Amino Acid Differences Research Articles

Evaluation of a Series of Turnip Mosaic Virus Chimeric Clones Reveals Two Amino Acid Sites Critical for Systemic Infection in Chinese Cabbage.

Two infectious clones of turnip mosaic virus (TuMV), pKBC-1 and pKBC-8, with differential infectivity in Chinese cabbage (Brassica rapa subsp. pekinensis), were obtained. Both infected Nicotiana benthamiana systemically, inducing similar symptoms, whereas only virus KBC-8 infected Chinese cabbage systemically. To identify the determinants affecting infectivity on Chinese cabbage, chimeric clones were constructed by restriction fragment exchange between the parental clones and tested on several Chinese cabbage cultivars. Chimeric clones p1N8C and p8N1C demonstrated that the C-terminal portion of the polyprotein determines systemic infection of Chinese cabbage despite only three amino acid differences in this region, in the cylindrical inclusion (CI), viral protein genome-linked (VPg), and coat protein (CP). A second pair of hybrid constructs, pHindIII-1N8C and pHindIII-8N1C, failed to infect cultivars CR Victory and Jinseonnorang systemically, yet pHindIII-1N8C caused hypersensitive response-like lesions on inoculated leaves of these cultivars, and could systemically infect cultivars CR Chusarang and Jeongsang; this suggests that R genes effective against TuMV may exist in the first two cultivars but not the latter two. Constructs with single amino acid changes in both VPg (K2045E) and CP (Y3095H) failed to infect Chinese cabbage, implying that at least one of these two amino acid substitutions is essential for successful infection on Chinese cabbage. Successful infection by mutant KBC-8-CP-H and delayed infection with mutant HJY1-VPg-E following mutation or reversion suggested that VPg (2045K) is the residue required for infection of Chinese cabbage and involved in the interaction between VPg and eukaryotic initiation factor eIF(iso)4E, confirmed by yeast two-hybrid assay.

Determination and Analysis of Composition, Structure, and Properties of Teff Protein Fractions.

To develop teff-based food products with acceptable quality, the composition, structure, and properties of teff protein fractions should be better understood. In this study, teff proteins were extracted, and their protein composition, structure, and properties were calculated, analyzed, and compared with those of wheat gliadin and glutenin. Results showed that teff flour contained 9.07% protein, with prolamin as its main protein fraction. The isoelectric points of albumin, globulin, prolamin, and glutelin were at pH 3.6, 3.0, 4.4, and 3.4, respectively. Teff prolamin and glutelin showed a significant difference in amino acids and free energy of hydration compared to wheat gliadins and glutenins. The protein chain length of teff prolamins was smaller than that of wheat gliadins, and teff glutelins lacked high molecular weight glutelin subunits. Teff prolamin had the highest α-helices content (27.08%), whereas no random coils were determined, which is different from wheat gliadin. Teff glutelin had a lower content of β-turn than wheat glutenin, and no α-helices were determined in it. Teff prolamin and glutelin had lower disulfide bond content and surface hydrophobicity. Teff prolamin had significantly higher thermal stability than wheat gliadin, whereas the thermal stability of teff glutelin was significantly lower than that of wheat glutenin.

Ancient duplication, coevolution, and selection at the MHC class IIA and IIB genes of birds.

The Major Histocompatibility Complex (MHC) of vertebrates is a dynamically evolving multigene family primarily responsible for recognizing non-self peptide antigens and triggering a pathogen-specific adaptive immune response. In birds, the MHC was previously thought to evolve via concerted evolution with high degree of gene homogenization and the rapid loss of orthology. However, the discovery of two ancient avian MHC-IIB gene lineages (DAB1 and DAB2) originating before the radiation of extant birds indicated that despite the action of concerted evolution, orthology may be detectable for long evolutionary periods. Here, we take advantage of rapidly accumulating digital genomic resources to search for the signal of an ancient duplication at the avian MHC-IIA genes, as well as to compare phylogenetic distribution and selection between MHC-IIA and IIB gene lineages. The analysis of MHC sequences from over 230 species representing ca. 70 bird families revealed the presence of two ancient MHC-IIA gene lineages (DAA1 and DAA2) and showed that their phylogenetic distribution matches exactly the distribution of DAB1 and DAB2 lineages, suggesting tight coevolution. The early post-duplication divergence of DAA1 and DAA2 was driven by positive selection fixing radical amino acid differences within the membrane-proximal domain and, most probably, being functionally related to the interactions between α2 and β2 chains of the MHC-II heterodimer. We detected no evidence for an overall (gene-wide) relaxation or intensification of selection at either DAA1/DAB1 or DAA2/DAB2, but codon-specific differences in selection signature were found at the peptide-binding sites between the two gene lineages, perhaps implying specialization to different pathogen regimes. Our results suggest that specific pairing of MHC-II α and β chains may have an adaptive significance, a conclusion that advances knowledge on the macroevolution of the avian MHC-II and opens exciting novel directions for future research.

Untargeted metabolomics provide new insights into the implication of Lactobacillus helveticus strains isolated from natural whey starter in methylglyoxal-mediated browning

Hard cheeses may occasionally show a brown discolouration during ripening due to multifactorial phenomena that involve bacteria and give rise to pyrazines arising from methylglyoxal. The present work aimed at developing a novel approach to investigate the role of natural starters in browning. To this object, 11 strains of L. helveticus were incubated in a medium containing 10 % rennet casein dissolved in whey, and then growth was monitored by measuring pH and number of genomes/mL. Browning was assessed through CIELab analysis, methylglyoxal production was determined by targeted mass spectrometry, and untargeted metabolomics was used to extrapolate marker compounds associated with browning discoloration. The medium allowed the growth of all the strains tested and differences in colour were observed, especially for strain A7 (ΔE* value 15.92 ± 0.27). Noteworthy, this strain was also the higher producer of methylglyoxal (2.44 µg/mL). Metabolomics highlighted pyrazines and β-carboline compounds as markers of browning at 42 °C and 16 °C, respectively. Moreover, multivariate statistics pointed out differences in free amino acids and oligopeptides linked to proteolysis, while 1,2-propanediol and S-Lactoylglutathione suggested specific detoxification route in methylglyoxal-producing strains. Our model allowed detecting differences in browning amid strains, paving the way towards the study of individual L. helveticus strains to identify the variables leading to discoloration or to study the interaction between different strains in natural whey starters.

Natural variation in a short region of the Acidovorax citrulli type III-secreted effector AopW1 is associated with differences in cytotoxicity and host adaptation.

Bacterial fruit blotch, caused by Acidovorax citrulli, is a serious disease of melon and watermelon. The strains of the pathogen belong to two major genetic groups: group I strains are strongly associated with melon, while group II strains are more aggressive on watermelon. A. citrulli secretes many protein effectors to the host cell via the type III secretion system. Here we characterized AopW1, an effector that shares similarity to the actin cytoskeleton-disrupting effector HopW1 of Pseudomonas syringae and with effectors from other plant-pathogenic bacterial species. AopW1 has a highly variable region (HVR) within amino acid positions 147 to 192, showing 14 amino acid differences between group I and II variants. We show that group I AopW1 is more toxic to yeast and Nicotiana benthamiana cells than group II AopW1, having stronger actin filament disruption activity, and increased ability to induce cell death and reduce callose deposition. We further demonstrated the importance of some amino acid positions within the HVR for AopW1 cytotoxicity. Cellular analyses revealed that AopW1 also localizes to the endoplasmic reticulum, chloroplasts, and plant endosomes. We also show that overexpression of the endosome-associated protein EHD1 attenuates AopW1-induced cell death and increases defense responses. Finally, we show that sequence variation in AopW1 plays a significant role in the adaptation of group I and II strains to their preferred hosts, melon and watermelon, respectively. This study provides new insights into the HopW1 family of bacterial effectors and provides first evidence on the involvement of EHD1 in response to biotic stress.

Genotypic diversity of staphylococcal enterotoxin B gene (seb) and its association with molecular characterization and antimicrobial resistance of Staphylococcus aureus from retail food

To investigate the expression pattern of staphylococcal enterotoxin B (SEB) in food and the genotypic diversity of SEB-encoding gene in association with molecular characteristics and antimicrobial resistance of S. aureus, 498 isolates from retail food were screened for seb gene and detected for SEB production in S. aureus. In addition, the seb nucleotide sequences, virulence genes, resistance genes, antimicrobial susceptibility and molecular characteristics of S. aureus were examined. A total of 45 (9.0 %) seb-positive S. aureus strains were identified, all of which expressed SEB. The detection rate of SEB-production strains was significantly higher from dairy-related sources than those from other sources (P < 0.05). In vitro simulations showed that S. aureus could grow and express SEB in both milk and pork, with SEB expression exceeding 20 ng/g after 1 day of storage at room temperature. There were 2 distinct SEB genotyping (SEB1 and SEB2) in the SEB amino acid sequences of the 45 isolates, including 4 amino acid differences (Ala-13Val, Ser14Ala, Asn192Ser, and Met222Leu). There was no significant difference (P > 0.05) in SEB production between SEB1 and SEB2 genotyping strains. Based on MLST clustering analysis, the same molecular type strains were found to have the same SEB genotyping, virulence gene profile, resistance gene profile and drug resistance profile. Among them, the dominant molecular types of SEB1 and SEB2 strains were CC1-ST188-t189 and CC59-ST59-t437, respectively. Compared to the CC1-ST188-t189 clonal strain, the CC59-ST59-t437 clonal strain carried a higher number of virulence and resistance genes and exhibited a broader resistance profile. Therefore, understanding the characteristics of the strains and their expression patterns in food can be effective in preventing food poisoning incidents.

Recognition of polymorphic Csd proteins determines sex in the honeybee.

Sex in honeybees, Apis mellifera, is genetically determined by heterozygous versus homo/hemizygous genotypes involving numerous alleles at the single complementary sex determination locus. The molecular mechanism of sex determination is however unknown because there are more than 4950 known possible allele combinations, but only two sexes in the species. We show how protein variants expressed from complementary sex determiner (csd) gene determine sex. In females, the amino acid differences between Csd variants at the potential-specifying domain (PSD) direct the selection of a conserved coiled-coil domain for binding and protein complexation. This recognition mechanism activates Csd proteins and, thus, the female pathway. In males, the absence of polymorphisms establishes other binding elements at PSD for binding and complexation of identical Csd proteins. This second recognition mechanism inactivates Csd proteins and commits male development via default pathway. Our results demonstrate that the recognition of different versus identical variants of a single protein is a mechanism to determine sex.

Polymorphism and mutational diversity of virulence (vcgCPI/vcgCPE) and resistance determinants (aac(3)-IIa, (aacC2, strA, Sul 1, and 11) among human pathogenic Vibrio species recovered from surface waters in South-Western districts of Uganda

BackgroundVibrio species are among the autochthonous bacterial populations found in surface waters and associated with various life-threatening extraintestinal diseases, especially in human populations with underlying illnesses and wound infections. Presently, very diminutive information exists regarding these species’ mutational diversity of virulence and resistance genes. This study evaluated variations in endonucleases and mutational diversity of the virulence and resistance genes of Vibrio isolates, harboring virulence-correlated gene (vcgCPI), dihydropteroate synthase type 1 and type II genes (Sul 1 and 11), (aadA) aminoglycoside (3′′) (9) adenylyltransferase gene, (aac(3)-IIa, (aacC2)a, aminoglycoside N(3)-acetyltransferase III, and (strA) aminoglycoside 3′-phosphotransferase resistance genes. MethodsUsing combinations of molecular biology techniques, bioinformatics tools, and sequence analysis. ResultsOur result revealed various nucleotide variations in virulence determinants of V. vulnificus (vcgCPI) at nucleotide positions (codon) 73–75 (A → G) and 300–302 (N → S). The aminoglycosides resistance gene (aadA) of Vibrio species depicts a nucleotide difference at position 482 (A → G), while the aminoglycosides resistance gene (sul 1 and 11) showed two variable regions of nucleotide polymorphism (102 and 140). The amino acid differences exist with the nucleotide polymorphism at position 140 (A → E). The banding patterns produced by the restriction enzymes HinP1I, MwoI, and StyD4I showed significant variations. Also, the restriction enzyme digestion of protein dihydropteroate synthase type 1 and type II genes (Sul 1 and 11) differed significantly, while enzymes DpnI and Hinf1 indicate no significant differences. The restriction enzyme NlaIV showed no band compared to reference isolates from the GenBank. However, the resistant determinants show significant point nucleotide mutation, which does not produce any amino acid change with diverse polymorphic regions, as revealed in the restriction digest profile. ConclusionThe described virulence and resistance determinants possess specific polymorphic locus relevant to pathogenomics studies, pharmacogenomic, and control of such water-associated strains.

FRI099 Advanced Lipoprotein Analysis In Patients With Cushing’s Syndrome

Abstract Disclosure: R.T. Varghese: None. R. McGlotten: None. M. Sampson: None. A. Prakash: None. R. Shamburek: None. A.T. Remaley: None. R. Muniyappa: None. L.K. Nieman: None. Introduction: Cushing syndrome (CS) patients have a higher mortality rate due to increased cardiovascular disease (CVD). Analysis of lipoprotein particles with nuclear magnetic resonance (NMR) spectroscopy provides additional insights into CVD risk beyond the standard lipid panel. The goal of the study was to better characterize the lipid abnormalities in patients with CS by using high-resolution separation of lipoprotein particles. Methods: We measured plasma lipids (Cholesterol, TC; calculated low density lipoprotein, LDL; triglycerides, TG; high density lipoprotein, HDL) by the traditional enzymatic assay and lipoprotein particles by NMR spectroscopy. Measures of inflammation and insulin resistance were also derived from NMR spectra. Values are shown as mean ± SEM or median [IQR]. Results: After initial evaluation, 42 subjects (mean age 47.4 ± 2.5 years; 28 females; weight 85.0 ± 3.1 kg) had Cushing's disease (n=23), ectopic ACTH secretion (n=17), or adrenal adenoma (n=2). Ten patients were on chronic statin therapy, and 14 subjects had type 2 diabetes mellitus (T2DM). The control group comprised 45 subjects without T2DM [mean age 45.4 ± 2.1 years and weight (83.5 ± 2.7) kg]. None in the control group were on statin therapy. Compared to controls, CS patients had significantly higher systolic (135 ±2 vs. 119 ±2 mm Hg, p&amp;lt;0.001) and diastolic blood pressure (79 ±2 vs. 71 ±1 mm Hg, p&amp;lt;0.001), HbA1c (6.1 [5.4-7.5] vs. 5.4 [5.0-5.7], p&amp;lt;0.001), plasma glucose (103 [90-137] vs 94 [89-100] mg/dL, p=0.003), and insulin (17[12-26] vs 11[7-15] uU/mL, p=0.01) levels. Compared to controls, patients with CS had significantly higher triglyceride concentration (116 [84 -146] vs 87 [62-120] mg/dL, p=0.02) and lower LDL concentration (90 [69 -113] vs 108 [90 -125] mg/dL, p=0.04). However, total cholesterol and HDL cholesterol concentrations were not different. LDL particle size in CS was smaller (20.5[20.0-21.0] vs 21.0 [20.7-21.2] nm, p=0.01) and VLDL particle size was larger (49.8 ± 0.9 vs 45.7 ± 1.0 nm, p=0.002) than in controls. No difference was noted between groups in HDL particle number or size or in LDL and VLDL particle number. In the group with CS, a marker of inflammation, GlycA (Glycan N-acetylglucosamine) (494.5 ± 18.2 vs. 382.5 ± 9.0 µmol/L, p&amp;lt;0.001) and Lipoprotein Insulin Resistance score (LPIR) (46.4 ± 3.1 vs. 36.2 ± 3.1, p=0.02) were significantly higher. There were no group differences in branch-chained amino acids (BCCA) levels. These differences remained even when patients with type 2 diabetes and those on statin therapy were excluded. Conclusions: These data suggest that the higher inflammatory and insulin resistance state, dysglycemia, hypertension, and altered lipoprotein profiles may contribute to the increased CV risk in patients with Cushing’s syndrome. Early detection and treatment for these abnormalities may attenuate CV risk. Presentation: Friday, June 16, 2023

Metabolomic comparison of meat quality and metabolites of geese breast muscle at different ages.

The purpose of this study was to distinguish the effect of age on the meat quality and chemical composition of Yangzhou goose breast meat. Nontargeted metabolomics analysis (UHPLC-MS/MS) was used to distinguish the metabolic composition of goose meat at different ages, and Pearson's correlations between differential metabolites and key meat parameters were assessed. Compared with goslings, adult geese had lighter, redder and chewier meat (p<0.05). Metabolite analysis revealed significant differences in nucleosides, organic acids, amino acids and sugars. Levels of IMP, xanthosine, pretyrosine and l-threonine were significantly higher in older meat (p<0.05) and positively correlated with meat freshness indicators. However, pyruvic acid, l-cysteine and glucose 6-phosphate were up-regulated in gosling meat (p<0.05), which were important flavor compounds. These results facilitate the further investigation of changes in goose meat composition and provide biomarkers for determining goose meat quality at different ages.

Multidimensional quantitative phenotypic and molecular analysis reveals neomorphic behaviors of p53 missense mutants

Mutations in the TP53 tumor suppressor gene occur in >80% of the triple-negative or basal-like breast cancer. To test whether neomorphic functions of specific TP53 missense mutations contribute to phenotypic heterogeneity, we characterized phenotypes of non-transformed MCF10A-derived cell lines expressing the ten most common missense mutant p53 proteins and observed a wide spectrum of phenotypic changes in cell survival, resistance to apoptosis and anoikis, cell migration, invasion and 3D mammosphere architecture. The p53 mutants R248W, R273C, R248Q, and Y220C are the most aggressive while G245S and Y234C are the least, which correlates with survival rates of basal-like breast cancer patients. Interestingly, a crucial amino acid difference at one position—R273C vs. R273H—has drastic changes on cellular phenotype. RNA-Seq and ChIP-Seq analyses show distinct DNA binding properties of different p53 mutants, yielding heterogeneous transcriptomics profiles, and MD simulation provided structural basis of differential DNA binding of different p53 mutants. Integrative statistical and machine-learning-based pathway analysis on gene expression profiles with phenotype vectors across the mutant cell lines identifies quantitative association of multiple pathways including the Hippo/YAP/TAZ pathway with phenotypic aggressiveness. Further, comparative analyses of large transcriptomics datasets on breast cancer cell lines and tumors suggest that dysregulation of the Hippo/YAP/TAZ pathway plays a key role in driving the cellular phenotypes towards basal-like in the presence of more aggressive p53 mutants. Overall, our study describes distinct gain-of-function impacts on protein functions, transcriptional profiles, and cellular behaviors of different p53 missense mutants, which contribute to clinical phenotypic heterogeneity of triple-negative breast tumors.

A novel non-centrifugal sugar prepared from tiger nut (Cyperus esculentus L.) meal: Preparation methods and comparison with sugarcane

The lack of research on the rich sucrose in tiger nut meal has been a major obstruction to the comprehensive utilization of tiger nut (Cyperus esculentus L.). In this study, for the first time, tiger nut meal was used to producing non-centrifugal sugar (NCS). Three samples − NCS-W1 (NCS prepared by water extraction and concentrated at 115 °C), NCS-W2 (NCS prepared by water extraction and concentrated at 135 °C), and NCS-E (NCS prepared by 70 % ethanol–water extraction and concentrated at 115 °C) were obtained, with yields of 14.25–14.59 %. These samples and sugarcane NCS products (NCS-C1, NCS-C2, NCS-L) were compared and analyzed in terms of color, pH, turbidity, soluble solid content, and proximate composition. Their Fourier-transformed infrared spectra, crystal patterns, and thermal stabilities were also analyzed. The NCS-W1, -W2, and -E showed excellent performance, and they were better than sugarcane NCS products in terms of free radical scavenging ability and cytoprotective effects. Differences in phenolic acid composition, flavonoid composition, amino acid, mineral content, and vitamins C and E content were also analyzed. This work demonstrates that tiger nut meal might be a new source of NCS. As such it would contribute to the full utilization of tiger nut.

The Biological Action and Structural Characterization of Eryngitin 3 and 4, Ribotoxin-like Proteins from Pleurotus eryngii Fruiting Bodies.

Ribotoxin-like proteins (RL-Ps) are specific ribonucleases found in mushrooms that are able to cleave a single phosphodiester bond located in the sarcin-ricin loop (SRL) of the large rRNA. The cleaved SRL interacts differently with some ribosomal proteins (P-stalk). This action blocks protein synthesis because the damaged ribosomes are unable to interact with elongation factors. Here, the amino acid sequences of eryngitin 3 and 4, RL-Ps isolated from Pleurotus eryngii fruiting bodies, were determined to (i) obtain structural information on this specific ribonuclease family from edible mushrooms and (ii) explore the structural determinants which justify their different biological and antipathogenic activities. Indeed, eryngitin 3 exhibited higher toxicity with respect to eryngitin 4 against tumoral cell lines and model fungi. Structurally, eryngitin 3 and 4 consist of 132 amino acids, most of them identical and exhibiting a single free cysteinyl residue. The amino acidic differences between the two toxins are (i) an additional phenylalanyl residue at the N-terminus of eryngitin 3, not retrieved in eryngitin 4, and (ii) an additional arginyl residue at the C-terminus of eryngitin 4, not retrieved in eryngitin 3. The 3D models of eryngitins show slight differences at the N- and C-terminal regions. In particular, the positive electrostatic surface at the C-terminal of eryngitin 4 is due to the additional arginyl residue not retrieved in eryngitin 3. This additional positive charge could interfere with the binding to the SRL (substrate) or with some ribosomal proteins (P-stalk structure) during substrate recognition.

Comparing the differences of physicochemical properties and volatiles in semi-dry Hakka rice wine and traditional sweet rice wine via HPLC, GC–MS and E-tongue analysis

This study aimed to explore effects of indica rice addition, rice soaking time and rice soup addition on total sugar and alcohol content of semi-dry Hakka rice wine (HRW) and compare its difference in physicochemical properties and volatiles with traditional sweet rice wine (TSRW) via HPLC, GC–MS and E-tongue. The optimal fermentation conditions of semi-dry HRW were 50 % indica rice addition, 12 h rice soaking time and 85 % rice soup addition. The total sugar (16.13 mg/mL) of semi-dry HRW was significantly lower than that of TSRW (135.79 mg/mL), especially the trehalose, glucose, sucrose and maltose. Its alcohol content was significantly higher than that of TSRW. There were significant differences in volatile components between semi-dry HRW and TSRW, especially esters, alcohols and ketones, but no significant differences in organic acids and amino acids. Results obtained could provide reference data for improving the fermentation process and quality of semi-dry HRW.

Biophysical Rationale for the Selective Inhibition of PTP1B over TCPTP by Nonpolar Terpenoids.

Protein tyrosine phosphatases (PTPs) are emerging drug targets for many diseases, including cancer, autoimmunity, and neurological disorders. A high degree of structural similarity between their catalytic domains, however, has hindered the development of selective pharmacological agents. Our previous research uncovered two unfunctionalized terpenoid inhibitors that selectively inhibit PTP1B over T-cell PTP (TCPTP), two PTPs with high sequence conservation. Here, we use molecular modeling, with supporting experimental validation, to study the molecular basis of this unusual selectivity. Molecular dynamics (MD) simulations suggest that PTP1B and TCPTP share a h-bond network that connects the active site to a distal allosteric pocket; this network stabilizes the closed conformation of the catalytically essential WPD loop, which it links to the L-11 loop and neighboring α3 and α7 helices on the other side of the catalytic domain. Terpenoid binding to either of two proximal C-terminal sites─an α site and a β site─can disrupt the allosteric network; however, binding to the α site forms a stable complex only in PTP1B. In TCPTP, two charged residues disfavor binding at the α site in favor of binding at the β site, which is conserved between the two proteins. Our findings thus indicate that minor amino acid differences at the poorly conserved α site enable selective binding, a property that might be enhanced with chemical elaboration, and illustrate more broadly how minor differences in the conservation of neighboring─yet functionally similar─allosteric sites can affect the selectivity of inhibitory scaffolds (e.g., fragments).

Liver and Muscle Transcriptomes Differ in Mid-Lactation Cows Divergent in Feed Efficiency in the Presence or Absence of Supplemental Rumen-Protected Choline.

Improving dairy cow feed efficiency is critical to the sustainability and profitability of dairy production, yet the underlying mechanisms that contribute to individual cow variation in feed efficiency are not fully understood. The objectives of this study were to (1) identify genes and associated pathways that are altered in cows with high- or low-residual feed intake (RFI) using RNA sequencing, and (2) determine if rumen-protected choline supplementation during mid-lactation would influence performance or feed efficiency. Mid-lactation (134 ± 20 days in milk) multiparous Holstein cows were randomly assigned to either supplementation of 0 g/d supplementation (CTL; n = 32) or 30 g/d of a rumen-protected choline product (RPC; 13.2 g choline ion; n = 32; Balchem Corp., New Hampton, NY, USA). Residual feed intake was determined as dry matter intake regressed on milk energy output, days in milk, body weight change, metabolic body weight, and dietary treatment. The 12 cows with the highest RFI (low feed efficient; LE) and 12 cows with the lowest RFI (high feed efficient; HE), balanced by dietary treatment, were selected for blood, liver, and muscle analysis. No differences in production or feed efficiency were detected with RPC supplementation, although albumin was greater and arachidonic acid tended to be greater in RPC cows. Concentrations of β-hydroxybutyrate were greater in HE cows. Between HE and LE, 268 and 315 differentially expressed genes in liver and muscle tissue, respectively, were identified through RNA sequencing. Pathway analysis indicated differences in cell cycling, oxidative stress, and immunity in liver and differences in glucose and fatty acid pathways in muscle. The current work indicates that unique differences in liver and muscle post-absorptive nutrient metabolism contribute to sources of variation in feed efficiency and that differences in amino acid and fatty acid oxidation, cell cycling, and immune function should be further examined.

Function-structure approach reveals novel insights on the interplay of Immunoglobulin G 1 proteoforms and Fc gamma receptor IIa allotypes.

Human Fc gamma receptor IIa (FcγRIIa) or CD32a has two major allotypes with a single amino acid difference at position 131 (histidine or arginine). Differences in FcγRIIa allotypes are known to impact immunological responses such as the clinical outcome of therapeutic monoclonal antibodies (mAbs). FcγRIIa is involved in antibody-dependent cellular phagocytosis (ADCP), which is an important contributor to the mechanism-of-action of mAbs by driving phagocytic clearance of cancer cells. Hence, understanding the impact of individual mAb proteoforms on the binding to FcγRIIa, and its different allotypes, is crucial for defining meaningful critical quality attributes (CQAs). Here, we report a function-structure based approach guided by novel FcγRIIa affinity chromatography-mass spectrometry (AC-MS) assays to assess individual IgG1 proteoforms. This allowed to unravel allotype-specific differences of IgG1 proteoforms on FcγRIIa binding. FcγRIIa AC-MS confirmed and refined structure-function relationships of IgG1 glycoform interactions. For example, the positive impact of afucosylation was higher than galactosylation for FcγRIIa Arg compared to FcγRIIa His. Moreover, we observed FcγRIIa allotype-opposing and IgG1 proteoform integrity-dependent differences in the binding response of stress-induced IgG1 proteoforms comprising asparagine 325 deamidation. The FcγRIIa-allotype dependent binding differences resolved by AC-MS were in line with functional ADCP-surrogate bioassay models. The molecular basis of the observed allotype specificity and proteoform selectivity upon asparagine 325 deamidation was elucidated using molecular dynamics. The observed differences were attributed to the contributions of an inter-molecular salt bridge between IgG1 and FcγRIIa Arg and the contribution of an intra-molecular hydrophobic pocket in IgG1. Our work highlights the unprecedented structural and functional resolution of AC-MS approaches along with predictive biological significance of observed affinity differences within relevant cell-based methods. This makes FcγRIIa AC-MS an invaluable tool to streamline the CQA assessment of therapeutic mAbs.

Investigation of circulating infectious agents in experimental Beagle dogs of a production colony and three research facilities in China from June 2021 to May 2022.

To understand the epizootiologic characteristics of pathogens and opportunistic infections in one Beagle dog production colony and three research facilities, viruses and mycoplasma were detected in 1777 samples collected from Beagle dogs in China by polymerase chain reaction/reverse transcription polymerase chain reaction, and bacteria were isolated and identified by 16S rRNA sequence analysis. In addition, genotyping of the major circulating viruses was carried out by amplification of gene fragments and homology analysis. Canine coronavirus (CCoV), Escherichia coli, canine parvovirus (CPV), Bordetella bronchiseptica, Clostridium perfringens, Mycoplasma cynos, Klebsiella pneumoniae, Streptococcus canis, canine astrovirus (CaAstV), canine kobuvirus (CaKV), Pseudomonas aeruginosa, Proteus mirabilis, Macrococcus canis, Pasteurella canis, canine bocavirus (CBoV) and canine adenovirus (CAdV) were detected in the samples. Single, double, triple and quadruple infections accounted for 6.6%, 1.4%, 1.2% and 0.96% of samples, respectively. CCoV strains in 81 samples included three genotypes, CCoV-I, CCoV-IIa and CCoV-IIb, by analysis of S gene. The rate of single infection of CCoV-I, CCoV-IIa or CCoV-IIb was 19%, 38% or 7.4% respectively. The double and triple infection rates of CCoV were 32.8% and 2.5% respectively. All CPV strains in 36 samples belonged to CPV-2c. There were three amino acid differences in the Fiber protein of CAdV-positive sample QD2022, compared with the reference strain Toronto A26/61 and the vaccine strain YCA-18. These results suggest that CCoV and CPV are primary infectious agents, and that these two viruses were often identified in mixed infections, or coinfections alongside mycoplasma or other bacteria. These results will provide the basis for improvements in prevention and control of naturally occurring infectious diseases in Beagle dog production colonies and research facilities.

Characterization of tigurilysin, a novel human CD59-specific cholesterol-dependent cytolysin, reveals a role for host specificity in augmenting toxin activity.

Cholesterol-dependent cytolysins (CDCs) are a large family of pore-forming toxins, produced by numerous Gram-positive pathogens. CDCs depend on host membrane cholesterol for pore formation; some CDCs also require surface-associated human CD59 (hCD59) for binding, conferring specificity for human cells. We purified a recombinant version of a putative CDC encoded in the genome of Streptococcus oralis subsp. tigurinus, tigurilysin (TGY), and used CRISPR/Cas9 to construct hCD59 knockout (KO) HeLa and JEG-3 cell lines. Cell viability assays with TGY on wild-type and hCD59 KO cells showed that TGY is a hCD59-dependent CDC. Two variants of TGY exist among S. oralis subsp. tigurinus genomes, only one of which is functional. We discovered that a single amino acid change between these two TGY variants determines its activity. Flow cytometry and oligomerization Western blots revealed that the single amino acid difference between the two TGY isoforms disrupts host cell binding and oligomerization. Furthermore, experiments with hCD59 KO cells and cholesterol-depleted cells demonstrated that TGY is fully dependent on both hCD59 and cholesterol for activity, unlike other known hCD59-dependent CDCs. Using full-length CDCs and toxin constructs differing only in the binding domain, we determined that having hCD59 dependence leads to increased lysis efficiency, conferring a potential advantage to organisms producing hCD59-dependent CDCs.

Discovery and identification of a novel canine coronavirus causing a diarrhea outbreak in Vulpes

Cross-species transmission of viruses from wildlife animal reservoirs, such as bats, poses a threat to human and domestic animal health. Previous studies have shown that domestic animals have important roles as intermediate hosts, enabling the transmission of genetically diverse coronaviruses from natural hosts to humans. Here, we report the identification and characterization of a novel canine coronavirus (VuCCoV), which caused an epidemic of acute diarrhea in Vulpes (foxes) in Shenyang, China. The epidemic started on November 8, 2019, and caused more than 39,600 deaths by January 1, 2022. Full-length viral genomic sequences were obtained from 15 foxes with diarrhea at the early stage of this outbreak. The VuCCoV genome shared more than 90% nucleotide identity with canine coronavirus (CCoV) for three of the four structural genes, with the S gene showing a larger amount of divergence. In addition, 67% (10/15) of the VuCCoV genomes contained an open reading frame (ORF3) gene, which was previously only detected in CCoV-I genomes. Notably, VuCCoV had only two to three amino acid differences at the partial RNA-dependent RNA polymerase (RdRp) level to bat CoV, suggesting a close genetic relationship. Therefore, these novel VuCCoV genomes represent a previously unsampled lineage of CCoVs. We also show that the VuCCoV spike protein binds to canine and fox aminopeptidase N (APN), which may allow this protein to serve as an entry receptor. In addition, cell lines were identified that are sensitive to VuCCoV using a pseudovirus system. These data highlight the importance of identifying the diversity and distribution of coronaviruses in domestic animals, which could mitigate future outbreaks that could threaten livestock, public health, and economic growth.