Search results for: cell and gene therapies

Authors: Melikhaya Skhephe, Robert Mawuli Kwasi Boadzo, Zanoxolo Berington Gobingca

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This research study sought to examine parental monitoring of learners’ cell phone use in the Eastern Cape, South Africa. To this end, the researchers employed a quantitative approach. Data were obtained through questionnaires, with a sample of 15 parents having been purposively selected. The findings revealed that parents are unaware that they have to monitor the learner’s cell phone. Another finding was that parents in the 21-century did not support the use of mobile phones in education. The researchers recommend that parent’s discussion forums be created to educate parents on how a cell phone can be used in education. Cellphone companies need to be encouraged to educate parents on how they monitor cell phones used by learners. Another recommendation was that network providers need to restrict access to searching on the internet according to age.

Keywords: parental monitoring, app blocking services, learner’s cell phone use, cell phone

Procedia PDF Downloads 154

Authors: Bo Ram Choi, Ji Su Kim, Juyeon Cho, Hyukjin Lee

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Food contaminated by bacteria (E.coli), causes food poisoning, which occurs to many patients worldwide annually. We have introduced an application of rolling circle amplification (RCA) as a versatile biosensor and developed a diagnostic platform composed of capillary tube and microbeads for rapid and easy detection of Escherichia coli (E. coli). When specific mRNA of E.coli is extracted from cell lysis, rolling circle amplification (RCA) of DNA template can be achieved and can be visualized by beads aggregation in capillary tube. In contrast, if there is no bacterial pathogen in sample, no beads aggregation can be seen. This assay is possible to detect visually target gene without specific equipment. It is likely to the development of a genetic kit for point of care testing (POCT) that can detect target gene using microbeads.

Keywords: rolling circle amplification (RCA), Escherichia coli (E. coli), point of care testing (POCT), beads aggregation, capillary tube

Procedia PDF Downloads 360

Authors: Emine Şahin, Murat Soner Balcıoğlu

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The aim of this study was to determine the growth hormone (bGH) gene polymorphism in the Holstein cattle growing around Antalya in Turkey. In order to determine the bGH-AluI polymorphism, polymerase chain reaction - restriction fragment length polymorphism (PCR-RFLP) method was performed. A 891 bp fragment of bGH was amplified and two types of alleles C and D for bGH were observed. In this study, the frequencies of C and D alleles were 0.8438 and 0.1562, respectively. The genotype frequencies for CC, CD and DD were 0.787, 0.191 and 0.022, respectively. According to the results of the chi-square test, a significant deviation from the Hardy-Weinberg equilibrium was not determined for the bGH locus in the population.

Keywords: Growth Hormone Gene, Holstein , Polymorphism, RFLP

Procedia PDF Downloads 363

Authors: Juliana A. Knocikova, Yann Bouret, Médéric Argentina, Laurent Counillon

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Cell volume, together with membrane potential and intracellular hydrogen ion concentration, is an essential biophysical parameter for normal cellular activity. Cell volumes can be altered by osmotically active compounds and extracellular tonicity. In this study, a simple mathematical model of osmotically induced cell swelling and shrinking is presented. Emphasis is given to water diffusion across the membrane. The mathematical description of the cellular behavior consists in a system of coupled ordinary differential equations. We compare experimental data of cell volume alterations driven by differences in osmotic pressure with mathematical simulations under hypotonic and hypertonic conditions. Implications for a future model are also discussed.

Keywords: eukaryotic cell, mathematical modeling, osmosis, volume alterations

Procedia PDF Downloads 457

Authors: Marco Avila Lopez, Hasnae Ait-Douchi, Silvia De Los Santos, Badr Eddine Lebrouhi, Pamela Ramírez Vidal

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In the context of the energy transition, fuel cell technology has emerged as a solution for harnessing hydrogen energy and mitigating greenhouse gas emissions. An in-depth study was conducted on a PEMFC-type fuel cell, with an initiation of an analysis of its operational principles and constituent components. Subsequently, the modelling of the fuel cell was undertaken using the Python programming language, encompassing both steady-state and transient regimes. In the case of the steady-state regime, the physical and electrochemical phenomena occurring within the fuel cell were modelled, with the assumption of uniform temperature throughout all cell compartments. Parametric identification was carried out, resulting in a remarkable mean error of only 1.62% when the model results were compared to experimental data documented in the literature. The dynamic model that was developed enabled the scrutiny of the fuel cell's response in terms of temperature and voltage under varying current conditions.

Keywords: fuel cell, modelling, dynamic, thermal model, PEMFC

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Authors: K. L. Virupaksha Gupta

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Ayurveda offers a holistic outlook regarding skin care. Most Initial step in Ayurveda is to identify the skin type and care accordingly which is highly personalized. Though dermatologically there are various skin type classifications such Baumann skin types (based on 4 parameters i) Oily Vs Dry ii) Sensitive Vs Resistant iii) Pigmented Vs Non-Pigmented iv) Wrinkled Vs Tight (Unwrinkled) etc but Skin typing in Ayurveda is mainly determined by the prakriti (constitution) of the individual as well as the status of Doshas (Humors) which are basically of 3 types – i.e Vata Pitta and Kapha,. Difference between them is mainly attributed to the qualities of each dosha (humor). All the above said skin types can be incorporated under these three types. The skin care modalities in each of the constitution vary greatly. Skin of an individual of Vata constitution would be lustreless, having rough texture and cracks due to dryness and thus should be given warm and unctuous therapies and oil massage for lubrication and natural moisturizers for hydration. Skin of an individual of Pitta constitution would look more vascular (pinkish), delicate and sensitive with a fair complexion, unctuous and tendency for wrinkles and greying of hair at an early age and hence should be given cooling and nurturing therapies and should avoid tanning treatments. Skin of an individual of kapha constitution will have oily skin, they are delicate and look beautiful and radiant and hence these individuals would require therapies to mainly combat oily skin. Hence, the skin typing and skin care in Ayurveda is highly rational and scientific.

Keywords: Ayurveda, dermatology, Dosha, skin types

Procedia PDF Downloads 402

Authors: Misa Nakao, Yuta Kurashina, Chikahiro Imashiro, Kenjiro Takemura

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The Research Goal: With the growing demand for regenerative medicine, an effective mass cell culture process is required. In a repetitive subculture process for proliferating cells, preparing single cell suspension which does not contain any cell aggregates is highly required because cell aggregates often raise various undesirable phenomena, e.g., apoptosis and decrease of cell proliferation. Since cell aggregates often occur in cell suspension during conventional subculture processes, this study proposes a single cell sorter driven by a resonance vibration of a cell culture substrate. The Method and the Result: The single cell sorter is simply composed of a cell culture substrate and a glass pipe vertically placed against the cell culture substrate with a certain gap corresponding to a cell diameter. The cell culture substrate is made of biocompatible stainless steel with a piezoelectric ceramic disk glued to the bottom side. Applying AC voltage to the piezoelectric ceramic disk, an out-of-plane resonance vibration with a single nodal circle of the cell culture substrate can be excited at 5.5 kHz. By doing so, acoustic radiation force is emitted, and then cell suspension containing only single cells is pumped into the pipe and collected. This single cell sorter is effective to collect single cells selectively in spite of its quite simple structure. We collected C2C12 myoblast cell suspension by the single cell sorter with the vibration amplitude of 12 µmp-p and evaluated the ratio of single cells in number against the entire cells in the suspension. Additionally, we cultured the collected cells for 72 hrs and measured the number of cells after the cultivation in order to evaluate their proliferation. As a control sample, we also collected cell suspension by conventional pipetting, and evaluated the ratio of single cells and the number of cells after the 72-hour cultivation. The ratio of single cells in the cell suspension collected by the single cell sorter was 98.2%. This ratio was 9.6% higher than that collected by conventional pipetting (statistically significant). Moreover, the number of cells cultured for 72 hrs after the collection by the single cell sorter yielded statistically more cells than that collected by pipetting, resulting in a 13.6% increase in proliferated cells. These results suggest that the cell suspension collected by the single cell sorter driven by the resonance vibration hardly contains cell aggregates whose diameter is larger than the gap between the cell culture substrate and the pipe. Consequently, the cell suspension collected by the single cell sorter maintains high cell proliferation. Conclusions: In this study, we developed a single cell sorter capable of sorting and pumping single cells by a resonance vibration of a cell culture substrate. The experimental results show the single cell sorter collects single cell suspension which hardly contains cell aggregates. Furthermore, the collected cells show higher proliferation than that of cells collected by conventional pipetting. This means the resonance vibration of the cell culture substrate can benefit us with the increase in efficiency of mass cell culture process for clinical applications.

Keywords: acoustic radiation force, cell proliferation, regenerative medicine, resonance vibration, single cell sorter

Procedia PDF Downloads 259

Authors: Pui Shan Wong, Kosuke Tashiro, Satoru Kuhara, Sachiyo Aburatani

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Functional genomics and gene regulation inference has readily expanded our knowledge and understanding of gene interactions with regards to expression regulation. With the advancement of transcriptome sequencing in time-series comes the ability to study the sequential changes of the transcriptome. This method presented here works to augment existing regulation networks accumulated in literature with transcriptome data gathered from time-series experiments to construct a sequential representation of transcription factor activity. This method is applied on a time-series RNA-Seq data set from Escherichia coli as it transitions from growth to stationary phase over five hours. Investigations are conducted on the various metabolic activities in gene regulation processes by taking advantage of the correlation between regulatory gene pairs to examine their activity on a dynamic network. Especially, the changes in metabolic activity during phase transition are analyzed with focus on the pagP gene as well as other associated transcription factors. The visualization of the sequential transcriptional activity is used to describe the change in metabolic pathway activity originating from the pagP transcription factor, phoP. The results show a shift from amino acid and nucleic acid metabolism, to energy metabolism during the transition to stationary phase in E. coli.

Keywords: Escherichia coli, gene regulation, network, time-series

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Authors: Yasser M. Saad, Heba El-Sebaie Abd El-Sadek

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Some Metapenaeus monoceros cox1 gene fragments were isolated, purified, sequenced, and comparatively analyzed with some other Crustacean Cox1 gene sequences (obtained from National Center for Biotechnology Information). This work was designed for testing the efficiency of this system in reconstruction of phylogenetic relations among some Crustacean species belonging to four genera (Metapenaeus, Artemia, Daphnia and Calanus). The single nucleotide polymorphism and haplotype diversity were calculated for all estimated mt-DNA fragments. The genetic distance values were 0.292, 0.015, 0.151, and 0.09 within Metapenaeus species, Calanus species, Artemia species, and Daphnia species, respectively. The reconstructed phylogenetic tree is clustered into some unique clades. Cytochrome oxidase subunit 1 gene (cox1) was a powerful system in reconstruction of phylogenetic relations among evaluated crustacean species.

Keywords: crustaceans, genetics, Cox1, phylogeny

Procedia PDF Downloads 357

Authors: Nunthawan Nowwarote, Waleerat Sukarawan, Prasit Pavasant, Thanaphum Osathanon

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Interleukin 6 (IL-6) is a multifunctional cytokine, regulating various biological responses in several tissues. A Recent study shows that IL-6 plays a role in stemness maintenance in stem cells isolated from human exfoliated deciduous teeth (SHEDs). However, the role of IL-6 on cell differentiation in SHEDs remains unknown. The present study investigated the effect of IL-6 on SHEDs differentiation. Cells were isolated from dental pulp tissues of human deciduous teeth. Flow cytometry was used to determined mesenchymal stem cell marker expression, and the multipotential differentiation (osteogenic, adipogenic and neurogenic lineage ) was also determined. The mRNA was determined using real-time quantitative polymerase chain reaction, and the phenotypes were confirmed by chemical and immunofluorescence staining. Results demonstrated that SHEDs expressed CD44, CD73, CD90, CD105 but not CD45. Further, the up-regulation of osteogenic, adipogenic and neurogenic marker genes was observed upon maintaining cells in osteogenic, adipogenic and neurogenic induction medium, respectively. The addition of IL-6 induced osteogenic by up-regulated osteogenic marker gene also increased in vitro mineralization. Under neurogenic medium supplement with IL-6, up-regulated neurogenic marker. Whereas, an addition of IL-6 attenuated adipogenic differentiation by SHEDs. In conclusion, this evidence implies that IL-6 may participate in cells differentiation ability of SHEDs.

Keywords: SHEDs, IL-6, cell differentiations, dental pulp

Procedia PDF Downloads 178

Authors: Tety Hartatik, Dian Kurniawati, Adiarto

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The aim of the research was to determine the associations between polymorphism of the bovine growth hormone (GH) gene (Leu/Val, L/V) and milk production of Friesian Holstein Cattle. A total of 62 cows which consist of two Friesian Holstein groups (cattle from New Zealand are 19 heads and cattle from Australia are 43 heads). We perform the PCR and RFLP method for analyzing the genotype of the target gene GH 211 bp in the part of intron 4 and exon 5 of GH gene. The frequencies of genotypes LL were higher than genotype LV. The number of genotype LL in New Zealand and Australia groups are 84% and 79%, respectively. The number of genotype LV in New Zealand and Australia groups are 16% and 21%, respectively. The association between Leu/Val polymorphism on milk production, fat and protein content in both groups does not show the significant effect. However base on the groups (cows from New Zealand compare with those from Australia) show the significant effect on fat and protein content.

Keywords: Friesian Holstein, fat content, growth hormone gene, milk production, PCR-RLFP, protein content

Procedia PDF Downloads 651

Authors: Babak Forouraghi

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A genetic circuit is a collection of interacting genes and proteins that enable individual cells to implement and perform vital biological functions such as cell division, growth, death, and signaling. In cell engineering, synthetic gene circuits are engineered networks of genes specifically designed to implement functionalities that are not evolved by nature. These engineered networks enable scientists to tackle complex problems such as engineering cells to produce therapeutics within the patient's body, altering T cells to target cancer-related antigens for treatment, improving antibody production using engineered cells, tissue engineering, and production of genetically modified plants and livestock. Construction of computational models to realize genetic circuits is an especially challenging task since it requires the discovery of flow of genetic information in complex biological systems. Building synthetic biological models is also a time-consuming process with relatively low prediction accuracy for highly complex genetic circuits. The primary goal of this study was to investigate the utility of a pre-trained bidirectional encoder transformer that can accurately predict gene expressions in genetic circuit designs. The main reason behind using transformers is their innate ability (attention mechanism) to take account of the semantic context present in long DNA chains that are heavily dependent on spatial representation of their constituent genes. Previous approaches to gene circuit design, such as CNN and RNN architectures, are unable to capture semantic dependencies in long contexts as required in most real-world applications of synthetic biology. For instance, RNN models (LSTM, GRU), although able to learn long-term dependencies, greatly suffer from vanishing gradient and low-efficiency problem when they sequentially process past states and compresses contextual information into a bottleneck with long input sequences. In other words, these architectures are not equipped with the necessary attention mechanisms to follow a long chain of genes with thousands of tokens. To address the above-mentioned limitations of previous approaches, a transformer model was built in this work as a variation to the existing DNA Bidirectional Encoder Representations from Transformers (DNABERT) model. It is shown that the proposed transformer is capable of capturing contextual information from long input sequences with attention mechanism. In a previous work on genetic circuit design, the traditional approaches to classification and regression, such as Random Forrest, Support Vector Machine, and Artificial Neural Networks, were able to achieve reasonably high R2 accuracy levels of 0.95 to 0.97. However, the transformer model utilized in this work with its attention-based mechanism, was able to achieve a perfect accuracy level of 100%. Further, it is demonstrated that the efficiency of the transformer-based gene expression classifier is not dependent on presence of large amounts of training examples, which may be difficult to compile in many real-world gene circuit designs.

Keywords: transformers, generative ai, gene expression design, classification

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Authors: Neslihan Taskale Karatug, Mustafa Akcelik

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Some literature data show that misL protein play a role on host immune response formed against Salmonella Typhimurium. The aim of the present study is to learn the role of the protein in S. Typhimurium pathogenicity. To describe certain functions of the protein, primarily recombinant misL protein was produced and purified. PCR was performed using a primer set targeted to passenger domain of the misL gene on S. Typhimurium LT2 genome. Amplicon and pet28a vector were enzymatically cleaved with EcoRI and NheI. The digested DNA materials were purified with High Pure PCR Product Purification Kit. The ligation reaction was achieved with the pure products. After preparation of competent Escherichia coli Dh5α, ligation mix was transformed into the cell by electroporation. To confirm the existence of insert gene, recombinant plasmid DNA of Dh5α was isolated with high pure plasmid DNA kit. Proved the correctness of recombinant plasmid was electroporated to BL21. The cell was induced by IPTG. After induction, the presence of recombinant protein was checked by SDS-PAGE. The recombinant misL protein was purified using HisPur Ni-NTA spin colon. The pure protein was shown by SDS-PAGE and western blot immünoassay. The concentration of the protein was measured BCA Protein Assay kit. In the wake of ligation with digested products (2 kb misL and 5.4 kb pet28a) visualised on gel size of the band was about 7.4 kb and was named as pNT01. The pNT01 recombinant plasmid was transformed into Dh5α and colonies were chosen in selective medium. Plasmid DNA isolation from them was carried out. PCR was achieved on the pNT01 to check misL and 2 kb band was observed on the agarose gel. After electroporation of the plasmid and induction of the cell, 68 kDa misL protein was seen. Subsequent to the purification of the protein, only a band was observed on SDS-PAGE. Association of the pure protein with anti-his antibody was verified by the western blot assay. The concentration of the pure misL protein was determined as 345 μg/mL. Production of polyclonal antibody will be achieved by using the obtained pure recombinant misL protein as next step. The role of the protein will come out on the immune system together some assays.

Keywords: cloning, Escherichia coli, recombinant protein purification, Salmonella Typhimurium

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Authors: Jacek Kabzinski, Karolina Przybylowska, Lukasz Dziki, Adam Dziki, Ireneusz Majsterek

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The incidence of colorectal cancer (CRC) is increasing from year to year. Despite intensive research CRC etiology remains unknown. Studies suggest that at the basis of the process of carcinogenesis can lie reduced efficiency of DNA repair mechanisms, often caused by polymorphisms in DNA repair genes. The aim of the study was to determine the relationship between gene polymorphisms Pro242Arg of PolB gene and Arg780His of Lig3 gene and modulation of the risk of colorectal cancer in the Polish population. Determination of the molecular basis of carcinogenesis process and predicting increased risk will allow qualifying patients to increased risk group and including them in preventive program. We used blood collected from 110 patients diagnosed with colorectal cancer. The control group consisted of equal number of healthy people. Genotyping was performed by TaqMan method. The obtained results indicate that the genotype 780Arg/His of Lig3 gene is associated with an increased risk of colorectal cancer. On the basis of these results, we conclude that Lig3 gene polymorphism Arg780His may be associated with an increased risk of colorectal cancer.

Keywords: BER, colorectal cancer, PolB, Lig3, polymorphisms

Procedia PDF Downloads 452

Authors: W. Loongyai, N. Saengsawang, W. Danvilai, C. Kridtayopas, P. Sopannarath, C. Bunchasak

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Betong chicken is a slow growing and a lean strain of chicken, while the rapid growth of broiler is accompanied by increased fat. We investigated the growth performance, fat accumulations, lipid serum biochemical levels and lipoprotein lipase (LPL) gene expression of female Betong (KU line) at the age of 4 and 6 weeks. A total of 80 female Betong chickens (KU line) and 80 female broiler chickens were reared under open system (each group had 4 replicates of 20 chicks per pen). The results showed that feed intake and average daily gain (ADG) of broiler chicken were significantly higher than Betong (KU line) (P < 0.01), while feed conversion ratio (FCR) of Betong (KU line) at week 6 were significantly lower than broiler chicken (P < 0.01) at 6 weeks. At 4 and 6 weeks, two birds per replicate were randomly selected and slaughtered. Carcass weight did not significantly differ between treatments; the percentage of abdominal fat and subcutaneous fat yield was higher in the broiler (P < 0.01) at 4 and 6 week. Total cholesterol and LDL level of broiler were higher than Betong (KU line) at 4 and 6 weeks (P < 0.05). Abdominal fat samples were collected for total RNA extraction. The cDNA was amplified using primers specific for LPL gene expression and analysed using real-time PCR. The results showed that the expression of LPL gene was not different when compared between Betong (KU line) and broiler chickens at the age of 4 and 6 weeks (P > 0.05). Our results indicated that broiler chickens had high growth rate and fat accumulation when compared with Betong (KU line) chickens, whereas LPL gene expression did not differ between breeds.

Keywords: lipoprotein lipase gene, Betong (KU line), broiler, abdominal fat, gene expression

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Authors: Imran Hashmi, Sipkaduwa Arachchige Sashika Sureni Wickramasooriya

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Gene-drive technology has emerged as a promising tool for disease control by influencing the population dynamics of disease-carrying organisms. Various gene drive mechanisms, derived from global laboratory experiments, aim to strategically manage and prevent the spread of targeted diseases. One prominent strategy involves population replacement, wherein genetically modified mosquitoes are introduced to replace the existing local wild population. To enhance our understanding and aid in the design of effective release strategies, we employ a comprehensive mathematical model. The utilized approach employs agent-based modeling, enabling the consideration of individual mosquito attributes and flexibility in parameter manipulation. Through the integration of an agent-based model and a meta-population spatial approach, the dynamics of gene drive mosquito spreading in a released site are simulated. The model's outcomes offer valuable insights into future population dynamics, providing guidance for the development of informed release strategies. This research significantly contributes to the ongoing discourse on the responsible and effective implementation of gene drive technology for disease vector control.

Keywords: gene drive, agent-based modeling, disease-carrying organisms, malaria

Procedia PDF Downloads 63

Authors: Igor Sukhotnik

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Objectives: Notch signaling is thought to act to drive cell versification in the lining of the small intestine. When Notch signaling is blocked, proliferation ceases, and epithelial cells become secretory. The purpose of the present study was to evaluate the role of Notch signaling pathway in stem cell differentiation in a rat model of intestinal ischemia-reperfusion (IR). Methods: Male Sprague-Dawley rats were randomly divided into four experimental groups: Sham-24 and Sham-48 rats underwent laparotomy and were killed 24 or 48 h later, respectively; IR-24 and IR-48 rats underwent occlusion of SMA and portal vein for 30 min followed by 24 or 48 h of reperfusion, respectively. Notch-related gene and protein expression were determined using Real Time PCR, Western blotting and immunohistochemistry. Wax histology and immunohistochemistry was used to determine cell differentiation toward absorptive (enterocytes) or secretory progenitors (goblet cells, enteroendocrine cells or Paneth cells). Results: IR-48 rats exhibited a significant decrease in Notch-1 protein expression (Western blot) that was coincided with a significant decrease in the number of Notch-1 positive cells (immunohistochemistry) in jejunum and ileum as well as Hes-1 positive cells in jejunum and ileum compared to Sham-48 rats. A significant down-regulation of Notch signaling related genes and proteins in IR animals was accompanied by a significant increase in the number of goblet and Paneth cells and decreased number of absorptive cells compared to control rats. Conclusions: Forty-eight hours following intestinal IR in rats, inhibited Notch signaling pathway was accompanied by intestinal stem cells differentiation toward secretory progenitors.

Keywords: Intestine, notch, ischemia-reperfusion, cell differentiation, secretory

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Authors: Omar S. Sharaf

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The mitochondrial 12s rRNA (mt-12s rDNA) gene for pig-specific was developed to detect material from pork species in different products collected from Jordanian market. The amplification PCR products of 359 bp and 531 bp were successfully amplified from the cyt b gene of pig the amplification product using mt-12S rDNA gene were successfully produced a single band with a molecular size of 456 bp. In the present work, the PCR amplification of mtDNA of cytochrome b has been shown as a suitable tool for rapid detection of pig DNA. 100 samples from different dairy, gelatin and chocolate based products and 50 samples from baby food formula were collected and tested to a presence of any pig derivatives. It was found that 10% of chocolate based products, 12% of gelatin and 56% from dairy products and 5.2% from baby food formula showed single band from mt-12S rDNA gene.

Keywords: halal food, baby infant formula, chocolate based products, PCR, Jordan

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Authors: Mahmoud Mabrok, Channarong Rodkhum

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Flavobacterium columanre is one of the devastating pathogen that causes noticeable economic losses in freshwater cultured fish. Like other filamentous bacteria, F. columanre tends to aggregate and fluctuate to all kind of media, thus revealing obstacles in recognition of its colonies. Since the molecular typing is the only fundamental tool for rapid and precise detection of this pathgen. The present study developed a species-specific PCR assay based on cslA unique gene of F. columnare. The cslA gene sequences of 13 F. columnare, strains retrieved from gene bank database, were aligned to identify a conserved homologous segment prior to primers design. The new primers yielded amplicons of 287 bp from F. columnare strains but not from relevant or other pathogens, unlike to other published set that showed no specificity and cross-reactivity with F. indicum. The primers were sensitive and detected as few as 7 CFUs of bacteria and 3 pg of gDNA template. The sensitivity was reduced ten times when using tissue samples. These primers precisely defined all field isolates in a double-blind study, proposing their applicable use for field detection.

Keywords: Columnaris infection, cslA gene, Flavobacterium columnare, PCR

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Authors: Ankush M. Dewle, Suditi Bhattacharya, Prachi R. Abhang, Savita Datar, Ajay J. Jog, Rupesh K. Srivastava, Geetanjali Tomar

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Objectives: The aim of this study was to investigate the quality of mesenchymal stem cells (MSCs) obtained from ageing gingival tissues, in order to suggest their potential role in autologous stem cell therapy for old individuals. Methods: MSCs were isolated from gingival tissues of young (18-45 years) and old (above 45 years) donors by enzymatic digestion. MSCs were analysed for cfu-f, surface marker expression by flow-cytometry and multilineage differentiation potential. The angiogenic potential was compared in a chick embryo yolk sac membrane model. The aging and differentiation markers including SA-β-galactosidase and p21 respectively were analysed by staining and flow-cytometry analysis. Additionally, osteogenic markers such as glucocorticoid receptor (GR), vitamin D receptor (VDR) were measured by flow-cytometry and RT-qPCR was performed for quantification of osteogenic gene expression. Alizarin Red S and alkaline phosphatase (ALP) activity were also quantitated. Results: Gingival MSCs (GMSCs) from both the age groups were similar in their morphology and displayed cfu-f. They had similar expression of MSC surface markers and p21, comparable rate of proliferation and differentiated to all the four lineages. GMSCs from young donors had a higher adipogenic differentiation potential as compared to the old GMSCs. Moreover, these cells did not display a significant difference in ALP activity probably due to comparable expression of GR, VDR, and osteogenic genes. Conclusions: Ageing of GMSCs occurs at a much slower rate than stem cells from other sources. Thus we suggest GMSCs as an excellent candidate for autologous stem cell therapy in degenerative diseases of elderly individuals. Clinical Significance: GMSCs could help overcome the setbacks in clinical implementation of autologous stem cell therapy for regenerative medicine in all age group of patient.

Keywords: bone regeneration, cell therapy, senescence, stem cell

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Authors: Mohammad Aladwan

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Alzheimer’s disease (AD) is a widespread dementing illness with a complex and poorly understood etiology. An important role in improving our understanding of the AD process is the modeling of disease-associated changes in tau protein phosphorylation, a protein known to mediate events essential to the onset and progression of AD. A main feature of AD is the abnormal phosphorylation of tau protein and the presence of neurofibrillary tangles. In order to evaluate the respective roles of the microtubule-binding region (MTBR) and alternatively spliced exons in the N-terminal projection domains in AD, we have constructed SHSY-5Y cell lines that stably overexpress four different species of tau protein (4R2N, 4R0N, N(E-2), N(E+2)). Since the toxicity and spreading of tau lesions in AD depends on the interactions of tau with other proteins, we have performed a proteomic analysis of exosome-fraction interactomes for cell lysates and media samples that were isolated from SHSY-5Y cell lines. Functional analysis of tau interactomes based on gene ontology (GO) terms was performed using the String 10.5 database program. The highest number of exosomes proteomes and tau associated proteins were found with 4R2N isoform (2771 and 159) in cell lysate and they have a high strength of connectivity (78%) between proteins, while N(E-2) isoform in the media proteomes has the highest number of proteins and tau associated protein (1829 and 205). Moreover, known AD markers were significantly enriched in secreted interactomes relative to lysate interactomes in the SHSY-5Y cells of tau isoforms lacking exons 2 and 3 in the N-terminal. The lack of exon 2 (E-2) from tau protein can be mediated by tau secretion and spreading to different cells. Enriched functions in the secreted E-2 interactome include signaling and developmental pathways that have been linked to a) tau misprocessing and lesion development and b) tau secretion and which, therefore, could play novel roles in AD pathogenesis.

Keywords: Alzheimer's disease, dementia, tau protein, neurodegenration disease

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Authors: Gwenyth C. Eichfeld

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Variation in global COVID-19 spread is partly explained by social and behavioral factors. Many of these behaviors are linked to genetics. The short polymorphism of the 5-HTTLPR promoter region of the SLC6A4 gene is linked to collectivism. The seven-repeat polymorphism of the DRD4 gene is linked to risk-taking, migration, sensation-seeking, and impulsivity. Fewer CAG repeats in the androgen receptor gene are linked to impulsivity. This study investigates an association between the country-level frequency of these variants and early Covid-19 spread. Results of regression analysis indicate a significant association between increased country-wide prevalence of the short allele of the SLC6A4 gene and decreased COVID-19 spread when other factors that have been linked to COVID-19 are controlled for. Additionally, results show that the short allele of the SLC6A4 gene is associated with COVID-19 spread through GDP and percent urbanization rather than collectivism. Results showed no significant association between the frequency of the DRD4 polymorphism nor the androgen receptor polymorphism with early COVID-19 spread.

Keywords: neuroscience, genetics, population sciences, Covid-19

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Authors: Almudena Espin Perez, Tyler Risom, Carl Pelz, Isabel English, Robert M. Angelo, Rosalie Sears, Andrew J. Gentles

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Pancreatic ductal adenocarcinoma (PDAC) is one of the most deadly malignancies. The role of the tumor microenvironment (TME) is gaining significant attention in cancer research. Despite ongoing efforts, the nature of the interactions between tumors, immune cells, and stromal cells remains poorly understood. The cell-intrinsic properties that govern cell lineage plasticity in PDAC and extrinsic influences of immune populations require technically challenging approaches due to the inherently heterogeneous nature of PDAC. Understanding the cell lineage plasticity of PDAC will improve the development of novel strategies that could be translated to the clinic. Members of the team have demonstrated that the acquisition of ductal to neuroendocrine lineage plasticity in PDAC confers therapeutic resistance and is a biomarker of poor outcomes in patients. Our approach combines computational methods for deconvolving bulk transcriptomic cancer data using CIBERSORTx and high-throughput single-cell imaging using Multiplexed Ion Beam Imaging (MIBI) to study lineage plasticity in PDAC and its relationship to the infiltrating immune system. The CIBERSORTx algorithm uses signature matrices from immune cells and stroma from sorted and single-cell data in order to 1) infer the fractions of different immune cell types and stromal cells in bulked gene expression data and 2) impute a representative transcriptome profile for each cell type. We studied a unique set of 300 genomically well-characterized primary PDAC samples with rich clinical annotation. We deconvolved the PDAC transcriptome profiles using CIBERSORTx, leveraging publicly available single-cell RNA-seq data from normal pancreatic tissue and PDAC to estimate cell type proportions in PDAC, and digitally reconstruct cell-specific transcriptional profiles from our study dataset. We built signature matrices and optimized by simulations and comparison to ground truth data. We identified cell-type-specific transcriptional programs that contribute to cancer cell lineage plasticity, especially in the ductal compartment. We also studied cell differentiation hierarchies using CytoTRACE and predict cell lineage trajectories for acinar and ductal cells that we believe are pinpointing relevant information on PDAC progression. Collaborators (Angelo lab, Stanford University) has led the development of the Multiplexed Ion Beam Imaging (MIBI) platform for spatial proteomics. We will use in the very near future MIBI from tissue microarray of 40 PDAC samples to understand the spatial relationship between cancer cell lineage plasticity and stromal cells focused on infiltrating immune cells, using the relevant markers of PDAC plasticity identified from the RNA-seq analysis.

Keywords: deconvolution, imaging, microenvironment, PDAC

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Authors: Mohamed Abdel-Hamid, Olfat Gamil Shaker, Doha El-Sayed Ellakwa, Eman Fathy Abdel-Maksoud

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Introduction: Despite advances in the knowledge of the molecular virology of hepatitis C virus (HCV), the mechanisms of hepatocellular injury in HCV infection are not completely understood. Hepatitis C viral infection (HCV) influences the susceptibility to apoptosis. This could lead to insufficient antiviral immune response and persistent viral infection. Aim of this study: was to examine whether BCL-2 gene polymorphism at codon 43 (+127G/A or Ala43Thr) has an impact on development of hepatocellular carcinoma caused by chronic hepatitis C Egyptian patients. Subjects and Methods: The study included three groups; group 1: composing of 30 patients with hepatocellular carcinoma (HCC), group 2 composing of 30 patients with HCV, group 3 composing of 30 healthy subjects matching the same age and socioeconomic status were taken as a control group. Gene polymorphism of BCL2 (Ala43Thr) were evaluated by PCR-RFLP technique and measured for all patients and controls. Results: The summed 43Thr genotype was more frequent and statistically significant in HCC patients as compared to control group. This genotype of BCL2 gene may inhibit the programmed cell death which leads to disturbance in tissue and cells homeostasis and reduction in immune regulation. This result leads to viral replication and HCV persistence. Moreover, virus produces variety of mechanisms to block genes participated in apoptosis. This mechanism proves that HCV patients who have 43Thr genotype are more susceptible to HCC. Conclusion: The data suggest for the first time that the BCL2 polymorphism is associated with the susceptibility to HCC in Egyptian populations and might be used as molecular markers for evaluating HCC risk. This study clearly demonstrated that Chronic HCV exhibit a deregulation of apoptosis with the disease progression. This provides an insight into the pathogenesis of chronic HCV infection, and may contribute to the therapy.

Keywords: BCL2 gene, Hepatitis C Virus, Hepatocellular carcinoma, sensitivity, specificity, apoptosis

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Authors: Babak Forouraghi

Abstract:

A genetic circuit is a collection of interacting genes and proteins that enable individual cells to implement and perform vital biological functions such as cell division, growth, death, and signaling. In cell engineering, synthetic gene circuits are engineered networks of genes specifically designed to implement functionalities that are not evolved by nature. These engineered networks enable scientists to tackle complex problems such as engineering cells to produce therapeutics within the patient's body, altering T cells to target cancer-related antigens for treatment, improving antibody production using engineered cells, tissue engineering, and production of genetically modified plants and livestock. Construction of computational models to realize genetic circuits is an especially challenging task since it requires the discovery of the flow of genetic information in complex biological systems. Building synthetic biological models is also a time-consuming process with relatively low prediction accuracy for highly complex genetic circuits. The primary goal of this study was to investigate the utility of a pre-trained bidirectional encoder transformer that can accurately predict gene expressions in genetic circuit designs. The main reason behind using transformers is their innate ability (attention mechanism) to take account of the semantic context present in long DNA chains that are heavily dependent on the spatial representation of their constituent genes. Previous approaches to gene circuit design, such as CNN and RNN architectures, are unable to capture semantic dependencies in long contexts, as required in most real-world applications of synthetic biology. For instance, RNN models (LSTM, GRU), although able to learn long-term dependencies, greatly suffer from vanishing gradient and low-efficiency problem when they sequentially process past states and compresses contextual information into a bottleneck with long input sequences. In other words, these architectures are not equipped with the necessary attention mechanisms to follow a long chain of genes with thousands of tokens. To address the above-mentioned limitations, a transformer model was built in this work as a variation to the existing DNA Bidirectional Encoder Representations from Transformers (DNABERT) model. It is shown that the proposed transformer is capable of capturing contextual information from long input sequences with an attention mechanism. In previous works on genetic circuit design, the traditional approaches to classification and regression, such as Random Forrest, Support Vector Machine, and Artificial Neural Networks, were able to achieve reasonably high R2 accuracy levels of 0.95 to 0.97. However, the transformer model utilized in this work, with its attention-based mechanism, was able to achieve a perfect accuracy level of 100%. Further, it is demonstrated that the efficiency of the transformer-based gene expression classifier is not dependent on the presence of large amounts of training examples, which may be difficult to compile in many real-world gene circuit designs.

Keywords: machine learning, classification and regression, gene circuit design, bidirectional transformers

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Authors: Wissem Abdaoui, Nizar M. Mhaidat, Ilhem Mokhtari, Adel Gouri

Abstract:

Colorectal cancer (CRC) is one of the most common tumors all over the world. Obesity, considered a risk factor of CRC, is characterized by a high level of secreted cytokines from adipose tissue. Among these inflammatory molecules, leptin is considered the key mediator for CRC cancer development and progression by activation of mitogenic and anti apoptotic signaling pathways. Gene expression can be significantly modulated by alterations in DNA methylation patterns. The aim of this study is to investigate the impact of leptin gene methylation on CRC prognosis and sensitivity to chemotherapy. The study involved 70 CRC tissue samples collected from King Abdullah University Hospital (KAUH) from which only 53 was analyzed because of bisulfate fragmentation and low yield of DNA extracted from FFPE tissues. A total of 22 blood samples were collected from healthy volunteers and enrolled as a control group. Leptin promoter methylation was analyzed by methylation specific PCR after bisulfate conversion. Results revealed that the incidence of leptin gene methylation was significantly higher in CRC patients in comparison to that of controls (P < 0.05). The correlation between patient’s demographics and leptin gene methylation was not significant (P < 0.05). However, a significant correlation between leptin gene methylation status and early cancer stages (I, II and III) was found in male but not in female (p < 0.05). Moreover, a significant correlation was found between leptin promoter methylation and early tumor localization T1-2 (p < 0.05). The correlation between epigenetic regulation of leptin and chemosensitivity was not significant. Taken together, these results suggest the possibility to use leptin gene methylation as a biomarker for the evaluation of CRC prognosis and metastasis.

Keywords: colorectal cancer, obesity, leptin, DNA methylation, disease prognosis, bisulfate conversion, chemoresistance

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Authors: Iman Farasat, Howard M. Salis

Abstract:

The CRISPR/Cas9 system has revolutionized genome engineering by enabling site-directed and high-throughput genome editing, genome insertion, and gene knockdowns in several species, including bacteria, yeast, flies, worms, and human cell lines. This technology has the potential to enable human gene therapy to treat genetic diseases and cancer at the molecular level; however, the current CRISPR/Cas9 system suffers from seemingly sporadic off-target genome mutagenesis that prevents its use in gene therapy. A comprehensive mechanistic model that explains how the CRISPR/Cas9 functions would enable the rational design of the guide-RNAs responsible for target site selection while minimizing unexpected genome mutagenesis. Here, we present the first quantitative model of the CRISPR/Cas9 genome mutagenesis system that predicts how guide-RNA sequences (crRNAs) control target site selection and cleavage activity. We used statistical thermodynamics and law of mass action to develop a five-step biophysical model of cas9 cleavage, and examined it in vivo and in vitro. To predict a crRNA's binding specificities and cleavage rates, we then compiled a nearest neighbor (NN) energy model that accounts for all possible base pairings and mismatches between the crRNA and the possible genomic DNA sites. These calculations correctly predicted crRNA specificity across 5518 sites. Our analysis reveals that cas9 activity and specificity are anti-correlated, and, the trade-off between them is the determining factor in performing an RNA-mediated cleavage with minimal off-targets. To find an optimal solution, we first created a scheme of safe-design criteria for Cas9 target selection by systematic analysis of available high throughput measurements. We then used our biophysical model to determine the optimal Cas9 expression levels and timing that maximizes on-target cleavage and minimizes off-target activity. We successfully applied this approach in bacterial and mammalian cell lines to reduce off-target activity to near background mutagenesis level while maintaining high on-target cleavage rate.

Keywords: biophysical model, CRISPR, Cas9, genome editing

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Authors: Saeedeh Salimi, Farzaneh Farajian- Mashhadi, Ehsan Tabatabaei, Mahnaz Shahrakipoor, Minoo Yaghmaei, Mojgan Mokhtari

Abstract:

Purpose: Estrogen receptor-α (ERα) plays an essential role in the adaptation of increased uterine blood flow during gestation. Therefore ERα gene could be a possible candidate for preeclampsia(PE) susceptibility. In the current study, we aimed to investigate the association of the ERα gene polymorphisms and PE in an Iranian population. Methods: One hundred ninety-two pregnant women with PE and 186 normotensive women were genotyped for ERα gene (PvuII and XbaI) polymorphisms by PCR-RFLP method. Results: The frequency of alleles and genotypes of ERα PvuII and XbaI polymorphisms were not different between PE and normotensive control women. However, higher frequency of GG genotype was observed in women with severe PE compared to mild PE (OR, 1.8 [95% CI, 1.1 to 3]; P = 0.02) and in severe PE compared to normotensive women [OR= 1.8(1.1-3), P=0.02] after adjusting for age, ethnicity and primiparity. Conclusions: The GG genotype of ERα XbaI polymorphism could be a genetic risk factor for PE predisposition.

Keywords: estrogen receptor-α, polymorphism, gene, preeclampsia

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Authors: D. J. Kalita

Abstract:

Cancer is one of the prime causes of early death worldwide. Mutation of the gene involve in DNA repair and damage, like BRCA2 (Breast cancer gene two) genes, can be detected efficiently by PCR-RFLP to early breast cancer diagnosis and adopt the suitable method of treatment. Host Defense Peptide can be used as blueprint for the design and synthesis of novel anticancer drugs to avoid the side effect of conventional chemotherapy and chemo resistance. The change at nucleotide position 392 of a -› c in the cancer sample of dog mammary tumour at BRCA2 (exon 7) gene lead the creation of a new restriction site for SsiI restriction enzyme. This SNP may be a marker for detection of canine mammary tumour. Support vector machine (SVM) algorithm was used to design and predict the anticancer peptide from the mature functional peptide. MTT assay of MCF-7 cell line after 48 hours of post treatment showed an increase in the number of rounded cells when compared with untreated control cells. The ability of the synthesized peptide to induce apoptosis in MCF-7 cells was further investigated by staining the cells with the fluorescent dye Hoechst stain solution, which allows the evaluation of the nuclear morphology. Numerous cells with dense, pyknotic nuclei (the brighter fluorescence) were observed in treated but not in control MCF-7 cells when viewed using an inverted phase-contrast microscope. Thus, PCR-RFLP is one of the attractive approach for early diagnosis, and synthetic cationic peptide can be used for the treatment of canine mammary tumour.

Keywords: cancer, cationic peptide, host defense peptides, Breast cancer genes

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Authors: Nazira B. Bekenova, Lydia A. Mukovozova, Andrej M. Grjibovski, Alma Z. Tokayeva, Yerbol M. Smail, Nurlan E. Aukenov

Abstract:

Erysipelas is an infectious disease with socio-economic significance and prone to prolonged recurrent course (30%). Contribution of genetic factors, in particular the gene polymorphism of cytokines, can be essential in disease etiology and pathogenesis. Interleukin – 17 A are produced by T helpers of 17 type and plays a key role in development of local inflammation process. Local inflammatory process is a dominant in the clinic of erysipelas. Established that the skin and mucosas are primary areas of migration (homing) Th17-cell and their cytokines are stimulate the barrier function of the epithelium. We studied associations between gene polymorphism of IL-17A (C737T) rs 8193036 and IL-17A level in patients with erysipelas Kazakh population. Altogether, 90 cases with erysipelas and 90 healthy controls from an ethnic Kazakh population comprised the sample. Cases were identified at Clinical Infectious Diseases Hospital of Semey (Kazakhstan). The IL-17A (rs8193036) polymorphism was analyzed by a real time polymerase chain reaction. Plasma levels of IL-17 A were assessed by immuneenzyme analysis method using ‘Vector-Best’ test-system (Russia). Differences in levels of IL-17 A between CC, TT, CT groups were studied using Kruskal — Wallis test. Pairwise comparisons were performed using Mann-Whitney tests with Bonferroni correction (New significance level was set to 0.025). We found, that in patients with erysipelas with CC genotype the level of IL-17 A was higher (p= 0, 010) compared to the carriers of CT genotype. When compared the level of IL – 17 A between the patients with TT genotype and patients with CC genotype, also between the patients with CT genotype and patients with TT genotype statistically significant differences are not revealed (p = 0.374 and p = 0.043, respectively). Comparisons of IL-17 A plasma levels between the CC and CT genotypes, between the CC and TT genotypes, and between the TT and CT in healthy patients did not reveal significant differences (p = 0, 291). Therefore, we are determined the associations of gene polymorphism of IL-17 A (C737T) with its level in patients erysipelas carriers CC genotype.

Keywords: erysipelas, interleukin – 17 A, Kazakh, polymorphism

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