Avicenna Journal of Medical Biotechnology
Year:2019 | Volume:11 | Issue:2|Papers Count:11

Autism Spectrum Disorder (ASD) represents a neurodevelopmental condition characterized by two main deficits: im-paired social communication and interaction; restricted and repetitive patterns of interests, behaviors or activities 1 with prevalence ranges from 2 to 20 per 1, 000, worldwide 1, 2. Presently, core symptoms of autism have no approved treatment. Autistic disorder management emphasis is on behavioral and educational modalities that target the core symptoms 3. Psychopharmacologic interventions are introduced to improve daily function and treat associated behavioral problems including hyperactivity, irritability, and aggression; leading to support the implementation of behavioral approaches through reducing the interfering symptoms. ...

The DNA motif discovery is a primary step in many systems for studying gene func-tion. Motif discovery plays a vital role in identification of Transcription Factor Binding Sites (TFBSs) that help in learning the mechanisms for regulation of gene expression. Over the past decades, different algorithms were used to design fast and accurate motif discovery tools. These algorithms are generally classified into consensus or prob-abilistic approaches that many of them are time-consuming and easily trapped in a local optimum. Nature-inspired algorithms and many of combinatorial algorithms are recently proposed to overcome these problems. This paper presents a general clas-sification of motif discovery algorithms with new sub-categories that facilitate build-ing a successful motif discovery algorithm. It also presents a summary of comparison between them.

Background: The Clustered, Regularly Interspaced, Short Palindromic Repeats (CRIS-PR) and CRISPR-associated protein (Cas) system has been used as a powerful tool for genome engineering. In this study, the application of this system is reported for target-ing Rag genes to produce mutant mouse NIH/3T3 cell line. The Rag1 and Rag2 genes are essential for generation of mature B and T lymphocytes. Disruption of Rag genes causes disease like Severe Combined Immunodeficiency syndrome (SCID). Here, the efficiency and specificity of CRISPR system were tested with highly active sgRNAs to generate novel mutations in the NIH/3T3 mouse cell line. Methods: Four single guide RNAs were designed to target sequences in the coding re-gion of the Rag1 and Rag2 genes. Four sgRNA-CAS9 plasmids were tested to target Rag1 and Rag2. Results: Based on T7 endonuclease assay and sequencing analysis, the expression of sgRNAs targeting two sites in Rag1 resulted in deletion of the intervening DNA frag-ment. The expression of sgRNAs with Cas9 targeting two sites in Rag2 gene resulted in indel mutations at both sites. In this report, fragment deletion in Rag1 gene was de-tected in about 50% of transfected cells. Conclusion: Therefore, CRISPR/Cas9 system can be highly efficient and specific when gRNAs are designed rationally and provides a powerful approach for genetic engi-neering of cells and model animals.

Background: Colon tumor is generated and maintained by a small subset of chemo-resistant cancer cells known as Cancer Stem-like Cells (CSCs) that are able to self-renew and differentiate into various cell types within the cancer milieu. CSCs are id-entified through expression of CD133 that is the most important surface marker of these cells. Epithelial Cell Adhesion Molecule (EpCAM) is another colon CSCs marker. Other markers that are probably involved in colon tumorigenesis are Leucine-rich re-peat-containing G-protein-coupled Receptor 5 (LGR5), B cell-specific Moloney murine leukemia virus insertion site 1 (BMI1) and Ten-Eleven Translocations (TETs). Methods: Here, mRNA expression rates of LGR5, BMI1 and TETs were surveyed by real-time PCR. After collection and digestion, colon samples were used to isolate CD133 and EpCAM positive CSCs through evaluation of AC133 EpCAM by Magnetic Activat-ed Cell Sorting (MACS) and flow cytometry. Real-time PCR was carried out for as-sessing expressions of LGR5, BMI1 and TETs. Results: High expressions for LGR5, BMI1, TET1 and TET2 in the CD133 and EpCAM posi-tive CSCs (p≤ 0. 05 vs. non-CSCs) were found. TET3, however, showed no significant changes for mRNA expression in the CSCs. Conclusion: In conclusion, high mRNA expressions for LGR5, BMI1, TET1 and TET2 in the CD133 and EpCAM positive CSCs may be a useful criterion for better identification of the cells involved in colon cancer in order to specify therapeutic targets against this type of cancer.

Background: Expression of heterologous proteins at large scale is often a challenging job due to plasmid instability, accumulation of acetate and oxidative damage in bio-reactors. Therefore, it is necessary to optimize parameters influencing cell growth and expression of recombinant protein. Methods: In the present study, the optimal culture conditions for expression of retep-lase by Escherichia coli (E. coli) BL21 (DE3) in a bench-top bioreactor was determined. Response Surface Methodology (RSM) based on Box-Behnken design was used to evaluate the effect of three variables (i. e., temperature, shaking speed and pH) and their interactions with cellular growth and protein production. The obtained data were analyzed by Design Expert software. Results: Based on results of 15 experiments, a response surface quadratic model was developed which was used to explain the relation between production of reteplase and three investigated variables. The high value of "R-Squared" (0. 9894) and F-value of 51. 99 confirmed the accuracy of this model. According to the developed model, the optimum fermentation conditions for reteplase expression were temperature of 32° C, shaking speed of 210 rpm, and pH of 8. 4. This predicted condition was applied for the production of reteplase in the bioreactor leading to a protein yield of 188 mg/l. Conclusion: Our results indicate the significant role of culture conditions (e. g., pH, temperature and oxygen supply) in protein expression at large scale and confirm the need for optimization. The proposed strategy here can also be applied to experi-mental set-up of optimization for fermentation of other proteins.

Background: The accurate and early diagnosis of tuberculosis is important for its ef-fective management. During the last decade, several molecular methods for detec-tion of Tuberculosis (TB) have been developed. Since RNA especially mRNA has a generally much shorter half-life than DNA, its detection may be useful for the assess-ment of viability of bacteria. This research is a Nucleic Acid Sequence Based Amplifi-cation-Enzyme Linked Immunosorbent Assay (NASBA-ELISA) which was designed and developed for rapid detection of viable Mycobacterium tuberculosis (M. tubercu-losis). Methods: Oligonucleotide primers targeting tuf gene encoding viability marker EF-Tu mRNAs were selected and used for the amplification of mycobacterial RNA by the isothermal NASBA Digoxigenin (DIG) labeling process and incorporated with DIG-UTP, reverse transcriptase and T7 RNA polymerase. Results: Using the NASBA-ELISA system, as little as 17. 5 pg of RNA of M. tuberculosis was detected within 4 hr and no interference was encountered in the amplification and detection of viable M. tuberculosis in the presence of non-target RNA or DNA. Results obtained from the clinical specimens showed 97 and 75% of sensitivity and specificity, respectively. Conclusion: The NASBA-ELISA system offers several advantages in terms of sensitivity, rapidity and simplicity for detection of M. tuberculosis. Furthermore, due to its simplic-ity and high sensitivity feature, it could be used in limited access laboratories in a cost-effective manner.

Background: In this study, bacteriostatic potency of the Iron oxide nanoparticles against Enterococcus faecalis (E. faecalis) (a clinical sample and the ATCC11700 strain) was investigated. Methods: Nanoparticles’ bacteriostatic concentration was determined and used to appraise the characteristics of the Iron Oxide (Fe2O3) against the isolates. Antimicro-bial examinations with 108cfu. ml-1 were performed at the baseline. Due to evaluation level of potency, after performing Minimum Inhibitory Concentration (MIC), the as-sessment of death kinetic and susceptibility constant of nanoparticles was done by suspension at two MICs in 0 to 360 min treatment time. Results: Fe2O3 nanoparticles in size range of 10-50 nm demonstrated the most effec-tive susceptibility reaction against E. faecalis and ATCC11700 strain in Z=78. 125 ml/μ g-1 and 39. 0625 ml/μ g-1, respectively. The kinetic reaction of E. faecalis against Fe2O3 sus-pension was supposed to be decreased through the elapse of treatment time, whereas increased concentration was along with bacteria growth after a certain time. So, the efficient concentration of nanoparticles was applied with semi-sensitive and antibiotic resistant for both strains. However, synergism of Fe2O3 nanoparticles with Ceftazidime and Clindamycin revealed a higher susceptibility compared with Fe2O3nanoparticles alone against E. faecalis. Conclusion: The experimental results reveal that Fe2O3 has a strong antimicrobial ef-fect at a certain concentration over the time so could potentially be used for bacterial inhibition and this feature will be strengthened in combination with antibiotics.

Background: Acinetobacter baumannii (A. baumannii) is a bothersome fatal patho-gen, particularly in healthcare system. Persistence and successful invasion of A. bau-mannii in vertebrate host cells largely depends on iron acquisition methods. Sidero-phore molecules and Iron-Regulated Outer Membrane Proteins (IROMPs) are the two essential members of iron acquisition system. Siderophores are secreted by bacteria to bind peripheral ferric iron and the IROMPs are expressed at the bacterial outer mem-brane as the receptor of ferric-siderophore complex. BauA is the corresponding sider-ophore receptor of A. baumannii. In this study, an attempt was made to assess the immunogenicity of antigenic domains of BauA which could be effective in iron up-take restriction and protection against bacterial invasion of the host cells. Methods: The antigenic domains of bauA were amplified from A. baumannii ATCC-19606. The PCR products were ligated into pET32a and expressed in Escherichia coli (E. coli) BL21 (DE3). Purification of recombinant domains was done by Nickel-Nitri-lotriacetic Acid (Ni-NTA) affinity chromatography. The recombinant domains were injected into BALB/C mice separately and in combination. Sero-reactivities of the re-combinant proteins and mouse challenge tests were carried out. Results: The antibodies raised in mice could successfully recognize and bind antigenic domains. Passive immunization studies accomplished by immune rabbit serum inhib-ited the establishment of infection in mice. Conclusion: The results adapted from the present study disclose the protective role of functional domains of BauA, especially the cork domain, suggesting a novel recombi-nant immunogen candidate.

Background: Rheumatoid Arthritis (RA) is a debilitating disorder in which the im-mune system mainly targets the synovial tissue. Janus kinase family including tyrosine kinase 2 (TYK2) is one of the crucial mediators of the downstream signaling pathway of inflammatory cytokines that further contributes to RA pathogenesis. In this study, the association of TYK2 gene rs34536443 polymorphism, which may affect the func-tion of TYK protein and, hence, the inflammatory settings, with RA susceptibility was investigated. Moreover, its correlation with demographic and serological features of the patients was assessed. Methods: In the present study, 700 RA patients and 700 sex, age and ethnicity-matched healthy individuals as the control group were included. MGB TaqMan real-time allelic discrimination method was used to determine the rs34536443 polymor-phism. Rheumatoid factor, anti-cyclic citrullinated peptide antibody, erythrocyte sed-imentation rate and C-reactive protein were also measured. Results: The frequency of rs34536443 minor allele (C allele) was not different between patients and control group [1. 7 vs. 2. 61 percent, OR (95% CI)=1. 35 (0. 78-2. 33); p=0. 27]. There was not a statistically significant association between rs34536443 genotypes and RA susceptibility. Genotypes of rs34536443 polymorphism were associated nor with demographic neither with serological features of RA patients. Conclusion: In the present study, there was not any association between TYK2 gene rs34536443 polymorphism with either disease susceptibility, demographic and serolog-ical features of Iranian RA patients. These findings are not compatible with previous works from other ethnicities, further supporting the role of genetics in disease suscep-tibility.

Background: Urinary Tract Infection (UTI) in patients with Chronic Kidney Disease (CKD) caused by multi-drug resistance and Extended Spectrum Beta Lactamase (ESBL)-producing gram-negative bacteria has been increased in different countries. The aim of the present study was to detect the antibiotic susceptibility patterns and the distribution of Bla-TEM, Bla-SHV and Bla-CTX-M genes in gram-negative bacte-ria isolated from outpatients infected with UTI, with and without CKD in Al-Najaf city, Iraq. Methods: A total of 120 non-duplicate urine samples were collected from outpatients (37 male and 83 female) infected with UTI in Al-Najaf city, Iraq; 60 samples from pa-tients Without Kidney Disease (WKD) and 60 samples from patients with CKD. The antibiotic susceptibility testing was done according to Kirby-Bauer method. PCR technique was performed to investigate the prevalence of Bla-TEM, Bla-SHV and Bla-CTX-M genes. Results: A total of 126 different gram-negative bacterial strains were isolated. Esche-richia coli (E. coli) was the most prevalent bacterium (49 isolates) followed by Idebsiella pneumonia (K. pneumonia) (35 isolates), Pseudomonas aeruginosa (P. ae-ruginosa) (18 isolates), Citrobacter freundii (C. freundii) (12 isolates), Enterobocter aer-ogenes (E. aerogenes) (8 isolates) and Proteus mirabilis (P. mirabilis) (4 isolates). All bacterial isolates from UTI patients with CKD were resistant to antibiotics and carried Bla-TEM, Bla-SHV and Bla-CTX-M genes more than isolates from UTI patients with WKD. Conclusion: This study demonstrated that all bacterial isolates from UTI patients with CKD were more virulent than isolates from UTI patients with WKD.

Background: Toll-Like Receptors (TLRs) are the cause of phagocytosis activation and destruction of the infection agents. In addition, new evidences support the idea that TLRs play a vital role in starting the acquired immunity reactions. Methods: In this study, it has been attempted to infect the BALB/c mice with Leish-mania major (L. major) and treat them using morphine and imiquimod; then the ex-pressions of TLR2, 4 from treated lesion were studied by using Real-Time PCR method. Treatment with morphine 1 mg/kg, imiquimod 5% and nalmefene 1 mg/kg began four weeks after the challenge. After treatment period, half of the mice of each group were killed and their lesions were isolated for RNA extraction and making cDNA. For the rest of mice, lesion size was measured weekly. Results: The results showed increase of expression of TLR2 gene among all treated groups relative to the control, and the difference was significant (p<0. 05). The expres-sion of TLR4 gene only was reduced in groups under treatment with morphine and morphine plus nalmefene relative to the control group and in the other groups in-creased. The highest expression of TLR2 was seen in the group treated by glucantime (p<0. 0001). Conclusion: However, in this study it was found that despite decreasing the size of le-sion in all treated groups, expression of TLR4 in the morphine, nalmefene, morphine plus nalmefene treated groups compared to the control group was decreased. There-fore, morphine may have a different function mechanism in treatment of the Leish-maniasis with the L. major.