Avicenna Journal of Medical Biotechnology
Volume:11 Issue: 1, Jan -Mar 2019

Major depressive disorder (MDD) is one of the prominent causes of disability in the world, affecting 15-20% of people over the period of a lifetime. Patients commonly experience continuous remaining symptoms, functional impairment, and diminished health 1.
Treatment of MDD is still far from optimal and drug resistance in MDD is still considered a serious clinical challenge. Only about one-third of patients completely respond to their first antidepressant medication, first with an approximate lag time of 2 months. This notable lag time for onset of therapeutic efficacy is associated with significant morbidity and suicidal risk. As a result, there is a widely accepted need for fast acting antidepressants. Another downside of the currently available treatments is their side effects which are documented in large proportion of patients 1,2.
Patients who do not respond to their first-line medication are generally treated by either switching to another treatment or with augmentation therapy to achieve favorable response. Combination therapy from start of treatment has been suggested recently to gain quicker and better response and remission rates, however, not all studies have supported this opinion. Most MDD pathophysiology etiological theories used to focus on brain modulatory monoamine systems (dopamine, serotonin and norepinephrine) 3,4. A more recent line of evidence points to glutamate, the brain’s principal excitatory neurotransmitter, as playing a role in MDD’s pathophysiology. Additionally, glutamate dysregulation is known to cause impairments in structural plasticity and cellular resilience, which seems to be implicated in mood disorders as well. It is therefore reasonable to hypothesize that medications which reduce glutamatergic tone may be able to play a role in treatment of depression 5.
Depression is demonstrated to be accompanied with parallel increases in the immuno-inflammatory biomarkers including significantly higher levels of pro-inflammatory cytokines interleukin (IL)-1, IL-6, tumor necrosis factor (TNF)-α, and C-reactive protein (CRP) in depressed patients compared to normal individuals. The dynamic interaction between pro-inflammatory cytokines, prostaglandin (PG)-E2 synthesis and depression has led to suggestions that anti-inflammatory agents could be useful for treatment of depression 6,7. Celecoxib, a nonsteroidal anti-inflammatory drug that acts via the selective inhibition of cyclooxygenase (COX)-2, has shown promising outcomes in several psychiatric diorders, including autistic disorder, schizophrenia, and depression. Antidepressive effects of celecoxib is suggested to be largely attributed to its inhibitory effect on the levels of pro-inflammatory cytokines. In line with this idea, baseline levels of serum IL-6 is demonstrated to be significantly correlated with the Hamilton Depression Rating Scale (HDRS) score. In the same article, adjunctive therapy with celecoxib, as an antidepressant, resulted in significant decreases in both serum IL-6 levels and HDRS scores. Although most of the previous research has focused on the antidepressant effects of celecoxib as an add-on treatment, few reports have investigated the safety and efficacy of celecoxib as a monotherapy 8-10.
In clinical trials, two new classes of anti-inflammatory drugs-anti-cytokine monoclonal antibodies and cytokine inhibitors have been shown to reduce inflammation in a range of autoimmune diseases, and these drugs have already started to be administered to patients who do not respond to standard treatments 11,12.

The design of Antibody Drug Conjugates (ADCs) as efficient targeting agents for tumor cell is still in its infancy for clinical applications. This approach incorporates the antibody specificity and cell killing activity of chemically conjugated cytotoxic agents. Antibody in ADC structure acts as a targeting agent and a nanoscale carrier to deliver a therapeutic dose of cytotoxic cargo into desired tumor cells. Early ADCs encountered major obstacles including, low blood residency time, low penetration capacity to tumor microenvironment, low payload potency, immunogenicity, unusual off-target toxicity, drug resistance, and the lack of stable linkage in blood circulation. Although extensive studies have been conducted to overcome these issues, the ADCs based therapies are still far from having high-efficient clinical outcomes. This review outlines the key characteristics of ADCs including tumor marker, antibody, cytotoxic payload, and linkage strategy with a focus on technical improvement and some future trends in the pipeline.

Archived bone marrow aspirate slides are almost infinite, readily available resource of biospecimens that enable retrospective molecular investigations of diseases. RNAs obtained from slides has limitations in utility because of their low quality and highly fragmented nature. MicroRNAs are small (<22 nt) noncoding RNAs with various cellular regulatory roles. Due to their small size, microRNAs are less prone to degradation and modification, therefore, can be preserved well in archived tissues. The current study investigated the efficacy of archived bone marrow aspirate slides for miRNA expression analysis in pediatric leukemia. Total RNA was isolated from air-dried unstained archived slides using High pure miRNA isolation Kit with some modifications and from fresh samples using TRizol. After cDNA synthesis, RT-qPCR was then carried out using specific hsa-miR-326 LNA primers. Finally, statistical analyses were conducted using GraphPad Prism 6 software. The difference observed in miRNA expression due to disease state was far greater than the differences between archived slides and their matching fresh bone marrow specimens. In fact, the expression of archival slide smears for the miR-326 closely mimicked that of fresh-frozen tissues (0.035±0.04 vs. 0.03±0.04) (Mean±SD, p>0.05). Differential expression of hsa-miR-326 was detected between leukemic and non-leukemic samples from archived slides or fresh frozen bone marrows. The demonstration that archived bone marrow aspirate slides can be utilized for miRNA expression studies offers tremendous potential for future investigations into the role that miRNAs play in the development and long term outcome of hematologic, as well as non-hematologic diseases.

The present study assessed the alteration of gene expression during transdifferentiation of Bone Marrow Stromal Cells (BMSCs) into oligodendrocyte in the presence of Cerebrospinal Fluid (CSF). BMSCs were collected from female Sprague-Dawley rats and were cultured in DMEM/F12 medium supplemented with Retinoic Acid (RA), basic Fibroblast Growth Factor (bFGF), and Epidermal Growth Factor (EGF). CSF was added daily to the culture media. The oligoprogenitor and oligodendrocyte generation was assessed by immunocytochemistry for Oligo 2, A2B5, CNP and MBP markers. The mean percentages of immunopositive cells for Olig2 and A2B5 were 52.1±1.74 and 56.34±2.55%, respectively. The number of immunopositive cells for glial markers CNP and MBP were 48.8±3.12 and 40.5±8.92%, respectively. Alteration of gene expression of Oct4, Olig 2, PDGFR-α and PLP were examined by real time PCR during transdifferentiation of BMSC to oligodendrocyte. Immunocytochemical results indicate that oligoprogenitor cells were immunopositive for Oligo2 and A2B5 markers. Also, oligodendrocytes expressed the mature glial markers of CNP and MBP indicating successful differentiation. In conclusion, CSF promotes the transdifferentiation of BMSC into mature oligodendrocyte via providing an appropriate niche for glial maturation.

Nowadays, transplantation of bone marrow-derived Mesenchymal Stromal Cells (BMSCs) is currently an important alternative therapy for patient’s type 1 diabetes mellitus. But a number of critical obstacles lie ahead of this new strategy including reducing stem cell homing to the damaged tissue due to oxidative stress. The purpose of the present study was to investigate whether preconditioning of BMSCs with SDF-1 could enhance their homing to the pancreas and promote regeneration of the pancreatic β cells after being intravenously injected. Mice BMSCs were isolated and expanded. Cell proliferation was assayed by MTT Assay. Preconditioning was performed with 10 ng/ml SDF-1α for 24 hr. Male NMRI mice were injected with high-dose STZ (150 mg/kg). The preconditioned or unpreconditioned BMSCs at a dose of 1×106 cells were infused via the tail vein. Blood and pancreatic tissue samples were taken from all mice for flow cytometry, biochemical and histological studies. Proliferation and homing of BMSCs to the pancreas were significantly increased in the BMSCs with SDF-1α preconditioning. Differentiation of transplanted BMSCs, were significantly increased in preconditioning group. Although BMSCs without SDF-1 preconditioning exhibited remarkable recovery of pancreatic islets structure but this recovery were significantly increased in the BMSCs with SDF-1α preconditioning. Our results showed the effectiveness of SDF-1αpreconditioning in BMSCs transplantation of STZ induced diabetes mice which might be achieved through improvement of BMSCs homing into the injured pancreas.

Recently, due to the steep increase in cancer lethality statistics, pharmaceutical societies seek approaches for designing drugs with higher efficiency and lower expenses. Plant-based drugs have therefore gained much attention, due to their abundance and ease of accessibility, and their higher effectiveness. Wild-grown caper [Capparis spinosa (C. spinosa)] was collected from northern Iran and next 100 g of the powder was added to 300 ml of a solvent (Ethanol 80), the solution was mixed for 72 hr and later filtered via Whatman filter papers. The solvent was taken out under vacuum conditions and extracts were then collected and stored in glass vials. The High Pressure Liquid Chromatography (HPLC) method was used to assay quercetin which consisted of the following specifications: C18 column, UV detector wavelength of 260 nm, mobile phase acetonitrile and water and flow rate of 1 ml/min. In this study, the anti-cancer effects of C. spinosa extract on HeLa, MCF7, Saos and Fibroblast cancer cell lines have been investigated. The amount of quercetin was assessed by HPLC. The anti-tumor activity and the antioxidant level of hydroalcoholic extract of C. spinosa have been evaluated with MTT assay and FRAP technique, respectively. HPLC data showed quercetin form the major component of C. spinosa extract. In addition, FRAP data indicated that C. spinosa extract had high antioxidant activity and MTT assay indicated that C. spinosa extract effectively decreased the cancer cell lines. The quercetin in C. spinosa extract had significant anti-tumor effects and may be regarded as an ideal natural drug for cancer therapy.

Staphylococcus aureus (S. aureus) causing numerous diseases in humans, have become resistant to antibiotics, hence, urging the need for alternative medicines. In this study, the Indian medicinal weed, Ruellia patula (R. patula) extracted and fractioned through column chromatography was subjected to antibacterial and anti-quorum sensing activity against S. aureus and Methicillin Resistant Staphylococcus aureus MRSA. The obtained results confirmed fraction F44 to have significant effect as antimicrobial and anti-biofilm agent against both the micro-organism. Therefore, few of such highly active fractions were chemical finger printed using GC-MS and the compounds identified were further docked with DNA binding (LytTR) domain of agrA, which revealed that compounds identified from fraction were interactive to the protein. R. patula is promising antimicrobial and anti-biofilm agent against S. aureus and MRSA.

The present study focused on the production of L-asparaginase using Solid State Fermentation (SSF) by Myroides gitamensis. Initially, five significant parameters (Carbon source; Nitrogen source, temperature, pH and incubation period) were identified that affect the production process of L-asparaginase using Classical One Factor at a Time (OFAT) optimization. An optimized L-asparaginase specific activity obtained by OFAT was recorded as 85.7 IU. Central Composite Design (CCD) was also employed successively to optimize the multiple parameters at a time and their results were compared. Maximum L-asparaginase enzyme specific activity obtained by CCD method was 295.6 IU under the hold values of carbon source (wheat bran) 12 g/L, nitrogen source (yeast extract) 7 g/L, temperature 37oC, pH=7.5 and incubation period 47 hr. Upon validation, the obtained results proved that there was a good relation existing between the experimental and the predicted model (p<0.05). L-asparaginase activity was enhanced in statistical method up to 3.4 folds compared to that of classical method. Utilization of wheat bran as a low cost carbon source in SSF for the production of L-asparaginase enzyme makes the process economical and in turn reduces the environmental pollution by biotransformation to commercially useful bio product.

Tax-1 protein of Human T-cell Leukemia Virus type 1(HTLV-1) serves as a key transcriptional regulatory gene product and has a crucial role in transactivating genes of infected cells by employing their transcriptional factors. This modulation includes induction of genes which encode CC-chemokines and their receptors. In this study, a recombinant vector containing Tax-1 gene was made and tested for its ability to induce CCR5 (CC chemokine receptor 5) expression in K562 cell line. In order to perform this research, two blood samples of HTLV-1 positive were obtained from Urmia blood transfusion center. After DNA extraction, a complete sequence of Tax-1 gene was amplified by specific primers. Recombinant vectors carrying Tax-1 gene were synthesized and transformed into Escherichia coli (E. coli). After bacteria transformation, bacteria containing recombinant plasmid were selected and purified. Then, the recombinant shuttle vectors, pCDNA3.1-TAX, were transfected into the cell culture (K562 cell line). Expression of CCR5 was measured after 72 hr by Syber Green Real-Time PCR method compared to control cell culture. Normalization was done with GAPDH as a standard gene. Cloning of Tax-1 gene in the vector, pCDNA3.1 was confirmed by colony PCR, restriction digestion, and sequencing methods. Expression of Tax-1 and CCR5 genes were confirmed by real time PCR and also, expression of CCR5 gene showed an 8-fold increase in comparison to mock-treated controls (p<0.05). Our data suggested that recombinant Tax-1 may have the enhancing effect on CCR5 expression rate at mRNA levels in K562 cell line. Further studies are necessary to evaluate the effect of pCDNA3.1-TAX on cell surface CCR5 expression.

Prostate cancer is the second most common cancer in men. Prostate-Specific Antigen (PSA) is a tumor-associated glycoprotein with enzymatic activity which is secreted by the prostate gland. Following entry to the blood, 70-90% of PSA forms complexes with protease inhibitors and its enzymatic activity is inhibited. The serum level of PSA is increased and the rate of free PSA (fPSA) to total PSA is decreased in prostate cancer patients. Therefore, measurement of PSA and fPSA in serum is very valuable for diagnosis and prognosis of prostate cancer. In the present study, five anti PSA monoclonal Antibodies (mAb) were characterized by Enzyme-Linked Immunosorbent Assay (ELISA) and immunoblotting. For designing a sandwich ELISA, epitope specificity of these antibodies was studied by a competition ELISA. Free PSA was purified by electroelution technique from seminal plasma and used to produce polyclonal anti-fPSA antibody in rabbit. Purified polyclonal antibody (pAb) and mAbs were conjugated with HRP enzyme and Biotin (Bio) to set up the sandwich ELISA. Three of the mAbs were found to recognize PSA similarly. One of these mAbs (2G3) was paired with anti-fPSA pAb to detect fPSA in serum. Eventually, serum fPSA concentration of 356 subjects was measured and compared by our designed ELISA and a commercial ELISA kit. Our results demonstrated a significant correlation (r=0.68; p<0.001) between the two assays. Sensitivity and specificity of our designed ELISA was 72.4 and 82.8%, respectively. These results imply suitability of our designed ELISA for detection of fPSA in patients with prostate cancer.

Periodontal disease, which can become a chronic condition, is an inflammatory disease that upsets the soft and hard structures supporting the teeth. The aim of the present study was to design and develop an in-house Line Probe Assay (LiPA), to detect putative periodontitis-related bacterial pathogens, and compare it with SYBR Green Real-time PCR. The LiPA method was launched using biotinylated 16s rRNA universal primers and specific probes for each of the five bacteria including Aggregatibacter actinomycetemcomitans, Prevotella intermedia, Tannerella forsythia, Porphyromonas gingivalis and Treponema denticola. For this, optimized quantities of the primers and specific probes were dotted onto nylon membrane stripes in a defined pattern. Hybridization was performed between the probes and the single-stranded biotinylated PCR products. The stripes were developed via biotin-streptavidin reaction. Ultimately, the analytical and diagnostic sensitivity and specificity of the in-house LiPA was evaluated and compared with SYBR Green Real-time PCR. The detection limit of the LiPA was 2760 copies of targeted genes. In testing analytical specificity, only signals corresponding to the specific biotinylated products were produced. The calculated diagnostic sensitivity of the LiPA for the five bacterial targets ranged from 96.4 to 100%, whereas the diagnostic specificity was between 90.9 and 100%. Comparing the results, no noticeable difference (p=0.4795) was observed between the two methods. To screen periodontal pathogens, a simple, inexpensive and accurate method is desirable. The in-house LiPA, having advantages such as high specificity and sensitivity, and the ability to detect five major periodontal pathogens, offers the option of evaluating samples without the need for a post-PCR platform.

Nitric Oxide (NO) which is synthesized by endothelial Nitric Oxide Synthase (eNOS) in both vascular tissues and platelets plays an important role as a protective mediator in the cardiovascular system. It modulates blood pressure, vasodilation and thrombosis. In this regard, eNOS activity and gene expression in platelets and NO levels in patients’ plasmas with Coronary Thrombosis (CT) and stenosis diseases were determined. Blood samples were collected from 60 subjects that where divided into three equal groups [without coronary artery disease, with CT and less than 70% Coronary Stenosis (CS)]. NO concentration in plasma was measured by the Griess reagent system. The eNOS activity was assessed based on a fluorimetric detection system in platelets and the gene expression was quantified by the real time-reverse transcription-polymerase chain reaction. There was a significant decrease in the amount of NO concentration in the plasma of subjects with CT (0.53±0.09 µM, p<0.01) and CS (1.31±0.11 µM, p<0.01) compared to the control group (2.6±0.10 µM). The activity levels of eNOS enzyme were significantly lower in patients’ platelets with CT (0.68±0.013 UF/mn, p<0.01) and CS (0.85±0.017 UF/mn, p<0.01) than the control cases (1.29±0.019 UF/mn). These data were consistent with the reduction of the expression levels of eNOS in patients with CT (75 folds) and CS (4 folds) lower than the control cases. Patients with CT and CS possessed reduced eNOS activity and gene expression in their platelets. Decreased plasma concentration of NO in these patients confirmed the potential significance of the diagnostic and prognostic value of NO in the subjects’ plasma with vascular disease risk.

Zika virus is the family member of flavivirus with no reported clinically approved drugs or vaccines in the market till date. This virus is spread by Aedes mosquitoes, and can also be transmitted through sexual contact or blood transfusions. There are reported medical conditions like microcephaly among new-borns delivered by infected pregnant women. The envelope protein of Zika virus is associated with virulence, tropism, mediation of receptor binding and membrane fusion. ED1-EDII domain (K1 loop pocket) is an integral part of the envelope protein and a potential drug target. In the present study, the purpose was to identify the potential lead molecules to dock against K1 loop which could be later considered as flavivirus entry inhibitors. Multiple sequence alignment method was considered for the analysis of indels in envelope protein. Phylogenetic tree was constructed based on the alignment. Aliphatic index, GRAVY scores and hydropathy plot of the envelope proteins were calculated for the flavivirus family members. Zika envelope protein was homology modeled and considered for protein-ligand docking analysis with chemical compounds of known functions. As per in silico based analysis, the envelope protein of Zika virus is highly hydrophilic with the least number of amino acid deletions compared to rest of the family members. During docking studies, it was observed that compounds like NITD, compound 6, P02, Doxytetracycline and Rolitetracycline show better binding affinity with Zika envelope protein compared to dengue virus. These better binding compounds could be the promising lead molecules for Zika envelope protein which could better block the viral entry.

Nucleic acid-binding proteins play major roles in different biological processes, such as transcription, splicing and translation. Therefore, the nucleic acid-binding function prediction of proteins is a step toward full functional annotation of proteins. The aim of our research was the improvement of nucleic-acid binding function prediction. In the current study, nine machine-learning algorithms were used to predict RNA- and DNA-binding proteins and also to discriminate between RNA-binding proteins and DNA-binding proteins. The electrostatic features were utilized for prediction of each function in corresponding adapted protein datasets. The leave-one-out cross-validation process was used to measure the performance of employed classifiers. Radial basis function classifier gave the best results in predicting RNA- and DNA-binding proteins in comparison with other classifiers applied. In discriminating between RNA- and DNA-binding proteins, multilayer perceptron classifier was the best one. Our findings show that the prediction of nucleic acid-binding function based on these simple electrostatic features can be improved by applied classifiers. Moreover, a reasonable progress to distinguish between RNA- and DNA-binding proteins has been achieved.

In spite of recent progress in mRNA technologies and their potential applications for treatment of human diseases, problems such as the transient nature of mRNA limit the stability of gene up-regulation and, thus, potentially reduce mRNA efficiency for gene therapy. Using human β-globin 5′ and 3′ untranslated regions (UTRs), this study aimed to develop the different chimeric constructs of mRNAs to increase the stability of destabilized green fluorescent protein (EGFPd2) in HEK 293 cells. Purified human β-globin (HBG) 5′-3′UTRs, and the coding sequence of destabilized green fluorescent protein (EGFPd2) were amplified separately and ligated to each other using SOEing PCR method in a different format. As controls, the original construct of EGFPd2 under the control of T7 promoter was used. Following in vitro transcription, HEK 293 cells were then transfected with several constructs and incubated at 37oC in a CO2 incubator. They were monitored under a fluorescence microscope every four hours for the first 24 hr, then every 12 hr afterwards. The resulting fluorescence was measured as a surrogate for translation efficiency and duration. By monitoring the HEK cells over 48 hr, cells transfected with mRNA with various HBG UTRs showed significantly different fluorescence intensity and stability in comparison with the pEGFPd2 prototype (control transcript) overtime. Overall, the images show that replacement of the 3′ UTR end of the prototype vector pGFPd2 with the 3′ end of β- globin mRNA increases the half-life of the chimeric mRNA for more than 32 hr. This result indicates that β-globin 3′ UTR would definitely increase the half-life of mRNA and may help to decrease the mRNA therapeutic dosage in the treatment of diseases associated with mRNA therapy.

The Angiotensin Converting Enzyme (ACE) Insertion/Deletion and rs4343 gene polymorphisms could be associated with pathogenesis of essential hypertension and cardiovascular disorders and Coronary Artery Disease (CAD). In the present study, a fast and novel approach of multiplex Tetra-Primer Amplification Refractory Mutation System-PCR (T-ARMS-PCR) was developed for simultaneous detection of two SNPs including ACE I/D (rs4340) and 2350A>G (rs4343) of Angiotensin Converting Enzyme (ACE) gene. The present research was performed using 148 blood samples taken from patients with CAD and 135 healthy individuals. One set of inner primers (for rs4343) and one set of outer primer pairs were designed for genotyping of Insertion/Deletion and rs4343 polymorphisms in single tube T-ARMS-PCR. Our results manifested that genotypes and alleles frequency of the ACE polymorphisms showed no statistically significant association between CAD patients and the control group. In addition, complete concordance was seen between sensitive Tetra-ARMS-PCR and sequencing method. The technique is the first work for simultaneous detection of Insertion/Deletion polymorphism and rs4343 SNPs in ACE gene and the results were entirely according to those from an independent procedure.

This report is about a pregnancy with a triploid fetus and underscores the potential of first trimester combined screening to detect this devastating chromosomal aberration earlier in pregnancy. This report is about a pregnancy with a triploid fetus identified from the first trimester combined screening and confirmed by amniocentesis. A 28 year old, G5P2AB2 woman was referred to our clinic at 15 weeks of gestation due to a remarkable decrease of her first trimester double biochemical markers and therefore in the high-risk range for trisomy 13 and 18. The woman underwent amniocentesis which revealed a karyotype of 69,XXX. The parents opted for termination and in post mortem physical examination, a hydrocephalus fetus with marked Intra-Uterine Growth Retardation (IUGR) in addition to syndactyly of third and fourth digits, low set malformed ears, micrognathia and club foot, was seen. Our results and previous reports highlight the need to consider a somewhat consistent pattern of the first trimester combined screening in a pregnancy with triploidy and underscore the potential of this screening strategy to detect this chromosomal aberration earlier in pregnancy. Early prenatal diagnosis of this syndrome would provide women an opportunity to terminate an affected pregnancy earlier. This is also important in preventing the risks of associated later induced abortion or obstetric complications.