Search results for: gene expression analysis

Authors: Ge Cui, Mei Fang Chien, Chihiro Inoue

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In this research, enrichment culture using an inorganic liquid medium collected soil contaminated with 1,2-dichlorobenzene (1,2-DCB) in Sendai, Japan, was added 1,2-DCB as the sole carbon source to create a stable consortium. The purpose of this research is to analysis dominant microorganisms in the stable consortium and enzyme system which play a role in the degradation of DCBs. The consortium is now at 30 generation and is still being cultured. By the result of PCR-DGGE and clone library, two bacteria are dominant. The bacteria named sk1 was isolated. 40mg/l of 1,2-DCB and 40mg/l of 1,4-DCB were completely degraded after 32 hours and 50 hours, respectively, but no degradation occurred in the case of 1,3-DCB. By PCR, tecA1 (α-subunit of DCB dioxygenase) gene which plays a role degrading DCB to DCB dihydrodiol, and tecB (dehydrogenase) gene which plays a role degrading DCB dihydrodiol to dichlorocatechol were amplified from strain sk1. Bacteria named sk100 was also isolated. 40mg/l of 1,2-DCB was completely degraded after 32 hours, but no degradation occurred in case of 1,3-DCB and 1,4-DCB. By the result of the catalytic core region of dioxygenase amplified by PCR, gene played a role degrading DCB was analyzed. The results of this study concluded that the isolated strains which have not been reported are able to degrade 1,2-DCB stably, and the characterization of degradation and the genomic analysis which is now in progress is helpful to have an overall view of this microbial degradation.

Keywords: DCB, 1, 2-DCB degrading strains, DCB dioxygenase, enrichment culture

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Authors: Sandeep Kumar Agrawal, Aditi Bhattacharya, Janvie Manhas, Krushna Bhatt, Yatin Kholakiya, Nupur Khera, Ajoy Roychoudhury, Sudip Sen

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Autologous cultured explants of human oral mucosal epithelial cells (OMEC) are a potential therapeutic modality for limbal stem cell deficiency (LSCD). Injury or inflammation of the ocular surface in the form of burns, chemicals, Stevens Johnson syndrome, ocular cicatricial pemphigoid etc. can lead to destruction and deficiency of limbal stem cells. LSCD manifests in the form of severe ocular surface diseases (OSD) characterized by persistent and recurrent epithelial defects, conjuntivalisation and neovascularisation of the corneal surface, scarring and ultimately opacity and blindness. Most of the cases of OSD are associated with severe dry eye pertaining to diminished mucin and aqueous secretion. Mycophenolate mofetil (MMF) has been shown to upregulate the mucin expression in conjunctival goblet cells in vitro. The aim of this study was to evaluate the effects of MMF on mucin expression in primary cultures of oral mucosal epithelial cells. With institutional ethics committee approval and written informed consent, thirty oral mucosal epithelial tissue samples were obtained from patients undergoing oral surgery for non-malignant conditions. OMEC were grown on human amniotic membrane (HAM, obtained from expecting mothers undergoing elective caesarean section) scaffold for 2 weeks in growth media containing DMEM & Ham’s F12 (1:1) with 10% FBS and growth factors. In vitro dosage of MMF was standardised by MTT assay. Analysis of stem cell markers was done using RT-PCR while mucin mRNA expression was quantified using RT-PCR and q-PCR before and after treating cultured OMEC with graded concentrations of MMF for 24 hours. Protein expression was validated using immunocytochemistry. Morphological studies revealed a confluent sheet of proliferating, stratified oral mucosal epithelial cells growing over the surface of HAM scaffold. The presence of progenitor stem cell markers (p63, p75, β1-Integrin and ABCG2) and cell surface associated mucins (MUC1, MUC15 and MUC16) were elucidated by RT-PCR. The mucin mRNA expression was found to be upregulated in MMF treated primary cultures of OMEC, compared to untreated controls as quantified by q-PCR with β-actin as internal reference gene. Increased MUC1 protein expression was validated by immunocytochemistry on representative samples. Our findings conclude that OMEC have the ability to form a multi-layered confluent sheet on the surface of HAM similar to a cornea, which is important for the reconstruction of the damaged ocular surface. Cultured OMEC has stem cell properties as demonstrated by stem cell markers. MMF can be a novel enhancer of mucin production in OMEC. It has the potential to improve dry eye in patients undergoing OMEC transplantation for bilateral OSD. Further clinical trials are required to establish the role of MMF in patients undergoing OMEC transplantation.

Keywords: limbal stem cell deficiency, mycophenolate mofetil, mucin, ocular surface disease

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Authors: Xuan Li, Xiaobing Cui, Nan Meng, Shuangshuang Guo, Lingling Wang

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Objective: To investigate the influence of Paeonol on diabetic nephropathy progression in streptozocin (STZ) -induced diabetic rats. Method Male Wistar rats were injected STZ 30mg.kg-1 combined with Freund's complete adjuvant (CFA) 0.1mL/rat once a week for three weeks. The diabetic rats were treated with Paenol for 13 weeks. At the end of the experiments, the rats were anesthetized. Serum and the kidney were collected. Serum superoxide dismutase (SOD) activity, malondialdehyde (MDA), blood urea nitrogen (BUN), creatinine (Cr) and total cholesterol (Chol) level were detected; kidney paraffin sections were prepared and HE and PAS staining sections were used to evaluate the pathology changes of the kidney. Immunohistochemical analysis was used to observe the expression of VEGF and fibernectin expression in the kidney. Result The blood glucose level remained over 16mmol. L-1 for 13 weeks and the ECM accumulated in the diabetic kidney apparently. Paeonol treatment increased serum SOD activity, however, MDA, BUN, Cr, and Chol level was decreased by paeonol treatment. VEGF and fibernectin expression were increased significantly in the DN rats and paeonol treatment ameliorated the overexpression. Conclusion: paeonol prevented the progression of DN.

Keywords: paeonol, STZ, diabetic nephropathy, fibernectin expression, kidney paraffin sections

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Authors: Malik SS, Masood N, Mubarik S, Khadim TM

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Introduction: Xeroderma pigmentosum group G (XPG) gene plays a crucial role in the correction of UV-induced DNA damage through nucleotide excision repair pathway. Single nucleotide polymorphisms in XPG gene have been reported to be associated with different cancers. Current case-control study was designed to evaluate the relationship between one of the most frequently found XPG (rs1047768 T>C) polymorphism and breast cancer risk. Methodology: A total of 200 individuals were screened for this polymorphism including 100 pathologically confirmed breast cancer cases and age-matched 100 controls. Genotyping was carried out using Tetra amplification-refractory mutation system (ARMS) PCR and results were confirmed by gel electrophoresis. Results: Conditional logistic regression analysis showed significant association between TC genotype (OR: 8.9, CI: 2.0 – 38.7) and increased breast cancer risk. Although homozygous CC genotype was more frequent in patients as compared to controls, but it was statistically non-significant (OR: 3.9, CI: 0.4 – 35.7). Conclusion: In conclusion, XPG (rs1047768 T>C) polymorphism may contribute towards increased risk of breast cancer but other polymorphisms may also be evaluated to elucidate their role in breast cancer.

Keywords: XPG, breast cancer, NER, ARMS-PCR

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

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Colorectal cancer (CRC) is one of the deadliest cancers. Every year we see an increase in the number of cases, and in spite of intensive research etiology of the disease remains unknown. For many years, researchers are seeking to associate genetic factors with an increased risk of CRC, so far it has proved to be a compelling link between the MMR system of DNA repair and hereditary nonpolyposis colorectal cancers (HNPCC). Currently, research is focused on finding the relationship between the remaining DNA repair systems and an increased risk of developing colorectal cancer. The aim of the study was to determine the relationship between gene polymorphisms Ser835Ser of XPF gene and Gly23Ala of XPA gene–elements of NER DNA repair system, 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 23Gly/Ala of XPA gene is associated with an increased risk of colorectal cancer, while 23Ala/Ala as well as TCT allele of Ser835Ser of XPF gene may reduce the risk of CRC.

Keywords: NER, colorectal cancer, XPA, XPF, polymorphisms

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Authors: Fereydoon Bondarian, Samira Shaygan

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Today, many nutritionists recommend the consumption of plants, fruits, and vegetables to provide the antioxidants needed by the body because the use of plant antioxidants usually causes fewer side effects and better treatment. Natural antioxidants increase the power of plasma antioxidants and reduce the incidence of some diseases, such as cancer. Bad lifestyles and environmental factors play an important role in increasing the incidence of cancer. In this study, different extracts of white teas taken from two types of tea available in Iran (clone 100 and Chinese hybrid) due to the presence of a hydroxyl functional group in their structure to inhibit free radicals and anticancer properties, using 3 aqueous, methanolic and aqueous-methanolic methods were used. The total polyphenolic content was calculated using the Folin-Ciocalcu method, and the percentage of inhibition and trapping of free radicals in each of the extracts was calculated using the DPPH method. With the help of high-performance liquid chromatography, a small amount of each catechin in the tea samples was obtained. Clone 100 white tea was found to be the best sample of tea in terms of all the examined attributes (total polyphenol content, antioxidant properties, and individual amount of each catechin). The results showed that aqueous and aqueous-methanolic extracts of Clone 100 white tea have the highest total polyphenol content with 27.59±0.08 and 36.67±0.54 (equivalent gallic acid per gram dry weight of leaves), respectively. Due to having the highest level of different groups of catechin compounds, these extracts have the highest property of inhibiting and trapping free radicals with 66.61±0.27 and 71.74±0.27% (mg/l) of the extracted sample against ascorbic acid). Using the MTT test, the inhibitory effect of clone 100 white tea extract in inhibiting the growth of HCT-116 colon cancer cells was investigated and the best time and concentration treatments were 500, 150 and 1000 micrograms in 8, 16 and 24 hours, respectively. To investigate gene expression changes, selected genes, including tumorigenic genes, proto-oncogenes, tumor suppressors, and genes involved in apoptosis, were selected and analyzed using the real-time PCR method and in the presence of concentrations obtained for white tea. White tea extract at a concentration of 1000 μg/ml 3 times 16, 8, and 24 hours showed the highest growth inhibition in cancer cells with 53.27, 55.8, and 86.06%. The concentration of 1000 μg/ml aqueous extract of white tea under 24-hour treatment increased the expression of tumor suppressor genes compared to the normal sample.

Keywords: catechin, gene expression, suppressor genes, colon cell line

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Authors: M. Sunitha, B. Nithyavani, Mathew Yohannan, S. Thiruvieni Balajji, M. A. Rathi, C. Arul Raj, P. Ragavendran, V. K. Gopalkrishnan

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Establishment of an early and reliable biomarker for ovarian carcinogenesis whose expression can be monitored through noninvasive techniques will enable early diagnosis of cancer. Carbonic anhydrases (CA) isozymes IX and XII have been suggested to play a role in oncogenic processes. In von Hippel-Lindau (VHL)-defective tumors, the cell surface transmembrane carbonic anhydrase (CA) CA XI and CA XII genes are overexpressed because of the absence of pVHL. These enzymes are involved in causing a hypoxia condition, thereby providing an environment for metastasis. Aberrant expression of the facilitative glucose transporter GLUT I is found in a wide spectrum of epithelial malignancies. Studying the mRNA expression of CA IX, CA XII and Glut I isozymes in ovarian cancer cell lines (OAW-42 and PA-1) revealed the expression of these hypoxia genes. Immunohistochemical staining of carbonic anhydrases was also performed in 40 ovarian cancer tissues. CA IX and CA XII were expressed at 540 bp and 520 bp in OAW42, PA1 in ovarian cancer cell lines. GLUT-1 was expressed at 325bp in OAW 42, PA1 genes in ovarian cancer cell lines. Immunohistochemistry revealed high to moderate levels of expression of these enzymes. The immuostaining was seen predominantly on the cell surface membrane. The study concluded that these genes CA IX, CA XII and Glut I are expressed under hypoxic condition in tumor cells. From the present results expression of CA IX, XII and Glut I may represent potential targets in ovarian cancer therapy.

Keywords: ovarian cancer, carbonic anhydrase IX, XII, Glut I, tumor markers

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Authors: Fangfang Wang, Fan Qu

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Polycystic ovary syndrome (PCOS), the most common endocrinopathy among women of reproductive age, is characterized by ovarian dysfunction, hyperandrogenism and reduced fecundity. The aim of this study is to investigate whether the circadian disruption is involved in pathogenesis of PCOS in androgen-induced animal model. We established a rat model of PCOS using single subcutaneous injection with testosterone propionate on the ninth day after birth, and confirmed their PCOS-like phenotypes with vaginal smears, ovarian hematoxylin and eosin (HE) staining and serum androgen measurement. The control group rats received the vehicle only. Gene expression was detected by real-time quantitative PCR. (1) Compared with control group, PCOS model rats of 10-week group showed persistently keratinized vaginal cells, while all the control rats showed at least two consecutive estrous cycles. (2) Ovarian HE staining and histological examination showed that PCOS model rats of 10-week group presented many cystic follicles with decreased numbers of granulosa cells and corpora lutea in their ovaries, while the control rats had follicles with normal layers of granulosa cells at various stages of development and several generations of corpora lutea. (3) In the 10-week group, serum free androgen index was notably higher in PCOS model rats than controls. (4) Disturbed mRNA expression patterns of core clock genes were found in ovaries of PCOS model rats of 10-week group. Abnormal expression of key genes associated with circadian rhythm in ovary may be one of the mechanisms for ovarian dysfunction in PCOS model rats induced by androgen.

Keywords: polycystic ovary syndrome, androgen, animal model, circadian disruption

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Authors: Christopher R. Triggle, Hong Ding, Khaled Machaca, Gnanapragasam Arunachalam

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The antidiabetic drug, metformin, has been in clinical use for over 50 years and remains the first choice drug for the treatment of type two diabetes. In addition to its effectiveness as an oral anti-hyperglycaemic drug metformin also possesses vasculoprotective effects that are assumed to be secondary to its ability to reduce insulin resistance and control glycated hemoglobin levels; however, recent data from our laboratory indicate that metformin also has direct vasoprotective effects that are mediated, at least in part, via the anti-ageing gene, SIRT1. Diabetes is a major risk factor for the development of cardiovascular disease (CVD) and it is also well established that tobacco use further enhances the risk of CVD; however, it is not known whether treatment with metformin can offset the negative effects of diabetes and tobacco use on cardiac function. The current study was therefore designed to investigate 1: the effects of hyperglycaemia (HG) either alone or in the presence of elevated fatty acids (palmitate) and nicotine on the protein expression levels of the deacetylase sirtuin 1 (the protein product of SIRT1), anti-apoptotic Bcl-2, pro-apoptotic BIM and the pro-apoptotic, tumour suppressor protein, acetylated p53 in cardiomyocytes. 2: the ability of metformin to prevent the detrimental effects of HG, palmitate and nicotine on cardiomyocyte survival. Cell culture protocols were designed using a rat cardiomyocyte cell line, H9c2, either under normal glycaemic (NG) conditions of 5.5mM glucose, or hyperglycaemic conditions (HG) of 25mM glucose with, or without, added palmitate (250μM) or nicotine (1.0mM) for 24h. Immuno-blotting was used to detect the expression of sirtuin 1, Bcl-2, BIM, acetylated (Ac)-p53, p53 with β-actin used as the reference protein. Exposure to HG, palmitate, or nicotine alone significantly reduced expression of sirtuin1, Bcl-2 and raised the expression levels of acetylated p53 and BIM; however, the combination of HG, palmitate and nicotine had a synergistic effect to significantly suppress the expression levels of sirtuin 1 and Bcl-2, but further enhanced the expression of Ac-p53, and BIM. The inclusion of 1000μM, but not 50μM, metformin in the H9c2 cell culture protocol prevented the effects of HG, palmitate and nicotine on the pro-apoptotic pathways. Collectively these data indicate that metformin, in addition to its anti-hyperglycaemic and vasculoprotective properties, also has direct cardioprotective actions that offset the negative effects of hyerglycaemia, elevated free fatty acids and nicotine on cardiac cell survival. These data are of particular significance for the treatment of patients with diabetes who are also smokers as the inclusion of metformin in their therapeutic treatment plan should help reduce cardiac-related morbidity and mortality.

Keywords: apoptosis, cardiac muscle, diabetes, metformin, nicotine

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Authors: Negin Parsamanesh, Saman Ameri-Mahabadi, Ali Nikfar, Mojdeh Mansouri, Hossein Chiti, Gita Fatemi Abhari

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Duchenne muscular dystrophy (DMD) is a severe progressive X-linked neuromuscular illness that affects movement through mutations in dystrophin gene. The mutation leads to insufficient, lack of or dysfunction of dystrophin. The cause of DMD was determined in an Iranian family. Exome sequencing was carried out along with a complete physical examination of the family. In silico methods were applied to find the alteration in the protein structure. The homozygous variant in DMD gene (NM-004006.2) was defined as c.2732-2733delTT (p.Phe911CysfsX8) in exon 21. In addition, phylogenetic conservation study of the human dystrophin protein sequence revealed that phenylalanine 911 is one of the evolutionarily conserved amino acids. In conclusion, our study indicated a new deletion in the DMD gene in the affected family. This deletion with an X-linked inheritance pattern is new in Iran. These findings could facilitate genetic counseling for this family and other patients in the future.

Keywords: duchenne muscular dystrophy, whole exome sequencing, iran, metabolic syndrome

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Authors: E. Ramprasath, P. Manojkumar, P. Veena

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Proposed paper dealt with the modelling and analysis of induction motor based on the mathematical expression using the graphical programming environment of Laboratory Virtual Instrument Engineering Workbench (LabVIEW). Induction motor modelling with the mathematical expression enables the motor to be simulated with the various required parameters. Owing to the invention of variable speed drives study about the induction motor characteristics became complex.In this simulation motor internal parameter such as stator resistance and reactance, rotor resistance and reactance, phase voltage, frequency and losses will be given as input. By varying the speed of motor corresponding parameters can be obtained they are input power, output power, efficiency, torque induced, slip and current.

Keywords: induction motor, LabVIEW software, modelling and analysi, electrical and mechanical characteristics of motor

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Authors: Debjyoti Bhakat, Indranil Mondal, Asish K. Mukhopadayay, Nabendu S. Chatterjee

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Enterotoxigenic Escherichia coli is one of the leading causes of diarrhea in infants and travelers in developing countries. Colonization factors play an important role in pathogenesis and are one of the main targets for Enterotoxigenic Escherichia coli (ETEC) vaccine development. However, ETEC vaccines had poorly performed in the past, as the prevalence of colonization factors is region-dependent. There are more than 25 classical colonization factors presently known to be expressed by ETEC, although all are not expressed together. Further, there are other multiple non-classical virulence factors that are also identified. Here the presence and expression of common classical and non-classical virulence factors were studied. Further studies were done on the expression of prevalent colonization factors in different strains. For the prevalence determination, multiplex polymerase chain reaction (PCR) was employed, which was confirmed by simplex PCR. Quantitative RT-PCR was done to study the RNA expression of these virulence factors. Strains negative for colonization factors expression were confirmed by SDS-PAGE. Among the clinical isolates, the most prevalent toxin was est+elt, followed by est and elt, while the pattern was reversed in the control strains. There were 29% and 40% strains negative for any classical colonization factors (CF) or non-classical virulence factors (NCVF) among the clinical and control strains, respectively. Among CF positive ETEC strains, CS6 and CS21 were the prevalent ones in the clinical strains, whereas in control strains, CS6 was the predominant one. For NCVF genes, eatA was the most prevalent among the clinical isolates and etpA for control. CS6 was the most expressed CF, and eatA was the predominantly expressed NCVF for both clinical and controlled ETEC isolates. CS6 expression was more in strains having CS6 alone. Different strains express CS6 at different levels. Not all strains expressed their respective virulence factors. Understanding the prevalent colonization factor, CS6, and its nature of expression will contribute to designing an effective vaccine against ETEC in this region of the globe. The expression pattern of CS6 also will help in examining the relatedness between the ETEC subtypes.

Keywords: classical virulence factors, CS6, diarrhea, enterotoxigenic escherichia coli, expression, non-classical virulence factors

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Authors: Lay Poh Tan, Chor Yong Tay, Haiyang Yu

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Micro-contact printing is a form of soft lithography that uses the relief patterns on a master polydimethylsiloxane (PDMS) stamp to form patterns of self-assembled monolayers (SAMs) of ink on the surface of a substrate through conformal contact technique. Here, we adopt this method to print proteins of different dimensions on our biodegradable polymer substrates. We started off with printing 20-500 μm scale lanes of fibronectin to engineer the shape of bone marrow derived human mesenchymal stem cell (hMSCs). After 8 hours of culture, the hMSCs adopted elongated shapes, and upon analysis of the gene expressions, genes commonly associated with myogenesis (GATA-4, MyoD1, cTnT and β-MHC) and neurogenesis (NeuroD, Nestin, GFAP, and MAP2) were up-regulated but gene expression associated to osteogenesis (ALPL, RUNX2, and SPARC) were either down modulated or remained at the nominal level. This is the first evidence that cellular morphology control via micropatterning could be used to modulate stem cell fate without external biochemical stimuli. We further our studies to modulate the focal adhesion (FA) instead of the macro shape of cells. Micro-contact printed islands of different smaller dimensions were investigated. We successfully regulated the FAs into dense FAs and elongated FAs by micropatterning. Additionally, the combined effects of hard (40.4 kPa), and intermediate (10.6 kPa) PA gel and FAs patterning on hMSCs differentiation were studied. Results showed that FA and matrix compliance plays an important role in hMSCs differentiation, and there is a cross-talk between different physical stimulants and the significance of these stimuli can only be realized if they are combined at the optimum level.

Keywords: micro-contact printing, polymer substrate, cell-material interaction, stem cell differentiation

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Authors: Rasoul Eslami, Reza Gharakhanlou

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NT-4/5 and TrkB have been proposed to be involved in the coordinated adaptations of the neuromuscular system to elevated level of activity. Despite the persistence of this neurotrophin and its receptor expression in adult skeletal muscle, little attention has been paid to the functional significance of this complex in the mature neuromuscular system. Therefore, the purpose of this research was to study the effect of one session of resistance exercise on mRNA expression of NT4/5 and TrkB proteins in slow and fast muscles of Wistar Rats. Male Wistar rats (10 mo of age, preparation of Pasteur Institute) were housed under similar living conditions in cages (in groups of four) at room temperature under a controlled light/dark (12-h) cycle with ad libitum access to food and water. A number of sixteen rats were randomly divided to two groups (resistance exercise (T) and control (C); n=8 for each group). The resistance training protocol consisted of climbing a 1-meter–long ladder, with a weight attached to a tail sleeve. Twenty-four hours following the main training session, rats of T and C groups were anaesthetized and the right soleus and flexor hallucis longus (FHL) muscles were removed under sterile conditions via an incision on the dorsolateral aspect of the hind limb. For NT-4/5 and TrkB expression, quantitative real time RT-PCR was used. SPSS software and independent-samples t-test were used for data analysis. The level of significance was set at P < 0.05. Data indicate that resistance training significantly (P<0.05) decreased mRNA expression of NT4/5 in soleus muscle. However, no significant alteration was detected in FHL muscle (P>0.05). Our results also indicate that no significant alterations were detected for TrkB mRNA expression in soleus and FHL muscles (P>0.05). Decrease in mRNA expression of NT4/5 in soleus muscle may be as result of post-translation regulation following resistance training. Also, non-alteration in TrkB mRNA expression was indicated in probable roll of P75 receptor.

Keywords: neurotrophin-4/5 (NT-4/5), TrkB receptor, resistance training, slow and fast muscles

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Authors: J. Bernasovska, I. Boronova, J. Poracova, M. Mydlarova Blascakova, V. Szabadosova, P. Ruzbarsky, E. Petrejcikova, I. Bernasovsky

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The paper presents the results of the molecular genetics analysis in sports research, with special emphasis to use genetic information in diagnosing of motoric predispositions in Roma boys from East Slovakia. The ability and move are the basic characteristics of all living organisms. The phenotypes are influenced by a combination of genetic and environmental factors. Genetic tests differ in principle from the traditional motoric tests, because the DNA of an individual does not change during life. The aim of the presented study was to examine motion abilities and to determine the frequency of ACTN3 (R577X) gene in Roma children. Genotype data were obtained from 138 Roma and 155 Slovak boys from 7 to 15 years old. Children were investigated on physical performance level in association with their genotype. Biological material for genetic analyses comprised samples of buccal swabs. Genotypes were determined using Real Time High resolution melting PCR method (Rotor-Gene 6000 Corbett and Light Cycler 480 Roche). The software allows creating reports of any analysis, where information of the specific analysis, normalized and differential graphs and many information of the samples are shown. Roma children of analyzed group legged to non-Romany children at the same age in all the compared tests. The % distribution of R and X alleles in Roma children was different from controls. The frequency of XX genotype was 9.26%, RX 46.33% and RR was 44.41%. The frequency of XX genotype was 9.26% which is comparable to a frequency of an Indian population. Data were analyzed with the ANOVA test.

Keywords: ACTN3 gene, R577X polymorphism, Roma children, sport performance, Slovakia

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Authors: Berengere Vire, Nicolas Salvetat, Yoann Lannay, Guillaume Marcellin, Siem Van Der Laan, Franck Molina, Dinah Weissmann

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Predicting suicidal behaviors is one of the most complex challenges of daily psychiatric practices. Today, suicide risk prediction using biological tools is not validated and is only based on subjective clinical reports of the at-risk individual. Therefore, there is a great need to identify biomarkers that would allow early identification of individuals at risk of suicide. Alterations of adenosine-to-inosine (A-to-I) RNA editing of neurotransmitter receptors and other proteins have been shown to be involved in etiology of different psychiatric disorders and linked to suicidal behavior. RNA editing is a co- or post-transcriptional process leading to a site-specific alteration in RNA sequences. It plays an important role in the epi transcriptomic regulation of RNA metabolism. On postmortem human brain tissue (prefrontal cortex) of depressed suicide victims, Alcediag found specific alterations of RNA editing activity on the mRNA coding for the serotonin 2C receptor (5-HT2cR). Additionally, an increase in expression levels of ADARs, the RNA editing enzymes, and modifications of RNA editing profiles of prime targets, such as phosphodiesterase 8A (PDE8A) mRNA, have also been observed. Interestingly, the PDE8A gene is located on chromosome 15q25.3, a genomic region that has recurrently been associated with the early-onset major depressive disorder (MDD). In the current study, we examined whether modifications in RNA editing profile of prime targets allow identifying disease-relevant blood biomarkers and evaluating suicide risk in patients. To address this question, we performed a clinical study to identify an RNA editing signature in blood of depressed patients with and without the history of suicide attempts. Patient’s samples were drawn in PAXgene tubes and analyzed on Alcediag’s proprietary RNA editing platform using next generation sequencing technology. In addition, gene expression analysis by quantitative PCR was performed. We generated a multivariate algorithm comprising various selected biomarkers to detect patients with a high risk to attempt suicide. We evaluated the diagnostic performance using the relative proportion of PDE8A mRNA editing at different sites and/or isoforms as well as the expression of PDE8A and the ADARs. The significance of these biomarkers for suicidality was evaluated using the area under the receiver-operating characteristic curve (AUC). The generated algorithm comprising the biomarkers was found to have strong diagnostic performances with high specificity and sensitivity. In conclusion, we developed tools to measure disease-specific biomarkers in blood samples of patients for identifying individuals at the greatest risk for future suicide attempts. This technology not only fosters patient management but is also suitable to predict the risk of drug-induced psychiatric side effects such as iatrogenic increase of suicidal ideas/behaviors.

Keywords: blood biomarker, next-generation-sequencing, RNA editing, suicide

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Authors: Helan Satish, M. Ramasubba Reddy

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The simulation of an endocardial cell for gene mutation in the cardiac sodium ion channel NaV1.5, encoded by SCN5A gene, is discussed. The characterization of Brugada Syndrome by loss of function effect on SCN5A mutation due to L812Q mutant present in the DII-S4 transmembrane region of the NaV1.5 channel protein and its effect in an endocardial cell is studied. Ten Tusscher model of human ventricular action potential is modified to incorporate the changes contributed by L812Q mutant in the endocardial cells. Results show that BrS-associated SCN5A mutation causes reduction in the inward sodium current by modifications in the channel gating dynamics such as delayed activation, enhanced inactivation, and slowed recovery from inactivation in the endocardial cell. A decrease in the inward sodium current was also observed, which affects depolarization phase (Phase 0) that leads to reduction in the spike amplitude of the cardiac action potential.

Keywords: SCN5A gene mutation, sodium channel, Brugada syndrome, cardiac arrhythmia, action potential

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Authors: Agustyas Tjiptaningrum, Intanri Kurniati, Fadilah Fadilah, Linda Erlina, Tiwuk Susantiningsih

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Coronavirus disease-19 (COVID-19) is still one of the health problems. It can be a severe condition that is caused by a cytokine storm. In a cytokine storm, several proinflammatory cytokines are released massively. It destroys epithelial cells, and subsequently, it can cause death. The anti-inflammatory agent can be used to decrease the number of severe Covid-19 conditions. Melaleuca cajuputi is a plant that has antiviral, antibiotic, antioxidant, and anti-inflammatory activities. This study was carried out to analyze the prediction mechanism of the M. cajuputi extract from Lampung, Indonesia, as an anti-inflammatory agent for COVID-19. This study constructed a database of protein host target that was involved in the inflammation process of COVID-19 using data retrieval from GeneCards with the keyword “SARS-CoV2”, “inflammation,” “cytokine storm,” and “acute respiratory distress syndrome.” Subsequent protein-protein interaction was generated by using Cytoscape version 3.9.1. It can predict the significant target protein. Then the analysis of the Gene Ontology (GO) and KEGG pathways was conducted to generate the genes and components that play a role in COVID-19. The result of this study was 30 nodes representing significant proteins, namely NF-κβ, IL-6, IL-6R, IL-2RA, IL-2, IFN2, C3, TRAF6, IFNAR1, and DOX58. From the KEGG pathway, we obtained the result that NF-κβ has a role in the production of proinflammatory cytokines, which play a role in the COVID-19 cytokine storm. It is an important factor for macrophage transcription; therefore, it will induce inflammatory gene expression that encodes proinflammatory cytokines such as IL-6, TNF-α, and IL-1β. In conclusion, the blocking of NF-κβ is the prediction mechanism of the M. cajuputi extract as an anti-inflammation agent for COVID-19.

Keywords: antiinflammation, COVID-19, cytokine storm, NF-κβ, M. cajuputi

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Authors: Jagdish S. Patel, Piyush Chudasama

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Context: Over-expression of P-glycoprotein (P-gp) plays critical role in absorption of many drug candidates which results into lower bioavailability of the drug. P-glycoprotein also over expresses in many pathological conditions like diabetes, affecting the drug therapy. Modulation of P-gp expression using inhibitors can help in designing novel formulation enhancing the bioavailability of the drug in question. Objectives: The main focus of the study was to develop advanced glycation end products (AGEs) induced P-gp over expression in Caco-2 cells. Curcumin, piperine and epigallocatechin gallate were used to evaluate their P-gp inhibitory action using combinatorial approach. Materials and methods: Methylglyoxal (MG) induced P-gp over expression was checked in Caco-2 cells using real time PCR. P-gp inhibitory effects of the phytochemicals were measured after induction with MG alone and in combination of any two compounds. Cytotoxicity of each of the phytochemical was evaluated using MTT assay. Results: Induction with MG (100mM) significantly induced the over expression of P-glycoprotein in Caco-2 cells after 24 hr. Curcumin, piperine and epigallocatechin gallate alone significantly reduced the level of P-gp within 6 hr of treatment period monitored by real time PCR. The combination of any two phytochemical also down regulated the expression of P-gp in cells. Combinations of Curcumin and epigallocatechin gallate have shown significant down regulation when compared with other two combinations. Conclusions: Combinatorial approach for down regulating the expression of P-gp, in pathological conditions like diabetes, has demonstrated promising approach for therapeutic purpose.

Keywords: p-glycoprotein, curcumin, piperine, epigallocatechin gallate, p-gp inhibition

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Authors: Sajad Haghshenas

Abstract:

Emerging evidence from clinical, genetic, and animal model studies suggests that the N-methyl-D-aspartate (NMDA) glutamate receptors (NMDAR) may contribute to the pathophysiology and aetiology of neurological and psychiatric disorders and the patients with impaired NMDR receptors experience psychological symptoms. Therefore, we hypothesised that NMDAR receptors play a key role in the development of attention deficit hyperactivity disorder (ADHD). In this comparative analytical study, we utilized western blotting method to assay the expression levels of NMDA subunits NR1 and NR2 in the blood plasma of 50 male individuals diagnosed with ADHD in comparison to 20 healthy controls. The findings from the western blotting analysis provide support for the hypothesis that individuals with ADHD exhibit significantly lower levels of NR1/2 receptors compared to those without the disorder. Further research is needed to explore the potential causal relationship between reduced NR1/NR2 receptor levels and the development of ADHD.

Keywords: expression, glutamate receptors, NR1, NR2, ADHD

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Authors: Karolis Leonavičius, Dalius Kučiauskas, Dangiras Lukošius, Arnoldas Jasiūnas, Kostas Zdanys, Rokas Stanislovas, Emilis Gegevičius, Žana Kapustina, Juozas Nainys

Abstract:

Screening throughput is a common bottleneck in many research areas, including functional genomics, drug discovery, and directed evolution. High-throughput screening techniques can be classified into two main categories: (i) affinity-based screening and (ii) functional screening. The first one relies on binding assays that provide information about the affinity of a test molecule for a target binding site. Binding assays are relatively easy to establish; however, they reveal no functional activity. In contrast, functional assays show an effect triggered by the interaction of a ligand at a target binding site. Functional assays might be based on a broad range of readouts, such as cell proliferation, reporter gene expression, downstream signaling, and other effects that are a consequence of ligand binding. Screening of large cell or gene libraries based on direct activity rather than binding affinity is now a preferred strategy in many areas of research as functional assays more closely resemble the context where entities of interest are anticipated to act. Droplet sorting is the basis of high-throughput functional biological screening, yet its applicability is limited due to the technical complexity of integrating high-performance droplet analysis and manipulation systems. As a solution, the Droplet Genomics Styx platform enables custom droplet sorting workflows, which are necessary for the development of early-stage or complex biological therapeutics or industrially important biocatalysts. The poster will focus on the technical design considerations of Styx in the context of its application spectra.

Keywords: functional screening, droplet microfluidics, droplet sorting, dielectrophoresis

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Authors: Min-Shiuan Liou, Yi Shan Yang, Yang-Zhan Huang, Chia-Wen Hsieh

Abstract:

A high speed growing and butanol-tolerant Clostridium acetobutylicum HOL1 mutant was screened throughout continuous adaption culture with C. acetobutylicum ATCC 824. The HOL1 strain can grow well in 10 g/L butanol contained CGM medium and can produce about 12.8 g /L butanol during 24 hrs. The C. acetobutylicum HOL1 strain was able to produce 166 mM butanol with 21 mM acetone at pH 4.8, resulting in a butanol selectivity (a molar ratio of butanol to total solvents) of 0.79, which is much higher than that (0.6) of the wild-type strain C. acetobutylicum ATCC 824. The acetate and butyrate accumulation were not observed during fermentation of the HOL1 strain. A hyper-butanol producing C. acetobutylicum HOL1 (pBPHS-3), which was created to overexpress the Bacillus psychrosaccharolyticus originated specific heat-shock protein gene, hspX, from a clostridial phosphotransbutyrylase promoter, was studied for its potential to produce a high titer of butanol. Overexpression of hspX resulted in increased final butanol yield 47% and 30% higher than those of the the ATCC824 and the HOL1 strains, respectively. The remarkable high-speed growth and butanol tolerance of strain HOL1 (pBPHS-3) demonstrates that overexpression of heterogeneous stress protein-encoding gene, hspX, could help C. acetobutylicum to effectively produce a high concentration of butanol.

Keywords: Clostridium acetobutylicum, butanol, heat-shock protein, resistance

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Authors: Neelma Ashraf

Abstract:

Actinomycetes, particularly genus Streptomyces is of great importance due to their role in the discovery of new natural products, particularly antimicrobial secondary metabolites in the medicinal science and biotechnology industry. Different Streptomyces strains were isolated from Helianthus annuus plants and tested for antibacterial and antifungal activities. The most promising five strains were chosen for further investigation, and growth conditions for antibiotic synthesis were optimised. The supernatants were extracted in different solvents, and the extracted products were analyzed using liquid chromatography-mass spectrometry (LC-MS) and biological testing. From one of the potent strains Streptomyces globusus sp. BR123, a compound lavendamycin was identified using these analytical techniques. In addition, this potent strain also produces a strong antifungal polyene compound with a quasimolecular ion of 2072. Streptomyces sp. BR123 was genome sequenced because of its promising antimicrobial potential in order to identify the gene cluster responsible for analyzed compound “lavendamycin”. The genome analysis yielded candidate genes responsible for the production of this potent compound. The genome sequence of 8.15 Mb of Streptomyces sp. isolate BR123 with a GC content of 72.63% and 8103 protein coding genes was attained. Many antimicrobial, antiparasitic, and anticancerous compounds were detected through multiple biosynthetic gene clusters predicted by in-Silico analysis. Though, the novelty of metabolites was determined through the insignificant resemblance with known biosynthetic gene clusters. The current study gives insight into the bioactive potential of Streptomyces sp. isolate BR123 with respect to the synthesis of bioactive secondary metabolites through genomic and spectrometric analysis. Moreover, the comparative genome study revealed the connection of isolate BR123 with other Streptomyces strains, which could expand the knowledge of this genus and the mechanism involved in the discovery of new antimicrobial metabolites.

Keywords: streptomyces, secondary metabolites, genome, biosynthetic gene clusters, high performance liquid chromatography, mass spectrometry

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Authors: Eric Bang

Abstract:

Interactions between receptors and ligands are crucial for many essential biological processes, including neurotransmission and metabolism. Ligand-receptor analyses that examine cell behavior and interactions often utilize cell type-specific RNA expressions from single-cell RNA sequencing (scRNA-seq) data. Using CellPhoneDB, a public repository consisting of ligands, receptors, and ligand-receptor interactions, the cell-cell interactions were explored in a specific scRNA-seq dataset from kidney tissue and portrayed the results with dot plots and heat maps. Depending on the type of cell, each ligand-receptor pair was aligned with the interacting cell type and calculated the positori probabilities of these associations, with corresponding P values reflecting average expression values between the triads and their significance. Using single-cell data (sample kidney cell references), genes in the dataset were cross-referenced with ones in the existing CellPhoneDB dataset. For example, a gene such as Pleiotrophin (PTN) present in the single-cell data also needed to be present in the CellPhoneDB dataset. Using the single-cell transcriptomics data via slide-seq and reference data, the CellPhoneDB program defines cell types and plots them in different formats, with the two main ones being dot plots and heat map plots. The dot plot displays derived measures of the cell to cell interaction scores and p values. For the dot plot, each row shows a ligand-receptor pair, and each column shows the two interacting cell types. CellPhoneDB defines interactions and interaction levels from the gene expression level, so since the p-value is on a -log10 scale, the larger dots represent more significant interactions. By performing an interaction analysis, a significant interaction was discovered for myeloid and T-cell ligand-receptor pairs, including those between Secreted Phosphoprotein 1 (SPP1) and Fibronectin 1 (FN1), which is consistent with previous findings. It was proposed that an effective protocol would involve a filtration step where cell types would be filtered out, depending on which ligand-receptor pair is activated in that part of the tissue, as well as the incorporation of the CellPhoneDB data in a streamlined workflow pipeline. The filtration step would be in the form of a Python script that expedites the manual process necessary for dataset filtration. Being in Python allows it to be integrated with the CellPhoneDB dataset for future workflow analysis. The manual process involves filtering cell types based on what ligand/receptor pair is activated in kidney cells. One limitation of this would be the fact that some pairings are activated in multiple cells at a time, so the manual manipulation of the data is reflected prior to analysis. Using the filtration script, accurate sorting is incorporated into the CellPhoneDB database rather than waiting until the output is produced and then subsequently applying spatial data. It was envisioned that this would reveal wherein the cell various ligands and receptors are interacting with different cell types, allowing for easier identification of which cells are being impacted and why, for the purpose of disease treatment. The hope is this new computational method utilizing spatially explicit ligand-receptor association data can be used to uncover previously unknown specific interactions within kidney tissue.

Keywords: bioinformatics, Ligands, kidney tissue, receptors, spatial transcriptome

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Authors: Manwendra Kumar Tripathi, Subhas Ganguly

Abstract:

A few glass forming ability expressions in terms of characteristic temperatures have been proposed in the literature. Attempts have been made to correlate the expression with the critical diameter of the bulk metallic glass composition. However, with the advent of new alloys, many exceptions have been noted and reported. In the present approach, a genetic programming based code which generates an expression in terms of input variables, i.e., three characteristic temperatures viz. glass transition temperature (Tg), onset crystallization temperature (Tx) and offset temperature of melting (Tl) with maximum correlation with a critical diameter (Dmax). The expression evolved shows improved correlation with the critical diameter. In addition, the expression can be explained on the basis of time-temperature transformation curve.

Keywords: glass forming ability, genetic programming, bulk metallic glass, critical diameter

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Authors: Hongping Lu, Kelbinur Tursun, Yaru Li, Yu Zhang, Shunai Liu, Ming Han

Abstract:

Objective: To investigate whether NS5ATP9 is involved in IL-6 mediated autophagy and the relationship between IL-6 and NS5ATP9 in liver cancer cells. Methods: 1. Detect the mRNA and protein levels of Beclin 1 after HepG2 cells were treated with or without recombinant human IL-6 protein. 2. Measure and compare of the changes of autophagy-related genes with their respective control, after IL-6 was silenced or neutralized with monoclonal antibody against human IL-6. 3. HepG2 cells were incubated with 50 ng/ml of IL-6 in the presence or absence of PDTC. The expression of NS5ATP9 was analyzed by Western blot after 48 h. 4. After NS5ATP9-silenced HepG2 cells had been treated with 50 ng/ml recombinant IL-6 protein, we detected the Beclin 1 and LC3B (LC3Ⅱ/Ⅰ) expression. 5. HepG2 cells were transfected with pNS5ATP9, si-NS5ATP9, and their respective control. Total RNA was isolated from cells and analyzed for IL-6. 6. Silence or neutralization of IL-6 in HepG2 cells which has been transfected with NS5ATP9. Beclin 1 and LC3 protein levels were analyzed by Western blot. Result: 1. After HepG2 were treated with recombinant human IL-6 protein, the expression of endogenous Beclin 1 was up-regulated at mRNA and protein level, and the conversion of endogenous LC3-I to LC3-II was also increased. These results indicated that IL-6 could induce autophagy. 2. When HepG2 cells were treated with IL-6 siRNA or monoclonal antibody against human IL-6, the expression of autophagy-related genes were decreased. 3. Exogenous human IL-6 recombinant protein up-regulated NS5ATP9 via NF-κB activation. 4. The expression of Beclin 1 and LC3B was down-regulated after IL-6 treated NS5ATP9-silenced HepG2 cells. 5. NS5ATP9 could reverse regulates IL-6 expression in HepG2 cells. 6. Silence or neutralization of IL-6 attenuates NS5ATP9-induced autophagy slightly. Conclusion: Our results implied that in HCC patients, maybe the higher level of IL-6 in the serum promoted the expression of NS5ATP9 and induced autophagy in cancer cells. And the over-expression of NS5ATP9 which induced by IL-6, in turn, increased IL-6 expression, further, promotes the IL-6/NS5ATP9-mediated autophagy and affects the progression of tumor. Therefore, NS5ATP9 silence might be a potential target for HCC therapy.

Keywords: autophagy, Hepatocellular carcinoma, IL-6, microenvironment, NS5ATP9

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Authors: Sandra Pietri, Helene Dubout, Sabrina Ena, Candice Hoste, Enrico Bastianelli

Abstract:

Bone Therapeutics is a bone cell therapy company addressing high unmet medical needs in the field of bone fracture repair, more specifically in non-union and delayed-union fractures where the bone repair process is impaired. The company has developed a unique allogeneic osteoblastic cell product (ALLOB®) derived from bone marrow which is currently tested in humans in the indication of delayed-union fractures. The purpose of our study was to directly compare ALLOB® vs. non-differentiated mesenchymal stem cells (MSC) for their in vitro osteogenic characteristics and their in vivo osteogenic potential in order to determine which cellular type would be the most adapted for bone fracture repair. Methods: Healthy volunteers’ bone marrow aspirates (n=6) were expended (i) into BM-MSCs using a complete MSC culture medium or (ii) into ALLOB® cells according to its manufacturing process. Cells were characterized in vitro by morphology, immunophenotype, gene expression and differentiation potential. Additionally, their osteogenic potential was assessed in vivo in the subperiosteal calvaria bone formation model in nude mice. Results: The in vitro side-by-side comparison studies showed that although ALLOB® and BM-MSC shared some common general characteristics such as the 3 minimal MSC criteria, ALLOB® expressed significantly higher levels of chondro/osteoblastic genes such as BMP2 (fold change (FC) > 100), ALPL (FC > 12), CBFA1 (FC > 3) and differentiated significantly earlier than BM-MSC toward the osteogenic lineage. Moreover the bone formation model in nude mice demonstrated that used at the same cellular concentration, ALLOB® was able to induce significantly more (160% vs.107% for control animals) bone formation than BM-MSC (118% vs. 107 % for control animals) two weeks after administration. Conclusion: Our side-by-side comparison studies demonstrated that in vitro and in vivo, ALLOB® displays superior osteogenic capacity to BM-MScs and is therefore a better candidate for the treatment of bone fractures.

Keywords: gene expression, histomorphometry, mesenchymal stem cells, osteogenic differentiation potential, preclinical

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Authors: Anjan Hazra, Nirjhar Dasgupta, Chandan Sengupta, Sauren Das

Abstract:

Tea (Camellia sinensis) has received substantial attention from the scientific world time to time, not only for its commercial importance, but also for its demand to the health-conscious people across the world for its extensive use as potential sources of antioxidant supplement. These health-benefit traits primarily rely on some regulatory networks of different metabolic pathways. Development of microsatellite markers from the conserved genomic regions is being worthwhile for studying the genetic diversity of closely related species or self-pollinated species. Although several SSR markers have been reported, in tea the trait-specific Simple Sequence Repeats (SSRs) are yet to be identified, which can be used for marker assisted breeding technique. MicroRNAs are endogenous, noncoding, short RNAs directly involved in regulating gene expressions at the post-transcriptional level. It has been found that diversity in miRNA gene interferes the formation of its characteristic hair pin structure and the subsequent function. In the present study, the precursors of small regulatory RNAs (microRNAs) has been fished out from tea Expressed Sequence Tag (EST) database. Furthermore, the simple sequence repeat motifs within the putative miRNA precursor genes are also identified in order to experimentally validate their existence and function. It is already known that genic-SSR markers are very adept and breeder-friendly source for genetic diversity analysis. So, the potential outcome of this in-silico study would provide some novel clues in understanding the miRNA-triggered polymorphic genic expression controlling specific metabolic pathways, accountable for tea quality.

Keywords: micro RNA, simple sequence repeats, tea quality, trait specific marker

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Authors: Annet Namusoke, Annet Namayanja, Peter Wasswa, Shakirah Nampijja

Abstract:

Common bean cultivar G2333 which offers broad resistance for anthracnose has been widely used as a source of resistance in breeding for anthracnose resistance. The cultivar is pyramided with three genes namely CO-4, CO-5 and CO-7 and of these three genes, the CO-4 gene has been found to offer the broadest resistance. The main aim of this work was to identify and validate easily assayable PCR based co-dominant molecular markers for selection of the CO-4 gene in segregating populations derived from crosses of G2333 with RWR 1946 and RWR 2075, two commercial Andean cultivars highly susceptible to anthracnose. Marker sequences for the study were obtained by blasting the sequence of the COK-4 gene in the Phaseolus gene database. Primer sequence pairs that were not provided from the Phaseolus gene database were designed by the use of Primer3 software. PCR conditions were optimized and the PCR products were run on 6% HPAGE gel. Results of the polymorphism test indicated that out of 18 identified markers, only two markers namely BM588 and BM211 behaved co-dominantly. Phenotypic evaluation for reaction to anthracnose disease was done by inoculating 21days old seedlings of three parents, F1 and F2 populations with race 7 of Colletotrichum lindemuthianum in the humid chamber. DNA testing of the BM588 marker onto the F2 segregating population of the crosses RWR 1946 x G 2333 and RWR 2075 x G2333 further revealed that the marker BM588 co-segregated with disease resistance with co-dominance of two alleles of 200bp and 400bp, fitting the expected segregation ratio of 1:2:1. The BM588 marker was significantly associated with disease resistance and gave promising results for marker assisted selection of the CO-4 gene in the breeding lines. Activities to validate the BM211 marker are also underway.

Keywords: codominant, Colletotrichum lindemuthianum, MAS, Phaseolus vulgaris

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Authors: Patricia M. B. Saint-Vincent, Anna Furches, Stephanie Galanie, Erica Teixeira Prates, Piet Jones, Nancy Engle, David Kainer, Wellington Muchero, Daniel Jacobson, Timothy J. Tschaplinski

Abstract:

Uridine diphosphate-glycosyltransferases (UGTs) are enzymes that catalyze sugar transfer to a variety of plant metabolites. UGT substrates, which include plant secondary metabolites involved in lignification, demonstrate new activities and incorporation when glycosylated. Knowledge of UGT function, substrate specificity, and enzyme products is important for plant engineering efforts, especially related to increasing plant biomass through lignification. UGTs in Populus trichocarpa, a biofuel feedstock, and model woody plant, were selected from a pool of gene candidates using rapid prioritization strategies. A functional genomics workflow, consisting of a metabolite genome-wide association study (mGWAS), expression of synthetic codon-optimized genes, and high-throughput biochemical assays with mass spectrometry-based analysis, was developed for determining the substrates and products of previously-uncharacterized enzymes. A total of 40 UGTs from P. trichocarpa were profiled, and the biochemical assay results were compared to predicted mGWAS connections. Assay results confirmed seven of 11 leaf mGWAS associations and demonstrated varying levels of substrate specificity among candidate UGTs. P. trichocarpa UGT substrate processing confirms the role of these newly-characterized enzymes in lignan, flavonoid, and phytohormone metabolism, with potential implications for cell wall biosynthesis, nitrogen uptake, and biotic and abiotic stress responses.

Keywords: Populus, metabolite-gene associations, GWAS, bio feedstocks, glycosyltransferase

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