imagine (Institute of molecular genetics and genetic engineering)
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Genetic variants in TNFA, LTA, TLR2 and TLR4 genes and risk of sepsis in patients with severe trauma: nested case-control study in a level-1 trauma centre in SERBIA
No full textIntroduction: Single nucleotide variants (SNVs) represent important genetic risk factors for susceptibility to posttraumatic sepsis and a potential target for immunotherapy. We aimed to evaluate the association between 8 different SNVs within tumor necrosis factor alpha (TNFA), lymphotoxin alpha (LTA) and Tolllike receptor (TLR2 and TLR4) genes and the risk of posttraumatic sepsis. Methods: Nested case-control study was conducted in the emergency department of the Clinical Centre of Serbia including 228 traumatized patients (44 with sepsis and 184 without sepsis). To compare the results of trauma subjects with the data from the general population, a control group of 101 healthy persons was included in the study. Genotyping of TNFA (rs1800629 and rs361525), LTA (rs909253), TLR2 (rs3804099, rs4696480 and rs3804100), and TLR4 (rs4986790 and rs4986791) was performed for all patients within all three groups using the real-time PCR method. MutationTaster database and in silico software SIFT were used to predict the variant pathogenic effect. Results: Carriage of the G allele of the TNFA rs1800629 gene variant (OR 2.1, 95%CI 1.06-4.16) and T allele-carriage of the TLR4 rs4986791 genetic variant (OR 3.02, 95%CI 1.31-6.57) were associated with significantly higher risk of sepsis in trauma patients when compared to the general population prone to sepsis and traumatized patients without developing a sepsis, respectively. Of these two variants, only variant in TLR4 gene (rs4986791) has been labeled as disease causing by both the MutationTaster database and the in-silico software SIFT, which further supports the role of this variant in various pathologies including sepsis. For the remaining six variants no significant association with the susceptibility to sepsis was detected. Conclusions: Carriage of the G allele of the TNFA rs1800629 gene variant and T allele-carriage of the TLR4 rs4986791 genetic variant confer significant risk of posttraumatic sepsis. TLR4 gene variants (rs4986790 and rs4986791) has been labelled as disease causing
Novel Transaminase and Laccase from Streptomyces spp. Using Combined Identification Approaches
Get PDFThree Streptomyces sp. strains with a multitude of target enzymatic activities confirmed by functional screening, namely BV129, BV286 and BV333, were subjected to genome sequencing aiming at the annotation of genes of interest, in-depth bioinformatics characterization and functional expression of the biocatalysts. A whole-genome shotgun sequencing followed by de novo genome assembly and annotation was performed revealing genomes of 6.4, 9.4 and 7.3 Mbp, respectively. Functional annotation of the proteins of interest resulted in between 2047 and 2763 putative targets. Among the various enzymatic activities that the three Streptomyces strains demonstrated to produce by functional screening, we focused our attention on transaminases (TAs) and laccases due to their high biocatalytic potential. Bioinformatics search allowed the identification of a putative TA from Streptomyces sp. BV333 as a potentially novel broad substrate scope TA and a putative laccase from Streptomyces sp. BV286 as potentially novel blue multicopper oxidase. The two sequences were cloned and overexpressed in Escherichia coli and the two novel enzymes, transaminase Sbv333-TA and laccase Sbv286-LAC, were characterized. Interestingly, both enzymes resulted to be exceptionally thermostable, Sbv333-TA showing a melting temperature (T-M = 85 degrees C) only slightly lower compared to the T-M of the most thermostable transaminases described to date (87-88 degrees C) and Sbv286-LAC being even thermoactivated at temperature gt 60 degrees C. Moreover, Sbv333-TA showed a broad substrate scope and remarkably demonstrated to be active in the transamination of beta-ketoesters, which are rarely accepted by currently known TAs. On the other hand, Sbv286-LAC showed an improved activity in the presence of the cosolvent acetonitrile. Overall, it was shown that a combination of approaches from standard microbiological and biochemical screens to genome sequencing and analysis is required to afford novel and functional biocatalysts
Halochromic cellulose textile obtained via dyeing with biocolorant isolated from Streptomyces sp. strain NP4
No full textHalochromic (pH-responsive) material was obtained by dyeing functionalized viscose fabric with a crude extract from Streptomyces sp. strain NP4. The functionalization of the fabric before dyeing was performed to make cellulose susceptible to coloration with NP4 extract. Two combined pre-treatment steps were used, oxidation to obtain dialdehyde cellulose and chitosan deposition after oxidation. Chitosan was deposited onto untreated fabric as well, while only oxidized viscose was also investigated for dyeing. Functionalization by both protocols made viscose susceptible to dyeing with the notion that the deposition of chitosan onto oxidized viscose produced the darkest shade on the material. Dyed fabrics showed visual pH responsiveness in the range pH 4-10, with a color change from pink to red (pH 4-pH 7) and a major color change from red to blue (pH 7-pH 10) whereby fabric was tested and could withstand 10 color-changing cycles. Cytotoxicity assay confirmed the non-toxic nature of dyed material, which indicates its possible use as wound dressing's indicators
Chemical profiling, antimicrobial, anti-enzymatic, and cytotoxic properties of Phlomis fruticosa L.
No full textStructural diversity of biologically active compounds identified in plants after many years of storage is rarely reported in literature. Herein, we studied chemical profile and biological activities of Phlomis fruticosa L. after plant material storage for 20 years. Chemical analyzes were performed by UHPLC-LTQ-Orbitrap/MS, and revealed presence of 44 compounds: including 13 phenolic acids, 9 phenylethanoids, 20 flavonoids and 2 phenolic related compounds (a phenolic acid derivative and an aliphatic alcohol). The extract showed antimicrobial activity, being the most potent against Aspergillus fumigatus with minimum inhibitory concentration of 0.31 mg/mL. Also, the extract was able to inhibit biofilm formed by Candida species and to inhibit biofilm formation by Staphylococcus aureus. Obtained results revealed that the extract has potential to interfere with the cell membrane permeability of Candida albicans and to suppress production of virulence factor staphyloxanthin in S. aureus. Furthermore, the extract inhibited the activity of a-amylase which is one of the therapeutic targets for diabetes type II. Also, the antiproliferative effect of the extract was demonstrated on human cancer cell lines, while the extract did not exhibit any cytotoxic effect on primary human cells. Based on the obtained results, P. fruticosa could be an interesting source of biologically active compounds even after long term storage
Molecular markers in systemic sclerosis: candidate genes and therapeutic modalities
Get PDFSistemska skleroza je izuzetno složeno i heterogeno oboljenje u pogledu kliničke prezentacije, progresije
bolesti, zahvaćenosti organa i ishodu. Na individualnom nivou, ova složenost predstavlja izazov u predviđanju
progresije bolesti i lečenju. U ovom radu će biti prikazana dosadašnja saznanja o molekularnim
markerima u sistemskoj sklerozi, signalnim putevima koji su uključeni u patogenezu, kao i njihova uloga u
kliničkoj prezentaciji bolesti. Biće predstavljeni novi terapijski pristupi u lečenju, sa posebnim osvrtom na biološku
terapiju zasnovanu na genima kandidatima, kao i mogućnosti koje regenerativna medicina nudi u
lečenju ove autoimune sistemske bolesti.Systemic sclerosis is an extremely complex and heterogeneous disease in terms of clinical presentation, disease
progression, organ involvement, and outcome. At the individual level, this complexity presents a challenge
in predicting disease progression and treatment. This paper will present the current knowledge about
molecular markers in systemic sclerosis, signaling pathways involved in pathogenesis, as well as their role
in the clinical presentation of the disease. New therapeutic approaches in treatment will be presented, with
special emphasis on biological therapy based on candidate genes, as well as the possibilities that regenerative
medicine offers in the treatment of this autoimmune systemic disease
De Novo Transcriptome Sequencing of Ramonda serbica: Identification of Late Embryogenesis Abundant Proteins
Get PDFAn extreme loss of cellular water or desiccation (5-10% of relative water content) leads to protein denaturation, aggregation and degradation, and affects the fluidity of membrane lipids resulting in loss of membrane integrity [1]. The essential constituents of vegetative desiccation tolerance in so-called resurrection plants are late embryogenesis abundant proteins (LEAPs). This heterogeneous group of anhydrobiosis-related intrinsically disordered proteins forms mostly random conformation when fully hydrated, turning into compact α-helices during desiccation [2]. Based on in vitro studies, LEAPs can be involved in water binding, ion sequestration, stabilization of both membrane and enzymes during freezing or drying, while by forming intracellular proteinaceous condensates they increase structural integrity and intracellular viscosity of cells during desiccation.Here, we identify 164 members of LEA gene family in endemic and relict resurrection species Ramonda serbica by integrating previously done de novo transcriptome and homologues protein motifs. Identified LEAPs were classification into six groups according to Protein family (PFAM) database and the most populated group was LEA4 containing 47% of total identified LEAPs. By using four secondary structure predictors, we showed that this group exhibited a high propensity to form amphipathic α-helices (81% of total sequence length is predicted to form α-helical structure). This implies that charged residues might be exposed to the solvent, while hydrophobic amino acids might interact with lipid bilayers or with other target proteins in the cell. In addition, as predicted by several bioinformatic tools, more than 70% of identified LEAPs were found to be highly disordered (~64%). Structural characterization of LEAPs is a key to understand their function and regulation of their intrinsic structural disorder-to-order transition during desiccation. These findings will promote transformative advancements in various fields, such as the development of new strategies in neurodegenerative disorders, cell preservation technology and the improvement of crop drought tolerance.Book of Abstracts; Belgrade BioInformatics Conference 2021, 21-25 June 2021, Vinča, Serbi
Twenty different late embryogenesis abundant proteins (LEAPs) accumulate in desiccated Ramonda serbica leaves
Get PDFResurrection plant Ramonda serbica Panc. survives desiccation for a long period and fully recovers metabolic functions already within one day upon watering [1]. Besides osmotic stress, desiccation provokes the accelerated generation of reactive oxygen species. The aim of our study was obtaining more insight into the mechanisms of desiccation tolerance in R. serbica by TMT labelled comparative quantitative proteomics of hydrated (HL) and desiccated leaves (DL). After de novo transcriptome analysis, 189456 transcripts with 189003 unigenes were annotated with seven common databases. Proteomic analysis allowed for the relative quantification of 895 different protein groups, 321 with a statistically significant difference in abundance between FL and DL. Among them, 25% referred to chloroplast and almost the same percentage were associated with desiccation and oxidative stress. Almost all differentially abundant proteins related to photosynthetic processes were down-regulated in DL, while those required for protein translation were more abundant in HL. Within differentially abundant proteins involved in antioxidative defence, the levels of enzymes involved in ascorbate-glutathione cycle, peroxiredoxins, Fe and Mn superoxide dismutase (SOD) were all reduced in DL, while germin-like proteins, three Cu/Zn SOD isoforms and polyphenol oxidases were more abundant in DL compared with HL. The protein family with the highest number of members showing the greatest accumulation upon desiccation comprised twenty different late embryogenesis abundant proteins (LEAPs), similarly as found by differential transcriptomic analysis. Taken together, our results imply a key role of LEAPs and Cu/Zn SOD in protective mechanism against desiccation in R. serbica, that may have significant implications on drought-related studies of crops grown in arid areas. This work was supported by the Science Fund of the Republic of Serbia (PROMIS project LEAPSyn-SCI, grant number 6039663). M.V. wishes to acknowledge the support of COST Action BM1405 for approving STSM in Padua during 2017 and 2018.Abstract:[https://imagine.imgge.bg.ac.rs/handle/123456789/1835
Pharmacogenomics landscape of COVID-19 therapy response in Serbian population and comparison with worldwide populations
No full textDrug repurposing became important when treating COVID-19 patients. With limited time to test individual pharmacogenomics markers, population pharmacogenomics could help in predicting a higher risk of developing adverse reactions and treatment failure in COVID-19 patients. Aim of our study was to identify pharmacogenomics markers associated with drugs recommended for COVID-19 treatment, chloroquine/hydroxychloroquine, azithromycin, lopinavir and ritonavir, in Serbian population and other world populations. Genotype information of 143 individuals of Serbian origin was extracted from database previously obtained using TruSight One Gene Panel (Illumina). Genotype data of individuals from different world populations were extracted from the 1000 Genome Project. Fisher’sexacttest was used for comparison of allele frequencies. We have identified 11 potential pharmacogenomics markers in 7 pharmacogenes relevant for COVID-19 treatment. Based on high alterative allele frequencies in population and the functional effect of the variants, ABCB1 rs1045642 and rs2032582 could be relevant for reduced clearance of azithromycin, lopinavir and ritonavir drugs and UGT1A7 rs17868323 for hyperbilirubinemia in ritonavir treated COVID-19 patients in Serbian population. SLCO1B1 rs4149056 is a potential marker of lopinavir response, especially in Italian population. Our results confirmed that pharmacogenomics profile of African population is different from the rest of the world. Considering population specific pharmacogenomics landscape, preemptive testing for pharmacogenes relevant for drugs used in COVID-19 treatment could contribute to better understanding of the inconsistency in therapyresponseandcouldbeappliedtoimprovetheoutcome of the COVID-19 patients. Ministry of Education, Science and Technological Development Republic of Serbia, (EB: 451-03-68/ 2020-14/200042) supported this work
Shotgun metagenomics reveals differences in antibiotic resistance genes among bacterial communities in Western Balkans glacial lakes sediments
Get PDFLong-term overuse of antibiotics has driven the propagation and spreading of antibiotic resistance genes (ARGs) such as efflux pumps in the environment, which can be transferred to clinically relevant pathogens. This study explored the abundance and diversity of ARGs and mobile genetic elements within bacterial communities from sediments of three Western Balkans glacial lakes: Plav Lake (high impact of human population), Black Lake (medium impact of human population) and Donje Bare Lake (remote lake, minimal impact of human population) via shotgun metagenomics. Assembled metagenomic sequences revealed that Resistance-Nodulation-Division (RND) efflux pumps genes were most abundant in metagenome from the Plav Lake. The Integron Finder bioinformatics tool detected 38clusters ofattCsiteslackingintegron-integrases (CALIN) elements: 20 from Plav Lake, four from Black Lake and 14 from Donje Bare Lake. A complete integron sequence was recovered only from the assembled metagenome from Plav Lake. Plasmid contents within the metagenomes were similar, with proportions of contigs being plasmid-related: 1.73% for Plav Lake, 1.59% for Black Lake and 1.64% for Donje Bare Lake. The investigation showed that RNDs and mobile genetic elements content correlated with human population impact
Cloning and expression profiling of muscle regulator ANKRD2 in domestic chickenGallus gallus
No full textStriated muscle signaling protein and transcriptional regulator ANKRD2 participates in myogenesis, myogenic differentiation, muscle adaptation and stress response. It is preferentially expressed in slow, oxidative fibers of mammalian skeletal muscle. In this study, we report on characterization of chickenANKRD2. The chickenANKRD2coding region contains 1002 bp and encodes a 334-amino acid protein which shares approximately 58% identity with human and mouse orthologs, mostly in the conserved region of ankyrin repeats. Comprehensive analysis of theANKRD2gene and protein expression in adult chicken demonstrated its predominant expression in red muscles of thigh and drumstick, compared to white muscle. It was not detected in heart and white pectoral muscle. Uneven expression of ANKRD2 in chicken skeletal muscles, observed by immunohistochemistry, was attributed to its selective expression in slow, oxidative, type I and fast, oxidative-glycolytic, type IIA myofibers. Association of chickenANKRD2with phenotypic differences between red and white muscles points to its potential role in the process of myofiber-type specification. In addition to expression in slow oxidative myofibers, as demonstrated for mammalian protein, chicken ANKRD2 was also detected in fast fibers with mixed oxidative and glycolytic metabolism. This finding suggests thatANKRD2is responsive to metabolic differences between types of avian myofibers and orientates future studies towards investigation of its role in molecular mechanisms of myofiber-type-specific gene expression