Journal of Applied Biotechnology Reports
Volume:9 Issue: 3, Summer 2022

Gastric cancer, known as one of the most frequent types of cancer, is associated with extensive mortality. Early detection could help to increase the survival rate of patients. Long non-coding RNAs (lncRNAs) as newly considered cancer probes were evaluated regarding as potential diagnostic candidates. LncRNAs NUTM2A-AS1, CTBP1-DT, THUMPD3-AS1, and LINC00667 were selected for wet-lab analysis based on literature review and pathway enrichment in-silico analysis. Four candidate lncRNAs in 70 gastric tissue samples including gastric tumors (n = 35) and paired Adjacent Normal Gastric Tissues (ANGTs) were collected for quantitative analysis. In the following, the expression level of four candidate lncRNAs was analyzed in gastric cancer samples compared to ANGTs and patient’s clinico-pathological data. Receiver Operating Characteristic (ROC) analysis was also performed to determine the diagnostic value of NUTM2A-AS1, CTBP1-DT, THUMPD3-AS1, and LINC00667 levels in tissue samples. Different pathways associated with NUTM2A-AS1, CTBP1-DT, THUMPD3-AS1, and LINC00667 lncRNAs were identified. The significantly elevated levels of all four lncRNAs were detected in GC tissue samples compared to ANGTs (P-value<0.05). The Aria Under Curve (AUC) of NUTM2A-AS1, CTBP1-DT, THUMPD3-AS1, and LINC00667 in the ROC curve were 0.825, 0.928, 0.688, and 0.649, respectively. Our results indicated that the expression levels of the four candidate lncRNAs were elevated in gastric tumors compared to ANGT samples. It might be helpful to illuminate the molecular mechanisms underlying gastric carcinogenesis which have been served as tumor-associated biomarkers for diagnosis.

The appalling environmental hazards associated with the use of a triphenylmethane dye i.e., Malachite Green (MG) was unveiled by the National Institute of Health in 2004. However, in spite of the successful ban of MG in the US, UK, and several European countries, it continues to be the most commonly used dye in microbiological laboratories and a few textile industries. In the present study, the bio-remediation potential of a bacterium isolated from a flower vase filled with traces of MG dye solution was investigated. The physicochemical parameter for degradation of MG was optimized. Also, considering the fact that the dyes are complex molecules and their breakdown products may be unsafe for environmental disposal, toxicity tests were carried out using an aquatic invertebrate (Daphnia magna) as a model organism. The bacterium was identified as Enterobacter cloacae NAM-9415 by 16S rRNA analysis. It showed 96% decolourization of MG at 500 ppm dye concentration when cultured at optimum conditions of incubation i.e., 15 h at 45 °C under shaker (120 rpm) conditions using nutrient broth medium (pH 7). In addition, it also showed tolerance to high salt concentrations of up to 6g%. Moreover, the breakdown products supported the growth of daphnids in our study. The above observations indicate the suitability of E. cloacae NAM-9415 for biodecolorization of textile effluents.

Shigella dysenteriae with a low infectious dose and its strains that are resistant to antibiotics is considered a health problem all over the world. IpaD as an important protein in the shigella type 3 secretion systems can be a convenient target for designing recombinant subunit vaccines against the bacteria due to its immunogenic properties. The present study aimed to evaluate the immunogenicity of a recombinant protein containing immunogenic regions of IpaD as a subunit recombinant vaccine candidate against Shigella dysenteriae. The gene encoding immunogenic segments of ipaD was sub-cloned into pET28a expression vector. The new plasmid (pET-IpaD) was transformed into E. coli strain Rosetta (DE3). The recombinant protein was then expressed and purified using affinity chromatography and confirmed by western blotting. The guinea pigs were then immunized with purified protein and the antibody titer and specificity of the sera were analyzed by ELISA. Finally, an animal challenge was performed using the Sereny test. According to the designed pET-IpaD plasmid, the expression of recombinant protein in E. coli caused the production of a recombinant protein with 22 kDa molecular weight, and the western blot technique indicated the reaction of recombinant protein with anti-histidine monoclonal antibody. The yield of the purified protein from the culture medium was estimated at about 0.57 mg/ml and the immunogenic effect of the produced protein was determined by using in vitro and in vivo studies. According to the findings of the present study, it can be concluded that the recombinant produced IpaD is a perfect peptide vaccine candidate for the development of a recombinant vaccine against Shigella dysenteriae.

Bacillus megaterium is a ubiquitous bacterial strain that produces the enzyme arsenate reductase that catalyzes the reduction of less toxic arsenate (V) to more toxic arsenite (III). Due to the functional significance of this enzyme, the present study was carried out to construct and validate the Three Dimensional (3D) structure of arsenate reductase of B. megaterium and study its interaction with arsenate. The 3D model was generated by MODELLER using the known crystal structure of the enzyme. The superimposition of the model with the template structures was done by PyMOL. PATCHDOCK was used to perform molecular docking of the enzyme with arsenate ion and Fire Dock was used to refining the docked complexes. The highest geometric score containing docked complex was visualized and the intra-molecular interaction within it was evaluated. The evaluation of the 3D computed model showed good qualities including fine stereochemical properties, satisfactory compatibility between the structure and its amino acid sequence, acceptable residues error value, etc. The model as well as its phylogenetic relatives (Bacillus and Staphylococcus) showed the same active site motif which is CTGNSCRS. The crucial amino acids involved in binding with the arsenate ion (AsO43-) were Cys10, Thr11, Gly12, Asn13, Ser14, Cys15, His43, and Asp106. Among these, the first six amino acids fell in the conservative motif (CTGNSCRS). Studying this interaction can be helpful for more research to inhibit the binding or any other approach that will stop the inimical conversion of less toxic arsenate to more toxic arsenite.

Colorectal Cancer (CRC) is a genetic disease with complex and diverse pathways. The p16 is a tumor-inhibiting gene that acts as a regulator of the cell cycle. Therefore, the present study aimed to investigate the expression of the p16 marker in CRC and its relationship with clinical and pathological parameters. In this retrospective study, paraffin blocks of tumors of consecutive CRC patients registered in the histopathology laboratory of hospitals under the auspices of Ahvaz Jundishapur University of Medical Sciences were used. Clinicopathological information such as the degree of tumor differentiation, tumor depth of invasion, lymph node involvement status, etc. were extracted from the patient's files and pathology reports and using paraffin blocks, specific staining for the p16 factor was performed using immunohistochemistry. Data were analyzed by SPSS software. In the immunohistochemistry technique from 38 samples, the staining rate of P16 marker: 13 samples (34.2%) scored 3, 12 samples (31.6%) scored 2, seven samples (18.4%) scored 1 and six samples (15.8%) scored zero. Also, the staining intensity was severe in 10 cases (26.3%), moderate in 14 cases (36.8%), mild in 8 cases (21.1%), and negative in 6 cases (15.8%). The amount and intensity of staining for the p16 factor in the immunohistochemistry technique were not associated with sex, age, tumor location, tumor differentiation rate, tumor depth of invasion, lymph node involvement, lymph vascular invasion, and perineural invasion (p>0.05). Tumor size was not significantly associated with staining rate but was significantly associated with staining intensity (p<0.05), so in cases with larger tumor size, staining intensity was lower. Despite the positive expression of P16 in 84.2% of colorectal cancer cases, its expression was not associated with clinical and pathological parameters.

Tomato (Solanum lycopersicum L.) is one of the essential vegetables worldwide, for consumption and mitigating malnutrition. Genetic transformation conceivably overcomes its yield challenges due to salinity, a crucial constraint for the economical use of 30% of cultivable lands in the coastal region of Bangladesh. Therefore, a robust and reproducible protocol has been established for in vitro regeneration and transformation to develop transgenic salt-tolerant tomato plants. During micropropagation, cotyledonary leaf explants were excised and cultured on MS media containing different combinations and concentrations of plant growth regulators. In transformation, the pre-cultured explants were inoculated and co-cultivated with Agrobacterium. Then they were transferred to the antibiotics-supplemented media to achieve salt-tolerant putative transformed plants. The transformation was confirmed by β-glucuronidase (GUS) assay and PCR for the antiporter gene. Maximum regeneration response was achieved from the explants abaxially positioned at a 1.5 cm distance apart. BARI Tomato 14 and BINA Tomato 3 showed the highest shoot regeneration response (%) on MS media supplemented with 2 mg/L BAP and with 0.1 mg/L IAA for BARI Tomato 2 and BARI Tomato 15. Bacterial culture of OD600 0.68 for 30 min and a Co-cultivation period of 48 h resulted in the highest transformation frequency (47%) in Agrobacterium-mediated transformation with pBI121 in BARI Tomato 3. The highest regeneration frequency (20.5%) was obtained in transformation with pH7WG2_OsNHX1_1.6. The optimized procedure is simple, efficient, and can be used for micro-propagation and the production of tolerant varieties to increase yield in saline areas.

Leukemia is one of the types of cancer that its most common treatments, including radiation and chemotherapy, are associated with many problems and side effects such as drug resistance. Therefore, researchers are looking for multi-factor combined therapies with fewer side effects and more anti-cancer effects to treat leukemia. The aim of this study was to analyze the combination effect of gold nanoparticles (Au NPs) and hyperthermia on the proliferation of chronic myeloid leukemia cells. To study the nanoparticle absorption by K562 cancer cells, various amounts of nanoparticles were used. The MTT and apoptosis assays were used to evaluate the anti-proliferation properties of NPs (20, 40, and 80 μg/ml) and hyperthermal (41 °C for an hour) on cancer cells. Our results revealed a significant decrease in the proliferation index (43 ± 3.311, p<0.01) and an increase in apoptosis (54 ± 3.605, p<0.01) of K562 cells in the group receiving a combination of 80 µg/ml of Au NPs and hyperthermia compared to the control and other treatment groups. According to the results, the combination of Au NPs and hyperthermia could significantly suppress the proliferation and induce apoptosis in K562 cells in comparison with Au NPs or hyperthermia, alone. Therefore, it confirms that the application of several anti-cancer strategies using NPs and hyperthermia is a useful strategy in cancer treatment.

It is believed that the identification of the differentially-expressed genes is extremely important for the clarification of the complex molecular mechanisms under drought conditions. This study aims to identify candidate genes in tomato genotypes under drought stress through transcriptomics analysis, investigate the expression of these genes, and also some physiological parameters. To the analysis of transcriptome profiles of sensitive and tolerant tomato genotypes under drought stress, three up-regulated genes were selected, including Chlorophyll a-b binding protein3 (CAB3), S-adenosylmethionine decarboxylase proenzyme (SAMDC), and 1-aminocyclopropane-1-carboxylate synthase 9 (ACS9). After bioinformatics analysis, the tomato genotypes were subjected to drought stress and gene expression was determined using Real-Time PCR. Physiological parameters of genotypes were also measured by spectrophotometer-based methods. According to the results, these three genes play a key role in stress tolerance. Expression of the CAB3 gene in both sensitive and tolerant genotypes was not significantly different compared to the control. This is while the SAMDC gene decreased in both genotypes, the ACS9 gene decreased in the sensitive genotype and also increased in the tolerant genotype. The physiological analysis also showed that under stress conditions, the photosynthetic system of the plant was disrupted and the chlorophyll content was reduced. However, proline content and antioxidant enzymes activity increased, in which their quantity in the tolerant genotype was significantly higher than sensitive. In accordance with the obtained findings, it can be stated that under drought stress, due to damage to the lipid membrane, malondialdehyde content also increased, in which the sensitive genotype was more affected.

Biotransformation can be an effective tool for the structural modification of bioactive natural and synthetic compounds to synthesize novel and more potent compounds. The present study describes the biotransformation of α-Pinene using two bacterial strains Peanibacillus popilliae 1C and Streptomyces rochei AB1, isolated and identified in our previous study. The inhibitory concentration of the target substance against both strains was evaluated as 15 mg/ml. However, for technical considerations, a concentration of the α-Pinene at 10 mg/ml as Biotransformation Assimilable Concentration (BAC) was used to carry out the biotransformation process. For the biotransformation highlighting, both strains were cultivated on the rich medium (Luria-Bertani (LB) for Peanibacillus popilliae 1C and International Streptomyces Project 9 (ISP9) for Streptomyces rochei AB1) and poor medium (Minimum Medium (MM) for 1C and ISP9 for AB1). The chemical composition and percentage content of each compound were performed by GC/MS and GC/FID, respectively. The GC/MS analysis revealed that all the biotransformation products were hydrocarbon and oxygenated monoterpenes, which can be divided into two groups. The first group of 11 compounds (Verbenene, Isocarveol, E-2,3-Epoxycarane, γ-Terpinene, Dehydrolinalool, α-Campholene aldehyde, Menthol, Carvacrol, Limonene dioxide, Piperitenone, Ocimenol) is described for the first time in the biotransformation of α-Pinene and a second group bringing together 8 compounds previously reported (Trans Verbenol, p-Cymen-8-ol, α-Terpineol, Myrtenol, Verbenone, Trans Sobrerol, D-Limonene, Pinocarvone). These bacterial strains possess distinctive biocatalytic capacities toward α-Pinene which leads to the production of other secondary metabolites.

Conus is the genus of toxic gastropods with pharmacologically active compounds in its venom that mostly lives in marine environments. Conus venom consists of a rich source of analgesic peptides. In the current study, the analgesic effects of Conus coronatus venom from the Persian Gulf were investigated in mice models. The venom ducts were extracted and homogenized. Deoxygenated cold aqueous acetonitrile solution (40%) was used in this study for conotoxin extraction. Purification was carried out using Sephadex G-25. Purified fractions were injected intraperitoneal (IP) in both formalin and hotplate tests with different doses. Following the pain response assessment, nicotine was used as the agonist of the acetylcholine receptor, and pain response to the co-injection of nicotine and conotoxin was calculated. Tricine-SDS-PAGE was used for molecular weight determination. Findings revealed that the action of purified fraction of C. coronatus venom (C2) at a dose of 0.1 mg/kg was comparable with morphine as a positive control (2.5 mg/kg). The analgesic potential of this fraction was observed in the hot plate test. However, the co-injection of nicotine and C2 decreased the analgesic effect. According to findings, it can be stated that conotoxins isolated from C. coronatus had analgesic effects and could be used for discovering and producing novel medicines. Moreover, the peptides observed in this study with less than 6.5 kDa probably are members of the antagonist conotoxins which have been reported for the first time in this study.

Malaria is a protozoan disease that is caused by different types of Plasmodium in humans and animals. Resistance to the main drugs in the treatment of malaria infections has led to studying alternative drugs. Therefore, in the present study, the effect of hydroalcoholic extract of wild garlic was studied on Plasmodium berghei malaria-infected mice. This experimental study was conducted on 45 male mice infected with Plasmodium berghei. The treatment with hydroalcoholic extract of wild garlic was performed using Peter’s proposed method. Statistical analysis of data was conducted using SPSS v.18 software. Findings showed that the wild garlic hydroalcoholic extract had the highest effect at the treatment dose of 800 mg/kg with 92.4% prevention of parasite growth compared to the control group (P<0.05). No significant difference was observed in the mean weight of the mice or the morphology of the liver and kidney in the group receiving wild garlic extract compared to the negative control group. The anti-malarial effects of the wild garlic plant observed in the present study, elicit the necessity for further research, evaluation, and comparison of different extraction methods such as aqueous and chloroform as well as higher therapeutic dosages.