An up-to-date review of biomedical applications of serratiopeptidase and its biobetter derivatives as a multi-potential metalloprotease.

Serratiopeptidase is a bacterial metalloprotease used in a variety of medical applications. The multidimensional properties of serratiopeptidase make it noticeable as a miraculous enzyme. Anti-coagulant, anti-inflammatory and anti-biofilm activity of serratiopeptidase making it useful in reducing pain and swelling associated with various conditions including arthritis, diabetes, cancer, swelling, pain and also thrombolytic disorders. It breaks down fibrin, thins the fluids formed during inflammation and due to its anti-biofilm activity, can be used in the combination of antibiotics to reduce development of antibiotic resistance. However, some drawbacks like sensitivity to environmental conditions and low penetration into cells due to its large size have limited its usage as a potent pharmaceutical agent. To overcome such limitations, improved versions of the enzyme were introduced using protein engineering in our previous studies. Novel functional serratiopeptidases with shorter length and higher stability have seemingly created a hope for using this enzyme as a more effective therapeutic enzyme. This review explains the structural properties and functional aspects of serratiopeptidase, its main characteristics and properties, pre-clinical and clinical applications of the enzyme, improved qualities of the modified forms, different formulations of the enzyme, and the potential future developments.

MicroRNA-561-3p indirectly regulates the PD-L1 expression by targeting ZEB1, HIF1A, and MYC genes in breast cancer.

Globally, breast cancer is the second most common cause of cancer-related deaths among women. In breast cancer, microRNAs (miRNAs) are essential for both the initiation and development of tumors. It has been suggested that the tumor suppressor microRNA-561-3p (miR-561-3p) is crucial in arresting the growth of cancer cells. Further research is necessary to fully understand the role and molecular mechanism of miR-561 in human BC. The aim of this study was to investigate the inhibitory effect of miR-561-3p on ZEB1, HIF1A, and MYC expression as oncogenes that have the most impact on PD-L1 overexpression and cellular processes such as proliferation, apoptosis, and cell cycle in breast cancer (BC) cell lines. The expression of ZEB1, HIF1A, and MYC genes and miR-561-3p were measured in BC clinical samples and cell lines via qRT-PCR. The luciferase assay, MTT, Annexin-PI staining, and cell cycle experiments were used to assess the effect of miR-561-3p on candidate gene expression, proliferation, apoptosis, and cell cycle progression. Flow cytometry was used to investigate the effects of miR-561 on PD-L1 suppression in the BC cell line. The luciferase assay showed that miRNA-561-3p targets the 3'-UTRs of ZEB1, HIF1A and MYC genes significantly. In BC tissues, the qRT-PCR results demonstrated that miR-561-3p expression was downregulated and the expression of ZEB1, HIF1A and MYC genes was up-regulated. It was shown that overexpression of miR-561-3p decreased PD-L1 expression and BC cell proliferation, and induced apoptosis and cell cycle arrest through downregulation of candidate oncogenes. Furthermore, inhibition of candidate genes by miR-561-3p reduced PD-L1 at both mRNA and protein levels. Our research investigated the impact of miR-561-3p on the expression of ZEB1, HIF1A and MYC in breast cancer cells for the first time. Our findings may help clarify the role of miR-561-3p in PD-L1 regulation and point to this miR as a potential biomarker and novel therapeutic target for cancer immunotherapy.

Anti-Acinetobacter Baumannii single-chain variable fragments provide therapeutic efficacy in an immunocompromised mouse pneumonia model.

BACKGROUND: The emergence of carbapenem-resistant and extensively drug-resistant (XDR) Acinetobacter baumannii as well as inadequate effective antibiotics calls for an urgent effort to find new antibacterial agents. The therapeutic efficacy of two human scFvs, EB211 and EB279, showing growth inhibitory activity against A. baumannii in vitro, was investigated in immunocompromised mice with A. baumannii pneumonia. RESULTS: The data revealed that infected mice treated with EB211, EB279, and a combination of the two scFvs showed better survival, reduced bacterial load in the lungs, and no marked pathological abnormalities in the kidneys, liver, and lungs when compared to the control groups receiving normal saline or an irrelevant scFv. CONCLUSIONS: The results from this study suggest that the scFvs with direct growth inhibitory activity could offer promising results in the treatment of pneumonia caused by XDR A. baumannii.

Targeting long non-coding RNA MALAT1 reverses cancerous phenotypes of breast cancer cells through microRNA-561-3p/TOP2A axis.

Non-coding RNAs, including Inc-RNA and miRNA, have been reported to regulate gene expression and are associated with cancer progression. MicroRNA-561-3p (miR-561-3p), as a tumor suppressor, has been reported to play a role in preventing cancer cell progression, and MALAT1 (Lnc-RNA) have also been demonstrated to promote malignancy in various cancers, such as breast cancer (BC). In this study, we aimed to determine the correlation between miR-561-3p and MALAT1 and their roles in breast cancer progression. The expression of MALAT1, mir-561-3p, and topoisomerase alpha 2 (TOP2A) as a target of miR-561-3p was determined in BC clinical samples and cell lines via qRT-PCR. The binding site between MALAT1, miR-561-3p, and TOP2A was investigated by performing the dual luciferase reporter assay. MALAT1 was knocked down by siRNA, and cell proliferation, apoptotic assays, and cell cycle arrest were evaluated. MALAT1 and TOP2A were significantly upregulated, while mir-561-3p expression was downregulated in BC samples and cell lines. MALAT1 knockdown significantly increased miR-561-3p expression, which was meaningfully inverted by co-transfection with the miR 561-3p inhibitor. Furthermore, the knockdown of MALAT1 by siRNA inhibited proliferation, induced apoptosis, and arrested the cell cycle at the G1 phase in BC cells. Notably, the mechanistic investigation revealed that MALAT1 predominantly acted as a competing endogenous RNA in BC by regulating the miR-561-3p/TOP2A axis. Based on our results, MALAT1 upregulation in BC may function as a tumor promoter in BC via directly sponging miRNA 561-3p, and MALAT1 knockdown serves a vital antitumor role in BC cell progression through the miR-561-3p/TOP2A axis.

Association of ABCB1, ABCG2 drug transporter polymorphisms and smoking with disease risk and cytogenetic response to imatinib in chronic myeloid leukemia patients.

BACKGROUND: Despite acceptable results of imatinib in the treatment of chronic myeloid leukemia (CML), some patients fail to acquire a complete cytogenetic response (CCyR), which may be caused by polymorphisms in the pharmacogenetic genes. The study aimed to evaluate the association of two polymorphisms in the ABCB1 and ABCG2 genes with cytogenetic response to imatinib and the risk of CML development. METHODS: We genotyped ABCB1 (c .2677G/T/A) and ABCG2 (c .421C/A) polymorphisms by PCR-RFLP, T-ARMS-PCR methods in 111 patients with CML and 102 sex- and age-matched healthy subjects. CCyR was determined by standard chromosome banding analysis (CBA). RESULTS: Analysis of polymorphisms showed significant association of ABCG2 c.421CA genotype (p < 0.0001; OR = 0. 17), and ABCG2c.421A allele (p < 0.0001; OR = 0.31) with decreased risk of CML. Moreover, ABCB1c.2677GT- ABCG2c.421CC combined genotype (p = 0.017; OR = 4.20) was associated with increased risk of CML. Analysis of the joint effect of SNP-smoking combination showed that smoker subjects with the ABCB1c.2677GG/GT (p = 0.001; OR = 15.96, p = 0.001; OR = 8.13, respectively) or ABCG2c.421CC genotypes (p = 0.001; OR = 5.82) had the increased risk of CML, while the risk of the CML in non-smokers carrying the ABCG2c.421CA (p < 0.0001; OR = 0. 18) genotype was strongly decreased compared with reference group. Regarding drug response, ABCG2c.421 CC/CA genotypes in the smoker patients were associated with an increased risk of resistance to imatinib (p < 0.0001; OR = 7.02, p = 0.018; OR = 4.67, respectively). CONCLUSION: Our results suggest the impact of ABCG2c .421C/A polymorphism on CML development, and smoking may have a synergistic role in the risk of CML and resistance to imatinib.

Aberrant promoter hypermethylation of miR-335 and miR-145 is involved in breast cancer PD-L1 overexpression.

PD-L1 is one of the most important immune checkpoint molecules in breast cancer that plays an important role in suppressing the immune system when confronted with tumor cells and is regulated by various microRNAs. Among them, microRNA-335-3p and microRNA-145-5p, regulated by DNA methylation, have tumor suppressor activities. We studied the role of miR-335 and -145 on PD-L1 suppression in breast cancer. The expression of miR-355 and miR-145 was significantly downregulated in BC tissues and cell lines compared to their controls, and their downregulation was negatively correlated with PD-L1 overexpression. In-silico and luciferase reporter systems confirmed that miR-335 and -145 target PD-L1. In BC tissues and cell lines, cancer-specific methylation was found in CpG-rich areas upstream of miR-335 and-145, and up-regulation of PD-L1 expression was connected with hypermethylation (r = 0.4089, P = 0.0147, and r = 0.3373, P = 0.0475, respectively). The higher levels of miR-355 and -145 in BC cells induced apoptosis, arrested the cell cycle, and reduced proliferation significantly. In summary, we found that miR-335 and -145 are novel tumor suppressors inactivated in BC, and these miRs may serve as potential therapeutic targets for breast cancer treatment.

Circulating LncRNAs landscape as potential biomarkers in breast cancer.

BACKGROUND: In Iran, the delay in diagnosis and treatment of breast cancer results in low survival rates. AIM: It is essential to characterize new therapeutic targets and prognostic breast cancer biomarkers. The rising evidence suggested that long non-coding RNAs (lncRNAs) expression levels are deregulated in human cancers and can use as biomarkers for the rapid diagnosis of breast cancer. METHODS: In the present study, a quantitative real-time polymerase chain reaction (qRT-PCR) technique was used to measure 20 oncogenic and tumor suppressor lncRNAs expression levels in whole blood samples of female breast cancer patients and healthy women. Receiver operating characteristic curve (ROC) was used to assess the diagnostic value of each selected lncRNA as a biomarker. RESULTS: The results revealed that some circulating lncRNAs (MEG3, NBAT1, NKILA, GAS5, EPB41L4A-AS2, Z38, and BC040587) were significantly down-regulated in breast cancer patients compared to healthy women. In contrast, other circulating lncRNAs (H19, SPRY4-IT1, XIST, UCA1, AC026904.1, CCAT1, CCAT2, ITGB2-AS, and AK058003) were significantly up-regulated in breast cancer patients compared to controls. It was shown that the expression levels of NKILA, and NBAT1 lncRNAs were related to tumor size, and BC040587 expression level related to age, node metastasis, tumor size, and grade (p < .05). The association between H19 and SPRY4-IT1 lncRNAs with HER-2 was confirmed statistically (p < .05). ROC curves illustrated that the blood levels of SPRY4-IT1, XIST, and H19 lncRNAs have excellent potential in discriminating breast cancer from the healthy controls, showing an AUC of 1.0 (95% CI 1.0-1.0, p = .00), 0.898 (95% CI 0.815-0.981, p = .00), and 0.848 (95% CI 0.701-0.995, p = .01), respectively. CONCLUSION: In conclusion, the expression levels of circulating H19 and SPRY4-IT1 lncRNAs in breast cancer patients could consider as the prognostic biomarkers and therapeutic targets in breast cancer, because of their excellent power in discriminating breast cancer from healthy individuals and the significant correlation of H19, and SPRY4-IT1 lncRNAs with clinicopathological traits. We also suggest the possible application of BC040587 lncRNA as a diagnostic and prognostic indicator to assess tumor progression in case of verification in larger patients' cohorts.

Targeted integration in CHO cells using CRIS-PITCh/Bxb1 recombinase-mediated cassette exchange hybrid system.

Recombinant Chinese hamster ovary (CHO) cell line development for complex biotherapeutic production is conventionally based on the random integration (RI) approach. Due to the lack of control over the integration site and copy number, RI-generated cell pools are always coupled with rigorous screening to find clones that satisfy requirements for production titers, quality, and stability. Targeted integration into a well-defined genomic site has been suggested as a possible strategy to mitigate the drawbacks associated with RI. In this work, we employed the CRISPR-mediated precise integration into target chromosome (CRIS-PITCh) system in combination with the Bxb1 recombinase-mediated cassette exchange (RMCE) system to generate an isogenic transgene-expressing cell line. We successfully utilized the CRIS-PITCh system to target a 2.6 kb Bxb1 landing pad with homology arms as short as 30 bp into the upstream region of the S100A gene cluster, achieving a targeting efficiency of 10.4%. The platform cell line (PCL) with a single copy of the landing pad was then employed for the Bxb1-mediated landing pad exchange with an EGFP encoding cassette to prove its functionality. Finally, to accomplish the main goal of our cell line development method, the PCL was applied for the expression of a secretory glycoprotein, human recombinant soluble angiotensin-converting enzyme 2 (hrsACE2). Taken together, on-target, single-copy, and stable expression of the transgene over long-term cultivation demonstrated our CRIS-PITCh/RMCE hybrid approach might possibly improve the cell line development process in terms of timeline, specificity, and stability. KEY POINTS: • CRIS-PITCh system is an efficient method for single copy targeted integration of the landing pad and generation of platform cell line • Upstream region of the S100A gene cluster of CHO-K1 is retargetable by recombinase-mediated cassette exchange (RMCE) approach and provides a stable expression of the transgene • CRIS-PITCh/Bxb1 RMCE hybrid system has the potential to overcome some limitations of the random integration approach and accelerate the cell line development timeline.

Anti-Acinetobacter baumannii single-chain variable fragments show direct bactericidal activity.

Objectives: The high resistance rate of Acinetobacter baumannii and the limited number of available antibiotics have prompted a worldwide effort to develop effective antimicrobial agents. Accordingly, identifying single-chain variable fragment antibodies (scFvs), capable of exerting direct antibacterial activity in an immune system-independent manner, may be making immunocompromised patients more susceptible to A. baumannii infections. Materials and Methods: To isolate bactericidal scFvs targeting A. baumannii, we panned a large human scFv phage display library against whole-cell extensively drug-resistant (XDR) A. baumannii strains grown as biofilm or cultured with human blood or human peripheral blood mononuclear cells plus plasma. The binding of scFv-phages to A. baumannii was assessed by the dot-blot assay. Soluble scFvs, derived from the selected phages, were assessed based on their ability to bind and inhibit the growth of A. baumannii. Results: Five phage clones showed the highest reactivity toward A. baumannii. Among five soluble scFvs, derived from positive phage clones, two scFvs, EB211 and EB279, had high expression yields and displayed strong binding to A. baumannii compared with the controls. Moreover, XDR A. baumannii strains treated with positively-charged scFvs, including EB211, EB279, or a cocktail of EB211 and EB279 (200 µg/ml), displayed lower viability (approximately 50%, 78%, and 40% viability, respectively) compared with PBS-treated bacteria. Conclusion: These results suggest that combining last-resort antibiotics with bactericidal scFvs could provide promising outcomes in immunocompromised individuals with A. baumannii infections.

Identification of a novel fully human anti-toxic shock syndrome toxin (TSST)-1 single-chain variable fragment antibody averting TSST-1-induced mitogenesis and cytokine secretion.

BACKGROUND: Staphylococcal superantigens are virulence factors that help the pathogen escape the immune system and develop an infection. Toxic shock syndrome toxin (TSST)-1 is one of the most studied superantigens whose role in toxic shock syndrome and some particular disorders have been demonstrated. Inhibiting TSST-1 production with antibiotics and targeting TSST-1 with monoclonal antibodies might be one of the best strategies to prevent TSST-1-induced cytokines storm followed by lethality. RESULTS: A novel single-chain variable fragment (scFv), MS473, against TSST-1 was identified by selecting an scFv phage library on the TSST-1 protein. The MS473 scFv showed high affinity and specificity for TSST-1. Moreover, MS473 could significantly prevent TSST-1-induced mitogenicity (the IC50 value: 1.5 µM) and cytokine production. CONCLUSION: Using traditional antibiotics with an anti-TSST-1 scFv as a safe and effective agent leads to deleting the infection source and preventing the detrimental effects of the toxin disseminated into the whole body.

Reducing Proteoglycan Synthesis and NOX Activity by ROCK Inhibitors: Therapeutic Targets in Atherosclerosis.

Atherosclerosis is a chronic inflammatory disease of the arteries characterized by the accumulation of inflammatory cells in the arterial wall. Hypertension, dyslipidemia, and hyperglycemia are major risk factors of atherosclerosis. Rho-associated protein kinase (ROCK), a serine/threonine kinase, is a downstream effector of the small GTPase RhoA. ROCK is involved in different stages of atherosclerosis. Accumulating evidence has demonstrated that ROCK signaling plays vital roles in various cellular functions, such as contraction, migration, and proliferation of smooth muscle cells. Dysregulation of the ROCK pathway is associated with atherosclerosis and hypertension. Experimental studies have shown that ROCK inhibitors may have favorable effects in ameliorating atherosclerosis. ROCK signaling has a role in proteoglycan synthesis through transactivation of the TGF-ß receptor Type I (TßRI) mediated by G-protein-coupled receptor (GPCR) agonists (endothelin-1, angiotensin II and …), and ROCK inhibitors could decrease proteoglycan synthesis and atherosclerotic plaque formation. Based on the hypothesis that targeting ROCK pathway may be effective in ameliorating atherosclerosis, we suggest that ROCK inhibitors may have a potential therapeutic role in inhibition or slowing atherogenesis. However, for this hypothesis more research is needed.

Novel serratiopeptidase exhibits different affinities to the substrates and inhibitors.

The clinical application of serratiopeptidase as an anti-biofilm and anti-inflammatory agent is restricted due to the enzyme sensitivity to the environmental conditions. In our previous study, six enzyme variants were designed by introducing different mutations and truncations that exhibited higher thermal stability. In the present study, the interaction pattern and affinity of variants to substrates and inhibitors were studied using molecular docking and in vitro studies. CABS-dock and Swiss-dock servers were used for substrate (Bradykinin and Substance-P) and inhibitor (Lisinopril and EDTA) docking, respectively. The interactions were analyzed using LigPlot, UCSF Chimera, and visual molecular dynamics packages. Free energy calculations were performed using PRODIGY. Finally, the native enzyme and the best variant in terms of interaction pattern and binding score were selected for in-vitro affinity analysis toward Bradykinin and EDTA using HPLC and casein hydrolysis test, respectively. Molecular docking revealed that T344 [8-339ss] variant showed a different pattern for both substrates and inhibitors in the way that none of the native active site residues were involved in the receptor binding. As revealed by in vitro studies, T344 [8-339ss] displayed the highest number of hydrogen bond formation in docking with Bradykinin and remarkable decrement in the binding affinity for EDTA. This was the first report on the design of novel serratiopeptidase with higher activity to Bradykinin and improved resistance to EDTA as an inhibitor.

The role of miRNA-377 as a tumor suppressor in lung cancer by negative regulation of genes belonging to ErbB signaling pathway.

BACKGROUND: The ErbB signaling pathway plays important role in the pathogenesis of lung cancer. We explored the role of miRNA-377 as a tumor suppressor in NSCLC through silencing of some genes in the ErbB pathway. METHODS AND RESULTS: The targeting effect of miRNA-377 on EGFR, MAPK1, ABL2, and PAK2 was evaluated. The expression levels of these genes and miRNA-377 were surveyed in NSCLC and normal human tissues, Calu-6, and A549 cells. Real-time PCR was used to figure out whether miRNA-377 could decrease the target genes mRNAs in transfected lung cancer cell lines. The effects of miRNA-377 on apoptosis cell and proliferation were analyzed. We showed that miRNA-377 targets EGFR, MAPK1, and PAK2 mRNAs in in-silico and luciferase reporter assay. The expression of miRNA-377 was significantly downregulated in human NSCLC tissues, Calu-6 and A549 cells compared to their controls. We observed a negative correlation between EGFR, MAPK1, PAK2, and miRNA-377 expression in human NSCLC tissues. A significant reduction in EGFR, MAPK1, and PAK2 mRNA levels was detected, following miRNA-377 transfection in Calu-6 and A549 cells. The higher levels of miRNA-377 in Calu-6, and A549 cells induced apoptosis and reduced proliferation, significantly. CONCLUSIONS: All these data reveal that miRNA-377 functions as a tumor suppressor in NSCLC and may serve as a potential therapeutic target for the treatment of NSCLC.

Bactericidal fully human single-chain fragment variable antibodies protect mice against methicillin-resistant Staphylococcus aureus bacteraemia.

OBJECTIVES: The increasing prevalence of antibiotic-resistant Staphylococcus aureus, besides the inadequate numbers of effective antibiotics, emphasises the need to find new therapeutic agents against this lethal pathogen. METHODS: In this study, to obtain antibody fragments against S. aureus, a human single-chain fragment variable (scFv) library was enriched against living methicillin-resistant S. aureus (MRSA) cells, grown in three different conditions, that is human peripheral blood mononuclear cells with plasma, whole blood and biofilm. The antibacterial activity of scFvs was evaluated by the growth inhibition assay in vitro. Furthermore, the therapeutic efficacy of anti-S. aureus scFvs was appraised in a mouse model of bacteraemia. RESULTS: Three scFv antibodies, that is MEH63, MEH158 and MEH183, with unique sequences, were found, which exhibited significant binding to S. aureus and reduced the viability of S. aureus in in vitro inhibition assays. Based on the results, MEH63, MEH158 and MEH183, in addition to their combination, could prolong the survival rate, reduce the bacterial burden in the blood and prevent inflammation and tissue destruction in the kidneys and spleen of mice with MRSA bacteraemia compared with the vehicle group (treated with normal saline). CONCLUSION: The combination therapy with anti-S. aureus scFvs and conventional antibiotics might shed light on the treatment of patients with S. aureus infections.

Quantitative evaluation of PpSP15-LmSTI1 fusion gene expression following transfection with an alphavirus-derived self-amplifying mRNA and conventional DNA vaccine platforms.

New vaccine platforms are crucial to address complex parasitic infections such as cutaneous leishmaniasis. Self-amplifying mRNA (SAM) based vaccines represent the next generation nucleic acid-based platform. In the present study, we compared the expression levels of PpSP15-LmSTI1 fusion gene in BHK-21 cells following transfection with Semliki Forest virus (SFV)-derived SAM, SFV-derived plasmid DNA (pSFV-PD) and conventional plasmid DNA (pcDNA3.1+). PpSP15-LmSTI1 fusion gene expression levels were evaluated at different time points, using quantitative Real-time PCR. All data were validated and normalized by two internal control genes. According to the results, mean values of relative expression were significantly higher for SFV-PD SAM/fusion than pcDNA/fusion and pSFV-PD/fusion at all concentrations and time points. Our results showed that higher levels of PpSp15-LmSTI1 antigen expression could be achieved using a SAM vector than pcDNA and pSFV-PD, making it a valuable and efficient alternative to conventional plasmid DNA-based vaccines against leishmaniasis.

LncRNAs AK058003 and MVIH Overexpression in the Blood Samples of Iranian Breast Cancer Patients.

Background: Long non-coding RNAs (LncRNAs) are considered as novel biological regulators and potential cancer biomarkers. LncRNAs microvascular invasion in hepatocellular carcinoma (HCC; microvascular invasion [MVIH]) and AK058003 are associated with MVIH in HCC. In breast cancer (BC), upregulated MVIH and AK058003 expression levels have been shown to promote cell proliferation, though LncRNA-AK058003 acts as a tumor suppressor in HCC. Methods: Blood samples were collected from 30 healthy women and 30 female BC patients. RNA was extracted from the blood of both groups, and cDNA was then synthesized. A real-time PCR technique was conducted to measure the expression level of LncRNA-AK058003 and MVIH. Results: The expression level of two LncRNAs in the blood samples of BC patients increased significantly compared with healthy individuals. The levels of AK058003 and MVIH were not associated with lymph node metastasis (p = 0.402 and p = 0.39), tumor size (p = 0.76 and p = 0.461), and tumor size; lymph nodes, metastasis stage (TNM; p = 0.574 and p = 0.711), respectively. Conclusion: As per our findings, LncRNA-AK058003 could serve as a suitable indicator for low stage of BC. In addition, the increased level of LncRNA-MVIH could be considered as a biomarker for BC, which needs more evaluation in the future.

Serum Calprotectin as a Blood-Based Biomarker for Monitoring Knee Osteoarthritis at Early but Not Late Stages.

OBJECTIVE: The identification of early-stage osteoarthritis (OA) is crucial for the deceleration of its progression; however, no reliable biomarker is available for this purpose. The current study aimed to determine the role of serum calprotectin in the detection of early-stage knee OA. DESIGN: In a case-control study, serum samples were collected from 84 patients with primary bilateral knee OA and 52 healthy controls. The radiographic grading of knee OA was performed using the Kellgren-Lawrence classification system. Serum concentrations of calprotectin were measured using an enzyme-linked immunosorbent assay. RESULTS: The mean serum calprotectin level was 2908 ± 2516 ng/mL in OA patients and 901 ± 875 ng/mL in healthy control subjects (P < 0.001). Mean serum calprotectin levels were significantly higher in the lower stages of OA: 3740 ± 2728 ng/mL in OA grade I, 3100 ± 2084 ng/mL in OA grade II, 2246 ± 1418 ng/mL in OA grade III, and 2035 ± 765 ng/mL in OA grade IV (P = 0.047). Serum calprotectin levels were significantly higher in patients with a disease duration <42 months compared with those with a disease duration >42 months (P = 0.043). CONCLUSION: Serum calprotectin level increases significantly in the early stages of OA and shows a reverse association with disease severity. Therefore, it could be suggested as a promising blood-based marker for early-stage knee OA.

The value of serum total oxidant to the antioxidant ratio as a biomarker of knee osteoarthritis.

BACKGROUND & AIMS: The balance between oxidants and antioxidants is known as oxidative balance, which is impaired in many disease conditions such as osteoarthritis (OA). In this study, we aimed to evaluate this balance in OA patients through the evaluation of the oxidant to the antioxidant ratio. MATERIALS AND METHODS: A total of 62 knee OA patients and 20 age, sex, and BMI-matched healthy controls were included in this cross-sectional study. Serum total oxidant status (TOS) and total antioxidant capacity (TAC) were evaluated using the oxidation-reduction colorimetric assay. The TOS to TAC ratio (TOS/TAC) was evaluated as an estimate of the oxidant to antioxidant balance. RESULTS: The mean TOS was 14.2 ± 2 µM in the healthy controls and 23.3 ± 7 µM in the OA patients (p < 0.001). The mean TAC was 38.8 ± 6.6 µM in the healthy subjects and 35.8 ± 12 µM in the OA patients (p = 0.33). The mean TOS/TAC was 0.38 ± 0.09 in the healthy subjects and 0.72 ± 0.3 in the OA patients (p < 0.0001). TOS/TAC value was capable of distinguishing OA patients from healthy controls with the sensitivity and specificity of 87.1% and 80%, respectively (p < 0.001). At the cutoff value of 0.46, positive TOS/TAC (>0.46) was identified in 100% of grade I patients, whereas it was negative in 27.3%, 16.7%, and 16.7% of grades II, III, and IV, respectively (p = 0.039). CONCLUSION: In the knee OA, an equation of the serum TOS to TAC could be a good representative of oxidative balance than each component individually.

Assessment of arsenic-induced modifications in the DNA methylation of insulin-related genes in rat pancreatic islets.

Extended exposure to inorganic arsenic through contaminated drinking water has been linked with increased incidence of diabetes mellitus. The most common exposure occurs through the consumption of contaminated drinking water mainly through geogenic sources of inorganic arsenic. Epigenetic modifications are important mechanisms through which environmental pollutants could exert their toxic effects. Bisulfite sequencing polymerase chain reaction method followed by Sanger sequencing was performed for DNA methylation analysis. Our results showed that sodium arsenite treatment significantly reduced insulin secretion in pancreatic islets. It was revealed that the methylation of glucose transporter 2 (Glut2) gene was changed at two cytosine-phosphate-guanine (CpG) sites (-1743, -1734) in the promoter region of the sodium arsenite-treated group comparing to the control. No changes were observed in the methylation status of peroxisome proliferator-activated receptor-gamma (PPARγ), pancreatic and duodenal homeobox 1 (Pdx1) and insulin 2 (Ins2) CpG sites in the targeted regions. Measuring the gene expression level showed increase in Glut2 expression, while the expression of insulin (INS) and Pdx1 were significantly affected by sodium arsenite treatment. This study revealed that exposure to sodium arsenite changed the DNA methylation pattern of Glut2, a key transporter of glucose entry into the pancreatic beta cells (ß-cells). Our data suggested possible epigenetic-mediated toxicity mechanism for arsenite-induced ß-cells dysfunction. Further studies are needed to dissect the precise epigenetic modulatory activity of sodium arsenite that affect the biogenesis of insulin.

MiR-377 reverses cancerous phenotypes of pancreatic cells via suppressing DNMT1 and demethylating tumor suppressor genes.

AIM: The aim was to investigate the effect of miR-377 on DNMT1 expression and cancer phenotype in pancreatic cancer cells. MATERIALS & METHODS: Real-time PCR, luciferase assay, MTT and Annexin-PI staining were used. RESULTS: Decreased miR-377 and increased DNMT1 (verified as a target for mir-377) levels in pancreatic cancer tissues and cell lines in comparison with normal tissues was confirmed to be influenced by promoter methylation. Also hypermethylation of BNIP3, SPARC, TFPI2 and PENK promoters was observed in tumor samples but not in normal tissues which negatively correlated with their expression. Restoration of miR-377 resulted in a reduction of the expression of DNMT1 and reactivation of BNIP3 and SPARC genes via promoter demethylation. Furthermore, enhanced expression of miR-377 could significantly inhibit cell proliferation and induce apoptosis. CONCLUSION: Our findings showed that miR-377 through targeting DNMT1 could reduce DNA methylation of some tumor suppressor genes and restore their expression in pancreatic cancer cells.