Amino Acid Sequence Divergence Research Articles

Characterization of Cucumber Mosaic Virus Isolated from Passion Fruit (<ii>Passiflora edulis) in Korea

In July 2020, total RNA was extracted from passion fruit (<i>Passiflora edulis</i>) leaves showing virus symptoms such as chlorotic spots and vein banding in Haenam, South Korea. Cucumber mosaic virus (CMV)-HN2 was identified through reverse transcription polymerase chain reaction and sequencing analysis. To confirm the biological characteristics of the CMV infecting passion fruit, 10 indicator plants were inoculated with CMV-HN2, and the results showed a typical CMV symptoms. Phylogenetic analysis based on the amino acid of the coat protein (CP) of CMVs revealed that the CMV passion fruit isolates belonged to subgroup I, among which CMV-HN2 belonged to subgroup IA. Additionally, CMVs isolated from passion fruit in Korea have amino acid sequence variation between the subgroup. Among them, CMV-HN2 had four to eight amino acid differences in CP from other CMV isolates from passion fruit. These results confirm the presence of genetic diversity in the CPs of passion fruit CMV isolates.

Conserved patterns of sequence diversification provide insight into the evolution of two-component systems in Enterobacteriaceae.

Two-component regulatory systems (TCSs) are a major mechanism used by bacteria to sense and respond to their environments. Many of the same TCSs are used by biologically diverse organisms with different regulatory needs, suggesting that the functions of TCS must evolve. To explore this topic, we analysed the amino acid sequence divergence patterns of a large set of broadly conserved TCS across different branches of Enterobacteriaceae, a family of Gram-negative bacteria that includes biomedically important genera such as Salmonella, Escherichia, Klebsiella and others. Our analysis revealed trends in how TCS sequences change across different proteins or functional domains of the TCS, and across different lineages. Based on these trends, we identified individual TCS that exhibit atypical evolutionary patterns. We observed that the relative extent to which the sequence of a given TCS varies across different lineages is generally well conserved, unveiling a hierarchy of TCS sequence conservation with EnvZ/OmpR as the most conserved TCS. We provide evidence that, for the most divergent of the TCS analysed, PmrA/PmrB, different alleles were horizontally acquired by different branches of this family, and that different PmrA/PmrB sequence variants have highly divergent signal-sensing domains. Collectively, this study sheds light on how TCS evolve, and serves as a compendium for how the sequences of the TCS in this family have diverged over the course of evolution.

Variations of the NodB Architecture Are Attuned to Functional Specificities into and beyond the Carbohydrate Esterase Family 4.

Enzymes of the carbohydrate esterase family 4 (CE4) deacetylate a broad range of substrates, including linear, branched and mesh-like polysaccharides. Although they are enzymes of variable amino acid sequence length, they all comprise the conserved catalytic domain NodB. NodB carries the metal binding and active site residues and is characterized by a set of conserved sequence motifs, which are linked to the deacetylation activity. Besides a non-structured, flexible peptide of variable length that precedes NodB, several members of the CE4 family contain additional domains whose function or contribution to substrate specificity are not efficiently characterized. Evidence suggests that CE4 family members comprising solely the NodB domain have developed features linked to a variety of substrate specificities. To understand the NodB-based substrate diversity within the CE4 family, we perform a comparative analysis of all NodB domains structurally characterized so far. We show that amino acid sequence variations, topology diversities and excursions away from the framework structure give rise to different NodB domain classes associated with different substrate specificities and particular functions within and beyond the CE4 family. Our work reveals a link between specific NodB domain characteristics and substrate recognition. Thus, the details of the fold are clarified, and the structural basis of its variations is deciphered and associated with function. The conclusions of this work are also used to make predictions and propose specific functions for biochemically/enzymatically uncharacterized NodB-containing proteins, which have generally been considered as putative CE4 deacetylases. We show that some of them probably belong to different enzymatic families.

Chromosome-scale reference genome of broccoli (Brassica oleracea var. italica Plenck) provides insights into glucosinolate biosynthesis.

Broccoli (Brassica oleracea var. italica Plenck) is an important vegetable crop, as it is rich in health-beneficial glucosinolates (GSLs). However, the genetic basis of the GSL diversity in Brassicaceae remains unclear. Here we report a chromosome-level genome assembly of broccoli generated using PacBio HiFi reads and Hi-C technology. The final genome assembly is 613.79Mb in size, with a contig N50 of 14.70Mb. The GSL profile and content analysis of different B. oleracea varieties, combined with a phylogenetic tree analysis, sequence alignment, and the construction of a 3D model of the methylthioalkylmalate synthase 1 (MAM1) protein, revealed that the gene copy number and amino acid sequence variation both contributed to the diversity of GSL biosynthesis in B. oleracea. The overexpression of BoMAM1 (BolI0108790) in broccoli resulted in high accumulation and a high ratio of C4-GSLs, demonstrating that BoMAM1 is the key enzyme in C4-GSL biosynthesis. These results provide valuable insights for future genetic studies and nutritive component applications of Brassica crops.

A Pilot Investigation of the Association Between Vpr Amino Acid Substitutions and Peripheral Immune Marker Levels in People With Human Immunodeficiency Virus: Implications for Neurocognitive Impairment.

Subtype-specific amino acid variations in viral proteins of human immunodeficiency virus type 1 (HIV-1) influence disease progression. Furthermore, Vpr sequence variation correlates with chronic inflammation, a central mechanism in HIV-1 (neuro)pathogenesis. Nevertheless, no clinical study has investigated the link between Vpr sequence variation and peripheral inflammation in people with HIV (PWH). The aim of this pilot study was to ascertain whether specific Vpr amino acid variants were associated with immune markers in PWH. We included a unique cohort of 48 treatment-naive South African PWH to determine the association between blood-derived Vpr sequence variation and peripheral immune marker levels using Sanger sequencing and enzyme-linked immunosorbent assay analysis, respectively. Our findings indicate that among the many neuropathogenic Vpr amino acid variants and immune markers examined, after applying Bonferroni corrections (P = .05/3) and adjusting for sex and locality, soluble urokinase plasminogen activator receptor (suPAR) was nearing significance for higher levels in participants with the G41 amino acid variant compared to those with the S41 variant (P = .035). Furthermore, amino acid variations at position 41 (between G41 and S41) exhibited a significant association with suPAR (adjusted R2 = 0.089, β = .386 [95% confidence interval, .125-3.251]; P = .035). These findings suggest that Vpr amino acid sequence variations might contribute to dysregulated inflammation, which could explain the observed association between specific Vpr variants and HIV-1 (neuro)pathogenesis found in prior research. These Vpr variants merit further investigation to fully understand their roles in HIV-1 pathogenesis and neuropathogenesis.

A pilot investigation of the association between HIV-1 Vpr amino acid sequence diversity and the tryptophan-kynurenine pathway as a potential mechanism for neurocognitive impairment

HIV infection compromises both the peripheral and central immune systems due to its pathogenic and neuropathogenic features. The mechanisms driving HIV-1 pathogenesis and neuropathogenesis involve a series of events, including metabolic dysregulation. Furthermore, HIV-subtype-specific variations, particularly alterations in the amino acid sequences of key viral proteins, are known to influence the severity of clinical outcomes in people living with HIV. However, the impact of amino acid sequence variations in specific viral proteins, such as Viral protein R (Vpr), on metabolites within the Tryptophan (Trp)-kynurenine (Kyn) pathway in people living with HIV remains unclear. Our research aimed to explore the relationship between variations in the Vpr amino acid sequence (specifically at positions 22, 41, 45, and 55, as these have been previously linked to neurocognitive function) and peripheral Trp-Kyn metabolites. Additionally, we sought to clarify the systems biology of Vpr sequence variation by examining the link between Trp-Kyn metabolism and peripheral inflammation, as a neuropathogenic mechanism. In this preliminary study, we analyzed a unique cohort of thirty-two (n = 32) South African cART naïve people living with HIV. We employed Sanger sequencing to ascertain blood-derived Vpr amino acid sequence variations and a targeted LC-MS/MS metabolomics platform to assess Trp-Kyn metabolites, such as Trp, Kyn, kynurenic acid (KA), and quinolinic acid (QUIN). Particle-enhanced turbidimetric assay and Enzyme-linked immunosorbent assays were used to measure immune markers, hsCRP, IL-6, suPAR, NGAL and sCD163. After applying Bonferroni corrections (p =.05/3) and adjusting for covariates (age and sex), only the Vpr G41 and A55 groups was nearing significance for higher levels of QUIN compared to the Vpr S41 and T55 groups, respectively (all p =.023). Multiple regression results revealed that Vpr amino acid variations at position 41 (adj R2 = 0.049, β = 0.505; p =.023), and 55 (adj R2 = 0.126, β = 0.444; p =.023) displayed significant associations with QUIN after adjusting for age and sex. Lastly, the higher QUIN levels observed in the Vpr G41 group were found to be correlated with suPAR (r =.588, p =.005). These results collectively underscore the importance of specific Vpr amino acid substitutions in influencing QUIN and inflammation (specifically suPAR levels), potentially contributing to our understanding of their roles in the pathogenesis and neuropathogenesis of HIV-1.

Identification of Potent siRNA Delivery Peptides Using Computer Modeling.

Peptides with suitable aggregation behavior and electrical properties are potential siRNA delivery vectors. However, identifying suitable peptides with ideal delivery and safety features is difficult owing to the variations in amino acid sequences. Here, a holistic program based on computer modeling and single-cell RNA sequencing (scRNA-seq)is used to identify ideal siRNA delivery peptides. Stage one of this program consists of a sequential screening process for candidates with ideal assembly and delivery ability; stage two is a cell subtype-level analysis program that screens for high in vivo tissue safety. The leading candidate peptide selected from a library containing 12 amino acids showed strong lung-targeted siRNA delivery capacity after hydrophobic modification. Systemic administration of these compounds caused the least damage to liver and lung tissues and has little impact on macrophage and neutrophil numbers. By loading STAT3 siRNA, strong anticancer effects are achieved in multiple models, including patient-derived xenografts (PDX). This screening procedure may facilitate the development of peptide-based RNA interference (RNAi) therapeutics.

Conformational ensembles of the human intrinsically disordered proteome.

Intrinsically disordered proteins and regions (collectively, IDRs) are pervasive across proteomes in all kingdoms of life, help to shape biological functions and are involved in numerous diseases. IDRs populate a diverse set of transiently formed structures and defy conventional sequence-structure-function relationships1. Developments in protein science have made it possible to predict the three-dimensional structures of folded proteins at the proteome scale2. By contrast, there is a lack of knowledge about the conformational properties of IDRs, partly because the sequences of disordered proteins are poorly conserved and also because only a few of these proteins have been characterized experimentally. The inability to predict structural properties of IDRs across the proteome has limited our understanding of the functional roles of IDRs and how evolution shapes them. As a supplement to previous structural studies of individual IDRs3, we developed an efficient molecular model to generate conformational ensembles of IDRs and thereby to predict their conformational properties from sequences4,5. Here we use this model to simulate nearly all of the IDRs in the human proteome. Examining conformational ensembles of 28,058 IDRs, we show how chain compaction is correlated with cellular function and localization. We provide insights into how sequence features relate to chain compaction and, using a machine-learning model trained on our simulation data, show the conservation of conformational properties across orthologues. Our results recapitulate observations from previous studies of individual protein systems and exemplify how to link-at the proteome scale-conformational ensembles with cellular function and localization, amino acid sequence, evolutionary conservation and disease variants. Our freely available database of conformational properties will encourage further experimental investigation and enable the generation ofhypotheses about the biological roles and evolution of IDRs.

Genomic Analysis of Sindbis Virus Reveals Uncharacterized Diversity within the Australasian Region, and Support for Revised SINV Taxonomy.

Sindbis virus (SINV) is a widely dispersed mosquito-borne alphavirus. Reports of Sindbis disease are largely restricted to northern Europe and South Africa. SINV is frequently sampled in Australian mosquito-based arbovirus surveillance programs, but human disease has rarely been reported. Molecular epidemiological studies have characterized six SINV genotypes (G1-G6) based on E2 gene phylogenies, mostly comprising viruses derived from the African-European zoogeographical region and with limited representation of Australasian SINV. In this study, we conducted whole genome sequencing of 66 SINV isolates sampled between 1960 and 2014 from countries of the Australasian region: Australia, Malaysia, and Papua New Guinea. G2 viruses were the most frequently and widely sampled, with three distinct sub-lineages defined. No new G6 SINV were identified, confirming geographic restriction of these viruses to south-western Australia. Comparison with global SINV characterized large-scale nucleotide and amino acid sequence divergence between African-European G1 viruses and viruses that circulate in Australasia (G2 and G3) of up to 26.83% and 14.55%, respectively, divergence that is sufficient for G2/G3 species demarcation. We propose G2 and G3 are collectively a single distinct alphavirus species that we name Argyle virus, supported by the inapparent or mild disease phenotype and the higher evolutionary rate compared with G1. Similarly, we propose G6, with 24.7% and 12.61% nucleotide and amino acid sequence divergence, is a distinct alphavirus species that we name Thomson's Lake virus.

Genetic Diversity and Sequence Conservation of Peptide-Binding Regions of MHC Class I Genes in Pig, Cattle, Chimpanzee, and Human.

Comparative analyses of MHC gene diversity and evolution across different species could offer valuable insights into the evolution of MHC genes. Intra- and inter-species sequence diversity and conservation of 12 classical major histocompatibility complex (MHC) class I genes from cattle, chimpanzees, pigs, and humans was analyzed using 20 representative allelic groups for each gene. The combined analysis of paralogous loci for each species revealed that intra-locus amino-acid sequence variations in the peptide-binding region (PBR) of MHC I genes did not differ significantly between species, ranging from 8.44% for SLA to 10.75% for BoLA class I genes. In contrast, intraspecies differences in the non-PBRs of these paralogous genes were more pronounced, varying from 4.59% for SLA to 16.89% for HLA. Interestingly, the Shannon diversity index and rate of nonsynonymous substitutions for PBR were significantly higher in SLA and BoLA than those in Patr and HLA. Analysis of peptide-binding pockets across all analyzed MHC class I genes of the four species indicated that pockets A and E showed the lowest and highest diversity, respectively. The estimated divergence times suggest that primate and artiodactyl MHC class I genes diverged 60.41 Mya, and BoLA and SLA genes diverged 35.34 Mya. These results offer new insights into the conservation and diversity of MHC class I genes in various mammalian species.

1334. Amino Acid Sequence Diversity of HIV-1 Viral Proteins Tat and Vpr in South African People Living with HIV: Implications for Neurocognitive Function

Abstract Background The transactivator of transcription (Tat) and Viral protein R (Vpr) of HIV-1 have functional roles in the neuropathophysiology of HIV-associated neurocognitive disorders (HAND). Pre-clinical studies have identified specific amino acid substitutions, including Tat T24K, C31S, R57S and Vpr H45Y, A55T and R77Q, as crucial influencers of the neurovirulence of HIV-1 subtype C infection. However, there is limited clinical data on the amino acid sequence diversity of Tat and Vpr, particularly in South African people living with HIV (PLWH). Methods In this study, amino acid sequence diversity in Tat and Vpr was investigated in n = 70 treatment-naïve South African PLWH. RNA was extracted from plasma, reverse transcribed and DNA was prepared for PCR analysis. The Tat exon 1/Vpr (HXB2 position 4900–6351) was amplified using primer pair Vif-1/ CATH-4R. PCR products were sequenced by BigDye Terminator v.3.1 Cycle Sequencing Ready Reaction Kit and analysed on the ABI Prism 3130xl automated DNA sequencer. Bioinformatic analyses were applied to identify key amino acids in Tat and Vpr. Results Tat sequence data were available for n = 53 participants and Vpr sequence data was available for n = 63 participants. The most common amino acid at position 24 of Tat was K (37%), followed by N (28%) and P (9%). At position 31, S was the most common amino acid (66%), followed by C (11%). At position 57, the most common amino acid was S (53%), followed by R (9%) and A (8%). Regarding Vpr, the most common amino acid at position 45 was Y (53%), followed by H (33%). At position 55 of Vpr, T was the most common amino acid (70%), followed by A (19%). At position 77 of Vpr, Q was the most common amino acid (84%), followed by R (6%) and H (5%). Conclusion Our understanding of the impact of HIV-1 sequence variation on neurovirulence primarily stems from preclinical studies and research conducted on participants with subtype B. However, this study presents evidence of sequence variations in Tat and Vpr among South African PLWH, who are predominantly infected with subtype C. The predominant amino acids observed in Tat and Vpr of the investigated participants are known to be linked to lower neurovirulence. These findings may help elucidate the differing neurocognitive outcomes observed when comparing PLWH with subtypes B and C. Disclosures All Authors: No reported disclosures

High prevalence of antibiotic-resistant and metal-tolerant cultivable bacteria in remote glacier environment

Studies of antibiotic-resistant bacteria (ARB) have mainly originated from anthropic-influenced environments, with limited information from pristine environments. Remote cold environments are major reservoirs of ARB and have been determined in polar regions; however, their abundance in non-polar cold habitats is underexplored. This study evaluated antibiotics and metals resistance profiles, prevalence of antibiotic resistance genes (ARGs) and metals tolerance genes (MTGs) in 38 ARB isolated from the glacier debris and meltwater from Baishui Glacier No 1, China. Molecular identification displayed Proteobacteria (39.3%) predominant in debris, while meltwater was dominated by Actinobacteria (30%) and Proteobacteria (30%). Bacterial isolates exhibited multiple antibiotic resistance index values > 0.2. Gram-negative bacteria displayed higher resistance to antibiotics and metals than Gram-positive. PCR amplification exhibited distinct ARGs in bacteria dominated by β-lactam genes blaCTX-M (21.1–71.1%), blaACC (21.1–60.5%), tetracycline-resistant gene tetA (21.1–60.5%), and sulfonamide-resistant gene sulI (18.4–52.6%). Moreover, different MTGs were reported in bacterial isolates, including mercury-resistant merA (21.1–63.2%), copper-resistant copB (18.4–57.9%), chromium-resistant chrA (15.8–44.7%) and arsenic-resistant arsB (10.5–44.7%). This highlights the co-selection and co-occurrence of MTGs and ARGs in remote glacier environments. Different bacteria shared same ARGs, signifying horizontal gene transfer between species. Strong positive correlation among ARGs and MTGs was reported. Metals tolerance range exhibited that Gram-negative and Gram-positive bacteria clustered distinctly. Gram-negative bacteria were significantly tolerant to metals. Amino acid sequences of blaACC,blaCTX-M,blaSHV,blaampC,qnrA, sulI, tetA and blaTEM revealed variations. This study presents promising ARB, harboring ARGs with variations in amino acid sequences, highlighting the need to assess the transcriptome study of glacier bacteria conferring ARGs and MTGs.

Adaptive evolution in virulence effectors of the rice blast fungus Pyricularia oryzae.

Plant pathogens secrete proteins called effectors that target host cellular processes to promote disease. Recently, structural genomics has identified several families of fungal effectors that share a similar three-dimensional structure despite remarkably variable amino-acid sequences and surface properties. To explore the selective forces that underlie the sequence variability of structurally-analogous effectors, we focused on MAX effectors, a structural family of effectors that are major determinants of virulence in the rice blast fungus Pyricularia oryzae. Using structure-informed gene annotation, we identified 58 to 78 MAX effector genes per genome in a set of 120 isolates representing seven host-associated lineages. The expression of MAX effector genes was primarily restricted to the early biotrophic phase of infection and strongly influenced by the host plant. Pangenome analyses of MAX effectors demonstrated extensive presence/absence polymorphism and identified gene loss events possibly involved in host range adaptation. However, gene knock-in experiments did not reveal a strong effect on virulence phenotypes suggesting that other evolutionary mechanisms are the main drivers of MAX effector losses. MAX effectors displayed high levels of standing variation and high rates of non-synonymous substitutions, pointing to widespread positive selection shaping the molecular diversity of MAX effectors. The combination of these analyses with structural data revealed that positive selection acts mostly on residues located in particular structural elements and at specific positions. By providing a comprehensive catalog of amino acid polymorphism, and by identifying the structural determinants of the sequence diversity, our work will inform future studies aimed at elucidating the function and mode of action of MAX effectors.

A robust nanoscale RP HPLC-MS approach for sensitive Fc proteoform profiling of IgG allotypes

The conserved region (Fc) of IgG antibodies dictates the interactions with designated receptors thus defining the immunological effector functions of IgG. Amino acid sequence variations in the Fc, recognized as subclasses and allotypes, as well as post-translational modifications (PTMs) modulate these interactions. Yet, the high similarity of Fc sequences hinders allotype-specific PTM analysis by state-of-the-art bottom-up methods and current subunit approaches lack sensitivity and face co-elution of near-isobaric allotypes.To circumvent these shortcomings, we present a nanoscale reversed-phase (RP) HPLC-MS workflow of intact Fc subunits for comprehensive characterization of Fc proteoforms in an allotype- and subclass-specific manner. Polyclonal IgGs were purified from individuals followed by enzymatic digestion releasing single chain Fc subunits (Fc/2) that were directly subjected to analysis. Chromatographic conditions were optimized to separate Fc/2 subunits of near-isobaric allotypes and subclasses allowing allotype and proteoform identification and quantification across all four IgG subclasses. The workflow was complemented by a semi-automated data analysis pipeline based on the open-source software Skyline followed by post-processing in R. The approach revealed pronounced differences in Fc glycosylation between donors, besides inter-subclass and inter-allotype variability within donors. Notably, partial occupancy of the N-glycosylation site in the CH3 domain of IgG3 was observed that is generally neglected by established approaches. The described method was benchmarked across several hundred runs and showed good precision and robustness.This methodology represents a first mature Fc subunit profiling approach allowing truly subclass- and allotype-specific Fc proteoform characterization beyond established approaches. The comprehensive information obtained paired with the high sensitivity provided by the miniaturization of the approach guarantees applicability to a broad range of research questions including clinically relevant (auto)antibody characterization or pharmacokinetics assessment of therapeutic IgGs.

Monitoring Drought Tolerance in Oil Palm: Choline Monooxygenase as a Novel Molecular Marker.

Water scarcity negatively impacts oil palm production, necessitating the development of drought-tolerant varieties. This study aimed to develop molecular markers for oil palm breeding programs focused on drought tolerance. Genes associated with drought tolerance were selected, and single nucleotide polymorphism (SNP)-based markers were developed. Genomic DNA was successfully extracted from 17 oil palm varieties, and 20 primers out of 44 were effectively amplified. Screening with single-strand conformation polymorphism (SSCP) revealed an informative SNP marker from the choline monooxygenase (CMO) gene, exhibiting CC, CT, and TT genotypes. Notably, the oil palm variety La Mé showed the CT genotype, while Surat Thani 2 (Deli × La Mé) exhibited the CT and CC genotypes in a 1:1 ratio. Gene expression analysis confirmed the association of the CMO gene with drought tolerance in commercial oil palm varieties. The full-length CMO gene was 1308 bp long and shared sequence similarities with other plant species. However, amino acid sequence variations were observed compared with existing databases. These findings highlight the potential utility of the CMO marker for drought tolerance selection, specifically within the La Mé parent of oil palm Surat Thani 2 varieties, and strongly confirm the La Mé S5 population and Surat Thani 2 as drought-tolerant varieties.

Distribution of large lungworms (Nematoda: Dictyocaulidae) in free-roaming populations of red deer Cervus elaphus (L.) with the description of Dictyocaulus skrjabini n. sp.

Lungworms of the genus Dictyocaulus are causative agents of parasitic bronchitis in domestic and wild ungulates. This study investigates the distribution, morphology and genetic diversity of D. cervi and a new lungworm species, Dictyocaulus skrjabini n. sp. infecting red deer Cervus elaphus, fallow deer Dama dama and moose Alces alces in Poland and Sweden. The study was conducted on 167 red deer from Poland and on the DNA of lungworms derived from 7 fallow deer, 4 red deer and 2 moose collected in Sweden. The prevalence of D. cervi and D. skrjabini n. sp. in dissected red deer in Poland was 31.1% and 7.2%, respectively. Moreover, D. skrjabini n. sp. was confirmed molecularly in 7 isolates of fallow deer lungworms and 1 isolate of red deer lungworms from Sweden. Dictyocaulus skrjabini n. sp. was established based on combination of their distinct molecular and morphological features; these included the length of cephalic vesicle, buccal capsule (BC), buccal capsule wall (BCW), distance from anterior extremity to the nerve ring, the width of head, oesophagus, cephalic vesicle, BC and BCW, as well as the dimensions of reproductive organs of male and female. Additionally, molecular analyses revealed 0.9% nucleotide sequence divergence for 1,605 bp SSU rDNA, and 16.5–17.3% nucleotide sequence divergence for 642 bp mitochondrial cytB between D. skrjabini n. sp. and D. cervi, respectively, and 18.7–19% between D. skrjabini n. sp. and D. eckerti, which translates into 18.2–18.7% amino acid sequence divergence between D. skrjabini n. sp. and both lungworms.

Cannabinoid Receptor 2 (CB2) Orthologue In Vitro Function and Perspectives for Preclinical to Clinical Translation.

Cannabinoid receptor 2 (CB2 ) agonists are in development as therapeutics, including for immune modulation and pain relief. Despite promising results in rodent preclinical studies, efficacy in human clinical trials has been marginal to date. Fundamental differences in ligand engagement and signalling responses between human CB2 and preclinical model species orthologues may contribute to mismatches in functional outcomes. This is a tangible possibility for CB2 in that there is a relatively large degree of primary amino acid sequence divergence between human and rodent. Here, we summarise CB2 gene and protein structure, assess comparative molecular pharmacology between CB2 orthologues, and review the current status of preclinical to clinical translation for CB2 -targeted drugs, focusing on comparisons between human, mouse and rat CB2 . We hope that raising wider awareness of, and proposing strategies to address, this additional challenge in CB2 drug development will assist in ongoing efforts toward successful therapeutic translation of CB2 -targeted drugs.

Molecular Population Genetics of Aspen Mosaic-Associated Virus in Finland and Sweden.

Aspen mosaic-associated virus (AsMaV) is a newly identified Emaravirus, in the family Fimoviridae, Bunyavirales, associated with mosaic symptoms in aspen trees (Populus tremula). Aspen trees are widely distributed in Europe and understanding the population structure of AsMaV may aid in the development of better management strategies. The virus genome consists of five negative-sense single-stranded RNA (-ssRNA) molecules. To investigate the genetic diversity and population parameters of AsMaV, different regions of the genome were amplified and analyzed and full-length sequence of the divergent isolates were cloned and sequenced. The results show that RNA3 or nucleoprotein is a good representative for studying genetic diversity in AsMaV. Developed RT-PCR-RFLP was able to identify areas with a higher number of haplotypes and could be applied for screening the large number of samples. In general, AsMaV has a conserved genome and based on the phylogenetic studies, geographical structuring was observed in AsMaV isolates from Sweden and Finland, which could be attributed to founder effects. The genome of AsMaV is under purifying selection but not distributed uniformly on genomic RNAs. Distant AsMaV isolates displayed amino acid sequence variations compared to other isolates, and bioinformatic analysis predicted potential post-translational modification sites in some viral proteins.

Identification of novel SNPs in Pun1 locus for pungency in Capsicum species.

Capsaicin and its analogues known as capsaicinoids are the principal sources of pungency in Capsicum spp. In this study, characterization of North-West Himalayan chilli germplasm and commercial landraces of different Indian states known for different pungency-color combinations was done based on capsaicin concentration. Moreover, molecular variation in pungency among high, medium and mild/not pungent Capsicum spp., especially those adapted to North-West Himalayas were elucidated. Forty-nine genotypes of chilli comprising breeding lines of Kashmiri origin, commercial landraces of Southern Indian origin and one of the world's hottest chilli Bhut Jolokia from Nagaland state of India were used as an experimental material. Wide variation in capsaicin content was observed among the genotypes, wherein, Bhut Jolokia (Capsicum chinense) expressed the highest capsaicin content (10,500.75µg/g). Further, molecular analysis of PunI gene was done for discovering SNPs responsible for variations in pungency. In the non-pungent Nishat-1 (Capsicum annuum var. grossum), the 650bp DNA fragment was not amplified due to 2.5kb deletion spanning the putative promoter and first exon of AT3. The amplified DNA product for high and medium pungent was sequencing. Sequence alignment among revealed SNPs which were further observed responsible for variations in amino acid sequence and protein structure. The observed variation in protein structure might be responsible for high capsaicin production in one genotype as compared to the other and hence the protein conformation determines its interaction with the substrate.

Continuous presence ofgenetically diverse rustrela virus lineages inyellow-necked field mouse reservoir populations innortheastern Germany.

Rustrela virus (RusV; species Rubivirus strelense, family Matonaviridae) was discovered in different zoo animal species affected by fatal encephalitis. Simultaneous RusV RNA detection in multiple yellow-necked field mice (Apodemus flavicollis) suggested this rodent as a reservoir of RusV. Here, we investigated 1,264 yellow-necked field mice and sympatric other small mammals from different regions in Germany for RusV RNA using an optimized reverse transcription-quantitative polymerase chain reaction (RT-qPCR) protocol and high-throughput sequencing. The investigation resulted in the detection of RusV RNA exclusively in 50 of 396 (12.6 per cent) yellow-necked field mice but absence in other sympatric species. RT-qPCR-determined tissue distribution of RusV RNA revealed the highest viral loads in the central nervous system, with other tissues being only very rarely affected. The histopathological evaluation did not reveal any hints of encephalitis in the brains of infected animals despite the detection of viral RNA in neurons by in situ hybridization (ISH). The positive association between the body mass of yellow-necked field mice and RusV RNA detection suggests a persistent infection. Phylogenetic analysis of partial E1 and full-genome sequences showed a high diversification with at least four RusV lineages (1A-1D) in northeastern Germany. Moreover, phylogenetic and isolation-by-distance analyses indicated evolutionary processes of RusV mostly in local reservoir populations. A comparison of complete genome sequences from all detected RusV lineages demonstrated a high level of amino acid and nucleotide sequence variability within a part of the p150 peptide of the non-structural polyprotein and its coding sequence, respectively. The location of this region within the RusV genome and its genetic properties were comparable to the hypervariable region of the rubella virus. The broad range of detected RusV spillover hosts in combination with its geographical distribution in northeastern Germany requires the assessment of its zoonotic potential and further analysis of encephalitis cases in mammals. Future studies have to prove a putative co-evolution scenario for RusV in the yellow-necked field mouse reservoir.