The P1 protein of wheat yellow mosaic virus exerts RNA silencing suppression activity to facilitate virus infection in wheat plants.

Wheat yellow mosaic virus (WYMV) causes severe wheat viral disease in Asia. However, the viral suppressor of RNA silencing (VSR) encoded by WYMV has not been identified. Here, the P1 protein encoded by WYMV RNA2 was shown to suppress RNA silencing in Nicotiana benthamiana. Mutagenesis assays revealed that the alanine substitution mutant G175A of P1 abolished VSR activity and mutant Y10A VSR activity remained only in younger leaves. P1, but not G175A, interacted with gene silencing-related protein, N. benthamiana calmodulin-like protein (NbCaM), and calmodulin-binding transcription activator 3 (NbCAMTA3), and Y10A interacted with NbCAMTA3 only. Competitive Bimolecular fluorescence complementation and co-immunoprecipitation assays showed that the ability of P1 disturbing the interaction between NbCaM and NbCAMTA3 was stronger than Y10A, Y10A was stronger than G175A. In vitro transcript inoculation of infectious WYMV clones further demonstrated that VSR-defective mutants G175A and Y10A reduced WYMV infection in wheat (Triticum aestivum L.), G175A had a more significant effect on virus accumulation in upper leaves of wheat than Y10A. Moreover, RNA silencing, temperature, and autophagy have significant effects on the accumulation of P1 in N. benthamiana. Taken together, WYMV P1 acts as VSR by interfering with calmodulin-associated antiviral RNAi defense to facilitate virus infection in wheat, which has provided clear insights into the function of P1 in the process of WYMV infection.

An Expeditious Neutralization Assay for Porcine Reproductive and Respiratory Syndrome Virus Based on a Recombinant Virus Expressing Green Fluorescent Protein.

Due to the extensive genetic and antigenic variation in Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), as well as its rapid mutability and evolution, PRRS prevention and control can be challenging. An expeditious and sensitive neutralization assay for PRRSV is presented to monitor neutralizing antibodies (NAbs) in serum during vaccine research. Here, a PRRSV expressing eGFP was successfully rescued with reverse genetics based on the infectious clone HuN4-F112-eGFP which we constructed. The fluorescent protein expressions of the reporter viruses remained stable for at least five passages. Based on this reporter virus, the neutralization assay can be easily used to evaluate the level of NAbs by counting cells with green fluorescence. Compared with the classical CPE assay, the newly developed assay increases sensitivity by one- to four-fold at the early antibody response stage, thus saving 2 days of assay waiting time. By using this assay to unveil the dynamics of neutralizing antibodies against PRRSV, priming immunity through either a single virulent challenge or only vaccination could produce limited NAbs, but re-infection with PRRSV would induce a faster and stronger NAb response. Overall, the novel HuN4-F112-eGFP-based neutralization assay holds the potential to provide a highly efficient platform for evaluating the next generation of PRRS vaccines.

Escherichia coli sequence type 410 with carbapenemases: a paradigm shift within E. coli toward multidrug resistance.

Escherichia coli sequence type ST410 is an emerging carbapenemase-producing multidrug-resistant (MDR) high-risk One-Health clone with the potential to significantly increase carbapenem resistance among E. coli. ST410 belongs to two clades (ST410-A and ST410-B) and three subclades (ST410-B1, ST410-B2, and ST410-B3). After a fimH switch between clades ST410-A and ST410-B1, ST410-B2 and ST410-B3 subclades showed a stepwise progression toward developing MDR. (i) ST410-B2 initially acquired fluoroquinolone resistance (via homologous recombination) in the 1980s. (ii) ST410-B2 then obtained CMY-2, CTX-M-15, and OXA-181 genes on different plasmid platforms during the 1990s. (iii) This was followed by the chromosomal integration of blaCMY-2, fstl YRIN insertion, and ompC/ompF mutations during the 2000s to create the ST410-B3 subclade. (iv) An IncF plasmid "replacement" scenario happened when ST410-B2 transformed into ST410-B3: F36:31:A4:B1 plasmids were replaced by F1:A1:B49 plasmids (both containing blaCTX-M-15) followed by blaNDM-5 incorporation during the 2010s. User-friendly cost-effective methods for the rapid identification of ST410 isolates and clades are needed because limited data are available about the frequencies and global distribution of ST410 clades. Basic mechanistic, evolutionary, surveillance, and clinical studies are urgently required to investigate the success of ST410 (including the ability to acquire successive MDR determinants). Such information will aid with management and prevention strategies to curb the spread of carbapenem-resistant E. coli. The medical community can ill afford to ignore the spread of a global E. coli clone with the potential to end the carbapenem era.

Genetic trans-complementation of L-protease fails to rescue the infectious foot-and-mouth disease virus from the Lbpro defective genome.

Outbreaks of the foot-and-mouth disease (FMD) have major economic impact on the global livestock industry by affecting the animal health and product safety. L-protease, a non-structural protein of FMDV, is a papain-like cysteine proteinase involved in viral protein processing as well as cleavage of host proteins for promoting the virus growth. FMDV synthesizes two forms of leader proteinase, Lpro (Labpro and Lbpro), where the deletion of Labpro is lethal and Lbpro deletion is reported to be attenuated. Defective replicons have been used by trans-complementing the deleted gene to produce one time replicating virus; thus, the bio-safety procedure can be compromised in the production units. Attempts are made to rescue of ΔLbproFMDV Asia1 virus by co-expressing the Lbpro protein carried in pcDNA plasmid. Mutant FMDV cDNA, pAsia-ΔLbpro, was constructed by PCR mediated mutagenesis using inverse primers. Transfection of BHK-21 cells with in-vitro transcribed RNA from the constructs failed to produce an infective mutant FMDV. Genetic trans-complementation of the Lbpro, which was done by co-transfecting the pcDNALbpro plasmid DNA along with the pAsia-ΔLbpro RNA in BHK-21 cells also failed to produce viable virus. Expression experiments of reporter genes and indirect immune-fluorescence confirmed the production of the viral proteins in wild type FMDV pAsiaWT; however, it was absent in the pAsia-ΔLbpro indicating that the leaderless virus was unable to produce infectious progeny and infect the cells. Failure to produce virus either by Lbpro deleted mutant clone or by genetic complementation suggests little chance of reversion of the disabled virus with large deletions of FMDV genome.

The role of bacterial metabolism in human gut colonization.

Can we anticipate the emergence of the next pandemic antibiotic-resistant bacterial clone? Addressing such an ambitious question relies on our ability to comprehensively understand the ecological and epidemiological factors fostering the evolution of high-risk clones. Among these factors, the ability to persistently colonize and thrive in the human gut is crucial for most high-risk clones. Nonetheless, the causes and mechanisms facilitating successful gut colonization remain obscure. Here, we review recent evidence that suggests that bacterial metabolism plays a pivotal role in determining the ability of high-risk clones to colonize the human gut. Subsequently, we outline novel approaches that enable the exploration of microbial metabolism at an unprecedented scale and level of detail. A thorough understanding of the constraints and opportunities of bacterial metabolism in gut colonization will foster our ability to predict the emergence of high-risk clones and take appropriate containment strategies.

Population-based genomic surveillance of Pseudomonas aeruginosa causing bloodstream infections in a large Canadian health region.

PURPOSE: Population-based surveillance was undertaken to determine clinical factors, susceptibility patterns, and incidence rates (IR) of Pseudomonas aeruginosa causing bloodstream infections (BSIs) in a Canadian region (2010-2018). METHODS: We combined clinical data with genomics to characterize P. aeruginosa (BSIs) (n = 167) in a well-defined Canadian (Calgary) human population over a 9-year period (2010-2018). RESULTS: The annual population IR per 100,000 patient years increased from 3.4/100,000 in 2010 to 5.9/100,000 in 2018, with the highest IRs in elderly males from the hospital setting. Over a quarter of patients presented with febrile neutropenia, followed by urinary tract infections and pneumonia. Antimicrobial resistance (AMR) rates and determinants were rare. The P. aeruginosa population was polyclonal consisting of three dominant sequence types (STs), namely ST244, ST111, and ST17. Antimicrobial-susceptible ST244 was the most common clone and belonged to three clades (A, B, C). The ST244 IR/100,000 increased over time due to the expansion of clade C. Multidrug-resistant ST111 was the second most common clone and IR/100,000 decreased over time. ST111 belonged to three clades (A, B, C) with clade C containing blaVIM-2. Different serotypes were linked to various STs. The IR/100,000 of P. aeruginosa that belonged to serotypes O6 increased significantly over time. CONCLUSION: An effective multivalent vaccine consisting of five serotypes (O1, O3, O5, O6, O11) would confer protection to > 70% of Calgary residents with P. aeruginosa BSIs. This study has provided a unique perspective of the population dynamics over time of P. aeruginosa STs, clades, and serotypes responsible for BSIs.

Co-occurrence of blaNDM-1 and blaOXA-23 in carbapenemase-producing Acinetobacter baumannii belonging to high-risk lineages isolated from burn patients in Tunisia.

AIMS: Carbapenem-resistant Acinetobacter baumannii (CR-Ab) is an important cause of infections in burn patients. This study aimed to characterize the antimicrobial susceptibility pattern of CR-Ab isolated from burns in Burn Intensive Care Unit (BICU) of the Trauma and Burn Centre of Ben Arous, to determine the prevalence of ß-lactamase-encoding genes and to search eventual genetic relatedness of CR-Ab strains. METHODS AND RESULTS: From 15 December 2016 to 2 April 2017, all nonduplicated CR-Ab isolated in burn patients in the BICU were screened by simplex Polymerase Chain Reaction (PCR) for the class A, B, C, and D ß-lactamase genes. Sequencing was performed for NDM gene only. Genetic relatedness was determined by using pulsed field gel electrophoresis (PFGE) and by multilocus sequence typing. During the study period, 34 strains of CR-Ab were isolated in burns, mainly in blood culture (n = 14) and central vascular catheter (n = 10). CR-Ab strains were susceptible to colistin but resistant to amikacin (91%), ciprofloxacin (100%), rifampicin (97%), and trimethoprim-sulfamethoxazole (100%). All strains harbored blaOXA-51-like and blaOXA-23 genes, only or associated to blaGES (n = 26; 76%), blaADC (n = 20; 59%), blaPER-1 (n = 6; 18%) or/and blaNDM-1 (n = 3; 9%). PFGE identified 16 different clusters and revealed that most strains belonged to one major cluster A (n = 15; 44.1%). Among NDM-1 isolates, two were clonally related in PFGE and belonged to two single locus variant sequence type ST-6 and ST-85. CONCLUSIONS: This is the first description of clonally related NDM-1 and OXA-23-producing A. baumannii strains in the largest Tunisian BICU associated with two single locus variant sequence types ST6 and ST85.

Genomic diversity of ß-lactamase producing Pseudomonas aeruginosa in Iran; the impact of global high-risk clones.

BACKGROUND: Hospital-acquired infections caused by multidrug-resistant Pseudomonas aeruginosa incline hospital stay and costs of treatment that resulted in an increased mortality rate. The frequency of P. aeruginosa high-risk clones producing carbapenemases was investigated in our clinical samples. METHODS: In this cross-sectional study, 155 non-repetitive P. aeruginosa isolates were included from different medical centers of Iran. Antibiotic susceptibility testing was determined, and the presence of ß-lactamases were sought by phenotypic and genotypic methods. The clonal relationship of all isolates was investigated, and multi-locus sequence typing (MLST) was used for finding the sequence types of carbapenemase-producers. RESULTS: The agent with highest percent susceptibility rate was recorded for colistin (94.9%). MOX and FOX were found both as low as 1.95% (3/155). The most frequent narrow spectrum ß-lactamase was SHV with 7.7% (12/155) followed by PER, OXA-1, and TEM with the frequency of 7.1% (11/155), 3.2% (5/155), and 1.3% (2/155), respectively. Carbapenemases were detected in 28 isolates (18%). The most frequent carbapenemase was IMP with 9% (14/155) followed by NDM, 8.4% (13/155). OXA-48 and VIM were also detected both per one isolate (0.65%). MLST of carbapenem resistant P. aeruginosa isolates revealed that ST244, ST664, ST235, and ST357 were spread in subjected clinical settings. REP-PCR uncovered high genomic diversity in our clinical setting. CONCLUSION: Clonal proliferation of ST235 strain plays a key role in the propagation of MDR pattern in P. aeruginosa. Our data showed that high-risk clones has distributed in Iran, and programs are required to limit spreading of these clones.

Treated municipal wastewater as a source of high-risk and emerging multidrug-resistant clones of E. coli and other Enterobacterales producing extended-spectrum ß-lactamases.

Extended-spectrum ß-lactamase (ESBL)-producing Enterobacterales are a major public health problem, and wastewater from municipal wastewater treatment plants (WWTPs) is a potential means of spreading them into the environment and community. Our objective was to isolate ESBL-producing E. coli and other Enterobacterales from wastewater after treatment at Croatia's largest WWTP and to characterize these isolates by phenotypic and genotypic testing. Of the 200 bacterial isolates, 140 were confirmed as Enterobacterales by MALDI-TOF MS, with Escherichia coli and Klebsiella spp. predominating (69% and 7%, respectively). All 140 enterobacterial isolates were multidrug-resistant (MDR) and produced ESBLs. The most prevalent ESBL genes among the isolates tested were blaCTX-M-15 (60%), blaTEM-116 (44%), and blaCTX-M-3 (13%). Most isolates (94%) carried more than one ESBL gene in addition to blaCTX-M. Genes encoding plasmid-mediated AmpC, most notably blaEBC, were detected in 22% of isolates, whereas genes encoding carbapenemases (blaOXA-48, blaNDM-1, blaVIM-1) were less represented (10%). In E. coli, 9 different sequence types (ST) were found, with the emerging high-risk clones ST361 (serotype A-O9:H30) and pandemic ST131 (serotype B2-O25:H4) predominating (32% and 15%, respectively). Other high-risk E. coli clones included ST405 (3%), ST410 (3%), CC10 (3%), ST10 (3%), and ST38 (2%), and emerging clones included ST1193 (2%) and ST635 (2%). Whole-genome sequencing of three representative E. coli from two dominant clone groups (ST361 and ST131) and one extensively drug-resistant K. oxytoca revealed the presence of multiple plasmids and resistance genes to several other antibiotic classes, as well as association of the blaCTX-M-15 gene with transposons and insertion sequences. Our findings indicate that treated municipal wastewater contributes to the spread of emerging and pandemic MDR E. coli clones and other enterobacterial strains of clinical importance into the aquatic environment, with the risk of reintroduction into humans.

Raw meat-based diet for pets: a neglected source of human exposure to Salmonella and pathogenic Escherichia coli clones carrying mcr, Portugal, September 2019 to January 2020.

BackgroundThe pet industry is expanding worldwide, particularly raw meat-based diets (RMBDs). There are concerns regarding the safety of RMBDs, especially their potential to spread clinically relevant antibiotic-resistant bacteria or zoonotic pathogens.AimWe aimed to investigate whether dog food, including RMBD, commercially available in Portugal can be a source of Salmonella and/or other Enterobacteriaceae strains resistant to last-line antibiotics such as colistin.MethodsFifty-five samples from 25 brands (21 international ones) of various dog food types from 12 suppliers were screened by standard cultural methods between September 2019 and January 2020. Isolates were characterised by phenotypic and genotypic methods, including whole genome sequencing and comparative genomics.ResultsOnly RMBD batches were contaminated, with 10 of 14 containing polyclonal multidrug-resistant (MDR) Escherichia coli and one MDR Salmonella. One turkey-based sample contained MDR Salmonella serotype 1,4,[5],12:i:- ST34/cgST142761 with similarity to human clinical isolates occurring worldwide. This Salmonella exhibited typical antibiotic resistance (bla TEM + strA-strB + sul2 + tet(B)) and metal tolerance profiles (pco + sil + ars) associated with the European epidemic clone. Two samples (turkey/veal) carried globally dispersed MDR E. coli (ST3997-complexST10/cgST95899 and ST297/cgST138377) with colistin resistance (minimum inhibitory concentration: 4 mg/L) and mcr-1 gene on IncX4 plasmids, which were identical to other IncX4 circulating worldwide.ConclusionSome RMBDs from European brands available in Portugal can be a vehicle for clinically relevant MDR Salmonella and pathogenic E. coli clones carrying genes encoding resistance to the last-line antibiotic colistin. Proactive actions within the One Health context, spanning regulatory, pet-food industry and consumer levels, are needed to mitigate these public health risks.

Physiological, Biochemical, and Molecular Analyses Reveal Dark Heartwood Formation Mechanism in Acacia melanoxylon.

Acacia melanoxylon is highly valued for its commercial applications, with the heartwood exhibiting a range of colors from dark to light among its various clones. The underlying mechanisms contributing to this color variation, however, have not been fully elucidated. In an effort to understand the factors that influence the development of dark heartwood, a comparative analysis was conducted on the microstructure, substance composition, differential gene expression, and metabolite profiles in the sapwood (SW), transition zone (TZ), and heartwood (HW) of two distinct clones, SR14 and SR25. A microscopic examination revealed that heartwood color variations are associated with an increased substance content within the ray parenchyma cells. A substance analysis indicated that the levels of starches, sugars, and lignin were more abundant in SP compared to HW, while the concentrations of phenols, flavonoids, and terpenoids were found to be higher in HW than in SP. Notably, the dark heartwood of the SR25 clone exhibited greater quantities of phenols and flavonoids compared to the SR14 clone, suggesting that these compounds are pivotal to the color distinction of the heartwood. An integrated analysis of transcriptome and metabolomics data uncovered a significant accumulation of sinapyl alcohol, sinapoyl aldehyde, hesperetin, 2', 3, 4, 4', 6'-peptahydroxychalcone 4'-O-glucoside, homoeriodictyol, and (2S)-liquiritigenin in the heartwood of SR25, which correlates with the up-regulated expression of CCRs (evm.TU.Chr3.1751, evm.TU.Chr4.654_667, evm.TU.Chr4.675, evm.TU.Chr4.699, and evm.TU.Chr4.704), COMTs (evm.TU.Chr13.3082, evm.TU.Chr13.3086, and evm.TU.Chr7.1411), CADs (evm.TU.Chr10.2175, evm.TU.Chr1.3453, and evm.TU.Chr8.1600), and HCTs (evm.TU.Chr4.1122, evm.TU.Chr4.1123, evm.TU.Chr8.1758, and evm.TU.Chr9.2960) in the TZ of A. melanoxylon. Furthermore, a marked differential expression of transcription factors (TFs), including MYBs, AP2/ERFs, bHLHs, bZIPs, C2H2s, and WRKYs, were observed to be closely linked to the phenols and flavonoids metabolites, highlighting the potential role of multiple TFs in regulating the biosynthesis of these metabolites and, consequently, influencing the color variation in the heartwood. This study facilitates molecular breeding for the accumulation of metabolites influencing the heartwood color in A. melanoxylon, and offers new insights into the molecular mechanisms underlying heartwood formation in woody plants.

Global Epidemiology and Mechanisms of Resistance of Acinetobacter baumannii-calcoaceticus Complex.

Acinetobacter baumannii-calcoaceticus complex is the most commonly identified species in the genus Acinetobacter and it accounts for a large percentage of nosocomial infections, including bacteremia, pneumonia, and infections of the skin and urinary tract. A few key clones of A. baumannii-calcoaceticus are currently responsible for the dissemination of these organisms worldwide. Unfortunately, multidrug resistance is a common trait among these clones due to their unrivalled adaptive nature. A. baumannii-calcoaceticus isolates can accumulate resistance traits by a plethora of mechanisms, including horizontal gene transfer, natural transformation, acquisition of mutations, and mobilization of genetic elements that modulate expression of intrinsic and acquired genes.

Emergence of Carbapenemase-Producing Hypervirulent Klebsiella pneumoniae in Switzerland.

Increasing occurrence of multidrug-resistant (MDR) and hypervirulent (hv) Klebsiella pneumoniae (MDR-hvKp) convergent clones is being observed. Those strains have the potential of causing difficult-to-treat infections in healthy adults with an increased capacity for mortality. It is therefore crucial to track their dissemination to prevent their further spread. The aim of our study was to investigate the occurrence of carbapenemase-producing hvKp isolates in Switzerland and to determine their genetic profile. A total of 279 MDR carbapenemase-producing K. pneumoniae from patients hospitalized all over Switzerland was investigated, and a rate of 9.0% K. pneumoniae presenting a virulence genotype was identified. Those isolates produced either KPC, NDM, or OXA-48 and had been either recovered from rectal swabs, urine, and blood. A series of previously reported K. pneumoniae clones such as ST23-K1, ST395-K2, and ST147-K20 or ST147-K64 were identified. All the isolates defined as MDR-hvKp (4.7%) possessed the aerobactin and the yersiniabactin clusters. The ST23-K1s were the only isolates presenting the colibactin cluster and achieved higher virulence scores. This study highlights the occurrence and circulation of worrisome MDR-hvKp and MDR nonhypervirulent K. pneumoniae (MDR-nhv-Kp) isolates in Switzerland. Our findings raise an alert regarding the need for active surveillance networks to track and monitor the spread of such successful hybrid clones representing a public health threat worldwide.

The Hinge Region of the Israeli Acute Paralysis Virus Internal Ribosome Entry Site Directs Ribosomal Positioning, Translational Activity, and Virus Infection.

All viruses must usurp host ribosomes for viral protein synthesis. Dicistroviruses utilize an intergenic region internal ribosome entry site (IGR IRES) to directly recruit ribosomes and mediate translation initiation from a non-AUG start codon. The IGR IRES adopts a three-pseudoknot structure that comprises a ribosome binding domain of pseudoknot II and III (PKII and PKIII), and a tRNA-like anticodon domain (PKI) connected via a short, one to three nucleotide hinge region. Recent cryo-EM structural analysis of the dicistrovirus Taura syndrome virus (TSV) IGR IRES bound to the ribosome suggests that the hinge region may facilitate translocation of the IRES from the ribosomal A to P site. In this study, we provide mechanistic and functional insights into the role of the hinge region in IGR IRES translation. Using the honeybee dicistrovirus, Israeli acute paralysis virus (IAPV), as a model, we demonstrate that mutations of the hinge region resulted in decreased IRES-dependent translation in vitro. Toeprinting primer extension analysis of mutant IRESs bound to purified ribosomes and in rabbit reticulocyte lysates showed defects in the initial ribosome positioning on the IRES. Finally, using a hybrid dicistrovirus clone, mutations in the hinge region of the IAPV IRES resulted in decreased viral yield. Our work reveals an unexpected role of the hinge region of the dicistrovirus IGR IRES coordinating the two independently folded domains of the IRES to properly position the ribosome to start translation. IMPORTANCE Viruses must use the host cell machinery to direct viral protein expression for productive infection. One such mechanism is an internal ribosome entry site that can directly recruit host cell machinery. In this study, we have identified a novel sequence in an IRES that provides insight into the mechanism of viral gene expression. Specifically, this novel sequence promotes viral IRES activity by directly guiding the host cell machinery to start gene expression at a specific site.

Establishing NK-Cell Receptor Restriction by Flow Cytometry and Detecting Potential NK-Cell Clones of Uncertain Significance.

Natural killer (NK) cells develop a complex inhibitory and/or activating NK-cell receptor system, including killer cell immunoglobulin-like receptors (KIRs or CD158) and CD94/NKG2 dimers, which are variably combined to generate the individual's NK-cell receptor repertoire. Establishing NK-cell receptor restriction by flow cytometric immunophenotyping is an important step in diagnosing NK-cell neoplasms, but reference interval (RI) data for interpreting these studies are lacking. Specimens from 145 donors and 63 patients with NK-cell neoplasms were used to identify discriminatory rules based on 95% and 99% nonparametric RIs for CD158a+, CD158b+, CD158e+, KIR-negative, and NKG2A+ NK-cell populations to establish NK-cell receptor restriction. These 99% upper RI limits (NKG2a >88% or CD158a >53% or CD158b >72% or CD158e >54% or KIR-negative >72%) provided optimal discrimination between NK-cell neoplasm cases and healthy donor controls with an accuracy of 100% compared with the clinicopathologic diagnosis. The selected rules were applied to 62 consecutive samples received in our flow cytometry laboratory that were reflexed to an NK-cell panel due to an expanded NK-cell percentage (exceeding 40% of total lymphocytes). Twenty-two (35%) of 62 samples were found to harbor a very small NK-cell population with restricted NK-cell receptor expression based on the rule combination, suggestive of NK-cell clonality. A thorough clinicopathologic evaluation for the 62 patients did not reveal diagnostic features of NK-cell neoplasms; therefore, these potential clonal populations of NK cells were designated as NK-cell clones of uncertain significance (NK-CUS). In this study, we established decision rules for NK-cell receptor restriction from the largest published cohorts of healthy donors and NK-cell neoplasms. The presence of small NK-cell populations with restricted NK-cell receptors does not appear to be an uncommon finding, and its significance requires further exploration.

Metabolomic profiling of different clones of vitis vinifera L. cv. "Glera" and "Glera lunga" grapes by high-resolution mass spectrometry.

INTRODUCTION: Prosecco wine production has been strongly extended in the last decade and several new clones have been introduced. "Glera" (minimum 85%) and "Glera lunga" are grape varieties of great economic impact used to produce Prosecco wines. Study of grape berry secondary metabolites is effective in the classification of vine varieties and clones. High-resolution mass spectrometry provides complete panorama of these metabolites in single analysis and coupling to statistical multivariate analysis is successfully applied in vine chemotaxonomy. OBJECTIVES: update and deepen the knowledge on the "Glera" and "Glera lunga" berry grapes chemotaxonomy and investigate some of the most produced and marketed clones by using the modern analytical and statistical tools. METHODS: five clones of "Glera" and two of "Glera lunga" grown in the same vineyard with same agronomical practices were studied for three vintages. Grape berry metabolomics was characterized by UHPLC/QTOF and multivariate statistical analysis was performed on the signals of main metabolites of oenological interest. RESULTS: "Glera" and "Glera lunga" showed different monoterpene profiles ("Glera" is richer in glycosidic linalool and nerol) and differences in polyphenols (catechin, epicatechin and procyanidins, trans-feruloyltartaric acid, E-ε-viniferin, isorhamnetin-glucoside, quercetin galactoside). Vintage affected the accumulation of these metabolites in berry. No statistical differentiation among the clones of each variety, was found. CONCLUSIONS: Coupling HRMS metabolomics/statistical multivariate analysis enabled clear differentiation between the two varieties. The examined clones of same variety showed similar metabolomic profiles and enological characteristics, but vineyard planting using different clones can result in more consistent final wines reducing the vintage variability linked to genotype × environment interaction.

Various arrangements of mobile genetic elements among CC147 subpopulations of Klebsiella pneumoniae harboring blaNDM-1: a comparative genomic analysis of carbapenem resistant strains.

BACKGROUND: Certain clonal complexes (CCs) of Klebsiella pneumoniae such as CC147 (ST147 and ST392) are major drivers of blaNDM dissemination across the world. ST147 has repeatedly reported from our geographical region, but its population dynamics and evolutionary trajectories need to be further studied. METHODS: Comparative genomic analysis of 51 carbapenem-nonsusceptible strains as well as three hypervirulent K. pneumoniae (hvKp) recovered during 16-months of surveillance was performed using various bioinformatics tools. We investigated the genetic proximity of our ST147 strains with publicly available corresponding genomes deposited globally and from neighbor countries in our geographic region. RESULTS: While IncL/M plasmid harboring blaOXA-48 was distributed among divergent clones, blaNDM-1 was circulated by twenty of the 25 CC147 dominant clone and were mostly recovered from the ICU. The NDM-1 core structure was bracketed by a single isoform of mobile genetic elements (MGEs) [ΔISKpn26-NDM-TnAs3-ΔIS3000-Tn5403] and was located on Col440I plasmid in 68.7% of ST392. However, various arrangements of MGEs including MITESen1/MITESen1 composite transposon or combination of MITESen1/ISSen4/IS903B/IS5/ISEhe3 on IncFIb (pB171) were identified in ST147. It seems that ST392 circulated blaNDM-1 in 2018 before being gradually replaced by ST147 from the middle to the end of sample collection in 2019. ST147 strains possessed the highest number of resistance markers and showed high genetic similarity with four public genomes that harbored blaNDM-1 on the same replicon type. Mainly, there was a convergence between clusters and isolated neighboring countries in the minimum-spanning tree. A conserved arrangement of resistance markers/MGEs was linked to methyltransferase armA which was embedded in class 1 integron in 8 isolates of ST147/ST48 high-risk clones. CONCLUSION: Our findings highlight the dynamic nature of blaNDM-1 transmission among K. pneumoniae in Iran that occurs both clonally and horizontally via various combinations of MGEs. This is the first analysis of Iranian ST147/NDM + clone in the global context.

A CHO stable pool production platform for rapid clinical development of trimeric SARS-CoV-2 spike subunit vaccine antigens.

Protein expression from stably transfected Chinese hamster ovary (CHO) clones is an established but time-consuming method for manufacturing therapeutic recombinant proteins. The use of faster, alternative approaches, such as non-clonal stable pools, has been restricted due to lower productivity and longstanding regulatory guidelines. Recently, the performance of stable pools has improved dramatically, making them a viable option for quickly producing drug substance for GLP-toxicology and early-phase clinical trials in scenarios such as pandemics that demand rapid production timelines. Compared to stable CHO clones which can take several months to generate and characterize, stable pool development can be completed in only a few weeks. Here, we compared the productivity and product quality of trimeric SARS-CoV-2 spike protein ectodomains produced from stable CHO pools or clones. Using a set of biophysical and biochemical assays we show that product quality is very similar and that CHO pools demonstrate sufficient productivity to generate vaccine candidates for early clinical trials. Based on these data, we propose that regulatory guidelines should be updated to permit production of early clinical trial material from CHO pools to enable more rapid and cost-effective clinical evaluation of potentially life-saving vaccines.

Occurrence of high-risk clones of Klebsiella pneumoniae ST11, ST340, and ST855 carrying the blaKPC-2, blaNDM-1, blaNDM-5, and blaNDM-7 genes from colonized and infected patients in Brazil.

AIMS: Determine which sequence type (ST) clones were carrying the blaKPC, blaNDM, blaVIM, blaIMP, and blaGES genes and their variants in clinical isolates of multidrug-resistant Klebsiella pneumoniae. METHODS AND RESULTS: Ten K. pneumoniae isolates were obtained from the colonized and infected patients in a public hospital in the city of Recife-PE, in northeastern Brazil, and were further analyzed. The detection of carbapenem resistance genes and the seven housekeeping genes [for multilocus sequence typing (MLST) detection] were done with PCR and sequencing. The blaKPC and blaNDM genes were detected concomitantly in all isolates, with variants being detected blaNDM-1, blaNDM-5, blaNDM-7, and blaKPC-2. The blaKPC-2 and blaNDM-1 combination being the most frequent. Molecular typing by MLST detected three types of high-risk ST clones, associated with the clonal complex 258, ST11/CC258 in eight isolates, and ST855/CC258 and ST340/CC258 in the other two isolates. CONCLUSIONS: These findings are worrying, as they have a negative impact on the scenario of antimicrobial resistance, and show the high genetic variability of K. pneumoniae and its ability to mutate resistance genes and risk of dissemination via different ST clones.

Mathematical Characterization of Private and Public Immune Receptor Sequences.

Diverse T and B cell repertoires play an important role in mounting effective immune responses against a wide range of pathogens and malignant cells. The number of unique T and B cell clones is characterized by T and B cell receptors (TCRs and BCRs), respectively. Although receptor sequences are generated probabilistically by recombination processes, clinical studies found a high degree of sharing of TCRs and BCRs among different individuals. In this work, we use a general probabilistic model for T/B cell receptor clone abundances to define "publicness" or "privateness" and information-theoretic measures for comparing the frequency of sampled sequences observed across different individuals. We derive mathematical formulae to quantify the mean and the variances of clone richness and overlap. Our results can be used to evaluate the effect of different sampling protocols on abundances of clones within an individual as well as the commonality of clones across individuals. Using synthetic and empirical TCR amino acid sequence data, we perform simulations to study expected clonal commonalities across multiple individuals. Based on our formulae, we compare these simulated results with the analytically predicted mean and variances of the repertoire overlap. Complementing the results on simulated repertoires, we derive explicit expressions for the richness and its uncertainty for specific, single-parameter truncated power-law probability distributions. Finally, the information loss associated with grouping together certain receptor sequences, as is done in spectratyping, is also evaluated. Our approach can be, in principle, applied under more general and mechanistically realistic clone generation models.