Acid Variations Research Articles

Abstract 5547: Evaluation of two clinically focused targeted NGS systems for liquid biopsy testing shows a high level of concordance in resulting actionable mutations

Abstract Introduction: The use of blood-based molecular diagnostics is increasingly becoming routine in clinical oncology practice. The purpose of this study was to evaluate variant resulting concordance for the four major somatic variant classes using two clinical next-generation sequencing (NGS) systems for conducting highly sensitive blood-based analyses. All donor specimens were de-identified remnants from patients previously diagnosed with advanced NSCLC. We utilized two independent panels, systems and bioinformatic pipelines that are focused on clinical testing for key actionable variants. The fifty-gene panel Oncomine Precision Assay (OPA) GX was performed on the Genexus Integrated Sequencer and the 52 gene GeneStrat NGS (GSNGS) test was run as a reference on the Ion GeneStudio S5 PRIME system. The four major classes of mutations evaluated included Single Nucleotide Variants (SNV), Insertions and Deletions (INDEL), copy number amplifications (CNAs) and gene fusions. Methods: For this study, de-identified reference GSNGS cell-free nucleic acid (cfNA) remnant specimens (n=33 variants) that passed all validated QC bioinformatics thresholds were blinded and tested on the Genexus. DNA concentration was measured by fluorometry, and specimens were diluted to an input of 6.6 ng to 53.4 ng for library preparation. The Genexus system is pre-programmed to process specimens to result generation. Variant calls for all mutation classes were conducted using the on-board bioinformatics pipelines, and results were compared to those generated by the GeneStrat NGS Test. We included specimens harboring somatic variant mutations in KRAS, NRAS, BRAF, EGFR, ERBB2, and FGFR; CNAs; EML4-ALK fusions; and MET exon 14 skipping. Results: All samples passed the OPA (Genexus) bioinformatic quality control criteria and mapped reads were highly consistent, demonstrating accuracy of calls between both the Ion Torrent platforms, informatic pipelines and panels. We observed 100% concordance in variant calls for SNVs, INDELs and fusions between the two platforms. There was one CNA variant observed on the S5 that was not called by the Genexus workflow, yielding overall concordance of 97% (32 of 33). Of note, 44 additional somatic variants (SNV and INDELs) and 1 additional fusion were detected using the Genexus workflow. This was due to the lower threshold for variant calling on this system. Conclusion: The high concordance of the independent workflows for the detection of nucleic acid variants in circulation demonstrates the capability of both systems to be used for testing of clinical specimens. Specifically, actionable variants in the four major mutation classes were successfully detected in the reference and test specimen set. As a part of clinical validation, orthogonal testing of variants will need to be conducted. Citation Format: Sarah Kreston, Claire Gould, Kylie Blair, Leisa Jackson, Janice Riley, Gary A. Pestano. Evaluation of two clinically focused targeted NGS systems for liquid biopsy testing shows a high level of concordance in resulting actionable mutations. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5547.

Polymorphism of the rs1815739 Locus of the <i>ACTN3</i> Gene and rs11227639 <i>cis</i>-eQTL Affecting Its Expression in Populations of Siberia

The polymorphism of the rs1815739 locus of the ACTN3 gene and the rs11227639 cis-eQTL that affects expression of this gene was studied in the populations of the northern (Chukchi, Koryaks, Evens, and Evenks) and southern (Buryats, Altaians, and Tuvinians) parts of Siberia. It was shown that the frequencies of the rs1815739-C allele of the ACTN3 gene (corresponding to the 577R amino acid variant) and the rs11227639-A upregulating ACTN3 allele, which increases the expression level of this gene, are significantly higher in the north than in the south of Siberia. Similarly, in the northern direction, the frequency of the combination of CC/AA genotypes at these loci increases. The revealed pattern of the geographical distribution of alleles and genotypes at the rs1815739 and rs11227639 loci in Siberian populations may be due to adaptation to cold and the processes of thermoregulation of the body when exposed to cold. It is assumed that an increase in the frequency of rs1815739-C and rs11227639-A alleles in the aboriginal populations of the north of Siberia is associated with an increase in the role of shivering thermogenesis during adaptation to cold exposure, and also contributes to an increase in muscle mass in their carriers, which reduces heat loss in the conditions of the North.

In-Depth Characterization of Acidic Variants Induced by Metal-Catalyzed Oxidation in a Recombinant Monoclonal Antibody.

Characterization of antibody charge heterogeneity is an important task for antibody drug development. Recently, a correlation between acidic charge heterogeneity and metal-catalyzed oxidation has been observed for antibody drugs. However, to date, the acidic variants induced by metal-catalyzed oxidation have not been elucidated. In addition, it is challenging to satisfactorily explain the induced acidic charge heterogeneity, as the existing analytical workflows, which relied on either untargeted or targeted peptide mapping analysis, could lead to incomplete identification of the acidic variants. In this work, we present a new characterization workflow by combining untargeted and targeted analyses to thoroughly identify and characterize the induced acidic variants in a highly oxidized IgG1 antibody. As a part of this workflow, a tryptic peptide mapping method was also developed for accurate determination of the relative extent of site-specific carbonylation, where a new hydrazone reduction procedure was established to minimize under-quantitation artifacts caused by incomplete reduction of hydrazones during sample preparation. In summary, we identified 28 site-specific oxidation products, which are located on 26 residues and of 11 different modification types, as the sources of the induced acidic charge heterogeneity. Many of the oxidation products were reported for the first time in antibody drugs. More importantly, this study provides new insights to understanding acidic charge heterogeneity of antibody drugs in the biotechnology industry. Additionally, the characterization workflow presented in this study can be applied as a platform approach in the biotechnology industry to better address the need for in-depth characterization of antibody charge variants.

The diversification of the antimicrobial peptides from marine worms is driven by environmental conditions

Antimicrobial peptides (AMPs) play a key role in the external immunity of animals, offering an interesting model for studying the influence of the environment on the diversification and evolution of immune effectors. Alvinellacin (ALV), arenicin (ARE) and polaricin (POL, a novel AMP identified here), characterized from three marine worms inhabiting contrasted habitats (‘hot’ vents, temperate and polar respectively), possess a well conserved BRICHOS domain in their precursor molecule despite a profound amino acid and structural diversification of the C-terminal part containing the core peptide. Data not only showed that ARE, ALV and POL display an optimal bactericidal activity against the bacteria typical of the habitat where each worm species lives but also that this killing efficacy is optimal under the thermochemical conditions encountered by their producers in their environment. Moreover, the correlation between species habitat and the cysteine contents of POL, ARE and ALV led us to investigate the importance of disulfide bridges in their biological efficacy as a function of abiotic pressures (pH and temperature). The construction of variants using non-proteinogenic residues instead of cysteines (α-aminobutyric acid variants) leading to AMPs devoid of disulfide bridges, provided evidence that the disulfide pattern of the three AMPs allows for a better bactericidal activity and suggests an adaptive way to sustain the fluctuations of the worm's environment. This work shows that the external immune effectors exemplified here by BRICHOS AMPs are evolving under strong diversifying environmental pressures to be structurally shaped and more efficient/specific under the ecological niche of their producer.

Botulinum Neurotoxin A4 Has a 1000-Fold Reduced Potency Due to Three Single Amino Acid Alterations in the Protein Receptor Binding Domain.

Botulinum neurotoxin subtype A4 (BoNT/A4) is ~1000-fold less potent than BoNT/A1. This study addresses the basis for low BoNT/A4 potency. Utilizing BoNT/A1-A4 and BoNT/A4-A1 Light Chain-Heavy Chain (LC-HC) chimeras, HC-A4 was responsible for low BoNT/A4 potency. Earlier studies showed BoNT/A1-receptor binding domain (Hcc) bound a β-strand peptide (556-564) and glycan-N559 within Luminal Domain 4 (LD4) of SV2C, the BoNT/A protein receptor. Relative to BoNT/A1, the Hcc of BoNT/A4 possesses two amino acid variants (D1141 and N1142) within the β-peptide binding interface and one amino acid variant (R1292) located near the SV2C glycan-N559. Introduction of BoNT/A4 β-strand peptide variant (D1141 and N1142) into BoNT/A1 reduced toxin potency 30-fold, and additional introduction of the BoNT/A4 glycan-N559 variant (D1141, N1142, and R1292) further reduced toxin potency to approach BoNT/A4. While introduction of BoNT/A1 glycan-N559 variant (G1292) into BoNT/A4 did not alter toxin potency, additional introduction of BoNT/A1 β-strand peptide variants (G1141, S1142, and G1292) resulted in potency approaching BoNT/A1 potency. Thus, outcomes from these functional and modeling studies indicate that in rodent models, disruption of Hcc -SV2C β-peptide and -glycan-N559 interactions mediate low BoNT/A4 potency, while in human motor neurons, disruption of Hcc-SV2C β-peptide alone mediates low BoNT/A4 potency, which link to a species-specific variation at SV2C563.

Circulation pattern and genetic variation of human respiratory syncytial virus in China during 2008-2021.

To better understand the circulation pattern and genetic characterization of human respiratory syncytial virus (HRSV) in China during 2008-2021, a total of 3967 HVR2 sequences were obtained from 20 provinces in China for phylogenetic analysis and sequence variation analysis. The results showed that the HRSV subtype presented the prevalence pattern of "ABBAABAABAAABB."Further genotyping identified sevengenotypes for HRSVA and ninegenotypes for HRSVB. Multiple genotypes of HRSV were cocirculating during 2008-2015, while ON1 and BA9 became the only predominant genotypes for HRSVA and HRSVB, respectively, since 2015. A genotype switch from NA1 to ON1 for HRSVA occurred in approximately 2014, while genotype BA9 of HRSVB had been the predominant genotype for at least 14 years. ON1 strains could be divided into four lineages with no temporal or geographical distribution tendency. In contrast, BA9 strains could be divided into three lineages with noticeable temporal clustering. Sequence variation analysis showed that two ON1 sequences in 2017 had 10 nucleotide deletion and compensatory extension at the C-terminal;15BA9 sequences during 2019-2021 had novel insertions between K225 and E226, along with 6 identical amino acid variant sites. This study further enriched the genetic data of HRSV circulating in China and provided an important basis for the development of HRSV vaccines and drugs as well as the formulation of prevention and control strategies.

Flagella-driven motility is a target of human Paneth cell defensin activity.

In the mammalian intestine, flagellar motility can provide microbes competitive advantage, but also threatens the spatial segregation established by the host at the epithelial surface. Unlike microbicidal defensins, previous studies indicated that the protective activities of human α-defensin 6 (HD6), a peptide secreted by Paneth cells of the small intestine, resides in its remarkable ability to bind microbial surface proteins and self-assemble into protective fibers and nets. Given its ability to bind flagellin, we proposed that HD6 might be an effective inhibitor of bacterial motility. Here, we utilized advanced automated live cell fluorescence imaging to assess the effects of HD6 on actively swimming Salmonella enterica in real time. We found that HD6 was able to effectively restrict flagellar motility of individual bacteria. Flagellin-specific antibody, a classic inhibitor of flagellar motility that utilizes a mechanism of agglutination, lost its activity at low bacterial densities, whereas HD6 activity was not diminished. A single amino acid variant of HD6 that was able to bind flagellin, but not self-assemble, lost ability to inhibit flagellar motility. Together, these results suggest a specialized role of HD6 self-assembly into polymers in targeting and restricting flagellar motility.

Two-Dimensional Capillary Zone Electrophoresis-Mass Spectrometry: Intact mAb Charge Variant Separation Followed by Peptide Level Analysis Using In-Capillary Digestion.

Characterization of charge heterogeneity is an essential pillar for pharmaceutical development and quality control of therapeutic monoclonal antibodies (mAbs). The highly selective and commonly applied capillary zone electrophoresis (CZE) method containing high amounts of ε-aminocaproic acid (EACA) provides a detailed and robust charge heterogeneity profile of intact mAb variants. Nevertheless, the exact location of protein modifications within these charge profiles remains ambiguous. Electrospray ionization mass spectrometry (ESI-MS) is a promising tool for this purpose; however, EACA is incompatible with electrospray. In this context, we present a two-dimensional CZE-CZE-MS system to combine efficient charge variant separation of intact mAbs with subsequent peptide analysis after in-capillary digestion of selected charge variants. The first dimension is based on a generic CZE(EACA) method in a fused silica capillary. In the second dimension, a neutral-coated capillary is used for in-capillary reduction and digestion with Tris(2-carboxyethyl)phosphine (TCEP) and pepsin, followed by CZE separation and MS/MS-characterization of the resulting peptides. The setup is demonstrated using stressed and nonstressed mAbs where peaks of basic, main, and acidic variants were transferred in a heart-cut fashion, digested, and characterized on the peptide level. Sequence coverages of more than 90% were obtained for heavy chain (HC) and light chain (LC) for four different mAbs, including low-abundance variants (<2% of the main peak). Frequently observed modifications (deamidation, oxidation, etc.) could be detected and localized. This study demonstrates a proof-of-concept for identification and localization of protein modifications from CZE charge heterogeneity profiles and, in this way, is expected to support the development and quality control testing of protein pharmaceuticals.

Homozygous Ser-1 to Pro-1 mutation in parathyroid hormone identified in hypocalcemic patients results in secretion of a biologically inactive pro-hormone

Like other secreted peptides, nascent parathyroid hormone (PTH) is synthesized with a pre- and a pro-sequence (25 and 6 amino acids, respectively). These precursor segments are sequentially removed in parathyroid cells before packaging into secretory granules. Three patients from two unrelated families who presented during infancy with symptomatic hypocalcemia were found to have a homozygous serine (S) to proline (P) change affecting the first amino acid of the mature PTH. Unexpectedly, biological activity of synthetic [P1]PTH(1-34) was indistinguishable from that of unmodified [S1]PTH(1-34). However, in contrast to conditioned medium from COS-7 cells expressing prepro[S1]PTH(1-84), medium from cells expressing prepro[P1]PTH(1-84) failed to stimulate cAMP production despite similar PTH levels when measured by an intact assay that detects PTH(1-84) and large amino-terminally truncated fragments thereof. Analysis of the secreted, but inactive PTH variant led to the identification of pro[P1]PTH(-6 to+84). Synthetic pro[P1]PTH(-6 to+34) and pro[S1]PTH(-6 to+34) had much less bioactivity than the corresponding PTH(1-34) analogs. Unlike pro[S1]PTH(-6 to+34), pro[P1]PTH(-6 to+34) was resistant to cleavage by furin suggesting that the amino acid variant impairs preproPTH processing. Consistent with this conclusion, plasma of patients with the homozygous P1 mutation had elevated proPTH levels, as determined with an in-house assay specific for pro[P1]PTH(-6 to+84). In fact, a large fraction of PTH detected by the commercial intact assay represented the secreted pro[P1]PTH. In contrast, two commercial biointact assays that use antibodies directed against the first few amino acid residues of PTH(1-84) for capture or detection failed to detect pro[P1]PTH.

Online Alkaline-pH Reverse Phase Nanoelectrospray-Tandem Mass Spectrometry Complements Conventional Low-pH Method for Global Proteomic Analysis.

The global proteome analysis was limited by the identification of peptides with low abundance or specific physiochemical properties. Here, a one-dimensional online alkaline-pH reverse phase nanoelectrospray-tandem mass spectrometry (alkaline-pH-MS/MS) method was developed and optimized for global proteomic analysis. In this method, peptides were separated on a nanoflow C18 column with an alkaline-pH mobile phase (pH = 8.0) and directly injected into the mass spectrometer. The unique peptides overlapped between alkaline-pH-MS/MS and conventional online low-pH reverse phase nanoelectrospray-tandem mass spectrometry (low-pH-MS/MS) were as low as 45%, strongly indicating that these two methods were complementary to each other. In addition, alkaline-pH-MS/MS showed identification capacity for a higher proportion of peptides with negative grand average of hydropathy (GRAVY) or high isoelectric point (pI). Compared to low-pH-MS/MS, alkaline-pH-MS/MS enabled enrichment preference toward histidine-, lysine-, methionine-, and proline-containing peptides. The complementarity of alkaline-pH-MS/MS and low-pH-MS/MS was further demonstrated for the analysis of tryptic digests from 15 intrahepatic cholangiocarcinoma (iCCA) cell lines. The alternating 60 min alkaline-pH-MS/MS plus 60 min low-pH-MS/MS method outperformed the conventional 120 min low-pH-MS/MS method in both the identification of amino acid variants and protein groups. Therefore, we established the alkaline-pH-MS/MS method as a simple, competitive, alternative method to low-pH-MS/MS for global proteomic analysis.

Effects of various pine needle extracts on Chinese hamster ovary cell growth and monoclonal antibody quality

Chinese hamster ovary (CHO) cells are commonly used as “bio-machines” to pro-duce monoclonal antibodies (mAb) because of their ability to produce very complex proteins. In this study, we evaluated the effects of pine needle water extract (PNWE), pine needle ethanol extract (PNEE), and pine needle polysaccharide extract (PNPE) on the CHO cell growth, mAb production and quality using a Fed-batch culture process. PNPE maintained high VCD and viability, and the titer increase was correlated with its concentration. Three extracts effectively reduced the acidic charge variant and modulated mAb glycosylation. PNPE had the most profound effect, with G0F decreasing by 8.7% and G1Fa increasing by 6.7%. The change in the glycoform was also closely related to the PNPE concentration. This study demonstrated that PNPE could facilitate CHO cell growth, increase the mAb production, decrease acidic charge variants, and regulate mAb glycoforms. To identify the components responsible for the above changes, the sugar and flavonoid contents in the extracts were determined, and the chemical compounds were identified by LC-MS, resulting in 38 compounds identified from PNPE. Rich in sugars and flavonoids in these three extracts may be related to increased CHO cell growth and productivity, and changes in glycoforms.

Mega-scale analysis of protein folding stability in biology and protein design.

Progress in DNA sequencing and machine learning have illuminated protein sequences and structures on an enormous scale, but protein thermodynamics remains invisible and largely unknown. New approaches are needed to reveal protein folding thermodynamics on the same enormous scale to overcome challenges in biology, medicine, and engineering. We present cDNA display proteolysis, a new method for measuring protein folding stability for up to 900,000 protein domains in a one-week experiment. From 1.7 million measurements in total, we curated a set of ∼800,000 high-quality folding stabilities comprehensively examining single amino acid variants of ∼350 natural and ∼250 de novo designed protein domains 40-72 amino acids in length. Using this immense dataset, we quantified (1) environmental factors influencing amino acid fitness, (2) thermodynamic couplings (including unexpected interactions) between protein sites, and (3) the global divergence between evolutionary amino acid usage and protein folding stability. We also demonstrated applications of our method and dataset to protein design. The unique scale, speed, and accuracy of cDNA display proteolysis hold the potential to reveal protein folding thermodynamics across the entire universe of protein domains.

Improving the genome and proteome annotations of the marine model diatom Thalassiosira pseudonana using a proteogenomics strategy

Diatoms are unicellular eukaryotic phytoplankton that account for approximately 20% of global carbon fixation and 40% of marine primary productivity; thus, they are essential for global carbon biogeochemical cycling and climate. The availability of ten diatom genome sequences has facilitated evolutionary, biological and ecological research over the past decade; however, a complimentary map of the diatom proteome with direct measurements of proteins and peptides is still lacking. Here, we present a proteome map of the model marine diatom Thalassiosira pseudonana using high-resolution mass spectrometry combined with a proteogenomic strategy. In-depth proteomic profiling of three different growth phases and three nutrient-deficient samples identified 9526 proteins, accounting for ~ 81% of the predicted protein-coding genes. Proteogenomic analysis identified 1235 novel genes, 975 revised genes, 104 splice variants and 234 single amino acid variants. Furthermore, our quantitative proteomic analysis experimentally demonstrated that a considerable number of novel genes were differentially translated under different nutrient conditions. These findings substantially improve the genome annotation of T. pseudonana and provide insights into new biological functions of diatoms. This relatively comprehensive diatom proteome catalog will complement available diatom genome and transcriptome data to advance biological and ecological research of marine diatoms.

Long term culturing of CHO cells: phenotypic drift and quality attributes of the expressed monoclonal antibody

Establishing cell lines with enhanced protein production requires a deep understanding of the cellular dynamics and cell line stability. The aim of the study is to investigate the impact of long term culturing (LTC) on cell morphology and altered cellular functions possibly leading to phenotypic drift, impacting product yield and quality. Study highlights the orthogonal cellular and analytical assay toolbox to define cell line stability for optimal culture performance and product quality. We investigated recombinant monoclonal antibody (mAb) expressing CHO cells for 60 passages or 180 generations and assessed the cell growth characteristics and morphology by confocal and scanning electron microscopy. Quality attributes of expressed mAb is accessed by performing charge variants, glycan, and host cell protein analysis. We observed a 1.65-fold increase in viable cell population and 1.3-fold increase in cell specific growth rate. A 2.5-fold decrease in antibody titer and abatement of actin filament indicate cellular phenotypic drift. Mitochondrial membrane potential (∆ΨM) signified cell health and metabolic activity during LTC. Host cell protein production is reduced by 1.8-fold. Charge heterogeneity was perturbed with 12.5% and 43% reduction in abundance of acidic and basic charge variants respectively. Glycan profile indicated a decline in fucosylation with 17% increase in galactosylated species as compared with early passaged cells. LTC impinges on cellular phenotype as well as the quality of the expressed antibody, suggesting a defined subculturing limit to retain stable protein expression and cell morphology to achieve consistent product quality. Study signifies the changes in cellular and metabolic markers, suggesting cellular and analytical toolbox which could play a significant role in defining cell characteristics and ensured product quality.

Physicochemical stability of durvalumab (Imfinzi®) concentrate for solution in original vials after first opening

Abstract Objectives Durvalumab (Imfinzi®), a PD-L1 monoclonal antibody (mAb) medication is available as concentrate (50 mg/mL) for solution for infusion. The summary of product characteristics provides information about the physicochemical stability of ready-to-administer durvalumab preparations (vehicle solution 0.9 % NaCl, G5%), but not about the concentrate after first opening. The objective of this study was to determine the physicochemical stability of durvalumab concentrate for solution after first opening over a period of 28 days. Methods Imfinzi® vials were punctured and stored refrigerated (2–8 °C) or at room temperature (20–25 °C) light protected. At predefined time points (day 0, 7, 14, 21, 28) the physicochemical stability of the concentrated solution was determined by ion-exchange/size-exclusion high-performance liquid chromatography (IE-/SE-HPLC) with photodiode array detection and pH measurement. Vials were inspected with regard to changes of color, clarity, and visible particles at any time point. Results Regardless of the storage temperature, durvalumab 50 mg/mL solutions remained physiochemically stable for 28 days in punctured vials. The concentrations of durvalumab monomer remained unchanged and no secondary peaks (fragments, aggregates) were observed in any of the SE-HPLC chromatograms. The IE-HPLC test results showed no substantial changes of the peak areas of the main peak and of the acidic and basic charge variants during the whole storage period. Appearance and pH of the test solutions remained unchanged until the end of the study. Conclusions Regardless of storage conditions none of the analytical methods indicated physicochemical instability of the intact durvalumab monomer over the 28 days of the study. To avoid microbiological instability storage under refrigeration is recommended.

SAV-Pred: A Freely Available Web Application for the Prediction of Pathogenic Amino Acid Substitutions for Monogenic Hereditary Diseases Studied in Newborn Screening.

Next Generation Sequencing (NGS) technologies are rapidly entering clinical practice. A promising area for their use lies in the field of newborn screening. The mass screening of newborns using NGS technology leads to the discovery of a large number of new missense variants that need to be assessed for association with the development of hereditary diseases. Currently, the primary analysis and identification of pathogenic variations is carried out using bioinformatic tools. Although extensive efforts have been made in the computational approach to variant interpretation, there is currently no generally accepted pathogenicity predictor. In this study, we used the sequence-structure-property relationships (SSPR) approach, based on the representation of protein fragments by molecular structural formula. The approach predicts the pathogenic effect of single amino acid substitutions in proteins related with twenty-five monogenic heritable diseases from the Uniform Screening Panel for Major Conditions recommended by the Advisory Committee on Hereditary Disorders in Newborns and Children. In order to create SSPR models of classification, we modified a piece of cheminformatics software, MultiPASS, that was originally developed for the prediction of activity spectra for drug-like substances. The created SSPR models were compared with traditional bioinformatic tools (SIFT 4G, Polyphen-2 HDIV, MutationAssessor, PROVEAN and FATHMM). The average AUC of our approach was 0.804 ± 0.040. Better quality scores were achieved for 15 from 25 proteins with a significantly higher accuracy for some proteins (IVD, HADHB, HBB). The best SSPR models of classification are freely available in the online resource SAV-Pred (Single Amino acid Variants Predictor).

Population Structure and Genomic Characteristics of Australian Erysipelothrix rhusiopathiae Reveals Unobserved Diversity in the Australian Pig Industry.

Erysipelothrix rhusiopathiae is a bacterial pathogen that is the causative agent of erysipelas in a variety of animals, including swine, emus, turkeys, muskox, caribou, moose, and humans. This study aims to investigate the population structure and genomic features of Australian isolates of E. rhusiopathiae in the Australian pig industry and compare them to the broader scope of isolates worldwide. A total of 178 isolates (154 Australian, seven vaccine isolates, six international isolates, and 11 of unknown origin) in this study were screened against an MLST scheme and publicly available reference isolates, identifying 59 new alleles, with isolates separating into two main single locus variant groups. Investigation with BLASTn revealed the presence of the spaA gene in 171 (96%) of the isolates, with three main groups of SpaA protein sequences observed amongst the isolates. Novel SpaA protein sequences, categorised here as group 3 sequences, consisted of two sequence types forming separate clades to groups 1 and 2, with amino acid variants at positions 195 (D/A), 303 (G/E) and 323(P/L). In addition to the newly identified groups, five new variant positions were identified, 124 (S/N), 307 (Q/R), 323 (P/L), 379 (M/I), and 400 (V/I). Resistance screening identified genes related to lincomycin, streptomycin, erythromycin, and tetracycline resistance. Of the 29 isolates carrying these resistance genes, 82% belonged to SpaA group 2-N101S (n = 22) or 2-N101S-I257L (n = 2). In addition, 79% (n = 23) of these 29 isolates belonged to MLST group ST 5. Our results illustrate that Australia appears to have a unique diversity of E. rhusiopathiae isolates in pig production industries within the wider global context of isolates.

Association of cytochrome P450 2D15 (CYP2D15) nonsynonymous polymorphisms and exon 3 deleted RNA splice variant with CYP2D15 protein content and enzyme function in dog liver microsomes.

CYP2D15 is a major drug metabolizing P450 in canine liver. Like the human orthologue (CYP2D6), this enzyme is highly polymorphic with at least five common nonsynonymous variants reported that result in amino acid changes, including p.Ile109Val, p.Leu115Phe, p.Gly186Ser, p.Ile250Phe and p.Ile307Val. Furthermore, a mRNA splice variant of CYP2D15 has been found in canine liver that lacks the exon 3 gene region resulting in an inactive enzyme. The objective of this study was to evaluate whether any of these amino acid variants or the exon 3 deletion mRNA variant (exon3-delta) was associated with differences in CYP2D15-selective activities or protein content in a bank of canine livers. Livers were obtained from 25 Beagles and 34 dogs of various other breeds. CYP2D15-selective activities measured included dextromethorphan o-demethylation and tramadol o-demethylation. Reverse transcription PCR showed that 76% of livers (44/58) expressed both exon3-delta and normally spliced CYP2D15 RNA, while the remaining 24% (14/58) expressed only normally spliced RNA. The presence of exon3-delta was not correlated with CYP2D15 activities or protein content. Compared with wild-type livers, Beagle dog livers heterozygous for the p.Ile109Val and p.Gly186Ser variants showed from 40 to 50% reductions in median enzyme activities, while heterozygous p.Gly186Ser livers were associated with a 41% reduction in median CYP2D15 protein content (p< .05; Dunn's test). In the entire liver bank, livers homozygous for p.Ile109Val were also associated with a 40% reduction in median dextromethorphan O-demethylation activities versus wild-type livers (p< .05). These results identify several nonsynonymous CYP2D15 gene variants associated with variable CYP2D15 metabolism in canine liver.

Baloxavir safety and clinical and virologic outcomes in influenza virus-infected pediatric patients by age group: age-based pooled analysis of two pediatric studies conducted in Japan

BackgroundAnti-influenza treatment is important for children and is recommended in many countries. This study assessed safety, clinical, and virologic outcomes of baloxavir marboxil (baloxavir) treatment in children based on age and influenza virus type/subtype.MethodsThis was a post hoc pooled analysis of two open-label non-controlled studies of a single weight-based oral dose of baloxavir (day 1) in influenza virus-infected Japanese patients aged < 6 years (n = 56) and ≥ 6 to < 12 years (n = 81). Safety, time to illness alleviation (TTIA), time to resolution of fever (TTRF), recurrence of influenza illness symptoms and fever (after day 4), virus titer, and outcomes by polymerase acidic protein variants at position I38 (PA/I38X) were evaluated.ResultsAdverse events were reported in 39.0 and 39.5% of patients < 6 years and ≥ 6 to < 12 years, respectively. Median (95% confidence interval) TTIA was 43.2 (36.3–68.4) and 45.4 (38.9–61.0) hours, and TTRF was 32.2 (26.8–37.8) and 20.7 (19.2–23.8) hours, for patients < 6 years and ≥ 6 to < 12 years, respectively. Symptom and fever recurrence was more common in patients < 6 years with influenza B (54.5 and 50.0%, respectively) compared with older patients (0 and 25.0%, respectively). Virus titers declined (day 2) for both age groups. Transient virus titer increase and PA/I38X-variants were more common for patients < 6 years.ConclusionsThe safety and effectiveness of single-dose baloxavir were observed in children across all age groups and influenza virus types. Higher rates of fever recurrence and transient virus titer increase were observed in children < 6 years.Trial registrationJapan Pharmaceutical Information Center Clinical Trials Information JapicCTI-163,417 (registered 02 November 2016) and JapicCTI-173,811 (registered 15 December 2017).

Targeted and explorative profiling of kallikrein proteases and global proteome biology of pancreatic ductal adenocarcinoma, chronic pancreatitis, and normal pancreas highlights disease-specific proteome remodelling

Pancreatic ductal adenocarcinoma (PDAC) represents one of the most aggressive and lethal malignancies worldwide with an urgent need for new diagnostic and therapeutic strategies. One major risk factor for PDAC is the pre-indication of chronic pancreatitis (CP), which represents highly inflammatory pancreatic tissue. Kallikreins (KLKs) are secreted serine proteases that play an important role in various cancers as components of the tumor microenvironment. Previous studies of KLKs in solid tumors largely relied on either transcriptomics or immunodetection. We present one of the first targeted mass spectrometry profiling of kallikrein proteases in PDAC, CP, and normal pancreas. We show that KLK6 and KLK10 are significantly upregulated in PDAC (n=14) but not in CP (n=7) when compared to normal pancreas (n=16), highlighting their specific intertwining with malignancy. Additional explorative proteome profiling identified 5936 proteins in our pancreatic cohort and observed disease-specific proteome rearrangements in PDAC and CP. As such, PDAC features an enriched proteome motif for extracellular matrix (ECM) and cell adhesion while there is depletion of mitochondrial energy metabolism proteins, reminiscent of the Warburg effect. Although often regarded as a PDAC hallmark, the ECM fingerprint was also observed in CP, alongside with a prototypical inflammatory proteome motif as well as with an increased wound healing process and proteolytic activity, thereby possibly illustrating tissue autolysis. Proteogenomic analysis based on publicly accessible data sources identified 112 PDAC-specific and 32 CP-specific single amino acid variants, which among others affect KRAS and ANKHD1. Our study emphasizes the diagnostic potential of kallikreins and provides novel insights into proteomic characteristics of PDAC and CP.