Association between Toxoplasma gondii and gestational diabetes with regard to serum leptin and tumor necrosis factor alpha, a preliminary study.

Gestational diabetes (GDM), the onset of any degree of glucose intolerance during pregnancy, increases a wide range of adverse health outcomes for both the mother and the fetus. The aim of the present study was to evaluate the association of Toxoplasma gondii infection with GDM in a case-control study with regard to the levels of leptin and tumor necrosis factor alpha (TNF-α) as two inflammatory biomarkers. Fifty-one pregnant diabetic cases and 109 controls were selected from a prenatal care clinic of a general hospital in Shiraz, southern Iran during July-November 2020. Cases and controls were similar in age, gestational age and number of parturitions. The presence of IgG antibodies against T. gondii, and serum concentrations of leptin and TNF-α were determined by ELISA. Anti-Toxoplasma antibodies were detected in 25 subjects (15.6 %, 95 % CI: 9.9-21.3). Nine (18 %) diabetic cases were infected with Toxoplasma compared to 16 (15 %) healthy controls (P = 0.63). Level of leptin was higher (P = 0.07) while TNF-α was lower in diabetic cases compared to healthy controls (P = 0.08). When subjects were classified according to the combination of GDM and T. gondii, leptin was significantly lower in healthy (non-diabetic, non-infected) subjects compared to diabetics (P = 0.026), and TNF-α was higher in healthy subjects compared to Toxoplasma-infected diabetics (P = 0.032). These findings can be interpreted as both comorbidities being individually associated with increasing serum leptin and decreasing TNF-α concentrations, with modifying effects on each other. The present study opens a new perspective on GDM and its complex pathophysiological mechanism. Future research in this area is needed to better understand the underlying pathway for the development of GDM and the role of T. gondii and inflammatory biomarkers.

A novel fusion protein candidate for the serodiagnosis of Mycoplasma agalactiae infection.

BACKGROUND: The aim of current study was to construct, express, purify and immunogenicity evaluate of a novel recombinant fusion protein including Pyruvate dehydrogenase beta subunit (PDHB) and high antigenic region of lipoprotein P80 of Mycoplasma agalactiae. Using bioinformatics tools, antigenicity and physiochemical properties of fused protein were assessed. MATERIAL AND METHODS: The recombinant fusion protein of GST-PDHB-P80 were expressed in pGEX4T-1 and purified then verified by Western blot assay. The purified protein was successfully used for immunization of mice. 30 female BALB/c mice were divided into three groups (10 mice per each group) injected with GST-PDHB-P80, inactivated bacteria vaccine and PBS as negative control, separately. RESULTS: Western blot analysis confirmed the interaction between the immunized mice serum and the blotted recombinant protein GST-PDHB-P80, demonstrating the immunogenicity of this protein. Moreover, the sera of vaccinated mice with inactivated bacteria vaccine, containing whole cell proteins, detected the recombinant protein GST-PDHB-P80 confirming the antigenicity of PDHB-P80. Negative control displayed no reactivity with GST-PDHB-P80. CONCLUSION: We proposed a novel designed chimeric protein of Mycoplasma agalactiae as a potential marker for serodiagnostic assays but still further field research is required.

Plasma miR-21 as a potential predictor in prediabetic individuals with a positive family history of type 2 diabetes mellitus.

Type 2 diabetes mellitus (T2DM) is a heritable metabolic perturbation, rapidly growing across the world. Primary recognition of susceptible individuals with a family history of type 2 diabetes (FHD) in the prediabetes stage could delay the onset of T2DM or reduce complications induced by diabetes. This study aims to evaluate the expression levels of miR-21, miR-126 as noninvasive predictive biomarkers in individuals with genetic predisposition and investigate the correlation of miRNAs and cardiometabolic risk factors. Our study demonstrated that miR-21 expression has a notable elevate in both groups of T2DM and pre-T2DM. miR-21 expression was distinguished in the pre-T2DM and T2DM from the nondiabetic individuals by ROC curve analysis with AUC of 0.77 (95% CI 0.65-0.90; p = 0.0004) and AUC of 0.78 (95% CI 0.64-0.92; p = 0.0042), respectively. The relative gene expression of miR-126 was nearly equal among groups. miR-21 expression was positively associated with glycosylated hemoglobin (HbA1c), fasting blood sugar (FBS), and triglyceride (TG) and might have diagnostic value for T2DM and pre-T2DM. This study has revealed that the expression level of miR-21 can be considered as a non-invasive and rapid tool for distinguishing pre-T2DM and T2DM counterparts from healthy individuals.

A novel approach of recombinant laterosporulin production using the N-SH2 domain of SHP-2.

BACKGROUND: The current study was aimed at evaluating the role of the N-SH2 domain of SHP-2 as a partner protein in the expression of a toxic peptide, laterosporulin (LTS). We also investigated its effects on the formation of the disulfide bond and functional folding of the peptide in vitro. The N-SH2-LTS protein was expressed as a His-tagged fusion protein, capable of undergoing enzymatic cleavage. RESULTS: Based on the data presented herein, the total yield of the folded fusion protein from inclusion bodies was found to be about 105 mg/l, demonstrating a high-level of heterologous expression. After enzymatic cleavage, 1.5 mg of the folded recombinant laterosporulin was obtained from each 10 mg of the fusion protein. The purity of the recombinant laterosporulin was analyzed by RP-HPLC, to yield peptides with suitable purity (85%). CONCLUSIONS: Our findings indicated the advantages of using the N-SH2 domain of SHP-2 as a rapid and easy approach not only in producing easy target proteins but also in its function as a chaperone. N-SH2 domain of SHP-2 can influence on the purification of laterosporulin at reasonable yield and in a cost-effective fashion. The N-SH2 domain of SHP-2 as a protein chaperone may be potentially favorable to produce other proteins with disulfide bonds.

A study on the potential role of autophagy-related protein 10 as a biomarker for ulcerative colitis.

PURPOSE: Ulcerative colitis (UC) is a lifelong disease with unclear etiology and increasing prevalence worldwide. Autophagy has been reported to play roles in the pathogenesis and progression of UC. Here, we aimed to analyze the expression of autophagy related protein 10 (ATG10) and its regulator, micro-RNA (miR) 519a, in UC patients. METHODS: The level of ATG10 in the serum, stool, and colon biopsies from 15 UC patients and 30 non-UC healthy individuals (HC) group was measured by ELISA. Also, the blood level of miR-519a was investigated by quantitative real-time PCR. RESULTS: We found 13.63 ng/ml versus 0.99 ng/ml, 11.01 ng/ml versus 1.11 ng/ml and 6.41 ng/ml versus 3.21 ng/ml of ATG10 in the stool, colon tissue, and serum of UC and HC, respectively. There was no significant difference in the expression of miR-519a in the blood samples of UC and HC. CONCLUSIONS: ATG10 might be a potential non-invasive diagnostic biomarker for UC.

A novel chimeric recombinant protein PDHB-P80 of Mycoplasma agalactiae as a potential diagnostic tool.

The aim of this study was to construct, expression of a novel recombinant chimeric protein consisting of Pyruvate dehydrogenase beta subunit (PDHB) and high antigenic region of integral membrane lipoprotein P80 of Mycoplasma agalactiae as a potential diagnostic tool. The full-length sequence of pdhb and a portion of antigenic regions of P80 were selected and analyzed by CLC main workbench 5.5 software. Several linkers and three dimensional structure of PDHB-P80 were compared to the native PDHB and analyzed to select a proper one for expression. The fusion gene sequence was optimized and synthesized in pMAT cloning vector. The synthetic pMAT-pdhb-p80 was digested using Bam HI and Sal I restriction enzymes and ligated into pMAL-p5X expression vector. The pMAL-pdhb-p80 construct was transfected into E.coli BL21 strain cells and expressed protein were purified using amylose resin. and the purified protein was analyzed in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. In silico analysis demonstrated that fusion proteins using IgG4 middle hinge (CPSCP) with TM-score of 0.99 showed the higher similarity between three dimensional structure of PDHB before and after fusion with high antigenic region of P80. Successful cloning verified by PCR colony, double digestion and sequence analysis. Besides, SDS-PAGE analysis and Western blotting indicated and confirmed the expression of intact recombinant chimeric protein MBP-PDHB-P80 along with some truncated forms of the recombinant protein. it could be concluded that the fusion construct has a potential for serodiagnostic assay in future studies.

Pathophysiological role of Atg5 in human ulcerative colitis.

BACKGROUND/AIMS: Ulcerative colitis (UC), along with Crohn's disease, is one of the main types of inflammatory bowel disease (IBD). On the other hand, deregulated autophagy is involved in many chronic diseases, including IBD. In this study, we aimed to investigate the role of Atg5 and microRNA-181a (miR-181a) in the pathophysiology of UC. METHODS: Colon biopsy, stool, and blood samples of 6 men and 9 women were confirmed for UC. Also, 13 men and 17 women were selected as healthy control (HC). Enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry were used to measure the Atg-5 content of the colon biopsies. Besides, the serum and stool levels of Atg5 were measured using ELISA. Moreover, the total RNA of blood cells was extracted and evaluated for the expression of miR-181a. RESULTS: We found 1.2 ng/mL versus 0.46 ng/mL, 0.34 ng/mL versus 0.24 ng/mL, and 0.082 ng/mL versus 0.062 ng/mL of Atg5 in stool, intestinal tissue, and serum of UC and HCs, respectively. There was no significant difference in the expression of miR-181a in the blood samples of UC and HCs. Immunohistochemistry showed high positivity without any significant difference between the 2 groups in the quantitative analysis. CONCLUSIONS: The significant difference observed between the stool Atg5 content of the HCs and UC patients may provide new insight into using this protein as a diagnostic biomarker, however, considering the small size of our studied population further studies are needed.

Quercetin conjugated with superparamagnetic iron oxide nanoparticles improves learning and memory better than free quercetin via interacting with proteins involved in LTP.

Biomedical application of quercetin (QT) as an effective flavonoid has limitations due to its low bioavailability. Superparamagnetic iron oxide nanoparticle (SPION) is a novel drug delivery system that enhances the bioavailability of quercetin. The effect of short time usage of quercetin on learning and memory function and its signaling pathways in the healthy rat is not well understood. The aim of this study was to investigate the effect of free quercetin and in conjugation with SPION on learning and memory in healthy rats and to find quercetin target proteins involved in learning and memory using Morris water maze (MWM) and computational methods respectively. Results of MWM show an improvement in learning and memory of rats treated with either quercetin or QT-SPION. Better learning and memory functions using QT-SPION reveal increased bioavailability of quercetin. Comparative molecular docking studies show the better binding affinity of quercetin to RSK2, MSK1, CytC, Cdc42, Apaf1, FADD, CRK proteins. Quercetin in comparison to specific inhibitors of each protein also demonstrates a better QT binding affinity. This suggests that quercetin binds to proteins leading to prevent neural cell apoptosis and improves learning and memory. Therefore, SPIONs could increase the bioavailability of quercetin and by this way improve learning and memory.

Using superparamagnetic iron oxide nanoparticles to enhance bioavailability of quercetin in the intact rat brain.

BACKGROUND: Quercetin (QT) as a bioactive flavonoid has a potential therapeutic activity for numerous neuronal injuries and neurodegenerative diseases. However, the low absorption rate of QT, especially through the blood-brain barrier, restricts its bioactivity in the body. The current research took the advantage of superparamagnetic iron oxide nanoparticles (SPIONs) to enhance the bioavailability of quercetin. METHODS: Quercetin conjugated with SPIONs was prepared by means of nanoprecipitation method and was characterized by X-ray diffractometer, scanning electron microscope, and Fourier transformed infrared spectrometer analyses. Wistar male rats were orally fed by gavage with QT and QT-SPION at 50 and 100 mg/kg daily doses for 7 days. Using high-performance liquid chromatography (HPLC) method, biodistribution of QT was evaluated in plasma and brain tissue. RESULTS: The outcomes of this research revealed a higher concentration in the plasma and brain of the rats fed with QT-SPION in comparison to free QT. CONCLUSION: The results of this study confirm that SPION as a targeted drug delivery system enhances the bioavailability of quercetin in the brain about ten folds higher than free quercetin and could be used for the treatment of neurodegenerative disorders.

Quercetin Prevents Body Weight Loss Due to the Using of Superparamagnetic Iron Oxide Nanoparticles in Rat.

BACKGROUND: Superparamagnetic iron oxide nanoparticles (SPION) have been largely considered for numerous applications in biomedicine such as magnetic resonance imaging, hyperthermia, cell tracking, anticancer treatment, and targeted delivery of drugs or genes. However, they may have side effects such body weight loss. Quercetin (QT), a strong antioxidant and free radical scavenger and a natural flavonoid, has a wide range of biological and therapeutic effects. In this study, the effect of QT on prevention of weight loss due to the using of SPION has been investigated. MATERIALS AND METHODS: SPION and QT-SPION were administered orally at 50 and 100 mg/kg for 7 days. Then, the body weight was measured at the beginning and the end of the study. RESULTS: Rats fed with 50 and 100 mg/kg SPION showed a significant weight loss, whereas those that fed with 50 mg/kg QT-SPION did not. A weight loss was observed in rats treated with 100 mg/kg of QT-SPION. CONCLUSIONS: The results of this study showed that quercetin could prevent weight loss due to the SPION.

Proteomic analysis of continuous 900-MHz radiofrequency electromagnetic field exposure in testicular tissue: a rat model of human cell phone exposure.

Although cell phones have been used worldwide, some adverse and toxic effects were reported for this communication technology apparatus. To analyze in vivo effects of exposure to radiofrequency-electromagnetic field (RF-EMF) on protein expression in rat testicular proteome, 20 Sprague-Dawley rats were exposed to 900 MHz RF-EMF for 0, 1, 2, or 4 h/day for 30 consecutive days. Protein content of rat testes was separated by high-resolution two-dimensional electrophoresis using immobilized pH gradient (pI 4-7, 7 cm) and 12% acrylamide and identified by MALDI-TOF/TOF-MS. Two protein spots were found differentially overexpressed (P < 0.05) in intensity and volume with induction factors 1.7 times greater after RF-EMF exposure. After 4 h of daily exposure for 30 consecutive days, ATP synthase beta subunit (ASBS) and hypoxia up-regulated protein 1 precursor (HYOU1) were found to be significantly up-regulated. These proteins affect signaling pathways in rat testes and spermatogenesis and play a critical role in protein folding and secretion in the endoplasmic reticulum. Our results indicate that exposure to RF-EMF produces increases in testicular proteins in adults that are related to carcinogenic risk and reproductive damage. In light of the widespread practice of men carrying phones in their pockets near their gonads, where exposures can exceed as-tested guidelines, further study of these effects should be a high priority.

Bacterial Secretome Analysis in Hunt for Novel Bacteriocins with Ability to Control Xanthomonas citri subsp. Citri.

BACKGROUND: Xanthomonas citri subsp. citri (Xcc), the causative agent of bacterial citrus canker, has affected citriculture worldwide. Varieties of means have been used to minimize its devastating effects, but no attention has been given to bacteriocins. OBJECTIVES: Here and for the first time, we report the isolation and characterization of two novel bacteriocins. MATERIALS AND METHODS: Secretome containing bacteriocins of isolated bacteria was separated via SDS-PAGE. Each isolated protein band was characterized and checked for its efficacy in controlling two pathogenic isolates of Xcc via disk diffusion assay. The effects of varieties of carbon, nitrogen and phosphate sources were evaluated on both bacterial growth and bacteriocin production via Taguchi orthogonal method. RESULTS: The two bacteriocins showed an activity up to 55ºC that were sensitive to proteases suggesting being protein in nature. Analysis of SDS-PAGE purified protein bands of bacterial secretomes with demonstrated potency against Xcc revealed the presence of peptides with relative molecular masses of 16.9 and 17 kDa for Cronobacter and Enterobacter, respectively. Sequence analysis of peptides revealed an HCP1 family VI secretion system homologue for Cronobacter (YP_001439956) and pilin FimA homologue for Enterobacter (CBK85798.1). A Taguchi orthogonal array was also implemented to determine the effect of temperature and eight other chemical factors on bacteriocin production for each bacterium. CONCLUSIONS: Two peptides with novel antibacterial activities effective against Xcc were isolated, characterized and conditions were optimized for their higher production.

Analysis of rat testicular proteome following 30-day exposure to 900 MHz electromagnetic field radiation.

The use of electromagnetic field (EMF) generating apparatuses such as cell phones is increasing, and has caused an interest in the investigations of its effects on human health. We analyzed proteome in preparations from the whole testis in adult male Sprague-Dawley rats that were exposed to 900 MHz EMF radiation for 1, 2, or 4 h/day for 30 consecutive days, simulating a range of possible human cell phone use. Subjects were sacrificed immediately after the end of the experiment and testes fractions were solubilized and separated via high-resolution 2D electrophoresis, and gel patterns were scanned, digitized, and processed. Thirteen proteins, which were found only in sham or in exposure groups, were identified by MALDI-TOF/TOF-MS. Among them, heat shock proteins, superoxide dismutase, peroxiredoxin-1, and other proteins related to misfolding of proteins and/or stress were identified. These results demonstrate significant effects of radio frequency modulated EMFs exposure on proteome, particularly in protein species in the rodent testis, and suggest that a 30-day exposure to EMF radiation induces nonthermal stress in testicular tissue. The functional implication of the identified proteins was discussed.

Mineral elements and essential oil contents of Scutellaria luteo-caerulea Bornm. & Snit.

OBJECTIVE: Scutellaria luteo-caerulea Bornm. & Snit. is one of the species of genus Scutellaria, within the family of the Lamiaceae, that is used for immune system stimulation and antibacterial effects in traditional medicine in Iran. The aims of this study were to analyze essential oils and mineral element contents of leaves of S. luteo-caerulea in flowering stage of development. MATERIALS AND METHODS: The essential oils were obtained by hydrodistillation of the leaves of S. luteo-caerulea and were analyzed by gas chromatography mass spectrometry (GC/MS). Moreover, microwave digestion with atomic absorption spectrophotometry were used for the mineral elements assay. RESULTS: Ninety-seven constituents were detected. Between them, the major components were trans-caryophyllene (25.4%), D-germacrene (7.9%), and linalool (7.4%). Determination of mineral elements showed that the highest minerals were Ca(2+) (65.14±1.95 µg/ml) and K(+) (64.67±3.10 µg/ml). CONCLUSION: Presence of different essential oils and rich sources of Ca(2+) and K(+) candidate this plant as an auxiliary medication in different diseases, but more complementary researches are needed about its potency and side effects.

Alpha-1 antitrypsin, retinol binding protein and keratin 10 alterations in patients with psoriasis vulgaris, a proteomic approach.

OBJECTIVES: Psoriasis is an autoimmune disease that appears on the skin. Although psoriasis is clinically and histologically well characterized, its pathogenesis is unknown in detail. The aims of this study were to evaluate the proteome of psoriatic patients' sera and to compare them with those of normal healthy human to find valuable biomarkers. MATERIALS AND METHODS: In a case-control study, twenty cases of white patients with psoriasis vulgaris, 10 males and 10 females and sixteen healthy controls, 8 males and 8 females were enrolled in the study. The serum protein expression patterns obtained after depletion of albumin were compared by using two dimensional gel electrophoresis (2-DE) coupled to MALDI/TOF-TOF to identify disease associated proteins. RESULTS: Differential expression of nine protein spots representing four unique proteins including alpha-1 antitrypsin, retinol binding protein, keratin 10 and an unknown protein (with pI 6.47 and molecular weight of 19941 Da), between psoriatic and healthy human serum were found. Furthermore, expression of four new alpha-1 antitrypsin isoforms with different molecular weight and isoelectric point were observed in psoriatic serums in this research for the first time. CONCLUSION: A unique proteomic profiling with abnormal expression of alpha-1 antitrypsin and presence of keratin 10 in sera of psoriasis patients were observed that may constitute new and useful findings of psoriasis and offer a clue to a better understanding of the inflammatory pathway.

Impact of 900 MHz electromagnetic field exposure on main male reproductive hormone levels: a Rattus norvegicus model.

This work analyzes the effects of radiofrequency-electromagnetic field (RF-EMF) exposure on the reproductive system of male rats, assessed by measuring circulating levels of FSH, LH, inhibin B, activin B, prolactin, and testosterone. Twenty adult male Sprague-Dawley rats (180 ± 10 g) were exposed to 900 MHz RF-EMF in four equal separated groups. The duration of exposure was 1, 2, and 4 h/day over a period of 30 days and sham-exposed animals were kept under the same environmental conditions as the exposed group except with no RF-EMF exposure. Before the exposure, at 15 and 30 days of exposure, determination of the abovementioned hormone levels was performed using ELISA. At the end of the experiment, FSH and LH values of the long time exposure (LTE) group were significantly higher than the sham-exposed group (p < 0.05). Serum activin B and prolactin in the LTE group showed significant increase and inhibin B showed significant decrease than sham and short time exposed (STE) groups after 30 days RF-EMF exposure (p < 0.05). Also, a significant decrease in serum testosterone levels in the LTE group was found compared to short and moderate time exposed (MTE) groups after 30 days RF-EMF exposure (p < 0.05). Results suggest that reproductive hormone levels are disturbed as a result of RF-EMF exposure and it may possibly affect reproductive functions. However, testosterone and inhibin B concentrations as a fertility marker and spermatogenesis were decreased significantly.