The CAR mRNA-Interaction Surface is a Zipper Extension of the Ribosome A Site

The ribosome CAR interaction surface behaves like an extension of the decoding center A site and has H-bond interactions with the +1 codon that is next in line to enter the A site. Through molecular dynamics simulations, we investigated the codon sequence specificity of this CAR-mRNA interaction and discovered a strong preference for GCN codons, suggesting that there may be a sequence-dependent layer of translational regulation dependent on the CAR interaction surface. Dissection of the CAR-mRNA interaction through nucleotide substitution experiments showed that the first nucleotide of the +1 codon dominates over the second nucleotide position, consistent with an energetically favorable zipper-like activity that emanates from the A site through the CAR-mRNA interface. The +1 codon/CAR interaction is also affected by the identity of nucleotide 3 of +1 GCN codons which influences the stacking of G and C. Clustering analysis suggests that the A site decoding center adopts different neighborhood substates that depend on the identity of the +1 codon.

Antibiotic resistance of Mycoplasma Synoviae strains isolated in China from 2016 to 2019

Background In the past decade, Mycoplasma synoviae (M. synoviae) infection has become widely prevalent in China, has caused serious economic losses and has become one of the most important diseases in the chicken industry. Medication is a general approach for the control of M. synoviae infection, but antibiotics are sometimes ineffective in clinical practice. To investigate the sensitivity of M. synoviae to antimicrobials commonly used in the treatment of M. synoviae infection, the antibiotic susceptibility of 32 M. synoviae strains isolated from China from 2016 to 2019 were determined using the minimum inhibitory concentration (MIC) method. Results All isolates had low MIC values for the combination of lincomycin and spectinomycin, pleuromutilin, and macrolides. However, the M. synoviae isolates displayed variance in MICs for doxycycline hydrochloride with a range of 0.25 to 8 μg/mL, and oxytetracycline hydrochloride with a range of 0.5 to 8 μg/mL. Three and one M. synoviae isolates showed intermediate MIC values to doxycycline hydrochloride and oxytetracycline hydrochloride, respectively. High MIC values for enrofloxacin were detected in all isolates with MICs ranging from 4 to 32 μg/mL. Furthermore, comparison of the parC QRDR identified a mutation at nucleotide position 254 (C254T) resulting in a Thr 85 Ile amino acid change in all M. synoviae isolates and the reference strain ATCC 25204 being resistant to enrofloxacin. Moreover, mutations at Glu 804 Gly and Thr 686 Ala of gyrA QRDR were identified in all M. synoviae isolates and ATCC 25204. The mutation in the QRDR of the parE gene resulted in amino acid changes at positions 197 (Pro to Ser) in 27/32 M. synoviae isolates. Conclusion Three nonsynonymous mutations in gyrA and parE were first identified to be related to enrofloxacin resistance. Our results showed that M. synoviae resistance to enrofloxacin is widespread.

Characterization of a Chickpea Mutant Resistant to Phelipanche aegyptiaca Pers. and Orobanche crenata Forsk

Chickpea (Cicer arietinum L.) is a major pulse crop in Israel grown on about 3000 ha spread, from the Upper Galilee in the north to the North-Negev desert in the south. In the last few years, there has been a gradual increase in broomrape infestation in chickpea fields in all regions of Israel. Resistant chickpea cultivars would be simple and effective solution to control broomrape. Thus, to develop resistant cultivars we screened an ethyl methanesulfonate (EMS) mutant population of F01 variety (Kabuli type) for broomrape resistance. One of the mutant lines (CCD7M14) was found to be highly resistant to both Phelipanche aegyptiaca and Orobanche crenata. The resistance mechanism is based on the inability of the mutant to produce strigolactones (SLs)—stimulants of broomrape seed germination. LC/MS/MS analysis revealed the SLs orobanchol, orobanchyl acetate, and didehydroorobanchol in root exudates of the wild type, but no SLs could be detected in the root exudates of CCD7M14. Sequence analyses revealed a point mutation (G-to-A transition at nucleotide position 210) in the Carotenoid Cleavage Dioxygenase 7 (CCD7) gene that is responsible for the production of key enzymes in the biosynthesis of SLs. This nonsense mutation resulted in a CCD7 stop codon at position 70 of the protein. The influences of the CCD7M14 mutation on chickpea phenotype and chlorophyll, carotenoid, and anthocyanin content were characterized.

Ceftazidime/Avibactam-Resistant Klebsiella pneumoniae subsp. pneumoniae Isolates in a Tertiary Italian Hospital: Identification of a New Mutation of the Carbapenemase Type 3 (KPC-3) Gene Conferring Ceftazidime/Avibactam Resistance

Several Klebsiella pneumoniae carpabenemase (KPC) gene mutations are associated with ceftazidime/avibactam (CAZ-AVI) resistance. Here, we describe four Klebsiella pneumoniae subsp. pneumoniae CAZ-AVI-resistant clinical isolates, collected at the University Hospital of Tor Vergata, Rome, Italy, from July 2019 to February 2020. These resistant strains were characterized as KPC-3, having the transition from cytosine to thymine (CAC-TAC) at nucleotide position 814, with histidine that replaces tyrosine (H272Y). In addition, two different types of KPC gene mutations were detected. The first one, common to three strains, was the D179Y (G532T), associated with CAZ-AVI resistance. The second mutation, found only in one strain, is a new mutation of the KPC-3 gene: a transversion from thymine to adenine (CTG-CAG) at nucleotide position 553. This mutation causes a KPC variant in which glutamine replaces leucine (Q168L). None of the isolates were detected by a rapid immunochromatographic assay for detection of carbapenemase (NG Biotech, Guipry, France) and were unable to grow on a selective chromogenic medium Carba SMART (bioMerieux, Firenze, Italy). Thus, they escaped common tests used for the prompt detection of Klebsiella pneumoniae KPC-producing.

In depth analysis of Cyprus-specific mutations of SARS-CoV-2 strains using computational approaches

Background This study aims to characterize SARS-CoV-2 mutations which are primarily prevalent in the Cypriot population. Moreover, using computational approaches, we assess whether these mutations are associated with changes in viral virulence. Methods We utilize genetic data from 144 sequences of SARS-CoV-2 strains from the Cypriot population obtained between March 2020 and January 2021, as well as all data available from GISAID. We combine this with countries’ regional information, such as deaths and cases per million, as well as COVID-19-related public health austerity measure response times. Initial indications of selective advantage of Cyprus-specific mutations are obtained by mutation tracking analysis. This entails calculating specific mutation frequencies within the Cypriot population and comparing these with their prevalence world-wide throughout the course of the pandemic. We further make use of linear regression models to extrapolate additional information that may be missed through standard statistical analysis. Results We report a single mutation found in the ORF1ab gene (nucleotide position 18,440) that appears to be significantly enriched within the Cypriot population. The amino acid change is denoted as S6059F, which maps to the SARS-CoV-2 NSP14 protein. We further analyse this mutation using regression models to investigate possible associations with increased deaths and cases per million. Moreover, protein structure prediction tools show that the mutation infers a conformational change to the protein that significantly alters its structure when compared to the reference protein. Conclusions Investigating Cyprus-specific mutations for SARS-CoV-2 can lead to a better understanding of viral pathogenicity. Researching these mutations can generate potential links between viral-specific mutations and the unique genomics of the Cypriot population. This can not only lead to important findings from which to battle the pandemic on a national level, but also provide insights into viral virulence worldwide.

Comprehensive Genomic Characterization of ASXL1 C.1934dupG (p.G646fs*12) Versus Other ASXL1 mutations in Myeloid Neoplasia

Introduction: ASXL1 mutations are frequently seen across the clinical spectrum of myeloid neoplasia. The most commonly identified ASXL1 mutation represents a single base duplication within an 8-guanine repeat at nucleotide position 1934 (c.1934dupG). Due to technical limitations of sequencing homopolymer regions, the ASXL1 c.1934dupG variant has been identified as potential artifact in some sequencing assays, though modern next generation sequencing assays and bioinformatics pipelines can generally accurately detect this mutation. However, a comprehensive comparison of ASXL1 c.1934dupG mutations versus non-c.1934dupG ASXL1 mutations have not been performed to date. Thus, we sought to explore a large dataset to determine if any biologic differences existed between these two groups. Methods: Comprehensive genomic profiling by FoundationOne ®Heme testing was performed on patient samples with known or suspected myeloid neoplasms (MN). All MN patients ≥18 years old with 1 or more mutation were identified by internal database query. Patients were categorized as acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), non-chronic myeloid leukemia myeloproliferative neoplasms (MPN), or MDS/MPN overlap based on mutation and outside clinical and pathology data. Mutations with variant allele fractions (VAF) >1% were included for analysis, except for the ASXL1 c.1934dupG variant, which was only reported if the VAF was ≥15%. Fisher's exact tests were used to evaluate proportional differences between categorical variables, and Mann-Whitney U tests were used for comparisons of continuous variables. Results: Truncating ASXL1 mutations were identified in 1,414 included patients, occurring in 18% of AML and 26% of chronic myeloid neoplasms. Twenty-eight (2%) patients had multiple ASXL1 mutations, and ASXL1 was the sole mutated gene in 52 patients (4%). The most common ASXL1 mutation was c.1934dupG (Figure 1A), and this was the sole or dominant ASXL1 mutation in 520 cases (37%). The remaining 894 patients (63%) had one or more mutations at other sites in the ASXL1 gene (ASXL1other), with p.E635Rfs, p.R693*, and codon 591 mutations being the most common. There were no significant differences in age, sex, or ancestry signatures between ASXL1c.1934dupG and ASXL1other. We noted slightly fewer ASXL1c.1934dupG mutations in patients with MDS (ASXL1c.1934dupG: ASXL1other 0.48:1) compared to AML (0.65:1, p = 0.03) and MPN (0.60:1, p = 0.01) and those in whom ASXL1 was the sole mutation (Figure 1B). However, these trends may have been due to VAF-based reporting thresholds, as ASXL1 VAFs were lower in singly mutated patients and those with an MDS diagnosis classification. Comparison of co-mutated genes with VAFs ≥15% between ASXL1c.1934dupG and ASXL1other revealed no significant difference in median non-ASXL1 mutations (each median 4, IQR 2-5, p = 0.74). When individual genes were assessed, co-mutation rates of STAG2 (p = 0.01) and KMT2A (p = 0.02) were higher in ASXL1c.1934dupG MNs, while SETBP1 (p = 0.01) mutations were more common with ASXL1other. In all MNs, the absolute differences in the frequency of mutations in ASXL1c.1934dupG versus ASXL1other were small. However, some differences emerged within phenotypic subgroups (Figure 1C). For instance, KMT2A rearrangements and STAG2 mutations were strongly associated with ASXL1c.1934dupG in MDS/MPN and MPN, with ASXL1c.1934dupG: ASXL1other ratios of 5:1 (p = 0.03) and 9:1 (p < 0.001), respectively. In contrast, AML patients with TP53 or SETBP1 mutations had a significantly higher mutation rate in ASXL1other (TP53: 11% vs. 3% in ASXL1c.1934dupG, p < 0.01; SETBP1: 14% vs. 7%, p=0.04). We further identified that other specific ASXL1 mutations were more commonly co-mutated in AML with TP53 (ASXL1 p.R693*, p < 0.001) or SETBP1 (ASXL1 p.R404*, p < 0.001). Conclusion: Our results confirm the ASXL1 c.1934dupG variant occurs in a similar patient population to other ASXL1 mutations, and further supports its pathogenicity in myeloid neoplasia. Subset analysis suggests that ASXL1c.1934dupG and ASXL1other may be associated with certain phenotypic and co-mutational tendencies. Thus, ASXL1 mutation site may be an important variable in some patients and should be considered in future mechanistic and clinical studies. Further study is warranted to determine whether clinical outcomes are affected by different ASXL1 mutations. Figure 1 Figure 1. Disclosures Haberberger: Foundation Medicine, Inc.: Current Employment. Ferguson: Foundation Medicine Inc: Current Employment, Other: ownership.

An Intergenic Noncoding Chromosome 18 Variant Suppresses Lethal Thrombosis in Mice By Normalizing Blood Coagulation and Reducing Platelet Reactivity

Background: Factor V Leiden (FVL) is a common thrombosis susceptibility variant in humans. It is incompletely penetrant; this indicates that there are modifiers of FVL that alter thrombosis susceptibility. We used mouse models of FVL (F5L) and heterozygous tissue factor pathway inhibitor deficiency (Tfpi+/-), to identify a perinatal lethal genetic interaction when mice inherited F5L/L Tfpi+/-. This phenotype was used as the basis for a sensitized genome wide ENU mutagenesis screen to identify mutations suppressing lethal thrombosis in F5L/L Tfpi+/- mice. From this screen, we generated multiple independent lines of thrombosuppressed mice, called MF5L, for Modifier of F5L. MF5L16 was a large, highly penetrant (77.2%), multigenerational pedigree containing 136 viable F5L/L Tfpi+/- mice. Aims: In the present study, we aimed to identify and functionally characterize the thrombosuppressor mutation present in MF5L16. Methods: Genomic analyses: We performed whole genome sequencing (WGS) on four MF5L16 F5L/L Tfpi+/- mice. We used comparative bioinformatic analyses to identify variants inherited by all four mice and compiled these variants into candidate variant list. PCR and Sanger sequencing were used to analyze the 136 F5L/L Tfpi+/- mice for inheritance of each of the candidate variants. Functional analyses: We performed biochemical blood coagulation and platelet assays of blood from the Chr18 A mice . Complete blood counts were measured using the Advia 2120 with settings optimized for C57BL/6 mouse blood. Platelet aggregation studies were performed using the Roche Multiplate Aggregometer with ADP and type 1 collagen as the aggregating agents. Results: We analyzed four MF5L16 mice by WGS and identified seven spontaneous mutations that arose in our F5L/L breeding colony that were introduced into MF5L16. Importantly, no coding variants were linked to these variants. Analysis of these seven mutations in all 136 MF5L16 F5L/L Tfpi+/- mice revealed a significant association between a Chromosome 18 intergenic variant (Chr18 G to A, Chr18 A) and F5L/L Tfpi+/- mouse survival (p=0.003). To re-create the suppression of the lethal F5L/L Tfpi+/- phenotype, we bred F5+/L Tfpi+/- Chr18 +/A triple heterozygous mice to F5L/L Chr18 A/A mice to observe the effects of Chr18 A on F5L/L Tfpi+/- mouse survival. Out of 109 mice from this cross, two F5L/L Tfpi+/- Chr18 +/A mice were produced (expected ratio ~1:8). This suggests that the Chr18 A variant suppresses F5L/L Tfpi+/- lethal thrombosis at ~15% penetrance. Complete blood count analysis on Chr18 +/+,Chr18 +/A, and Chr18 A/A mice determined that Chr18 A/A mice had reduced platelet count and distribution width and increased variability in red blood cell (RBC) mean corpuscular volume (n≥4; p<0.05). The Chr18 A/A mice did not display differences in PT or aPTT assays, but had significantly reduced platelet aggregation velocity when stimulated by both ADP and collagen agonists (n≥4; p=0.0002). Additionally, blood smears revealed the presence of poikilocytic RBCs in the Chr18 A/A mice. Conclusions and future directions: Our results establish that a noncoding intergenic Chr18 variant at nucleotide position 62,970,011 (G>A, Chr18 A) contributes to thrombosuppression by reducing platelet reactivity. The observed platelet and RBC phenotypes suggest that a major mechanism of Chr18 A thrombosuppression could be through regulation of gene expression in cells of the myeloid lineage. We are performing additional platelet and blood coagulation analyses to refine the phenotypic differences due to the Chr18 A variant. Comparative transcriptomic analyses are also being performed to identify the genetic pathways involved. Understanding the mechanism in which this intergenic mutation suppresses thrombosis could provide insights into human thrombosis regulation. Disclosures No relevant conflicts of interest to declare.

​POU1F1 Gene Polymorphism and its Associations with Body Weight of Osmanabadi Goat

Background: Osmanabadi is the premium meat type goat breed of Maharashtra, known for its adaptability and reproductive efficiency. Identification of superior germplasm and incorporating them into breeding programme is the need of hour for augmenting productivity of Osmanabadi as well as other indigenous goat breeds. Hence, the present research was aimed to study Pituitary Specific Transcription Factor I (POU1F1) gene polymorphism and its associations with body weight of Osmanabadi goat. Methods: Genomic DNA was extracted from 217 blood samples of randomly selected Osmanabadi goats. The 450 bp fragment of POU1F1 gene encompassing exon-6 and its flanking region (3’UTR) was PCR amplified. The PCR amplicon was subjected to RFLP using AluI and PstI restriction enzymes to identify polymorphism at nucleotide position 174 (T greater than C) in exon 6 and at nucleotide position 110 (T greater than C) in the 3’UTR of POU1F1 gene, respectively. The association of the observed allelic variants / genotypes with body weight of the animals was performed using One-way ANOVA. Result: The AluI PCR-RFLP of 450 bp amplicon of POU1F1 gene in Osmanabadi goats revealed polymorphism with three different genotypes viz., ‘CC’, ‘TT’ and ‘TC’. The observed frequencies for TT, TC and CC genotypes were 0.604, 0.355 and 0.041 respectively. The frequencies of T and C alleles were found to be 0.781 and 0.219, respectively. However, PstI PCR-RFLP revealed monomorphic 3’UTR of POU1F1 gene with single TT genotype. We observed significant differences (P less than 0.01) between various genotypes at AluI exon-6 locus of POU1F1 gene and body weights at birth, 3 and 6 months of Osmanabadi goat. Highest body weight was recorded for TT genotype followed by TC and CC genotypes in all three age groups. Our findings indicate that the AluI PCR-RFLP locus of the exon-6 of POU1F1 gene can be used as a potent molecular marker for selection of superior stock of Osmanabadi goats.

Research on Phylogenetic Relationship of Lotus Populations Collected in Thua Thien Hue Province, Vietnam based on the Chloroplast Genome by DNA Barcode

Background: The DNA barcoding is currently an effective and widely used tool that enables rapid and accurate identification of plant species. Methods: DNA barcoding of 9 chloroplast genes (rbcL, matK, trnH-psbA, accD-psaI, ndhA, psbE-petL, Rpl32-trnL, trnW-psaJ, trnSGCU-trnGGCC) were used to provide the theoretical basis for species identification, genetic diversity analysis of lotus population collected in Thua Thien Hue province, Vietnam. Universal primers were used and sequence products were analyzed using the MEGA X program. Result: The results showed that high levels of haplotype diversity (Hd), ranging from 0.618-0.869 and low levels of nucleotide diversity (Pi), ranging from 0.180 × 10-3-3.280 × 10-3 base on a total of nine gene regions of chloroplast genome. The neutrality tests show an excess of rare nucleotide position variations in individuals’ white lotus and derived haplotypes recent expansion. While the evolution of the individuals in the pink lotus may have to decrease. The phylogenetic analyses indicated that combined sequences were not insufficient to make a difference to the DNA barcoding in the individual’s lotus of the N. nucifera species this is in the study. The standardized and accurate barcode information of lotus is provided for researchers. It lays the foundation for the conservation, evaluation, innovative utilization and protection of Nelumbonaceae germplasm resources.

Potential Targets for Evaluation of Sugarcane Yellow Leaf Virus Resistance in Sugarcane Cultivars: In silico Sugarcane miRNA and Target Network Prediction

The Sugarcane yellow leaf virus (SCYLV) is associated with sugarcane yellow leaf disease (SCYLD) and is considered to be the most economically deleterious emerging pathogen that represents a potential threat and danger to sugarcane cultivation in China. Over the last two decades, high genetic diversity in the SCYLV genotypes was observed worldwide, with a greater chance of YLD incidence for sugarcane injury. SCYLV infection has significantly damaged its economic traits and is responsible for substantial losses in biomass production in sugarcane cultivars. This study aims to identify and comprehensively analyze sugarcane microRNAs (miRNAs) as therapeutic targets against SCYLV using plant miRNA prediction tools. Mature sugarcane miRNAs are retrieved and are used for hybridization of the SCYLV. A total of seven common sugarcane miRNAs were selected based on consensus genomic positions. The biologically significant, top ranked ssp-miR528 was consensually predicted to have a potentially unique hybridization site at nucleotide position 4162 for targeting the ORF5 of the SCYLV genome; this was predicted by all the algorithms used in this study. Then, the miRNA–mRNA regulatory network was generated using the Circos algorithm, which was used to predict novel targets. There are no acceptable commercial SCYLV-resistant sugarcane varieties available at present. Therefore, the predicted biological data offer valuable evidence for the generation of SCYLV-resistant sugarcane plants.