions was 50 %. Over time, 4 persons (10.3 %) had shedding rate increase by at least 30 % on an absolute scale, 7 persons (18.0 %) had shedding rate decrease by the same amount, and 28 (71.8 %) had no consistent pattern or a stable shedding rate Fig. . Using vThis is the largest study of HHV-8 shedding published to date, evaluating more than 10,000 oral swab samples collected from geographically and demographically diverse HHV-8 seropositive persons not receiving antiviral or antiretroviral therapy. HHV-8 shedding was a common event, although nearly half of participants never shed while under observation. We found a strong correlation between shedding rate, quantity, and duration of oropharyngeal shedding within an individual, but significant variability between individuals.Our findings are consistent with earlier studies of HHV-8 oropharyngeal shedding that observed 15 %-32 % shedding rates , 17. In HHV-8 shedding in relationship to KS and HIV has been studied and results are inconsistent. In Uganda, KS patients were more likely to have HHV-8 detected than those with asymptomatic HHV-8 infection, while HIV serostatus was not associated with detection of HHV-8 in the oral mucosa . AnotherOur study was limited by performance characteristics of the HHV-8 serologic assays used to include and exclude participants, which have sub-optimal sensitivity and specificity , 27. We We did not detect a consistent or singular pattern to HHV-8 shedding, and substantial variability was observed between persons. We demonstrated that rate and quantity of HHV-8 shedding are highly correlated and that persons experiencing longer and higher quantity shedding episodes can be predicted to shed more frequently in the future. Patterns of HHV-8 shedding are distinctly different from other well-characterized herpesvirus infections, such as HSV-2. This work provides a foundation to assess mucosal shedding of HHV-8 as a possible biomarker to assess risk of HHV-8 transmission or disease. Longitudinal studies in asymptomatically infected persons are needed to further understand clinical correlates to patterns of HHV-8 shedding, and if replication is associated with HHV-8 related diseases.We evaluated 22 prospective cohorts in which HHV-8 DNA quantification had been performed on oropharyngeal swabs, including 18 cohorts from the Virology Research Clinic, University of Washington , 28–32, 233 region of orf26 orf73 (latency-associated nuclear antigen-1)orf73 plus T07-K12 (kaposin)orf73 as the gold standard, orf26 had sensitivity 83.1 %, specificity 98.8 %. To minimize the variance in sensitivity at low end of detectable range, a cut-off of ≥150 copies/mL (≥3 copies/PCR reaction) were considered positive for HHV-8 detection [2 = 0.75. Compared to qPCR with orf73 and T07-K12 multiplex as the gold standard, orf73 had a sensitivity of 94.1 %, specificity of 97.5 %. Applying the minimum cut-off for positivity, the correlation between the latter two primer sets was R2 = 0.96. Quantitation standard using the cloned PCR product of KS330Bam233 primers was consistent throughout all cohorts. Quality control using standardization and positive and negative controls for each batch were supervised by M.L.H. in the same Seattle-based reference laboratory for all cohorts. Overall, 92 % of samples were run with the orf73 assay, 5 % with the orf26 assay and 3 % with the orf73 and T07.K12 multiplex assay.HHV-8 seropositivity was determined by positive test result on either immunofluorescence assay (IFA) against latent or lytic antibodies  or wholeetection . Applyin10 transformed for analysis and was assessed as maximum and median log10 copies/mL. We used Pearson correlations to evaluate the association of shedding rate with maximum and median log10 copy number within sessions.Sessions are defined as discrete periods of contiguous swab collection ≥14 days and separated in time from other observation periods. For participants contributing multiple sessions, the first and second sessions were included in the primary analysis. For each session, shedding rate was defined as number of swabs with HHV-8 DNA detection, divided by total swabs. Quantity of HHV-8 was logTo test the association of episode duration with time between episodes, we classified episodes as “short” or “long” by dichotomizing at the median length of all episodes and used log rank tests. First, we assumed episodes not observed to terminate were “long” and then then performed a sensitivity analysis reclassifying these episodes as “short”. We excluded Cameroonian samples from episode duration analysis since swabbing was not conducted daily.Participants with two to five sessions were included in the longitudinal shedding analysis. The proportion of variability in shedding rate ascribed to the individual was evaluated with variance components.


Inflammation constitutes one of the important components of colorectal cancer (CRC) pathogenesis. Tumor necrosis factor-α (TNF-α), a cytokine and an important inflammatory mediator plays a pivotal role in the malignant cellular proliferation, angiogenesis, tissue invasion and metastasis in CRC. The studies on association of various polymorphisms in human TNF-α gene including TNF-α-308G/A single nucleotide polymorphism (SNP) are limited, mixed and inconclusive.The aim of this study was to analyze the association of TNF-α-308G/A promoter SNP with colorectal cancer (CRC) susceptibility and development risk and also to evaluate the modifying effects of possible TNF-α-308G/A genotypes on different risk factors of CRC in ethnic population of Kashmir, India through a case–control setup. The genotype frequencies of TNF-α-308G/A promoter SNP were compared between 142 CRC patients and 184 individually matched healthy controls by using polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) method. The associations between the TNF-α-308G/A SNP and CRC risk were examined through conditional logistic regression models adjusted for multiple possible confounding (third) variables. Further, the associations between this SNP and various clinico-pathological parameters, demographic variables and environmental factors within the case group subjects with regard to CRC risk were also evaluated.The association between the TNF-α-308G/A SNP and the modulation of risk of CRC was not found to be significant . The effect of less common TNF-α-308A allele on the risk of colorectal cancer was also not found to be significant . The variant genotype (AA) was nonexistent in the study population. Further, we found no significant effect modulation of CRC risk by wild and heterozygous TNF-α-308G/A SNP genotypes in presence of different possible risk factors (p > 0.05). We also found no significant association of TNF-α-308G/A SNP with the subsets of various characteristics of the case group subjects under study (p > 0.05).This study indicates that there is no significant association between the TNF-α-308G/A promoter SNP and the risk of developing CRC in ethnic Kashmiri population. However, in order to substantiate our findings, this study needs to be replicated with bigger sample size and should involve other ethnically defined populations with high CRC risk. GloballThe CRC development is influenced by or contributed to by multiple factors that include dietary and lifestyle habits, demographic factors, various pathological conditions and genetic predispositions . Each ofCytokines play a key role in the mediation and regulation of immune response including inflammation. Several of these cytokines including pro-inflammatory and anti-inflammatory have been implicated in the process of tumorigenesis . Tumor nSeveral studies have reported the involvement of TNF-α and the important role it plays in the pathogenesis and progression of various cancers . TNF-α hThe functional significance of many of these SNPs such as those at positions − 238, − 308, − 376, − 857, − 863 and − 1031 has been reported and elucidated and have been related to the modulation of TNF-α production mostly through their effect on transcriptional activity that in turn results in differential gene expression . Among tVarious meta-analyses and other studies have reported the association of TNF-α-308G/A polymorphism with differences in susceptibility between different individuals and on a larger scale between different populations to several diseases including various types of neoplastic diseases . SeveralIn this study, we systematically conducted a case–control evaluation of the possible association between TNF-α-308G/A SNP and susceptibility to CRC in Kashmiri population. We also evaluated the possible effect modulation of CRC risk by age, gender and smoking status. Further, we investigated the possible relationship of this SNP with various clinico-pathological parameters, demographic variables and environmental factors including smoking habit and hence their role in modulating the risk of colorectal cancer in the population under study.22.1The present study involved two subject groups: case and control. The case group included 142 consecutively recruited patients with histopathologically confirmed primary colorectal cancer who underwent surgical resection for primary CRC tumors at the Department of General Surgery, Sher-I-Kashmir Institute of Medical Sciences, the largest and the only tertiary care hospital in the Kashmir valley. The tumor stage and tumor grade were classified according to the 8th edition of TNM classification of Union International Control of Cancer (UICC). Only those cases who had not received any chemo or radiotherapy were chosen for this study. All the cases were more than 18 years old and had no prior history of any malignancy. Blood and tissue samples were obtained from these CRC patients. The control group included 184 healthy individuals with no history or prior diagnosis of any malignant disorder or any other serious disease from whom blood was collected and used as control for the present study. The control group included both population based subjects and hospital-based subjects. The control group subjects were matched to the case group subjects individually for age (± 5 years), sex, place of residence  and smoking habit. Both the case and control subjects chosen for this study were ethnic Kashmiris.2.2The data relevant to the study concerning all the CRC patients including various clinico-pathological parameters, demographic variables and environmental factors including smoking habit were obtained and evaluated from patient medical records (files), pathology reports and also from the personal interviews with patients and/or their guardians (for those who were illiterate). The data collected included tumor location, Dukes Stage, lymph node status, sex, age, place of residence, smoking habits among several other relevant parameters. The relevant data was also obtained for each of the recruited controls mostly through personal interviews. All the patients and/or their guardians were informed about the study and their willingness to participate in this study was documented using a predesigned questionnaire. Same procedure was followed for the controls. The study was approved by the Institutional Ethics Committee. The work was carried out in accordance with The Code of Ethics of the World Medical Association (Declaration of Helsinki) for experiments in humans.2.3The tumor tissue samples collected after surgical resection were immediately snap frozen in liquid nitrogen and then stored at − 80 °C until further use for DNA extraction and other research purposes. Prior to DNA extraction, tumor tissue samples were washed 2–3 times in phosphate buffered saline (PBS) and adipose and connective tissue portions if any were dissected away. Peripheral blood sample, 3–5 mL from each case and control group individual was collected into EDTA-blood vacutainer collection tubes and stored at − 80 °C until further use. Genomic DNA was extracted from both the tumor tissue and blood specimens using DNeasy Blood and Tissue Kit  and Quick-gDNA™ MiniPrep kit  according to the manufacturers' instructions. The extracted DNA was stored at − 20 °C until further use. The qualitative and the quantitative assessments of the extracted genomic DNA samples were carried out by absorbance measurements at 260 nm and 280 nm using UV–visible spectrophotometeric analysis and also by agarose gel electrophoresis. The DNA extracted from blood samples of case and control group subjects was used for this study.2.4The TNF-α-308G/A (rs1800629) SNP was genotyped using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay.2.52; 0.28 mM deoxynucleotide triphosphate mix ; 0.56 μM forward and revere oligonucleotide primers  and nuclease-protease free water  added up to a final volume of 25 μL.The PCR for TNF-α-308G/A SNP was carried out in a total volume of 25 μL containing 100 ng–1 μg of genomic DNA, 0.7-1 U Taq DNA polymerase with lX Standard Taq reaction buffer , 2.1 mM MgClCCAT-3′ (forward) and 5′-CTGTCT-CGGTTTCTTCTCCATGGCG-3′ (reverse). The underlined base at the 3′-end of the forward primer was incorporated to generate an NcoI restriction site in the polymorphic region under study . The digestion was carried out according to the manufacturers' instructions in a 30 μL reaction volume containing 10 μL of PCR product and 10 U of NcoI enzyme and incubated at 37 °C overnight. NcoI enzyme cleaved the TNF-α-308G (TNF1) allele into two fragments of 173 bp and 22 bp but not the TNF-α-308A (TNF2) allele that gave a single undigested fragment of 195 bp. The digestion products were separated on 3.6–4.0% agarose gel stained with ethidium bromide . The statistical power of 80% is used widely to avoid false negative associations and to determine a cost-effective sample size under the assumption of 25% variant allele frequency, 1:1 case-to-control ratio, and 5% type I error rate (α).The effective sample size and statistical power were computed by using “Genetic Power Calculator” developed by Purcell et al. , median and inter-quartile range for continuous variables. Conditional logistic regression analysis was carried out to calculate unadjusted and adjusted odds ratios (ORs) and corresponding 95% confidence intervals (CIs) to assess the possible association of relevant TNF-α-308G/A SNP genotypes with CRC risk and to assess the possible gene-environment interactions. In order to eliminate the possible confounding (third) variables, the conditional logistic regression models were adjusted for known risk factors like gender, age and smoking habit and also with place of residence. The comparison of genotype and allele distributions of TNF-α-308G/A SNP between the CRC patient group and control group using conditional logistic regression involved two-sided chi-square test. The correlation between genotypes and clinico-pathological parameters, demographic variables and environmental factors including smoking habit within the case group was analyzed by Fisher exact test. The fitness of the genotype distributions to Hardy-Weinberg equilibrium for allele and genotype frequencies in the population under study was tested using the chi–square test. A two sided probability value of or less than 5% (p ≤ 0.05) was considered statistically significant for all types of analyses. All statistical analyses were performed using IBM SPSS Statistics v21 software.4A total of 142 primary CRC patients and 184 control subjects were included in this study with prior consent of each individual. The frequencies of various clinico-pathological parameters, demographic variables and environmental factors in colorectal cancer case subjects and relevant parameters in control subjects are listed in 2 = 0.65; p value = 0.420 and controls: χ2 = 1.91; p value = 0.167).The frequencies of the genotypes for TNF-α-308G/A SNP for both the case and the control groups are listed in The possible effect modulation of CRC risk by age, gender and smoking status is summarized in The number and the frequencies of the subsets of various characteristics of the case group subjects under study i.e. age, gender, dwelling, smoking status, tumor location, tumor grade and lymph node status for the TNF-α-308G/A SNP are listed in 5CRC is one of the most prevalent cancers worldwide and is associated with a high degree of cancer related mortality and morbidity. CRC is one of the few cancers in which inflammation is mostly evident at the earliest stages of tumor progression as a basic pathological incident and is capable of transforming the incipient tumors into full blown malignancy . CRC devTumor necrosis factor-α (TNF-α) gene is located on short arm of chromosome 6 (region p21.33) and is expressed as a 26 kDa membrane bound protein which through proteolytic cleavage by the enzyme metalloproteinase disintegrin called TNF-α converting enzyme (TACE) produces the final 17 kDa soluble active TNF-α molecule. The regulation of TNF-α expression mostly occurs at the transcriptional level . The 5′-Several meta-analyses studies have shown a moderate to strong association of TNF-α-308G/A SNP with an increased risk of and in many cases the progression of various cancers including hepatocellular carcinoma , prostathaploinsufficiency effect whereby the elevated CRC risk associated with homozygous variant (AA) genotype may be significantly decreased when only heterozygous (GA) genotype is present. At the protein level this means that TNF-α expression associated with heterozygous (GA) genotype though will be high compared to wild genotype (GG) but this increased expression resulting from a single TNF-α-308A allele might be insufficient to influence the CRC risk. It should be noted, however, that this haploinsufficiency effect though seems plausible here does not stand true for this SNP or any other SNP in every population studied as the final outcome may be influenced by several other intricate factors. Another plausible explanation of this lack of association with CRC risk is related to the location of TNF-α gene within the human leukocyte antigen (HLA) gene complex. TNF-α-308G/A SNP is located in the major histocompatibility complex (MHC) class III, telemetric to the MHC class II and centrometric to MHC class I gene. This is the most polymorphic region of the genome and there is strong linkage disequilibrium (LD) between the alleles across the HLA gene complex including that between TNF-α and other HLA genes. The direct effect of this linkage disequilibrium is that TNF-α gene and the polymorphisms within may also be linked to another gene, region or haplotype. As a result, the association or disassociation of TNF-α haplotypes itself and of other HLA haplotypes with TNF-α phenotypes and further with various pathological conditions might be due to the variation in a linked gene that regulates the expression of this cytokine rather than due to polymorphism within the TNF-α gene itself. Further, due to the wide differences in the distribution of HLA alleles across different populations in different geographical areas, the associations between TNF-α polymorphisms including TNF-α-308G/A SNP and various pathological conditions including cancer shows a considerable variation which makes it important to evaluate and provide a direct functional link of any polymorphism with the disease or condition under study so as to nullify the possibility of observed association or disassociation due to the existence of linkage disequilibrium with the actual causative gene. Therefore, further mechanistic studies are required to fully evaluate and validate the effect of this SNP on CRC risk in our population. The nonexistence of variant genotype (AA) in our population can be explained through the wide variation in the distribution of TNF-α-308A allele across different ethnically defined populations and geographical areas worldwide. The frequency of TNF-α-308A allele is significantly lower in the Asian population compared to most other world populations. The TNF-α-308A is present in 30% of white Caucasians in the United Kingdom, 10–23% of Europeans, 8–10% of South Americans, 5% of South Africans and 2–9% of East Asians  variables. The major limitations of this study are modest sample size of the study to detect gene-gene and gene-environment interactions which usually requires much larger sample size. Further studies incorporating a larger sample size and/or another ethnic population in our study are needed to confirm in depth the role of TNF-α-308G/A SNP in relation CRC susceptibility. However, these limitations are unlikely to affect the final outcome of this study.6We have demonstrated through this study that there is no significant association between the TNF-α-308G/A promoter single-nucleotide polymorphism and the risk of developing CRC in our ethnic Kashmiri population. Further, we have also shown that there is no significant association between this SNP and various clinico-pathological parameters, demographic variables and environmental factors. However, it is important to emphasize that this study does not nullify the role of TNF-α itself in the pathogenesis of CRC but only indicates that this particular and important TNF-α SNP may not be associated with CRC risk in the population studied. Further, our findings need to be replicated with bigger sample size and should involve other ethnically defined populations with high CRC risk.None exist between any parties.MZB acknowledges University Grants Commission (UGC), GOI for UGC-BSR fellowship.


Heart failure with preserved ejection fraction (HFpEF) is characterised by a high incidence of metabolic comorbidities that share the potential to induce both systemic and coronary microvascular inflammation and oxidative stress. These pathophysiological alterations contribute to increased passive stiffness of the myocardium and to diastolic dysfunction, both hallmarks of HFpEF. Passive myocardial stiffness depends mainly on two components: the extracellular matrix (ECM) and the cardiomyocytes. Quantitative and qualitative changes in collagen metabolism leading to myocardial fibrosis determine the ECM-based stiffness of the myocardium. Different noninvasive diagnostic tools to assess myocardial fibrosis are being developed, some of which have demonstrated to correlate with clinical status and prognosis. Cardiomyocytes mainly alter the passive stiffness through alterations in the giant myofilament titin, which serves as a spring. By modifying its phosphorylation state or by direct oxidative effects, titin determines cardiomyocyte-based passive stiffness. Probably the relative importance of cardiomyocyte-based changes is more important in the beginning of the disease, whereas ECM-based changes become more prominent in the more advanced stages. The present review focuses on these changes in ECM and cardiomyocytes in HFpEF and their potential prognostic and therapeutic implications. In contrast to heart failure with a reduced ejection fraction (HFrEF), patients with heart failure with a preserved ejection fraction (HFpEF) still do not benefit from evidence-based treatment options in the absence of a profound knowledge about its pathophysiology. Therefore, HFpEF therapy is aimed at comorbidities and at reducing signs and symptoms of congestion . Indeed,Although there are many other pathophysiological findings in HFpEF, as recently discussed comprehensively elsewhere , this reThe signs and symptoms of HFpEF are based on increased myocardial stiffness, leading to diastolic left ventricular (LV) dysfunction, which is defined as the inability of the heart to fill to an adequate end-diastolic volume at acceptably low pressures in the absence of endocardial or pericardial disease . AlthougLV diastole can be subdivided into two components: myocardial inactivation and myocardial stiffness. This myocardial inactivation is the consequence of dissociating contractile myofilaments and calcium reuptake into the sarcoplasmic reticulum. Myocardial stiffness can be attributed to the viscoelastic properties of the myocardium  in HFpEF patients compared with controls . These fHowever, quantification of the total collagen content with CVF seems to have less functional implications than the relative amount of the stiffer collagen type I over the more compliant collagen type III, or the amount of cross-linked collagen by lysyl oxidase  risk of complications and sampling error, both inherent to the procurement of an endomyocardial biopsy. Cardiac magnetic resonance (CMR) imaging, for example, allows for quantification of diffuse myocardial fibrosis by measurement of longitudinal relaxation time (T1 mapping). Several T1 mapping methods have been validated with endomyocardial biopsies to assess diffuse myocardial fibrosis. These techniques include post-contrast T1 mapping, calculation of extracellular volume fraction using MOLLI (Modified Look-Locker inversion recovery) sequences, and equilibrium contrast CMR . Indeed,After fibrosis is detected, its clinical or prognostic consequences need to be established. As mentioned above, extracellular volume fraction can be determined with CMR in HFpEF patients as a measure of diffuse myocardial fibrosis . ExtraceThe number of biomarkers that are currently being studied or developed reflecting myocardial fibrosis in HFpEF is growing exponentially and a detailed discussion of this topic is beyond the scope of this review. The most important and promising biomarkers were recently discussed elsewhere . EspeciaAssociations of myocardial CVF with parameters related to diastolic dysfunction such as LV end-diastolic pressure  or the Epassive) of single isolated, membrane-permeabilised cardiomyocytes was shown to be significantly higher during muscle lengthening than in HFrEF, despite increased CVF in HFrEF versus HFpEF [passive. More recently, functional experiments on small myocardial muscle strips allowed the differentiation between cardiomyocyte- and ECM-based Fpassive. In an HFpEF patient population undergoing coronary artery bypass surgery, force measurements were performed on epicardial biopsy samples obtained during surgery [passive account for more than two-thirds of total Fpassive in HFpEF [passive was also shown to be increased and to correlate with left atrial diameter [passive are therefore dependent on sarcomere length, but the actual operating range of sarcomere length in HFpEF patients is unknown. The giant protein titin forms a unique filament network in cardiomyocytes, which engages in both mechanical and signalling functions of the heart and will be discussed in more detail later [In this regard, in HFpEF patients, passive stiffness . However, CVF was not increased in these animals compared with the control group and the increase in Fpassive was attributed to a stiffer cardiomyocyte compartment [Of interest, the functional relevance of myocardial collagen on diastolic dysfunction may depend on the stage of the disease as illustrated in a ZSF1 (Zucker diabetic fatty/Spontaneously hypertensive heart failure F1 hybrid) rat model in which HFpEF is induced by obesity and diabetes mellitus on top of arterial hypertension . Twenty-partment .passive in HFpEF patients [Finally, as previously mentioned, not only the quantity of collagen but also some qualitative aspects such as the degree of collagen cross-linking  or the cpatients . On the patients .passive upon stretch [passive by means of isoform switching, phosphorylation and oxidative modifications [Next to the observed ECM changes, cardiomyocytes also undergo changes in HFpEF. When compared with HFrEF, cardiomyocytes in HFpEF patients are larger and stiffer with higher F stretch . In card stretch . Titin d stretch . Titin iications . In the ications . Howeverications .in vitro administration of PKA decreased Fpassive in isolated cardiomyocytes, suggesting a titin phosphorylation deficit in HFpEF [in vitro administration of PKA or PKG [passivein vitro [Titin can be divided into certain regions, and especially the I-band is known to contain two spring elements: the N2-B unique sequence (N2-Bus) and a region rich in proline, glutamate, valine, and lysine (PEVK) . Many sein HFpEF . Indeed,A or PKG , but alsA or PKG , 46. On in vitro , but in in vitro . It was in vitro . The clipassive and cardiomyocyte hypertrophy [For HFpEF, especially the relative hypophosphorylation of PKG-dependent titin sites, is an interesting finding that offers potential therapeutic targets Fig. . Not onlertrophy . On top ertrophy . Inflammertrophy . Also, dertrophy . The finertrophy . This capassive [Besides indirect effects via decreased NO-sGC-cGMP-PKG signalling, oxidative stress can also have direct effects on titin-based stiffness Fig. . The N2-passive .passive decreased, whereas incubation with reduced glutathione increased Fpassive, suggesting that also the redox state plays a modifying role in titin-based stiffness that needs to be studied in more detail  domains, which make up the majority of elastic titin. Usually parts of these Ig domains are folded into crypts and they can become unfolded and expose cryptic cysteines to disulphide bonding or S-glutathionylation during stretch . A recenail Fig. .in vitro and myocardial inflammation and fibrosis in vivo [Cardiomyocytes and myocardial ECM are not two completely independent compartments and a close interaction can be expected. Both have been shown to contribute to myocardial stiffness  and to b in vivo . Since c in vivo , future  in vivo . The imp in vivo .passive without any change in ECM turnover at 20 weeks of age [This interplay between endothelium, ECM and cardiomyocytes has several implications. Firstly, therapeutic options that target either the ECM (e.g. spironolactone) or the cardiomyocytes (e.g. sildenafil), cannot be expected to ‘cure’ HFpEF. Secondly, changes in cardiomyocyte function due to inflammation or oxidative stress may trigger ECM changes and, vice versa, ECM changes caused by chemical or oxidative endothelial-ECM signalling or by mechanical stress can be expected to disturb normal cardiomyocyte functioning . Thirdlys of age . On the s of age , 28.passive [This hypothesis has important therapeutic implications. If cardiomyoctes predominate the initial phases of HFpEF, it is probably useful to improve cGMP-PKG signalling to reduce cardiomyocyte Fpassive . Howeverpassive . In the passive. However, further mechanistic studies are necessary to elucidate the relative contribution and interplay between the two mechanisms, which may depend on the aetiology and comorbidities as well as on the stage of the disease.HFpEF is a complex disease of which the pathophysiology is gradually becoming unravelled. Both structural and functional alterations in cardiomyocytes and the ECM have been reported, leading to increased myocardial FOn the other hand, it is essential to develop noninvasive biomarkers for the early identification of the alterations in these two components. For instance, noninvasive techniques such as CMR with T1 mapping and circulating ECM biomarkers are promising, although future studies are needed to assess their potential to truly diagnose, stage or predict outcome and response to therapeutic strategies in HFpEF.This work was supported by a grant from the European Commission .


Our molecular study further demonstrated that miR-489 inhibited silica-induced pulmonary fibrosis primarily by repressing its target genes MyD88 and Smad3. Moreover, the up-regulated lncRNA cardiac hypertrophy-related factor (CHRF) reversed the inhibitory effect of miR-489 on MyD88 and Smad3 and then triggered the inflammation and fibrotic signaling pathways. Overall, our data indicate that the CHRF-miR-489-MyD88 Smad3 signaling axis exerts key functions in silica-induced pulmonary fibrosis and may represent a therapeutic target for silicosis.Silicosis is an incurable occupational disease associated with inflammation, fibroblast proliferation and the accumulation of extracellular matrix in lung tissues. The dysregulation of lncRNAs and miRNAs has been implicated in many complex diseases; however, the current understanding of their roles in fibrotic lung diseases, especially silicosis, remains limited. Our previous microRNA  microarray data have indicated decreased expression levels of miR-489 in lung tissues of silica-induced pulmonary fibrosis. Here, we further explored the role of miR-489 in a mouse model of silicosis. Interestingly, miR-489 levels were reduced in both macrophages that were exposed to silica and fibroblasts that were exposed to TGF-β1. Additionally, the overexpressed miR-489 carried out its anti-fibrotic role by attenuating inflammation and fibrotic progression  Silicosis, caused by the inhalation of crystalline silicon dioxide or silica, is one of the most severe occupational diseases14MicroRNAs  play a key role in gene regulation by inhibiting the translation of or promoting the degradation of target mRNAs6Long non-coding RNAs (lncRNAs) are non-protein coding transcripts longer than 200 nucleotides. Numerous molecular functions of lncRNAs have been observed, including modulation of proliferation and metastasis1014in vivo and in vitro by regulating its target genes MyD88 and Smad3, which are critical mediators in the inflammation and fibrotic signaling pathways, respectively. One interesting study has shown that the cardiac hypertrophy-associated lncRNA CHRF acts as an endogenous ‘sponge’ of miR-489, which represses miR-489 activity and functions in cardiac hypertrophyOur previous miRNA microarray study has shown that miR-489 expression is decreased in lung tissues of silica-induced lung fibrosisOur previous microarray data have shown that miR-489 levels in mouse lung tissues harvested on day 3, 7, 14, 28 and 56 after the silica injection are lower than that those observed after injection of a saline control 20. Interin vivo, as shown in P < 0.01). Furthermore, the silica-induced elevations in the protein expression of α-SMA and Vimentin were significantly reduced in the presence of miR-489 agomir. Additionally, the protein expression of E-cadherin was restored in the miR-489 agomir treatment group compared with the silica plus miR-NC group . One predicted target of miR-489 is MyD88, which is an important upstream molecule of IL-1β in the inflammatory pathway; moreover, previous studies have reported that miR-489 directly regulates MyD88The inflammatory and fibrotic signaling pathways act as important drivers in the development of pulmonary fibrosis. Having demonstrated that miR-489 blocked silica-induced pulmonary fibrosis in vivo partially abolished the elevation of IL-1β and TGF-β1  with silica and observed that miR-489 levels rapidly decreased . As expeConsidering the suppression of miR-489 in inflammatory signaling events, we attempted to determine whether miR-489 influences the fibrotic signaling pathways induced by silica. Using bioinformatics databases , we focused on another predicted target of miR-489, Smad3, a key molecule in the TGF-β1 pathway. Therefore, a luciferase reporter assay was used to determine whether Smad3 is a direct target of miR-489. In the presence of miR-489, the relative luciferase activity of Smad3-wt in NIH/3T3 cells was significantly reduced, whereas the Smad3-mut was unaffected . These rin vivo. We determined the total Smad3 and p-Smad3 levels in fibrotic lung tissue and found that total Smad3 and p-Smad3 expression both increased after silica injection compared with the expression in the day 28 saline group  was used in NIH/3T3 cells. We found that the total Smad3 and p-Smad3 levels were significantly decreased  and thatMounting evidence suggests that lncRNAs may participate in competing endogenous RNA (ceRNA) regulatory networks. The lncRNA CHRF has been reported to be an endogenous ‘sponge’ of miR-489 that inhibits the repression of miR-489 on its target genes in cardiac hypertrophyWe next determined whether CHRF is associated with the progression of silica-induced pulmonary fibrosis. Because miR-489 directly regulates MyD88 and Smad3, the potential effect of CHRF on the expression levels of MyD88 and Smad3 warranted investigation. We found that the overexpression of CHRF induced significant increases in MyD88, TGF-β1, IL-1β , Smad3, To confirm that miR-489 was a mediator of CHRF in the pulmonary fibrosis network, the co-transfection methods were used in following experiments. When co-transfecting miR-489 mimic and CHRF plasmid, we observed that the overexpression of CHRF counteracted the repression of miR-489 on its target genes   and promA more complete understanding of miRNAs has prompted researchers to focus on their roles in fibrotic diseases. For example, let-7i negatively regulates angiotensin II-induced cardiac fibrosis by suppressing the expression of interleukin-6 and multiple collagens in the heartmiR-489 has previously been identified as a key regulator of EMT progression, and the overexpression of miR-489 significantly inhibits the migration of Adriamycin (ADM)-resistant human breast cancer cells by blocking EMT signalingInflammation and the fibrotic process are two primary characteristics of pulmonary fibrosis3035in-vitro study has further shown that TGF-β1 induces EMT in bladder cancer cells and increases EMT-associated gene expressionAdditionally, when silica particulates enter the alveoli during silicosis, macrophages may be activated and injured. During this scenario, type I alveolar epithelial cells may be injured, and type II alveolar epithelial cells may be activated and differentiated into type I cells, to repair the injured alveoli. In these processes, (with the exception for the release of inflammatory cytokines), macrophages can also derive many fibrogenic factors such as TGF-β1in vivo. It is unknown which cell type or types had decreased miR-489 expression in the present study. This issue will be explored in our future study.In this study, we further demonstrated that Smad3 is another target of miR-489; this finding is supported by previous reports39LncRNAs have recently been reported to act as miRNA sponges or miRNA inhibitors (antagomirs), which interact with miRNAs and modulate the expression of miRNA target genes41However, there are some limitations to the present study: first, the regulation of miR-489 by CHRF has not yet been validated in humans because the human and mouse sequences have limited homology; second, other factors that may contribute to lung inflammation and fibrosis have not been explored. Therefore, further mechanism studies are needed to determine the relationships and regulatory interactions among CHRF, miR-489 and key molecules.In conclusion, as shown in All animal procedures were conducted in accordance with humane animal care standards, and all experimental protocol were approved by the Nanjing Medical University Ethics Committee .n = 8 in each group): day 7, 14, and 28 saline groups (control) and day 7, 14, and 28 silica groups. The mice were anesthetized using pentobarbital sodium  through intraperitoneal injection. The mice were instilled with 50 mg/kg of silica  in 0.05 ml sterile saline or 0.05 ml sterile saline intratracheally. The mice were sacrificed on day 7, 14 or 28 after instillation, and the lungs were harvested and stored at −80 °C immediately for further analysis.For the mouse model of pulmonary fibrosis, C57BL/6 male mice (4–6 weeks of age) were purchased from the Shanghai Laboratory Animal Center . A total of 48 C57BL/6 mice were randomly divided into 6 groups (n = 8 in each group): saline, silica, silica plus miR-NC and silica plus miR-489 agomir. The methods of anesthesia, saline and silica treatment were the same as mentioned above. Either 5 nmol of miR-489 agomir or miR-NC  was co-administered with silica-suspended saline. Subsequently, 2.5 nmol of miR-489 agomir or miR-NC was injected via the tail vein into each mouse weekly. The mice were sacrificed on day 28 after silica administration, and the lungs were isolated and stored at −80 °C.A total of 32 C57BL/6 male mice were randomly divided into 4 groups  for 48–72 h.The mouse macrophages (RAW 264.7) and fibroblasts (NIH/3T3), as well as the human monocytes (THP-1) and fibroblasts (MRC-5) were purchased from the American Type Culture Collection . All cell lines were maintained in Dulbecco’s modified Eagle’s medium  or RPMI Medium 1640 basic  containing 10% fetal calf serum , 100 U/ml penicillin and 100 μg/ml streptomycin . The cell lines were cultured in a humidified atmosphere containing 5% CO5 cells) into 6-well plates overnight. The macrophages were treated with 100 μg/ml silica for 12 h. The fibroblasts were treated with 1 ng/ml TGF-β1 (Sigma-Aldrich) for 48 h, and the total RNA or protein was prepared according to the experimental instructions.For western blot or qRT-PCR analysis, macrophages and fibroblasts were plated  at 4 °C overnight. Specimens were incubated for 30 min with secondary antibody, then diaminobenzidine (DAB) solution was used. Counterstaining was performed with hematoxylin. The 3D HISTECH panoramic viewer was used for imaging histologic sections and immunohistological sections.The blank slices of mouse lung tissues underwent blocking of endogenous peroxidase activity by incubation in 3% HThe degrees of severity and distribution were used to determine lesion injury including alveolar wall thickening, inflammatory lesions, collagen expression and fibrosis. A grading system was utilized for each group of animals, as described in a previous report. For lesion severity: 0 = nothing/zero, 1 = marginal, 2 = slight, 3 = moderate, 4 = severe and 5 = very severe. For lesion distribution: 0 = absent, 1 = rare/occasional (10% of the lung area), 2 = sparse/limited (10–25% of the lung area), 3 = moderate (25–50% of the lung area), 4 = extensive/widespread (50–75% of the lung area) and 5 = very extensive/predominant (over 75% of the lung area)TM RT reagent Kit . The SYBR® Premix Ex TaqTM II kit  was used for CHRF and miR-489 amplification. The primers for CHRF and the bulge-loop™ miRNA qRT-PCR Primer Sets (one RT primer and a pair of qPCR primers for each set) specific for miR-489 were designed by RiboBio Co., Ltd. . The sequences of CHRF primers were forward: 5′-GTGTTAGCCACCACTACCCAGC-3′; reverse: 5′-CCACAGCCCACAACTTTCA AG-3′. qRT-PCR analysis was performed using an ABI 7900HT Real-Time PCR System (Applied Biosystems). miR-489 expression was normalized to U6. CHRF expression was normalized to that of GAPDH. The relative expression levels were analyzed using the 2−ΔΔCT method.Total RNA from cultured cells and mouse tissues was extracted using Trizol  according to the manufacturer’s protocol. The concentration and quality of the RNA were confirmed using a Thermo NanoDrop 2000 spectrophotometer. To quantify CHRF, 500 ng of total RNA was reverse-transcribed using random primers in a 10-μl reaction. To measure miR-489, 500 ng of total RNA was reverse-transcribed using specific primers in a 10-μl reaction. Reverse transcription was performed as described using the PrimeScriptThe proteins in lung tissues and cultured cells were extracted in lysis buffer . A total of 80 μg protein extracts was electrophoresed on 12.5% polyacrylamide gradient gels and then transferred to nitrocellulose membranes. The membranes were incubated in blocking buffer (5% milk) prior to incubation with primary antibodies at 4 °C overnight. After incubation with the corresponding secondary antibody , membrane analysis was performed using a ChemiDoc XRS+ imaging system .Antibodies specific to Smad3 , p-Smad3 , α-SMA  and IL-1β  were purchased from Abcam. Antibodies specific to MyD88 , Vimentin , E-cadherin  and GAPDH  were obtained from Cell Signaling Technology. Anti-TGF-β1  was obtained from Santa Cruz.4 NIH/3T3 cells per well were seeded into 96-well plates in triplicate, then 0.4 μg of smad3 3′UTR wt or mut psiCHECK-2 plasmid was co-transfected with 20 nM miR-489 mimic (miR-489) or mimic control (mimic-NC) into NIH/3T3 cells using transfection reagent  according to the manufacturer’s instructions. Firefly and Renilla luciferase activities in cell lysates were determined using the dual-luciferase assay system (Promega) according to the manufacturer’s protocol and normalized as the quotient of Renilla/firefly luciferase activities.The wild type 3′UTR sequence of Smad3, which contains miR-489 binding sites (Smad3 3′UTR-wt), and its mutant sequence (Smad3 3′UTR-mut) were chemically synthesized  and inserted into the psiCHECK-2 plasmid (Promega). Approximately 1 × 104 macrophages or fibroblasts were seeded in 6-well plates containing 2 ml of DMEM or 1640 for 24 h. Subsequently, 25 nM of miR-489 or mimic-NC  was incubated with cells in each well for another 24 h, and this was followed by silica or TGF-β1 treatment. Cells were harvested for assays at 12 hours after silica treatment or 48 hours after TGF-β1 treatment.Approximately 1 × 10MyD88 siRNA (siMyD88) and Smad3 siRNA (siSmad3)  were diluted as suggested by the protocol. Non-targeting siRNA (siRNA-NC) was used as the negative control. SiMyD88 or siSmad3 and siRNA-NC were transfected into macrophages or fibroblasts and then treated with silica or TGF-β1 according to the previously described methods.NIH/3T3 cells were plated onto coverslips and placed in a laser scanning confocal vessel. After TGF-β1 treatment, the cells were fixed with 4% (m/v) carbinol, washed with 0.1% PBST, and blocked with 5% BSA. They were then incubated with primary antibody overnight at 4 °C. The primary antibody was specific for α-SMA . Finally, the cells were stained with anti-rabbit secondary antibody and DAPI in the dark, after being washed with 0.1% PBST. A 3D HISTECH panoramic viewer was used for cell imaging.5 cells were cultured on a 6-well plate, transfected with 5 μg/well CHRF plasmids or pUC57-NC for 24 h, and subjected to silica or TGF-β1 treatment. For siCHRF transfection, the cells were seeded into 6-well plates, transfected with 150 nM/well siCHRF or siRNA-NC for 24 h, then subjected to silica or TGF-β1 treatment. The transfection reagent  was used according to the manufacturer’s instructions.The CHRF lncRNA sequence was synthesized and subcloned into pUC57 to generate CHRF plasmids . The overexpression of CHRF was achieved via transfection with CHRF plasmids into RAW 264.7 and NIH/3T3 cells. An empty pUC57 (pUC57-NC) was used as the control. Small interfering RNAs targeting CHRF (siCHRF) were designed and synthesized by RiboBio Co., Ltd. Non-targeting siRNA (siRNA-NC) was used as a negative control. For CHRF plasmid transfection, 1 × 10t tests between two groups and one-way analysis of variance (ANOVA) for more groups with Dunnett’s test (dose 0 as the control group). A value of p < 0.05 was considered significant.All data are expressed as the means ± SD of at least three independent experiments. Data were analyzed using independent-samples How to cite this article: Wu, Q. et al. miR-489 inhibits silica-induced pulmonary fibrosis by targeting MyD88 and Smad3 and is negatively regulated by lncRNA CHRF. Sci. Rep.6, 30921; doi: 10.1038/srep30921 (2016).


TNF) is central to the immune response to Plasmodium infection. Its plasma concentration is influenced by allele variants in the promoter region of TNF. The study’s objectives were to assess TNF allele variants : (1) modulation of malaria rates in young Tanzanian children; (2) modulation of the severity of malaria as indicated by haemoglobin concentrations at the time of presentation with febrile episodes; and (3) the association between Plasmodium infection and haemoglobin concentration in symptomless parasite carriers.Tumour necrosis factor  in children reported sick. Linkage disequilibrium (LD) pattern assessment as well as haplotype analysis was conducted using HAPLOVIEW. Cox regression models used in the primary analysis accounted for multiple episodes per child.Data from a placebo-controlled trial in which 612 Tanzanian children aged 6–60 months with height-for-age z-score in the range −3 SD to 1.5 SD was utilised. Those with TNF−1031 (TNF−1031T>C); allele frequency was 0.39. Corresponding values for rs1800629 (TNF−308G>A) were 95.4% (584/612) and 0.17. Compared to the wild type genotype (TT), malaria rates were increased in the TNF−1031CC genotype  but decreased in those with the TNF−308AA genotype (corresponding HR: 0.13 [0.02‒0.63] and 0.16 [0.04‒0.67]). These associations were weaker when analysing first episodes of malaria . No evidence that allele variants of TNF−1031 and TNF−308 affected haemoglobin concentration at first episode of malaria, or that they modified the association between Plasmodium infection and haemoglobin concentrations at baseline was observed.Genotyping of 94.9% (581/612) children for TNF−1031CC genotype was associated with increased rates of malarial episodes, whereas the TNF−308AA genotype was associated with decreased rates.In this cohort of Tanzanian children, the  TNF) is central to the immune response to Plasmodium infection because of its pyrogenic properties and its key role in triggering the cascade of pro-inflammatory cytokines that regulate immune cells. It is secreted predominantly by activated macrophages following exposure to Plasmodium antigens, and acts to suppress parasitaemia.Tumour necrosis factor (TNF promoter at positions 1031T>C (rs1799964), −863C>A (rs1800630), −857C>T (rs1799724), −575G>A, −376G>A (rs1800750), −308G>A (rs1800629), −244G>A, and −238G>A (rs361525) relative to the transcription start site [TNF variants with various infectious diseases, but others failed to show an association [Eight variants have been described within the 31T>C rs199964, −8TNF1031−C allele has been associated with decreased risk of severe malaria in a child cohort of Gambian malaria patients but this observation was not made in similar cohorts from Kenya and Malawi [TNF1031−C allele is associated with elevated plasma TNF concentrations and increased susceptibility to severe falciparum malaria [TNF−308AA has been associated with severe cerebral malaria in Gambian children, and with a seven-fold increased risk of death and severe cerebral malaria [TNF−308A allele to be associated with shorter intervals of malaria reinfection [TNF−308AA was associated with both severe malaria and other infections [TNF−308AA and malaria. A study of malaria patients from Thailand found no association between TNF−308AA and disease severity. This finding is similar to that of Ubalee et al. [TNF−308AA [TNF−308GA heterozygotes but did not observe major differences in other malaria related indices [In relation to malaria, the d Malawi . Also ob malaria . Furthernfection . A Kenyanfection . Anotherfections . Some ste et al. , which dNF−308AA . Another indices .TNF allele variants on malaria outcomes. Children with an elevated TNF response to Plasmodium infection are more likely to become sicker than their peers with a lower TNF response capacity, so that the former may experience more frequent episodes of malaria. On the other hand, in children exposed to chronic or repeated Plasmodium infections, an increased TNF response associated with the TNF−308AA genotype may intensify a hepcidin-mediated block in iron absorption and consequently induce or exacerbate iron deficiency [There have been few cohort studies to longitudinally assess the association of ficiency , which pficiency , 19.TNF−1031 and TNF−308 allele variants with malaria rates in young Tanzanian children. Additionally, the study aimed to investigate the association of these variants on the severity of malaria as indicated by haemoglobin concentrations at the time of presentation with febrile episodes. Lastly, how these variants modulated the association between Plasmodium infection and haemoglobin concentration in symptomless parasite carriers was assessed.The aim of this study was therefore to assess the associations of Plasmodium falciparum [Data from a randomized trial that aimed to assess the effect of supplementation with zinc and other micronutrients on malaria rates was used. The trial was conducted between February 2008 and March 2009 in four rural villages in Handeni District, northeastern Tanzania. Residents primarily belong to the Wazigua and Wabondei Bantu tribes, but settlement of migrant plantation workers has resulted in a mixture of tribes with different origins and much intermarriage. The area is mainly populated by poor farmer families involved in subsistence farming. Malaria transmission is intense and perennial, with nearly all infections being due to lciparum .Plasmodium infection was detected in fresh blood by rapid dipstick test . Children with a positive test result were treated immediately with artemether-lumefantrine. The location of the child’s homestead was determined using a global positioning system. Further details about recruitment procedures are reported elsewhere [All resident children were eligible for randomization when aged 6–59 months and with a height-for-age z-score in the range −3 SD to 1.5 SD. Children with haemoglobin concentration <70 g/L, signs of chronic illness, and those unlikely to remain permanently resident or comply with the supplementation for the duration of the trial, or whose parents or guardians declined consent, were excluded from the study. Venous blood samples were collected in EDTA tubes and centrifuged immediately. An aliquot of 90 μL erythrocyte sediment with the buffy coat was mixed with 90 μL phosphate-buffered saline and 180 μL of DNA stabilizing buffer  and stored at 4°C for subsequent genotyping. Plasma samples were stored in liquid nitrogen in the field and at −80°C during transport and subsequent storage until biochemical analysis in The Netherlands. Haemoglobin concentration was measured in an aliquot of whole blood by a haematology analyser . Plasmodium infection (binary) and age class  and randomly permuted blocks with size randomly selected of four or eight. They then received daily supplements with either zinc alone (10 mg as gluconate), multi-nutrients without zinc, zinc combined with multi-nutrients or placebo. Supplements, in the form of powder in colour-coded capsules, were contained in blister packs, and administered orally after suspending capsule contents in clean water or breastmilk. Supplementation was performed by local community volunteers, who reported adherence daily to field staff at the research dispensary.Children were randomized within six strata defined by P. falciparum and other Plasmodium species) was detected by rapid tests . Axillary temperature was measured using an electronic thermometer and dipstick tests were administered for children with guardian-reported fever; for those with positive test results, plasma samples were collected and measured whole-blood C-reactive protein concentrations using a point-of-care test. Plasma was stored as described for the recruitment procedure. Artemether-lumefantrine  was administered to any child with current Plasmodium infection upon enrolment, or with reported fever and a positive dipstick test result during the follow-up period.A clinical officer was on duty at the research clinic day and night. At recruitment, parents or guardians were requested to bring participating children to the dispensary immediately when detecting fever or any other illness during the intervention period. In samples collected at baseline and from sick children, the presence of parasite-specific lactate dehydrogenase  in samples collected during the first malaria episode were measured using a commercial enzyme-linked immunosorbent assay kit . Plasma concentrations of C-reactive protein were measured  on a Beckman Coulter Unicel DxC880i system according to the manufacturer’s instructions. Genotypes were determined using Illumina’s VeraCode™ GoldenGate Genotyping Assay on a BeadXpress™ platform. TNF variant allele clustering was assessed visually to determine success of genotyping. Further quality control cut-offs were: a GenCall Score of >0.5 and a call rate of ≥0.95 [TNF−1031, the reference allele was T and the alternate C. Individuals homozygous for the TNF−1031 reference allele (T) are hereon described as wild type while those homozygous for the alternate allele (C) are referred to as homozygote mutant. For the variant TNF−308, the reference allele was G and the alternate was A. Individuals homozygous for the TNF−308 reference allele (G) are hereon described as wild type while those homozygous for the alternate allele (A) are referred to as homozygote mutant.For children who presented with malarial episodes, whole-blood concentrations of haemoglobin and C-reactive protein were measured using point-of-care tests . Plasma concentrations of of ≥0.95 . For theLinkage disequilibrium (LD) pattern assessment as well as haplotype analysis was conducted using HAPLOVIEW . AnthropTNF genotype and incidence ratios based on time to first episodes, with wild-type homozygotes as reference group was calculated. In the primary analysis, group rates were compared using Cox regression with robust estimates of the standard error to account for multiple episodes within children. The extent to which supplementation with either zinc or multi-nutrients including iron modulated the magnitude of the association of TNF genotype on malaria rates was explored. The extent to which adjustment for baseline factors that were a priori expected to be prognostic for malaria  and experimental intervention modulated the estimated association of genotypes were explored. In this adjusted analysis, experimental intervention as a binary variable indicating pooled groups receiving multi-nutrients (with or without zinc) and pooled groups receiving no multi-nutrients (with or without zinc) was included. Kaplan–Meier analysis with Peto tests were used to assess associations of TNF genotypes with time-to-first episode of malaria.The primary outcome, an episode of malaria, was predefined as a positive result for either a pLDH or a HRP2 dipstick test with any of the following: (a) confirmed fever (axillary temperature ≥37.5°C as measured by electronic thermometer); or, (b) guardian-reported but unconfirmed 24-h history of fever in the presence of inflammation (whole blood C-reactive protein concentrations >8 mg/L), separated by at least 14 days from a previous malaria episode. Incidence per P. falciparum and haemoglobin concentration. Main terms for P. falciparum infection and genotype dummies were included, and adjusted as pre planned for age class, mosquito net use, height-for-age z-score, and distance between the child’s homestead and the dispensary in regression analyses.Since the additive genetic model was considered, for each genotype, two dummy variables were used to indicate the three classes, resulting in two interaction terms per variant. Regression models were then used to assess the modulation of genotypes on the associations between P. falciparum infection and haemoglobin concentration at first episode of malaria was then assessed by analysis of variance (ANOVA) and adjusted for age class, height-for-age z-score and distance between the child’s homestead and the dispensary and treatment group.The additive genetic model was considered so for each genotype, two dummy variables were used to indicate the three classes. TNF genotype modulation of the association between TNF−1031 and TNF−308, respectively. Only 3% (20/612) of children failed to complete the trial. Minor allele frequencies were 0.39 (TNF−1031) and 0.17 (TNF−308), respectively. Neither of the TNF variants showed a departure from Hardy–Weinberg equilibrium . Additionally, the variants TNF−1031 is not in linkage disequilibrium with TNF−308 (r2 = 0.03). No haplotype blocks were identified.Of 612 children recruited in the original study, 581 (94.9%) and 584 (95.4%) had DNA typed for TNF−1031C allele seemed to have larger weight-for-age z-scores than their peers with the wild type variant  . No evidence of other factors being associated with the TNF genotypes investigated was observed.Children with TNF−1031C allele (TNF−1031CC) were elevated by 41% . In the analysis of first episodes, malaria rates also seemed elevated for this genotype . Only one episode of malaria occurred in homozygotes for the TNF−308A allele (TNF−308AA). This genotype was nonetheless associated with a decrease in malaria rates by 87% . Malaria rates were similarly decreased in the analysis of first episodes, but the statistical evidence was weak (P = 0.38). Kaplan–Meier analysis did not indicate marked group-specific differences in malaria rates for specific time periods of follow-up for either of the two TNF genotypes investigated . Of 581 children, 489 children (84%) experienced at least one malaria episode while recurrent episodes occurred in 406 (69%) children Table . CompareTNF−1031 was observed  was observed. Similarly, no evidence that this association was modified by homozygosity for TNF−1031C  was found  and decreased in homozygotes for the TNF−308A allele (TNF−308AA). No evidence that allele variants of TNF−1031 and TNF−308 were associated with haemoglobin concentration at first episode of malaria, or that they modified the association between Plasmodium infection and haemoglobin concentrations at baseline was found.In this cohort of Tanzanian children, malaria rates due to TNF allele variants and malaria rates were generally more pronounced in the analysis of all episodes than in the analysis of first episodes , genotyping was successful for almost all (95%) children, and the study is believed to have captured virtually all malaria cases [Findings described here add to the growing but conflicting evidence that allele variants of the  malaria , 30, 31. malaria , 33. Suc malaria –37. A be malaria  selectedr tissue . In thisia cases .P. falciparum malaria.Secondly, both false positives (type I errors) and false negatives (type II errors) may occur especially among studies including relatively small numbers of subjects. Indeed our study had small numbers compared to studies that have previously been conducted to investigate these kind of associations. Due to this deficiency, this study may have had reduced chance of detecting a true effect due to low power. Furthermore, the likelihood that the statistically significant results reflect a true effect may be in question. To try and reduce the possibility of sample size affecting study conclusions we have avoided other possible sources of bias by having rigorous genotyping quality measures as well as diagnosis of TNF allele variants that are associated with an enhanced TNF response to chronic or repeated infections can possibly protect against newly acquired Plasmodium infections through an altered iron metabolism. Such protection is conceivably more effective in children who already have marginal iron status than in their iron-replete peers, and in children who are frequently exposed to infection. There may also be population-specific masking of gene variant effects (epistasis) or gene–environment interactions specific to certain populations. Additionally, haplotype heterogeneity between populations studied may exist. It has been proposed that the causal allele variants regulating TNF responses may actually be located downstream of the TNF gene and that TNF allele variants considered are in linkage disequilibrium with the causal variant in some populations, but not in others [TNF promoter polymorphisms have any functional effect on TNF transcription or influence disease susceptibility appears to report negative results, emphasizing the conclusion that polymorphisms at this locus are functionally silent and exist only due to linkage disequilibrium with selectable human leukocyte antigen (HLA) alleles. Due to this, recent studies have increasingly included more markers in the HLA classes. The relevance of HLA antigens to disease is well recognized [P.falciparum malaria [P.falciparum malaria in different geographical areas. With the increasing evidence of HLA associations with malaria, it may well be that TNF−1031 and TNF−308 are not functional variants but markers of causal variants located elsewhere.Thirdly, the relationship between TNF genotype and susceptibility to malaria may depend on population-specific factors. For example, as noted in the introduction, n others . Much ofcognized , 41 and cognized , 43. Sev malaria –46. It iTNF−1031CC genotype was associated with increased rates of malarial episodes, whereas the TNF−308AA genotype was associated with decreased rates. Further studies are needed to confirm these findings. Additionally, further studies considering associations with the wider MHC should be conducted so as to identify candidate functional variants associated with severity of malaria.In pre-school Tanzanian children living in an area of intense transmission, the


The E. coli mutant with ackA-pta, poxB, ldhA, and adhE deletions accumulated 0.67 g/L P3HB, up to 78.84% of cell dry weight in tube cultivation. The deletion of pyruvate formate-lyase gene pflB drastically decreased P3HB production and P3HB content to 0.09 g/L and 24.44%, respectively. Overexpressing pflB via the plasmid in its knocked out mutant restored cell growth and P3HB accumulation, indicating the importance of the pyruvate formate-lyase in microaerobic carbon metabolism. The engineered E. coli BWapld (pWYC09) produced 5.00 g/L P3HB from 16.50 g/L glucose in 24 h batch fermentation, and P3HB production yield from glucose was 0.30 g/g, which reached up to 63% of maximal theoretical yield.The alcohol dehydrogenase promoter P Thus, microbial production of P3HB via fermentation has attracted extensive attentions during the past few decades [Poly(3-hydroxybutyrate) (P3HB) is a polyester of 3-hydroxybutyrate that is synthesized by a variety of microorganisms as intracellular carbon and energy storages under unbalanced nutrients conditions . The mecAlthough considerable efforts have been devoted to decreasing the production cost of P3HB to make it economically competitive, the present price of P3HB is still not feasible to replace traditional petro-based plastics. Generally, P3HB was mainly produced from renewable carbon sources through aerobic fermentation , 5. Give phaCAB from Ralstonia eutropha was changed to the anaerobic alcohol dehydrogenase promoter PadhE, which was reported to be upregulated under oxygen limited condition. As a result, P3HB content was improved from 30% to 48% of cell dry weight [ E. coli undertakes mixed acid fermentation pathway, producing lactate, succinate, acetate, formate, and ethanol under oxygen limited conditions, which decreases the carbon flux into P3HB accumulation. E. coli mutant with deletions of ackA-pta, poxB, ldhA, and adhE was constructed and doubled the cell dry weight and improved P3HB production by 3.5-folds compared to the control [To achieve high P3HB content under microaerobic condition, the native promoter of P3HB biosynthesis operony weight . Moreove control .adhE promoter to drive the expression of phaCAB operon in E. coli mutants with defected mixed acid fermentation pathway. Moreover, the pyruvate formate-lyase, which catalyzes the coenzyme A-dependent, nonoxidative cleavage of pyruvate to acetyl-CoA and formate under anaerobic or microaerobic conditions, was overexpressed to investigate its effect on cell growth and P3HB accumulation  medium supplemented with 10 g/L glucose and then maintained at 37°C for 48 h as static cultures. For 5.5 liter bioreactor cultivation, 50 mL seed culture was transferred to the bioreactor containing 3 L LB medium supplemented with 20 g/L glucose. The pH was maintained at 7.0 via automatic addition of 5 M sodium hydroxide solution. Batch cultures were performed without air supply; the agitation was set at 75 rpm to prevent biomass sedimentation and maintain heat transfer and substrate exchange.2SO4 and then assayed with a gas chromatograph (GC) to measure the P3HB content. For glucose and by-products lactate, succinate, acetate, formate, and ethanol measurements, the supernatant of cell cultures was filtered through a 0.2 μm syringe filter and then analyzed by high-performance liquid chromatography (HPLC) equipped with an ion exchange column  and a refractive index detector.Cells were harvested by centrifugation at 8,000 ×g and 4°C for 10 min. The cell pellets were washed twice with distilled water and then lyophilized for 12 h for cell dry weight (CDW) assay. Lyophilized cells were subjected to methanolysis at 100°C for 4 h in the presence of 3% (v/v) HadhE has been demonstrated to improve the expression level of phaCAB operon under microaerobic condition [adhE controlled phaCAB was transformed into the wild type E. coli and a series of mixed acid fermentation mutants BWa, BWap, BWapl, BWapld, and BWapldf  and BWapld (pWYC09 + pMCS2pflB) were cultivated in oxygen limited 5.5 liter bioreactor to investigate cell growth and P3HB production profile. After 24 h cultivation, the recombinant BWapld (pWYC09) consumed 16.50 g/L glucose and produced 5.00 g/L P3HB; the P3HB content reached 73.58% of cell dry weight. The by-product formation was maintained at low level during the process, and finally 0.23 g/L succinate, 0.86 g/L acetate, 0.17 g/L formate, and 0.03 g/L ethanol were detected ( pflB was overexpressed, the final CDW and P3HB content were 5.88 g/L and 84.43%, respectively, accompanied with a total amount of 1.72 g/L mixed acid ( pflB helped to further improve P3HB content to 84.43%, which will facilitate downstream biopolymer purification processes.The recombinantdetected . When pfxed acid . Previou pta mutant JW2294 generated 16.60 g/L mixed acids ( pta mutant showed a 70% increase to 0.17 g/g. BWapld mutant reached up to 0.30 g/g, which was 2 times higher than that obtained from the wild type ( arcA mutant showed 46% of maximal yield under optimized microaerobic fermentation condition [ E. coli constructed here showed superior ability for microaerobic P3HB production compared to previous strains. Further optimization of different degrees of oxygen limitation in bioreactor batch cultivations should be performed to obtain much higher P3HB production titer and productivity. Since the overall redox balance is important to P3HB biosynthesis and the metabolic response [In terms of byproduct formation and P3HB production yield, the wild type strain BW25113 produced 13.60 g/L mixed acids, anded acids . In thisild type . The max pflB gene significantly decreased cell biomass and P3HB production and overexpressing pflB from the plasmid restored the cell growth and P3HB accumulation, indicating the importance of pyruvate formate-lyase for microaerobic metabolism. The engineered strain BWapld harboring pWYC09 accumulated 5.00 g/L P3HB, up to 73.58% of cell dry weight in 5.5 liter bioreactor fermentation. The P3HB production yield from glucose was 0.30 g/g, up to 63% of maximal theoretical yield. The optimization of phaCAB expression in mixed acid fermentation mutants was proved to be effective for improving microaerobically P3HB production.The alcohol dehydrogenase promoter and mixed acid fermentation mutants were combined to improve P3HB production under microaerobic condition. The deletion of


Ethyl pyruvate (EP) is a simple aliphatic ester of the metabolic intermediate pyruvate that has been demonstrated to be a potent anti-inflammatory agent in a variety of in vivo and in vitro model systems. However, the protective effects and mechanisms underlying the actions of EP against endothelial cell (EC) inflammatory injury are not fully understood. Previous studies have confirmed that endoplasmic reticulum stress (ERS) plays an important role in regulating the pathological process of EC inflammation. In this study, our aim was to explore the effects of EP on tumor necrosis factor-α (TNF-α)-induced inflammatory injury in human umbilical vein endothelial cells (HUVECs) and to explore the role of ERS in this process. TNF-α treatment not only significantly increased the adhesion of monocytes to HUVECs and inflammatory cytokine  production in cell culture supernatants but it also increased ICAM and MMP9 protein expression in HUVECs. TNF-α also effectively increased the ERS-related molecules in HUVECs . EP treatment effectively reversed the effects of the TNF-α-induced adhesion of monocytes on HUVECs, inflammatory cytokines and ERS-related molecules. Furthermore, thapsigargin  attenuated the protective effects of EP against TNF-α-induced inflammatory injury and ERS. The PERK siRNA treatment not only inhibited ERS-related molecules but also mimicked the protective effects of EP to decrease TNF-α-induced inflammatory injury. In summary, we have demonstrated for the first time that EP can effectively reduce vascular endothelial inflammation and that this effect at least in part depends on the attenuation of ERS. The incidence of cardiovascular diseases, such as atherosclerosis (AS), is increasing globally and has become a costly public health issue Pyruvate, which is the anionic form of a simple alpha-keto acid, plays a key role in intermediary metabolism as a product of glycolysis and as the starting substrate for the tricarboxylic acid (TCA) cycle 2+ imbalances or perturbations in protein glycosylation, leading to the accumulation of unfolded proteins in the ER and the activation of the unfolded protein response (UPR) pathway The endoplasmic reticulum (ER) is an organelle involved in protein folding and modification, and it acts as a major intracellular calcium store Most importantly, we assessed the anti-inflammatory effects of EP in human umbilical vein endothelial cells (HUVECs) and explored the role of ERS in this study.EP, thapsigargin , tumor necrosis factor-α (TNF-α), dimethyl sulfoxide (DMSO) and the antibody against TNF-α were purchased from the Sigma-Aldrich Company . The Cell Counting Kit-8 (CCK8) was purchased from Dojindo . PERK siRNA and the antibodies against GRP78, intercellular cell adhesion molecule 1 (ICAM-1), matrix metalloproteinase 9 (MMP9) and caspase12 were obtained from Santa Cruz Biotechnology . Human soluble adhesion molecule ICAM-1 (sICAM-1), soluble E-selectin (sE-selectin), interleukin (IL-8) and monocyte chemoattractant protein-1 (MCP-1) ELISA kits were obtained from R&D Systems . The antibodies against phosphorylated-PERK (p-PERK), PERK, ATF4 and β-actin were obtained from Cell Signaling Technology . The rabbit anti-goat, goat anti-rabbit and goat anti-mouse secondary antibodies were purchased from the Zhongshan Company .2 and 95% air. The EP solution was prepared in DMSO and diluted with culture medium immediately prior to the experiment. DMSO (0.01%) was used as the control group. As described in previous studies HUVECs  were cultured in RPMI 1640 medium  supplemented with 10% fetal calf serum, 2 mM L-glutamine, 100 U/ml penicillin, and 100 g/ml streptomycin at 37°C in 5% COCCK-8 was used to measure cell viability. Cells were cultured in a 96-well plate and exposed to various treatments, according to the manufacturer's protocols. The control group was treated with 0.1% DMSO. Then, 10 µl of CCK-8 was added to each well, and the plate was incubated at 37°C for 2 h. Optical density (OD) values were assessed at 450 nm using a microplate reader , and cell viability was expressed in terms of the OD value.4/ml DMEM medium containing 1% FBS) were then added. After incubation for 1 h, the HUVEC monolayer was gently washed with phosphate-buffered saline (PBS) to remove unbound monocytes. The fluorescence was measured to determine the levels of bound monocytes using a microplate reader  at excitation and emission wavelengths of 496 and 520 nm, respectively.Monocyte adhesion to ECs was determined using U937 cells  as previously described by our group After treatment, the cell culture supernatants were collected, and the production of sICAM1, sE-selectin, MCP-1 and IL-8 by the HUVECs was measured using special ELISA kits.For siRNA transfections, HUVECs were plated onto 6-well, 24-well or 96-well plates and allowed to grow to subconfluency. The cells were transiently transfected with the negative control or PERK siRNA at 100 pM for 24 h using the Lipofectamine RNAiMAX reagent  in OPTI-MEM media . The cells were subsequently prepared for use in further experiments.After treatment, the HUVECs were lysed in sample buffer , sonicated, boiled, run through an 8–12% Bis/Tris gel using 5× MES buffer (Invitrogen) and transferred to an Immobilon NC membrane . The membranes were blocked with 5% nonfat milk in TBST  and then probed with antibodies against GRP78, ICAM1, caspase12 and MMP9 (1∶500) and against p-PERK, TNF-α, ATF4 and β-actin (1∶1000) overnight at 4°C. The membranes were then placed in blocking buffer, washed with TBST, probed with secondary antibodies (1∶5000) in blocking buffer at room temperature for 90 min and washed. Fluorescence was detected using a Bio-Rad imaging system , and the signals were quantified using the Image Lab Software .t-test. A difference of P<0.05 was considered to be statistically significant. All of the experiments were repeated three times.All of the values are presented as the mean ± standard deviation (SD). Group comparisons were performed using ANOVA (SPSS 13.0). All of the groups were analyzed simultaneously using the LSD P>0.05). However, TNF-α increased sICAM-1 production in the cell culture supernatants to 6.14±0.41, 11.93±0.85 and 15.01±1.26 pg/well, for each respective trial . In addition, TNF-α increased the expression of the ERS-related protein GRP78, as well as ATF4, cleaved-caspase12 and p-PERK  for 6 h to explore the effects of TNF-α on cell viability, inflammatory cytokines and ERS-related molecules. As shown in P>0.05). Exposure of the HUVECs to TNF-α significantly increased their binding to the U937 monocytes to 874.02±51.69% . In addition, exposure of the HUVECs to TNF-α significantly increased the production of sICAM1, IL-8, MCP-1 and sE-selectin to 14.59±1.30 pg/well, 9.17±0.75 ng/well, 14.71±1.21 ng/well and 1.715±0.128 ng/well in the cell culture supernatants , and the effects were the most obvious at 10 mM EP, which led to decreased sICAM1, IL-8, MCP-1 and sE-selectin production to 6.95±0.57 pg/well, 4.15±0.41 ng/well, 6.94±0.65 ng/well and 0.810±0.064 ng/well. TNF-α (10 ng/mL) significantly up-regulated the expression of the ICAM-1 and MMP9 proteins to 496.15±32.87% and 320.47±19.95% in the HUVECs HUVECs were treated with EP  for 2 h and then subjected to TNF-α(10 ng/mL) treatment. As shown in P<0.01). EP treatment reversed the effects of TNF-α, decreasing the GRP78, cleaved-caspase12, ATF4 and p-PERK expression to 96.44±5.50%, 52.94±4.26%, 67.44±4.31% and 128.60±6.88% at 10 mM EP .As shown in P<0.01, P<0.01). In addition, EP treatment also decreased ICAM-1, MMP9 and TNF-α protein expression to 31.09±3.80%, 25.14±3.30% and 45.61±4.50% in the HUVECs . Compared with the TNF-α group, THA+TNF-α treatment not only significantly increased the binding of U937 monocytes to HUVECs and increased sICAM1 and MCP-1 production in the cell culture supernatants  for 2 h in the absence or presence of THA  and then subjected to TNF-α (10 ng/mL) treatment. EP treatment significantly inhibited the TNF-α-induced binding of U937 monocytes to the HUVECs to 39.81±4.05% and decreased the production of sICAM1 and MCP-1 in the cell culture supernatants to 6.80±0.57 pg/well and 7.03±0.60 ng/well , while THA and EP co-treatment reversed the down-regulation of GRP78, ATF4, cleaved-caspase12, and p-PERK protein expression to 78.07±4.35%, 32.29±3.75%, 24.80±3.48% and 138.14±7.02% . Compared with the TNF-α group, THA+TNF-α treatment significantly increased GRP78, ATF4, cleaved-caspase12 and p-PERK protein expression to 168.42±8.90%, 167.14±9.01%, 269.66±15.89% and 205.60±11.87% in the HUVECs .As shown in P<0.01, P<0.01, P>0.05, P>0.05, To further explore the role of ERS in the anti-inflammatory effects, HUVECs were treated with PERK siRNA for 24 h and then subjected to TNF-α (10 ng/mL) treatment. PERK siRNA treatment significantly inhibited the TNF-α-induced binding of U937 monocytes to HUVECs to 619.47±36.92% and decreased sICAM1 and MCP-1 production in the cell culture supernatants to 8.41±0.66 pg/well and 8.52±0.71 ng/well , while PERK siRNA+TNF-α treatment significantly decreased GRP78, ATF4, cleaved-caspase12 and p-PERK protein expression to 191.28±10.37%, 235.71±14.02%, 293.20±16.07% and 125.09±7.80% . Compared with the control siRNA group, PERK siRNA treatment alone significantly decreased GRP78 and p-PERK protein expression to 41.52±4.70%, 82.95±5.27%, 70.28±4.81% and 22.13±3.46% .As shown in Although the pathogenesis of atherosclerotic vascular disease involves multifactorial processes, accumulating evidence demonstrates that inflammation and subsequent endothelial dysfunction play fundamental roles in the initiation and progression of atherosclerosis Accumulating evidence suggests that the inflammatory cytokine TNF-α, which is a pleiotropic proinflammatory cytokine, plays an important role in the disruption of vascular function and the subsequent development of vascular disease EP has been demonstrated to protect against the inflammatory response in some cardiovascular diseases. For example, it has the ability to inhibit neutrophil activation, inflammatory cytokine release, and nuclear factor κB translocation. EP has been associated with a delayed myocardial protective effect after regional ischemia and reperfusion (IR) injury in an in vivo rat heart model Previous studies have confirmed that ERS plays an important role in regulating the pathological process of EC inflammation. Shinozaki and colleagues have confirmed that a deficiency in Herp (an ERS-related protein) suppresses atherosclerosis in apolipoprotein E knockout mice by attenuating the inflammatory response In summary, we have demonstrated for the first time that EP can effectively reduce vascular endothelial inflammation and that this effect at least in part depends on the attenuation of ERS. Furthermore, the inhibition of the ERS pathway confers a protective effect against endothelial inflammatory injury, indicating that ERS is a crucial mediator of endothelial inflammation.