Testing assembly strategies of Francisella tularensis genomes to infer an evolutionary conservation analysis of genomic structures

Background We benchmarked sequencing technology and assembly strategies for short-read, long-read, and hybrid assemblers in respect to correctness, contiguity, and completeness of assemblies in genomes of Francisella tularensis. Benchmarking allowed in-depth analyses of genomic structures of the Francisella pathogenicity islands and insertion sequences. Five major high-throughput sequencing technologies were applied, including next-generation “short-read” and third-generation “long-read” sequencing methods. Results We focused on short-read assemblers, hybrid assemblers, and analysis of the genomic structure with particular emphasis on insertion sequences and the Francisella pathogenicity island. The A5-miseq pipeline performed best for MiSeq data, Mira for Ion Torrent data, and ABySS for HiSeq data from eight short-read assembly methods. Two approaches were applied to benchmark long-read and hybrid assembly strategies: long-read-first assembly followed by correction with short reads (Canu/Pilon, Flye/Pilon) and short-read-first assembly along with scaffolding based on long reads (Unicyler, SPAdes). Hybrid assembly can resolve large repetitive regions best with a “long-read first” approach. Conclusions Genomic structures of the Francisella pathogenicity islands frequently showed misassembly. Insertion sequences (IS) could be used to perform an evolutionary conservation analysis. A phylogenetic structure of insertion sequences and the evolution within the clades elucidated the clade structure of the highly conservative F. tularensis.

Molecular Modelling of nsSNPs in GABRA2 Gene in Epilepsy and Study of Their Impact On Structure and Stability of GABRA2 Protein

Epilepsy is a neurological condition characterized by abrupt, unprovoked, and recurrent seizures that are unpredictable in frequency. The objective of this analysis was to identify novel non-synonymous polymorphisms in the GABRA2 gene and determine their effect on protein structure and stability. Most pathogenic/deleterious nsSNPs were predicted using six different bioinformatic tools. Mutpred2, Mupro was used to check the impact of identified nsSNPS on protein structure and stability. The pathogenic score of SNPs was predicted using the FATHMM tool. The CONSURF webserver was used for conservation analysis of pathogenic SNPs.3-D structure of the protein was realized using SWISS-MODEL and residue position in protein visualized by Lite Mol. GeneMANIA and STRING databases were used to predict the function of interlinked gene interactions. Out of 228 nsSNPs retrieved from the dbSNP database, we identified a novel missense variant, F285S (rs41310789), as most deleterious and its possible association with epilepsy syndrome focal epilepsy. Stability and conservation analysis results interpret rs41310789 present in evolutionarily conserved regions of a gene and affect its structure. This analysis provides information regarding the impact of nsSNPs that might affect the structure and activity of GABRA2 protein. Thus, these coding variants should be taken into scrutiny while genetic screening of epileptic patients.

Molecular dynamics and protein frustration analysis of human fused in Sarcoma protein variants in Amyotrophic Lateral Sclerosis type 6: An In Silico approach

Amyotrophic lateral sclerosis (ALS) is the most frequent adult-onset motor neuron disorder. The disease is characterized by degeneration of upper and lower motor neurons, leading to death usually within five years after the onset of symptoms. While most cases are sporadic, 5%-10% of cases can be associated with familial inheritance, including ALS type 6, which is associated with mutations in the Fused in Sarcoma (FUS) gene. This work aimed to evaluate how the most frequent ALS-related mutations in FUS, R521C, R521H, and P525L affect the protein structure and function. We used prediction algorithms to analyze the effects of the non-synonymous single nucleotide polymorphisms and performed evolutionary conservation analysis, protein frustration analysis, and molecular dynamics simulations. Most of the prediction algorithms classified the three mutations as deleterious. All three mutations were predicted to reduce protein stability, especially the mutation R521C, which was also predicted to increase chaperone binding tendency. The protein frustration analysis showed an increase in frustration in the interactions involving the mutated residue 521C. Evolutionary conservation analysis showed that residues 521 and 525 of human FUS are highly conserved sites. The molecular dynamics results indicate that protein stability could be compromised in all three mutations. They also affected the exposed surface area and protein compactness. The analyzed mutations also displayed high flexibility in most residues in all variants, most notably in the interaction site with the nuclear import protein of FUS.

Novel FGFR1 Variants Are Associated with Congenital Scoliosis

FGFR1 encodes a transmembrane cytokine receptor, which is involved in the early development of the human embryo and plays an important role in gastrulation, organ specification and patterning of various tissues. Pathogenic FGFR1 variants have been associated with Kallmann syndrome and hypogonadotropic hypogonadism. In our congenital scoliosis (CS) patient series of 424 sporadic CS patients under the framework of the Deciphering disorders Involving Scoliosis and COmorbidities (DISCO) study, we identified four unrelated patients harboring FGFR1 variants, including one frameshift and three missense variants. These variants were predicted to be deleterious by in silico prediction and conservation analysis. Signaling activities and expression levels of the mutated protein were evaluated in vitro and compared to that of the wild type (WT) FGFR1. As a result, the overall protein expressions of c.2334dupC, c.2339T>C and c.1261A>G were reduced to 43.9%, 63.4% and 77.4%, respectively. By the reporter gene assay, we observed significantly reduced activity for c.2334dupC, c.2339T>C and c.1261A>G, indicating the diminished FGFR1 signaling pathway. In conclusion, FGFR1 variants identified in our patients led to only mild disruption to protein function, caused milder skeletal and cardiac phenotypes than those reported previously.

Short communication: Production, distribution and conservation analysis of Cherax crayfish endemic to Papua and West Papua Provinces, Indonesia

Widyasari F, Sayuti M, Salampessy RBS. 2021. Short communication: Production, distribution and conservation analysis of Cherax crayfish endemic to Papua and West Papua Provinces, Indonesia. Biodiversitas 22: 3271-3276. Freshwater crayfish is a species from the Parastacidae family that is indigenous to Papua and West Papua Indonesia. This study analyzes the amount of production, economic value, distribution, and conservation of freshwater crayfish endemic to Papua and West Papua. The production data were obtained from Stasiun Karantina Ikan or Fish Quarantine Inspection Agency in Papua and West Papua, Indonesia. Data regarding the price of Cherax crayfish were determined based on interviews with freshwater crayfish sellers. Furthermore, the distribution of freshwater crayfish was explained based on a literature review, while the data of its conservation were retrieved from the IUCN Red List of Threatened Species. Cherax crayfish caught from wild populations in West Papua is known higher than in Papua. Increased sales revenue could support the economy of the local community. There have been 25 species of freshwater crayfish identified, nine of which were from West Papua and 16 from Papua. Three species were under Endangered (EN), Least Concern (LC), Vulnerable (VU) status, respectively. Four species were under Data Deficient (DD) status, while the rest were unidentified. Overfishing of Cherax crayfish causes decline Cherax crayfish stock in the wild, future researchers are expected to conduct more specific studies that include relevant stakeholders regarding the conservation of Cherax crayfish that are endemic to Papua and West Papua.

Identification of Novel Candidate CD8+ T Cell Epitopes of the SARS-CoV2 with Homology to Other Seasonal Coronaviruses

Cross-reactive T cell immunity to seasonal coronaviruses (HCoVs) may lead to immunopathology or protection during SARS-CoV2 infection. To understand the influence of cross-reactive T cell responses, we used IEDB (Immune epitope database) and NetMHCpan (ver. 4.1) to identify candidate CD8+ T cell epitopes, restricted through HLA-A and B alleles. Conservation analysis was carried out for these epitopes with HCoVs, OC43, HKU1, and NL63. 12/18 the candidate CD8+ T cell epitopes (binding score of ≥0.90), which had a high degree of homology (>75%) with the other three HCoVs were within the NSP12 and NSP13 proteins. They were predicted to be restricted through HLA-A*2402, HLA-A*201, HLA-A*206, and HLA-B alleles B*3501. Thirty-one candidate CD8+ T cell epitopes that were specific to SARS-CoV2 virus (<25% homology with other HCoVs) were predominantly identified within the structural proteins (spike, envelop, membrane, and nucleocapsid) and the NSP1, NSP2, and NSP3. They were predominantly restricted through HLA-B*3501 (6/31), HLA-B*4001 (6/31), HLA-B*4403 (7/31), and HLA-A*2402 (8/31). It would be crucial to understand T cell responses that associate with protection, and the differences in the functionality and phenotype of epitope specific T cell responses, presented through different HLA alleles common in different geographical groups, to understand disease pathogenesis.