Direct and macrophage stimulation mediated effects of active, inactive, and cell-free supernatant forms of Akkermansia muciniphila and Faecalibacterium duncaniae on hepcidin gene expression in HepG2 cells.

Hepcidin is a crucial regulator of iron homeostasis with protective effects on liver fibrosis. Additionally, gut microbiota can also affect liver fibrosis and iron metabolism. Although the hepatoprotective potential of Akkermansia muciniphila and Faecalibacterium duncaniae, formerly known as F. prausnitzii, has been reported, however, their effects on hepcidin expression remain unknown. We investigated the direct and macrophage stimulation-mediated effects of active, heat-inactivated, and cell-free supernatant (CFS) forms of A. muciniphila and F. duncaniae on hepcidin expression in HepG2 cells by RT-qPCR analysis. Following stimulation of phorbol-12-myristate-13-acetate (PMA) -differentiated THP-1 cells with A. muciniphila and F. duncaniae, IL-6 concentration was assessed via ELISA. Additionally, the resulting supernatant was treated with HepG2 cells to evaluate the effect of macrophage stimulation on hepcidin gene expression. The expression of genes mediating iron absorption and export was also examined in HepG2 and Caco-2 cells via RT-qPCR. All forms of F. duncaniae increased hepcidin expression while active and heat-inactivated/CFS forms of A. muciniphila upregulated and downregulated its expression, respectively. Active, heat-inactivated, and CFS forms of A. muciniphila and F. duncaniae upregulated hepcidin expression, consistent with the elevation of IL-6 released from THP-1-stimulated cells as a macrophage stimulation effect in HepG2 cells. A. muciniphila and F. duncaniae in active, inactive, and CFS forms altered the expression of hepatocyte and intestinal iron-mediated absorption /exporter genes, namely dcytb and dmt1, and fpn in HepG2 and Caco-2 cells, respectively. In conclusion, A. muciniphila and F. duncaniae affect not only directly but also through macrophage stimulation the expression of hepcidin gene in HepG2 cells. These findings underscore the potential of A. muciniphila and F. duncaniae as a potential therapeutic target for liver fibrosis by modulating hepcidin and intestinal and hepatocyte iron metabolism mediated gene expression.

Two fluorimetric determinations of acid α-glucosidase activity in dried blood spot: Pompe disease in Iranian population.

INTRODUCTION: Pompe disease is a lysosomal storage disorder. This study aimed to validate and compare 2 fluorimetric methods for measuring α-glucosidase acid activity in dried blood spot sample (DBS), with potential applications in neonatal screening, and disease follow-up of Pompe patients among the Iranian population for the first time. MATERIALS AND METHODS: The evaluation involved 3 enzyme levels and 7 parameters. The analysis included 141 Healthy individuals, 8 Pompe patients, and 10 obligate heterozygotes using reference and modified methods. RESULTS: Both methods exhibited highly linear calibration curves. The limit of detection (LOD) and limit of quantification (LOQ) were obtained in the micromolar concentration range in 2 methods. Inter-day and intra-day precision, expressed as relative standard deviations (RSD%) were calculated. The normal ranges were determined in healthy individuals. Receiver operating characteristic (ROC) curves were analyzed, and 2 parameters, total neutral α-glucosidase (NAG)/acid α-glucosidase (GAA) and pH ratio, were identified as cut-off values with excellent accuracy, sensitivity, and specificity for evaluating Pompe disease in both methods. CONCLUSIONS: Establishing and implementing these 2 methods for the Iranian population effectively differentiated between healthy and patient individuals. Method II, with its shorter incubation time, demonstrated practicality in the clinical setting.

The impact of Akkermansia muciniphila and its extracellular vesicles in the regulation of serotonergic gene expression in a small intestine of mice.

OBJECTIVES: A better understanding of host-microbe interactions as a cross-talk between the gastrointestinal (GI) tract and the gut microbiota can help treat and prevent GI disorders by improving the maintenance of GI homeostasis. The gut microbiota can affect signaling molecules, such as serotonin, which regulates endocrine systems through the GI tract. Moreover, studying the effects of gut microbiota in the small intestine on the human GI tract health is pivotal. METHODS: Male C57BL/6J mice (n = 30, 10 mice per group) were orally gavaged with 200 µL of PBS (control group); mice in group II were orally gavaged with 109 colony-forming units (CFU)/200 µL of viable A. muciniphila, suspended in PBS (A. muciniphila group); and mice in group III were orally gavaged with 10 µg of protein/200 µL of EVs (A. muciniphila-EV group) once daily for four weeks. The gene expression of serotonin system-related genes (Slc6a4, Tph1, Mao, Htr3, Htr4, and Htr7) was examined by quantitative real-time PCR (qPCR) method. RESULTS: Based on the results, A. muciniphila significantly affected the mRNA expression of genes related to the serotonin system (Tph1, Mao, Htr3B, and Htr7) in the duodenum and (Htr3B, Htr4 and Htr7) in the ileum of mice (P < 0.05). Moreover, A. muciniphila-derived EVs affected the expression of major genes related to the serotonin system (Tph1, slc6a4a, Mao, Htr3B, Htr4, and Htr7) in the duodenum and ileum of mice (P < 0.05). CONCLUSIONS: The present findings may pave the way for further investigation of the effects of strain-specific probiotics on the serotonergic system, which is currently in its infancy.

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.

Downregulation of ACE, AGTR1, and ACE2 genes mediating SARS-CoV-2 pathogenesis by gut microbiota members and their postbiotics on Caco-2 cells.

INTRODUCTION: Coronavirus disease-2019 (COVID-19) is a complex infection caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that can cause also gastrointestinal symptoms. There are various factors that determine the host susceptibility and severity of infection, including the renin-angiotensin system, the immune response, and the gut microbiota. In this regard, we aimed to investigate the gene expression of ACE, AGTR1, ACE2, and TMPRSS2, which mediate SARS-CoV-2 pathogenesis by Akkermansia muciniphila, Faecalibacterium prausnitzii, Bacteroides thetaiotaomicron, and Bacteroides fragilis on Caco-2 cells. Also, the enrichment analysis considering the studied genes was analyzed on raw data from the microarray analysis of COVID-19 patients. MATERIALS AND METHODS: Caco-2 cells were treated with live, heat-inactivated form and cell free supernatants of A. muciniphila, F. prausnitzii, B. thetaiotaomicron and B. fragilis for overnight. After RNA extraction and cDNA synthesis, the expression of studied genes was assessed by RT-qPCR. DNA methylation of studied genes was analyzed by Partek® Genomics Suite® software on the GSE174818 dataset. We used GSE164805 and GSE166552 datasets from COVID-19 patients to perform enrichment analysis by considering the mentioned genes via GEO2R, DAVID. Finally, the related microRNAs to GO terms concerned on the studied genes were identified by miRPath. RESULTS: The downregulation of ACE, AGTR1, and ACE2 genes by A. muciniphila, F. prausnitzii, B. thetaiotaomicron, and B. fragilis in live, heat-inactivated, and cell-free supernatants was reported for the first time. These genes had hypomethylated DNA status in COVID-19 patients' raw data. The highest fold enrichment in upregulated RAS pathways and immune responses belonged to ACE, AGTR1, and ACE2 by considering the protein-protein interaction network. The common miRNAs targeting the studied genes were reported as miR-124-3p and miR-26b-5p. CONCLUSION: In combination with our experimental data and bioinformatic analysis, we showed the potential of A. muciniphila, F. prausnitzii, B. thetaiotaomicron, and B. fragilis and their postbiotics to reduce ACE, ATR1, and ACE2 expression, which are essential genes that drive upregulated biological processes in COVID-19 patients. Accordingly, due to the potential of studied bacteria on the alteration of ACE, AGTR1, ACE2 genes expression, understanding their correlation with demonstrated miRNAs expression could be valuable. These findings suggest the importance of considering targeted gut microbiota intervention when designing the possible therapeutic strategy for controlling the COVID-19.

Studies in lung cancer cytokine proteomics: a review.

INTRODUCTION: Proteins are molecules that have role in the progression of the diseases. Proteomics is a tool that can play an effective role in identifying diagnostic and therapeutic biomarkers for lung cancer. Cytokines are proteins that play a decisive role in activating body's immune system in lung cancer. They can increase the growth of the tumor (oncogenic cytokines) or limit tumor growth (anti-tumor cytokines) by regulating related signaling pathways such as proliferation, growth, metastasis, and apoptosis. AREAS COVERED: In the present study, a total of 223 papers including 196 research papers and 27 review papers, extracted from PubMed and Scopus and published from 1997 to present, are reviewed. The most important involved-cytokines in lung cancer including TNF-α, IFN- γ, TGF-ß, VEGF and interleukins such as IL-6, IL-17, IL-8, IL-10, IL-22, IL-1ß and IL-18 are introduced. Also, the pathological and biological role of such cytokines in cancer signaling pathways is explained. EXPERT OPINION: In lung cancer, the cytokine expression changes under the physiological conditions of the immune system, and inflammatory cytokines are associated with the progression of lung cancer. Therefore, the cytokine expression profile can be used in the diagnosis, prognosis, prediction of therapeutic responses, and survival of patients with lung cancer.

Extracellular vesicles and pasteurized cells derived from Akkermansia muciniphila protect against high-fat induced obesity in mice.

BACKGROUND: Several studies have shown that probiotics have beneficial effects on weight control and metabolic health. In addition to probiotics, recent studies have investigated the effects of paraprobiotics and postbiotics. Therefore, we evaluated the preventive effects of live and pasteurized Akkermansia muciniphila MucT (A. muciniphila) and its extracellular vesicles (EVs) on HFD-induced obesity. RESULTS: The results showed that body weight, metabolic tissues weight, food consumption, and plasma metabolic parameters were increased in the HFD group, whereas A. muciniphila preventive treatments inhibited these HFD. The effects of pasteurized A. muciniphila and its extracellular vesicles were more noticeable than its active form. The HFD led to an increase in the colonic, adipose tissue, and liver inflammations and increased the expression of genes involved in lipid metabolism and homeostasis. Nevertheless, these effects were inhibited in mice that were administered A. muciniphila and its EVs. The assessment of the gut microbiota revealed significant differences in the microbiota composition after feeding with HFD. However, all treatments restored the alterations in some bacterial genera and closely resemble the control group. Also, the correlation analysis indicated that some gut microbiota might be associated with obesity-related indices. CONCLUSIONS: Pasteurized A. muciniphila and its EVs, as paraprobiotic and postbiotic agents, were found to play a key role in the regulation of metabolic functions to prevent obesity, probably by affecting the gut-adipose-liver axis.

Evaluation of autoantibodies against vimentin and α-enolase in rheumatoid arthritis patients.

INTRODUCTION: Rheumatoid arthritis (RA) is categorized as an autoimmune disease with a frequency of 0.2-1% worldwide. It is reported that various autoantibodies are produced in the RA population, particularly against citrullinated peptides. Among various candidate markers for RA diagnosis, the citrullinated proteins have the highest specificity and sensitivity for both diagnosis and prognosis of RA. Anti-mutated citrullinated vimentin and α-enolase constitute a new class of autoantibodies for early detection of RA. MATERIAL AND METHODS: 45 serum samples and 19 synovial fluid (SF) specimens collected from RA patients were considered for American College of Rheumatology criteria and 20 serum samples and 10 SF specimens were provided from healthy subjects as a control group. To assess the quantity of anti-citrullinated protein antibodies (ACPA), anti-mutated citrullinated vimentin (MCV) and anti-α-enolase in the serum and SF of RA patients were determined by the enzyme-linked immunosorbent assay (ELISA) method. For the evaluation of disease activity and joint destruction, we used the Disease Activity Score of 28 joints based on erythrocyte sedimentation rate (ESR) Disease Activity Score 28 (DAS28). Furthermore, to measure the molecular weight of vimentin and α-enolase, electrophoresis on 10% SDS-PAGE was performed as described before. RESULTS: The anti-α-enolase level among serum samples from RA patients was significantly higher than in healthy subjects (4.49 ±0.20 ng/ml vs. 0.76 ±0.12 ng/ml) (p < 0.001). There was a direct relation between α-enolase quantity and (rheumatoid factor) RF and C-reactive protein (CRP) levels. The mean ESR value in positive and negative ACPA patients was 38.2 ±22.6 mm/h and 9.2 ±5.8 mm/h respectively (p < 0.0001). The mean DAS28-ESR was 3.3. The level of anti-MCV in the serum of RA patients (244.6 ±53.3 U/ml) was higher than in serum of the healthy group (148.73 ±71.8) (p < 0.0001). The level of anti-MCV in the SF of patients was 687.5 ±148.4 U/ml. CONCLUSIONS: In conclusion, both autoantibodies against MCV and α-enolase are two important markers that increase in serum and SF of RA patients and are specific for diagnosis of RA disease.

Comparative study of interruption of signaling pathways in lung epithelial cell by two different Mycobacterium tuberculosis lineages.

Alveolar epithelial cell (AEC) provides a replication niche for Mycobacterium tuberculosis. Based on the role of AEC in M. tuberculosis pathogenesis and existence of genetic diversity within this bacterium, we investigated interactions between AEC II and two different M. tuberculosis lineages. We have compared the transcriptome and cytokines/chemokines levels of A549 infected by M. tuberculosis lineage three and four using qRT-PCR and ELISA arrays, respectively. We showed different M.  tuberculosis strains induced changes in different effectors that involved in TLRs and NF-κB signaling pathways. We observed different reaction of the studied lineages specifically in pathogenesis, immune evasion mechanism, IL-12/IFN-γ axis, and autophagy. Similar behavior was detected in regarding to apoptosis, necroptosis, anti-inflammatory responses, and canonical inflammasome. Our findings contribute to elucidate more details in pathogenesis, immune evasion strategies, novel target and druggable pathway for therapeutic intervention, and host directed therapy in tuberculosis infection. Also, different M.  tuberculosis lineages-dependent host-pathogen interactions suggested using only one strain for this kind of research will be controversial.

Mutation analysis and clinical characterization of Iranian patients with mucopolysaccharidosis type I.

BACKGROUND: Mucopolysaccharidosis type I (MPSI) is a rare autosomal recessive disorder caused by a deficiency of α-l-iduronidase (IDUA) encoded by the IDUA gene. We examined the mutation spectrum of the IDUA gene to explain the clinical, biochemical, and molecular features in 21 Iranian patients with MPSI. METHODS: Sanger sequencing was used to measure the IDUA gene sequence in the coding region and exon-intron boundaries. We recorded the clinical findings of studied patients at the first diagnosis of disease and then during the treatment and follow-up. RESULTS: Five different missense disease-causing mutations were determined in our patient groups, indicating 90.48% of detection rate. The most widespread mutation was the p.Y109H, occurring in 15.625% of all alleles, which was reported for the first time in our study. Other frequent mutations were as follows: p.Ser157Pro (12.5%), p.Gly84Arg (12.5%), p.Asp257His (9.375%), and p.Asp301Glu (9.375%). Three ones of them were new missense mutations: p.Ser157Pro, p.Asp257His, and p.Asp301Glu. DISCUSSION: The results of this study explain the different spectrum of IDUA gene mutations in our patients with MPSI. We introduced here 32 different variants including four new variants: p.Y109H (15.625%), p.S157P (12.5%), p.D257H (9.375%), and p.D301E (9.375%). In this series, there was no relationship between the happening of clinical features and genotype variations and biochemical findings.

The Comparison of Iodine-Type and MnO2-Type Oxidation for Measuring the Levels of Urine Neopterin and Biopterin in Patients with Hyperphenylalaninemia: A Descriptive-Analytic Study in Iran.

Fresh urine sample is required for measurement of pterins in patients with hyperphenylalaninemia (HPA) for detection of tetrahydrobiopterin (BH4) coenzyme deficiency. The present study aimed to measure the levels of neopterin and biopterin in the urine of patients with HPA using manganese dioxide (MnO2) and iodine oxidation methods. The levels of neopterin and biopterin in the urine of 124 patients were measured using two methods of oxidation (MnO2 and iodine) and the results were statistically analyzed using correlation test, Roc-Curve and ANOVA in SPSS-20 software. The level of significant was determined to be P > 0.05. Patients age mean ± SD = 4.96 year, neopterin and biopterin mean ± SD in 124 patients were 2.75 mmol/molcr and 2.49 mmol/molcr. Correlation regression between two methods (iodine and MnO2 oxidation) was 0.99. There was no significant difference between the two oxidation methods (P > 0.05). The results of this study showed that MnO2 oxidation can be a suitable alternative method instead of iodine oxidation for measurement of urine neopterin, biopterin and diagnosis of BH4 deficiency.

Sequence and kinetic analyses of streptokinase from two group G streptococci with high fibrin-dependent plasminogen activities and the identification of novel altered amino acids as potential hot spots.

OBJECTIVE: To gain insights on the degree of heterogeneity and kinetic differences of streptokinase (SK) from group G (SKG) Streptococci compared with standard SK from group C (SKC) and identification of potentially contributing critical residues (hotspots). RESULTS: DNA and sequencing analyses confirmed the proper construction of all SK encoding vectors (two SKGs and one standard SKC). SDS-PAGE and western blot analyses confirmed the expression and proper purification of the recombinant SKs from E.coli with the expected size of 47 kDa. Kinetic analyses of two SKGs, compared with SKC, showed higher levels of specific [(×103 IU/mg) of 725 and 715 vs. 536] and fibrin-dependent proteolytic activities [Kcat/KM (min-1/µM) of 37 and 30 vs. 23], accompanied by declined fibrin-independent amidolytic activities [Kcat/KM (min-1/mM) of 109 and 84 vs. 113], respectively. Sequence alignments identified 10 novel residual substitutions scattered in SKα (I33F, R45Q, SKG132, A47D, and G55 N), SKß (N228 K, F287I), and SKγ domains (L335 V, V396A, T403S) of SKGs, as potential hotspots. CONCLUSION: The residue substitutions identified might critically contribute as hot spots to different kinetic parameters of SKGs and might assist in further elucidation of structure/function relations and rational design of SKs with improved (fibrin-dependent) therapeutic properties.

SH2 domain-containing inositol 5-phosphatase (SHIP2) regulates de-novo lipogenesis and secretion of apoB100 containing lipoproteins in HepG2 cells.

Hepatic de-novo lipogenesis and production of triglyceride rich VLDL are regulated via the phosphoinositide 3-kinase cascade, however, the role of a negative regulator of this pathway, the SH2 domain-containing inositol 5-phosphatase (SHIP2) in this process, remains unknown. In the present study, we investigated the molecular link between SHIP2 expression and metabolic dyslipidemia using overexpression or suppression of SHIP2 gene in HepG2 cells. The results showed that overexpression of the wild type SHIP2 gene (SHIP2-WT) led to a higher total lipid content (28%) compared to control, whereas overexpression of the dominant negative SHIP2 gene (SHIP2-DN) reduced total lipid content in oleate treated cells by 40%. Overexpression of SHIP2-WT also led to a significant increase in both secretion of apoB100 containing lipoproteins and de-novo lipogenesis, as demonstrated by an enhancement in secreted apoB100 and MTP expression, increased intra and extracellular triglyceride levels and enhanced expression of lipogenic genes such as SREBP1c, FAS and ACC. On the other hand, overexpression of the SHIP2-DN gene prevented oleate-induced de-novo lipogenesis and secretion of apoB100 containing lipoproteins in HepG2 cells. Collectively, these findings suggest that SHIP2 expression level is a key determinant of hepatic lipogenesis and lipoprotein secretion, and its inhibition could be considered as a potential target for treatment of dyslipidemia.

Determination of Biological Variance and Validation of a Fluorometric Assay for Measurement of α-l-Iduronidase Activity in Dried Blood Spots Samples: The First Experience in Iran.

Methods for assaying lysosomal diseases in dried blood samples are very useful today due to its several advantages related to the stability of samples, its transportation, handled and analysis, and its potential use for newborn screening compared to traditional methods in leucocytes samples. For this reason, it is important to validate these assays before being used in routine laboratory. Because of different in biological markers based on ethnicity, we aimed this study to validation a DBS-based fluorometric assay for measurement of α-l-Iduronidase activity for diagnosis of MPS I patients in Iran. DBS samples were collected from 15 MPS I patients and 60 healthy age matched subjects. Diagnostic value, biological variance and α-l-Iduronidase activity were determined. DBS α-l-Iduronidase activity was significantly higher in male subjects than in female group. Using a cut-off level of 1.08 µmol/spot 20 h, sensitivity and specificity were 100 and 98 %. The linearity of test was proved and we showed that within-run and between run precision were 5.6 and 14.66 %. Measurement of α-l-Iduronidase activity in DBS samples is an accurate test for diagnosis of MPS I and because of its rapid shipping and simplicity to keeping, DBS-based enzyme activity could be considered as a useful diagnostic tool in this disease.

Interplay between gut microbiota and the master iron regulator, hepcidin, in the pathogenesis of liver fibrosis.

INTRODUCTION: There is a proven role for hepcidin and the composition of gut microbiota and its derivatives in the pathophysiology of liver fibrosis. AREA COVERED: This review focuses on the literature search regarding the effect of hepcidin and gut microbiota on regulating liver physiology. We presented the regulating mechanisms of hepcidin expression and discussed the possible interaction between gut microbiota and hepcidin regulation. Furthermore, we investigated the importance of the hepcidin gene in biological processes and bacterial interactions using bioinformatics analysis. EXPERT OPINION: One of the main features of liver fibrosis is iron accumulation in hepatic cells, including hepatocytes. This accumulation can induce an oxidative stress response, inflammation, and activation of hepatic stellate cells. Hepcidin is a crucial regulator of iron by targeting ferroportin expressed on hepatocytes, macrophages, and enterocytes. Various stimuli, such as iron load and inflammatory signals, control hepcidin regulation. Furthermore, a bidirectional relationship exists between iron and the composition and metabolic activity of gut microbiota. We explored the potential of gut microbiota to influence hepcidin expression and potentially manage liver fibrosis, as the regulation of iron metabolism plays a crucial role in this context.

Association Between Altered Microbiota Composition and Immune System-Related Genes in COVID-19 Infection.

Microbiota and immunity affect the host's susceptibility to SARS-CoV-2 infection and the severity of COVID-19. This study aimed to identify significant alterations in the microbiota composition, immune signaling pathways, their potential association, and candidate microRNA in COVID-19 patients using an in silico study model. Enrichment online databases and Python programming were utilized to analyze GSE164805, GSE180594, and GSE182279, as well as NGS data of microbiota composition (PRJNA650244 and PRJNA660302) associated with COVID-19, employing amplicon-based/marker gene sequencing methods. C1, TNF, C2, IL1, and CFH genes were found to have a significant impact on immune signaling pathways. Additionally, we observed a notable decrease in Bacteroides spp. and Faecalibacterium sp., while Escherichia coli, Streptococcus spp., and Akkermansia muciniphila showed increased abundance in COVID-19. Notably, A. muciniphila demonstrated an association with immunity through C1 and TNF, while Faecalibacterium sp. was linked to C2 and IL1. The correlation between E. coli and CFH, as well as IL1 and Streptococcus spp. with C2, was identified. hsa-let-7b-5p was identified as a potential candidate that may be involved in the interaction between the microbiota composition, immune response, and COVID-19. In conclusion, integrative in silico analysis shows that these microbiota members are potentially crucial in the immune responses against COVID-19.

The comparative anti-oxidant and anti-inflammatory efficacy of postbiotics and probiotics through Nrf-2 and NF-kB pathways in DSS-induced colitis model.

IBD is a disorder which could be caused by oxidative stress. This investigation aims to determine if probiotics and postbiotics can control oxidative stress and inflammation and compare the effectiveness of these two probiotic and postbiotic mixtures of substances. 88 strains of Lactobacillus and Bifidobacterium were tested for antioxidant activity. Male wild-type C57BL/6 mice were divided into four experimental groups, namely high fat diet (HFD) + PBS, HFD + DSS, HFD + DSS + 109 cfu/ml of probiotics, and HFD + DSS + 109 cfu/ml of postbiotics. The phenotypical indices and pathological scores were assessed. The expression of genes related to NF-kB and Nrf2 signaling pathways and enzymes associated with oxidant/anti-oxidant activities, and proinflammatory/inflammatory cytokines were assessed. In contrast to the groups exposed to DSS, mice treated with probiotics mixture and postbiotics mixture alongside DSS displayed alleviation of DSS-induced adverse effects on phenotypical characteristics, as well as molecular indices such as the Nrf2 and NF-kB related genes, with a greater emphasis on the postbiotics component. In accordance with the findings of the present investigation, it can be inferred that even in using a high-fat dietary regimen as an inducer of oxidative stress, the emergence of inflammation can be effectively addressed through the utilization of probiotics and, more specifically, postbiotics.

Pitfalls in screening streptococci for retrieving superior streptokinase (SK) genes: no activity correlation for streptococcal culture supernatant and recombinant SK.

Streptokinase (SK), the heterogeneous protein family secreted by some groups of ß-hemolytic streptococci (ßHS), is a plasminogen activator and well-known drug for thrombolytic therapy. Differences in plasminogen activation property of streptococcal culture supernatants (SCS) have been traditionally used to identify superior producer strains and SK genes (skc) for recombinant SK (rSK) production. However, the role of SK heterogeneity and whether SK activities in SCS correlate with that of their corresponding rSK is a matter of debate. To address these concerns, SCS of nine group C streptococci (GCS) screened among 252 ßHS clinical isolates were compared for plasminogen activation using S-2251 chromogenic assay. The GCS (Streptococcus equisimilis) showing the highest (GCS-S87) and lowest (GCS-S131) activities were selected for PCR-based isolation of skc, cloning and rSK production in Escherichia coli. The 6×His-tagged rSK proteins were purified by NI-NTA chromatography, analyzed by SDS-PAGE and Western blotting and their activities were determined. While SCS of GCS-S87 and GCS-S131 showed different plasminogen activations (95 and 35 %, respectively) compared to that of the reference strain (GCS-9542), but interestingly rSK of all three strains showed close specific activities (1.33, 1.70, and 1.55 × 10(4) IU mg(-1)). Accordingly, SKS87 and SKS131 had more than 90 % sequence identity at the amino acids level compared to SK9542. Therefore, SK heterogeneity by itself may not contribute to the differences in plasminogen activation properties of SCS and evaluation of this activity in SCS might not be a proper assay for screening superior skc.

Evaluation and comparison of soluble transferrin receptor in thalassemia carriers and iron deficient patients.

Iron is an essential component in the structure of certain molecules such as hemoglobin (Hb), myoglobin, cytochrome C and some enzymes. The iron gateway to cells is transferrin receptor (TfR). Soluble transferrin receptor (sTfR) is a product of the TfR that circulates in plasma, its concentration therefore, is proportional to the total concentration of cellular TfR. Expression of TfR is elevated in anemia. The aim of this study was to evaluate the efficacy of sTfR determination in the diagnosis of iron deficiency in thalassemia carriers. In this cross-sectional study, 168 cases were examined. The subdivision of cases included: iron deficiency, concurrent thalassemia and iron deficiency, severe α-thalassemia (α-thal) (α-thal-1), mild α-thal (α-thal-2) and ß-thal minor. Hematological and biochemical variables were analyzed by the Statistical Package for Social Sciences (SPSS) version 16 statistical software. Analysis of variance was carried out using the non parametric Kruskal-Wallis test. Significant differences were observed in median values in sTfR concentration and sTfR/log ferritin (sTfR-F index) iron deficient groups, compared to thalassemia groups (p value <0.001), with both variables having higher values in iron deficient groups. In this study it was demonstrated that in iron deficient thalassemic patients, high sTfR can be a good indicator of iron deficiency anemia. The result of the sTfR test can be used for calculation of the sTfR-log ferritin index (sTfR-F index) (obtained by division of sTfR into log ferritin). This is a more sensitive parameter for iron deficiency diagnosis because in its calculation, two valuable amounts of sTfR and ferritin were taken into consideration.

The proportion of tetrahydrobiopterin deficiency and PAH gene deficiency variants among cases with hyperphenyalaninemia in Western Iran.

BACKGROUND: Defects either in phenylalanine hydroxylase (PheOH) or in the production and recycling of its cofactor (tetrahydrobiopterin [BH4]) are the causes of primary hyperphenylalaninemia (HPA). The aim of our study was to investigate the current status of different variants of HPA Kurdish patients in Kermanshah province, Iran. MATERIALS AND METHODS: From 33 cases enrolled in our study, 32 were identified as HPA patients. Reassessing of pre-treatment phenylalanine concentrations and the analysis of urinary pterins was done by high-performance liquid chromatography method. RESULTS: A total of 30 patients showed PAH deficiency and two patients were diagnosed with BH4 deficiency (BH4/HPA ratio = 6.25%). Both of these two BH4-deficient patients were assigned to severe variant of dihydropteridine reductase (DHPR) deficiency. More than 75% of patients with PAH deficiency classified as classic phenylketonuria (PKU) according their levels of pre-treatment phenylalanine concentrations. CONCLUSION: Based on the performed study, we think that the frequency of milder forms of PKU is higher than those was estimated before and/or our findings here. Furthermore, the frequency of DHPR deficiency seems to be relatively high in our province. Since the clinical symptoms of DHPR deficiency are confusingly similar to that of classic PKU and its prognosis are much worse than classical PKU and cannot be solely treated with the PKU regime, our pilot study support that it is crucial to set up screening for BH4 deficiency, along with PAH deficiency, among all HPA patients diagnosed with HPA.