Search results for: genotyping by sequencing

Authors: Lydia Tan, Philippe Lemey, Lieselot Houspie, Marco Viveen, Darren Martin, Frank Coenjaerts

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Introduction: Human respiratory syncytial virus (hRSV) is the leading cause of severe respiratory tract infections in infants under the age of two. Genomic substitutions and related evolutionary dynamics of hRSV are of great influence on virus transmission behavior. The evolutionary patterns formed are due to a precarious interplay between the host immune response and RSV, thereby selecting the most viable and less immunogenic strains. Studying genomic profiles can teach us which genes and consequent proteins play an important role in RSV survival and transmission dynamics. Study design: In this study, genetic diversity and evolutionary rate analysis were conducted on 36 RSV subgroup B whole genome sequences and 37 subgroup A genome sequences. Clinical RSV isolates were obtained from nasopharyngeal aspirates and swabs of children between 2 weeks and 5 years old of age. These strains, collected during epidemic seasons from 2001 to 2011 in the Netherlands and Belgium by either conventional or 454-sequencing. Sequences were analyzed for genetic diversity, recombination events, synonymous/non-synonymous substitution ratios, epistasis, and translational consequences of mutations were mapped to known 3D protein structures. We used Bayesian statistical inference to estimate the rate of RSV genome evolution and the rate of variability across the genome. Results: The A and B profiles were described in detail and compared to each other. Overall, the majority of the whole RSV genome is highly conserved among all strains. The attachment protein G was the most variable protein and its gene had, similar to the non-coding regions in RSV, more elevated (two-fold) substitution rates than other genes. In addition, the G gene has been identified as the major target for diversifying selection. Overall, less gene and protein variability was found within RSV-B compared to RSV-A and most protein variation between the subgroups was found in the F, G, SH and M2-2 proteins. For the F protein mutations and correlated amino acid changes are largely located in the F2 ligand-binding domain. The small hydrophobic phosphoprotein and nucleoprotein are the most conserved proteins. The evolutionary rates were similar in both subgroups (A: 6.47E-04, B: 7.76E-04 substitution/site/yr), but estimates of the time to the most recent common ancestor were much lower for RSV-B (B: 19, A: 46.8 yrs), indicating that there is more turnover in this subgroup. Conclusion: This study provides a detailed description of whole RSV genome mutations, the effect on translation products and the first estimate of the RSV genome evolution tempo. The immunogenic G protein seems to require high substitution rates in order to select less immunogenic strains and other conserved proteins are most likely essential to preserve RSV viability. The resulting G gene variability makes its protein a less interesting target for RSV intervention methods. The more conserved RSV F protein with less antigenic epitope shedding is, therefore, more suitable for developing therapeutic strategies or vaccines.

Keywords: drug target selection, epidemiology, respiratory syncytial virus, RSV

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Authors: Shaista Suhail

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As cancer populations is increasing sharply, the incidence of oral squamous cell carcinoma (OSCC) has also been expected to increase. Oral carcinogenesis is a highly complex, multistep process which involves accumulation of genetic alterations that lead to the induction of proteins promoting cell growth (encoded by oncogenes), increased enzymatic (telomerase) activity promoting cancer cell proliferation. The global increase in frequency and mortality, as well as the poor prognosis of oral squamous cell carcinoma, has intensified current research efforts in the field of prevention and early detection of this disease. The advances in the understanding of the molecular basis of oral cancer should help in the identification of new markers. The study of the carcinogenic process of the oral cancer, including continued analysis of new genetic alterations, along with their temporal sequencing during initiation, promotion and progression, will allow us to identify new diagnostic and prognostic factors, which will provide a promising basis for the application of more rational and efficient treatments. Telomerase activity has been readily found in most cancer biopsies, in premalignant lesions or germ cells. Activity of telomerase is generally absent in normal tissues. It is known to be induced upon immortalization or malignant transformation of human cells such as in oral cancer cells. Maintenance of telomeres plays an essential role during transformation of precancer to malignant stage. Mammalian telomeres, a specialized nucleoprotein structures are composed of large conctamers of the guanine-rich sequence 5_-TTAGGG-3_. The roles of telomeres in regulating both stability of genome and replicative immortality seem to contribute in essential ways in cancer initiation and progression. It is concluded that activity of telomerase can be used as a biomarker for diagnosis of malignant oral cancer and a target for inactivation in chemotherapy or gene therapy. Its expression will also prove to be an important diagnostic tool as well as a novel target for cancer therapy. The activation of telomerase may be an important step in tumorgenesis which can be controlled by inactivating its activity during chemotherapy. The expression and activity of telomerase are indispensable for cancer development. There are no drugs which can effect extremely to treat oral cancers. There is a general call for new emerging drugs or methods that are highly effective towards cancer treatment, possess low toxicity, and have a minor environment impact. Some novel natural products also offer opportunities for innovation in drug discovery. Natural compounds isolated from medicinal plants, as rich sources of novel anticancer drugs, have been of increasing interest with some enzyme (telomerase) blockage property. The alarming reports of cancer cases increase the awareness amongst the clinicians and researchers pertaining to investigate newer drug with low toxicity.

Keywords: oral carcinoma, telomere, telomerase, blockage

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Authors: Anastasia V. Kovaleva, Alena A. Ryabova, Vladimir N. Kasatkin

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Background: The most simple form of entrainment to a sensory (typically auditory) rhythmic stimulus involves perceiving and synchronizing movements with an isochronous beat with one level of periodicity, such as that produced by a metronome. Children with pediatric cancer usually treated with chemo- and radiotherapy. Because of such treatment, psychologists and health professionals declare cognitive and motor abilities decline in cancer patients. The purpose of our study was to measure working memory characteristics with association with audio-motor synchronization tasks, also involved some memory resources, in children with different degrees of central nervous system lesions: posterior fossa tumors, acute lymphoblastic leukemia, and healthy controls. Methods: Our sample consisted of three groups of children: children treated for posterior fossa tumors (PFT-group, n=42, mean age 12.23), children treated for acute lymphoblastic leukemia (ALL-group, n=11, mean age 11.57) and neurologically healthy children (control group, n=36, mean age 11.67). Participants were tested for working memory characteristics with Cambridge Neuropsychological Test Automated Battery (CANTAB). Pattern recognition memory (PRM) and spatial working memory (SWM) tests were applied. Outcome measures of PRM test include the number and percentage of correct trials and latency (speed of participant’s response), and measures of SWM include errors, strategy, and latency. In the synchronization tests, the instruction was to tap out a regular beat (40, 60, 90 and 120 beats per minute) in synchrony with the rhythmic sequences that were played. This meant that for the sequences with an isochronous beat, participants were required to tap into every auditory event. Variations of inter-tap-intervals and deviations of children’s taps from the metronome were assessed. Results: Analysis of variance revealed the significant effect of group (ALL, PFT and control) on such parameters as short-term PRM, SWM strategy and errors. Healthy controls demonstrated more correctly retained elements, better working memory strategy, compared to cancer patients. Interestingly that ALL patients chose the bad strategy, but committed significantly less errors in SWM test then PFT and controls did. As to rhythmic ability, significant associations of working memory were found out only with 40 bpm rhythm: the less variable were inter-tap-intervals of the child, the more elements in memory he/she could retain. The ability to audio-motor synchronization may be related to working memory processes mediated by the prefrontal cortex whereby each sensory event is actively retrieved and monitored during rhythmic sequencing. Conclusion: Our results suggest that working memory, tested with appropriate cognitive methods, is associated with the ability to synchronize movements with rhythmic sounds, especially in sub-second intervals (40 per minute).

Keywords: acute lymphoblastic leukemia (ALL), audio-motor synchronization, posterior fossa tumor, working memory

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Authors: Reham Khalaf-Nazzal, Imad Dweikat, Paula Gimenez, Iker Oyenarte, Alfonso Martinez-Cruz, Domonik Muller

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Metal cation transport mediator (CNNM) gene family comprises 4 isoforms that are expressed in various human tissues. Structurally, CNNMs are complex proteins that contain an extracellular N-terminal domain preceding a DUF21 transmembrane domain, a ‘Bateman module’ and a C-terminal cNMP-binding domain. Mutations in CNNM2 cause familial dominant hypomagnesaemia. Growing evidence highlights the role of CNNM2 in neurodevelopment. Mutations in CNNM2 have been implicated in epilepsy, intellectual disability, schizophrenia, and others. In the present study, we aim to elucidate the function of CNNM2 in the developing brain. Thus, we present the genetic origin of symptoms in two family cohorts. In the first family, three siblings of a consanguineous Palestinian family in which parents are first cousins, and consanguinity ran over several generations, presented a varying degree of intellectual disability, cone-rod dystrophy, and autism spectrum disorder. Exome sequencing and segregation analysis revealed the presence of homozygous pathogenic mutation in the CNNM2 gene, the parents were heterozygous for that gene mutation. Magnesium blood levels were normal in the three children and their parents in several measurements. They had no symptoms of hypomagnesemia. The CNNM2 mutation in this family was found to locate in the CBS1 domain of the CNNM2 protein. The crystal structure of the mutated CNNM2 protein was not significantly different from the wild-type protein, and the binding of AMP or MgATP was not dramatically affected. This suggests that the CBS1 domain could be involved in pure neurodevelopmental functions independent of its magnesium-handling role, and this mutation could have affected a protein partner binding or other functions in this protein. In the second family, another autosomal dominant CNNM2 mutation was found to run in a large family with multiple individuals over three generations. All affected family members had hypomagnesemia and hypermagnesuria. Oral supplementation of magnesium did not increase the levels of magnesium in serum significantly. Some affected members of this family have defects in fine motor skills such as dyslexia and dyslalia. The detected mutation is located in the N-terminal part, which contains a signal peptide thought to be involved in the sorting and routing of the protein. In this project, we describe heterogenous clinical phenotypes related to CNNM2 mutations and protein functions. In the first family, and up to the authors’ knowledge, we report for the first time the involvement of CNNM2 in retinal photoreceptor development and function. In addition, we report the presence of a neurophenotype independent of magnesium status related to the CNNM2 protein mutation. Taking into account the different modes of inheritance and the different positions of the mutations within CNNM2 and its different structural and functional domains, it is likely that CNNM2 might be involved in a wide spectrum of neuropsychiatric comorbidities with considerable varying phenotypes.

Keywords: magnesium transport, autosomal recessive, autism, neurodevelopment, CBS domain

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Authors: Saiyed M Ishtiaque, Priyal Dixit

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Objectives: An approach is being suggested to design the filter media to maximize the filtration efficiency with minimum possible pressure drop of composite nonwoven by incorporating the layers of different packing densities induced by fibre of different deniers and punching parameters by using the concept of sequential punching technique in specific order in layered composite nonwoven structure. X-ray computed tomography technique is used to measure the packing density along the thickness of layered nonwoven structure composed by placing the layer of differently oriented fibres influenced by fibres of different deniers and punching parameters in various combinations to minimize the pressure drop at maximum possible filtration efficiency. Methodology Used: This work involves preparation of needle punched layered structure with batts 100g/m2 basis weight having fibre denier, punch density and needle penetration depth as variables to produce 300 g/m2 basis weight nonwoven composite. X-ray computed tomography technique is used to measure the packing density along the thickness of layered nonwoven structure composed by placing the layers of differently oriented fibres influenced by considered variables in various combinations. to minimize the pressure drop at maximum possible filtration efficiencyFor developing layered nonwoven fabrics, batts made of fibre of different deniers having 100g/m2 each basis weight were placed in various combinations. For second set of experiment, the composite nonwoven fabrics were prepared by using 3 denier circular cross section polyester fibre having 64 mm length on needle punched nonwoven machine by using the sequential punching technique to prepare the composite nonwoven fabrics. In this technique, three semi punched fabrics of 100 g/m2 each having either different punch densities or needle penetration depths were prepared for first phase of fabric preparation. These fabrics were later punched altogether to obtain the overall basis weight of 300 g/m2. The total punch density of the composite nonwoven fabric was kept at 200 punches/ cm2 with a needle penetration depth of 10 mm. The layered structures so formed were subcategorised into two groups- homogeneous layered structure in which all the three batts comprising the nonwoven fabric were made from same denier of fibre, punch density and needle penetration depth and were placed in different positions in respective fabric and heterogeneous layered structure in which batts were made from fibres of different deniers, punch densities and needle penetration depths and were placed in different positions. Contributions: The results concluded that reduction in pressure drop is not derived by the overall packing density of the layered nonwoven fabric rather sequencing of layers of specific packing density in layered structure decides the pressure drop. Accordingly, creation of inverse gradient of packing density in layered structure provided maximum filtration efficiency with least pressure drop. This study paves the way for the possibility of customising the composite nonwoven fabrics by the incorporation of differently oriented fibres in constituent layers induced by considered variablres for desired filtration properties.

Keywords: filtration efficiency, layered nonwoven structure, packing density, pressure drop

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Authors: Benjamin Glasner, Carlos Henriquez, Fernando Alfaro, Nicole Trefault, Santiago Andrade, Rodrigo De La Iglesia

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A central question in ecology refers to the relative importance that local-scale variables have over community composition, when compared with regional-scale variables. In coastal environments, strong seasonal abiotic influence dominates these systems, weakening the impact of other parameters like micronutrients. After the industrial revolution, micronutrients like trace metals have increased in ocean as pollutants, with strong effects upon biotic entities and biological processes in coastal regions. Coastal picoplankton communities had been characterized as a cyanobacterial dominated fraction, but in recent years the eukaryotic component of this size fraction has gained relevance due to their high influence in carbon cycle, although, diversity patterns and responses to disturbances are poorly understood. South Pacific upwelling coastal environments represent an excellent model to study seasonal changes due to a strong influence in the availability of macro- and micronutrients between seasons. In addition, some well constrained coastal bays of this region have been subjected to strong disturbances due to trace metal inputs. In this study, we aim to compare the influence of seasonality and trace metals concentrations, on the community structure of planktonic picoeukaryotes. To describe seasonal patterns in the study area, satellite data in a 6 years time series and in-situ measurements with a traditional oceanographic approach such as CTDO equipment were performed. In addition, trace metal concentrations were analyzed trough ICP-MS analysis, for the same region. For biological data collection, field campaigns were performed in 2011-2012 and the picoplankton community was described by flow cytometry and taxonomical characterization with next-generation sequencing of ribosomal genes. The relation between the abiotic and biotic components was finally determined by multivariate statistical analysis. Our data show strong seasonal fluctuations in abiotic parameters such as photosynthetic active radiation and superficial sea temperature, with a clear differentiation of seasons. However, trace metal analysis allows identifying strong differentiation within the study area, dividing it into two zones based on trace metals concentration. Biological data indicate that there are no major changes in diversity but a significant fluctuation in evenness and community structure. These changes are related mainly with regional parameters, like temperature, but by analyzing the metal influence in picoplankton community structure, we identify a differential response of some plankton taxa to metal pollution. We propose that some picoeukaryotic plankton groups respond differentially to metal inputs, by changing their nutritional status and/or requirements under disturbances as a derived outcome of toxic effects and tolerance.

Keywords: Picoeukaryotes, plankton communities, trace metals, seasonal patterns

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Authors: K. S. Vimal, D. Bodhini, K. Ramya, N. Lakshmipriya, R. M. Anjana, V. Sudha, J. A. Lovegrove, V. Mohan, V. Radha

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Gene-lifestyle interaction studies have been carried out in various populations. However, to date there are no studies in an Asian Indian population. Hence, we examined whether lifestyle factors such as diet and physical activity modify the association between fat mass and obesity–associated (FTO) gene variants and obesity and type 2 diabetes (T2D) in an Asian Indian population. We studied 734 unrelated T2D and 884 normal glucose-tolerant (NGT) participants randomly selected from the Chennai Urban Rural Epidemiology Study (CURES) in Southern India. Obesity was defined according to the World Health Organization Asia Pacific Guidelines (non-obese, BMI < 25 kg/m2; obese, BMI ≥ 25 kg/m2). Six single nucleotide polymorphisms (SNPs) in the FTO gene (rs9940128, rs7193144, rs8050136, rs918031, rs1588413 and rs11076023) identified from recent genome-wide association studies for T2D were genotyped by polymerase chain reaction-restriction fragment length polymorphism and direct sequencing. Dietary assessment was carried out using a validated food frequency questionnaire and physical activity was based upon the self-report. Interaction analyses were performed by including the interaction terms in the model. A joint likelihood ratio test of the main SNP effects and the SNP-diet/physical activity interaction effects was used in the linear regression analyses to maximize statistical power. Statistical analyses were performed using STATA version 13. There was a significant interaction between FTO SNP rs8050136 and carbohydrate energy percentage (Pinteraction=0.04) on obesity, where the ‘A’ allele carriers of the SNP rs8050136 had 2.46 times higher risk of obesity than those with ‘CC’ genotype (P=3.0x10-5) among individuals in the highest tertile of carbohydrate energy percentage. Furthermore, among those who had lower levels of physical activity, the ‘A’ allele carriers of the SNP rs8050136 had 1.89 times higher risk of obesity than those with ‘CC’ genotype (P=4.0x10-5). We also found a borderline interaction between SNP rs11076023 and carbohydrate energy percentage (Pinteraction=0.08) on T2D, where the ‘A’ allele carriers in the highest tertile of carbohydrate energy percentage, had 1.57 times higher risk of T2D than those with ‘TT’ genotype (P=0.002). There was also a significant interaction between SNP rs11076023 and physical activity (Pinteraction=0.03) on T2D. No further significant interactions between SNPs and macronutrient intake or physical activity on obesity and T2D were observed. In conclusion, this is the first study to provide evidence for a gene-diet and gene-physical activity interaction on obesity and T2D in an Asian Indian population. These findings suggest that the association between FTO gene variants and obesity and T2D is influenced by carbohydrate intake and physical activity levels. Greater understanding of how FTO gene influences obesity and T2D through dietary and exercise interventions will advance the development of behavioral intervention and personalised lifestyle strategies predicted to reduce the development of metabolic diseases in ‘A’ allele carriers of both SNPs in this Asian Indian population.

Keywords: dietary intake, FTO, obesity, physical activity, type 2 diabetes, Asian Indian.

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Authors: Wei Xiao, Gangzhi Cai, Xingliang Qin, Hongyan Ren, Zaidong Hua, Zhe Zhu, Hongwei Xiao, Ximin Zheng, Jie Yao, Yanzhen Bi

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Myostatin (MSTN) is the master regulator of double muscling phenotype with overgrown muscle and decreased fatness in animals, but its action mode to regulate fat deposition remains to be elucidated. In this study a swin-specific pathway through which MSTN acts to regulate the fat deposition was deciphered. Deep sequenincing of the mRNA and miRNA of fat tissues of MSTN knockout (KO) and wildtype (WT) pigs discovered the positive correlation of myocyte enhancer factor 2C (MEF2C) and fat-inhibiting miR222 expression, and the inverse correlation of miR222 and stearoyl-CoA desaturase 5 (SCD5) expression. SCD5 is rodent-absent and expressed only in pig, sheep and cattle. Fatty acid spectrum of fat tissues revealed a lower percentage of oleoyl-CoA (18:1) and palmitoleyl CoA (16:1) in MSTN KO pigs, which are the catalyzing products of SCD5-mediated desaturation of steroyl CoA (18:0) and palmitoyl CoA (16:0). Blood metrics demonstrated a 45% decline of triglyceride (TG) content in MSTN KO pigs. In light of these observations we hypothesized that MSTN might act through MEF2C/miR222/SCD5 pathway to regulate desaturation of fatty acid as well as triglyceride synthesis in pigs. To this end, real-time PCR and Western blotting were carried out to detect the expression of the three genes stated above. These experiments showed that MEF2C expression was up-regulated by nearly 2-fold, miR222 up-regulated by nearly 3-fold and SCD5 down-regulated by nearly 50% in MSTN KO pigs. These data were consistent with the expression change in deep sequencing analysis. Dual luciferase reporter was then used to confirm the regulation of MEF2C upon the promoter of miR222. Ecotopic expression of MEF2C in preadipocyte cells enhanced miR222 expression by 3.48-fold. CHIP-PCR identified a putative binding site of MEF2C on -2077 to -2066 region of miR222 promoter. Electrophoretic mobility shift assay (EMSA) demonstrated the interaction of MEF2C and miR222 promoter in vitro. These data indicated that MEF2C transcriptionally regulates the expression of miR222. Next, the regulation of miR222 on SCD5 mRNA as well as its physiological consequences were examined. Dual luciferase reporter testing revealed the translational inhibition of miR222 upon the 3´ UTR (untranslated region) of SCD5 in preadipocyte cells. Transfection of miR222 mimics and inhibitors resulted in the down-regulation and up-regulation of SCD5 in preadipocyte cells respectively, consistent with the results from reporter testing. RNA interference of SCD5 in preadipocyte cells caused 26.2% reduction of TG, in agreement with the results of TG content in MSTN KO pigs. In summary, the results above supported the existence of a molecular pathway that MSTN signals through MEF2C/miR222/SCD5 to regulate the fat deposition in pigs. This swine-specific pathway offers potential molecular markers for the development and breeding of a new pig line with optimised fatty acid composition. This would benefit human health by decreasing the takeup of saturated fatty acid.

Keywords: fat deposition, MEF2C, miR222, myostatin, SCD5, pig

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Authors: Md. M. Hossain, Regan Roat, Jenica Christopherson, Colette Free, Zhiguang Guo

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Long noncoding RNA (lncRNA) mediated post-transcriptional gene regulation, and their epigenetic landscapes have been shown to be involved in many human diseases. However, their regulation in diabetes through governing islet’s β-cell function and survival needs to be elucidated. Due to the technical and ethical constraints, it is difficult to study their role in β-cell function and survival in human under in vivo condition. In this study, humanized mice have been developed through transplanting human pancreatic islet under the kidney capsule of NOD.SCID mice and induced β-cell death leading to diabetes condition to study lncRNA mediated regulation. For this, human islets from 3 donors (3000 IEQ, purity > 80%) were transplanted under the kidney capsule of STZ induced diabetic NOD.scid mice. After at least 2 weeks of normoglycecemia, lymphocytes from diabetic NOD mice were adoptively transferred and islet grafts were collected once blood glucose reached > 200 mg/dl. RNA from human donor islets, islet grafts from humanized mice with either adoptive lymphocyte transfer (ALT) or PBS control (CTL) were ribodepleted; barcoded fragment libraries were constructed and sequenced on the Ion Proton sequencer. lncRNA expression in isolated human islets, islet grafts from humanized mice with and without induced β-cell death and their regulation in human islets function in vitro under glucose challenge, cytokine mediated inflammation and induced apoptotic condition were investigated. Out of 3155 detected lncRNAs, 299 that highly expressed in islets were found to be significantly downregulated and 224 upregulated in ALT compared to CTL. Most of these are found to be collocated within 5 kb upstream and 1 kb downstream of 788 up- and 624 down-regulated mRNAs. Genomic Regions Enrichment of Annotations Analysis revealed deregulated and collocated genes are related to pancreas endocrine development; insulin synthesis, processing, and secretion; pancreatitis and diabetes. Many of them, that found to be located within enhancer domains for islet specific gene activity, are associated to the deregulation of known islet/βcell specific transcription factors and genes that are important for β-cell differentiation, identity, and function. RNA sequencing analysis revealed aberrant lncRNA expression which is associated to the deregulated mRNAs in β-cell function as well as in molecular pathways related to diabetes. A distinct set of candidate lncRNA isoforms were identified as highly enriched and specific to human islets, which are deregulated in human islets from donors with different BMIs and with type 2 diabetes. These RNAs show an interesting regulation in cultured human islets under glucose stimulation and with induced β-cell death by cytokines. Aberrant expression of these lncRNAs was detected in the exosomes from the media of islets cultured with cytokines. Results of this study suggest that the islet specific lncRNAs are deregulated in human islet with β-cell death, hence important in diabetes. These lncRNAs might be important for human β-cell function and survival thus could be used as biomarkers and novel therapeutic targets for diabetes.

Keywords: β-cell, humanized mouse, pancreatic islet, LncRNAs

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Authors: Oleg Reva, Ilya Korotetskiy, Marina Lankina, Murat Kulmanov, Aleksandr Ilin

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Molecular docking approaches are widely used for design of new antibiotics and modeling of antibacterial activities of numerous ligands which bind specifically to active centers of indispensable enzymes and/or key signaling proteins of pathogens. Widespread drug resistance among pathogenic microorganisms calls for development of new antibiotics specifically targeting important metabolic and information pathways. A generally recognized problem is that almost all molecular targets have been identified already and it is getting more and more difficult to design innovative antibacterial compounds to combat the drug resistance. A promising way to overcome the drug resistance problem is an induction of reversion of drug resistance by supplementary medicines to improve the efficacy of the conventional antibiotics. In contrast to well established computer-based drug design, modeling of drug resistance reversion still is in its infancy. In this work, we proposed an approach to identification of compensatory genetic variants reducing the fitness cost associated with the acquisition of drug resistance by pathogenic bacteria. The approach was based on an analysis of the population genetic of Mycobacterium tuberculosis and on results of experimental modeling of the drug resistance reversion induced by a new anti-tuberculosis drug FS-1. The latter drug is an iodine-containing nanomolecular complex that passed clinical trials and was admitted as a new medicine against MDR-TB in Kazakhstan. Isolates of M. tuberculosis obtained on different stages of the clinical trials and also from laboratory animals infected with MDR-TB strain were characterized by antibiotic resistance, and their genomes were sequenced by the paired-end Illumina HiSeq 2000 technology. A steady increase in sensitivity to conventional anti-tuberculosis antibiotics in series of isolated treated with FS-1 was registered despite the fact that the canonical drug resistance mutations identified in the genomes of these isolates remained intact. It was hypothesized that the drug resistance phenotype in M. tuberculosis requires an adjustment of activities of many genes to compensate the fitness cost of the drug resistance mutations. FS-1 cased an aggravation of the fitness cost and removal of the drug-resistant variants of M. tuberculosis from the population. This process caused a significant increase in genetic heterogeneity of the Mtb population that was not observed in the positive and negative controls (infected laboratory animals left untreated and treated solely with the antibiotics). A large-scale search for linkage disequilibrium associations between the drug resistance mutations and genetic variants in other genomic loci allowed identification of target proteins, which could be influenced by supplementary drugs to increase the fitness cost of the drug resistance and deprive the drug-resistant bacterial variants of their competitiveness in the population. The approach will be used to improve the efficacy of FS-1 and also for computer-based design of new drugs to combat drug-resistant infections.

Keywords: complete genome sequencing, computational modeling, drug resistance reversion, Mycobacterium tuberculosis

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Authors: M. Kazou, A. Gavriil, O. Kalagkatsi, T. Paschos, E. Tsakalidou

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Kopanisti cheese is a Greek soft Protected Designation of Origin (PDO) cheese made of raw cow, sheep or goat milk, or mixtures of them, with similar organoleptic characteristics to that of Roquefort cheese. Traditional manufacturing of Kopanisti cheese is limited in small-scale dairies, without the addition of starter cultures. Instead, an amount of over-mature Kopanisti cheese, called Mana Kopanisti, is used to initiate ripening. Therefore, the selection of proper starter cultures and the understanding of the contribution of various microbial groups to its overall quality is crucial for the production of a high-quality final product with standardized organoleptic and physicochemical characteristics. Taking the above into account, the aim of the present study was the investigation of Kopanisti cheese microbiota and its role in cheese quality. For this purpose, four different types of Kopanisti were produced in triplicates, all with pasteurized cow milk, with the addition of (A) the typical mesophilic species Lactococcus lactis and Lactobacillus paracasei used as starters in the production of soft spread cheeses, (B) strains of Lactobacillus acidipiscis and Lactobacillus rennini previously isolated from Kopanisti and Mana Kopanisti, (C) all the species from (A) and (B) as inoculum, and finally (D) the species from (A) and Mana Kopanisti. Physicochemical and microbiological analysis was performed for milk and cheese samples during ripening. Enumeration was performed for major groups of lactic acid bacteria (LAB), total mesophilic bacteria, yeasts as well as hygiene indicator microorganisms. Bacterial isolates from all the different LAB groups, apart from enterococci, alongside yeasts isolates, were initially grouped using repetitive sequence-based polymerase chain reaction (rep-PCR) and then identified at the species level using 16S rRNA gene and internal transcribed spacer (ITS) DNA region sequencing, respectively. Sensory evaluation was also performed for final cheese samples at the end of the ripening period (35 days). Based on the results of the classical microbiological analysis, the average counts of the total mesophilic bacteria and LAB, apart from enterococci, ranged between 7 and 10 log colony forming unit (CFU) g⁻¹, phychrotrophic bacteria, and yeast extract glucose chloramphenicol (YGC) isolates between 4 and 8 log CFU g⁻¹, while coliforms and enterococci up to 2 log CFU g⁻¹ throughout ripening in cheese samples A, C and D. In contrast, in cheese sample B, the average counts of the total mesophilic bacteria and LAB, apart from enterococci, phychrotrophic bacteria, and YGC isolates ranged between 0 and 10 log CFU g⁻¹ and coliforms and enterococci up to 2 log CFU g⁻¹. Although the microbial counts were not that different among samples, identification of the bacterial and yeasts isolates revealed the complex microbial community structure present in each cheese sample. Differences in the physicochemical characteristics among the cheese samples were also observed, with pH ranging from 4.3 to 5.3 and moisture from 49.6 to 58.0 % in the final cheese products. Interestingly, the sensory evaluation also revealed differences among samples, with cheese sample B ranking first based on the total score. Overall, the combination of these analyses highlighted the impact of different starter cultures on the Kopanisti microbiota as well as on the physicochemical and sensory characteristics of the final product.

Keywords: Kopanisti cheese, microbiota, classical microbiological analysis, physicochemical analysis

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Authors: Alvarez M., Chapela M. J., Balboa E., Rubianes D., Sinde E., Fernandez de Ana C., Rodríguez-Blanco A.

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The gut microbiota plays an important role as gut inflammation could contribute to colorectal cancer development; however, this role is still not fully understood, and tools able to prevent this progression are yet to be developed. The main objective of this study was to monitor the effects of a mushroom extracts formulation in gut microbial community composition of an Azoxymethane (AOM)/Dextran sodium sulfate (DSS) mice model of inflammation-linked carcinogenesis. For the in vivo study, 41 adult male mice of the C57BL / 6 strain were obtained. 36 of them have been induced in a state of colon carcinogenesis by a single intraperitoneal administration of AOM at a dose of 12.5 mg/kg; the control group animals received instead of the same volume of 0.9% saline. DSS is an extremely toxic polysaccharide sulfate that causes chronic inflammation of the colon mucosa, favoring the appearance of severe colitis and the production of tumors induced by AOM. Induction by AOM/DSS is an interesting platform for chemopreventive intervention studies. This time the model was used to monitor gut microbiota changes as a result of supplementation with a specific mushroom extracts formulation previously shown to have prebiotic activity. The animals have been divided into three groups: (i) Cancer + mushroom extracts formulation experimental group: to which the MicoDigest2.0 mushroom extracts formulation developed by Hifas da Terra S.L has been administered dissolved in drinking water at an estimated concentration of 100 mg / ml. (ii) Control group of animals with Cancer: to which normal water has been administered without any type of treatment. (iii) Control group of healthy animals: these are the animals that have not been induced cancer or have not received any treatment in drinking water. This treatment has been maintained for a period of 3 months, after which the animals were sacrificed to obtain tissues that were subsequently analyzed to verify the effects of the mushroom extract formulation. A microbiological analysis has been carried out to compare the microbial communities present in the intestines of the mice belonging to each of the study groups. For this, the methodology of massive sequencing by molecular analysis of the 16S gene has been used (Ion Torrent technology). Initially, DNA extraction and metagenomics libraries were prepared using the 16S Metagenomics kit, always following the manufacturer's instructions. This kit amplifies 7 of the 9 hypervariable regions of the 16S gene that will then be sequenced. Finally, the data obtained will be compared with a database that makes it possible to determine the degree of similarity of the sequences obtained with a wide range of bacterial genomes. Results obtained showed that, similarly to certain natural compounds preventing colorectal tumorigenesis, a mushroom formulation enriched the Firmicutes and Proteobacteria phyla and depleted Bacteroidetes. Therefore, it was demonstrated that the consumption of the mushroom extracts’ formulation developed could promote the recovery of the microbial balance that is disrupted in the mice model of carcinogenesis. More preclinical and clinical studies are needed to validate this promising approach.

Keywords: carcinogenesis, microbiota, mushroom extracts, inflammation

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Authors: Anna Solovyeva, Ivan Levakin, Nickolai Galaktionov, Olga Podgornaya

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Animals that reproduce asexually were thought to have the same genotypes within generations for a long time. However, some refuting examples were found, and mobile genetic elements (MGEs) or transposons are considered to be the most probable source of genetic instability. Dispersed nature and the ability to change their genomic localization enables MGEs to be efficient mutators. Hence the study of MGEs genomic impact requires an appropriate object which comprehends both representative amounts of various MGEs and options to evaluate the genomic influence of MGEs. Animals that reproduce asexually seem to be a decent model to study MGEs impact in genomic variability. We found a small marine trematode Himasthla elongata (Himasthlidae) to be a good model for such investigation as it has a small genome size, diverse MGEs and parthenogenetic stages in the lifecycle. In the current work, clonal diversity of cercaria was traced with an AFLP (Amplified fragment length polymorphism) method, diverse zones from electrophoretic patterns were cloned, and the nature of the fragments explored. Polymorphic patterns of individual cercariae AFLP-based fingerprints are enriched with retrotransposons of different families. The bulk of those sequences are represented by open reading frames of non-Long Terminal Repeats containing elements(non-LTR) yet Long-Terminal Repeats containing elements (LTR), to a lesser extent in variable figments of AFLP array. The CR1 elements expose both in polymorphic and conservative patterns are remarkably more frequent than the other non-LTR retrotransposons. This data was confirmed with shotgun sequencing-based on Illumina HiSeq 2500 platform. Individual cercaria of the same clone (i.e., originated from a single miracidium and inhabiting one host) has a various distribution of MGE families detected in sequenced AFLP patterns. The most numerous are CR1 and RTE-Bov retrotransposons, typical for trematode genomes. Also, we identified LTR-retrotransposons of Pao and Gypsy families among DNA transposons of CMC-EnSpm, Tc1/Mariner, MuLE-MuDR and Merlin families. We detected many of them in H. elongata transcriptome. Such uneven MGEs distribution in AFLP sequences’ sets reflects the different patterns of transposons spreading in cercarial genomes as transposons affect the genome in many ways (ectopic recombination, gene structure interruption, epigenetic silencing). It is considered that they play a key role in the origins of trematode clonal polymorphism. The authors greatly appreciate the help received at the Kartesh White Sea Biological Station of the Russian Academy of Sciences Zoological Institute. This work is funded with RSF 19-74-20102 and RFBR 17-04-02161 grants and the research program of the Zoological Institute of the Russian Academy of Sciences (project number AAAA-A19-119020690109-2).

Keywords: AFLP, clonal polymorphism, Himasthla elongata, mobile genetic elements, NGS

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Authors: Shagufta Jabeen, Faez Jesse Firdaus Abdullah, Zunita Zakaria, Nurulfiza Mat Isa, Yung Chie Tan, Wai Yan Yee, Abdul Rahman Omar

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Pasteurella multocida is associated with acute, as well as, chronic infections in avian and bovine such as pasteurellosis and hemorrhagic septicemia (HS) in cattle and buffaloes. Iron is one of the most important nutrients for pathogenic bacteria including Pasteurella and acts as a cofactor or prosthetic group in several essential enzymes and is needed for amino acid, pyrimidine, and DNA biosynthesis. In our recent study, we showed that 2% of Pasteurella multocida serotype A strain PMTB2.1 encode for iron regulating genes (Accession number CP007205.1). Genome sequencing of other Pasteurella multocida serotypes namely PM70 and HB01 also indicated up to 2.5% of the respective genome encode for iron regulating genes, suggesting that Pasteurella multocida genome comprises of multiple systems for iron uptake. Since P. multocida PMTB2.1 has more than 40 CDs out of 2097 CDs (approximately 2%), encode for iron-regulated. The gene expression profiling of four iron-regulating genes namely fbpb, yfea, fece and fur were characterized under iron-restricted environment. The P. multocida strain PMTB2.1 was grown in broth with and without iron chelating agent and samples were collected at different time points. Relative mRNA expression profile of these genes was determined using Taqman probe based real-time PCR assay. The data analysis, normalization with two house-keeping genes and the quantification of fold changes were carried out using Bio-Rad CFX manager software version 3.1. Results of this study reflect that iron reduced environment has significant effect on expression profile of iron regulating genes (p < 0.05) when compared to control (normal broth) and all evaluated genes act differently with response to iron reduction in media. The highest relative fold change of fece gene was observed at early stage of treatment indicating that PMTB2.1 may utilize its periplasmic protein at early stage to acquire iron. Furthermore, down-regulation expression of fece with the elevated expression of other genes at later time points suggests that PMTB2.1 control their iron requirements in response to iron availability by down-regulating the expression of iron proteins. Moreover, significantly high relative fold change (p ≤ 0.05) of fbpb gene is probably associated with the ability of P. multocida to directly use host iron complex such as hem, hemoglobin. In addition, the significant increase (p ≤ 0.05) in fbpb and yfea expressions also reflects the utilization of multiple iron systems in P. multocida strain PMTB2.1. The findings of this study are very much important as relative scarcity of free iron within hosts creates a major barrier to microbial growth inside host and utilization of outer-membrane proteins system in iron acquisition probably occurred at early stage of infection with P. multocida. In conclusion, the presence and utilization of multiple iron system in P. multocida strain PMTB2.1 revealed the importance of iron in the survival of P. multocida.

Keywords: iron-related genes, real-time PCR, gene expression profiling, fold changes

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Authors: Binder Hans

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Cancer is no longer seen as solely a genetic disease where genetic defects such as mutations and copy number variations affect gene regulation and eventually lead to aberrant cell functioning which can be monitored by transcriptome analysis. It has become obvious that epigenetic alterations represent a further important layer of (de-)regulation of gene activity. For example, aberrant DNA methylation is a hallmark of many cancer types, and methylation patterns were successfully used to subtype cancer heterogeneity. Hence, unraveling the interplay between different omics levels such as genome, transcriptome and epigenome is inevitable for a mechanistic understanding of molecular deregulation causing complex diseases such as cancer. This objective requires powerful downstream integrative bioinformatics methods as an essential prerequisite to discover the whole genome mutational, transcriptome and epigenome landscapes of cancer specimen and to discover cancer genesis, progression and heterogeneity. Basic challenges and tasks arise ‘beyond sequencing’ because of the big size of the data, their complexity, the need to search for hidden structures in the data, for knowledge mining to discover biological function and also systems biology conceptual models to deduce developmental interrelations between different cancer states. These tasks are tightly related to cancer biology as an (epi-)genetic disease giving rise to aberrant genomic regulation under micro-environmental control and clonal evolution which leads to heterogeneous cellular states. Machine learning algorithms such as self organizing maps (SOM) represent one interesting option to tackle these bioinformatics tasks. The SOMmethod enables recognizing complex patterns in large-scale data generated by highthroughput omics technologies. It portrays molecular phenotypes by generating individualized, easy to interpret images of the data landscape in combination with comprehensive analysis options. Our image-based, reductionist machine learning methods provide one interesting perspective how to deal with massive data in the discovery of complex diseases, gliomas, melanomas and colon cancer on molecular level. As an important new challenge, we address the combined portrayal of different omics data such as genome-wide genomic, transcriptomic and methylomic ones. The integrative-omics portrayal approach is based on the joint training of the data and it provides separate personalized data portraits for each patient and data type which can be analyzed by visual inspection as one option. The new method enables an integrative genome-wide view on the omics data types and the underlying regulatory modes. It is applied to high and low-grade gliomas and to melanomas where it disentangles transversal and longitudinal molecular heterogeneity in terms of distinct molecular subtypes and progression paths with prognostic impact.

Keywords: integrative bioinformatics, machine learning, molecular mechanisms of cancer, gliomas and melanomas

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Authors: Bello Gonzalez T. D. J., Setten Van M., Essen Van A., Brouwer M., Veldman K. T.

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Resistance to fluoroquinolones (FQ) has evolved increasingly over the years, posing a significant challenge for the treatment of human infections, particularly gastrointestinal tract infections caused by zoonotic bacteria transmitted through the food chain and environment. In broiler chickens, a relatively high proportion of FQ resistance has been observed in Escherichia coli indicator, Salmonella and Campylobacter isolates. We hypothesize that flumequine (Flu), used as a secondary choice for the treatment of poultry infections, could potentially be associated with a high proportion of FQ resistance. To evaluate this hypothesis, we used an in vitro fermentation chicken caeca model. Two continuous single-stage fermenters were used to simulate in real time the physiological conditions of the chicken caeca microbial content (temperature, pH, caecal content mixing, and anoxic environment). A pool of chicken caecal content containing FQ-resistant E. coli obtained from chickens at slaughter age was used as inoculum along with a spiked FQ-susceptible Campylobacter jejuni strain isolated from broilers. Flu was added to one of the fermenters (Flu-fermenter) every 24 hours for two days to evaluate the selection and maintenance of FQ resistance over time, while the other served as a control (C-Fermenter). The experiment duration was 5 days. Samples were collected at three different time points: before, during and after Flu administration. Serial dilutions were plated on Butzler culture media with and without Flu (8mg/L) and enrofloxacin (4mg/L) and on MacConkey culture media with and without Flu (4mg/L) and enrofloxacin (1mg/L) to determine the proportion of resistant strains over time. Positive cultures were identified by mass spectrometry and matrix-assisted laser desorption/ionization (MALDI). A subset of the obtained isolates were used for Whole Genome Sequencing analysis. Over time, E. coli exhibited positive growth in both fermenters, while C. jejuni growth was detected up to day 3. The proportion of Flu-resistant E. coli strains recovered remained consistent over time after antibiotic selective pressure, while in the C-fermenter, a decrease was observed at day 5; a similar pattern was observed in the enrofloxacin-resistant E. coli strains. This suggests that Flu might play a role in the selection and persistence of enrofloxacin resistance, compared to C-fermenter, where enrofloxacin-resistant E. coli strains appear at a later time. Furthermore, positive growth was detected from both fermenters only on Butzler plates without antibiotics. A subset of C. jejuni strains from the Flu-fermenter revealed that those strains were susceptible to ciprofloxacin (MIC < 0.12 μg/mL). A selection of E. coli strains from both fermenters revealed the presence of plasmid-mediated quinolone resistance (PMQR) (qnr-B19) in only one strain from the C-fermenter belonging to sequence type (ST) 48, and in all from Flu-fermenter belonged to ST189. Our results showed that Flu selective impact on PMQR-positive E. coli strains, while no effect was observed in C. jejuni. Maintenance of Flu-resistance was correlated with antibiotic selective pressure. Further studies into antibiotic resistance gene transfer among commensal and zoonotic bacteria in the chicken caeca content may help to elucidate the resistance spread mechanisms.

Keywords: fluoroquinolone-resistance, escherichia coli, campylobacter jejuni, in vitro model

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Authors: Dalia Ambrazaitienė, Monika Vilkienė, Danute Karcauskienė, Gintaras Siaudinis

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The soil is a complex ecosystem that is part of our biosphere. The ability of soil to provide ecosystem services is dependent on microbial diversity. T Tillage is one of the major factors that affect soil properties. The no-till systems or shallow ploughless tillage are opposite of traditional deep ploughing, no-tillage systems, for instance, increase soil organic matter by reducing mineralization rates and stimulating litter concentrations of the top soil layer, whereas deep ploughing increases the biological activity of arable soil layer and reduces the incidence of weeds. The role of soil organisms is central to soil processes. Although the number of microbial species in soil is still being debated, the metagenomic approach to estimate microbial diversity predicted about 2000 – 18 000 bacterial genomes in 1 g of soil. Despite the key role of bacteria in soil processes, there is still lack of information about the bacterial diversity of soils as affected by tillage practices. This study focused on metagenomic analysis of bacterial diversity in long-term experimental plots of Dystric Epihypogleyic Albeluvisols in western part of Lithuania. The experiment was set up in 2013 and had a split-plot design where the whole-plot treatments were laid out in a randomized design with three replicates. The whole-plot treatments consisted of two tillage methods - deep ploughing (22-25 cm) (DP), ploughless tillage (7-10 cm) (PT). Three subsamples (0-20 cm) were collected on October 22, 2015 for each of the three replicates. Subsamples from the DP and PT systems were pooled together wise to make two composition samples, one representing deep ploughing (DP) and the other ploughless tillage (PT). Genomic DNA from soil sample was extracted from approximately 200 mg field-moist soil by using the D6005 Fungal/Bacterial Miniprep set (Zymo Research®) following the manufacturer’s instructions. To determine bacterial diversity and community composition, we employed a culture – independent approach of high-throughput pyrosequencing of the 16S rRNA gene. Metagenomic sequencing was made with Illumina MiSeq platform in Base Clear Company. The microbial component of soil plays a crucial role in cycling of nutrients in biosphere. Our study was a preliminary attempt at observing bacterial diversity in soil under two common but contrasting tillage practices. The number of sequenced reads obtained for PT (161 917) was higher than DP (131 194). The 10 most abundant genus in soil sample were the same (Arthrobacter, Candidatus Saccharibacteria, Actinobacteria, Acidobacterium, Mycobacterium, Bacillus, Alphaproteobacteria, Longilinea, Gemmatimonas, Solirubrobacter), just the percent of community part was different. In DP the Arthrobacter and Acidobacterium consist respectively 8.4 % and 2.5%, meanwhile in PT just 5.8% and 2.1% of all community. The Nocardioides and Terrabacter were observed just in PT. This work was supported by the project VP1-3.1-ŠMM-01-V-03-001 NKPDOKT and National Science Program: The effect of long-term, different-intensity management of resources on the soils of different genesis and on other components of the agro-ecosystems [grant number SIT-9/2015] funded by the Research Council of Lithuania.

Keywords: deep ploughing, metagenomics, ploughless tillage, soil community analysis

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Authors: N. Perini, M. C. Thaller, F. Mercuri, S. Orlanducci, A. Rubechini, L. Migliore

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The preservation of cultural heritage is one of the major challenges of today’s society, because of the fundamental right of future generations to inherit it as the continuity with their historical and cultural identity. Parchments, consisting of a semi-solid matrix of collagen produced from animal skin (i.e., sheep or goats), are a significant part of the cultural heritage, being used as writing material for many centuries. Due to their animal origin, parchments easily undergo biodeterioration. The most common biological damage is characterized by isolated or coalescent purple spots that often leads to the detachment of the superficial layer and the loss of the written historical content of the document. Although many parchments with the same biodegradative features were analyzed, no common causative agent has been found so far. Very recently, a study was performed on a purple-damaged parchment roll dated back 1244 A.D, the A.A. Arm. I-XVIII 3328, belonging to the oldest collection of the Vatican Secret Archive (Fondo 'Archivum Arcis'), by comparing uncolored undamaged and purple damaged areas of the same document. As a whole, the study gave interesting results to hypothesize a model of biodeterioration, consisting of a microbial succession acting in two main phases: the first one, common to all the damaged parchments, is characterized by halophilic and halotolerant bacteria fostered by the salty environment within the parchment maybe induced by bringing of the hides; the second one, changing with the individual history of each parchment, determines the identity of its colonizers. The design of this model was pivotal to this study, performed by different labs of the Tor Vergata University (Rome, Italy), in collaboration with the Vatican Secret Archive. Three documents, belonging to a collection of dramatically damaged parchments archived as 'Faldone Patrizi A 19' (dated back XVII century A.D.), were analyzed through a multidisciplinary approach, including three updated technologies: (i) Next Generation Sequencing (NGS, Illumina) to describe the microbial communities colonizing the damaged and undamaged areas, (ii) RAMAN spectroscopy to analyze the purple pigments, (iii) Light Transmitted Analysis (LTA) to evaluate the kind and entity of the damage to native collagen. The metagenomic analysis obtained from NGS revealed DNA sequences belonging to Halobacterium salinarum mainly in the undamaged areas. RAMAN spectroscopy detected pigments within the purple spots, mainly bacteriorhodopsine/rhodopsin-like pigments, a purple transmembrane protein containing retinal and present in Halobacteria. The LTA technique revealed extremely damaged collagen structures in both damaged and undamaged areas of the parchments. In the light of these data, the study represents a first confirmation of the microbial succession model described above. The demonstration of this model is pivotal to start any possible new restoration strategy to bring back historical parchments to their original beauty, but also to open opportunities for intervention on a huge amount of documents.

Keywords: biodeterioration, parchments, purple spots, ecological succession

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Authors: Yuli Hou, Jianping Sun, Mengdan Gao, Hui Liu, Ling Qin, Ang Li, Dongfu Li, Yonghong Zhang, Yan Zhao

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Background and Aims: Interferon-induced transmembrane protein 3 (IFITM3) is a component of ISG (Interferon-Stimulated Gene) family. IFITM3 has been recognized as a key signal molecule regulating cell growth in some tumors. However, the function of IFITM3 rs12252-CC genotype in the hepatocellular carcinoma (HCC) remains unknown to author’s best knowledge. A cohort study was employed to clarify the relationship between IFITM3 rs12252-CC genotype and HCC progression, and cellular experiments were used to investigate the correlation of function of IFITM3 and the progress of HCC. Methods: 336 candidates were enrolled in study, including 156 with HBV related HCC and 180 with chronic Hepatitis B infections or liver cirrhosis. Polymerase chain reaction (PCR) was employed to determine the gene polymorphism of IFITM3. The functions of IFITM3 were detected in PLC/PRF/5 cell with different treated:LV-IFITM3 transfected with lentivirus to knockdown the expression of IFITM3 and LV-NC transfected with empty lentivirus as negative control. The IFITM3 expression, proliferation and migration were detected by Quantitative reverse transcription polymerase chain reaction (qRT-PCR), QuantiGene Plex 2.0 assay, western blotting, immunohistochemistry, Cell Counting Kit(CCK)-8 and wound healing respectively. Six samples (three infected with empty lentiviral as control; three infected with LV-IFITM3 vector lentiviral as experimental group ) of PLC/PRF/5 were sequenced at BGI (Beijing Genomics Institute, Shenzhen,China) using RNA-seq technology to identify the IFITM3-related signaling pathways and chose PI3K/AKT pathway as related signaling to verify. Results: The patients with HCC had a significantly higher proportion of IFITM3 rs12252-CC compared with the patients with chronic HBV infection or liver cirrhosis. The distribution of CC genotype in HCC patients with low differentiation was significantly higher than that in those with high differentiation. Patients with CC genotype found with bigger tumor size, higher percentage of vascular thrombosis, higher distribution of low differentiation and higher 5-year relapse rate than those with CT/TT genotypes. The expression of IFITM3 was higher in HCC tissues than adjacent normal tissues, and the level of IFITM3 was higher in HCC tissues with low differentiation and metastatic than high/medium differentiation and without metastatic. Higher RNA level of IFITM3 was found in CC genotype than TT genotype. In PLC/PRF/5 cell with knockdown, the ability of cell proliferation and migration was inhibited. Analysis RNA sequencing and verification of RT-PCR found out the phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin(PI3K/AKT/mTOR) pathway was associated with knockdown IFITM3.With the inhibition of IFITM3, the expression of PI3K/AKT/mTOR signaling pathway was blocked and the expression of vimentin was decreased. Conclusions: IFITM3 rs12252-CC with the higher expression plays a vital role in the progress of HCC by regulating HCC cell proliferation and migration. These effects are associated with PI3K/AKT/mTOR signaling pathway.

Keywords: IFITM3, interferon-induced transmembrane protein 3, HCC, hepatocellular carcinoma, PI3K/ AKT/mTOR, phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin

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Authors: H. I. Obadiah, S. N. Wieser, E. A. Omudu, B. O. Atu, O. Byanet, L. Schnittger, M. Florin-Christensen

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Sarcocystis sp. are Apicomplexan protozoan parasites with a life cycle that involves a predator and a prey as final and intermediate hosts, respectively. In tissues of the intermediate hosts, the parasites produce sarcocysts that vary in size and morphology according to the species. When a suitable predator ingests sarcocyst-containing meat, the parasites are released in the intestine and undergo sexual reproduction producing infective sporocysts, which are excreted with the feces into the environment. The cycle is closed when a prey ingests sporocyst-contaminated water or pasture; the parasites gain access to the circulation, and eventually invade tissues and reproduce asexually yielding sarcocysts. Pig farming is a common practice in Nigeria as well as in many countries around the world. In addition to its importance as protein source, pork is also a source of several pathogens relevant to humans. In the case of Sarcocystis, three species have been described both in domestic and wild pigs, namely, S. miescheriana, S. porcifelis and S. suihominis. Humans can act both as final and aberrant intermediate hosts of S. suihominis, after ingesting undercooked sarcocyst-infested pork. Infections are usually asymptomatic but can be associated with inappetence, nausea, vomiting and diarrhea, or with muscle pain, fever, eosinophilia and bronchospasm, in humans acting as final or intermediate hosts, respectively. Moreover, excretion of infective forms with human feces leads to further dissemination of the infection. In this study, macroscopic sarcocysts of white color, oval shape and a size range of approximately 3-5 mm were observed in the skeletal muscle of a slaughtered pig in an abattoir in Makurdi, Benue State, Nigeria, destined to human consumption. Sarcocysts were excised and washed in distilled water, and genomic DNA was extracted using a commercial kit. The near-complete length of the 18S rRNA gene was analyzed after PCR amplification of two overlapping fragments, each of which were submitted to direct sequencing. In addition, the mitochondrial cytochrome oxidase (cox-1) gene was PCR-amplified and directly sequenced. Two phylogenetic trees containing the obtained sequences along with available relevant 18S rRNA and cox-1 sequences were constructed by neighbor joining after alignment, using the corresponding sequences of Toxoplasma gondii as outgroup. The results showed in both cases that the analyzed sequences grouped with S. suihominis with high bootstrap value, confirming the identity of this macroscopic sarcocyst-forming parasite as S. suihominis. To the best of our knowledge, these results represent the first demonstration of this parasite in pigs of Nigeria and the largest sarcocysts described so far for S. suihominis. The close proximity between pigs and humans in pig farms, and the frequent poor sanitary conditions in human dwellings strongly suggest that the parasite undergoes the sexual stages of its life cycle in humans as final hosts. These findings provide an important reference for the examination and control of Sarcocystis species in pigs of Nigeria.

Keywords: nigeria, pork, sarcocystis suihominis, zoonotic parasite

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Authors: Beihai Xiong, Dengke Hua, Wouter Hendriks, Wilbert Pellikaan

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To improve the energy status of dairy cows in the early lactation, lots of jobs have been done on adjusting the starch to fiber ratio in the diet. As a complex ecosystem, the rumen contains a large population of microorganisms which plays a crucial role in feed degradation. Further study on the microbiota alterations and metabolic changes under different dietary energy sources is essential and valuable to better understand the function of the ruminal microorganisms and thereby to optimize the rumen function and enlarge feed efficiency. The present study will focus on the effects of two glucogenic diets (G: ground corn and corn silage; S: steam-flaked corn and corn silage) and a lipogenic diet (L: sugar beet pulp and alfalfa silage) on rumen fermentation, gas production, the ruminal microbiota and metabolome, and also their correlations in vitro. The gas production was recorded consistently, and the gas volume and producing rate at times 6, 12, 24, 48 h were calculated separately. The fermentation end-products were measured after fermenting for 48 h. The ruminal bacteria and archaea communities were determined by 16S RNA sequencing technique, the metabolome profile was tested through LC-MS methods. Compared to the diet G and S, the L diet had a lower dry matter digestibility, propionate production, and ammonia-nitrogen concentration. The two glucogenic diets performed worse in controlling methane and lactic acid production compared to the L diet. The S diet produced the greatest cumulative gas volume at any time points during incubation compared to the G and L diet. The metabolic analysis revealed that the lipid digestion was up-regulated by the diet L than other diets. On the subclass level, most metabolites belonging to the fatty acids and conjugates were higher, but most metabolites belonging to the amino acid, peptides, and analogs were lower in diet L than others. Differences in rumen fermentation characteristics were associated with (or resulting from) changes in the relative abundance of bacterial and archaeal genera. Most highly abundant bacteria were stable or slightly influenced by diets, while several amylolytic and cellulolytic bacteria were sensitive to the dietary changes. The L diet had a significantly higher number of cellulolytic bacteria, including the genera of Ruminococcus, Butyrivibrio, Eubacterium, Lachnospira, unclassified Lachnospiraceae, and unclassified Ruminococcaceae. The relative abundances of amylolytic bacteria genera including Selenomonas_1, Ruminobacter, and Succinivibrionaceae_UCG-002 were higher in diet G and S. These affected bacteria was also proved to have high associations with certain metabolites. The Selenomonas_1 and Succinivibrionaceae_UCG-002 may contribute to the higher propionate production in the diet G and S through enhancing the succinate pathway. The results indicated that the two glucogenic diets had a greater extent of gas production, a higher dry matter digestibility, and produced more propionate than diet L. The steam-flaked corn did not show a better performance on fermentation end-products than ground corn. This study has offered a deeper understanding of ruminal microbial functions which could assistant the improvement in rumen functions and thereby in the ruminant production.

Keywords: gas production, metabolome, microbiota, rumen fermentation

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Authors: Alla Ovsyannikova, Oksana Rymar, Elena Shakhtshneider, Mikhail Voevoda

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In Siberia endocrinologists increasingly noted young patients with the course of diabetes mellitus differing from 1 and 2 types. Therefore we did a molecular genetic study for this group of patients to verify the monogenic forms of diabetes mellitus in them and researched the characteristics of this pathology. When confirming the monogenic form of diabetes, we performed a correction therapy for many patients (transfer from insulin to tablets), prevented specific complications, examined relatives and diagnosed their diabetes at the preclinical stage, revealed phenotypic characteristics of the pathology which led to the high significance of this work. Materials and Methods: We observed 5 patients (4 families). We diagnosed MODY (Maturity Onset Diabetes of the Young) during the molecular genetic testing (direct automatic sequencing). All patients had a full clinical examination, blood samples for biochemical research, determination of C-peptide and TSH, antibodies to b-cells, microalbuminuria, abdominal ultrasound, heart and thyroid ultrasound, examination of ophthalmologist. Results: We diagnosed 3 rare types of MODY: two women had MODY8, one man – MODY6 and man and his mother - MODY12. Patients with types 8 and 12 had clinical features. Age of onset hyperglycemia ranged from 26 to 34 years. In a patient with MODY6 fasting hyperglycemia was detected during a routine examination. Clinical symptoms, complications were not diagnosed. The patient observes a diet. In the first patient MODY8 was detected during first pregnancy, she had itchy skin and mostly postprandial hyperglycemia. Upon examination we determined glycated hemoglobin 7.5%, retinopathy, non-proliferative stage, peripheral neuropathy. She uses a basic bolus insulin therapy. The second patient with MODY8 also had clinical manifestations of hyperglycemia (pruritus, thirst), postprandial hyperglycemia and diabetic nephropathy, a stage of microalbuminuria. The patient was diagnosed autoimmune thyroiditis. She used inhibitors of DPP-4. The patient with MODY12 had an aggressive course. In the detection of hyperglycemia he had complaints of visual impairment, intense headaches, leg cramps. The patient had a history of childhood convulsive seizures of non-epileptic genesis, without organic pathology, which themselves were stopped at the age of 12 years. When we diagnosed diabetes a patient was 28 years, he had hypertriglyceridemia, atherosclerotic plaque in the carotid artery, proliferative retinopathy (lacerocoagulation). Diabetes and early myocardial infarction were observed in three cases in family. We prescribe therapy with sulfonylureas and SGLT-2 inhibitors with a positive effect. At the patient's mother diabetes began at a later age (30 years) and a less aggressive course was observed. She also has hypertriglyceridemia and uses oral hypoglycemic drugs. Conclusions: 1) When young patients with hyperglycemia have extrapancreatic pathologies and diabetic complications with a short duration of diabetes we can assume they have one of type of MODY diabetes. 2) In patients with monogenic forms of diabetes mellitus, the clinical manifestations of hyperglycemia in each succeeding generation are revealed at an earlier age. Research had increased our knowledge of the monogenic forms of diabetes. The reported study was supported by RSCF, research project No. 14-15-00496-P.

Keywords: diabetes mellitus, MODY diabetes, monogenic forms, young patients

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Authors: Kaomud Tyagi, Rajasree Chakraborty, Shantanu Kundu, Devkant Singha, Kailash Chandra, Vikas Kumar

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The melon thrips, Thrips palmi is a serious pest of a wide range of agriculture crops and also act as vectors for plant viruses (genus Tospovirus, family Bunyaviridae). More molecular data on this species is required to understand the cryptic speciation and evolutionary affiliations. Mitochondrial genomes have been widely used in phylogenetic and evolutionary studies in insect. So far, mitogenomes of five thrips species (Anaphothrips obscurus, Frankliniella intonsa, Frankliniella occidentalis, Scirtothrips dorsalis and Thrips imaginis) is available in the GenBank database. In this study, we sequenced the first complete mitogenome T. palmi and compared it with available thrips mitogenomes. We assembled the mitogenome from the whole genome sequencing data generated using Illumina Hiseq2500. Annotation was performed using MITOS web-server to estimate the location of protein coding genes (PCGs), transfer RNA (tRNAs), ribosomal RNAs (rRNAs) and their secondary structures. The boundaries of PCGs and rRNAs was confirmed manually in NCBI. Phylogenetic analyses were performed using the 13 PCGs data using maximum likelihood (ML) in PAUP, and Bayesian inference (BI) in MrBayes 3.2. The complete mitogenome of T. palmi was 15,333 base pairs (bp), which was greater than the genomes of A. obscurus (14,890bp), F. intonsa (15,215 bp), F. occidentalis (14,889 bp) and S. dorsalis South Asia strain (SA1) (14,283 bp), but smaller than the genomes of T. imaginis (15,407 bp) and S. dorsalis East Asia strain (EA1) (15,343bp). Like in other thrips species, the mitochondrial genome of T. palmi was represented by 37 genes, including 13 PCGs, large and small ribosomal RNA (rrnL and rrnS) genes, 22 transfer RNA (tRNAs) genes (with one extra gene for trn-Serine) and two A+T-rich control regions (CR1 and CR2). Thirty one genes were observed on heavy (H) strand and six genes on the light (L) strand. The six tRNA genes (trnG,trnK, trnY, trnW, trnF, and trnH) were found to be conserved in all thrips species mitogenomes in their locations relative to a protein-coding or rRNA gene upstream or downstream. The gene arrangements of T. palmi is very close to T. imaginis except the rearrangements in tRNAs genes: trnR (arginine), and trnE (glutamic acid) were found to be located between cox3 and CR2 in T. imaginis which were translocated between atp6 and CR1 in T. palmi; trnL1 (Leucine) and trnS1(Serine) were located between atp6 and CR1 in T. imaginis which were translocated between cox3 and CR2 in T. palmi. The location of CR1 upstream of nad5 gene was suggested to be ancestral condition of the thrips species in subfamily Thripinae, was also observed in T. palmi. Both the Maximum likelihood (ML) and Bayesian Inference (BI) phylogenetic trees generated resulted in similar topologies. The T. palmi was clustered with T. imaginis. We concluded that more molecular data on the diverse thrips species from different hierarchical level is needed, to understand the phylogenetic and evolutionary relationships among them.

Keywords: thrips, comparative mitogenomics, gene rearrangements, phylogenetic analysis

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Authors: Okolie Pius Ifeanyi, Emerenini Emilymary Chima

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Aims: The study investigated the diversity and identities of Lactic Acid Bacteria (LAB) isolated from different fresh fruits using Molecular Nested PCR analysis and the efficacy of cell free supernatants from Lactic Acid Bacteria (LAB) isolated from fresh fruits for in vitro control of some tomato pathogens. Study Design: Nested PCR approach was used in this study employing universal 16S rRNA gene primers in the first round PCR and LAB specific Primers in the second round PCR with the view of generating specific Nested PCR products for the LAB diversity present in the samples. The inhibitory potentials of supernatant obtained from LAB isolates of fruits origin that were molecularly characterized were investigated against some tomato phytopathogens using agar-well method with the view to develop biological agents for some tomato disease causing organisms. Methodology: Gram positive, catalase negative strains of LAB were isolated from fresh fruits on Man Rogosa and Sharpe agar (Lab M) using streaking method. Isolates obtained were molecularly characterized by means of genomic DNA extraction kit (Norgen Biotek, Canada) method. Standard methods were used for Nested Polymerase Chain Reaction (PCR) amplification targeting the 16S rRNA gene using universal 16S rRNA gene and LAB specific primers, agarose gel electrophoresis, purification and sequencing of generated Nested PCR products (Macrogen Inc., USA). The partial sequences obtained were identified by blasting in the non-redundant nucleotide database of National Center for Biotechnology Information (NCBI). The antimicrobial activities of characterized LAB against some tomato phytopathogenic bacteria which include (Xanthomonas campestries, Erwinia caratovora, and Pseudomonas syringae) were obtained by using the agar well diffusion method. Results: The partial sequences obtained were deposited in the database of National Centre for Biotechnology Information (NCBI). Isolates were identified based upon the sequences as Weissella cibaria (4, 18.18%), Weissella confusa (3, 13.64%), Leuconostoc paramensenteroides (1, 4.55%), Lactobacillus plantarum (8, 36.36%), Lactobacillus paraplantarum (1, 4.55%) and Lactobacillus pentosus (1, 4.55%). The cell free supernatants of LAB from fresh fruits origin (Weissella cibaria, Weissella confusa, Leuconostoc paramensenteroides, Lactobacillus plantarum, Lactobacillus paraplantarum and Lactobacillus pentosus) can inhibits these bacteria by creating clear zones of inhibition around the wells containing cell free supernatants of the above mentioned strains of lactic acid bacteria. Conclusion: This study shows that potentially LAB can be quickly characterized by molecular methods to specie level by nested PCR analysis of the bacteria isolate genomic DNA using universal 16S rRNA primers and LAB specific primer. Tomato disease causing organisms can be most likely biologically controlled by using extracts from LAB. This finding will reduce the potential hazard from the use of chemical herbicides on plant.

Keywords: nested pcr, molecular characterization, 16s rRNA gene, lactic acid bacteria

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Authors: Ricardo Godoy, Herbert Venthur, Hector Jimenez, Andres Quiroz, Ana Mutis

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In agriculture, grape production has great economic importance at global level, considering that in 2013 it reached 7.4 million hectares (ha) covered by plantations of this fruit worldwide. Chile is the number one exporter in the world with 800,000 tons. However, these values have been threatened by the attack of the grapevine moth, Lobesia botrana (Denis & Schiffermuller) (Lepidoptera: Tortricidae), since its detection in 2008. Nowadays, the use of semiochemicals, in particular the major component of the sex pheromone, (E,Z)-7.9-dodecadienil acetate, are part of mating disruption methods to control L. botrana. How insect pests can recognize these molecules, is being part of huge efforts to deorphanize their olfactory mechanism at molecular level. Thus, an interesting group of proteins has been identified in the antennae of insects, where odorant-binding proteins (OBPs) are known by transporting molecules to odorant receptors (ORs) and a co-receptor (ORCO) causing a behavioral change in the insect. Other proteins such as chemosensory proteins (CSPs), ionotropic receptors (IRs), odorant degrading enzymes (ODEs) and sensory neuron membrane proteins (SNMPs) seem to be involved, but few studies have been performed so far. The above has led to an increasing interest in insect communication at a molecular level, which has contributed to both a better understanding of the olfaction process and the design of new pest management strategies. To date, it has been reported that the ORs can detect one or a small group of odorants in a specific way. Therefore, the objective of this study is the identification of genes that encode these ORs using the antennal transcriptome of L. botrana. Total RNA was extracted for females and males of L. botrana, and the antennal transcriptome sequenced by Next Generation Sequencing service using an Illumina HiSeq2500 platform with 50 million reads per sample. Unigenes were assembled using Trinity v2.4.0 package and transcript abundance was obtained using edgeR. Genes were identified using BLASTN and BLASTX locally installed in a Unix system and based on our own Tortricidae database. Those Unigenes related to ORs were characterized using ORFfinder and protein Blastp server. Finally, a phylogenetic analysis was performed with the candidate amino acid sequences for LbotORs including amino acid sequences of other moths ORs, such as Bombyx mori, Cydia pomonella, among others. Our findings suggest 61 genes encoding ORs and one gene encoding an ORCO in both sexes, where the greatest difference was found in the OR6 because of the transcript abundance according to the value of FPKM in females and males was 1.48 versus 324.00. In addition, according to phylogenetic analysis OR6 is closely related to OR1 in Cydia pomonella and OR6, OR7 in Epiphyas postvittana, which have been described as pheromonal receptors (PRs). These results represent the first evidence of ORs present in the antennae of L. botrana and a suitable starting point for further functional studies with selected ORs, such as OR6, which is potentially related to pheromonal recognition.

Keywords: antennal transcriptome, lobesia botrana, odorant receptors (ORs), phylogenetic analysis

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Authors: Tumer Orhun Aykut, Robin Michael Crucitti-Thoo, Agnieszka Rudak, Iwona Jasser

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Due to eutrophication connected to the growing human population, intensive agriculture, industrialization, and reinforcement of global warming, freshwater resources are changing negatively in every region of the World. This change also concerns the replacement of native species by invasive ones that can spread in many ways. Biological invasions are a developing problem to ecosystem continuity and their presence is mostly common in freshwater bodies. The occurrence and potential invasion of the species depends on associations between abiotic and biotic variables. Due to climate change, many species can extend their range from low to high latitudes and differ in their geographic ranges. In addition, the hydrological issues strongly influence the physicochemical parameters and biological processes, especially the growth rates of species and bloom formation of Cyanobacteria. Among tropical invasive species noted in temperate Europe, Raphidiopsis raciborskii, Chrysosporum bergii, and Sphaerospermopsis aphanizomenoides are considered a serious threat. R. raciborskii being the most important one as it is already known as a highly invasive species in almost all around the World, is a freshwater, planktonic, filamentous, potentially toxic, and nitrogen-fixing Cyanobacteria. This study aimed to investigate the presence of invasive cyanobacterial species in temperate lakes in Northeastern Poland, reveal the composition of phytoplankton communities, determine the effect of environmental variables, and identify the toxic properties of invasive Cyanobacteria and other phytoplankton groups. Our study was conducted in twenty-five lakes in August 2023. The lakes represent a geographical gradient from central Poland to the Northeast and have different depths, sizes, and trophic statuses. According to performed analyses, the presence of R. raciborskii was recorded in five lakes: Szczęśliwickie (Warsaw), Mikołajskie, Rekąty, Sztynorckie (Masurian Lakeland), and further East, in Pobondzie (Suwałki Lakeland). On the other hand, C. bergii was found in three lakes: Rekąty (Masurian Lakeland), Żabinki, and Pobondzie (Suwałki Lakeland), while S. aphanizomenoides only in Pobondzie (Suwałki Lakeland). Maximum phytoplankton diversity was found in Lake Rekąty, a small and shallow lake mentioned above. The highest phytoplankton biomass was detected in highly eutrophic Lake Suskie, followed by Lake Sztynorckie. In this last lake, which is also strongly eutrophic, the highest biomass of R. raciborskii was found. Cyanophyceae had the highest biovolume and was followed by Chlorophyceae in the entire study. Numerous environmental parameters, including nutrients, were studied, and their relationships with the invasive species and the whole phytoplankton community will be presented. In addition, toxic properties of environmental DNA results from each lake will also be shown. In conclusion, investigated invasive cyanobacterial species were found in a few Northeastern Polish temperate lakes, but the number of individuals was quite low, so the biomass was quite low. It has been observed that the structure of phytoplankton changed based on lakes and environmental parameters.

Keywords: biological invasion, cyanobacteria, cyanotoxins, phytoplankton ecology, sanger sequencing

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Authors: Arsyam Mawardi, Sony Suhandono, Azzania Fibriani, Fifi Fitriyah Masduki

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Malaria is an infectious disease caused by Plasmodium sp. This disease has a high prevalence in Indonesia, especially in Jayapura. The vaccine that is currently being developed has not been effective in overcoming malaria. This is due to the high polymorphism in the Plasmodium genome especially in areas that encode Plasmodium surface proteins. Merozoite Surface Protein 1 (MSP1) Plasmodium falciparum is a surface protein that plays a role in the invasion process in human erythrocytes through the interaction of Glycophorin A protein receptors and sialic acid in erythrocytes with Reticulocyte Binding Proteins (RBP) and Duffy Adhesion Protein (DAP) ligands in merozoites. MSP1 can be targeted to be a specific antigen and predicted epitope area which will be used for the development of diagnostic and malaria vaccine therapy. MSP1 consists of 17 blocks, each block is dimorphic, and has been marked as the K1 and MAD20 alleles. Exceptions only in block 2, because it has 3 alleles, among others K1, MAD20 and RO33. These polymorphisms cause allelic variations and implicate the severity of patients infected P. falciparum. In addition, polymorphism of MSP1 in Jayapura isolates has not been reported so it is interesting to be further identified and projected as a specific antigen. Therefore, in this study, we analyzed the allele polymorphism as well as detected the MSP1 epitope antigen candidate on block 2 P. falciparum. Clinical samples of selected malaria patients followed the consecutive sampling method, examining malaria parasites with blood preparations on glass objects observed through a microscope. Plasmodium DNA was isolated from the blood of malarial positive patients. The block 2 MSP1 gene was amplified using PCR method and cloned using the pGEM-T easy vector then transformed to TOP'10 E.coli. Positive colonies selection was performed with blue-white screening. The existence of target DNA was confirmed by PCR colonies and DNA sequencing methods. Furthermore, DNA sequence analysis was done through alignment and formation of a phylogenetic tree using MEGA 6 software and insilico analysis using IEDB software to predict epitope candidate for P. falciparum. A total of 15 patient samples have been isolated from Plasmodium DNA. PCR amplification results show the target gene size about ± 1049 bp. The results of MSP1 nucleotide alignment analysis reveal that block 2 MSP1 genes derived from the sample of malarial patients were distributed in four different allele family groups, K1 (7), MAD20 (1), RO33 (0) and MSP1_Jayapura (10) alleles. The most commonly appears of the detected allele is MSP1_Jayapura single allele. There was no significant association between sex variables, age, the density of parasitemia and alel variation (Mann Whitney, U > 0.05), while symptomatic signs have a significant difference as a trigger of detectable allele variation (U < 0.05). In this research, insilico study shows that there is a new epitope antigen candidate from the MSP1_Jayapura allele and it is predicted to be recognized by B cells with 17 amino acid lengths in the amino acid sequence 187 to 203.

Keywords: epitope candidate, insilico analysis, MSP1 P. falciparum, polymorphism

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Authors: Eric Bang

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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: Niklas Ravn-Boess, Nainita Bhowmick, Takamitsu Hattori, Shohei Koide, Christopher Park, Dimitris Placantonakis

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Background: Glioblastoma (GBM) is the most common and deadly primary brain malignancy in adults. Tumor propagation, brain invasion, and resistance to therapy critically depend on GBM stem-like cells (GSCs); however, the mechanisms that regulate GSC self-renewal are incompletely understood. Given the aggressiveness and poor prognosis of GBM, it is imperative to find biomarkers that could also translate into novel drug targets. Along these lines, we have identified a cell surface antigen, CD97 (ADGRE5), an adhesion G protein-coupled receptor (GPCR), that is expressed on GBM cells but is absent from non-neoplastic brain tissue. CD97 has been shown to promote invasiveness, angiogenesis, and migration in several human cancers, but its frequency of expression and functional role in regulating GBM growth and survival, and its potential as a therapeutic target has not been investigated. Design: We assessed CD97 mRNA and protein expression in patient derived GBM samples and cell lines using publicly available RNA-sequencing datasets and flow cytometry, respectively. To assess CD97 function, we generated shRNA lentiviral constructs that target a sequence in the CD97 extracellular domain (ECD). A scrambled shRNA (scr) with no predicted targets in the genome was used as a control. We evaluated CD97 shRNA lentivirally transduced GBM cells for Ki67, Annexin V, and DAPI. We also tested CD97 KD cells for their ability to self-renew using clonogenic tumorsphere formation assays. Further, we utilized synthetic Abs (sAbs) generated against the ECD of CD97 to test for potential antitumor effects using patient-derived GBM cell lines. Results: CD97 mRNA expression was expressed at high levels in all GBM samples available in the TCGA cohort. We found high levels of surface CD97 protein expression in 6/6 patient-derived GBM cell cultures, but not human neural stem cells. Flow cytometry confirmed downregulation of CD97 in CD97 shRNA lentivirally transduced cells. CD97 KD induced a significant reduction in cell growth in 3 independent GBM cell lines representing mesenchymal and proneural subtypes, which was accompanied by reduced (~20%) Ki67 staining and increased (~30%) apoptosis. Incubation of GBM cells with sAbs (20 ug/ ml) against the ECD of CD97 for 3 days induced GSC differentiation, as determined by the expression of GFAP and Tubulin. Using three unique GBM patient derived cultures, we found that CD97 KD attenuated the ability of GBM cells to initiate sphere formation by over 300 fold, consistent with an impairment in GSC self-renewal. Conclusion: Loss of CD97 expression in patient-derived GBM cells markedly decreases proliferation, induces cell death, and reduces tumorsphere formation. sAbs against the ECD of CD97 reduce tumorsphere formation, recapitulating the phenotype of CD97 KD, suggesting that sAbs that inhibit CD97 function exhibit anti-tumor activity. Collectively, these findings indicate that CD97 is necessary for the proliferation and survival of human GBM cells and identify CD97 as a promising therapeutically targetable vulnerability in GBM.

Keywords: adhesion GPCR, CD97, GBM stem cell, glioblastoma

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Authors: Julia Kapr, Andrea Rossi, Haribaskar Ramachandran, Marius Pollet, Ilka Egger, Selina Dangeleit, Katharina Koch, Jean Krutmann, Ellen Fritsche

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It is widely acknowledged that animal models do not always represent human disease. Especially human brain development is difficult to model in animals due to a variety of structural and functional species-specificities. This causes significant discrepancies between predicted and apparent drug efficacies in clinical trials and their subsequent failure. Emerging alternatives based on 3D in vitro approaches, such as human brain spheres or organoids, may in the future reduce and ultimately replace animal models. Here, we present a human induced pluripotent stem cell (hiPSC)-based 3D neural in a vitro disease model for the Cockayne Syndrome B (CSB). CSB is a rare hereditary disease and is accompanied by severe neurologic defects, such as microcephaly, ataxia and intellectual disability, with currently no treatment options. Therefore, the aim of this study is to investigate the molecular and cellular defects found in neural hiPSC-derived CSB models. Understanding the underlying pathology of CSB enables the development of treatment options. The two CSB models used in this study comprise a patient-derived hiPSC line and its isogenic control as well as a CSB-deficient cell line based on a healthy hiPSC line (IMR90-4) background thereby excluding genetic background-related effects. Neurally induced and differentiated brain sphere cultures were characterized via RNA Sequencing, western blot (WB), immunocytochemistry (ICC) and multielectrode arrays (MEAs). CSB-deficiency leads to an altered gene expression of markers for autophagy, focal adhesion and neural network formation. Cell migration was significantly reduced and electrical activity was significantly increased in the disease cell lines. These data hint that the cellular pathologies is possibly underlying CSB. By induction of autophagy, the migration phenotype could be partially rescued, suggesting a crucial role of disturbed autophagy in defective neural migration of the disease lines. Altered autophagy may also lead to inefficient mitophagy. Accordingly, disease cell lines were shown to have a lower mitochondrial base activity and a higher susceptibility to mitochondrial stress induced by rotenone. Since mitochondria play an important role in neurotransmitter cycling, we suggest that defective mitochondria may lead to altered electrical activity in the disease cell lines. Failure to clear the defective mitochondria by mitophagy and thus missing initiation cues for new mitochondrial production could potentiate this problem. With our data, we aim at establishing a disease adverse outcome pathway (AOP), thereby adding to the in-depth understanding of this multi-faced disorder and subsequently contributing to alternative drug development.

Keywords: autophagy, disease modeling, in vitro, pluripotent stem cells

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