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In the Baltic Sea, the dinoflagellates Apocalathium malmogiense, Biecheleria baltica, and Gymnodinium corollarium are important contributors to the spring bloom. However, their relative contribution to the bloom community cannot be unambiguously determined by conventional light microscopy due to a lack of resolution of distinctive morphological features of the three species. Here, we describe a molecular approach based on a quantitative real-time polymerase chain reaction (qPCR) primer and probe system, targeting the ITS1 and ITS2 regions of the rRNA gene for all three species and enabling their quantification. The specificity of the method was demonstrated using monocultures of A. malmogiense, B. baltica, G. corollarium as well as three other dinoflagellate species co-occurring in the Baltic Sea during spring and validated using field-collected phytoplankton samples. [ABSTRACT FROM AUTHOR]

Potato (Solanum tuberosum) is one of the most important crops facing threats from different diseases. Rapid and accurate diagnosis is essential to control disease development and spread. Quantitative real-time PCR (qRT-PCR) has been widely used in potato disease diagnosis. In this study, we evaluated the specificity of 19 probe-based and four SYBR Green-based qPCR protocols for 17 potato diseases using in silico analysis. Primers and probes of those protocols were subjected to BLASTn analysis against the nucleotide collection (nr/nt) database and the whole-genome shotgun contigs (wgs) database of NCBI for the presence of primer/probe sequences in non-target species. Results showed that 12 of 23 qPCR protocols were not specific to the target pathogens. A qPCR experiment indicated that even nine single nucleotide polymorphisms (SNPs) are present on the sequences of the primer/probe binding sites between the potato silver scurf pathogen Helminthosporium solani and its close-related species H. velutinum, the primers/probe specific to the former could amplify signals from the latter. These findings highlight the need for additional methods to enhance the diagnostic accuracy and new sequencing technologies such as next generation sequencing could provide useful information to develop specific diagnostic protocols for these pathogens. [ABSTRACT FROM AUTHOR]

This study presents a large-scale interlaboratory comparison (ILC) aimed at detecting and quantifying DNA from two European anglerfish (Lophius budegassa, Lophius piscatorius), pike (Esox lucius) and sea bream (Spondyliosoma cantharus) using real-time qPCR. To detect amplification of the parvalbumin genetic marker, single and multiplex qPCR assays using EvaGreen® dye or TaqMan™ probes were used. Genomic DNA isolated from target fish species and an advanced DNA calibrator, gBlocks® gene fragments, were used as standards. The DNA of anglerfish, pike and sea bream as well as their mixtures were analysed together with 14 other non-target fish species. All target fish samples were correctly identified by the participating laboratories. Qualitative assessment of anglerfish and seabream DNA showed an accuracy rate of 100%, while pike DNA achieved a match rate of 99%. Validation of quantitative protocols in four different laboratories consistently achieved z-scores below 2, indicating satisfactory performance and confirming the high degree of similarity of laboratory results. Furthermore, high accuracy and efficiency were demonstrated for the quantification of anglerfish and seabream DNA by triplex qPCR using TaqMan™ probes. Regarding the selected gene marker, the major fish allergenic protein parvalbumin enables indirect detection and quantification of the allergen in the sample. Therefore, the use of proposed protocols can significantly contribute to protecting the health of consumers and to controlling the food market. [ABSTRACT FROM AUTHOR]

Background: Accurate differentiation between lung adenocarcinoma (AC) and lung squamous cell carcinoma (SCC) is crucial owing to their distinct therapeutic approaches. MicroRNAs (miRNAs) exhibit variable expression across subtypes, making them promising biomarkers for discrimination. This study aimed to identify miRNAs with robust discriminatory potential between AC and SCC and elucidate their clinical significance. Methods: MiRNA expression profiles for AC and SCC patients were obtained from The Cancer Genome Atlas (TCGA) database. Differential expression analysis and supervised machine learning methods (Support Vector Machine, Decision trees and Naïve Bayes) were employed. Clinical significance was assessed through receiver operating characteristic (ROC) curve analysis, survival analysis, and correlation with clinicopathological features. Validation was conducted using reverse transcription quantitative polymerase chain reaction (RT-qPCR). Furthermore, signaling pathway and gene ontology enrichment analyses were conducted to unveil biological functions. Results: Five miRNAs (miR-205-3p, miR-205-5p, miR-944, miR-375 and miR326) emerged as potential discriminative markers. The combination of miR-944 and miR-326 yielded an impressive area under the curve of 0.985. RT-qPCR validation confirmed their biomarker potential. miR-326 and miR-375 were identified as prognostic factors in AC, while miR-326 and miR-944 correlated significantly with survival outcomes in SCC. Additionally, exploration of signaling pathways implicated their involvement in key pathways including PI3K-Akt, MAPK, FoxO, and Ras. Conclusion: This study enhances our understanding of miRNAs as discriminative markers between AC and SCC, shedding light on their role as prognostic indicators and their association with clinicopathological characteristics. Moreover, it highlights their potential involvement in signaling pathways crucial in non-small cell lung cancer pathogenesis. [ABSTRACT FROM AUTHOR]

Introduction: In clinical diagnosis of porcine diseases, co-infection with multiple viruses often leads to similar clinical symptoms. Postweaning multisystemic wasting syndrome (PMWS) can be caused by infections with TTSuV or PCV2, while PCV2, PRV, and PPV can cause respiratory and reproductive disorders in pigs. The overlapping clinical and pathological features of these infections necessitate the development of a rapid and specific method for differentiating and detecting these four DNA viruses. Methods: In this study, four pairs of primers and TaqMan probes were designed targeting the conserved sequence of TTSuV, the Rep gene of PCV2, the gE gene of PRV, and the VP2 gene of PPV. After optimizing reaction conditions, including annealing temperature, primer concentration, and probe concentration, a quadruplex real-time PCR method was developed. Results: This method can specifically detect TTSuV1, PCV2, PRV, and PPV simultaneously, with no cross-reactivity with ASFV, CSFV, PRRSV, PEDV, PSV, and TGEV. The minimum detection limit for each virus was 10 copies/ml, and the inter-assay and intra-assay coefficients of variation ranged from 0.33% to 1.43%. Subsequently, 150 clinical samples were tested to evaluate the practical applicability of this method. The positive rates for TTSuV1, PCV2, PRV, and PPV were 8.6% (13/150), 10.67% (16/150), 14% (21/150), and 11.33% (17/150), respectively. Discussion: The results indicate that the established quadruplex real-time PCR method can assist in the accurate and rapid diagnosis of TTSuV1, PCV2, PRV, and PPV in clinical settings, providing robust support for the prevention and control of these infections. [ABSTRACT FROM AUTHOR]

Background: Toxocara canis and Toxocara cati are parasitic nematodes that occur worldwide. As embryonated Toxocara spp. eggs in the environment pose a zoonotic risk, especially for children, optimal diagnostic approaches are necessary for effective disease response and management, including surveillance. However, little is known about the performance of different diagnostic protocols for detecting Toxocara spp. in the faeces of cats and dogs, hampering movement towards an optimal diagnostic process. This study aimed to compare detection methods, including a newly developed sequential sieving protocol (SF-SSV) and a high-throughput multiplex qPCR-based method to facilitate epidemiological studies. Methods: Species-specific Toxocara spp. egg suspensions and canine and feline faecal samples from the field were used to estimate analytical and diagnostic sensitivity of the protocols. The performance of two automated DNA extraction protocols using enzymatic and mechanical lysis were compared by multiplex qPCR, targeting both T. canis and T. cati-specific genomic sequences. All samples were examined by microscopy-based techniques, the sedimentation flotation technique (SF) and a newly developed SF-SSV for the detection, enrichment and purification of parasite eggs. The costs and processing times necessary for all protocols were estimated and compared for both single samples and sets of 100 samples. Results: To detect Toxocara spp. eggs, SF-SSV showed the highest analytical sensitivity and a significantly higher diagnostic sensitivity than the DNA detection methods. Mechanical lysis performed better than enzymatic lysis for automated DNA extraction. In automated DNA extraction, 96-well plates performed better than 24-well plates. DNA detection and microscopy-based parasitological methods showed substantial agreement between the results generated by each method. Microscopy-based techniques required the lowest costs and least hands-on time for a single sample. However, when costs and labour were estimated for a set of 100 samples, the DNA detection protocol using 96-well plates for extraction revealed costs similar to SF-SSV and the fastest processing times. Conclusions: SF-SSV was superior in terms of analytical and diagnostic sensitivity for the detection of Toxocara spp. eggs. For larger sets of samples, multiplex qPCR-based DNA detection represents an alternative to microscopy-based methods, based on the possibility of faster sample processing at similar costs to SF-SSV, and the ability to provide species-specific diagnoses. [ABSTRACT FROM AUTHOR]

Background: Biofilm formation is an essential virulence factor that creates a highly protected growth mode for Staphylococcus aureus (S. aureus) to survive in any hostile environment. Antibiotic sub-minimal inhibitory concentration (sub-MIC) may modulate the biofilm formation ability of bacterial pathogens, thereby affecting bacterial pathogenesis and infection outcomes. Intense antimicrobial therapy to treat biofilm-associated infections can control the pathogenic infection aggravation but cannot guarantee its complete eradication. Objective: This study aimed to assess the sub-MICs effect of 5 different antimicrobial classes on biofilm-forming capacity among Staphylococcus aureus clinical isolates using three different biofilm quantitation techniques. Methods: In this study, the effects of 5 different antimicrobial agents, namely, azithromycin, gentamicin, ciprofloxacin, doxycycline, and imipenem, at sub-MICs of 12.5%, 25%, and 50% were tested on 5 different clinical isolates of S. aureus. The biofilms formed in the absence and presence of different antimicrobial sub-MICs were then assessed using the following three different techniques: the crystal violet (CV) staining method, the quantitative PCR (qPCR) method, and the spread plate method (SPM). Results: Biofilm formation was significantly induced in 64% of the tested conditions using the CV technique. On the other hand, the qPCR quantifying the total bacterial count and the SPM quantifying the viable bacterial count showed significant induction only in 24% and 17.3%, respectively (Fig. 1). The difference between CV and the other techniques indicates an increase in biofilm biomass without an increase in bacterial growth. As expected, sub-MICs did not reduce the viable cell count, as shown by the SPM. The CV staining method revealed that sub-MICs of imipenem and ciprofloxacin had the highest significance rate (80%) showing an inductive effect on the biofilm development. On the other hand, doxycycline, azithromycin, and gentamicin displayed lower significance rates of 73%, 53%, and 47%, respectively. Conclusion: Exposure to sub-MIC doses of antimicrobial agents induces the biofilm-forming capacity of S. aureus via increasing the total biomass without significantly affecting the bacterial growth of viable count. [ABSTRACT FROM AUTHOR]

Background: Studying the molecular mechanisms of lipodystrophy can provide valuable insights into the pathophysiology of insulin resistance (IR), type 2 diabetes (T2D), and other clustering diseases [metabolic syndrome (MetS)] and its underlying adipocentric disease (MetS disease). Methods: A high-confidence lipodystrophy gene panel comprising 50 genes was created, and their expressions were measured in the visceral and subcutaneous (both peripheral and abdominal) adipose depots of MetS and non-MetS individuals at a tertiary care medical facility. Results: Most lipodystrophy genes showed significant downregulation in MetS individuals compared to non-MetS individuals in both subcutaneous and visceral depots. In the abdominal compartment, all the genes showed relatively higher expression in visceral depot as compared to their subcutaneous counterpart, and this difference narrowed with increasing severity of MetS. Their expression level shows an inverse correlation with T2D, MetS, and HOMA-IR and with other T2Drelated intermediate traits. Results also demonstrated that individualization of MetS patients could be done based on adipose tissue expression of just 12 genes. Conclusion: Adipose tissue expression of lipodystrophy genes shows an association with MetS and its intermediate phenotypic traits. Mutations of these genes are known to cause congenital lipodystrophy syndromes, whereas their altered expression in adipose tissue contributes to the pathogenesis of IR, T2D, and MetS. [ABSTRACT FROM AUTHOR]

Background: Biofilm formation is an essential virulence factor that creates a highly protected growth mode for Staphylococcus aureus (S. aureus) to survive in any hostile environment. Antibiotic sub-minimal inhibitory concentration (sub-MIC) may modulate the biofilm formation ability of bacterial pathogens, thereby affecting bacterial pathogenesis and infection outcomes. Intense antimicrobial therapy to treat biofilm-associated infections can control the pathogenic infection aggravation but cannot guarantee its complete eradication. Objective: This study aimed to assess the sub-MICs effect of 5 different antimicrobial classes on biofilm-forming capacity among Staphylococcus aureus clinical isolates using three different biofilm quantitation techniques. Methods: In this study, the effects of 5 different antimicrobial agents, namely, azithromycin, gentamicin, ciprofloxacin, doxycycline, and imipenem, at sub-MICs of 12.5%, 25%, and 50% were tested on 5 different clinical isolates of S. aureus. The biofilms formed in the absence and presence of different antimicrobial sub-MICs were then assessed using the following three different techniques: the crystal violet (CV) staining method, the quantitative PCR (qPCR) method, and the spread plate method (SPM). Results: Biofilm formation was significantly induced in 64% of the tested conditions using the CV technique. On the other hand, the qPCR quantifying the total bacterial count and the SPM quantifying the viable bacterial count showed significant induction only in 24% and 17.3%, respectively (Fig. 1). The difference between CV and the other techniques indicates an increase in biofilm biomass without an increase in bacterial growth. As expected, sub-MICs did not reduce the viable cell count, as shown by the SPM. The CV staining method revealed that sub-MICs of imipenem and ciprofloxacin had the highest significance rate (80%) showing an inductive effect on the biofilm development. On the other hand, doxycycline, azithromycin, and gentamicin displayed lower significance rates of 73%, 53%, and 47%, respectively. Conclusion: Exposure to sub-MIC doses of antimicrobial agents induces the biofilm-forming capacity of S. aureus via increasing the total biomass without significantly affecting the bacterial growth of viable count. [ABSTRACT FROM AUTHOR]

Telomeres are protective structures at the end of eukaryotic chromosomes that are strongly implicated in ageing and ill health. They attrition upon every cellular reproductive cycle. Evidence suggests that short telomeres trigger DNA damage responses that lead to cellular senescence. Accurate methods for measuring telomeres are required to fully investigate the roles that shortening telomeres play in the biology of disease and human ageing. The last two decades have brought forth several techniques that are used for measuring telomeres. This editorial highlights strengths and limitations of traditional and emerging techniques, guiding researchers to choose the most appropriate methodology for their research needs. These methods include Quantitative Polymerase Chain Reaction (qPCR), Omega qPCR (Ω-qPCR), Terminal Restriction Fragment analysis (TRF), Single Telomere Absolute-length Rapid (STAR) assays, Single TElomere Length Analysis (STELA), TElomere Shortest Length Assays (TESLA), Telomere Combing Assays (TCA), and Long-Read Telomere Sequencing. Challenges include replicating telomere measurement within and across cohorts, measuring the length of telomeres on individual chromosomes, and standardised reporting for publications. Areas of current and future focus have been highlighted, with recent methodical advancements, such as long-read sequencing, providing significant scope to study telomeres at an individual chromosome level. [ABSTRACT FROM AUTHOR]

This study investigates the potential of ellagic acid (EA) to mitigate the effects of drought and aluminum (Al3+) stresses in maize by examining various morpho-physiochemical parameters and gene expressions. Maize (Zea mays L.) serves as a crucial global food source, but its growth and productivity are significantly hindered by drought and aluminum (Al3+) stresses, which lead to impaired root development, elevated levels of reactive oxygen species (ROS), diminished photosynthetic efficiency, and reduced water and mineral absorption. Recently, ellagic acid (EA), a polyphenolic compound with potent antioxidant properties, has been identified for its role in regulating plant growth and enhancing stress tolerance mechanisms. However, the specific mechanisms through which EA contributes to Al3+ and/or drought tolerance in plants remain largely unknown. The present study was conducted to examine the defensive role of EA (100 μg/mL) in some morpho-physiochemical parameters and the expression profiles of some stress-related genes (ZmCPK22, ZmXTH1, ZmHIPP4, ZmSGR, ZmpsbA, ZmAPX1, and ZmGST1) in drought (polyethylene glycol-6000 (PEG-6000), − 0.6 MPa) and aluminum chloride (AlCl3, 60 μM) stressed Zea mays Ada 523 grown in nutrient solution. Our results indicated that drought and aluminum chloride stresses affected root length, shoot height, H2O2 content, chlorophyll content (SPAD), electrolyte leakage (EL), and relative water content (RWC) of maize with several significant (P < 0.05) shifts up and down. Conversely, EA (100 μg/mL) treatment had a mitigating effect on these parameters. Moreover, EA also mitigated the antioxidant enzyme activities (superoxide dismutase (SOD), peroxidase (POD), and ascorbate peroxidase (APX)), and regulated the expressions of aforementioned genes. These findings determined that EA treatment could efficiently improve the gene expressions and morpho-physiochemical parameters under drought and/or Al3+ stresses, thereby increasing the seedlings' adaptability to these stresses. [ABSTRACT FROM AUTHOR]

Aquatic systems have traditionally played a key role in the development of human life, providing multiple ecosystem services to society and being a reservoir for a wide biodiversity of organisms. Among them, bacteria belonging to Legionella stand out, mainly because they are of great interest both in the field of microbial ecology and public health, since some of them turn out to be pathogenic for humans. The aim of this work was to study the monthly temporal dynamics of Legionella spp. and its relationship with the environmental variables measured in two Pampean shallow lakes (Gómez and Carpincho, Buenos Aires Province, Argentina). The analysis was carried out using a quantitative approach by real-time polymerase chain reaction (qPCR) and a non-quantitative approach using bacterial diversity data obtained by next-generation sequencing (NGS), using the Illumina MiSeq platform. Our results showed that the overall Legionella abundance was very high in the studied Pampean shallow lakes. Notably, fluctuations in dissolved organic carbon and temperature influenced the dynamics shifts in Legionella abundances. Correlation analyses between Legionella reads from NGS and copy numbers obtained through qPCR revealed positive relationships, unveiling distinctions attributable to the diverse sequence processing algorithms employed in the analysis of NGS data. [ABSTRACT FROM AUTHOR]

The isolation of circulating tumoral DNA (ctDNA) present in the bloodstream brings about the opportunity to detect genomic aberrations from the tumor of origin. However, the low amounts of ctDNA present in liquid biopsy samples makes the development of highly sensitive techniques necessary to detect targetable mutations for the diagnosis, prognosis, and monitoring of cancer patients. Here, we employ standard genomic DNA (gDNA) and eight liquid biopsy samples from different cancer patients to examine the newly described CRISPR-Cas13a-based technology in the detection of the BRAF p.V600E actionable point mutation and appraise its diagnostic capacity with two PCR-based techniques: quantitative Real-Time PCR (qPCR) and droplet digital PCR (ddPCR). Regardless of its lower specificity compared to the qPCR and ddPCR techniques, the CRISPR-Cas13a-guided complex was able to detect inputs as low as 10 pM. Even though the PCR-based techniques have similar target limits of detection (LoDs), only the ddPCR achieved a 0.1% variant allele frequency (VAF) detection with elevated reproducibility, thus standing out as the most powerful and suitable tool for clinical diagnosis purposes. Our results also demonstrate how the CRISPR-Cas13a can detect low amounts of the target of interest, but its base-pair specificity failed in the detection of actionable point mutations at a low VAF; therefore, the ddPCR is still the most powerful and suitable technique for these purposes. [ABSTRACT FROM AUTHOR]

In the Baltic Sea, the dinoflagellates Apocalathium malmogiense, Biecheleria baltica, and Gymnodinium corollarium are important contributors to the spring bloom. However, their relative contribution to the bloom community cannot be unambiguously determined by conventional light microscopy due to a lack of resolution of distinctive morphological features of the three species. Here, we describe a molecular approach based on a quantitative real-time polymerase chain reaction (qPCR) primer and probe system, targeting the ITS1 and ITS2 regions of the rRNA gene for all three species and enabling their quantification. The specificity of the method was demonstrated using monocultures of A. malmogiense, B. baltica, G. corollarium as well as three other dinoflagellate species co-occurring in the Baltic Sea during spring and validated using field-collected phytoplankton samples. [ABSTRACT FROM AUTHOR]

Background: The dilution-replicate experimental design for qPCR assays is especially efficient. It is based on multiple linear regression of multiple 3-point standard curves that are derived from the experimental samples themselves and thus obviates the need for a separate standard curve produced by serial dilution of a standard. The method minimizes the total number of reactions and guarantees that Cq values are within the linear dynamic range of the dilution-replicate standard curves. However, the lack of specialized software has so far precluded the widespread use of the dilution-replicate approach. Results: Here we present repDilPCR, the first tool that utilizes the dilution-replicate method and extends it by adding the possibility to use multiple reference genes. repDilPCR offers extensive statistical and graphical functions that can also be used with preprocessed data (relative expression values) obtained by usual assay designs and evaluation methods. repDilPCR has been designed with the philosophy to automate and speed up data analysis (typically less than a minute from Cq values to publication-ready plots), and features automatic selection and performance of appropriate statistical tests, at least in the case of one-factor experimental designs. Nevertheless, the program also allows users to export intermediate data and perform more sophisticated analyses with external statistical software, e.g. if two-way ANOVA is necessary. Conclusions: repDilPCR is a user-friendly tool that can contribute to more efficient planning of qPCR experiments and their robust analysis. A public web server is freely accessible at https://repdilpcr.eu without registration. The program can also be used as an R script or as a locally installed Shiny app, which can be downloaded from https://github.com/deyanyosifov/repDilPCR where also the source code is available. [ABSTRACT FROM AUTHOR]

Background: Different responses in human coronary arteries (HCA) and human middle meningeal arteries (HMMA) were observed for some of the novel CGRP receptor antagonists, the gepants, for inhibiting CGRP-induced relaxation. These differences could be explained by the presence of different receptor populations in the two vascular beds. Here, we aim to elucidate which receptors are involved in the relaxation to calcitonin gene-related peptide (CGRP), adrenomedullin (AM) and adrenomedullin 2 (AM2) in HCA and HMMA. Methods: RNA was isolated from homogenized human arteries (23 HCAs; 12 F, 11 M, age 50 ± 3 years and 26 HMMAs; 14 F, 12 M, age 51 ± 3 years) and qPCR was performed for different receptor subunits. Additionally, relaxation responses to CGRP, AM or AM2 of the human arteries were quantified using a Mulvany myograph system, in the presence or absence of the adrenomedullin 1 receptor antagonist AM22-52 and/or olcegepant. Results: Calcitonin-like receptor (CLR) mRNA was expressed equally in both vascular beds, while calcitonin receptor (CTR) and receptor activity-modifying protein 3 (RAMP3) expression was low and could not be detected in all samples. RAMP1 expression was similar in HCA and HMMA, while RAMP2 expression was higher in HMMA. Moreover, receptor component protein (RCP) expression was higher in HMMA than in HCA. Functional experiments showed that olcegepant inhibits relaxation to all three agonists in both vascular beds. In HCA, antagonist AM22-52 did not inhibit relaxation to any of the agonists, while a trend for blocking relaxation to AM and AM2 could be observed in HMMA. Conclusion: Based on the combined results from receptor subunit mRNA expression and the functional responses in both vascular tissues, relaxation of HCA is mainly mediated via the canonical CGRP receptor (CLR-RAMP1), while relaxation of HMMA can be mediated via both the canonical CGRP receptor and the adrenomedullin 1 receptor (CLR-RAMP2). Future research should investigate whether RAMP2 predominance over RAMP1 in the meningeal vasculature results in altered migraine susceptibility or in a different response to anti-migraine medication in these patients. Moreover, the exact role of RCP in CGRP receptor signalling should be elucidated in future research. [ABSTRACT FROM AUTHOR]

Background: Porcine reproductive and respiratory syndrome virus (PRRSV) has significant productivity and economic impacts in swine herds. Accurately determining the PRRSV status at the herd level is crucial for producers and veterinarians to implement strategies to control and eliminate the virus from infected herds. This study collected oropharyngeal swabs (OSs), nasal swabs (NSs), oral fluid swabs (OFs), rectal swabs (RSs), and serum samples continuously from PRRSV challenged pigs under experimental conditions and growing pigs under field conditions. Additionally, OSs and serum samples were collected from individual sows from 50 large-scale breeding farms, and the collection of OSs does not require the sows to be restrained. Ct values of PRRSV were detected in all samples using real-time reverse transcriptase-polymerase chain reaction (RT-qPCR). Results: In PRRSV challenged pigs, OSs showed a higher PRRSV-positive rate until the end of the observation period. The Ct values of OSs were significantly lower than those of NSs, OFs, and RSs at 2, 8, 12, 14 and 20 days post-challenge (DPC) (P < 0.05). For growing pigs, the positivity rate of PRRSV in OSs was higher than that in other sample types at 30, 70, and 110 days of age. In sows, 24,718 OSs and 6259 serum samples were collected, with PRRSV-positive rate in OSs (9.4%) being significantly higher than in serum (4.1%) (P < 0.05). However, the Ct values of PRRSV RNA in serum were significantly lower than those in OSs (P < 0.001). Conclusions: The OSs sample type yielded higher PRRSV-positive rates for longer periods compared to NSs, RSs, OFs and serum samples for PRRSV detection in infected pigs. Therefore, OSs has a good potential to be a convenient, practical, and reliable sample type for implementing mass sampling and testing of PRRSV in large-scale pig farms. [ABSTRACT FROM AUTHOR]

Background: Identification of the opportunistic fungus Pneumocystis jirovecii in respiratory specimens presents challenges, particularly in differentiating between colonization and active infection. The present study assessed a probe-based real time PCR (qPCR) diagnostic effectiveness in patients with diverse underlying conditions, particularly those with COVID-19 and pulmonary insufficiency. Methods: To set up the qPCR, clinical samples from 281 patients with respiratory ailments were tested. Subsequently, a descriptive study was conducted on 112 patients with pulmonary insufficiency with and without COVID-19 suspected of P. jirovecii infection. All specimens were subjected to DNA extraction followed by nested PCR and qPCR targeting the mitochondrial large subunit (mtLSU)- rRNA gene. Results: Based on nested PCR and qPCR, P. jirovecii was identified in 40 out of 281 patients, with slight variations in positive samples observed across dilutions. Three patients who tested positive in nested PCR yielded negative results in probe-based qPCR. Conversely, three patients who tested positive in probebased qPCR yielded negative results in nested PCR. Considering nested PCR as the golden standard, probe-based qPCR demonstrated good diagnostic performance, with 92.5% sensitivity and 98.7% specificity. Based on cycle threshold (Ct) values, the positive cases were categorized: =32 as infection, >35 as colonization, and a grey zone between these values (32 < X = 35). The analysis of 112 PCP-suspected patients revealed a prevalence ranging from 6.25% (nested PCR) to 7% (probe-based qPCR). Conclusions: This study suggested Ct values to differentiate Pneumocystis pneumonia/colonization in immunocompromised patients. To further augment the diagnostic sensitivity, it is recommended to integrate qPCR results with clinical parameters and biomarkers to offer a more precise understanding of Pneumocystis-related conditions. [ABSTRACT FROM AUTHOR]

Background: Latency remains a major obstacle to finding a cure for HIV despite the availability of antiretroviral therapy. Due to virus dormancy, limited biomarkers are available to identify latent HIV-infected cells. Profiling of individual HIV-infected cells is needed to explore potential latency biomarkers and to study the mechanisms of persistence that maintain the HIV reservoir. Methods: Single cell spatial transcriptomic characterization using the CosMx Spatial Molecular Imager platform was conducted to analyze HIV-infected cells in formalin-fixed paraffin-embedded sections of splenic tissue surgically obtained from an HIV-infected humanized mouse model. Regulation of over a thousand human genes was quantified in both viremic and aviremic specimens. In addition, in situ hybridization and immunohistochemistry were performed in parallel to identify HIV viral RNA- and p24-containing cells, respectively. Finally, initial findings from CosMx gene profiling were confirmed by isolating RNA from CD4 + T cells obtained from a person living with HIV on antiretroviral therapy following either PMA/Ionomycin or DMSO treatment. RNA was quantified using qPCR for a panel of targeted human host genes. Results: Supervised cell typing revealed that most of the HIV-infected cells in the mouse spleen sections were differentiated CD4 + T cells. A significantly higher number of infected cells, 2781 (1.61%) in comparison to 112 (0.06%), and total HIV transcripts per infected cell were observed in viremic samples compared to aviremic samples, respectively, which was consistent with the data obtained from ISH and IHC. Notably, the expression of 55 genes was different in infected cells within tissue from aviremic animals compared to viremic. In particular, both spleen tyrosine kinase (SYK) and CXCL17, were expressed approximately 100-fold higher. This data was further evaluated against bulk RNA isolated from HIV-infected human primary CD4 + T cells. A nearly 6-fold higher expression of SYK mRNA was observed in DMSO-treated CD4 + T cells compared to those stimulated with PMA/Ionomycin. Conclusion: This study found that the CosMx SMI platform is valuable for assessing HIV infection and providing insights into host biomarkers associated with HIV reservoirs. Higher relative expression of the SYK gene in aviremic-infected cells from the humanized mouse HIV model was consistent with levels found in CD4 + T cells of aviremic donors. [ABSTRACT FROM AUTHOR]

Summary: Developing early maturing lentil has the potential to minimize yield losses, mainly during terminal drought. Whole‐genome resequencing (WGRS) based QTL‐seq identified the loci governing earliness in lentil. The genetic analysis for maturity duration provided a good fit to 3:1 segregation (F2), indicating earliness as a recessive trait. WGRS of Globe Mutant (late parent), late‐flowering, and early‐flowering bulks (from RILs) has generated 1124.57, 1052.24 million raw and clean reads, respectively. The QTL‐Seq identified three QTLs (LcqDTF3.1, LcqDTF3.2, and LcqDTF3.3) on chromosome 3 having 246244 SNPs and 15577 insertions/deletions (InDels) and 13 flowering pathway genes. Of these, 11 exhibited sequence variations between bulks and validation (qPCR) revealed a significant difference in the expression of nine candidate genes (LcGA20oxG, LcFRI, LcLFY, LcSPL13a, Lcu.2RBY.3g060720, Lcu.2RBY.3g062540, Lcu.2RBY.3g062760, LcELF3a, and LcEMF1). Interestingly, the LcELF3a gene showed significantly higher expression in late‐flowering genotype and exhibited substantial involvement in promoting lateness. Subsequently, an InDel marker (I‐SP‐383.9; LcELF3a gene) developed from LcqDTF3.2 QTL region showed 82.35% PVE (phenotypic variation explained) for earliness. The cloning, sequencing, and comparative analysis of the LcELF3a gene from both parents revealed 23 SNPs and InDels. Interestingly, a 52 bp deletion was recorded in the LcELF3a gene of L4775, predicted to cause premature termination of protein synthesis after 4 missense amino acids beyond the 351st amino acid due to the frameshift during translation. The identified InDel marker holds significant potential for breeding early maturing lentil varieties. [ABSTRACT FROM AUTHOR]

This study aims to explore the spatial distribution of false kelpfish (Sebastiscus marmoratus) in the mussel farming area, artificial reef areas of Gouqi Island (Shengsi, China), and natural areas using eDNA detection methods. Surface and bottom water samples were collected at 12 stations in November 2022 and April 2023, totaling 52 samples. We used species-specific primers and probes for quantitative PCR (qPCR) detection of Sebastiscus marmoratus eDNA. The eDNA concentrations differed seasonally (p < 0.05) and did not differ (p > 0.05) among the three sampling areas and two water layers. The greatest eDNA concentrations occurred in the surface layer during the spring. Higher concentrations of Sebastiscus marmoratus eDNA were also found in the mussel aquaculture area. Temperature exhibited a significant positive correlation with Sebastiscus marmoratus eDNA concentration (p < 0.05). Additionally, we developed linear equations predicting the relationship between environmental factors and environmental factors, providing a reference for future fishery resource surveys. [ABSTRACT FROM AUTHOR]

Roots play an important role in plant growth, including providing essential mechanical support, water uptake, and nutrient absorption. Nanomaterials play a positive role in improving plant root development, but there is limited knowledge of how nanomaterials affect lateral root (LR) formation. Poly (acrylic) acid coated nanoceria (cerium oxide nanoparticles, PNC) are commonly used to improve plant stress tolerance due to their ability to scavenge reactive oxygen species (ROS). However, its impact on LR formation remains unclear. In this study, we investigated the effects of PNC on LR formation in Arabidopsis thaliana by monitoring ROS levels and Ca2+ distribution in roots. Our results demonstrate that PNC significantly promote LR formation, increasing LR numbers by 26.2%. Compared to controls, PNC-treated Arabidopsis seedlings exhibited reduced H2O2 levels by 18.9% in primary roots (PRs) and 40.6% in LRs, as well as decreased O 2 · − levels by 47.7% in PRs and 88.5% in LRs. When compared with control plants, Ca2+ levels were reduced by 35.7% in PRs and 22.7% in LRs of PNC-treated plants. Overall, these results indicate that PNC could enhance LR development by modulating ROS and Ca2+ levels in roots. In this study, we showed that cerium oxide nanoparticles (PNC) promoted lateral root formation in Arabidopsis thaliana by modulating reactive oxygen species and Ca2+ distribution in both primary and lateral roots. Results indicated that applying PNC to plants have potential to improve the development of root system and thus overall plant growth performance. Adopting nanobiotechnology to stimulate plant root system may be beneficial to sustainable agriculture. [ABSTRACT FROM AUTHOR]

The rapid advancement of biotechnology over the recent decades has amplified the importance of DNA detection and quantification assays. Many of these assays, such as gel electrophoresis, microscopy, flow cytometry, and the detection of amplification in quantitative polymerase chain reaction (qPCR), rely on the use of DNA‐binding fluorescent dyes. This article presents a comprehensive study of six Thiazole‐Orange‐based fluorescent DNA‐binding dyes: SYBR Safe, SYBR Green, Pico Green, SYTO‐16, SYTO‐9, and the benzothiazole‐based analogue (TOPhBu) of the latter. The selected DNA markers were synthesized at a 10‐milligram scale and characterised spectroscopically to quantify their fluorescence enhancement upon binding to double‐stranded DNA. The ability of the dyes to detect DNA at low concentrations was evaluated using two new metrics, absolute fluorescence enhancement (AFE) and relative fluorescence enhancement (RFE). Quantum chemical calculations shed new light on the mechanism of their fluorogenicity through modelling the excited state behaviour and DNA binding of the dyes. Their analytical performance was further tested in qPCR experiments. The experimental results of this work highlight some important differences in the sensitivity and qPCR efficiency of the studied DNA‐binding dyes which will facilitate the DNA marker selection for analytical purposes and the future development of novel DNA sensors. [ABSTRACT FROM AUTHOR]

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Introduction: The cheese microbiota is very complex and is made up of technologically-relevant, spoilage, opportunistic and pathogenic microorganisms. Among them lactic acid bacteria and yeasts are the main ones. One of the most interesting dairy yeasts is Kluyveromyces marxianus because of its technological properties including the ability to produce aroma compounds. Methods: This study investigated the contribution of Kluyveromyces marxianus to the gross composition and aroma profile of cow cheeses. Experimental cheeses were prepared by inoculating a co-culture of K. marxianus FM09 and a commercial strain of Lacticaseibacillus casei and compared with cheeses obtained with only L. casei. The gross composition was determined by a FoodScan™ 2 Dairy Analyser, and free amino acids were evaluated at 507 nm after reaction with Cd-ninhydrin. The volatile organic compounds were extracted by head-space solid phase micro-extraction and analyzed by gas chromatography-mass spectrometry coupled with odor activity values. qRT-PCR was applied to determine the expression of genes involved in esters synthesis and degradation. Results: The inoculation of K. marxianus induced an increase of pH and a reduction of protein content of cheeses, in agreement with the stronger proteolysis detected in these cheeses. K. marxianus influenced the content of aroma compounds both quantitatively and qualitatively. In particular, an increase of higher alcohols, esters and organic acids was observed. Moreover, 12 compounds were detected only in cheeses obtained with the co-culture. These differences were in agreement with the odor activity values (OAV). In fact, only 11 compounds showed OAV > 1 in cheeses obtained with the commercial strain, and 24 in those obtained with the co-culture. The qPCR analysis revealed an over expression of ATF1, EAT1, and IAH1 genes. Conclusion: Kluyveromyces marxianus could act as an important auxiliary starter for cheese production through the development and diversification of compounds related to flavor in short-aged cow cheeses. [ABSTRACT FROM AUTHOR]

Many species are threatened by climate change and must rapidly respond to survive in changing environments. Epigenetic modifications, such as DNA methylation, can facilitate plastic responses by regulating gene expression in response to environmental cues. Understanding epigenetic responses is therefore essential for predicting species' ability to rapidly adapt in the context of global environmental change. Here, we investigated the functional significance of different methylation‐associated cellular processes on temperature‐dependent life history in seed beetles, Callosobruchus maculatus Fabricius 1775 (Coleoptera: Bruchidae). We assessed changes under thermal stress in (1) DNA methyltransferase (Dnmt1 and Dnmt2) expression levels, (2) genome‐wide methylation and (3) reproductive performance, with (2) and (3) following treatment with 3‐aminobenzamide (3AB) and zebularine (Zeb) over two generations. These drugs are well‐documented to alter DNA methylation across the tree of life. We found that Dnmt1 and Dnmt2 were expressed throughout the body in males and females, but were highly expressed in females compared with males and exhibited temperature dependence. However, whole‐genome methylation did not significantly vary with temperature, and only marginally or inconclusively with drug treatment. Both 3AB and Zeb led to profound temperature‐dependent shifts in female reproductive life history trade‐off allocation, often increasing fitness compared with control beetles. Mismatch between magnitude of treatment effects on DNA methylation versus life history effects suggest potential of 3AB and Zeb to alter reproductive trade‐offs via changes in DNA repair and recycling processes, rather than or in addition to (subtle) changes in DNA methylation. Together, our results suggest that epigenetic mechanisms relating to Dnmt expression, DNA repair and recycling pathways, and possibly DNA methylation, are strongly implicated in modulating insect life history trade‐offs in response to temperature change. [ABSTRACT FROM AUTHOR]

Heat therapy has been reported as a safe, effective, and readily available treatment method for heat-tolerant frogs infected with Batrachochytrium dendrobatidis (Bd). We treated wild-caught Australian green tree frogs (Litoria caerulea) infected with Bd using two periods of elevated ambient room temperature (28.2–30.3 °C for 7 weeks followed by 28.9–34.1 °C for 4 weeks). Frogs exhibited persistent and even increasing infection loads in the first treatment period despite prolonged exposure to elevated temperatures, likely due to the presence of cooler microenvironments within their enclosure (25.5–27.0 °C). All frogs eventually returned negative qPCR tests for Bd at the end of the second treatment period, but detectable infections reoccurred one month after frogs were returned to standard housing temperatures (21.2–28.7 °C). Our findings suggest that elevated ambient temperature alone might not eliminate Bd in vivo but can reduce infections loads such that they are undetectable by qPCR analysis of skin swabs. Additional factors, such as cooler microenvironments within enclosures or relative humidity, may influence the success of heat therapy. We recommend further research into the combined effects of temperature and humidity during heat therapy and emphasize the importance of accurate temperature measurements as well as post-treatment monitoring at Bd-permissive temperatures to confirm successful clearance of infections. [ABSTRACT FROM AUTHOR]

Background: Normalization with respect to stable housekeeping genes is important to facilitate gene transcription regulation research and acquire more accurate quantitative polymerase chain reaction (qPCR) data. In the current study, five candidates housekeeping genes of the cotton leafworm, Spodoptera littoralis encoding for Actin (Actin), elongation factor 1-alpha (EF1α), ribosomal protein S3 (RPS3), ribosomal protein 49 (RP49), and Ubiquitin (Ubi), were evaluated as normalization housekeeping genes under Spodoptera littoralis nucleopolyhedrovirus (SpliNPV) viral infection. Methods and Results: The qPCR results confirmed the expression of all five housekeeping genes in S. littoralis viral infected larvae. The expression profiles of the housekeeping genes showed that the EF1α, Actin, and RP49 had the minimum average Ct values of 18.41 ± 0.66, 18.84 ± 0.90 and 19.01 ± 0.87 in all infected samples, respectively. While RPS3 and Ubi showed the maximum average Ct of 21.61 ± 0.51 and 21.11 ± 0.82, respectively. According to the results of ΔCt and geNorm analysis, EF1α was ranked as the most stable housekeeping gene during infection time-course. While by using BestKeeper, geNorm and NormFinder, the Ubi, RP49, and RPS3 showed the most genes transcription stability. The obtained results were also validated using the Cytochrome c oxidase (COX) gene transcripts in response to SpliNPV infection. Conclusions: The results revealed that EF1α and Ubi were the most stable housekeeping genes to be used for normalizing S. littoralis gene transcription regulation under SpliNPV infection. These findings, provide a significant addition for gene transcription regulation studies of S. littoralis upon infection using SpliNPV as a bio-agent. [ABSTRACT FROM AUTHOR]

Background: Biofilm formation is an essential virulence factor that creates a highly protected growth mode for Staphylococcus aureus (S. aureus) to survive in any hostile environment. Antibiotic sub-minimal inhibitory concentration (sub-MIC) may modulate the biofilm formation ability of bacterial pathogens, thereby affecting bacterial pathogenesis and infection outcomes. Intense antimicrobial therapy to treat biofilm-associated infections can control the pathogenic infection aggravation but cannot guarantee its complete eradication. Objective: This study aimed to assess the sub-MICs effect of 5 different antimicrobial classes on biofilm-forming capacity among Staphylococcus aureus clinical isolates using three different biofilm quantitation techniques. Methods: In this study, the effects of 5 different antimicrobial agents, namely, azithromycin, gentamicin, ciprofloxacin, doxycycline, and imipenem, at sub-MICs of 12.5%, 25%, and 50% were tested on 5 different clinical isolates of S. aureus. The biofilms formed in the absence and presence of different antimicrobial sub-MICs were then assessed using the following three different techniques: the crystal violet (CV) staining method, the quantitative PCR (qPCR) method, and the spread plate method (SPM). Results: Biofilm formation was significantly induced in 64% of the tested conditions using the CV technique. On the other hand, the qPCR quantifying the total bacterial count and the SPM quantifying the viable bacterial count showed significant induction only in 24% and 17.3%, respectively (Fig. 1). The difference between CV and the other techniques indicates an increase in biofilm biomass without an increase in bacterial growth. As expected, sub-MICs did not reduce the viable cell count, as shown by the SPM. The CV staining method revealed that sub-MICs of imipenem and ciprofloxacin had the highest significance rate (80%) showing an inductive effect on the biofilm development. On the other hand, doxycycline, azithromycin, and gentamicin displayed lower significance rates of 73%, 53%, and 47%, respectively. Conclusion: Exposure to sub-MIC doses of antimicrobial agents induces the biofilm-forming capacity of S. aureus via increasing the total biomass without significantly affecting the bacterial growth of viable count. [ABSTRACT FROM AUTHOR]

Background: Limited crop production due to lower rainfall has a major impact on the supply and demand of food for the human population. In potato (Solanum tuberosum L.), one of the major crops, there is also concern about a lack of production due to drought stress. Especially the cultivar "Toyoshiro" suitable for processing, has significant reduction in drought yield. Therefore, it is necessary to understand the mechanism of gene expression changes that occur in potato "Toyoshiro" plants and tubers during drought. Methods and results: Seed potatoes were split in half and one was used as a control plant (CT), and the other was used as a drought-stressed plant (DS). CT was watered daily, and DS watered off to mimic the weather conditions of the Tokachi-Obihiro region in 2021. These tubers were harvested at week 14 and the transcriptome was analyzed. DS plants showed 423 downregulated genes and 197 upregulated genes compared to CT. Factors related to cell wall modification, heat stress response, and phytosterol metabolism were detected among the genes whose expression changed. Moreover, the expression of "Abscisic acid and environmental stress-inducible protein TAS14 like (TAS14)," a molecule reported to be upregulated under drought stress, was also upregulated, and was upregulated expression in all strains that reproduced drought. The localization of this molecule in the nucleus and plasma membrane was confirmed in a mCherry-tagged TAS14 mutant line. Conclusions: Our findings contribute to understanding the survival strategy system of Japanese processing potatoes in response to drought stress. [ABSTRACT FROM AUTHOR]

Objective: Environmental DNA (eDNA) methods are crucial for monitoring populations, particularly rare and cryptic species. For confident eDNA application, rigorous assay validation is required including specificity testing with genomic DNA (gDNA). However, this critical step is often difficult to achieve as obtaining fresh tissue samples from at-risk species can be difficult, highly limited, or impossible. Natural history museum collections could serve as a valuable and ethical voucher specimen resource for eDNA assay validation. The present study demonstrates the effectiveness of whole genome amplification (WGA) in providing enough gDNA to assemble high quality mitogenomes from which robust targeted eDNA assays can be designed. Results: Using fresh and historical museum tissue samples from six species spanning fish, birds, and mammals, we successfully developed a WGA method with an average yield of 380 to 1,268 ng gDNA per 20 µL reaction. This gDNA was used for whole genome shotgun sequencing and subsequent assembly of high quality mitogenomes using mtGrasp. These mitogenomes were then used to develop six new robust, targeted quantitative real time polymerase chain reaction-based eDNA assays and 200 ng WGA-enriched yielded satisfactory Cq values and near 100% detection frequencies for all assays tested. This approach offers a cost-effective and non-invasive alternative, streamlining eDNA research processes and aiding in conservation efforts. [ABSTRACT FROM AUTHOR]

Introduction: Camellia anthracnose is caused by multiple Colletotrichum species, resulting in severe yield losses of oil-tea Camellia. Colletotrichum fructicola is one of the major anthracnose pathogens of oil-tea Camellia worldwide. However, developing unique molecular markers for the rapid and accurate detection of Colletotrichum fructicola from diverse Colletotrichum species, as well as early monitoring and effective control of the disease, remains largely unexplored. Methods: C. fructicola-specific genes were obtained using a BLAST search of the sequences of predicted genes in C. fructicola against the genome sequences of Colletotrichum fungal pathogens. In this study, Colletotrichum fructicola- specific molecular markers were developed for rapid and accurate detection of C. fructicola among Camellia anthracnose causing fungal pathogens. Results: Using genomic DNA-based end-point PCR and qPCR, three C. fructicola-specific genes with the ability to distinguish C. fructicola from other oil-tea Camellia anthracnose-related Colletotrichum species, including Colletotrichum camelliae, Colletotrichum gloeosporioides, and Colletotrichum siamense, and oil-tea Camellia fungal pathogens belonging to the genus Neopestalotiopsis, Pestalotiopsis, and Alternaria, were validated as molecular markers. In addition, these three molecular markers were highly sensitive to detecting C. fructicola using DNA extracted fromthe inoculated leaves of oil-tea Camellia. Discussion: These findings enable us to rapidly and uniquely detect the Camellia anthracnose disease caused by Colletotrichum fructicola, which will equip farmers with an effective tool for monitoring Camellia anthracnose disease in the field and taking timely control measurements in advance. [ABSTRACT FROM AUTHOR]

Background: Next-generation sequencing (NGS) approaches have revolutionized gut microbiome research and can provide strain-level resolution, but these techniques have limitations in that they are only semi-quantitative, suffer from high detection limits, and generate data that is compositional. The present study aimed to systematically compare quantitative PCR (qPCR) and droplet digital PCR (ddPCR) for the absolute quantification of Limosilactobacillus reuteri strains in human fecal samples and to develop an optimized protocol for the absolute quantification of bacterial strains in fecal samples. Results: Using strain-specific PCR primers for L. reuteri 17938, ddPCR showed slightly better reproducibility, but qPCR was almost as reproducible and showed comparable sensitivity (limit of detection [LOD] around 104 cells/g feces) and linearity (R2 > 0.98) when kit-based DNA isolation methods were used. qPCR further had a wider dynamic range and is cheaper and faster. Based on these findings, we conclude that qPCR has advantages over ddPCR for the absolute quantification of bacterial strains in fecal samples. We provide an optimized and easy-to-follow step-by-step protocol for the design of strain-specific qPCR assays, starting from primer design from genome sequences to the calibration of the PCR system. Validation of this protocol to design PCR assays for two L. reuteri strains, PB-W1 and DSM 20016 T, resulted in a highly accurate qPCR with a detection limit in spiked fecal samples of around 103 cells/g feces. Applying our strain-specific qPCR assays to fecal samples collected from human subjects who received live L. reuteri PB-W1 or DSM 20016 T during a human trial demonstrated a highly accurate quantification and sensitive detection of these two strains, with a much lower LOD and a broader dynamic range compared to NGS approaches (16S rRNA gene sequencing and whole metagenome sequencing). Conclusions: Based on our analyses, we consider qPCR with kit-based DNA extraction approaches the best approach to accurately quantify gut bacteria at the strain level in fecal samples. The provided step-by-step protocol will allow scientists to design highly sensitive strain-specific PCR systems for the accurate quantification of bacterial strains of not only L. reuteri but also other bacterial taxa in a broad range of applications and sample types. -oGjUmj5e8MXZDxyDjzcm- Video Abstract [ABSTRACT FROM AUTHOR]

Introduction: Muscovy duck parvovirus (MDPV), Goose parvovirus (GPV), Duck circovirus, (DuCV) and Duck adenovirus 3 (DAdV-3) are important pathogens that cause high morbidity and mortality in ducks, causing huge economic loss for the duck industry. Methods: The present study, a quadruplex one-step real time quantitative PCR method for the detection of MDPV, GPV, DuCV, and DAdV-3 was developed. Results: The results showed that assay had no cross-reactivity with other poultry pathogens [Duck plague virus (DPV), Duck tembusu virus (DTMUV), H6 avian influenza virus (H6 AIV), New duck reovirus (NDRV), Newcastle disease virus (NDV), H4 avian influenza virus (H4 AIV), Escherichia coli (E. coli), Muscovy duck reovirus (MDRV), Egg drop syndrome virus (EDSV), Pasteurella multocida (P. multocida)]. The sensitivity result showed that the limits of detection for MDPV, GPV, DuCV, and DAdV-3 were 10, 10, 1 and 10 copies/µl, respectively; The coefficients of variation intra- and inter-method was 1-2%; The range of linear (109 to 103 copies/µL) demonstrated the R2 values for MDPV, GPV, DuCV, and DAdV-3 as 0.9975, 0.998, 0.9964, and 0.996, respectively. The quadruplex real time quantitative PCR method efficiency was 90.30%, 101.10%, 90.72%, and 90.57% for MDPV, GPV, DuCV, and DAdV-3, respectively. 396 clinical specimens collected in some duck sausages from June 2022 to July 2023 were simultaneously detected using the established quadruplex real time quantitative PCR method and the reported assays. The detection rates for MDPV, GPV, DuCV, and DAdV-3 were 8.33% (33/396), 17.93% (71/396), 33.58% (133/396), and 29.04% (115/396), respectively. The agreement between these assays was greater than 99.56%. Discussion: The developed quadruplex real-time quantitative PCR assay can accurately detect these four viruses infecting ducks, providing a rapid, sensitive, specific and accurate technique for clinical testing. [ABSTRACT FROM AUTHOR]

Aims: Protozoan and helminth parasitic infections pose significant public health challenges, especially in developing countries with rural populations marked by suboptimal hygiene practices and socio‐economic constraints. The parasites are the etiological agents of these infections and have a notably elevated global prevalence. Therefore, this study focuses on estimating the frequency and transmission dynamics of several parasitic species, including Blastocystis, Giardia, Cryptosporidium spp., Entamoeba histolytica, Ascaris lumbricoides, Trichuris trichiura, Taenia spp. and hookworms, within a rural community in southwest Colombia with a particular emphasis on the One Health framework, considering environmental and zoonotic transmission potentials. Methods and Results: This study involved the analysis of 125 samples, encompassing human participants (n = 99), their domestic pets (dogs) (n = 24) and water sources (n = 2). Parasite detection was carried out utilizing a combination of microscopy and molecular techniques. Furthermore, the characterization of Blastocystis subtypes (STs) was achieved through Oxford Nanopore sequencing of the rRNA‐18S gene. The investigation also entailed the examination of potential associations between intestinal parasitism and various sociodemographic factors. Results revealed a high frequency of parasitic infections when employing molecular methods, with Blastocystis (n = 109/87%), Giardia (n = 20/16%), Ancylostoma duodenale (n = 28/22%), Ancylostoma ceylanicum (n = 7/5.6%), E. histolytica (n = 6/4.8%), Cryptosporidium spp. (n = 12/9.6%) and even Taenia (n = 1/0.8%) detected. Cryptosporidium spp. was also identified in water samples. Coinfections were prevalent, with 57% (n = 70) of samples exhibiting single‐parasite infections and 43% (n = 53) showing various degrees of polyparasitism, emphasizing the complexity of transmission dynamics. Blastocystis subtyping, conducted via Oxford Nanopore sequencing, revealed a diversity of subtypes and coexistence patterns, with ST2 being the most prevalent. Conclusions: This research underscores the importance of using molecular techniques for frequency estimation, particularly emphasizing the relevance of zoonotic transmission in parasitic infections. It highlights the significance of the One Health approach in comprehending the circulation of parasites among animals, humans and environmental sources, thereby directly impacting public health and epidemiological surveillance. [ABSTRACT FROM AUTHOR]

In the deciduous fruit industry, orchards are often excavated and trees chipped. The organic material is then used as mulch for soil conservation, a practice that form part of sustainable agricultural. The presence and possible transmission of plant pathogens are not considered when trees are removed, chipped and used for mulch. Young apple trees can develop cankers due to Diplodia seriata of which the inoculum source might come from fruiting structures present on apple wood mulch. Therefore, the presence of D. seriata, on chipped apple tree wood pieces used for mulch in younger orchards was investigated. To be able to detect D. seriata, qPCR primers were designed from a previously identified unique sequence characterised amplified region (SCAR). The qPCR primers were specific for D. seriata (Cq ≤ 35 and Tm = 85 0.17 °C) when compared with DNA from nineteen other fungal taxa associated with canker or wood rot of apple trees tested, excluding Botryosphaeria dothidea (Cq = 38 and Tm = 85.25 °C). The qPCR assay was sensitive and had a limit of quantification of 2859 fg/µl and limit of detection of 571 fg/µl. Wood chips were collected at two time periods (from heaps and 6 months after it was spread in tree rows) in three apple orchards in the Western Cape of South Africa. DNA was extracted from water-washes of 120 wood piece samples and D. seriata was detected from 101 of these samples. This study showed that the newly developed primers was able to successfully detect D. seriata from mulched apple wood. The presence of D. seriata on apple tree wood chips indicates that there is a risk involved in using wood chips made from old apple trees. [ABSTRACT FROM AUTHOR]

Quantitative PCR is a molecular technique for DNA quantification that depends on reaction efficiency and the Ct value ("cycle threshold"). However, the results are dependent on laboratory conditions and mathematical approaches. Thus, the data of 16 genes from Pseudomonas aeruginosa strain AG1 were generated using qPCR to assess the effect of DNA concentration and three mathematical methods (a standard curve and two individual-curve-based approaches called exponential and sigmoidal models) on efficiency and DNA quantification. Differences in efficiency were revealed depending on the mathematical method used; the values were 100% in three out of the four standard curves, but estimations of the expected fold change in DNA serial dilutions were not achieved, indicating the possible overestimation of efficiency. Moreover, when efficiency was compared to DNA concentration, a decreasing trend in efficiency as DNA concentration increased in the reaction was observed in most cases, which is probably related to PCR inhibitors. For all 16 genes at a single DNA concentration, the efficiencies for the exponential model were found in the range of 1.5–2.79 (50–79%), and for the sigmoidal approach, the range was 1.52–1.75 (52–75%), with similar impact on normalized expression values, as indicated by the genes for standard curves. Jointly, DNA concentration and mathematical model choice were demonstrated to impact the estimation of reaction efficiency and, subsequently, DNA quantification when using qPCR. [ABSTRACT FROM AUTHOR]

Escherichia coli serotype O157:H7 is a diarrheal agent and a significant cause of hemorrhagic colitis and the development of hemolytic uremic syndrome (HUS), mainly in infants. Early detection of contaminated food and water using reliable and fast tests is one of the strategies to prevent infections from E. coli O157:H7. Methods: Four quantitative polymerase chain reaction protocols (SYBR Green qPCR) were developed and validated to determine the presence of the bacteria according to its rfbE, stx1, and stx2 genes. Results: The results of the efficiencies were between 80% and 97% with a high linearity (R2 0.99). The cut-off limits for each primer sequence were 3.1667 × 10−2 ng µL−1 for two sequences of the serogroup O157 (primers rfbE and O157), 1.7228 × 10−3 ng µL−1 for stx1, and 3.5185 × 10−3 ng µL−1 for stx2. The inclusivity and the exclusivity of each gene, as well as the analytical precision and the positive and negative predictive value, were 100%. A contaminated meat matrix was evaluated, detecting up to 4 CFU g−1. Conclusions: SYBR Green qPCR protocols could be implemented to trace the presence of E. coli O157 in a routine analysis of ground beef or as an easy, rapid, sensitive, and specific diagnostic test while still considering microbiological tests to validate any inconclusive results. [ABSTRACT FROM AUTHOR]

Human adenovirus (HAdV) F40/41 is an important pathogen in pediatric acute gastroenteritis cases. However, the diversity of study designs and diagnostic methods often leads to misinterpretations of their impact. Our study explored the genetic diversity of HAdV-F40/41 in Brazil using a specific qPCR assay for HAdV species F, combined with a phylogenetic analysis of the partial hexon and fiber genes. Our results demonstrated that HAdV-F41 strains predominated and exhibited higher diversity than HAdV-F40 strains. Based on the hexon gene, Brazilian HAdV-F41 strains were grouped into two genome type clusters (GTC), further divided into subclusters, with most strains clusteringto GTC2. The partial shaft region of the fiber gene exhibited higher conservation among HAdV-F41. The specific qPCR assay for HAdV species F identified HAdV-F in an additional 31.5% (34/108) of previously uncharacterized HAdV-positive samples detected using a non-specific HAdV qPCR assay. Both assays strongly correlated in detecting HAdV-F, and the specific qPCR assay for enteric types can enhance HAdV surveillance, especially when sequencing is not possible. Our study provides novel insights regarding the genetic diversity of HAdV-F species in Brazil. [ABSTRACT FROM AUTHOR]

During the COVID-19 pandemic, the surge in disinfectant use emphasised their pivotal role in infection control. While the majority of antimicrobial resistance research focuses on antibiotics, resistance to biocides, which are present in disinfectants and sanitisers, is escalating. Serratia sp. HRI is a highly resistant isolate, and through the study of this organism, the molecular mechanisms of resistance may be uncovered. Serratia sp. HRI was treated with the disinfectant benzalkonium chloride in preparation for RNA sequencing. Through mining of the RNA-Seq differential expression data, an uncharacterised Major Facilitator Superfamily (MFS) efflux pump gene was found to be up-regulated at least four-fold at four different time points of exposure. Real-time PCR revealed this uncharacterised MFS efflux gene was up-regulated after exposure to benzalkonium chloride and two additional disinfectants, didecyldimethylammonium chloride (DDAC) and VirukillTM. Additionally, expression of this gene was found to be higher at 20 min versus 90 min of exposure, indicating that the up-regulation of this gene is an initial response to biocide treatment that decreases over time. This suggests that MFS efflux pumps may be an initial survival mechanism for microorganisms, allowing time for longer-term resistance mechanisms. This work puts forward a novel biocide resistance gene that could have a major impact on biocide susceptibility and resistance. [ABSTRACT FROM AUTHOR]

Fusarium Head Blight (FHB), predominantly caused by Fusarium species, is a devastating cereal disease worldwide. While considerable research has focused on Fusarium communities in grains, less attention has been given to residues and soil, the primary inoculum sources. Knowledge of Fusarium spp. diversity, dynamics, and mycotoxin accumulation in these substrates is crucial for assessing their contribution to wheat head infection and the complex interactions among Fusarium communities throughout the wheat cycle. We monitored six minimum-tillage wheat fields, with maize as the preceding crop, over two years. Soils, maize residues, and wheat grains were sampled at four stages. Fusarium composition was analyzed using a culture-dependent method, species-specific qPCR, and EF1α region metabarcoding sequencing, enabling species-level resolution. The Fusarium communities were primarily influenced by substrate type, accounting for 35.8% of variance, followed by sampling location (8.1%) and sampling stage (3.2%). Among the 32 identified species, F. poae and F. graminearum dominated grains, with mean relative abundances of 47% and 29%, respectively. Conversely, residues were mainly contaminated by F. graminearum, with a low presence of F. poae, as confirmed by species-specific qPCR. Notably, during periods of high FHB pressure, such as in 2021, F. graminearum was the dominant species in grains. However, in the following year, F. poae outcompeted F. graminearum, resulting in reduced disease pressure, consistent with the lower pathogenicity of F. poae. Source Tracker analysis indicated that residues were a more significant source of Fusarium contamination on wheat in 2021 compared to 2022, suggesting that F. graminearum in 2021 primarily originated from residues, whereas F. poae's sources of infection need further investigation. Additionally, multiple mycotoxins were detected and quantified in maize residues during the wheat cycle, raising the question of their ecological role and impact on the soil microbiota. [ABSTRACT FROM AUTHOR]

It is pivotal to avoid cross-sample contamination in forensic genetic laboratories and optimal cleaning protocols for the removal of DNA are essential. A survey was performed, and ten forensic genetic laboratories shared their cleaning protocols in pre-PCR and post-PCR laboratories. The cleaning frequencies on different surface areas were somewhat similar, whereas none of the laboratories used the same cleaning reagents. Therefore, the efficiencies of the cleaning protocol utilised were tested and compared. The results showed that freshly made household bleach and Virkon® removed all amplifiable DNA from the surfaces, whereas DNA AWAY™ and the disinfection reagents ethanol, isopropanol, and ChemGene HLD4L did not. [ABSTRACT FROM AUTHOR]

The increasing significance of the aquaculture sector and commercially valuable species underscores the need to develop alternatives for controlling diseases such as Ichthyophthirius multifiliis‐induced ichthyophthiriasis. This ciliated protozoan parasite threatens nearly all freshwater fish species, causing substantial losses in the fishery industry. Despite this, effective large‐scale treatments are lacking, emphasizing the necessity of adopting preventive strategies. While the pathogenesis of ichthyophthiriasis and its immune stimulation allows for vaccination strategies, precise adjustments are crucial to ensure the production of an effective vaccine compound. Therefore, this study aimed to evaluate the impact of immunizing Astyanax lacustris with a genetic vaccine containing IAG52A from I. multifiliis and the molecular adjuvant IL‐8 from A. lacustris. Transcript analysis in immunized A. lacustris indicated mRNA production in fish muscles, demonstrating an expression of this mRNA. Fish were divided into five groups, receiving different vaccine formulations, and all groups received a booster dose 14 days after the initial immunization. Samples from vaccinated fish showed increased IL‐1β mRNA expression in the spleen within 6 h post the second dose and after 14 days. In the head kidney, IL‐1β mRNA expression showed no significant difference at 6 and 24 h but an increase was noted in fish injected with IAG and IAG + IL‐8 after 14 days. IL‐8 mRNA expression in the spleen and kidney did not significantly differ from the control group. Histological analysis revealed no variation in leukocyte concentration at 6 and 24 h post‐vaccination; however, after 14 days, the groups injected with IAG and IAG + IL‐8 exhibited a higher leukocyte density at the application sites than the control. The obtained data suggest that the used vaccine is transcribed, indicating its potential to stimulate innate immune response parameters through mRNA cytokine expression and leukocyte migration. [ABSTRACT FROM AUTHOR]

Cotija and Bola de Ocosingo are artisanal ripened cheeses produced in Mexico. Both are made with raw bovine milk from free-grazing cows and with no starter cultures. Unlike culture-based techniques, molecular methods for pathogen detection in food allow a shorter turnaround time, higher detection specificity, and represent a lower microbiological risk for the analyst. In the present investigation, we analyzed 111 cheese samples (95 Cotija and 16 Bola de Ocosingo) by qPCR (TaqMan®) after an enrichment-culture step specific to each foodborne bacterium. The results showed that 100% of the samples were free of DNA from Listeria monocytogenes, Brucella spp., Escherichia coli enterotoxigenic (ETEC), and O157:H7; 9% amplified Salmonella spp. DNA; and 11.7%, Staphylococcus aureus DNA. However, the threshold cycle (Ct) values of the amplified targets ranged between 23 and 30, indicating DNA from non-viable microorganisms. Plate counts supported this assumption. In conclusion, 100% of the cheeses analyzed were safe to consume, and the enrichment step before DNA extraction proved essential to discern between viable and non-viable microorganisms. Hygienic milking, milk handling, cheese manufacturing, and ripening are crucial to achieve an adequate microbiological quality of cheeses made with raw milk. [ABSTRACT FROM AUTHOR]

Estimating sex ratios is important for understanding population demographics. Here, we present a direct comparison of paired sex ratio estimates from visual field surveys and molecular sex identification using scat in sexually dimorphic Steller sea lions (Eumetopias jubatus). For this purpose, we designed novel qPCR Taqman gene expression assays to determine sex from scat of both Steller and California (Zalophus californianus) sea lions targeting the zinc-finger X-linked gene (ZFX) and sex-determining region Y gene (SRY). The assay pair was validated using scat samples from California sea lions of known sex with 100% success rate of signal amplification. Our assays yielded a 94.1% success rate in identifying sea lion sex from field-collected scats. In addition, the sex ratio determined via molecular analysis was closely correlated to the adult male and adult female proportions observed at the haul-out site for each month scat samples and demographic counts were collected in tandem. While visual counts cannot determine the sex of juvenile sea lions, scat collections are assumed to represent all age classes present at the haulout, suggesting that the juvenile sex ratios likely mirrored the adult sex ratio. The protocol described here could be implemented to identify the sex of Steller and California sea lions from their feces in free-ranging populations in other non-invasive, scat-based studies. Further, the concordance with visual observations suggests that molecular sex identification from scat collected at haul-outs is a robust methodology in sea lions and possibly in other pinniped species that lack distinctive sexual dimorphism. [ABSTRACT FROM AUTHOR]

Floodplain forests as one of the most endangered ecosystems in Europe have recently been impacted by fungal pathogens Phytophtora sp. and Hymenoscyphus fraxineus (chalara). Grey poplar (Populus × canescens Aiton Sm.) can be used as one of well adapted tree species for the reforestation of withering stands of ash (Fraxinus sp.). A unique population of grey poplar (Populus × canescens Aiton Sm.) characterized by desirable phenotypic traits was used for this purpose. The gender distribution was asymmetric; out of 155 individuals, 113 were female. Out of 33 different genotypes determined in the population, 15 were used as a source of approved forest reproductive material. Vegetative reproduction methods (ex situ clonal reproduction by micropropagation and cuttings) were developed and used, to rapidly initiate the recovery of forest stands of grey poplar. In total, 940 explants were successfully micropropagated and adapted to natural conditions, to ensure a genetically diverse source of viable plants used for reforestation. Moreover, we used methodological procedures of micropropagation for setting up cryopreservation technique. With respect to long-term storage of valuable grey poplar genotypes, modulation of gene expression by cold hardening during cryopreservation revealed significant changes in a few candidate genes involved in plant cellular processes. [ABSTRACT FROM AUTHOR]

The COVID-19 pandemic, caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), began in 2019. One of the strategies for pandemic control was mass vaccination. In Brazil, the recombinant COVID-19 vaccine (RCV) was produced on a large scale and offered at no charge to the population. The specifications for quality control analyses of RCV included identity and infectivity determination. To validate the results, a reference material (RM) must be analyzed in parallel with the sample vaccine. This research aimed to establish the RM for use in the identity and infectivity assay for RCV. The candidate RM was analyzed using homogeneity and stability studies. The RM was considered homogeneous for identity (cycle threshold (Ct) ≤ 25.19) and infectivity (average x - was 9.25 log10 infectious units/mL). The RM was considered adequately stable for identity during the total period in all studies, being stable at −70, 5, and 22.5 °C for 380, 313, and 14 days, respectively (Ct ≤ 21.81). For infectivity, the RM was stable at −70, 5, and 22.5 °C for 380, 97, and three days, respectively. Since the property identity and infectivity values of the RM were established, the new RM could be used in quality control analysis. [ABSTRACT FROM AUTHOR]

Purpose: This study aims to reveal the mechanism of fibroblast‐related mitochondrial genes on keloid formation and explore promising signature genes for keloid diagnosis. Method: The distribution of fibroblasts between the keloid sample and control sample based on three keloid datasets, followed by the differentially expressed genes (DEGs) investigation and associated enrichment analysis. Then, hub genes were explored based on DEGs, mitochondrial genes from an online database, as well as fibroblast‐related genes that were revealed by WCGNA. Subsequently, signature genes were screened through machine learning, and their diagnostic value was validated by nomogram. Moreover, the targeted drugs and related transcriptional regulation of these genes were analyzed. Finally, the verification analysis was performed on signature genes using qPCR analysis. Result: A total of totally 329 DEGs were revealed based on three datasets, followed by enrichment analysis. WGCNA revealed a total of 258 fibroblast‐related genes, which were primarily assembled in functions like muscle tissue development. By using machine learning, we screened four signature genes (ACSF2, ALDH1B1, OCIAD2, and SIRT4) based on eight hub genes (fibroblast‐related mitochondrial genes). Nomogram and validation analyses confirmed the well‐diagnostic performance of these four genes in keloid. Immune infiltration and drug correlation analyses showed that SIRT4 was significantly associated with immune cell type 2 T helper cells and molecular drug cyclosporin. All these findings provided new perspectives for the clinical diagnosis and therapy of keloid. Conclusion: The fibroblast‐related mitochondrial genes including SIRT4, OCIAD2, ALDH1B1, and ACSF2 were novel signature genes for keloid diagnosis, offering novel targets and strategies for diagnosis and therapy of keloid. [ABSTRACT FROM AUTHOR]

Purpose: The objectives of the present study are to determine the molecular prevalence of Leishmania spp. in the owned domestic cats in the Black Sea Region of Türkiye and analyze the associated risk factors in FeL. Methods: Conjunctival swabs (CS), blood, demographic, and clinical data were collected from 150 owned cats brought to the Veterinary Teaching Hospital during 2020–2022. Leishmania kinetoplast DNA (kDNA) from CS was screened by TaqMan Real-Time PCR (qPCR) with the genus-specific primers and a probe. Results: All qPCR positive products were also amplified and sequenced to identify Leishmania species by ITS1 primers. Molecular prevalence of L. infantum found as 12.6% (19/150) in the observed cats in the Black Sea Region of Türkiye. There was a significant difference (p < 0.05) between neutered and intact cats with regarding to L. infantum positivity. Intact cats found to be 0.368 times more prone to be L. infantum-positive (L+). Dermatological lesions were found the most common (26.3%) problems in the L + cats. The median leucocyte count was the only parameter that was found statistically (p < 0.05) lower in the L + group (6.60) than the negative group (L−) (8.96), when comparing the WBC, NEU/LYM, MONO/LYM, EOS/LYM and PLT/LYM values. Conclusion: This study presented the molecular occurrence of FeL in the Black Sea Region of Türkiye for the first time indicating that the carrier status of the cats makes them alternative reservoirs for possible zoonotic transmission of L. infantum in this zone. [ABSTRACT FROM AUTHOR]

The COVID-19 epidemic had a profound impact on global health and the economy and Ghana was no exception to its far-reaching consequences. Regarding detection of the causative agent—the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), reverse-transcription–qPCR (RT–qPCR) is widely recognized as a very sensitive and reliable diagnostic technique used globally. There are, however, high operational costs in acquiring test kits, equipment, and accessories for RT–qPCR testing, which pose significant challenges in resource-limited settings. Hence, this proof-of-concept study set out to develop a more affordable COVID-19 protocol for use in low or lower-middle-income settings, such as Ghana, that would bypass the traditional extraction process using inexpensive reagents and evaluate the possibility of processing samples collected using wooden shaft swabs. Several less expensive media were used for the extraction-free process. Results demonstrated that direct RT–qPCR assay after 5 min heat inactivation of virus at 95 °C in 0.1× PBS or molecular grade water resulted in viral detection with quantification cycle (Cq) values that are comparable to results obtained following the extraction process. Also, wooden shaft swabs could be used for sampling if incubation times are kept to less than 6 h. The study demonstrates that extraction-free protocols are one way to minimize the cost of COVID-19 testing by RT–qPCR. [ABSTRACT FROM AUTHOR]