Search results for: differentially expressed gene

Authors: Karolina Rutkowska, Hubert Pausch, Jolanta Oprzadek, Krzysztof Flisikowski

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The placenta is one of the most important organs that plays a crucial role in the fetal growth and development. Placenta dysfunction is one of the primary cause of the intrauterine growth restriction (IUGR). Cattle have the cotyledonary placenta which consists of two anatomical parts: fetal and maternal. In the case of cattle during the first months of pregnancy, it is very easy to separate maternal caruncle from fetal cotyledon tissue, easier in fact than removing an ordinary glove from one's hand. Which in fact make easier to conduct tissue-specific molecular studies. Typically, animal models for the study of IUGR are created using surgical methods and malnutrition of the pregnant mother or in the case of mice by genetic modifications. However, proposed cattle model with MIMT1Del/WT deletion is unique because it was created without any surgical methods what significantly distinguish it from the other animal models. The primary objective of the study was to identify differential expression of selected miRNAs in the placenta from normal and intrauterine growth restricted fetuses. There was examined the expression of miRNA in the fetal and maternal part of the placenta from 24 fetuses (12 samples from the fetal part of the placenta and 12 samples from maternal part of the placenta). In the study, there was done miRNAs sequencing in the placenta of MIMT1Del/WT fetuses and MIMT1WT/WT fetuses. Then, there were selected miRNAs that are involved in fetal growth and development. Analysis of miRNAs expression was conducted on ABI7500 machine. miRNAs expression was analyzed by reverse-transcription polymerase chain reaction (RT-PCR). As the reference gene was used SNORD47. The results were expressed as 2ΔΔCt: ΔΔCt = (Ctij − CtSNORD47j) − (Cti1 − CtSNORD471). Where Ctij and CtSNORD47j are the Ct values for gene i and for SNORD47 in a sample (named j); Cti1 and CtSNORD471 are the Ct values in sample 1. Differences between groups were evaluated with analysis of variance by using One-Way ANOVA. Bonferroni’s tests were used for interpretation of the data. All normalised miRNA expression values are expressed on a value of natural logarithm. The data were expressed as least squares mean with standard errors. Significance was declared when P < 0.05. The study shows that miRNAs expression depends on the part of the placenta where they origin (fetal or maternal) and on the genotype of the animal. miRNAs offer a particularly new approach to study IUGR. Corresponding tissue samples were collected according to the standard veterinary protocols according to the European Union Normative for Care and Use of Experimental Animals. All animal experiments were approved by the Animal Ethics Committee of the State Provincial Office of Southern Finland (ESAVI-2010-08583/YM-23).

Keywords: placenta, intrauterine growth restriction, miRNA, cattle

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

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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: Farrah Putri Salmanida, Mei-Li Wu, Rika Wahyuningtyas, Wen-Bin Chung, Hso-Chi Chaung, Ko-Tung Chang

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Within the field of immunotherapy, oncolytic virotherapy (OVT) employs dual approaches that directly eliminate tumor cells while preserving healthy ones and indirectly reprogram the tumor microenvironment (TME) to elicit antitumor responses. Within the TME, tumor associated macrophages (TAMs) manifest characteristics akin to those of anti-inflammatory M2 macrophages, thus earning the designation of M2-like TAMs. In prior research, two antigens denoted as A1 (g6Ld10T) and A3 (ORF6L5), derived from a complete sequence of ORF5 with partial sequence of ORF6 in Porcine Reproductive and Respiratory Syndrome Virus Genotype 2 (PRRSV-2), demonstrated the capacity to repolarize M2-type porcine alveolar macrophages (PAMs) into M1 phenotypes. In this study, we sought for utilizing OVT strategies by introducing A1 or A3 on TAMs to endow them with the anti-tumor traits of M1 macrophages while retaining their capacity to target cancer cells. Upon exposing human THP-1-derived M2 macrophages to a cross-species test with 2 µg/ml of either A1 or A3 for 24 hours, real time PCR revealed that A3, but not A1, treated cells exhibited upregulated gene expressions of M1 markers (CCR7, IL-1ß, CCL2, Cox2, CD80). These cells reacted to virus-derived antigen, as evidenced by increased expression of pattern-recognition receptors TLR3, TLR7, and TLR9, subsequently providing feedback in the form of type I interferon responses like IFNAR1, IFN-ß, IRF3, IRF7, OAS1, Mx1, and ISG15. Through an MTT assay, only after 15 µg/ml of A3 treatment could the cell viability decrease, with a predicted IC50 of 16.96 µg/ml. Interestingly, A3 caused dose-dependent toxicity to a rat C6 glial cancer cell line even at doses as low as 2.5 µg/ml and reached its IC50 at 9.419 µg/ml. Using Annexin V/7AAD staining and PCR test, we deduced that a significant proportion of C6 cells were undergoing the early apoptosis phase predominantly through the intrinsic apoptosis cascade involving Bcl-2 family proteins. Following this stage, we conducted a test on A3’s repolarization ability, which revealed a significant rise in M1 gene expression markers, such as TNF, CD80, and IL-1ß, in M2-like TAMs generated in vitro from murine RAW264.7 macrophages grown with conditioned medium of 4T1 breast cancer cells. This was corroborated by the results of transcriptome analysis, which revealed that the primary subset among the top 10 to top 30 significantly upregulated differentially expressed genes (DEGs) dominantly consisted of M1 macrophages profiles, including Ccl3, Ccl4, Csf3, TNF, Bcl6b, Stc1, and Dusp2. Our findings unveiled the remarkable potential of the PRRSV-derived antigen A3 to repolarize macrophages while also being capable of selectively inducing apoptosis in cancerous cells. While further in vivo study is needed for A3, it holds promise as an adjuvant by its dual effects in cancer therapy modalities.

Keywords: cancer cell apoptosis, interferon responses, macrophage repolarization, recombinant protein

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

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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: Yan Zeng, Li Tao, Liujun Han, Tianye Zhang, Yongan Yu, Kaijun Ma, Long Chen

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Mechanical asphyxia is one of the main cause of death; however, death by mechanical asphyxia may be difficult to prove in court, particularly in cases in which corpses exhibit no obvious signs of asphyxia. To identify a credible biomarker of asphyxia, we first examined the expression levels of all the mRNAs in human cardiac tissue specimens subjected to mechanical asphyxia and compared these expression levels with those of the corresponding mRNAs in specimens subjected to craniocerebral injury. A total of 119 differentially expressed mRNAs were selected and the expression levels of these mRNAs were examined in 44 human cardiac tissue specimens subjected to mechanical asphyxia, craniocerebral injury, hemorrhagic shock and other causes of death. We found that DUSP1 and KCNJ2 were up-regulated in tissue specimens of mechanical asphyxia compared with control tissues, with no significant correlation between age, environmental temperature and PMI, indicating that DUSP1 and KCNJ2 may associate with mechanical asphyxia-induced death and can thus serve as useful biomarkers of death by mechanical asphyxia.

Keywords: mechanical asphyxia, biomarkers, DUSP1, KCNJ2, cardiac tissue

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Authors: Narayan Moger, Divya B., Preethi Jambagi, Krishnaveni C. K., Apsana M. R., B. R. Patil, Basvaraj Bagewadi

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Salinity is one of the major threats to world food security. Considering the requirement for salt tolerant crop plants in the present study was undertaken to clone and transferred the salt tolerant ectA gene from marine ecosystem into agriculture crop system to impart salinity tolerance. Ectoine is the compatible solute which accumulates in the cell membrane, is known to be involved in salt tolerance activity in most of the Halophiles. The present situation is insisting to development of salt tolerant transgenic lines to combat abiotic stress. In this background, the investigation was conducted to develop transgenic tomato lines by cloning and transferring of ectA gene is an ectoine derivative capable of enzymatic action for the production of acetyl-diaminobutyric acid. The gene ectA is involved in maintaining the osmotic balance of plants. The PCR amplified ectA gene (579bp) was cloned into T/A cloning vector (pTZ57R/T). The construct pDBJ26 containing ectA gene was sequenced by using gene specific forward and reverse primers. Sequence was analyzed using BLAST algorithm to check similarity of ectA gene with other isolates. Highest homology of 99.66 per cent was found with ectA gene sequences of isolates Halomonas elongata with the available sequence information in NCBI database. The ectA gene was further sub cloned into pRI101-AN plant expression vector and transferred into E. coli DH5α for its maintenance. Further pDNM27 was mobilized into A. tumefaciens LBA4404 through tri-parental mating system. The recombinant Agrobacterium containing pDNM27 was transferred into tomato plants through In planta plant transformation method. Out of 300 seedlings, co-cultivated only twenty-seven plants were able to well establish under the greenhouse condition. Among twenty-seven transformants only twelve plants showed amplification with gene specific primers. Further work must be extended to evaluate the transformants at T1 and T2 generations for ectoine accumulation, salinity tolerance, plant growth and development and yield.

Keywords: salinity, computable solutes, ectA, transgenic, in planta transformation

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Authors: Scott A. Ochsner, Christopher M. Watkins, Apollo McOwiti, David L. Steffen Lauren B. Becnel, Neil J. McKenna

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Understanding signaling by nuclear receptors (NRs) requires an appreciation of their cognate ligand- and tissue-specific transcriptomes. While target gene regulation data are abundant in this field, they reside in hundreds of discrete publications in formats refractory to routine query and analysis and, accordingly, their full value to the NR signaling community has not been realized. One of the mandates of the Nuclear Receptor Signaling Atlas (NURSA) is to facilitate access of the community to existing public datasets. Pursuant to this mandate we are developing a freely-accessible community web resource, Transcriptomine, to bring together the sum total of available expression array and RNA-Seq data points generated by the field in a single location. Transcriptomine currently contains over 25,000,000 gene fold change datapoints from over 1200 contrasts relevant to over 100 NRs, ligands and coregulators in over 200 tissues and cell lines. Transcriptomine is designed to accommodate a spectrum of end users ranging from the bench researcher to those with advanced bioinformatic training. Visualization tools allow users to build custom charts to compare and contrast patterns of gene regulation across different tissues and in response to different ligands. Our resource affords an entirely new paradigm for leveraging gene expression data in the NR signaling field, empowering users to query gene fold changes across diverse regulatory molecules, tissues and cell lines, target genes, biological functions and disease associations, and that would otherwise be prohibitive in terms of time and effort. Transcriptomine will be regularly updated with gene lists from future genome-wide expression array and expression-sequencing datasets in the NR signaling field.

Keywords: target gene database, informatics, gene expression, transcriptomics

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Authors: Feng Zhang

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Kashin-Beck disease (KBD) is a chronic osteochondropathy. The mechanism of hand growth and development failure of KBD remains elusive now. In this study, we conducted a two-stage genome-wide association study (GWAS) of palmar length-width ratio (LWR) of KBD, totally involving 493 Chinese Han KBD patients. Affymetrix Genome Wide Human SNP Array 6.0 was applied for SNP genotyping. Association analysis was conducted by PLINK software. Imputation analysis was performed by IMPUTE against the reference panel of the 1000 genome project. In the GWAS, the most significant association was observed between palmar LWR and rs2071358 of COL2A1 gene (P value = 4.68×10-8). Imputation analysis identified 3 SNPs surrounding rs2071358 with significant or suggestive association signals. Replication study observed additional significant association signals at both rs2071358 (P value = 0.017) and rs4760608 (P value = 0.002) of COL2A1 gene after Bonferroni correction. Our results suggest that COL2A1 gene was a novel susceptibility gene involved in the growth and development failure of hand of KBD.

Keywords: Kashin-Beck disease, genome-wide association study, COL2A1, hand

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Authors: Suliman Khan, Rabeea Siddique, Mengzhou Xue

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Background: Patients with neurological disorders often display altered circadian rhythms. The disrupted circadian rhythms through chronic jetlag or shiftwork are thought to increase the risk and severity of human disease, including cancer, psychiatric, and related brain diseases. In this study, we investigated the impact of shiftwork or chronic jetlag (CJL) like conditions on mice’s brains. Transcriptome profiling based on RNA sequencing revealed that genes associated with serious neurological disorders were differentially expressed in the nucleus accumbens (NAc) and prefrontal cortex (PFC). According to the qPCR analysis, several key regulatory genes associated with neurological disorders were significantly altered in the NAc, PFC, hypothalamus, hippocampus, and striatum. Serotonin levels and the expression levels of serotonin transporters and receptors were significantly altered in mice treated with CJL. Overall, these results indicate that CJL may increase the risk of neurological disorders by disrupting the key regulatory genes, biological functions, serotonin, and corticosterone. These molecular linkages can further be studied to investigate the mechanism underlying CJL or shiftwork-mediated neurological disorders in order to develop treatment strategies.

Keywords: chronic jetlag, molecular profiles, brain disorders, circadian rhythms

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Authors: Fei Xu, Guofan Zhang, Xiao Liu

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Many major marine invertebrate phyla are characterized by indirect development. These animals transit from planktonic larvae to benthic adults via settlement and metamorphosis, which has many advantages for organisms to adapt marine environment. Studying the biological process of metamorphosis is thus a key to understand the origin and evolution of indirect development. Although the mechanism of metamorphosis has been largely studied on their relationships with the marine environment, microorganisms, as well as the neurohormones, little is known on the gene regulation network (GRN) during metamorphosis. We treated competent oyster pediveligers with epinephrine, which was known to be able to effectively induce oyster metamorphosis, and analyzed the dynamics of gene and proteins with transcriptomics and proteomics methods. The result indicated significant upregulation of protein synthesis system, as well as some transcription factors including Homeobox, basic helix-loop-helix, and nuclear receptors. The result suggested the GRN complexity of the transition stage during oyster metamorphosis.

Keywords: indirect development, gene regulation network, protein synthesis, transcription factors

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Authors: Anida Causevic-Ramosevac, Sabina Semiz

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The aim of this study was to investigate the association of four single nucleotide polymorphisms (SNPs) - rs673548, rs693 in ApoB gene, rs1800775 in CETP gene and rs4846914 in GALNT2 gene with parameters of type 2 diabetes (T2D) and diabetic dyslipidemia in the population of Bosnia and Herzegovina (BH). Materials and methods: Our study involved 352 patients with T2D and 156 healthy subjects. Biochemical and anthropometric parameters were measured in all participants. DNA was extracted from the peripheral blood for the purpose of genetic testing. Polymorphisms in ApoB (rs673548, rs693), CETP (rs1800775) and GALNT2 (rs4846914) genes were analyzed by using Sequenom IPLEX platform. Results: Our results demonstrated significant associations for rs180075 polymorphism in CETP gene with levels of fasting insulin (p = 0.020; p = 0.027; p = 0.044), triglycerides (p = 0.046) and ALT (p = 0.031) activity in control group. In group of diabetic patients, results showed a significant association of rs673548 in ApoB gene with levels of fasting insulin (p = 0.008), HOMA-IR (p = 0.013), VLDL-C (p = 0.037) and CRP (p = 0.029) and rs693 in ApoB gene with BMI (p = 0.025), systolic blood pressure (p = 0.027), fasting insulin (p = 0.037) and HOMA-IR (p = 0.023) levels. Significant associations were also observed for rs1800775 in CETP gene with triglyceride (p = 0.023) levels and rs4846914 in GALNT2 gene with HbA1C (p = 0.013) and triglyceride (p = 0.043) levels. Conclusion: In conclusion, this is the first study that examined the impact of variations of candidate genes on a wide range of metabolic parameters in BH population. Our results suggest an association of variations of ApoB, CETP and GALNT2 genes with specific markers of T2D and dyslipidemia. Further studies would be needed in order to confirm these genetic effects in other ethnic groups as well.

Keywords: ApoB, CETP, dyslipidemia, GALNT2, type 2 diabetes

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Authors: Sharon Yunger, Liat Altman, Yuval Garini, Yaron Shav-Tal

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Understanding the dynamics of transcription in living cells has improved since the development of quantitative fluorescence-based imaging techniques. We established a method for following transcription from a single copy gene in living cells. A gene tagged with MS2 repeats, used for mRNA tagging, in its 3' UTR was integrated into a single genomic locus. The actively transcribing gene was detected and analyzed by fluorescence in situ hybridization (FISH) and live-cell imaging. Several cell clones were created that differed in the promoter regulating the gene. Thus, comparative analysis could be obtained without the risk of different position effects at each integration site. Cells in S/G2 phases could be detected exhibiting two adjacent transcription sites on sister chromatids. A sharp reduction in the transcription levels was observed as cells progressed along the cell cycle. We hypothesized that a change in chromatin structure acts as a general mechanism during the cell cycle leading to down-regulation in the activity of some genes. We addressed this question by treating the cells with chromatin decondensing agents. Quantifying and imaging the treated cells suggests that chromatin structure plays a role both in regulating transcriptional levels along the cell cycle, as well as in limiting an active gene from reaching its maximum transcription potential at any given time. These results contribute to understanding the role of chromatin as a regulator of gene expression.

Keywords: cell cycle, living cells, nucleus, transcription

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Authors: Selisha A. Sooklal, Phelelani Mpangase, Shaun Aron, Karl Rumbold

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Organically bound fluorine (C-F bond) is extremely rare in nature. Despite this, the first fluorinated secondary metabolite, fluoroacetate, was isolated from the plant Dichapetalum cymosum (commonly known as Gifblaar). However, the enzyme responsible for fluorination (fluorinase) in Gifblaar was never isolated and very little progress has been achieved in understanding this process in higher plants. Fluorinated compounds have vast applications in the pharmaceutical, agrochemical and fine chemicals industries. Consequently, an enzyme capable of catalysing a C-F bond has great potential as a biocatalyst in the industry considering that the field of fluorination is virtually synthetic. As with any biocatalyst, a range of these enzymes are required. Therefore, it is imperative to expand the exploration for novel fluorinases. This study aimed to gain molecular insights into secondary metabolite biosynthesis in Gifblaar using a high-throughput sequencing-based approach. Mechanical wounding studies were performed using Gifblaar leaf tissue in order to induce expression of the fluorinase. The transcriptome of the wounded and unwounded plant was then sequenced on the Illumina HiSeq platform. A total of 26.4 million short sequence reads were assembled into 77 845 transcripts using Trinity. Overall, 68.6 % of transcripts were annotated with gene identities using public databases (SwissProt, TrEMBL, GO, COG, Pfam, EC) with an E-value threshold of 1E-05. Sequences exhibited the greatest homology to the model plant, Arabidopsis thaliana (27 %). A total of 244 annotated transcripts were found to be differentially expressed between the wounded and unwounded plant. In addition, secondary metabolic pathways present in Gifblaar were successfully reconstructed using Pathway tools. Due to lack of genetic information for plant fluorinases, a transcript failed to be annotated as a fluorinating enzyme. Thus, a local database containing the 5 existing bacterial fluorinases was created. Fifteen transcripts having homology to partial regions of existing fluorinases were found. In efforts to obtain the full coding sequence of the Gifblaar fluorinase, primers were designed targeting the regions of homology and genome walking will be performed to amplify the unknown regions. This is the first genetic data available for Gifblaar. It has provided novel insights into the mechanisms of metabolite biosynthesis and will allow for the discovery of the first eukaryotic fluorinase.

Keywords: biocatalyst, fluorinase, gifblaar, transcriptome

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Authors: Marija Ratkova Manovska, Mirko Prodanov, Dean Jankuloski, Katerina Blagoevska

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Staphylococci, which are widely found in the environment, animals, humans, and food products, include Staphylococcus aureus (S. aureus), the most significant pathogenic species in this genus. The virulence and toxicity of S. aureus are primarily attributed to the presence of specific genes responsible for producing toxins, biofilms, invasive components, and antibiotic resistance. Staphylococcal food poisoning, caused by the production of staphylococcal enterotoxins (SEs) by these strains in food, is a common occurrence. Globally, S. aureus food intoxications are typically ranked as the third or fourth most prevalent foodborne intoxications. For this study, a total of 333 milk samples and 1160 dairy product samples were analyzed between 2016 and 2020. The strains were isolated and confirmed using the ISO 6888-1:1999 "Horizontal method for enumeration of coagulase-positive staphylococci." Molecular analysis of the isolates, conducted using conventional PCR, involved detecting the 23s gene of S. aureus, the nuc gene, the mecA gene, and 11 genes responsible for producing enterotoxins (sea, seb, sec, sed, see, seg, seh, sei, ser, sej, and sep). The 23s gene was found in 93 (75.6%) out of 123 isolates of Staphylococcus spp. obtained from milk. Among the 76 isolates from dairy products, either S. aureus or the 23s gene was detected in 49 (64.5%) of them. The mecA gene was identified in three isolates from raw milk and five isolates from cheese samples. The nuc gene was present in 98.9% of S. aureus strains from milk and 97.9% from dairy products. Other Staphylococcus strains carried the nuc gene in 26.7% of milk strains and 14.8% of dairy product strains. Genes associated with SEs production were detected in 85 (69.1%) strains from milk and 38 (50%) strains from dairy products. In this study, 10 out of the 11 SEs genes were found, with no isolates carrying the see gene. The most prevalent genes detected were seg and sei, with some isolates containing up to five different SEs genes. These findings indicate the presence of enterotoxigenic staphylococci strains in the tested samples, emphasizing the importance of implementing proper sanitation and hygienic practices, utilizing safe raw materials, and ensuring adequate handling of finished products. Continued monitoring for the presence of SEs is necessary to ensure food safety and prevent intoxication.

Keywords: dairy products, milk, Staphylococci, enterotoxins, SE genes

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Authors: M. L. Mangena, N. Mokoena, K. Rashamuse, M. G. Tlou

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Tertiary alcohol ester (TAE) hydrolases are a group of esterases (EC 3.1.1.-) that catalyze the kinetic resolution of TAEs and as a result, they are sought-after for the production of optically pure tertiary alcohols (TAs) which are useful as building blocks for number biologically active compounds. What sets these enzymes apart is, the presence of a GGG(A)X-motif in the active site which appears to be the main reason behind their activity towards the sterically demanding TAEs. The genome of Pseudomonas syringae pv. maculicola (Psm) comprises a multitude of genes that encode esterases. We therefore, hypothesize that some of these genes encode TAE hydrolases. In this study, Psm was screened for TAE hydrolase activity using the linalyl acetate (LA) plate assay and a positive reaction was observed. As a result, the genome of Psm was screened for esterases with a GGG(A)X-motif using the motif search tool and two potential TAE hydrolase genes (PsmEST1 and 2, 1100 and 1000bp, respectively) were identified, PsmEST1 was amplified by PCR and the gene sequenced for confirmation. Analysis of the sequence data with the SingnalP 4.1 server revealed that the protein comprises a signal peptide (22 amino acid residues) on the N-terminus. Primers specific for the gene encoding the mature protein (without the signal peptide) were designed such that they contain NdeI and XhoI restriction sites for directional cloning of the PCR products into pET28a. The gene was expressed in E. coli JM109 (DE3) and the clones screened for TAE hydrolase activity using the LA plate assay. A positive clone was selected, overexpressed and the protein purified using nickel affinity chromatography. The activity of the esterase towards LA was confirmed using thin layer chromatography.

Keywords: hydrolases, tertiary alcohol esters, tertiary alcohols, screening, Pseudomonas syringae pv., maculicola genome, esterase activity, linalyl acetate

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

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CS6 is the prevalent CF in our region and deciphering its molecular regulators would play a pivotal role in reducing the burden of ETEC pathogenesis. In prokaryotes, most of the genes are under the control of one operon and the promoter present upstream of the gene regulates the transcription of that gene. Here the promoter of CS6 was characterized by computational method and further analyzed by β-galactosidase assay and sequencing. Promoter constructs and deletions were prepared as required to analyze promoter activity. The effect of different additives on the CS6 promoter was analysed by the β-galactosidase assay. Bioinformatics analysis done by Softberry/BPROM predicted fur, lrp, and crp boxes, -10 and -35 region upstream of the CS6 gene. The promoter construction in no promoter plasmid pTL61T showed that region -573 to +1 is actually the promoter region as predicted. Sequential deletion of the region upstream of CS6 revealed that promoter activity remains the same when -573bp to -350bp is deleted. But after the deletion of the upstream region -350 bp to -255bp, promoter expression decreases drastically to 26%. Further deletion also decreases promoter activity up to a little range. So the region -355bp to -255bp holds the promoter sequence for the CS6 gene. Additives like iron, NaCl, etc., modulate promoter activity in a dose-dependent manner. From the promoter analysis, it can be said that the minimum region lies between -254 and +1. Important region(s) lies between -350 bp to -255 bp upstream in the promoter, which might have important elements needed to control CS6 gene expression.

Keywords: microbiology, promoter, colonization factor, ETEC

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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: Maria Ostroukhova, Zhanneta Zalutskaya, Elena Ermilova

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

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

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Authors: D. Leibman, D. Wolf, A. Gal-On

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Viral diseases of tomato crops result in heavy yield losses and may even jeopardize the production of these crops. Classical tomato breeding for disease resistance against Tomato yellow leaf curl virus (TYLCV), leads to partial resistance associated with a number of recessive genes. To author’s best knowledge Pepino mosaic virus (PepMV) genetic resistance is not yet available. The generation of viral resistance by means of genetic engineering was reported and implemented for many crops, including tomato. Transgenic resistance against viruses is based, in most cases, on Post Transcriptional Gene Silencing (PTGS), an endogenous mechanism which destroys the virus genome. In this work, we developed immunity against PepMV and TYLCV in a tomato based on a PTGS mechanism. Tomato plants were transformed with a hairpin-construct-expressed transgene-derived double-strand-RNA (tr-dsRNA). In the case of PepMV, the binary construct harbored three consecutive fragments of the replicase gene from three different PepMV strains (Italian, Spanish and American), to provide resistance against a range of virus strains. In the case of TYLCV, the binary vector included three consecutive fragments of the IR, V2 and C2 viral genes constructed in a hairpin configuration. Selected transgenic lines (T0) showed a high accumulation of transgene siRNA of 21-24 bases, and T1 transgenic lines showed complete immunity to PepMV and TYLCV. Graft inoculation displayed immunity of the transgenic scion against PepMV and TYLCV. The study presents the engineering of resistance in tomato against two serious diseases, which will help in the production of high-quality tomato. However, unfortunately, these resistant plants have not been implemented due to public ignorance and opposition against breeding by genetic engineering.

Keywords: PepMV, PTGS, TYLCV, tr-dsRNA

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Authors: Rasha Ahmadi

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In December 2019, a coronavirus, currently identified as SARS-CoV-2, produced a series of acute atypical respiratory illnesses in Wuhan, Hubei Province, China. The sickness induced by this virus was named COVID-19. The virus is transmittable between humans and has caused pandemics worldwide. The number of death tolls continues to climb and a huge number of countries have been obliged to perform social isolation and lockdown. Lack of focused therapy continues to be a problem. Epidemiological research showed that senior patients were more susceptible to severe diseases, whereas children tend to have milder symptoms. In this study, we focus on other possible side effects of COVID-19 and more detailed treatment strategies. Using bioinformatics analysis, we first isolated the gene expression profile of patients with severe COVID-19 from the GEO database. Patients' blood samples were used in the GSE183071 dataset. We then categorized the genes with high and low expression. In the next step, we uploaded the genes separately to the Enrichr database and evaluated our data for signs and symptoms as well as related medication regimens. The results showed that 138 genes with high expression and 108 genes with low expression were observed differentially in the severe COVID-19 VS control group. Symptoms and diseases such as embolism and thrombosis of the abdominal aorta, ankylosing spondylitis, suicidal ideation or attempt, regional enteritis were observed in genes with high expression and in genes with low expression of acute and subacute forms of ischemic heart, CNS infection and poliomyelitis, synovitis and tenosynovitis. Following the detection of diseases and possible signs and symptoms, Carmustine, Bithionol, Leflunomide were evaluated more significantly for high-expression genes and Chlorambucil, Ifosfamide, Hydroxyurea, Bisphenol for low-expression genes. In general, examining the different and invisible aspects of COVID-19 and identifying possible treatments can help us significantly in the emergency and hospitalization of patients.

Keywords: phenotypes, drug regimens, gene expression profiles, bioinformatics analysis, severe COVID-19

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Authors: Zintle Kolo, Ndiko Ludidi

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The enzymatic activity of p-coumarate 3-hydroxylase (C3H) synthesize caffeic acid from p-coumaric acid. We recently showed that exogenously applied caffeic acid confers salinity tolerance in soybean (Glycine max) by inducing antioxidant enzymatic activity to promote enhanced scavenging or reactive oxygen species, thus limiting salinity-induced oxidative stress. Recent evidence also establishes that pre-treatment of plants with exogenously supplied caffeic acid improves plant tolerance to osmotic stress by improving plant antioxidant capacity and enhancing biosynthesis of compatible solutes. We aimed to identify a C3H in maize (Zea mays) and evaluate the effect of drought on the spatial and temporal expression of the gene encoding the candidate maize C3H (ZmC3H). Primary sequence analysis shows that ZmC3H shares 71% identity with an Arabidopsis thaliana C3H that is implicated in the control of Arabidopsis cell expansion, growth, and responses to stress. In silico ZmC3H promoter analysis reveals the presence of cis-acting elements that interact with transcription factors implicated in plant responses to drought. Spatial expression analysis by semi-quantitative RT-PCR shows that ZmC3H is expressed in both leaves and roots under normal conditions. However, drought represses the expression of ZmC3H in leaves whereas it up-regulates its expression in roots. These changes in ZmC3H expression correlate with the changes in the content of caffeic acid in maize in response to drought. We illustrate the implications of these changes in the expression of the gene in relation to maize responses to drought and discuss the potential of regulating caffeic acid biosynthesis towards genetic improvement of maize tolerance to drought stress. These findings have implications for food security because of the potential of the implications of the study for drought tolerance in maize.

Keywords: caffeic acid, drought-responsive expression, maize drought tolerance, p-coumarate 3-hydroxylase

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Authors: Irshad Ali Khan, Jian-Ping Lu, Xiao-Hong Liu, Fu-Cheng Lin

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This study reports the functional analysis of a gene MoNUC1 in M. oryzae, which is homologous to the Saccharomyces cerevisiae NUC1 encoding a mitochondrial nuclease protein. The MoNUC1 having a gene locus MGG_05324 is 1002-bp in length and encodes an identical protein of 333 amino acids. We disrupted the gene through gene disruption strategy and isolated two mutants confirmed by southern blotting. The deleted mutants were then used for phenotypic studies and their phenotypes were compared to those of the Guy-11 strain. The mutants were first grown on CM medium to find the effect of MoNUC1 gene disruption on colony growth and the mutants were found to show normal culture colony growth similar to that of the Guy-11 strain. Conidial germination and appressorial formation were also similar in both the mutants and Guy-11 strains showing that this gene plays no significant role in these phenotypes. For pathogenicity, the mutants and Guy-11 mycelium blocks were inoculated on blast susceptible barley seedlings and it was found that both the strains exhibited full pathogenicity showing coalesced and necrotic blast lesions suggesting that this gene is not involved in pathogenicity. Mating of the mutants with 2539 strain formed numerous perithecia showing that MoNUC1 is not essential for sexual reproduction in M. oryzae. However, the mutants were found to form reduced conidia (1.06±8.03B and 1.08±9.80B) than those of the Guy-11 strain (1.46±10.61A) and we conclude that this protein is not required for the blast fungus to cause pathogenicity but plays significant role in conidiation. Proteins of signal transduction pathways that could be disrupted/ intervened genetically or chemically could lead to antifungal products of important fungal cereal diseases and reduce rice yield losses. Tipping the balance toward understanding the whole of pathogenesis, rather than simply conidiation will take some time, but clearly presents the most exciting challenge of all.

Keywords: appressorium formation, conidiation, NUC1, Magnaporthe oryzae, pathogenicity

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Authors: Biswabandhu Bankura, Srikanta Guria, Madhusudan Das

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Purpose: Thyroid peroxidase (TPO) is the key enzyme in the biosynthesis of thyroid hormones. We aimed to identify the spectrum of mutations in the TPO gene leading to hypothyroidism in the population of West Bengal to establish the genetic etiology of the disease. Methods: 200 hypothyroid patients (case) and their corresponding sex and age matched 200 normal individuals (control) were screened depending on their clinical manifestations. Genomic DNA was isolated from peripheral blood samples and TPO gene (Exon 7 to Exon 14) was amplified by PCR. The PCR products were subjected to sequencing to identify mutations. Results: Single nucleotide changes such as Glu 641 Lys, Asp 668 Asn, Thr 725 Pro, Asp 620 Asn, Ser 398 Thr, and Ala 373 Ser were found. Changes in the TPO were assayed in vitro to compare mutant and wild-type activities. Five mutants were enzymatically inactive in the guaiacol and iodide assays. This is a strong indication that the mutations are present at crucial positions of the TPO gene, resulting in inactivated TPO. Key Findings: The results of this study may help to develop a genetic screening protocol for goiter and hypothyroidism in the population of West Bengal.

Keywords: thyroid peroxidase, hypothyroidism, mutation, in vitro assay, transfection

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Authors: Biswabandhu Bankura, Srikanta Guria, Madhusudan Das

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Purpose: Thyroid peroxidase (TPO) is the key enzyme in the biosynthesis of thyroid hormones. We aimed to identify the spectrum of mutations in the TPO gene leading to hypothyroidism in the population of West Bengal to establish the genetic etiology of the disease. Methods: 200 hypothyroid patients (case) and their corresponding sex and age matched 200 normal individuals (control) were screened depending on their clinical manifestations. Genomic DNA was isolated from peripheral blood samples and TPO gene (Exon 7 to Exon 14) was amplified by PCR. The PCR products were subjected to sequencing to identify mutations. Results: Single nucleotide changes such as Glu 641 Lys, Asp 668 Asn, Thr 725 Pro, Asp 620 Asn, Ser 398 Thr, and Ala 373 Ser were found. Changes in the TPO were assayed in vitro to compare mutant and wild-type activities. Five mutants were enzymatically inactive in the guaiacol and iodide assays. This is a strong indication that the mutations are present at crucial positions of the TPO gene, resulting in inactivated TPO. Key Findings: The results of this study may help to develop a genetic screening protocol for goiter and hypothyroidism in the population of West Bengal.

Keywords: thyroid peroxidase, hypothyroidism, mutation, in vitro assay, transfection

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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: Yakup Ulusu, Faruk Ozel, Numan Eczacioglu, Abdurrahman Ozen, Sabriye Acikgoz

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GFP discovered in the mid-1970s, has been used as a marker after replicated genetic study by scientists. In biotechnology, cell, molecular biology, the GFP gene is frequently used as a reporter of expression. In modified forms, it has been used to make biosensors. Many animals have been created that express GFP as an evidence that a gene can be expressed throughout a given organism. Proteins labeled with GFP identified locations are determined. And so, cell connections can be monitored, gene expression can be reported, protein-protein interactions can be observed and signals that create events can be detected. Additionally, monitoring GFP is noninvasive; it can be detected by under UV-light because of simply generating fluorescence. Moreover, GFP is a relatively small and inert molecule, that does not seem to treat any biological processes of interest. The synthesis of GFP has some steps like, to construct the plasmid system, transformation in E. coli, production and purification of protein. GFP carrying plasmid vector pBAD–GFPuv was digested using two different restriction endonuclease enzymes (NheI and Eco RI) and DNA fragment of GFP was gel purified before cloning. The GFP-encoding DNA fragment was ligated into pET28a plasmid using NheI and Eco RI restriction sites. The final plasmid was named pETGFP and DNA sequencing of this plasmid indicated that the hexa histidine-tagged GFP was correctly inserted. Histidine-tagged GFP was expressed in an Escherichia coli BL21 DE3 (pLysE) strain. The strain was transformed with pETGFP plasmid and grown on LuiraBertoni (LB) plates with kanamycin and chloramphenicol selection. E. coli cells were grown up to an optical density (OD 600) of 0.8 and induced by the addition of a final concentration of 1mM isopropyl-thiogalactopyranoside (IPTG) and then grown for additional 4 h. The amino-terminal hexa-histidine-tag facilitated purification of the GFP by using a His Bind affinity chromatography resin (Novagen). Purity of GFP protein was analyzed by a 12 % sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The concentration of protein was determined by UV absorption at 280 nm (Varian Cary 50 Scan UV/VIS spectrophotometer). Synthesis of GFP-Polymer composite nanofibers was produced by using GFP solution (10mg/mL) and polymer precursor Polyvinylpyrrolidone, (PVP, Mw=1300000) as starting materials and template, respectively. For the fabrication of nanofibers with the different fiber diameter; a sol–gel solution comprising of 0.40, 0.60 and 0.80 g PVP (depending upon the desired fiber diameter) and 100 mg GFP in 10 mL water: ethanol (3:2) mixtures were prepared and then the solution was covered on collecting plate via electro spinning at 10 kV with a feed-rate of 0.25 mL h-1 using Spellman electro spinning system. Results show that GFP-based nano-fiber can be used plenty of biomedical applications such as bio-imaging, bio-mechanic, bio-material and tissue engineering.

Keywords: biomaterial, GFP, nano-fibers, protein expression

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Authors: H. Chassaigne, S. Gioria, J. Lobo Vicente, D. Carpi, P. Barboro, G. Tomasi, A. Kinsner-Ovaskainen, F. Rossi

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Emerging approaches in the area of exposure to nanomaterials and assessment of human health effects combine the use of in vitro systems and analytical techniques to study the perturbation of the proteome and/or the metabolome. We investigated the modification in the cytoplasmic compartment of the Balb/3T3 cell line exposed to gold nanoparticles. On one hand, the proteomic approach is quite standardized even if it requires precautions when dealing with in vitro systems. On the other hand, metabolomic analysis is challenging due to the chemical diversity of cellular metabolites that complicate data elaboration and interpretation. Differentially expressed proteins were found to cover a range of functions including stress response, cell metabolism, cell growth and cytoskeleton organization. In addition, de-regulated metabolites were annotated using the HMDB database. The "omics" fields hold huge promises in the interaction of nanoparticles with biological systems. The combination of proteomics and metabolomics data is possible however challenging.

Keywords: data processing, gold nanoparticles, in vitro systems, metabolomics, proteomics

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Authors: Alyaa Abdlwahab

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Cutaneous leishmaniasis represents a serious health problem in Syria; this problem has become noticeably aggravated after the civil war in the country. Leishmania tropica parasite is the main cause of cutaneous leishmaniasis in Syria. In order to control the disease, we need an effective vaccine against leishmania parasite. DNA vaccination remains one of the favorable approaches that have been used to face cutaneous leishmaniasis. Ribosomal protein S4 is responsible for important roles in Leishmania parasite life. DNA vaccine based on S4 gene has been used against infections by many species of Leishmania parasite but leishmania tropica parasite, so this gene represents a good candidate for DNA vaccine construction. After proving the existence of ribosomal protein S4 gene in a Syrian strain of Leishmania tropica (LCED Syrian 01), sequencing it and cloning it into pCI plasmid, BALB/C mice were inoculated with pCI-S4 DNA vaccine. The immune response was determined by monitoring the lesion progression in inoculated BALB/C mice for six weeks after challenging mice with Leishmania tropica (LCED Syrian 01) parasites. IL-12, IFN-γ, and IL-4 were quantified in draining lymph nodes (DLNa) of the immunized BALB/C mice by using the RT-qPCR technique. The parasite burden was calculated in the final week for the footpad lesion and the DLNs of the mice. This study proved the existence and the expression of the ribosomal protein S4 gene in Leishmania tropica (LCED Syrian 01) promastigotes. The sequence of ribosomal protein cDNA S4 gene was determined and published in Genbank; the gene size was 822 bp. Expression was also demonstrated at the level of cDNA. Also, this study revealed that pCI-S4 DNA vaccine induces TH1\TH2 response in immunized mice; this response prevents partially developing a dermal lesion of Leishmania.

Keywords: ribosomal protein S4, DNA vaccine, Leishmania tropica, BALB\c

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Authors: Khiem M. Nguyen, Ming C. Yang

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Chloroplast pigments are extremely important during photosynthesis since they play essential roles in light absorption and energy transfer. Therefore, understanding the efficiency of chlorophyll (Chl) biosynthesis could facilitate enhancement in photo-assimilates accumulation, and ultimately, in crop yield. The Chl-deficient mutants have been used extensively to study the Chl biosynthetic pathways and the biogenesis of the photosynthetic apparatus. Rice (Oryza sativa L.) is one of the most leading food crops, serving as staple food for many parts of the world. To author’s best knowledge, Chl b–lacking rice has been found; however the molecular mechanism of Chl biosynthesis still remains unclear compared to wild-type rice. In this study, the ultrastructure analysis, photosynthetic properties, and transcriptome profile of wild-type rice (Norin No.8, N8) and its Chl b-lacking mutant (Chlorina 1, C1) were examined. The finding concluded that total Chl content and Chl b content in the C1 leaves were strongly reduced compared to N8 leaves, suggesting that reduction in the total Chl content contributes to leaf color variation at the physiological level. Plastid ultrastructure of C1 possessed abnormal thylakoid membranes with loss of starch granule, large number of vesicles, and numerous plastoglobuli. The C1 rice also exhibited thinner stacked grana, which was caused by a reduction in the number of thylakoid membranes per granum. Thus, the different Chl a/b ratio of C1 may reflect the abnormal plastid development and function. Transcriptional analysis identified 23 differentially expressed genes (DEGs) and 671 transcription factors (TFs) that were involved in Chl metabolism, chloroplast development, cell division, and photosynthesis. The transcriptome profile and DEGs revealed that the gene encoding PsbR (PSII core protein) was down-regulated, therefore suggesting that the lower in light-harvesting complex proteins are responsible for the lower photosynthetic capacity in C1. In addition, expression level of cell division protein (FtsZ) genes were significantly reduced in C1, causing chloroplast division defect. A total of 19 DEGs were identified based on KEGG pathway assignment involving Chl biosynthesis pathway. Among these DEGs, the GluTR gene was down-regulated, whereas the UROD, CPOX, and MgCH genes were up-regulated. Observation through qPCR suggested that later stages of Chl biosynthesis were enhanced in C1, whereas the early stages were inhibited. Plastid structure analysis together with transcriptomic analysis suggested that the Chl a/b ratio was amplified both by the reduction in Chl contents accumulation, owning to abnormal chloroplast development, and by the enhanced conversion of Chl b to Chl a. Moreover, the results indicated the same Chl-cycle pattern in the wild-type and C1 rice, indicating another Chl b degradation pathway. Furthermore, the results demonstrated that normal grana stacking, along with the absence of Chl b and greatly reduced levels of Chl a in C1, provide evidence to support the conclusion that other factors along with LHCII proteins are involved in grana stacking. The findings of this study provide insight into the molecular mechanisms that underlie different Chl a/b ratios in rice.

Keywords: Chl-deficient mutant, grana stacked, photosynthesis, RNA-Seq, transcriptomic analysis

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Authors: Haruka Ito, Toru Maruyama, Michihiro Ito, Chuya Shinzato, Hiroyuki Fujimura, Yoshikatsu Nakano, Shoichiro Suda, Sachiyo Aburatani, Haruko Takeyama

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Clarifying symbiotic relationships is one of the most important theme for understanding the marine eco-system. Coral reef has been regarded as an important environmental resource. Coral holobiont composed by coral, symbiotic microalgae zooxanthellae, and bacteria have complexed relationship. Zooxanthellae mainly supply organic matter to the host corals through their photosynthetic activity. The symbiotic relationship is indispensable for corals but may easily collapses due to the rise of seawater temperature. However, the molecular mechanism how seawater temperature influences their relationships still remain unclear. In this study, the transcriptomic analysis has applied to elucidate the coral-zooxanthellae relationships under high seawater temperature stress. To observe reactions of corals and zooxanthellae against the rise of seawater temperature, meta-gene expression in coral have been analyzed. The branches from six different colonies of a stony coral, Acropora tenuis, were sampled at nine times by 2016 at two locations, Ishikawabaru and South of Sesoko Island, Okinawa, Japan. The mRNAs extracted from the branches including zooxanthellae were sequenced by illumina HiSeq. Gene Set Enrichment Analysis (GSEA) based on hyper geometric distribution was performed. The seawater temperature at 2016 summer was unusually high, which was caused by El Niño event, and the number of zooxanthellae in coral was decreased in August. GSEA derived the several specific genes expressed in A. tenuis under heat stress conditions. The upregulated genes under heat stress highly related with infection immunity. The downregulated genes significantly contained cell cycle related genes. Thu, it is considered that heat stress cause disorder in cell metabolism of A. tenuis, resulting in serious influence to coral holobiont.

Keywords: coral, symbiosis, thermal stress response, transcriptome analysis

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