Search results for: beta 2 toxin gene

Authors: Sunil Kumar, Uday C. Ghoshal

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Background and aims: Serotonin (5-hydroxtryptamine, 5-HT) is an important factor in gut function, playing key roles in intestinal peristalsis and secretion, and in sensory signaling in the brain-gut axis. Removal from its sites of action is mediated by a specific protein called the serotonin reuptake transporter (SERT). Polymorphisms in the promoter region of the SERT gene have effects on transcriptional activity, resulting in altered 5-HT reuptake efficiency. Functional polymorphisms may underlie disturbance in gut function in individuals suffering with disorders such as irritable bowel syndrome (IBS). The aim of this study was to assess the potential association between SERT polymorphisms and the diarrhea predominant IBS (D-IBS) phenotype Subjects: A total of 36 northern Indian female patients and 55 female northern Indian healthy controls (HC) were subjected to genotyping. Methods: Leucocyte DNA of all subjects was analyzed by polymerase chain reaction based technologies for SERT polymorphisms, specifically the insertion/deletion polymorphism in the promoter (SERT-P). Statistical analysis was performed to assess association of SERT polymorphism allele with the D-IBS phenotype. Results: The frequency of distribution of SERT-P gene was comparable between female patients with IBS and HC (p = 0.086). However, frequency of SERT-P deletion/deletion genotype was significantly higher in female patients with D-IBS compared to C-IBS and A-IBS [17/19 (89.5%) vs. 4/12 (33.3%) vs. 1/5 (20%), p=0.001, respectively]. The mucosal level of serotonin was higher in D-IBS compared to C-IBS and A-IBS [Median, range (159.26, 98.78–212.1) vs. 110.4, 67.87–143.53 vs. 92.34, 78.8–166.3 pmol/mL, p=0.001, respectively]. The mucosal level of serotonin was higher in female patients with IBS with SERT-P deletion/deletion genotype compared deletion/insertion and insertion/insertion [157.65, 67.87–212.1 vs. 110.4, 78.1–143.32 vs. 100.5, 69.1–132.03 pmol/mL, p=0.001, respectively]. Patients with D-IBS with deletion/deletion genotype more often reported symptoms of abdominal pain, discomfort (p=0.025) and bloating (p=0.039). Symptoms development following lactose ingestion was strongly associated with D-IBS and SERT-P deletion/deletion genotype (p=0.004). Conclusions: Significant association was observed between D-IBS and the SERT-P deletion/deletion genotype, suggesting that the serotonin transporter is a potential candidate gene for D-IBS in women.

Keywords: serotonin, SERT, inflammatory bowel disease, genetic polymorphism

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Authors: Sahar Nasr, Lin Li, Edwin Wang

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Bladder cancer (BLCA) is known as the 13th cause of death among cancer patients worldwide, and ~575,000 new BLCA cases are diagnosed each year. Urothelial carcinoma (UC) is the most prevalent subtype among BLCA patients, which can be categorized into muscle-invasive bladder cancer (MIBC) and non-muscle-invasive bladder cancer (NMIBC). Currently, various therapeutic options are available for UC patients, including (1) transurethral resection followed by intravesical instillation of chemotherapeutics or Bacillus Calmette-Guérin for NMIBC patients, (2) neoadjuvant platinum-based chemotherapy (NAC) plus radical cystectomy is the standard of care for localized MIBC patients, and (3) systematic chemotherapy for metastatic UC. However, conventional treatments may lead to several challenges for treating patients. As an illustration, some patients may suffer from recurrence of the disease after the first line of treatment. Recently, immune checkpoint therapy (ICT) has been introduced as an alternative treatment strategy for the first or second line of treatment in advanced or metastatic BLCA patients. Although ICT showed lucrative results for a fraction of BLCA patients, ~80% of patients were not responsive to it. Therefore, novel treatment methods are required to augment the ICI response rate within BLCA patients. It has been shown that the infiltration of T-cells into the tumor microenvironment (TME) is positively correlated with the response to ICT within cancerous patients. Therefore, the goal of this study is to enhance the infiltration of cytotoxic T-cells into TME through the identification of target genes within the tumor that are responsible for the non-T-cell inflamed TME and their inhibition. BLCA bulk RNA-sequencing data from The Cancer Genome Atlas (TCGA) and immune score for TCGA samples were used to determine the Pearson correlation score between the expression of different genes and immune score for each sample. The genes with strong negative correlations were selected (r < -0.2). Thereafter, the correlation between the expression of each gene and survival in BLCA patients was calculated using the TCGA data and Cox regression method. The genes that are common in both selected gene lists were chosen for further analysis. Afterward, BLCA bulk and single-cell RNA-sequencing data were ranked based on the expression of each selected gene and the top and bottom 25% samples were used for pathway enrichment analysis. If the pathways related to the T-cell infiltration (e.g., antigen presentation, interferon, or chemokine pathways) were enriched within the low-expression group, the gene was included for downstream analysis. Finally, the selected genes will be used to calculate the correlation between their expression and the infiltration rate of the activated CD+8 T-cells, natural killer cells and the activated dendric cells. A list of potential target genes has been identified and ranked based on the above-mentioned analysis and criteria. SUN-1 got the highest score within the gene list and other identified genes in the literature as benchmarks. In conclusion, inhibition of SUN1 may increase the tumor-infiltrating lymphocytes and the efficacy of ICI in BLCA patients. BLCA tumor cells with and without SUN-1 CRISPR/Cas9 knockout will be injected into the syngeneic mouse model to validate the predicted SUN-1 effect on increasing tumor-infiltrating lymphocytes.

Keywords: data analysis, gene expression analysis, gene identification, immunoinformatic, functional genomics, transcriptomics

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Authors: B. M. Md-Zain, N. A. Rahman, M. A. B. Abdul-Latiff, W. M. R. Idris

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We conducted molecular research to portray phylogenetic relationships of Malaysian primates particularly in the genus of Macaca. We have sequenced cytochrome C oxidase subunit I (COI) of mitochondrial DNA of several individuals from M. fascicularis and M. arctoides. PCR amplifications were performed and COI DNA sequences were aligned using ClustalW. Phylogenetic trees were constructed using distance analyses by employing neighbor-joining algorithm (NJ). We managed to sequence 700 bp of COI DNA sequences. The tree topology showed that M. fascicularis did not clump based on phyleogeography division in Peninsular Malaysia. Individuals from Negeri Sembilan merged together with samples from Perak and Penang into one clade. In addition, phylogenetic analyses indicated that M. arctoides was classified into sinica group instead of fascicularis group supported by genetic distance data. COI gene is an effective locus to clarify phylogenetic position of M. arctoides but not in discriminating M. fascicularis population in Peninsular Malaysia.

Keywords: cercopithecine, long-tailed macaque, Macaca fascicularis, Macaca arctoides

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Authors: Raviporn Madarasmi, Anjalee Vacharaksa, Pravej Serichetaphongse

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The host response in peri-implant tissue may differ from that in periodontal tissue in a healthy individual. The purpose of this study is to investigate the expression of inflammatory cytokines in peri-implant crevicular fluid (PICF) from single implant with different abutment types in comparison to healthy periodontal tissue. 19 participants with healthy implants and teeth were recruited according to inclusion and exclusion criteria. PICF and gingival crevicular fluid (GCF) was collected using sterile paper points. The expression level of inflammatory cytokines including IL-1α, IL-1β, TNF-α, IFN-γ, IL-6, and IL-8 was assessed using enzyme-linked immunosorbent assay (ELISA). Paired t test was used to compare the expression levels of inflammatory cytokines around natural teeth and peri-implant in PICF and GCF of the same individual. The Independent t-test was used to compare the expression levels of inflammatory cytokines in PICF from titanium and UCLA abutment. Expression of IL-6, TNF-α, and IFN-γ in PICF was not statistically different from GCF among titanium and UCLA abutment group. However, the level of IL-1α in the PICF from the implants with UCLA abutment was significantly higher than GCF (P=0.030). In addition, the level of IL-1β in PICF from the implants with titanium abutment was significantly higher than GCF (P=0.032). When different abutment types was compared, IL-8 expression in PICF from implants with UCLA abutment was significantly higher than titanium abutment (P=0.003).

Keywords: abutment, dental implant, gingival crevicular fluid and peri-implant crevicular fluid

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Authors: Eisha Vienna M. Fernandez, Cristan Q. Cabanilla, Hiyasmin Lim, John Donnie A. Ramos

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Allergy is a multifactorial disease affecting a significant proportion of the population. It is developed through the interaction of allergens and the presence of certain polymorphisms in various susceptibility genes. In this study, the correlation of the 105A/C single nucleotide polymorphism (SNP) of the IL-18 gene and house dust mite-specific IgE among Filipino allergic and non-allergic population was investigated. Atopic status was defined by serum total IgE concentration of ≥100 IU/mL, while house dust mite allergy was defined by specific IgE value ≥ +1SD of IgE of nonatopic participants. Two hundred twenty match-paired Filipino cases and controls aged 6-60 were the subjects of this investigation. The level of total IgE and Specific IgE were measured using Enzyme-Linked Immunosorbent Assay (ELISA) while Polymerase Chain Reaction – Restriction Fragment Length Polymorphism (PCR-RFLP) analysis was used in the SNP detection. Sensitization profiles of the allergic patients revealed that 97.3% were sensitized to Blomia tropicalis, 40.0% to Dermatophagoides farinae, and 29.1% to Dermatophagoides pteronyssinus. Multiple sensitization to HDMs was also observed among the 47.27% of the atopic participants. Any of the allergy classes of the atopic triad were exhibited by the cases (allergic asthma: 48.18%; allergic rhinitis: 62.73%; atopic dermatitis: 19.09%), and two or all of these atopic states are concurrently occurring in 26.36% of the cases. A greater proportion of the atopic participants with allergic asthma and allergic rhinitis were sensitized to D. farinae, and D. pteronyssinus, while more of those with atopic dermatitis were sensitized to D. pteronyssinus than D. farinae. Results show that there is overrepresentation of the allele “A” of the 105A/C IL-18 gene SNP in both cases and control groups of the population. The genotype that predominate the population is the heterozygous “AC”, followed by the homozygous wild “AA”, and the homozygous variant “CC” being the least. The study confirmed a positive association between serum specific IgE against B. tropicalis and D. pteronyssinus and the allele “C” (Bt P=0.021, Dp P=0.027) and “AC” (Bt P=0.003, Dp P=0.026) genotype. Findings also revealed that the genotypes “AA” (OR:1.217; 95% CI: 0.701-2.113) and “CC” (OR, 3.5; 95% CI: 0.727-16.849) increase the risk of developing allergy. This indicates that the 105A/C IL-18 gene SNP is a candidate genetic marker for HDM allergy among Filipino patients.

Keywords: house dust mite allergy, interleukin-18 (IL-18), single nucleotide polymorphism,

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Authors: Morello Sara, Pederiva Sabina, Bianchi Manila, Martucci Francesca, Marchis Daniela, Decastelli Lucia

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Vegetables represent an integral part of a healthy diet due to their valuable nutritional properties and the growth in consumer demand in recent years is particularly remarkable for a diet rich in vitamins and micronutrients. However, plant-based products are involved in several food outbreaks connected to various sources of contamination and quite often, bacteria responsible for side effects showed high resistance to antibiotics. The abuse of antibiotics can be one of the main mechanisms responsible for increasing antibiotic resistance (AR). Plants grown for food use can be contaminated directly by spraying antibiotics on crops or indirectly by treatments with antibiotics due to the use of manure, which may contain both antibiotics and genes of antibiotic resistance (ARG). Antibiotic residues could represent a potential way of human health risk due to exposure through the consumption of plant-based foods. The presence of antibiotic-resistant bacteria might pose a particular risk to consumers. The present work aims to investigate through a multidisciplinary approach the occurrence of ARG by means of a biomolecular approach (PCR) and the prevalence of antibiotic residues using a multi residues LC-MS/MS method, both in different plant-based products. During the period from July 2020 to October 2021, a total of 74 plant samples (33 lettuces and 41 tomatoes) were collected from 57 farms located throughout the Piedmont area, and18 out of 74 samples (11 lettuces and 7 tomatoes) were selected to LC-MS/MS analyses. DNA extracted (ExtractME, Blirt, Poland) from plants used on crops and isolated bacteria were analyzed with 6 sets of end-point multiplex PCR (Qiagen, Germany) to detect the presence of resistance genes of the main antibiotic families, such as tet genes (tetracyclines), bla (β-lactams) and mcr (colistin). Simultaneous detection of 43 molecules of antibiotics belonging to 10 different classes (tetracyclines, sulphonamides, quinolones, penicillins, amphenicols, macrolides, pleuromotilines, lincosamides, diaminopyrimidines) was performed using Exion LC system AB SCIEX coupled to a triple quadrupole mass spectrometer QTRAP 5500 from AB SCIEX. The PCR assays showed the presence of ARG in 57% (n=42): tetB (4.8%; n=2), tetA (9.5%; n=4), tetE (2.4%; n=1), tetL (12%; n=5), tetM (26%; n=11), blaSHV (21.5%; n=9), blaTEM (4.8%; n =2) and blaCTX-M (19%; n=8). In none of the analyzed samples was the mcr gene responsible for colistin resistance detected. Results obtained from LC-MS/MS analyses showed that none of the tested antibiotics appear to exceed the LOQ (100 ppb). Data obtained confirmed the presence of bacterial populations containing antibiotic resistance determinants such as tet gene (tetracycline) and bla genes (beta-lactams), widely used in human medicine, which can join the food chain and represent a risk for consumers, especially with raw products. The presence of traces of antibiotic residues in vegetables, in concentration below the LOQ of the LC-MS/MS method applied, cannot be excluded. In conclusion, traces of antibiotic residues could be a health risk to the consumer due to potential involvement in the spread of AR. PCR represents a useful and effective approach to characterize and monitor AR carried by bacteria from the entire food chain.

Keywords: plant-based products, ARG, PCR, antibiotic residues

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Authors: Sonia Tamanna, Akramul Hassan, Mohammad Shakil Mahmood, Farzana Ansari, Gowhar Rashid, Mir Fahim Faisal, M. Zakir Hossain Howlader

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Background: Preeclampsia (PE), a pregnancy-specific hypertensive disorder, significantly impacts maternal and fetal health. It is particularly prevalent in underdeveloped countries and is linked to preterm delivery and fetal growth. The renin-angiotensin system (RAS) plays a crucial role in ensuring a successful pregnancy outcome, with Angiotensin-Converting Enzyme 2 (ACE2) being a key component. ACE2 converts ANG II to Ang-(1-7), offering protection against ANG II-induced stress and inflammation while regulating blood pressure and osmotic balance during pregnancy. The reduced maternal plasma angiotensin-converting enzyme 2 (ACE2) seen in preeclampsia might contribute to its pathogenesis. However, there has been a dearth of comprehensive research into the association between ACE2 gene polymorphism and preeclampsia. In the South Asian population, hypertension is strongly linked to two SNPs: rs2285666 and rs879922. This genotype was therefore considered, and the possible association of maternal and fetal ACE2 gene polymorphism with preeclampsia within the Bangladeshi population was evaluated. Method: DNA was extracted from peripheral white blood cells (WBCs) using the organic method, and SNP genotyping was done via PCR-RFLP. Odds ratios (OR) with 95% confidence intervals (95% CI) were calculated using logistic regression to determine relative risk. Result: A comprehensive case-control study was conducted on 51 PE patients and their infants, along with 56 control subjects and their infants. Maternal single nuvleotide polymorphisms (SNP) (rs2285666) analysis revealed a strong association between the TT genotype and preeclampsia, with a four-fold increased risk in mothers (P=0.024, OR=4.00, 95% CI=1.36-11.37) compared to their ancestral genotype CC. However, the CT genotype (rs2285666) showed no significant difference (P=0.46, OR=1.54, 95% CI=0.57-4.14). Notably, no significant correlation was found in infants, regardless of their gender. For rs879922, no significant association was observed in both mothers and infants. This pioneering study suggests that mothers carrying the ACE2 gene variant rs2285666 (TT allele) may be at higher risk for preeclampsia, potentially influencing hypertension characteristics, whereas rs879922 does not appear to be associated with developing preeclampsia. Conclusion: This study sheds light on the role of ACE2 gene polymorphism, particularly the rs2285666 TT allele, in maternal susceptibility to preeclampsia. However, rs879922 does not appear to be linked to the risk of PE. This research contributes to our understanding of the genetic underpinnings of preeclampsia, offering insights into potential avenues for prevention and management.

Keywords: ACE2, PCR-RFLP, preeclampsia, single nuvleotide polymorphisms (SNPs)

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Authors: Mohamed M. Bumadian, D. James Gilmour

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Three bacterial species were isolated from the River Wye (Derbyshire, England) and identified using 16S rRNA gene sequencing as Serratia proteamaculans, Aeromonas hydrophila and Klebsiella pneumoniae. Respiration rates of the strains were measured in order to determine the metabolic activity under salt stress. The highest respiration rates of all three strains were found at 0.17 M and 0.5 M NaCl and then the respiration rate decreased with increasing concentrations of NaCl. In addition, the effect of increasing concentrations of NaCl on malate dehydrogenase activity was determined using cell-free extracts of the three strains. Malate dehydrogenase activity was stimulated at NaCl concentrations up to 0.5 M, and a small level of activity remained even at 3.5 M NaCl. The pH optimum of the malate dehydrogenase in cell-free extracts of all strains was higher than pH 7.5.

Keywords: fresh water, halotolerant pathogenic bacteria, 16S rRNA gene, cell-free extracts, respiration rates, malate dehydrogenase

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Authors: O. H. Gebril, N. A. Meguid

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Background: Iron had been a focus of interest recently as a main exaggerating factor for oxidative stresses in the central nervous system and a link to various neurological disorders is suspected. Many studies with various techniques showed evidence of disturbed iron-related proteins in the cell in human and animal models of neurodegenerative disorders. Also, linkage to significant pathological changes had been evidenced e.g. apoptosis and cell signaling. On the other hand, the role of iron in neurodevelopmental disorders is still unclear. With increasing prevalence of autism worldwide, some changes in iron parameters and its stores were documented in many studies. This study includes Haemochromatosis HFE gene polymorphisms (p.H63D and p.C282Y) and ferroportin gene (SLC40A1) Q248H polymorphism in autism and control children. Materials and Methods: Whole genome DNA was extracted; p.H63D and p.C282Y genotyping was studied using specific sequence amplification followed by restriction enzyme digestion on a sample of autism patients (25 cases) and twenty controls. Results: The p.H63D is seen more than the C282Y among both autism and control samples, with no significant association of p.H63D or p.C282Y polymorphism and autism was revealed. Also, no association with Q248H polymorphism was evidenced. Conclusion: The study results do not prove the role of cellular iron genes polymorphisms as risk factors for neurodevelopmental disorders, and in turn highlights the specificity of cellular iron related pathways in neurodegeneration. These results demand further gene expression studies to elucidate the main pathophysiological pathways that are disturbed in autism and other neurodevelopmental disorders.

Keywords: iron, neurodevelopmental, oxidative stress, haemohromatosis, ferroportin, genes

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Authors: Christina Killian, Katie Wall, Séamus Fanning, Guerrino Macori

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Deep-level metagenomics offers a useful technical approach to explore the three dynamic One Health axes: healthcare, domiciliary and veterinary. There is currently limited understanding of the composition of complex biofilms, natural abundance of AMR genes and gene transfer occurrence in these ecological niches. By using a newly established small-scale complex biofilm model, COMBAT has the potential to provide new information on microbial diversity, antimicrobial resistance (AMR)-encoding gene abundance, and their transfer in complex biofilms of importance to these three One Health axes. Shotgun metagenomics has been used to sample the genomes of all microbes comprising the complex communities found in each biofilm source. A comparative analysis between untreated and biocide-treated biofilms is described. The basic steps include the purification of genomic DNA, followed by library preparation, sequencing, and finally, data analysis. The use of long-read sequencing facilitates the completion of metagenome-assembled genomes (MAG). Samples were sequenced using a PromethION platform, and following quality checks, binning methods, and bespoke bioinformatics pipelines, we describe the recovery of individual MAGs to identify mobile gene elements (MGE) and the corresponding AMR genotypes that map to these structures. High-throughput sequencing strategies have been deployed to characterize these communities. Accurately defining the profiles of these niches is an essential step towards elucidating the impact of the microbiota on each niche biofilm environment and their evolution.

Keywords: COMBAT, biofilm, metagenomics, high-throughput sequencing

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Authors: Sadegh Lotfieblisofla, Arash Khodabakhshi

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Tissue plasminogen activator (tPA) as a serine protease plays an important role in the fibrinolytic system and the dissolution of fibrin clots in human body. The production of this drug in plants such as tobacco could reduce its production costs. In this study, expression of tPA gene and protein targeting to different plant cell compartments, using various signal peptides has been investigated. For high level of expression, Kozak sequence was used after CaMV35S in the beginning of the gene. In order to design the final construction, Extensin, KDEL (amino acid sequence including Lys-Asp-Glu-Leu) and SP (γ-zein signal peptide coding sequence) were used as leader signals to conduct this protein into apoplast, endoplasmic reticulum and cytosol spaces, respectively. Cloned human tPA gene under the CaMV (Cauliflower mosaic virus) 35S promoter and NOS (Nopaline Synthase) terminator into pBI121 plasmid was transferred into tobacco explants by Agrobacterium tumefaciens strain LBA₄₄₀₄. The presence and copy number of genes in transgenic tobacco was proved by Southern blotting. Enzymatic activity of the rt-PA protein in transgenic plants compared to non-transgenic plants was confirmed by Zymography assay. The presence and amount of rt-PA recombinant protein in plants was estimated by ELISA analysis on crude protein extract of transgenic tobacco using a specific antibody. The yield of recombinant tPA in transgenic tobacco for SP, KDEL, Extensin signals were counted 0.50, 0.68, 0.69 microgram per milligram of total soluble proteins.

Keywords: tPA, recombinant, transgenic, tobacco

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Authors: Jie Gao

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The combination of chemotherapy with gene therapy is highly effective in cancer therapy. To achieve combined therapeutic effects in human gastric cancer over expressing EGFR, we developed targeted LPD (liposome-polycation-DNA complex) conjugated with anti-EGFR (epidermal growth factor receptor) Fab’ for co-delivery of doxorubicin (DOX) and ribonucleotide reductase M2 (RRM2) siRNA (DOX-RRM2-TLPD). The results showed that EGFR was over expressed in several gastric cancer cell lines and gastric cancer tissues. Gene Expression Omnibus (GEO) results showed that RRM2 expression was significantly higher in gastric cancer than in non-gastric cancer tissue, and RRM2 siRNA inhibited the proliferation of several gastric cancer cells, indicating that RRM2 is a candidate target for gastric cancer therapy. Confocal studies and flow cytometry showed that DOX-RRM2-TLPD delivered DOX and RRM2 siRNA to EGFR over expressing gastric cancer cells specifically and efficiently both in vitro and in vivo, resulting in enhanced therapeutic effects (cytotoxicity and apoptosis) compared with single-drug loaded or non-targeted controls, including DOX-NC-TLPD (targeted LPD co-delivering DOX and negative control siRNA), RRM2-TLPD (targeted LPD delivering RRM2 siRNA) and DOX-RRM2-NTLPD (non-targeted LPD co-delivering DOX and RRM2 siRNA). The in vivo antitumor assay showed that the average weight of the gastric cancer in mice treated with DOX-RRM2-TLPD was significantly lighter than that of mice treated with other controls. DOX-RRM2-TLPD represents an effective approach for combined therapy of gastric cancer over expressing EGFR.

Keywords: gene therapy, chemotherapy, immunoliposomes, gastric cancer

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Authors: Ali Asghar Saki, Sayed Ali Hosseini Siyar, Abbass Ashoori

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This study was conducted to evaluate the effect of Echinacea purpurea on the expression of cyclooxygenase-2 (COX-2), interleukin-17F (IL-17F) in seven-day-old broiler chickens. Four groups were fed with concentration of 0 g/kg, 5 g/kg, 10 g/kg and 20 g/kg from the root of E. purpurea in the basal diet and two other groups were only fed with the basal diet for 21 days. At the 28^th day, lipopolysaccharide (LPS, 2 mg/kg diet) was injected in four groups and the basal diet group was injected by saline as control. The chickens’ spleen RNA expression was measured for the COX-2 and IL-17F genes by Real-Time PCR. The results have shown that chickens which were fed E. purpurea had a lower COX-2 and IL-17F mRNA expression. The chickens who have received LPS only, lymphocyte was lower than other treatments. Vital organ weights were not significantly different, but body weight loss was recovered by dietary herbs inclusion. The results of this study have shown the positive effect of an anti-inflammatory herb to prevent the undesirable effect of inflammation.

Keywords: broiler chickens, Echinacea purporea, gene expression, lipopolysaccharide

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Authors: Debora P. Arce, Flavia J. Krsticevic, Marco R. Bertolaccini, Joaquín Ezpeleta, Estela M. Valle, Sergio D. Ponce, Elizabeth Tapia

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Small Heat Shock Proteins (sHSPs) are low molecular weight chaperones that play an important role during stress response and development in all living organisms. Fruit maturation and oxidative stress can induce sHSP synthesis both in Arabidopsis and tomato plants. RNA-Seq technology is becoming widely used in various transcriptomics studies; however, analyzing and interpreting the RNA-Seq data face serious challenges. In the present work, we de novo assembled the Solanum lycopersicum transcriptome for three different maturation stages (mature green, breaker and red ripe). Differential gene expression analysis was carried out during tomato fruit development. We identified 12 sHSPs differentially expressed that might be involved in breaker and red ripe fruit maturation. Interestingly, these sHSPs have different subcellular localization and suggest a complex regulation of the fruit maturation network process.

Keywords: sHSPs, maturation, tomato, RNA-Seq, assembly

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Authors: Sarita Agarwal, Deepika Delsa Dean

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Like the task of transferring photo images to artistic images, image-to-image translation aims to translate the data to the imitated data which belongs to the target domain. Neural Style Transfer and CycleGAN are two well-known deep learning architectures used for photo image-to-art image transfer. However, studies involving these two models concentrate on one-to-one domain translation, not one-to-multi domains translation. Our study tries to investigate deep learning architectures, which can be controlled to yield multiple artistic style translation only by adding a conditional vector. We have expanded CycleGAN and constructed Conditional CycleGAN for 5 kinds of categories translation. Our study found that the architecture inserting conditional vector into the middle layer of the Generator could output multiple artistic images.

Keywords: genetic counseling, FMR1 gene, fragile x-associated primary ovarian insufficiency, premutation

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Authors: Randa M. T. Mohamed

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Spices have long been used as traditional ingredients in the kitchen for seasoning, coloring, aromatic and food preservative properties. Besides, spices are equally used for therapeutic purposes. The objective of this study was to survey and document the medicinal properties of spices commonly used in the Sudanese kitchen for different food preparations. Also, extracts from reported spices were screened for the presence of secondary metabolites as well as their antioxidant and beta-lactamase inhibitory properties. This study was conducted in the Rekabbya Quartier in Omdurman, Khartoum State, Sudan. Information was collected by carrying out semi-structured interviews. All informants (30) in the present study were women. Spices were purchased from Attareen shop in Omdurman. Essential oils from spices were extracted by hydrodistillation, and ethanolic extracts by maceration. Phytochemical screening was performed by thin-layer chromatography (TLC). The antioxidant capacity of essential oils and ethanolic extracts was investigated through TLC bioautography. Beta lactamase inhibitory activity was performed by the acidimetric test. Ethnobotany study showed that a total of 16 spices were found to treat 36 ailments belonging to 10 categories. The most frequently claimed medicinal uses were for the digestive system diseases treated by 14 spices and respiratory system diseases treated by 8 spices. Gynecological problems were treated with 4 spices. Dermatological diseases were cured by 5 spices, while infections caused by tapeworms and other microbes causing dysentery were treated by 3 spices. 4 spices were used to treat bad breath, bleeding gum and toothache. Headache, eyes infection, cardiac stimulation and epilepsy were treated with one spice each. Other health problems like fatigue and loss of appetite, and low breast milk production were treated by 1, 3 and 2 spices, respectively. The majority (69%, 11/16) of spices were exported from different countries like India, China, Indonesia, Ethiopia, Egypt and Nigeria, while 31% (5/16) was cultivated in Sudan. Essential oils of all spices were rich in terpenes, while ethanolic extracts contained variable classes of secondary metabolites. Both essential oils and ethanolic extracts of all spices exerted considerable antioxidant activity. Only one extract, Syzygium aromaticum, possessed beta-lactamase inhibitory activity. In conclusion, this study could contribute to conserving information on traditional medicinal uses of spices in Sudan. Also, the results demonstrated the potential of some of these spices to exert beneficial antimicrobial and antioxidant effects. Detailed phytochemical and biological assays of these spices are recommended.

Keywords: spices, enthnobotany, antioxidant, betalactamase inhibition

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Authors: Maria Ostroukhova, Zhanneta Zalutskaya, Elena Ermilova

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The alternative oxidase (AOX) mediates cyanide-resistant respiration, which bypasses proton-pumping complexes III and IV of the cytochrome pathway to directly transfer electrons from reduced ubiquinone to molecular oxygen. In Chlamydomonas reinhardtii, AOX is a monomeric protein that is encoded by two genes of discrete subfamilies, AOX1 and AOX2. Although AOX has been proposed to play essential roles in stress tolerance of organisms, the role of subfamily AOX2 is largely unknown. In C. reinhardtii, AOX2 was initially identified as one of constitutively low expressed genes. Like other photosynthetic organisms C. reinhardtii cells frequently experience periods of hypoxia. To examine AOX2 transcriptional regulation and role of AOX2 in hypoxia adaptation, real-time PCR analysis and artificial microRNA method were employed. Two experimental approaches have been used to induce the anoxic conditions: dark-anaerobic and light-anaerobic conditions. C. reinhardtii cells exposed to the oxygen deprivation have shown increased AOX2 mRNA levels. By contrast, AOX1 was not an anoxia-responsive gene. In C. reinhardtii, a subset of genes is regulated by transcription factor CRR1 in anaerobic conditions. Notable, the AOX2 promoter region contains the potential motif for CRR1 binding. Therefore, the role of CRR1 in the control of AOX2 transcription was tested. The CRR1-underexpressing strains, that were generated and characterized in this work, exhibited low levels of AOX2 transcripts under anoxic conditions. However, the transformants still slightly induced AOX2 gene expression in the darkness. These confirmed our suggestions that darkness is a regulatory stimulus for AOX genes in C. reinhardtii. Thus, other factors must contribute to AOX2 promoter activity under dark-anoxic conditions. Moreover, knock-down of CRR1 caused a complete reduction of AOX2 expression under light-anoxic conditions. These results indicate that (1) CRR1 is required for AOX2 expression during hypoxia, and (2) AOX2 gene is regulated by CRR1 together with yet-unknown regulatory factor(s). In addition, the AOX2-underexpressing strains were generated. The analysis of amiRNA-AOX2 strains suggested a role of this alternative oxidase in hypoxia adaptation of the alga. In conclusion, the results reported here show that C. reinhardtii AOX2 gene is stress inducible. CRR1 transcriptional factor is involved in the regulation of the AOX2 gene expression in the absence of oxygen. Moreover, AOX2 but not AOX1 functions under oxygen deprivation. This work was supported by Russian Science Foundation (research grant № 16-14-10004).

Keywords: alternative oxidase 2, artificial microRNA approach, chlamydomonas reinhardtii, hypoxia

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Authors: Sundru Manjulata Devi, Prakash M. Halami

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The immemorial use of fermented foods from vegetables, dairy and other biological sources are of great demand in India because of their health benefits. However, the diversity of Lactobacillus plantarum group (LPG) of vegetable origin has not been revealed yet, particularly with reference to their probiotic functionalities. In the present study, the different species of probiotic Lactobacillus plantarum group (LPG) i.e., L. plantarum subsp. plantarum MTCC 5422 (from fermented cereals), L. plantarum subsp. argentoratensis FG16 (from fermented bamboo shoot) and L. paraplantarum MTCC 9483 (from fermented gundruk) (as characterized by multiplex recA PCR assay) were considered to investigate their relative expression of MUB domains of mub gene (mucin binding protein) by Real time PCR. Initially, the allelic variation in the mub gene was assessed and found to encode three different variants (Type I, II and III). All the three types had 8, 9 and 10 MUB domains respectively (as analysed by Pfam database) and were found to be responsible for adhesion of bacteria to the host intestinal epithelial cells. These domains either get inserted or deleted during speciation or evolutionary events and lead to divergence. The reverse transcriptase qPCR analysis with mubLPF1+R1 primer pair supported variation in amplicon sizes with 300, 500 and 700 bp among different LPG strains. The relative expression of these MUB domains significantly unregulated in the presence of 1% mucin in overnight grown cultures. Simultaneously, the mub gene expressed efficiently by 7 fold in the culture L. paraplantarum MTCC 9483 with 10 MUB domains. An increase in the expression levels for L. plantarum subsp. plantarum MTCC 5422 and L. plantarum subsp. argentoratensis FG16 (MCC 2974) with 9 and 8 repetitive domains was around 4 and 2 fold, respectively. The detection and expression of an integrase (int) gene in the upstream region of mub gene reveals the excision and integration of these repetitive domains. Concurrently, an in vitro adhesion assay to mucin and exclusion of pathogens (such as Listeria monocytogenes and Micrococcus leuteus) was investigated and observed that the L. paraplantarum MTCC 9483 with more adhesion domains has more ability to adhere to mucin and inhibited the growth of pathogens. The production and expression of plantaricin-like bacteriocin (plnNC8 type) in MTCC 9483 suggests the pathogen inhibition. Hence, the expression of MUB domains can act as potential biomarkers in the screening of a novel probiotic LPG strain with adherence property. The present study provides a platform for an easy, rapid, less time consuming, low-cost methodology for the detection of potential probiotic bacteria. It was known that the traditional practices followed in the preparation of fermented bamboo shoots/gundruk/cereals of Indian foods contain different kinds of neutraceuticals for functional food and novel compounds with health promoting factors. In future, a detailed study of these food products can add more nutritive value, consumption and suitable for commercialization.

Keywords: adhesion gene, fermented foods, MUB domains, probiotics

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Authors: Aslı Şalcıoğlu, Grigorous Krey, Raşit Bilgin

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In this study, the effect of the Turkish Straits System (TSS), comprising a biogeographical boundary that forms the connection between the Mediterranean and the Black Sea, on the evolutionary history, phylogeography and intraspecific gene flow of the whiting (Merlangius merlangus) a demersal fish species, was investigated. For these purposes, the mitochondrial DNA (CO1, cyt-b) genes were used. In addition, genetic comparisons samples from other regions (Greece, France, Atlantic) obtained from GenBank and Barcode of Life Database were made to better understand the phylogeographic history of the species at a larger geographic scale. Within this study, high level of genetic differentiation was observed along the Turkish coastal waters based on cyt-b gene, suggesting that TSS is a barrier to dispersal. Two different sub-species were also observed based on mitochondrial DNA, one found in Turkish coastal waters and Greece (M.m euxinus) and other (M.m. merlangus) in Atlantic, France.

Keywords: genetic, phylogeography, TSS, whiting

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Authors: Gerardo Arias, Richard Wall, Jamie Stevens

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Lucilia bufonivora Moniez, is an obligate parasite of toads and frogs widely distributed in Europe. Its sister taxon Lucilia silvarum Meigen behaves mainly as a carrion breeder in Europe, however it has been reported as a facultative parasite of amphibians. These two closely related species are morphologically almost identical, which has led to misidentification, and in fact, it has been suggested that the amphibian myiasis cases by L. silvarum reported in Europe should be attributed to L. bufonivora. Both species remain poorly studied and their taxonomic relationships are still unclear. The identification of the larval specimens involved in amphibian myiasis with molecular tools and phylogenetic analysis of these two closely related species may resolve this problem. In this work seventeen unidentified larval specimens extracted from toad myiasis cases of the UK, the Netherlands and Switzerland were obtained, their COX1 (mtDNA) and EF1-α (Nuclear DNA) gene regions were amplified and then sequenced. The 17 larval samples were identified with both molecular markers as L. bufonivora. Phylogenetic analysis was carried out with 10 other blowfly species, including L. silvarum samples from the UK and USA. Bayesian Inference trees of COX1 and a combined-gene dataset suggested that L. silvarum and L. bufonivora are separate sister species. However, the nuclear gene EF1-α does not appear to resolve their relationships, suggesting that the rates of evolution of the mtDNA are much faster than those of the nuclear DNA. This work provides the molecular evidence for successful identification of L. bufonivora and a molecular analysis of the populations of this obligate parasite from different locations across Europe. The relationships with L. silvarum are discussed.

Keywords: calliphoridae, molecular evolution, myiasis, obligate parasitism

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Authors: B. Grygalewicz, R. Woroniecka, J. Rygier, K. Borkowska, A. Labak, B. Nowakowska, B. Pienkowska-Grela

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Introduction: Chronic lymphocytic leukemia (CLL) is the most common form of adult leukemia in the Western world. Hemizygous and or homozygous loss at 13q14 occur in more than half of cases and constitute the most frequent chromosomal abnormality in CLL. It is believed that deletions 13q14 play a role in CLL pathogenesis. Two microRNA genes miR-15a and miR- 16-1 are targets of 13q14 deletions and plays a tumor suppressor role by targeting antiapoptotic BCL2 gene. Deletion size, as a single change detected in FISH analysis, has haprognostic significance. Patients with small deletions, without RB1 gene involvement, have the best prognosis and the longest overall survival time (OS 133 months). In patients with bigger deletion region, containing RB1 gene, prognosis drops to intermediate, like in patients with normal karyotype and without changes in FISH with overall survival 111 months. Aim: Precise delineation of 13q14 deletions regions in two groups of CLL patients, with mono- and biallelic deletions and qualifications of their prognostic significance. Methods: Detection of 13q14 deletions was performed by FISH analysis with CLL probe panel (D13S319, LAMP1, TP53, ATM, CEP-12). Accurate deletion size detection was performed by CGH Haematological Cancer and SNP array (8x60K). Results: Our investigated group of CLL patients with the 13q14 deletion, detected by FISH analysis, comprised two groups: 18 patients with monoallelic deletions and 18 patients with biallelic deletions. In FISH analysis, in the monoallelic group the range of cells with deletion, was 43% to 97%, while in biallelic group deletion was detected in 11% to 94% of cells. Microarray analysis revealed precise deletion regions. In the monoallelic group, the range of size was 348,12 Kb to 34,82 Mb, with median deletion size 7,93 Mb. In biallelic group discrepancy of total deletions, size was 135,27 Kb to 33,33 Mb, with median deletion size 2,52 Mb. The median size of smaller deletion regions on one copy chromosome 13 was 1,08 Mb while the average region of bigger deletion on the second chromosome 13 was 4,04 Mb. In the monoallelic group, in 8/18 deletion region covered RB1 gene. In the biallelic group, in 4/18 cases, revealed deletion on one copy of biallelic deletion and in 2/18 showed deletion of RB1 gene on both deleted 13q14 regions. All minimal deleted regions included miR-15a and miR-16-1 genes. Genetic results will be correlated with clinical data. Conclusions: Application of CGH microarrays technique in CLL allows accurately delineate the size of 13q14 deletion regions, what have a prognostic value. All deleted regions included miR15a and miR-16-1, what confirms the essential role of these genes in CLL pathogenesis. In our investigated groups of CLL patients with mono- and biallelic 13q14 deletions, patients with biallelic deletion presented smaller deletion sizes (2,52 Mb vs 7,93 Mb), what is connected with better prognosis.

Keywords: CLL, deletion 13q14, CGH microarrays, SNP array

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Authors: R. Sulakshana, R. Lakshmi

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Clustered Regularly Interspaced Short Palindromic Repeats, CRISPR associate (CRISPR/Cas) is an adaptive immunity system found in bacteria and archaea. It has been modified to serve as a potent gene editing tool. Moreover, it has found widespread use in the field of genome research because of its accessibility and low cost. Several bioinformatics methods have been created to aid in the construction of specific single guide RNA (sgRNA), which is highly active and crucial to CRISPR/Cas performance. Various Cas proteins, including Cas1, Cas2, Cas9, and Cas12, have been used to create genome engineering tools because of their programmable sequence specificity. Class 1 and 2 CRISPR/Cas systems, as well as the processes of all known Cas proteins (including Cas9 and Cas12), are discussed in this review paper. In addition, the various CRISPR methodologies and their tools so far discovered are discussed. Finally, the challenges and issues in the CRISPR system along with future works, are presented.

Keywords: gene editing tool, Cas proteins, CRISPR, guideRNA, programmable sequence

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Authors: M. F. Miah, A. Chakrabarty

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Two popular and highly valued fish, Stinging catfish (Heteropneustes fossilis) and Asian catfish (Clarias batrachus) are considered for observing genetic enhancement. Cross breeding was performed considering wild and farmed fish through inducing agent. Five RAPD markers were used to assess genetic diversity among parents and offspring of these two catfish for evaluating genetic enhancement in F1 generation. Considering different genetic data such as banding pattern of DNA, polymorphic loci, polymorphic information content (PIC), inter individual pair wise similarity, Nei genetic similarity, genetic distance, phylogenetic relationships, allele frequency, genotype frequency, intra locus gene diversity and average gene diversity of parents and offspring of these two fish were analyzed and finally in both cases higher genetic diversity was found in F1 generation than the parents.

Keywords: Heteropneustes fossilis, Clarias batrachus, cross breeding, genetic enhancement

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Authors: Hossein Rassi, Alaheh Gholami Roud-Majany, Zahra Razavi, Massoud Hoshmand

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Cervical cancer is multi step disease that is thought to result from an interaction between genetic background and environmental factors. Human papillomavirus (HPV) infection is the leading risk factor for cervical intraepithelial neoplasia (CIN)and cervical cancer. In other hand, some of hTERT and miRNA polymorphism may plays an important role in carcinogenesis. This study attempts to clarify the relation of hTERT genotypes and miR-146a genotypes in cervical cancer. Forty two archival samples with cervical lesion retired from Khatam hospital and 40 sample from healthy persons used as control group. A simple and rapid method was used to detect the simultaneous amplification of the HPV consensus L1 region and HPV-16,-18, -11, -31, 33 and -35 along with the b-globin gene as an internal control. We use Multiplex PCR for detection of hTERT and miR-146a rs2910164 genotypes in our lab. Finally, data analysis was performed using the 7 version of the Epi Info(TM) 2012 software and test chi-square(x2) for trend. Cervix lesions were collected from 42 patients with Squamous metaplasia, cervical intraepithelial neoplasia, and cervical carcinoma. Successful DNA extraction was assessed by PCR amplification of b-actin gene (99bp). According to the results, hTERT ( rs 2736098) GG genotype and miR-146a rs2910164 CC genotype was significantly associated with increased risk of cervical cancer in the study population. In this study, we detected 13 HPV 18 from 42 cervical cancer. The connection between several SNP polymorphism and human virus papilloma in rare researches were seen. The reason of these differences in researches' findings can result in different kinds of races and geographic situations and also differences in life grooves in every region. The present study provided preliminary evidence that a p53 GG genotype and miR-146a rs2910164 CC genotype may effect cervical cancer risk in the study population, interacting synergistically with HPV 18 genotype. Our results demonstrate that the testing of hTERT rs 2736098 genotypes and miR-146a rs2910164 genotypes in combination with HPV18 can serve as major risk factors in the early identification of cervical cancers. Furthermore, the results indicate the possibility of primary prevention of cervical cancer by vaccination against HPV18 in Iran.

Keywords: polymorphism of hTERT gene, miR-146a rs2910164 polymorphism, cervical cancer, virus

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Authors: T. N. Goh, M. Hashmi, O. Hussain

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Temporomandibular joint (TMJ) dysfunction (TMD) is a condition that may affect patients via restricted mouth opening, significant pain during normal functioning, and/or reproducible joint noise. TMD includes myofascial pain, TMJ functional derangements (internal derangement, dislocation), and TMJ degenerative/inflammatory joint disease. Internal derangement (ID) is the most common cause of TMD-related clicking and locking. These patients are managed in a stepwise approach, from patient education (homecare advice and analgesia), splint therapy, physiotherapy, botulinum toxin treatment, to arthrocentesis. Arthrotomy is offered when the aforementioned treatment options fail to alleviate symptoms and improve quality of life. The aim of this prospective study was to review the outcomes of jaw joint open surgery in TMD patients. Patients who presented from 2015-2022 at the Oral and Maxillofacial Surgery Department in the Doncaster NHS Foundation Trust, UK, with a Wilkes classification of III -V were included. These patients underwent either i) discopexy with bone-anchoring suture (9); ii) intrapositional temporalis flap (ITF) with bone-anchoring suture (3); iii) eminoplasty and discopexy with suturing to the capsule (3); iii) discectomy + ITF with bone-anchoring suture (1); iv) discoplasty + bone-anchoring suture (1); v) ITF (1). Maximum incisal opening (MIO) was assessed pre-operatively and at each follow-up. Pain score, determined via the visual analogue scale (VAS, with 0 being no pain and 10 being the worst pain), was also recorded. A total of 18 eligible patients were identified with a mean age of 45 (range 22 - 79), of which 16 were female. The patients were scored by Wilkes Classification as III (14), IV (1), or V (4). Twelve patients had anterior disc displacement without reduction (66%) and six had degenerative/arthritic changes (33%) to the TMJ. The open joint procedure resulted in an increase in MIO and reduction in pain VAS and for the majority of patients, across all Wilkes Classifications. Pre-procedural MIO was 22.9 ± 7.4 mm and VAS was 7.8 ± 1.5. At three months post-procedure there was an increase in MIO to 34.4 ± 10.4 mm (p < 0.01) and a decrease in the VAS to 1.5 ± 2.9 (p < 0.01). Three patients were lost to follow-up prior to six months. Six were discharged at six month review and five patients were discharged at 12 months review as they were asymptomatic with good mouth opening. Four patients are still attending for annual botulinum toxin treatment. Two patients (Wilkes III and V) subsequently underwent TMJ replacement (11%). One of these patients (Wilkes III) had improvement initially to MIO of 40 mm, but subsequently relapsed to less than 20 mm due to lack of compliance with jaw rehabilitation device post-operatively. Clinical improvements in 89% of patients within the study group were found, with a return to near normal MIO range and reduced pain score. Intraoperatively, the operator found bone-anchoring suture used for discopexy/discoplasty more secure than the soft tissue anchoring suturing technique.

Keywords: bone anchoring suture, open temporomandibular joint surgery, temporomandibular joint, temporomandibular joint dysfunction

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Authors: Nicola S. Schutte, John M. Malouff

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Introduction: Curiosity is a positive emotion and motivational state that consists of the desire to know. Curiosity consists of several related dimensions, including a desire for exploration, deprivation sensitivity, and stress tolerance. Creativity involves generating novel and valuable ideas or products. How curiosity may prompt greater creativity remains to be investigated. The phenomena of flow may link curiosity and creativity. Flow is characterized by intense concentration and absorption and gives rise to optimal performance. Objective of Study: The objective of the present study was to investigate whether the phenomenon of flow may link curiosity with creativity. Methods and Design: Fifty-seven individuals from Australia (45 women and 12 men, mean age of 35.33, SD=9.4) participated. Participants were asked to design a program encouraging residents in a local community to conserve water and to record the elements of their program in writing. Participants were then asked to rate their experience as they developed and wrote about their program. Participants rated their experience on the Dimensional Curiosity Measure sub-scales assessing the exploration, deprivation sensitivity, and stress tolerance facets of curiosity, and the Flow Short Scale. Reliability of the measures as assessed by Cronbach's alpha was as follows: Exploration Curiosity =.92, Deprivation Sensitivity Curiosity =.66, Stress Tolerance Curiosity =.93, and Flow=.96. Two raters independently coded each participant’s water conservation program description on creativity. The mixed-model intraclass correlation coefficient for the two sets of ratings was .73. The mean of the two ratings produced the final creativity score for each participant. Results: During the experience of designing the program, all three types of curiosity were significantly associated with the flow. Pearson r correlations were as follows: Exploration Curiosity and flow, r =.68 (higher Exploration Curiosity was associated with more flow); Deprivation Sensitivity Curiosity and flow, r =.39 (higher Deprivation Sensitivity Curiosity was associated with more flow); and Stress Tolerance Curiosity and flow, r = .44 (more stress tolerance in relation to novelty and exploration was associated with more flow). Greater experience of flow was significantly associated with greater creativity in designing the water conservation program, r =.39. The associations between dimensions of curiosity and creativity did not reach significance. Even though the direct relationships between dimensions of curiosity and creativity were not significant, indirect relationships through the mediating effect of the experience of flow between dimensions of curiosity and creativity were significant. Mediation analysis using PROCESS showed that flow linked Exploration Curiosity with creativity, standardized beta=.23, 95%CI [.02,.25] for the indirect effect; Deprivation Sensitivity Curiosity with creativity, standardized beta=.14, 95%CI [.04,.29] for the indirect effect; and Stress Tolerance Curiosity with creativity, standardized beta=.13, 95%CI [.02,.27] for the indirect effect. Conclusions: When engaging in an activity, higher levels of curiosity are associated with greater flow. More flow is associated with higher levels of creativity. Programs intended to increase flow or creativity might build on these findings and also explore causal relationships.

Keywords: creativity, curiosity, flow, motivation

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Authors: Anyaphat Srithanasuwan, Noppason Pangprasit, Montira Intanon, Phongsakorn Chuammitri, Witaya Suriyasathaporn, Ynte H. Schukken

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Streptococcus uberis is one of the most common mastitis-causing pathogens, with a wide range of intramammary infection (IMI) durations and pathogenicity. This study aimed to compare shared or unique virulence factor gene clusters distinguishing persistent and transient strains of S. uberis. A total of 139 S. uberis strains were isolated from three small-holder dairy herds with a high prevalence of S. uberis mastitis. The duration of IMI was used to categorize bacteria into two groups: transient and persistent strains with an IMI duration of less than 1 month and longer than 2 months, respectively. Six representative S. uberis strains, three from each group (transience and persistence) were selected for analysis. All transient strains exhibited multi-locus sequence types (MLST), indicating a highly diverse population of transient S. uberis. In contrast, MLST of persistent strains was available in an online database (pubMLST). Identification of virulence genes was performed using whole-genome sequencing (WGS) data. Differences in genomic size and number of virulent genes were found. For example, the BCA gene or alpha-c protein and the gene associated with capsule formation (hasAB), found in persistent strains, are important for attachment and invasion, as well as the evasion of the antimicrobial mechanisms and survival persistence, respectively. These findings suggest a genetic-level difference between the two strain types. Consequently, a comprehensive study of 139 S. uberis isolates will be conducted to perform an in-depth genetic assessment through WGS analysis on an Illumina platform.

Keywords: Streptococcus Uberis, mastitis, whole genome sequence, intramammary infection, persistent S. Uberis, transient s. Uberis

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Authors: Lemar Blake, Chris Oura, Ayanna C. N. Phillips Savage

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Improved DNA sequencing technology has greatly enhanced bacterial identification, especially for organisms that are difficult to culture. Mycobacteriosis with consistent hyphema, bilateral exophthalmia, open mouth gape and ocular lesions, were observed in various fish populations at the School of Veterinary Medicine, Aquaculture/Aquatic Animal Health Unit. Objective: To identify the species of Mycobacterium that is affecting aquarium fish at the School of Veterinary Medicine, Aquaculture/Aquatic Animal Health Unit. Method: A total of 13 fish samples were collected and analyzed via: Ziehl-Neelsen, conventional polymerase chain reaction (PCR) and real-time PCR. These tests were carried out simultaneously for confirmation. The following combination of conventional primers: 16s rRNA (564 bp), rpoB (396 bp), sod (408 bp) were used. Concatenation of the gene fragments was carried out to phylogenetically classify the organism. Results: Acid fast non-branching bacilli were detected in all samples from homogenized internal organs. All 13 acid fast samples were positive for Mycobacterium via real-time PCR. Partial gene sequences using all three primer sets were obtained from two samples and demonstrated a novel strain. A strain 99% related to Mycobacterium marinum was also confirmed in one sample, using 16srRNA and rpoB genes. The two novel strains were clustered with the rapid growers and strains that are known to affect humans. Conclusions: Phylogenetic analysis demonstrated two novel Mycobacterium strains with the potential of being zoonotic and one strain 99% related to Mycobacterium marinum.

Keywords: polymerase chain reaction, phylogenetic, DNA sequencing, zoonotic

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Authors: Hossein Rassi, Marjan Moradi Fard, Samaneh Niko

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Human papillomavirus type 16 and 18 are closely associated with the development of human cervical carcinoma, which is one of the most common causes of cancer death in women worldwide. At present, HPV type 18 accounts for about 34 % of all HPV infections in Iran and the most promising vaccine against HPV infection is based on the L1 major capsid protein. The L1 protein of HPV18 has the capacity to self-assemble into capsomers or virus-like particles (VLPs) that are non-infectious, highly immunogenic and allowing their use in vaccine production. The methylotrophic yeast Pichia pastoris is an efficient and inexpensive expression system used to produce high levels of heterologous proteins. In this study we expressed HPV18 L1 VLPs in P. pastoris. The gene encoding the major capsid protein L1 of the high-risk HPV type 18 was isolated from Iranian patient by PCR and inserted into pTG19-T vector to obtain the recombinant expression vector pTG19-HPV18-L1. Then, the pTG19-HPV18-L1 was transformed into E. coli strain DH5α and the recombinant protein HPV18 L1 was expressed under IPTG induction in soluble form. The HPV18 L1 gene was excised from recombinant plasmid with XhoI and EcoRI enzymes and ligated into the yeast expression vector pPICZα linearized with the same enzymes, and transformed into P. pastoris. Induction and expression of HPV18 L1 protein was demonstrated by BMGY/BMMY and RT PCR. The parameters for induced cultivation for strain in P. pastoris KM71 with HPV16L1 were investigated in shaking flask cultures. After induced cultivation BMMY (pH 7.0) medium supplemented with methanol to a final concentration of 1.0% every 24 h at 37 degrees C for 96 h, the recombinant produced 78.6 mg/L of L1 protein. This work offers the possibility for the production of prophylactic vaccine for cervical carcinoma by P. pastoris for HPV-18 L1 gene. The VLP-based HPV vaccines can prevent persistent HPV18 infections and cervical cancer in Iran. The HPV-18 L1 gene was expressed successfully in E.coli, which provides necessary basis for preparing HPV-18 L1 vaccine in human. Also, HPV type 6 L1 proteins expressed in Pichia pastoris will facilitate the HPV vaccine development and structure-function study.

Keywords: Pichia pastoris, L1 virus-like particles, human papillomavirus type 18, biotechnology

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Authors: Ayesha Imtiaz, Darlina Md. Naim

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Family Nemipteridae is among the most abundantly distributed family in Malaysian fish markets due to its high contribution to landing sites of Malaysia. Using an advanced molecular approach that used two mitochondrial (Cytochrome oxidase c I and Cytochrome oxidase b) and one nuclear gene (Recombination activating gene, RAGI) to expose cryptic diversity and phylogenetic relationships among commercially important species of family Nemipteridae. Our research covered all genera (including 31 species out total 45 species) of family Nemipteridae, distributed in Malaysia. We also found certain type of geographical barriers in the South China sea that reduces dispersal and stops a few species to intermix. Northside of the South China Sea (near Vietnam) does not allow genetic diversity to mix with the Southern side of the South China sea (Sarawak) and reduces dispersal. Straits of Malacca reduce the intermixing genetic diversity of South China Sea and the Indian Ocean.

Keywords: Nemipteridae, RAG I, south east Asia, Malaysia

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