PCR-based Analysis Research Articles

Degraded RNA from Human Anterior Cruciate Ligaments Yields Valid Gene Expression Profiles.

Correlating gene expression patterns with biomechanical properties of connective tissues provides insights into the molecular processes underlying the tissue growth and repair. Cadaveric specimens such as human knees are widely considered suitable for biomechanical studies, but their usefulness for gene expression experiments is potentially limited by the unavoidable, nuclease-mediated degradation of RNA. Here, we tested whether valid gene expression profiles can be obtained using degraded RNA from human anterior cruciate ligaments (ACLs). Human ACL RNA (N = 6) degraded in vitro by limited ribonuclease digestion resemble highly degraded RNA isolated from cadaveric tissue. PCR threshold cycle (Ct) values for 90 transcripts (84 extracellular matrix, 6 housekeeping) in degraded RNAs variably ranged higher than values obtained from their corresponding non-degraded RNAs, reflecting both the expected loss of target templates in the degraded preparations as well as differences in the extent of degradation. Relative Ct values obtained for mRNAs in degraded preparations strongly correlated with the corresponding levels in non-degraded RNA, both for each ACL as well as for the pooled results from all six ACLs. Nuclease-mediated degradation produced similar, strongly correlated losses of housekeeping and non-housekeeping gene mRNAs. RNA degraded in situ yielded comparable results, confirming that in vitro digestion effectively modeled degradation by endogenous ribonucleases in frozen and thawed ACL. We conclude that, contrary to conventional wisdom, PCR-based expression analyses can yield valid mRNA profiles even from RNA preparations that are more than 90% degraded, such as those obtained from connective tissues subjected to biomechanical studies. Furthermore, legitimate quantitative comparisons between variably degraded tissues can be made by normalizing data to appropriate housekeeping transcripts.

Listeria monocytogenes from food and food industry environments with reduced susceptibility to benzalkonium chloride, sodium hypochlorite, and peracetic acid.

The growing number of Listeria monocytogenes strains displaying increased tolerance to sanitizers widely applied in the food industry is becoming a problem. The aims of this study were to evaluate the susceptibility of L. monocytogenes isolates from food and food industry environments to sanitizers (benzalkonium chloride, sodium hypochlorite, peracetic acid, and chlorhexidine) and heavy metals (cadmium chloride), as well as to investigate the presence of the main genes related to efflux pumps. All 82 isolates showed reduced susceptibility to benzalkonium chloride (MIC from 16 to 128 µg mL-1), sodium hypochlorite (MIC of ≥ 2048 µg mL-1), and peracetic acid (MIC from 512 to ≥ 2048 µg mL-1), while 22 isolates showed reduced susceptibility to cadmium chloride (MIC > 70 µg mL-1). Susceptibility to chlorhexidine was found (MIC from 2 to 16 µg mL-1). PCR-based analysis revealed that mdrl and lde genes were harbored by 14.6% (12/82) and 40.2% (33/82) of the isolates, respectively. This study demonstrates the presence of L. monocytogenes from food and food industry environments with reduced susceptibility to sanitizers commonly used in food processing environments, highlighting the importance of continuous monitoring of the tolerance profile of this microorganism to sanitizers, as well as the need for strict control of sanitation conditions in food industries.

Evaluation of Microsatellite Instability Molecular Analysis versus Immuno-Histochemical Interpretation in Malignant Neoplasms with Different Localizations.

MMR gene germline mutations are considered a major genetic disorder in patients with hereditary nonpolyposis colon cancer (HNPCC) or Lynch syndrome; A total of 15% of sporadic colon carcinomas are MSI-High. MSI has also been observed in other cancers, such as endometrial, gastric, and ovarian cancer. The aim of the current study was to correlate and outline the optimal method between the molecular testing of the instability of microsatellite DNA regions (MSI status) and the loss of protein expression by immunehistochemistry (MMR). A total of 242 paraffin-embedded tissues from gastrointestinal, gynecological, genitourinary, lung, breast, and unknown primary cancer patients were analyzed for the expression of MLH1/MSH2/MSH6/PMS2 by immunohistochemistry, as well as for the molecular analysis of MSI status using PCR-based molecular fragment analysis. A total of 29 MSI-High patients were detected molecularly, while 23 patients were detected by immunohistochemistry, with rates that are comparable according to the literature. Based on the agreement coefficient of the two methods, a substantial agreement emerged (Kappa = 0.675 with standard error = 0.081, p < 0.001). Despite the substantial agreement, both methods ought to be established to determine MSI-H/dMMR status in all cancer types as a first-line screening test.

512. The relationship of Clostridioides difficile in-vitro antibiotic resistance, prior exposure to antibiotics and ribotype: A single center study

Abstract Background Antibiotic exposure is the most important risk factor for Clostridioides difficile associated diarrhea (CDAD). Little information has been reported on the relationship between prior antibiotic exposure, antibiotic resistance of C. difficile, and isolate ribotype. Methods C. difficile isolates were collected from the stools of patients with CDAD. Isolates were ribotyped by PCR-based fragment analysis. Susceptibility testing was performed using Clinical & Laboratory Standards Institute (CLSI) recommended methods. MIC results were interpreted using European Committee on Antimicrobial Susceptibility Testing (EUCAST) epidemiologic cut off values or the CLSI breakpoints. Demographic data and antibiotic exposure in the 30 days period prior to C. difficile isolation were collected via retrospective chart review. Results Of a total of 490 C. difficile isolates, 335 (67.3%) isolates were resistant to one or more antibiotics (Table 1). Patients who had higher Charlson Comorbidity Score (CCI) and who had hospital associated CDAD were more likely to receive antibiotics (Table 2). Moxifloxacin resistance was significantly associated with prior fluoroquinolone exposure (OR 2.5, CI 95% 1.5-4.1). Fluoroquinolone use was associated with vancomycin resistance (OR 2.7, CI 95% 1.3-5.6) and rifampin resistance (OR 1.9, CI 95% 1.2-3). Infection with a clindamycin resistant isolate was more likely to be hospital acquired (p = 0.006). We identified 54 different ribotypes (Figure 1). Prior treatment with aminopenicillins increased the odds of acquiring ribotype 053-163 (OR 2.9, 95% CI 1.0-8.2) and ribotype 002 (OR 3.2; CI 95% 1.0-10) (Table 3). .Prior treatment with cephalosporins had an increased risk of 014-020 (OR 2.0, CI 95% 1.2-3.3); prior treatment with carbapenems was associated with ribotype 106 (OR 2.3, CI 95% 1.2-4.4). Isolation of ribotype 027 following prior treatment to fluroquinolones was marginally significant (p = 0.054). Conclusion Specific antibiotic exposure may select for infection by specific C. difficile ribotypes, which can often lead to infection with resistant strains. Prior fluoroquinolone treatment, in particular, was associated with an increased risk of isolating multi-drug resistant strains. Disclosures Cheleste M. Thorpe, MD, Actelion: Grant/Research Support|Deinove: Advisor/Consultant|General Mills: Grant/Research Support|Merck: Grant/Research Support|Pfizer: Grant/Research Support|Summit: Advisor/Consultant|Summit: Grant/Research Support David R. Snydman, MD, Merck: Advisor/Consultant|Merck and Company: Grant/Research Support|Prolacta: Advisor/Consultant|Prolacta: Grant/Research Support|Seres: Advisor/Consultant|Seres Health: Grant/Research Support|Summit Plc: Grant/Research Support|Takeda: Advisor/Consultant|Takeda: Grant/Research Support|Visterra: Advisor/Consultant.

Predicting potential transmission risk of Everglades virus in Florida using mosquito blood meal identifications.

The overlap between arbovirus host, arthropod vectors, and pathogen distributions in environmentally suitable habitats represents a nidus where risk for pathogen transmission may occur. Everglades virus (EVEV), subtype II Venezuelan equine encephalitis virus (VEEV), is endemic to southern Florida where it is transmitted by the endemic vector Culex cedecei between muroid rodent hosts. We developed an ecological niche model (ENM) to predict areas in Florida suitable for EVEV transmission based upon georeferenced vector-host interactions from PCR-based blood meal analysis from blood-engorged female Cx. cedecei females. Thirteen environmental variables were used for model calibration, including bioclimatic variables derived from Daymet 1 km daily temperature and precipitation values, and land use and land cover data representing percent land cover derived within a 2.5 km buffer from 2019 National Land Cover Database (NLCD) program. Maximum temperature of the warmest month, minimum temperature of the coldest month, and precipitation of the driest month contributed 31.6%, 28.5% and 19.9% to ENM performance. The land cover types contributing the greatest to the model performance were percent landcover of emergent herbaceous and woody wetlands which contributed 5.2% and 4.3% to model performance, respectively. Results of the model output showed high suitability for Cx. cedecei feeding on rodents throughout the southwestern portion of the state and pockets of high suitability along the northern east coast of Florida, while areas with low suitability included the Miami-Dade metropolitan area and most of northern Florida and the Panhandle. Comparing predicted distributions of Cx. cedecei feeding upon rodent hosts in the present study to historical human cases of EVEV disease, as well as antibodies in wildlife show substantial overlap with areas predicted moderate to highly suitable for these vector/host associations. As such, the findings of this study likely predict the most accurate distribution of the nidus of EVEV to date, indicating that this method allows for better inference of potential transmission areas than models which only consider the vector or vertebrate host species individually. A similar approach using host blood meals of other arboviruses can be used to predict potential areas of virus transmission for other vector-borne diseases.

A Rapid and Simple Method for DNA Preparation of Magnaporthe oryzae from Single Rice Blast Lesions for PCR-Based Molecular Analysis.

Rice blast is one of the most destructive diseases of rice worldwide, and the causative agent is the filamentous ascomycete Magnaporthe oryzae. With the successful cloning of more and more avirulence genes from M. oryzae, the direct extraction of M. oryzae genomic DNA from infected rice tissue would be useful alternative for rapid monitoring of changes of avirulence genes without isolation and cultivation of the pathogen. In this study, a fast, low-cost and reliable method for DNA preparation of M. oryzae from a small piece of infected single rice leaf or neck lesion was established. This single step method only required 10 min for DNA preparation and conventional chemical reagents commonly found in the laboratory. The AvrPik and AvrPi9 genes were successfully amplified with the prepared DNA. The expected DNA fragments from 570 bp to 1,139 bp could be amplified even three months after DNA preparation. This method was also suitable for DNA preparation from M. oryzae strains stored on the filter paper. All together these results indicate that the DNA preparation method established in this study is reliable, and could meet the basic needs for polymerase chain reaction-based analysis of M. oryzae.

Evaluation of ERIC-PCR and MALDI-TOF as typing tools for multidrug resistant Klebsiella pneumoniae clinical isolates from a tertiary care center in India.

Multidrug resistant Klebsiella pneumoniae is associated with nosocomial infections in both outbreak and non-outbreak situations. The study intends to evaluate the potential of enterobacterial repetitive intergenic consensus- polymerase chain reaction (ERIC-PCR), a genomic based typing and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) proteomic-based typing techniques for clonal relatedness among multidrug resistant Klebsiella pneumoniae isolates. Multidrug resistant clinical isolates of Klebsiella pneumoniae (n = 137) were collected from March 2019 to February 2020. Identification and protein-based phylogenetic analysis were performed by MALDI-TOF MS. Genomic typing was done by ERIC-PCR and analyzed by an online data analysis service (PyElph). Dice method with unweighted pair group method with arithmetic mean (UPGMA) program was used to compare the ERIC profiles. The samples were also evaluated by PCR for the presence of genes encoding carbapenemases, extended spectrum beta lactamases (ESBLs) and mobile colistin resistance-1 (mcr1). The study presents ERIC-PCR as more robust and better discriminatory typing tool in comparison to MALDI-TOF for clonal relatedness in multidrug resistant K. pneumoniae clinical isolates. Isolates were typed into 40 ERIC types, and six groups by MALDI-TOF-MS. PCR-based analysis revealed that all the strains harbored two or more ESBL and carbapenemase genes. None of the isolates revealed the presence of the plasmid mediated mcr-1 gene for colistin resistance.

Identification and Characterization of a QTL for Growth of Fusarium circinatum on Pine-Based Medium.

Fusarium circinatum is an economically important pathogen of pine and resides in the Fusarium fujikuroi species complex. Here we investigated the molecular processes underlying growth in F. circinatum by exploring the association between growth and the nutritional environment provided by the pine host. For this purpose, we subjected a mapping population consisting of F. circinatum X F. temperatum hybrid progeny to an analysis of growth rate on a pine-tissue derived medium. These data, together with the available genetic linkage map for F. circinatum, were then used to identify Quantitative Trait Loci (QTLs) associated with growth. The single significant QTL identified was then characterized using the available genome sequences for the hybrid progeny's parental isolates. This revealed that the QTL localized to two non-homologous regions in the F. circinatum and F. temperatum genomes. For one of these, the F. circinatum parent contained a two-gene deletion relative to the F. temperatum parent. For the other region, the two parental isolates encoded different protein products. Analysis of repeats, G+C content, and repeat-induced point (RIP) mutations further suggested a retrotransposon origin for the two-gene deletion in F. circinatum. Nevertheless, subsequent genome and PCR-based analyses showed that both regions were similarly polymorphic within a collection of diverse F. circinatum. However, we observed no clear correlation between the respective polymorphism patterns and growth rate in culture. These findings support the notion that growth is a complex multilocus trait and raise the possibility that the identified QTL contains multiple small-effect QTLs, of which some might be dependent on the genetic backgrounds. This study improved our current knowledge of the genetic determinants of vegetative growth in F. circinatum and provided an important foundation for determining the genes and processes underpinning its ability to colonize its host environment.

Exploration of the regulatory relationship between KRAB-Zfp clusters and their target transposable elements via a gene editing strategy at the cluster specific linker-associated sequences by CRISPR-Cas9

BackgroundKrüppel Associated Box-containing Zinc Finger Proteins (KRAB-ZFPs), representing the largest superfamily of transcription factors in mammals, are predicted to primarily target and repress transposable elements (TEs). It is challenging to dissect the distinct functions of these transcription regulators due to their sequence similarity and diversity, and also the complicated repetitiveness of their targeting TE sequences.ResultsMouse KRAB-Zfps are mainly organized into clusters genomewide. In this study, we revealed that the intra-cluster members had a close evolutionary relationship, and a similar preference for zinc finger (ZnF) usage. KRAB-Zfps were expressed in a cell type- or tissue type specific manner and they tended to be actively transcribed together with other cluster members. Further sequence analyses pointed out the linker sequences in between ZnFs were conserved, and meanwhile had distinct cluster specificity. Based on these unique characteristics of KRAB-Zfp clusters, sgRNAs were designed to edit cluster-specific linkers to abolish the functions of the targeted cluster(s). Using mouse embryonic stem cells (mESC) as a model, we screened and obtained a series of sgRNAs targeting various highly expressed KRAB-Zfp clusters. The effectiveness of sgRNAs were verified in a reporter assay exclusively developed for multi-target sgRNAs and further confirmed by PCR-based analyses. Using mESC cell lines inducibly expressing Cas9 and these sgRNAs, we found that editing different KRAB-Zfp clusters resulted in the transcriptional changes of distinct categories of TEs.ConclusionsCollectively, the intrinsic sequence correlations of intra-cluster KRAB-Zfp members discovered in this study suggest that the conserved cluster specific linkers played crucial roles in diversifying the tandem ZnF array and the related target specificity of KRAB-Zfps during clusters’ evolution. On this basis, an effective CRISPR-Cas9 based approach against the linker sequences is developed and verified for rapidly editing KRAB-Zfp clusters to identify the regulatory correlation between the cluster members and their potential TE targets.

Determination of critical concentration for drug susceptibility testing of Mycobacterium tuberculosis against para-aminosalicylic acid with clinical isolates with thyA, folC and dfrA mutations

Background & ObjectivesAccurate determination of antimicrobial resistance profiles is of great importance to formulate optimal regimens against multidrug-resistant tuberculosis (MDR-TB). Although para-aminosalicylic acid (PAS) has been widely used clinically, the reliable testing methods for PAS susceptibility were not established. Herein, we aimed to establish critical test concentration for PAS on the Mycobacterial Growth Indicator Tube (MGIT) 960 in our laboratory settings.MethodsA total of 102 clinical isolates were included in this study, including 82 wild-type and 20 resistotype isolates. Minimum inhibitory concentration (MIC) was determined by MGIT 960. Whole-genome sequencing was used to identify the mutation patterns potentially conferring PAS resistance. Sequence alignment and structure modelling were carried out to analyze potential drug-resistant mechanism of folC mutant.ResultsOverall, the Minimum inhibitory concentration (MIC) distribution demonstrated excellent separation between wild-type and resistotype isolates. The wild-type population were all at least 1 dilution below 4 μg/ml, and the resistotype population were no lower than 4 μg/ml, indicating that 4 μg/ml was appropriate critical concentration to separate these two populations. Of 20 mutant isolates, 12 (60.0%) harbored thyA mutations, 2 (10%) had a mutation on upstream of dfrA, and the remaining isolates had folC mutations. Overall, thyA and folC mutations were scattered throughout the whole gene without any one mutation predominating. All mutations within thyA resulted in high-level resistance to PAS (MIC > 32 μg/ml); whereas the MICs of isolates with folC mutations exhibited great diversity, ranged from 4 to > 32 μg/ml, and sequence and structure analysis partially provided the possible reasons for this diversity.ConclusionsWe propose 4 μg/ml as tentative critical concentration for MGIT 960. The major mechanism of PAS resistance is mutations within thyA and folC in MTB isolations. The whole-gene deletion of thyA locus confers high-level resistance to PAS. The diversity of many distinct mutations scattered throughout the full-length folC gene challenges the PCR-based mutation analysis for PAS susceptibility.

Seasonal distribution of Cryptosporidium spp., Giardia duodenalis and Enterocytozoon bieneusi in Tibetan sheep in Qinghai, China

BackgroundCryptosporidium spp., Giardia duodenalis and Enterocytozoon bieneusi can cause important intestinal diseases in ruminants. However, data on the distribution of these three protozoan pathogens in Tibetan sheep are limited.MethodsWe collected 761 fecal samples from Tibetan sheep across four seasons in Qinghai Province, China, and screened the samples for Cryptosporidium spp., G. duodenalis and E. bieneusi using PCR-based sequence analysis of the genes encoding 18S ribosomal RNA, triosephosphate isomerase and the internal transcribed spacer, respectively.ResultsThe positivity rates of Cryptosporidium spp., G. duodenalis and E. bieneusi in Tibetan sheep were 3.68% (28/761 samples), 1.58% (12/761) and 6.44% (49/761), respectively. Four species of Cryptosporidium were identified: C. xiaoi (n = 13 samples), C. ubiquitum (n = 8), C. bovis (n = 6) and C. ryanae (n = 1). Two G. duodenalis assemblages, namely the A (n = 2 samples) and E (n = 10) assemblages, were detected. Five zoonotic E. bieneusi genotypes were found: BEB6 (n = 21 samples), COS-I (n = 14), CHS3 (n = 11) and CGS1 (n = 2) from group 2, and PIGEBITS5 (n = 1) from group 1. Geographic differences in the distribution of E. bieneusi, and seasonal differences for all the three protozoan pathogens were noted.ConclusionsOur results elucidate the prevalence and genetic diversity of these three pathogens in Tibetan sheep across different regions and seasons, including zoonotic pathogens such as C. ubiquitum, C. ryanae, G. duodenalis assemblage A and five genotypes of E. bieneusi.Graphical

Coronary artery disease ameliorates extracellular vesicle lncRNA PUNISHER regulates angiogenic response and endothelial cells function via NFkB-dependent mechanism

Abstract Augmenting evidence indicates that long noncoding RNAs are playing a crucial role in diverse cellular/pathological processes. Intercellular transfer of extracellular vesicles transmitted lncRNA regulates vascular health and diseases. However, whether lncRNA expression in EVs is regulated in patients with coronary artery disease, is unknown. A PCR-based lncRNA array analysis revealed that EV-PUNISHER was significantly upregulated in patients with CAD (n=221) compared to healthy subjects. Our data showed that PUNISHER is the most upregulated lncRNA in CAD in accordance with other highly expressed lncRNAs. To examine the specific role of PUNISHER in EC phenotypic regulation, siRNA-mediated silencing in ECs revealed that depletion of PUNISHER suppresses the migration and proliferation of ECs. Moreover, depletion of PUNISHER decreases cell survival by reducing cell viability and proliferation through increased apoptosis and cytotoxicity. In order to investigate EC function in PUNISHER depleted cells, in vitro sprouting and tube formation assay revealed that PUNISHER is an important mediator EC functions, which acts as an anti-angiogenic factor. In vitro atherosclerotic stimuli increased PUNISHER expression in EV/EC in a dose-dependent way. Microarray analysis identified a series of pro- and anti-angiogenic genes as well genes directly involved in cell viability that are differentially regulated. We confirmed that PUNISHER is incorporated into endothelial microvesicles (EMVs) and transferred to recipient cells by using different experiments. By using lncRNA-FISH and vesicle degradation assays, we showed that PUNISHER is incorporated into EMVs, augmented recipient EC function in vitro upon transfer via EMVs. To examine whether EMV-PUNISHER promotes the EC function, different in vitro functional experiments with ECs confirmed that PUNISHER is an important regulator. Mass spectrometry analysis has revealed EMV contains numerous proteins, including RNA binding proteins such as hnRNPU, hnRNPK, hnRNPA2/B1, etc. By using RNA-pulldown and RNA immunoprecipitation (RIP), we confirmed that PUNISHER interacts with hnRNPU, which facilitates packaging into EMV prior to transfer to recipient EC. The interaction acts as an important positive regulator of cell viability and survival of recipient cells, identified using functional assay (migration, viability, proliferation, and angiogenesis). Transcription factor array has shown that PUNISHER regulates NFkB to control cellular viability and apoptosis. A murine re-endothelialization model revealed that EV-PUNISHER promotes reendothelialization. Ex vivo internalization/uptake of PKH-67-labeled EVs revealed that EVs are detectable in the cross-section of EV-perfused carotid artery. Our study unveiled for the first time that EV-incorporated PUNISHER exerts its function in endothelial cells which might be beneficial in cardiovascular pathologies where the endothelial function is dysregulated. Funding Acknowledgement Type of funding sources: Foundation. Main funding source(s): German Research Foundation (DFG)German Society of Cardiology (DGK)

Lynch/Turcot Syndrome and Sporadic Mismatch Repair Deficient High-Grade Gliomas – A Case Series

Abstract AIMS The DNA mismatch repair (MMR) system is critical for genomic stability correcting errors generated during DNA replication with germline pathogenic variants (PVs) causing Lynch syndrome. Although acquired MMR deficiency is associated with temozolomide resistance, the incidence in treatment naïve tumours is unknown and not routinely screened for. Here we present three MMR deficient gliomas and describe their comprehensive molecular profile. METHOD Thirteen gliomas; 9/34 that had undergone sequencing of 50 cancer-associated genes and found to have frameshift/multiple point PVs, indicative of defective MMR, and four MSI-high tumours, identified following microsatellite analysis for 1p/19q co-deletion as part of routine care, were selected for analysis. MMR expression was then assessed by immunohistochemistry. Seven tumours showed loss of one or more MMR proteins and were molecularly characterised using PCR-based Promega MSI analysis and NGS-based Illumina TruSight500 panel. RESULTS Pathogenic variants in MMR genes and hypermutated phenotypes (TMB > 10mutations/Mb) were identified in three cases. One individual harboured an additional POLE PV resulting in an ultra-hypermutated phenotype (>100mutations/Mb). Two MMR PVs were germline, leading to a new diagnosis of Lynch syndrome. Only one of three tumours was found to be MSI-high using the Promega MSI analysis whilst all three were identified as MSI-high using TruSight500 MSI calling algorithms. CONCLUSION We describe the comprehensive molecular profile of seven treatment naïve gliomas, three being MMR deficient. We show TruSight500 to be more sensitive and reliable for detecting MSI compared to Promega. Given the implications, this additional analysis should be considered as part of the molecular report on all gliomas.

Molecular identification of nontuberculous mycobacteria using the rpoB, argH and cya genes analysis

Nontuberculous mycobacterial (NTM) infections are growing worldwide especially in immunocompromised individuals. Since treatment of NTM infections is species-specific, the precise identification of NTM to species level is critical for an optimal treatment. This study was aimed to identify different NTM species by sequencing the rpoB gene and evaluating the effectiveness of argH and cya gene markers. In total 64 clinical isolates suspected to NTM were collected. The identification of the isolates was done by standard conventional methods and PCR-based rpoB gene and sequence analysis. PCR sequencing of argH and cya genes was performed to evaluate the efficacy of these genes in identifying and differentiating different species and subspecies of NTM. Among 64 isolates tested, 51 (79.68%) were detected by conventional tests as NTM. The results of rpoB sequence analysis revealed that the 56 clinical isolates were identified in 10 species of NTM and 8 remaining isolates which showed ambiguous results by rpoB sequencing, application of argH and cya sequencing could detect these isolates. Furthermore, by using cya gene sequencing, M. abscessus subspecies were properly differentiated. Although the rpoB sequencing as a standard method, is beneficial for detecting various species of NTM, however, based on our findings, argH and cya gene markers have a superb ability to discriminate closely related species. Further investigations are required to verify our outcomes.

Multiomics reveals an essential role of long-distance translocation in regulating plant cadmium resistance and grain accumulation in allohexaploid wheat (Triticum aestivum).

Cadmium (Cd) is a highly toxic heavy metal that readily enters cereals, such as wheat, via the roots and is translocated to the shoots and grains, thereby posing high risks to human health. However, the vast and complex genome of allohexaploid wheat makes it challenging to understand Cd resistance and accumulation. In this study, a Cd-resistant cultivar of wheat, 'ZM1860', and a Cd-sensitive cultivar, 'ZM32', selected from a panel of 442 accessions, exhibited significantly different plant resistance and grain accumulation. We performed an integrated comparative analysis of the morpho-physiological traits, ionomic and phytohormone profiles, genomic variations, transcriptomic landscapes, and gene functionality in order to identify the mechanisms underlying these differences. Under Cd toxicity, 'ZM1860' outperformed 'ZM32', which showed more severe leaf chlorosis, poorer root architecture, higher accumulation of reactive oxygen species, and disordered phytohormone homeostasis. Ionomics showed that 'ZM32' had a higher root-to-shoot translocation coefficient of Cd and accumulated more Cd in the grains than 'ZM1860'. Whole-genome re-sequencing (WGS) and transcriptome sequencing identified numerous DNA variants and differentially expressed genes involved in abiotic stress responses and ion transport between the two genotypes. Combined ionomics, transcriptomics, and functional gene analysis identified the plasma membrane-localized heavy metal ATPase TaHMA2b-7A as a crucial Cd exporter regulating long-distance Cd translocation in wheat. WGS- and PCR-based analysis of sequence polymorphisms revealed a 25-bp InDel site in the promoter region of TaHMA2b-7A, and this was probably responsible for the differential expression. Our multiomics approach thus enabled the identification of a core transporter involved in long-distance Cd translocation in wheat, and it may provide an elite genetic resource for improving plant Cd resistance and reducing grain Cd accumulation in wheat and other cereal crops.

Rapid detection of kdr mutation F1534C in Aedes aegypti using recombinase polymerase amplification and lateral flow dipsticks

Insecticide resistance monitoring is essential in assessing the efficacy of vector control measures. However, gold standard PCR-based molecular analyses for insecticide resistance detection are often hindered by time-consuming sample processing, as well as considerable infrastructure and resourcing requirements. In this study, we combined a novel one-step sample preparation reagent with a rapid isothermal molecular test that detects a knock down resistance (kdr) mutation (F1534C) that enables pyrethroid resistance in Aedes aegypti mosquitoes. We trialled the rapid F1534C pyrethroid resistance test using insecticide resistant Ae. aegypti mosquito bodies and compared results to a conventional, allele-specific quantitative PCR (AS-qPCR) coupled with melt curve genotyping in corresponding mosquito heads. From a strain of Ae. aegypti established from an insecticide resistant population in Merida, Mexico (n = 27), all the mosquito bodies (n = 27) tested positive with the rapid F1534C test regardless of whether they were homozygous or heterozygous. To assess diagnostic test specificity, we confirmed that F1534 was not detected in laboratory-reared, fully susceptible Ae. aegypti mosquito bodies (n = 28) using the rapid F1534C test or the conventional AS-qPCR melt curve analysis. All corresponding mosquito heads (n = 28) were homozygous wild-type FF1534. The rapid F1534C test thus demonstrated 100% diagnostic sensitivity (95% CI: 87.23% to 100%) and 100% diagnostic specificity (95% CI: 87.66% to 100.00%) for detection of the F1534C pyrethroid resistant single nucleotide polymorphism (SNP) in both heterozygous and homozygous Ae. aegypti. In the collection of mutant mosquitoes from Mexico, CC1534 homozygous mutants occurred at a frequency of 74.1% (n = 20) and FC heterozygous mutants at a frequency of 25.9% (n = 7). The rapid F1534C test significantly reduced the sample processing and testing time from approximately 6 h for the AS-qPCR melt curve analysis to only 25 min. These results demonstrate significant potential for our approach to resistance testing as a field-based, low-resource, rapid alternative to time-consuming and expensive laboratory-based detection.

Identification of Novel Broad-Spectrum Leaf Rust Resistance Sources from Khapli Wheat Landraces.

Wheat leaf rust caused by Puccinia triticina Eriks is an important disease that causes yield losses of up to 40% in susceptible varieties. Tetraploid emmer wheat (T. turgidum ssp. Dicoccum), commonly called Khapli wheat in India, is known to have evolved from wild emmer (Triticum turgidum var. dicoccoides), and harbors a good number of leaf rust resistance genes. In the present study, we are reporting on the screening of one hundred and twenty-three dicoccum wheat germplasm accessions against the leaf rust pathotype 77-5. Among these, an average of 45.50% of the germplasms were resistant, 46.74% were susceptible, and 8.53% had mesothetic reactions. Further, selected germplasm lines with accession numbers IC138898, IC47022, IC535116, IC535133, IC535139, IC551396, and IC534144 showed high level of resistance against the eighteen prevalent pathotypes. The infection type varied from “;”, “;N”, “;N1” to “;NC”. PCR-based analysis of the resistant dicoccum lines with SSR marker gwm508 linked to the Lr53 gene, a leaf rust resistance gene effective against all the prevalent pathotypes of leaf rust in India and identified from a T. turgidum var. dicoccoides germplasm, indicated that Lr53 is not present in the selected accessions. Moreover, we have also generated 35K SNP genotyping data of seven lines and the susceptible control, Mandsaur Local, to study their relationships. The GDIRT tool based on homozygous genotypic differences revealed that the seven genotypes are unique to each other and may carry different resistance genes for leaf rust.

Chlamydia trachomatis L2/434/Bu Favors Hypoxia for its Growth in Human Lymphoid Jurkat Cells While Maintaining Production of Proinflammatory Cytokines.

The role of lymphocytes as a cornerstone of the inflammatory response in the invasive pathogenesis of Chlamydia trachomatis (Ct) LGV (L1-3) infection is unclear. Therefore, we assessed whether the adaptation of CtL2 to immortal lymphoid Jurkat cells under hypoxic conditions occurred through proinflammatory cytokine profile modification. The quantities of inclusion-forming units with chlamydial 16S rDNA confirmed that CtL2 grew well under hypoxic rather than normoxic conditions in the cells. Confocal microscopic imaging and transmission electron microscopy revealed the presence of bacterial progeny in the inclusions and showed that the inclusions were larger under hypoxic rather than normoxic conditions; this was supported by the results of 3D image construction. Furthermore, PCR-based analysis of proinflammatory cytokines revealed that the gene expression levels under hypoxic conditions were significantly higher than those under normoxic conditions. In particular, the expression of two genes (CXCL8 and CXCR3) was significantly diminished under normoxic conditions. Taken together, the results indicated that hypoxia promoted CtL2 growth in Jurkat cells while maintaining the levels of proinflammatory cytokines. Thus, Ct LGV infection in lymphocytes under hypoxic conditions might be crucial to a complete understanding of the invasive pathogenesis.

Viral load of SARS-CoV-2 Omicron is not high despite its high infectivity.

Patients infected with the Omicron variant of severe acute respiratory syndrome coronavirus 2 has increased worldwide since the beginning of 2022 and the variant has spread more rapidly than the Delta variant, which spread in the summer of 2021. It is important to clarify the cause of the strong transmissibility of the Omicron variant to control its spread. In 694 patients with coronavirus disease 2019, the copy numbers of virus in nasopharyngeal swab‐soaked samples and the viral genotypes were examined using quantitative polymerase chain reaction (PCR) and PCR‐based melting curve analysis, respectively. Whole‐genome sequencing was also performed to verify the viral genotyping data. There was no significant difference (p = 0.052) in the copy numbers between the Delta variant cases (median 1.5 × 105 copies/μl, n = 174) and Omicron variant cases (median 1.2 × 105 copies/μl, n = 328). During this study, Omicron BA.1 cases (median 1.1 ×105 copies/μl, n = 275) began to be replaced by BA.2 cases (median 2.3 × 105 copies/μl, n = 53), and there was no significant difference between the two groups (p = 0.33). Our results suggest that increased infectivity of the Omicron variant and its derivative BA.2 is not caused by higher viral loads but by other factors, such as increased affinity to cell receptors or immune escape.

The extent of the abundance of exosomal and non‐exosomal extracellular miRNAs in the bovine follicular fluid

Follicular fluid (FF) plays an important role during follicular development and it contains several bioactive molecules including extracellular microRNAs (ECmiRNAs) that may mediate cell-cell communication during follicular development. Yet, the distribution patterns of ECmiRNAs in FF is not well characterized. This study aims to investigate the distribution of ECmiRNAs in two major fractions, namely exosomal and non-exosomal, of bovine follicular fluid (bFF). Exosomal and non-exosomal fractions from bFF were separated using Exoquick™ exosomes precipitation kit. miRNA expression was evaluated using the human miRCURY LNA™ Universal RT miRNA PCR array system. Transmission electron microscopy and immunoblotting revealed that the isolated vesicles were exosomes. The real-time PCR-based expression analysis revealed that 516 miRNAs were detected in the exosomal fraction of bFF, while 393 miRNAs were detected in the non-exosomal fraction. Among the detected miRNAs, a total of 370 miRNAs were detected in both fractions, while 145 miRNAs and 23 miRNAs were solely detected in exosomal and non-exosomal fractions, respectively. Exploratory pathway analysis showed that the genes targeted by exosomal and non-exosomal miRNAs to be involved in MAPK, Wnt, FoxO, TGF-beta, Oxytocin, ErbB, PI3K-Akt, Neurotrophin signalling pathways which are believed to be involved in follicular development, cell proliferation, and meiotic resumption. The results of our study demonstrated that besides the exosomal fraction, non-exosomal fractions can carry a significant amount of miRNAs in bFF where the exosomal fraction carries a significantly higher number of detectable miRNAs.