Reports of Biochemistry and Molecular Biology
Volume:6 Issue: 2, Apr 2018

Infectious diseases such as ventilator- associated pneumonia (VAP) are one of the serious problems in intensive care units (ICU) of hospitals. To date, there has been no appropriate clinical and diagnostic marker for early detection of this disease. In this study, expression of PIK3R3 and ATp2A1 genes in patients with VAP were assessed to be used as biomarkers to identify and confirm the disease.
This study was conducted by using peripheral blood samples of 60 individuals, including 30 patients with VAP and 30 healthy volunteers. First, the peripheral blood samples were taken and then RNA was extracted and converted into cDNA. Finally, the assessment of genes was performed by Real-time PCR.
In peripheral blood samples, 46.6% and 30% were positive for PIK3R3 expression in patients and healthy groups, respectively. The ATp2A1 expression in patients and healthy controls were found 40% and 23.3%, respectively. Comparing the ΔCT obtained for the PIK3R3 and ATp2A1 genes showed statistically significant differences between the two groups of patients and healthy subjects (p=0.042, p=0.036).
ATp2A1 and PIK3R3 may be used as biomarkers for early detection of VAP disease. However, further studies are required.

Subunit vaccines are appropriate vaccine candidates for the prevention of some infections. In this study, three immunogenic proteins of Mycobacterium tuberculosis, including HspX, Ppe44, and EsxV as a new construction, were expressed alone and as a fusion protein to develop a new vaccine candidate against tuberculosis infection.
To make the fusion protein, the three genes were linked together by AEAAAKEAAAKA linkers and inserted into pET21b and pET32b vectors. Escherichia coli (E. coli) Top10 cells were transformed with the plasmid, and the purified plasmid was used to transform E. coli BL21 cells. Protein expression was induced with IPTG. After optimizing protein expression, the recombinant proteins were purified by Ni-NTA chromatography. Protein purification was confirmed by SDS-PAGE and Western blotting with an anti-poly histidine-peroxidase monoclonal antibody against the 6His–tags at the proteins’ C termini.
Directional cloning was confirmed by polymerase chain reaction (PCR), restriction enzyme digestion, and sequencing. The highest expression of the tri-fusion protein and HspX were obtained by the addition of 0.2 mM of IPTG to E. coli BL-21 cells at 37 ˚C and 18 h of incubation. For Ppe44 and EsxV, the optimum expression conditions were 18 ˚C and 16 h of incubation. SDS-PAGE and Western blots confirmed that the desired proteins were produced.
The three desired proteins and the fusion protein were successfully expressed and the conditions for optimum expression determined. These recombinant proteins will be evaluated as vaccine candidates against tuberculosis. Further studies are needed to evaluate the abilities of these proteins to induce strong immunological responses.

Lead (Pb) is a heavy metal that has devastating effects on many animal tissues. In this study we investigated the effects of orally-dosed lead acetate II on osteocalcin gene (osteocalcin) expression in mesenchymal stem cells grown in an osteogenic medium. Osteocalcin is an abundant bone matrix differentiation protein.
Twelve male Wistar rats were divided into three groups of four rats each. Two groups were fed orally with 50 or 100 ppm of lead acetate II with libitum feed and water for two months. The control group was fed with libitum feed and water only. Rats were euthanized and femoral bone marrow mesenchymal stem cells (BM-MSCs) were extracted. The cells were cultured in osteogenic medium and osteocalcin expression was determined by real-time PCR.
Real-time PCR showed that osteocalcin expression was significantly less in the BM-MSCs of rats that received 100 ppm of lead acetate II than in the BM-MSCs of the other groups (P Doses of 50 and 100 ppm of lead acetate II in rats caused a significant decrease in osteocalcin expression in BM-MSCs grown in osteogenic medium.

The incidence of esophageal squamous cell carcinoma (ESCC) is increasing, causing catastrophic health burdens on communities. Curcumin has shown promise as a therapeutic agent in the treatment of colon, colorectal, pancreatic, and esophageal cancers but it has very poor bioavailability. The application of nano-carriers as drug delivery systems increases curcumin's bioavailability. Cyclin D1 is overexpressed in ESCC and curcumin may change its expression.
In this study, the effect of SinaCurcumin®, a novel nano-micelle product containing 80 mg curcumin, on the growth of KYSE-30 cells and expression of cyclin D1, was investigated. Paclitaxel and Carboplatin served as reference drugs.
Nano-curcumin increased cell cytotoxicity, decreased IC50, and down-regulated of cyclin D1. However, treatment of cells with nano-curcumin might result in multidrug resistance.
Nano-curcumin suppressed proliferation of KYSE-30 cells and expression of cyclin D1 although its use in combination with other chemotherapeutic agents requires further testing.

Vascular endothelial growth factor-A (VEGF-A), an endothelial cell-specific mitogen produced by various cell types, plays important roles in cell differentiation and proliferation. In this study we investigated the effect of recombinant VEGF-A on differentiation of mesenchymal stem cells (MSCs) to endothelial cells (ECs).
VEGF-A was expressed in E. coli BL21 (DE3) and BL21 pLysS competent cells with the pET32a expression vector. Recombinant VEGF-A protein expression was verified by SDS-PAGE and western blotting. Mesenchymal stem cell differentiation to ECs in the presence of VEGF-A was evaluated by flow cytometry and fluorescence microscopy.
Recombinant VEGF-A was produced in E. coli BL21 (DE3) cells at 0.8 mg/mL concentration. Expression of CD31 and CD 144 was significantly greater, while expression of CD90, CD73, and CD44 was significantly less, in MSCs treated with our recombinant VEGF-A than in those treated with the commercial protein (p Recombinant VEGF-A expressed in a prokaryotic system can induce MSCs differentiation to ECs and can be used in research and likely therapeutic applications.

It was proposed that probiotics may influence immune system through direct or indirect exposure. Direct exposure is mostly mediated by surface receptors. Toll-like receptors (TLRs) are conserved molecular sensors which could be triggered via some pathogen associated structures, hence, modulate the immune responses. This study was conducted to elucidate the impact of lactobacillus acidophilus as a common probiotic on the expression level of TLRs in the chicken’s cecal tonsil.
Thirty one-day-old chicken were selected and separated into three groups as probiotic-fed, dairy-fed and control. In addition to commercial powder supply, each chicken in the probiotic-fed group received 109 CFU/Kg of L. acidophilus daily. While, chickens in the dairy-fed group were provided with commercial powder feed and sterile dairy milk. After 14 and 21 days of oral feeding the cecal tonsil was removed and the expression of TLR2, TLR4 and TLR5 were examined by real-time PCR.
At the age of 14-day, there was a slight upregulation in the expression levels of TLR2 (118.9%), TLR4 (129.6%) and TLR5 (123.7%) of the cecal tonsil in the probiotic-fed group; however, these alterations were not statistically significant. At the age of 21-day, a non-significant downregulation was observed in TLR expression level of both dairy-fed (TLR2, 85%; TLR4, 79.5%; and TLR5, 86.5%) and probiotic-fed (TLR2, 88.8%; TLR4, 81%; and TLR5, 87.2%) groups in comparison to controls.
The findings revealed that although the probiotic supplementation could be useful but it did not significantly affect innate immunity state through alteration of TLRs.

Betatrophin is a member of the angiopoietin-like (ANGPTL) family that has been implicated in both triglyceride and glucose metabolism. The physiological functions and molecular targets of this protein remain largely unknown; hence, a purified available protein would aid study of the exact role of betatrophin in lipid or glucose metabolism. In this study, we cloned the full-length cDNA of betatrophin from a human liver cDNA library. Betatrophin was expressed in the pET-21b-E. coli Bl21 (DE3) system and purified by immobilized metal-affinity chromatography and ion-exchange chromatography. Circular dichroism spectroscopy revealed α-helix as the major regular secondary structure in recombinant betatrophin. The production method is based on commonly available resources; therefore, it can be readily implemented.

Cystic echinococcosis (CE), known as hydatid cyst, is a zoonotic parasitic infection caused by the larval stage of Echinococcus granulosus (E. granulosus). Antigen B, the major component of hydatid cyst fluid, is encoded by members of a multigene family. The present study aimed to evaluate the genetic diversity of the gene encoding antigen B8/1 (EgAgB8/1) among the main intermediate hosts of E. granulosus.
Twenty-eight hydatid cyst isolates (10 sheep, 9 human, and 9 cattle) were collected in Fars province, Iran. DNA was extracted from each cyst and PCR, followed by DNA sequencing was used to identify potential EgAgB8/1 sequence variation and polymorphism. A phylogenetic tree was constructed using MEGA 7.0 software and the maximum likelihood method.
Using EgAgB8/1 primers, an approximately 315 bp band was amplified from all the isolates. The PCR products were sequenced, and the sequences were deposited in GenBank (accession numbers, KY709266-KY709293). The polymorphism variation among the isolates was 0.0, while intra-species variation within the isolates and related sequences in GenBank was 0.5-1%. Analysis of the phylogenetic tree revealed that the isolates from humans, sheep, and cattle all cluster in one group and are homologous to the EgAgB8/1 M1 allele.
Findings of this study revealed close similarity between the EgAgB8/1 of human, sheep, and cattle E. granulosus isolates. However, differences were found between the EgAgB8/1 sequences in our study and those reported from other CE endemic areas. Whether such similarities and differences exist in other subunits AgB subunits require further study.

Several lines of evidence suggest that oxidized LDL (Ox-LDL) scavenger receptors play a crucial role in the genesis and progression of diabetic atherosclerosis. This study aimed to elucidate the effect of vitamin D3 on gene expression of lectin-like oxidized LDL receptor-1 (LOX-1), scavenger receptor-A (SR-A), Cluster of Differentiation 36 (CD36), and Cluster of Differentiation 68 (CD68) as the main Ox-LDL receptors in streptozotocin (STZ)-induced diabetic rat aortas.
Eighteen Sprague-Dawley rats were randomly divided into three groups of six rats each. Two rats died during the study so five rats from each group were analyzed at the study’s end. Diabetes was induced in overnight starved rats in two of the groups by intraperitoneal injections of 60 mg/kg of STZ. The vitamin D3/diabetic group then received weekly intraperitoneal injections of 5000 IU/kg of vitamin D3 dissolved in cottonseed oil for four weeks, diabetic controls received cottonseed oil, and healthy controls received sterile saline weekly for the same period. At the end of the four-week study period the animals were killed and the aortas were collected to examine the mRNA expression using real-time polymerase chain reaction (RT-PCR).
SR-A and CD36 mRNA expression were significantly greater in the vitamin D3/diabetic rats than in both the diabetic control and healthy control rats. CD68 and LOX-1 expression were greater in the vitamin D3/diabetic rats than in the diabetic control and healthy control rats, respectively.
Vitamin D3 may increase the risk of diabetic atherosclerosis by inducing scavenger receptors expression.

Streptavidin is a protein produced by Streptomyces avidinii with strong biotin-binding ability. The non-covalent, yet strong bond between these two molecules has made it a preferable option in biological detection systems. Due to its extensive use, considerable attention is focused on streptavidin production by recombinant methods.
In this study, streptavidin was expressed in Escherichia coli (E. coli) BL21 (DE3) pLysS cells and purified by affinity chromatography. Various dialysis methods were employed to enable the protein to refold to its natural form and create a strong bond with biotin.
Streptavidin was efficiently expressed in E. coli. Streptavidin attained its natural form during the dialysis phase and the refolded protein bound biotin. The addition of proline or arginine to the dialysis buffer resulted in a refolded streptavidin with greater affinity for biotin than refolding in dialysis buffer with no added amino acids.
Dialysis of recombinant streptavidin in the presence of arginine or proline resulted in proper refolding of the protein. The recombinant dialyzed streptavidin bound biotin with affinity as great as that of a commercial streptavidin.

We explored the effect of vitamin D receptor gene (VDR) polymorphisms in response to PEG-IFN treatment in Egyptian chronic hepatitis B (CHB) patients.
Two hundred hepatitis B virus (HBV) patients (42.3±10.7 years) on PEG-IFN α-2a (180 μg /kg for 48 weeks) and one hundred control subjects (37.3 ±12 years) were enrolled in the study. Vitamin D levels and hepatitis B surface antigen (HBsAg) expression were assessed by ELISA. VDR polymorphisms FokI T>C (rs 10735810), BsmI A>G (rs 1544410), ApaI (rs7975253), and TaqI C>T (rs 731236), were genotyped using real-time PCR.
Hepatitis B virus patients expressed significantly greater AST (p= VDR gene polymorphisms may be used as treatment response predictors in HBV patients receiving PEG-IFN. FokI SNP and bAt haplotype are independent factors that that can be used to determine PEG-IFN treatment responses in HBV-infected patients.

Until recently, a gene polymorphism in the promoter region of endothelial nitric oxide synthase has been suggested as a risk factor for thromboangiitis obliterans (TAO) development. The aim of this study was to compare the metabolites of nitric oxide (NO) and its backup, heme-oxygenase-1 (HMOX1), between TAO patients and those of a smoking control group matched by race, age, sex, and smoking habits.
Twenty-four male Caucasian TAO patients and 20 male Caucasian controls enrolled in the study. Their smoking habits were matched based on the serum cotinine levels of 17 of the TAO patients and the 20 controls. A colorimetric kit was used to measure NO, and an enzyme-linked immunosorbent assay kit was used to measure cotinine and HMOX1 levels.
The mean serum level of NO metabolites in the TAO group was significantly less than in the controls (p = 0.03) and also significantly less in the patients with below-knee amputations than in non-amputees (p= 0.018). Also, HMOX1 was significantly greater in the TAO patients than in the controls (p= 0.01). No significant correlation was found between NO and HMOX1 (p = 0.054).
Nitric oxide may play a pivotal role in TAO development and its outcome. However, the intact HMOX1 pathway may demonstrate the unique role of NO, which cannot be compensated for by HMOX1 and whose absence may make patients susceptible to developing TAO. In addition, another pathway besides NO, with influence on vascular tone and hemostasis, might be involved in TAO development, such as the autonomic nervous system. Further studies are suggested regarding these issues.

Rhinitis, which occurs most commonly as allergic rhinitis and affects 20% of the world’s population, is a major health care burden causing significant morbidity. Considering the high prevalence of allergic rhinitis and anti-inflammatory effects of thyme, a favorite condiment, we performed a randomized clinical trial to determine whether thyme can relieve allergic rhinitis symptoms and affect the expression of TH17- and T-regulatory cell- (Treg) related cytokines IL-17, TGF-β, FOXP3, and IL-10.
Thirty patients with allergic rhinitis symptoms and positive skin prick test for common aero allergens were randomly assigned to experimental or control groups. The experimental group received thyme or Zataria multiflora (ZM) extracts and the control group received placebo for two months. Expression of IL-17, TGF-b, FOXP3, and IL-10 was evaluated in all subjects by real-time PCR before and after intervention.
After treatment IL-17 expression was significantly less in the ZM group than in controls (p Given the significant effect of thyme in reducing symptoms of allergic rhinitis and decrease IL-17 gene expression and because allergic rhinitis is a multifactorial disease, the administration of thyme extract along with conventional treatments may benefit allergic rhinitis sufferers.

Pasteurella multocida continues to pose a danger to prone farm and wild animals all over the world. Chemotherapeutic treatments are progressively losing their effectiveness, last for long time, and cost a lot of money, as well as being toxic to human consumers. Therefore, clearing the way for immunization as a big-wheel alternative against the economic grain. Yet, the vaccines available in the market do not confer the necessary protection against the pathogen. The integration of the well adjuvanted killed vaccine with the attenuated vaccines proved to offer an effective protection to the host animals. However, the bare use of the killed bacterin to provide protection from the possible harm of the live attenuated vaccine was doubtful.
In the present study, propolis extracts were used to ameliorate the immunogenicity of the Pasteurella bacterin. The cellular and humoral activities were assessed for the different bacterin formulations.
Propolis extracts adjuvants proved to broaden and extend the IgG potency, as well as to induce a unique mucosal protection against the bacterium. Simultaneously it offered an anti-inflammatory effect that increased the tolerability to the bacterin. While the cellular activity was relatively reduced with propolis extracts.
These results confirm the effectiveness of the formulation of the bacterin with propolis to offer a potent homologous primary protection to the animals against the long-life use of the attenuated Pasteurella vaccines.

Asthma and allergic diseases cases have risen in recent decades. Plant pollen is considered as the main aeroallergen causing allergic reactions. According to available data, urban residents experience more respiratory allergies than rural residents mainly due to the interaction between chemical air pollutants and pollen grains. This interaction can occur through several mechanisms; chemical pollutants might facilitate pollen allergen release, act as adjuvants to stimulate IgE-mediated responses, modify allergenic potential, and enhance the expression of some allergens in pollen grains. This review focuses on the most recent theories explaining how air pollutants can interact with pollen grains and allergens.