Reports of Biochemistry and Molecular Biology
Volume:8 Issue: 3, Oct 2019

Abnormal DNA methylation leading to altered transcription of certain genes occurs frequently in colorectal cancer (CRC). As with protein-coding genes, microRNAs (miRNAs) may be targeted for methylation in CRC; however, the methylation state of miRNA genes in CRC, especially in primary lesions, has not yet been completely elucidated. To understand the impact of DNA methylation on the miR-200c/141 cluster promoter, we investigated the methylation and expression of miR-141 in precancerous lesions and colorectal cancer.

In this cross-sectional study, 208 colorectal tissue samples, including 34 tumor tissue samples, 60 precancerous lesions with matched normal adjacent tissues, and 20 normal tissue samples, were collected. Promoter methylation of the miR-200c/141 cluster was studied using methylation-specific PCR. MiR-141 expression was examined using quantitative real-time PCR.

Our findings showed that the miR-200c/141 cluster promoter region was most frequently hypermethylated in colorectal tumors and adenomatous polyps, but unmethylated in hyperplastic polyp tissues (P < 0.001). DNA methylation of the miR-200c/141 cluster and the tumor stage were significantly correlated (P = 0.002); however, miR-141 expression difference between the tumor and polyp samples was not significant (p = 0.6).

The DNA methylation status of the miR-200c/141 cluster could serve as a progression marker from benign polyps to colorectal cancer.

Selenium is a mineral that showed both pro- and anti-oxidant activities in various disease models. In this study, we evaluated the anti-tumor effect of selenium against 1,2-dimethylhydrazine (DMH)-induced colorectal cancer in BALB/C mice and its effect on apoptosis and angiogenesis.

Colorectal cancer was induced by subcutaneous injection of DMH (20 mg/kg body weight) in BALB/C mice once weekly for 20 weeks. Selenium (200 mg/L) was given to DMH plus selenium-treated group in the drinking water for the next 3 months.

The DMH plus selenium-treated group exhibited significantly lower expression of cloned caudal-type homebox gene -2 (CDX-2) and vascular endothelial growth factor (VEGF) but higher caspase-3 expression level at p<0.001 compared to the DMH-treated group. Moreover, a decrease in the reduced glutathione content and glutathione peroxidase activity but an increase in the malondialdehyde content were observed at p<0.001. Both macroscopic and microscopic examination of the colorectal tissues confirmed the results.

The anti-tumor effect of selenium against an induced colorectal cancer in mice is attributed to its pro-oxidant, anti-angiogenic and apoptotic effects.

Inclusion body formation in E. coli is a significant problem in recombinant protein production. The aim of this study was to improve the solubility of recombinant bovine sex determining region Y protein (SRY) in BL21 (DE3) E. coli cells.

In this research two recombinant bovine SRY (rbSRY) sequences were analyzed; these were wild-type SRY (wtbSRY) and codon-optimized SRY (cobSRY). Their expression in various culture conditions was examined; these differences included IPTG concentrations, temperatures, and media stabilizers.

IPTG and temperature significantly affected rbSRY solubility (P < 0.001). The optimum IPTG concentration and temperatures for wtbSRY and cobSRY induction were 0.3 mM at 27 and 32 °C, respectively. In addition, arginine and sorbitol concentrations significantly affected rbSRY solubility (P < 0.01). Solubility of rbSRY protein was highest from the cobSRY construct in the presence 0.2 M arginine and 0.3 M sorbitol. The highest inclusion body production occurred with high glucose concentrations.

We found that modifications in temperature and IPTG and stabilizer concentrations affected rbSRY solubility.

Diabetes mellitus and metabolic disorders are a major burden on the healthcare system. Irisin is a novel myokine reported to have beneficial effects on glucose and lipid metabolism. Vitamin D deficiency has been implicated in the development of diabetes and hold a critical role in diabetes-related complications. In the present study, we examined the efficacy of vitamin D supplementation on serum irisin levels, skeletal muscle irisin levels, and the expression of the irisin precursor, FNDC5 (fibronectin-type III domain-containing 5) in type I diabetes mellitus rats.

Thirty-six adult male Sprague-Dawley rats (150 – 250 g) were randomly divided into four groups: group I: healthy control rats with no treatment (n=8), group II: healthy control rats receiving sesame oil as a placebo (n=8), group III: diabetic rats receiving sesame oil as placebo (n=10), group IV: diabetic rats treated with 4300 IU/kg/week vitamin D (n=10). Diabetes was induced by intraperitoneal (IP) injection of streptozotocin. At the end of the vitamin D intervention blood and triceps muscle samples were collected. RNA was extracted from muscle and real-time PCR was performed to examine FNDC5 gene expression.

Our study showed that the administration of vitamin D (4300 IU/kg/week) in a streptozotocin-diabetic rat model resulted in increased serum vitamin D levels, FNDC5 gene expression and muscle irisin levels. However, the levels of serum irisin were not significantly changed by the administration of vitamin D.

In conclusion, we show that vitamin D supplementation enhances serum vitamin D levels, FDNC5 gene expression and muscle irisin levels in the streptozotocin-diabetic rat model. Our study highlights the potential therapeutic effect of vitamin D supplementation for diabetes mellitus.

Colorectal cancer (CRC) is one of the most commonly-diagnosed malignancies throughout the world and the fourth-leading cause of cancer deaths globally. Angiogenesis and the resultant tumor neovascularization is a well-known cancer hallmark. Here we investigated the expression of FLT1 and KDR, the influential genes in angiogenesis regulation, in CRC patients.

We assessed FLT1 and KDR mRNA expression in 47 CRC samples and matched adjacent non-cancerous tissues (ANCT) by quantitative real-time PCR. The Spearmen correlation coefficient and receiver operating characteristic (ROC) curves were also examined.

Both genes were expressed at significantly greater levels in CRC tissues than in ANCT (p < 0.05). A significant association was found between KDR expression and disease stage and lymph status in CRC patients. Furthermore, the Spearman correlation demonstrated a moderate correlation between FLT1 and KDR expression in CRC samples. Finally, ROC curve analysis demonstrated that FLT1 had the greatest sensitivity (85.1%), while the greatest specificity was achieved by a combination of the two genes.

The dysregulated FLT1 and KDR expression, in addition to the observed correlation and ROC curve results, indicate the critical importance of angiogenesis among the cancer pathways in CRC. These data can broaden our current knowledge of angiogenesis in CRC to improve disease diagnosis and patient treatment.

The study aims at the comparison and correlation of serum levels of fructosamine and erythrocyte Na+/ K+ ATPase in Gestational diabetes mellitus (GDM) and Non Gestational Diabetes Mellitus (Non GDM).

A total of 326 samples were divided into 4 groups. Pregnant women between the age group of 20-40 years who gave samples for Oral Glucose Tolerance Test (OGTT) were included as the subjects. Anonymized and left over fasting and 2 hours’ samples were collected from biochemistry laboratory, Kasturba Hospital, Manipal.

In the comparison of fructosamine levels in GDM and Non GDM, fructosamine was found to be significant (p value<0.001) in both fasting and 2 hours (G2) blood glucose condition. Na+/ K+ ATPase did not show any significant variation between the groups. Correlation was not significant between the parameters.

Fructosamine showed significant increase when compared between the groups, whereas significant correlation is not obtained between the parameters. Thus, the use fructosamine as a diagnosis tool becomes inconclusive. Further studies must be carried out to identify a marker which reduces the interferences observed in fructosamine and to find out the exact relationship between hyperglycaemia and Na+/ K+ ATPase activity.

In recent years, prostate cancer prevails as one of the lead cancers affecting men. Currently, prostate cancer research involves the phytochemical study of plants with anti-tumour effects. This study compares the anti-tumour effects of three plant species indigenous to Iran and their interaction with cluster of differentiation (CD)-82 protein, a therapeutic target found in prostate cancer cells.

The extracts of Hypericum perforatum, Achillea millefolium, and Aloe vera were prepared and their toxicological, cellular and gene expression responses were evaluated in PC-3 human prostate cancer cells and normal human chondrocyte cell line C28/I2. They were exposed to different concentrations of the plants (10 mg/mL, 5 mg/mL, 1 mg/mL, 100 µg/mL, 10 µg/mL, and 1 µg/mL) at three exposure time points (24, 48, 72 hours) to determine cancer cell cytotoxicity and gene expression profiles.

Half-maximal inhibitory concentration (IC50) in PC-3 cells ranged from 0.6 to 8.5 mg/mL for H. perforatum extract, from 0.4 to 7.5 mg/mL for A. Millefolium extract, and from 0.2 to 8.0 mg/mL for A. vera extract in a time-dependent manner. A. vera extract caused the highest cell death levels in PC-3 cells (94%) and C28/I2 cells (57%) after 48 hours. A 1.97-, 3.00-, and 3.48-fold increase in relative gene expression of CD82 was observed for H. perforatum, A. millefolium, and A. vera extracts, respectively

A. vera and A. millefolium extracts are a selective inhibitor of prostate cancer cells and a potent activator of CD82 expression.

Gastric cancer is still the main health threat being the third leading cause of deaths from cancers in the world. The major risk behind the gastric cancer is that it remains asymptomatic in the early stages and in (97%) cases it metastasizes to other organs. Gastric cancer is a multifactorial disease in which Helicobacter pylori (H. pylori) has been known as a risk factor. However, patients with gastritis, especially atrophic gastritis and gastric ulcer have been shown to be at an increased risk for developing gastric cancer.

This study included measuring the serum levels of E-Cadherin protein, carbohydrate antigen 19-9 (CA19-9) and carcinoembryonic antigen (CEA) in 30 patients diagnosed with gastritis, 20 gastric ulcer patients, 20 gastric cancer patients and in 20 healthy volunteers serving as the control group.

Infection with H. pylori was diagnosed by serology (IgA and IgG antibodies) as well as by rapid urease test (RUT) and histology. The results showed that 50 (71.4%) of the patients were positive for H. pylori. Levels of E-Cadherin were increased significantly in all patients in comparison to the control group with a large significant increase in the gastric cancer group. The levels of E-Cadherin were also significantly increased in H. pylori infected patients compared to H. pylori negative patients. A non-significant difference in the levels of CA19-9 and CEA was observed in all patients in comparison to healthy controls.

This study concluded that serum E-Cadherin could be considered as a potential marker in diagnosis of gastric cancer.

Dextran is a commercially available bacterial exopolysaccharide (EPS) with several industrial applications in the food industry and in the biomedical industry as an adjuvant, emulsifier, carrier, and stabilizer. The production of dextran at the industrial level occurs through the fermentation of a sucrose-rich medium. Research to optimize dextran production has found that the yield of dextran varies depending the specific conditions for production. The aim of this study was to produce dextran and establish the optimal conditions for dextran biosynthesis from different Lactobacillus species isolated from healthy vaginal and infant stool samples.

Lactobacillus spp. were isolated and identified from vaginal and infant stool samples via the VITEK 2 system. The presence of dextran biosynthesis from the different Lactobacillus spp. isolates was determined by a screening test for mucoid colonies and confirmed via theethanol precipitation method. To optimize for the maximum yield of dextran, the effects of various parameters such as temperature, incubation time, pH, inoculum size, aeration, and sucrose concentration were examined.

All Lactobacillus spp. isolates were able to produce dextran. The optimal conditions for dextran production was at 24 hours of incubation at 30 °Cwith 15% sucrose, 4% inoculation size at pH 7.0 in aerobic conditions. This yielded a dextran dry weight of 580 mg/100 mL.

Dextran production from Lactobacillus species isolates vagina and infant stool had the ability to produce dextran.

The DNA methylation status of the miR-200c/141 cluster could serve as a progressionmarker frombenignpolypstocolorectalcancer.

Neuroprotective mechanisms triggered by peroxisome proliferator-activated receptor-gamma agonist: pioglitazone (PIO) and glucagon-like peptide 1 analog: exendin-4 (Ex-4) in neurological diseases were reported, but whether mitochondrial biogenesis is involved or not in their neuro-protective mechanisms in type 1 Diabetes Mellitus (T1DM); has not been studied before. To bridge this gap, we investigated the effect of PIO and Ex-4 on brain mitochondrial biogenesis in streptozotocin-induced diabetes in rats.

Seven weeks after induction of diabetes in rats, serum fasting glucose and insulin were measured in studied groups. The brain was removed for histological analysis and assessment of: mitochondrial complexes I and II, ATP, H2O2, brain derived neurotrophic factor (BDNF), cytochrome c and hemeoxygenase (HO)-1 activity, and relative gene expression of the nuclear factor; Nrf2and the apoptotic markers: bax& bcl2and mitochondrial biogenesis markers; peroxisome proliferator–activated receptor γ coactivator (PGC) 1-α and sirtuin 1 (SIRT-1) and AMP-activated protein kinase (AMPK) and c-Jun-N-terminal kinase (JNK) proteins.

Brain in untreated rats showed neurodegeneration area and significantly rising H2O2and JNK, up-regulation of bax, down-regulation of bcl2. These changes were paralleled with significant reduction in Nrf2, HO-1, BDNF, complex I, II and ATP and SIRT-1/ PGC1-αexpression. PIO and Ex-4 significantly improved the reported changes. Combined modality showed better improvement relative to each drug alone.

PIO and Ex-4 may have neuroprotective effects in T1DM, via targeting altered mitochondrial biogenesis probably due to modulation of brain SIRT-1signaling, improvement of oxidative stress and equilibrating the balance between pro-apoptotic and anti-apoptotic mediators.

Immunotherapies using monoclonal antibodies against influenza A hemagglutinin (HA) has been an effective means for controlling Influenza spread. An alternative method for viral prophylaxis and treatment is the development of human single-chain variable fragment (scFv) antibodies with no human anti-mouse antibody (HAMA) response and high specificity. In the present study, two highly conserved sequences of HA were used to select specific neutralizing scFvs againstH3N2 strain of influenza A virus.

Biopanning process was performed to isolate specific scFv antibodies against highly conserved HA sequences, aa173-181 and 227-239, of the influenza A H3N2 strain from a scFvlibrary. The peptide-binding specificity of the selected clones was examined via phage ELISA. The soluble forms of the clones were prepared and assessed using western blot analysis and neutralization efficiency of the selected clones were examined by TCID50 neutralizing assay and real-time PCR.

scFv 1 and scFv 2 were selected against HA of H3N2 influenza A virus with frequencies of 95% and 30% in the panning process, respectively. Western blot analysis confirmed the scFv band size. Significant neutralization in the presence of scFv 1 and scFv 2 were obtained. Real time PCR revealed significant decrease in viral copy number.

Two specific neutralizing scFvsagainst two highly conserved neutralizing epitopes of the influenza A virus HA glycoprotein were selected. A strong neutralization effect of scFv1, showed the potential of this antibody for H3N2 influenza A controlling in the viral spread

Blocking of gp41 of HIV virus, which is involved in the virus entry has been introduced as an effective strategy against HIV infection. In this study we used phage display technology to select specific single chain antibody (scFv) against gp41 HIV for its application in clinical use.

Single chain antibodies against an epitope located in C- terminal part of gp41 were selected using the panning process which enriched a phage antibody display library of scFv. Following panning, 20 clones were amplified by PCR and fingerprinted. To test the specificity of the selected antibodies phage ELISA was performed.

PCR of the library clones demonstrated the presence of VH-linker-VL inserts. Fingerprinting of the clones showed a diverse library with different patterns. Fingerprinting of selected clones after panning revealed two specific single chain antibodies with frequency of 25% and 20%. These clones were preserved for further investigations. Phage ELISA results showed specificity of the two scFvs against the immunodominant epitope of gp41. The absorbance of the scFv1 and scFv2 were 0.72 and 0.63 while the absorbance of the no peptide were 0.18 and 0.12, respectively.

In this study we successfully selected two specific recombinant antibodies against gp41. These libraries are human antibodies with high affinity and specificity and have the potential to be used for diagnosis and treatment. Further investigations are needed to show the effects of the antibodies in vitro and in vivo.

Inappropriate activation of the proto-oncogene LIN28Band inactivation of the p53 tumor suppressor, have been shown to have a critical role in tumorigenesis. Previousresearch has shown therapeutic potential for the use of herbal plants as an alternative strategyforcancer treatment. Achillae wilhelmsii C. Kochis a plant that has been traditionally used for its medicinal properties. The aim of this study was to investigate the cytotoxic and apoptosis-inducing effect of Achillea wilhelmsiiC. Kochhydroalcoholicextract (AWHE) on HeLa cervical cancer cells and its effect on LIN28Band p53 expression.

The cytotoxic activity of AWHEwas evaluated on HeLa cells using a trypan blue exclusion assay. The Annexin V/PI double staining assay was used to evaluate the apoptosis-inducing effect of the extract. The expressionof LIN28Band p53mRNA was measured using the real-time-PCRmethod.

Treatment with AWHEwas shown to induce cytotoxicity in both time and concentration-dependent manners(P<0.05). The propositionof HeLa cells undergoing apoptosis increased with increasing concentrations of AWHE(P<0.05). The mRNA levels of p53increased following 12, 24, and 48 hours of AWHEtreatmentwhereasthe mRNA levels of LIN28Bwere significantly decreased after4 to 12 hours of AWHEtreatment (p<0.05).

Our findings confirmed the pro-apoptotic function of AWHEon the cervical cancer HeLa cell line. This indicates that targeting the LIN28Bsignaling cascade may be a promising therapeutic strategy for cervical cancer. Further research is required to understand the therapeutic effects of AWHEin primary human cervical cancer cells and a pre-clinicalcervical cancer model.

Sex selection of sperm by separating X- and Y-chromosome bearing spermatozoa is critical for efficiently obtaining the desired sex of animal offspring in the livestock industry. The purpose of this study was to produce a goat polyclonal antibody (pAb) against the bovine Sex Determining Region Y
chromosome (bSRY) to separate female- and male-bearing spermatozoa.

To produce a goat polyclonal antibody against bSRY, a female goat was subcutaneously immunized with 27 kDa of recombinant bSRY (rbSRY) protein as the antigen. The anti-bSRY pAb was purified by ion-exchange chromatography. The purity of the pAb was determined using the SDS-PAGE
method. The biological activity of the anti-bSRY pAb was examined using PCR to assess the binding affinity of pAb for the bSRY antigen and commercially sexed bull sperm.

The total amount of purified anti-bSRY pAb was approximately 650 mg/goat serum (13 mg/mL). Interestingly, our data showed that the binding affinity of our pAb to the Y bearing was high, while the binding affinity of that to the X-chromosome bearing sperm was similar to the negative control.

In conclusion, our findings show that the goat anti-SRY pAb specifically binds to Ychromosome bearing sperm that suggesting its potential use for sex selection.

Tuberculosis (TB) still remains endemic worldwide making epidemiological studies essential to mitigating efforts implicated in identifying its source,controlling, and preventing the spread of dangerous strains amongst humans such as Mycobacterium tuberculosis (Mtb).

In this study, we sought to determine the 6 Mycobacterial Interspersed Repetitive Unit-Variable-Number Tandem Repeat (MIRU-VNTR) loci with high discriminatory powers for Mtb genotypingas well as the loci with the highest and the lowest discriminatory powers for MIRU-VNTR.To conduct our search, we used several databases such as science direct, Embase (Elsevier), Web of Science, Scopus and Medline via PubMed. Searches were performed using key words including: Mycobacterium tuberculosis, MIRU–VNTR, Allele diversity, Genetic diversity and human patient. Finally, 56 articles were selected after filtering out titles, abstracts and full texts.

Loci with high discriminatory powers included MIRU10 and MIRU26, while MIRU2, MIRU20, MIRU24 and ETRD had poor discriminatory powers. According to previous data in the literature, the loci MIRU10, MIRU26, MIRU40, QUB 26, QUB 11b and Mtub21 have high discriminatory powers.

Therefore, these loci recommended for genotyping Mtb to save time and cost and to ensure the production of reliable results.