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Seasonal floodplains in the Amazon basin are important sources of methane (CH4), while upland forests are known for their sink capacity. Climate change effects, including shifts in rainfall patterns and rising temperatures, may alter the functionality of soil microbial communities, leading to uncertain changes in CH4 cycling dynamics. To investigate the microbial feedback under climate change scenarios, we performed a microcosm experiment using soils from two floodplains (i.e., Amazonas and Tapajós rivers) and one upland forest. We employed a two-factorial experimental design comprising flooding (with non-flooded control) and temperature (at 27 °C and 30 °C, representing a 3 °C increase) as variables. We assessed prokaryotic community dynamics over 30 days using 16S rRNA gene sequencing and qPCR. These data were integrated with chemical properties, CH4 fluxes, and isotopic values and signatures. In the floodplains, temperature changes did not significantly affect the overall microbial composition and CH4 fluxes. CH4 emissions and uptake in response to flooding and non-flooding conditions, respectively, were observed in the floodplain soils. By contrast, in the upland forest, the higher temperature caused a sink-to-source shift under flooding conditions and reduced CH4 sink capability under dry conditions. The upland soil microbial communities also changed in response to increased temperature, with a higher percentage of specialist microbes observed. Floodplains showed higher total and relative abundances of methanogenic and methanotrophic microbes compared to forest soils. Isotopic data from some flooded samples from the Amazonas river floodplain indicated CH4 oxidation metabolism. This floodplain also showed a high relative abundance of aerobic and anaerobic CH4 oxidizing Bacteria and Archaea. Taken together, our data indicate that CH4 cycle dynamics and microbial communities in Amazonian floodplain and upland forest soils may respond differently to climate change effects. We also highlight the potential role of CH4 oxidation pathways in mitigating CH4 emissions in Amazonian floodplains.

Most pneumococcal disease occurs among infants and older adults and is thought to be driven by the transmission of Streptococcus pneumoniae from young children to these vulnerable age groups. However, pneumococcal disease outbreaks also affect non-elderly adults living or working in congregate, close-contact settings. Little is known about pneumococcal carriage in such populations. From July to November 2020, we collected saliva from low-income adult farmworkers in Monterey County, California, and tested for pneumococcal carriage following culture enrichment via quantitative PCR assays targeting the pneumococcal lytA and piaB genes. Participants were considered to carry pneumococci if lytA and piaB cycle threshold values were both below 40. Among 1,283 participants enrolled in our study, 117 (9.1%) carried pneumococci. Carriers tended more often than non-carriers to be exposed to children aged 2-4 and >4 persons per bedroom vs ≤2 persons per bedroom]. Household overcrowding remained associated with increased risk of carriage among participants not exposed to children aged 2 vs ≤2 persons per bedroom]. Exposure to children aged

In populations of healthy show horses, the subclinical transmission and circulation of respiratory pathogens can lead to disease outbreaks. Due to recent outbreaks of equine herpesvirus-1 myeloencephalopathy (EHM) in the USA and Europe, many show organizers have instituted various biosecurity protocols such as individual horse testing, monitoring for early clinical disease and increasing hygiene and cleanliness protocols. The aim of this study was to determine the accuracy of detecting EHV-1 in the various environmental samples collected from the stalls of subclinical shedders. Four healthy adult horses were vaccinated intranasally with a modified-live EHV-1 vaccine in order to mimic subclinical shedding. Three additional horses served as non-vaccinated controls. All the horses were stabled in the same barn in individual stalls. Each vaccinated horse had nose-to-nose contact with at least one other horse. Prior to the vaccine administration, and daily thereafter for 10 days, various samples were collected, including a 6 rayon-tipped nasal swab, an environmental sponge, a cloth strip placed above the automatic waterer and an air sample. The various samples were processed for nucleic acid purification and analyzed for the presence of EHV-1 via quantitative PCR (qPCR). EHV-1 in nasal secretions was only detected in the vaccinated horses for 1-2 days post-vaccine administration. The environmental sponges tested EHV-1 qPCR-positive for 2-5 days (median 3.5 days) in the vaccinated horses and 1 day for a single control horse. EHV-1 was detected by qPCR in stall strips from three out of four vaccinated horses and from two out of three controls for only one day. EHV-1 qPCR-positive air samples were only detected in three out of four vaccinated horses for one single day. For the vaccinated horses, a total of 25% of the nasal swabs, 35% of the environmental stall sponges, 7.5% of the strips and 7.5% of the air samples tested qPCR positive for EHV-1 during the 10 study days. When monitoring the subclinical EHV-1 shedders, the collection and testing of the environmental sponges were able to detect EHV-1 in the environment with greater frequency as compared to nasal swabs, stationary strips and air samples.

BACKGROUND: Malaria remains a significant cause of morbidity and mortality in Ethiopia with an estimated 3.8 million cases in 2021 and 61% of the population living in areas at risk of malaria transmission. Throughout the country Plasmodium vivax and Plasmodium falciparum are co-endemic, and Duffy expression is highly heterogeneous. The public health significance of Duffy negativity in relation to P. vivax malaria in Ethiopia, however, remains unclear. This study seeks to explore the prevalence and rates of P. vivax malaria infection across Duffy phenotypes in clinical and community settings. METHODS: A total of 9580 and 4667 subjects from community and health facilities from a malaria endemic site and an epidemic-prone site in western Ethiopia were enrolled and examined for P. vivax infection and Duffy expression from February 2018 to April 2021. Association between Duffy expression, P. vivax and P. falciparum infections were examined for samples collected from asymptomatic community volunteers and symptomatic subjects from health centres. RESULTS: Infection rate of P. vivax among Duffy positives was 2-22 fold higher than Duffy negatives in asymptomatic volunteers from the community. Parasite positivity rate was 10-50 fold higher in Duffy positives than Duffy negatives among samples collected from febrile patients attending health centres and mixed P. vivax and P. falciparum infections were significantly more common than P. vivax mono infections among Duffy negative individuals. Plasmodium vivax parasitaemia measured by 18sRNA parasite gene copy number was similar between Duffy positives and Duffy negatives. CONCLUSIONS: Duffy negativity does not offer complete protection against infection by P. vivax, and cases of P. vivax in Duffy negatives are widespread in Ethiopia, being found in asymptomatic volunteers from communities and in febrile patients from health centres. These findings offer evidence for consideration when developing control and intervention strategies in areas of endemic P. vivax and Duffy heterogeneity.

Background: Leptospira is a genus of bacteria that causes the zoonotic disease known as leptospirosis, which mainly affects countries with tropical and subtropical climates. Its prevalence may be underestimated because the initial stage of the infection is characterized by presenting a febrile condition that is easily confused with other diseases, such as dengue. This work reports the frequency of leptospirosis in the blood of patients with febrile symptoms of unknown origin. Materials and Methods: A total of 218 peripheral blood samples were analyzed from volunteer participants from Culiacan Sinaloa in June 2019, one half corresponded to patients with undiagnosed febrile symptoms and the other half to asymptomatic volunteers. Data collected included the age and sex of the participants. Leptospira was detected by qPCR using a fragment of the lipL32 gene from the bacteria's genome as a target. Fisher's exact test was used as a statistical method to estimate the relationship between the infection and the data collected. Results: The study group comprised 134 female and 84 male patients ranging from ages 1 to 92 years, averaging 41 years. In this study, Leptospira infection was identified in the blood of 22/218 participating volunteers (10.09%), of which 20/109 (18.34%) presented febrile symptoms, whereas 2/109 (1.83%) were asymptomatic. The most affected participants were women with ages between 27 and 59 years. However, the analysis of the relationship between infection and the variables studied did not show statistical significance. Conclusions: Leptospirosis was detected in blood samples from patients with undiagnosed febrile illness and asymptomatic symptoms in Sinaloa. The lipL32 gene is useful as a target in identifying Leptospira in human blood in the acute phase of the disease. [ABSTRACT FROM AUTHOR]

Using technologies and enforcing national reuse guidelines helps reduce health risks from recycled water used in irrigation. Analysing viral presence in raw and treated sewage is crucial for preventing pandemics. This study used qPCR to evaluate three urban wastewater treatment plants in Ramallah: Al-Tireh MBR, Al-Bireh AS, and Rawabi MBBR. Samples collected in March 2019 were treated and analysed using real-time PCR with specific primers and probes. Results showed Al-Tireh MBR achieved 100% removal of enterovirus, adenovirus, norovirus, and rotavirus. Al-Bireh WWTP achieved 33% removal for adenovirus and rotavirus. Rawabi MBBR achieved varying removal rates: 100% for adenovirus, 33% for norovirus, and 66% for rotavirus. Chlorinated samples showed no direct correlation between faecal coliforms and viral presence. These findings suggest retrofitting Al-Bireh WWTP and Rawabi MBBR with ultrafiltration or MBR units to mitigate viral outbreaks. [ABSTRACT FROM AUTHOR]

Contagious agalactia is an infectious disease of small ruminants caused by several Mycoplasma species with leading role of M. agalactiae. Clinical signs are manifested by mastitis, arthritis, keratoconjunctivitis, abortion, pneumonia and septicaemia often in various combinations. The aim of the study was to tested milk samples from sheep and goats in herds with different health status by a rapid, sensitive, specific method for the detection and quantification of DNA of M. agalactiae. A total of 67 milk samples from sheep and goats were tested by real-time polymerase chain reaction (qPCR). Thirteen were positive for DNA and 54 were negative. The present study is the first report of detection of M. agalactiae DNA in Bulgaria. [ABSTRACT FROM AUTHOR]

Cold environments are the most widespread extreme habitats in the world. However, the role of wastewater treatment plants (WWTPs) in the cryosphere as hotspots in antibiotic resistance dissemination has not been well established. Hence, a snapshot of the resistomes of WWTPs in cold environments, below 5 °C, was provided to elucidate their role in disseminating antibiotic resistance genes (ARGs) to the receiving waterbodies. The resistomes of two natural environments from the cold biosphere were also determined. Quantitative PCR analysis of the aadA, aadB, ampC, blaSHV, blaTEM, dfrA1, ermB, fosA, mecA, qnrS, and tetA(A) genes indicated strong prevalences of these genetic determinants in the selected environments, except for the mecA gene, which was not found in any of the samples. Notably, high abundances of the aadA, ermB, and tetA(A) genes were found in the influents and activated sludge, highlighting that WWTPs of the cryosphere are critical hotspots for disseminating ARGs, potentially worsening the resistance of bacteria to some of the most commonly prescribed antibiotics. Besides, the samples from non-disturbed cold environments had large quantities of ARGs, although their ARG profiles were highly dissimilar. Hence, the high prevalences of ARGs lend support to the fact that antibiotic resistance is a common issue worldwide, including environmentally fragile cold ecosystems. [ABSTRACT FROM AUTHOR]

Roe deer meat is a prized game product in many European countries. However, concerns exist regarding the accuracy of the amount of declared roe deer in processed game meat foods. This study aimed to develop a reliable method for the detection and quantification of roe deer in commercialized game meat products. A TaqMan probe-based quantitative real-time PCR (qPCR) assay was designed, targeting a single-copy 120-bp region of the roe deer agouti signaling protein (ASIP) encoding gene. The method employed the normalized ∆Cq approach to establish a calibration curve for roe deer detection and quantification within 0.05–50% (w/w) in complex raw and processed matrices. The method proved to be specific for roe deer identification, achieving limits of detection and quantification of 0.04 ng of roe deer DNA and 0.05% (w/w) of roe deer in simulated pâté. Following validation with blind samples, highlighting the precision and trueness of the approach, the assay was applied to 46 market samples from four European origins (Poland, Portugal, France, and Spain). The analysis revealed significant discrepancies between declared roe deer content and actual levels in all roe deer labeled products. The global analysis of results, combining the previous survey on red deer species with present roe deer data, identified 61% of mislabeled/adulterated samples due to the absence of deer species, substitution of roe deer with red deer, substitution of fallow deer with other deer species and red deer with pork, and undeclared addition of roe deer. This study demonstrates the effectiveness of the developed qPCR method for accurate roe deer meat authentication in foods, showing its usefulness as a tool for routine food inspection to ensure labeling compliance. [ABSTRACT FROM AUTHOR]

Recent progress in gene editing has enabled development of gene therapies for many genetic diseases, but also made gene doping an emerging risk in sports and competitions. By delivery of exogenous transgenes into human body, gene doping not only challenges competition fairness but also places health risk on athletes. World Anti‐Doping Agency (WADA) has clearly inhibited the use of gene and cell doping in sports, and many techniques have been developed for gene doping detection. In this review, we will summarize the main tools for gene doping detection at present, highlight the main challenges for current tools, and elaborate future utilizations of high‐throughput sequencing for unbiased, sensitive, economic and large‐scale gene doping detections. Quantitative real‐time PCR assays are the widely used detection methods at present, which are useful for detection of known targets but are vulnerable to codon optimization at exon–exon junction sites of the transgenes. High‐throughput sequencing has become a powerful tool for various applications in life and health research, and the era of genomics has made it possible for sensitive and large‐scale gene doping detections. Non‐biased genomic profiling could efficiently detect new doping targets, and low‐input genomics amplification and long‐read third‐generation sequencing also have application potentials for more efficient and straightforward gene doping detection. By closely monitoring scientific advancements in gene editing and sport genetics, high‐throughput sequencing could play a more and more important role in gene detection and hopefully contribute to doping‐free sports in the future. [ABSTRACT FROM AUTHOR]

Circulating tumor cells (CTCs) are an established prognostic marker in metastatic prostate cancer (PrC) but have received little attention in localized high-risk disease. Peripheral blood was obtained from patients with early intermediate and high-risk PrC (n = 15) at baseline, after radiotherapy, and during follow-up, as well as from metastatic PrC patients (n = 23). CTCs were enriched using the microfluidic Parsortix® technology. CTC-related marker were quantified with qPCR and RNA in-situ hybridization (ISH). Positivity and associations to clinical parameters were assessed using McNemar test, Fisher Exact test or log-rank test. The overall positivity was high in both cohorts (87.0% metastatic vs. 66.7% early at baseline). A high concordance of qPCR and RNA ISH was achieved. In metastatic PrC, PSA and PSMA were prognostic for shorter overall survival. In early PrC patients, an increase of positive transcripts per blood sample was observed from before to after radiation therapy, while a decrease of positive markers was observed during follow-up. CTC analysis using the investigated qPCR marker panel serves as tool for achieving high detection rates of PrC patient samples even in localized disease. RNA ISH offers the advantage of confirming these markers at the single cell level. Employing the clinically relevant marker PSMA, our CTC approach can be used for diagnostic purposes to screen patients profiting from PSMA-directed PET-CT or PSMA-targeted therapy. [ABSTRACT FROM AUTHOR]

The ABC1K gene family (activity of BC1 complex kinase), found in bacteria, archaea, and eukaryotes, plays a vital role in regulating cellular metabolism. Despite its significance, the evolutionary history and functional diversity of the ABC1K gene family little in Brassica crops, are not well understood. Chinese cabbage, a crop of Brassica genus extensively cultivated and consumed in East Asia, has a complex evolutionary history that includes polyploidization and whole-genome duplication. Through comprehensive analyses of gene structures, phylogenetic relationships, chromosomal locations, microsynteny, gene duplication, and gene expression, the ABC1K gene family in Chinese cabbage was characterized. We identified 25 members of the Chinese cabbage ABC1K gene family, all of which harbor the AarF domain. Notably, we further reported the occurrence of gene fusion, resulting in the formation of the BraraABC1K15-2 gene in Brassica. Additionally, we observed that the ABC1K genes displayed highly tissue-specific patterns, with significantly higher levels observed in flowers and leaves compared to other tissues. Remarkably, genes BraraABC1K4 and BraraABC1K14 were found for the first time to participate in plant photosynthesis. The findings presented here will help to understand the functions of ABC1K genes in plant development, growth, and stress response. [ABSTRACT FROM AUTHOR]

Duck adeno-associated Virus (DAAV) is a novel pathogen that was recently discovered in ducks. To establish a molecular detection assay for DAAV for further epidemiological investigation and pathogenic mechanism. Here, we designed specific primers and probes according to the sequence characteristics of the newly discovered DAAV and then established a TaqMan real-time PCR method (TaqMan-qPCR) for the detection of DAAV. Our data showed that the established TaqMan-qPCR for detecting DAAV had high sensitivity, with the lowest detection limit of 29.1 copies/μL. No cross reaction was found with duck circovirus (DuCV), H9N2 subtype avian influenza virus (AIV), avian Tembusu virus (ATmV). duck hepatitis A virus 1 and 3 (DHAV-1 and DHAV-3), duck adenovirus A (DAdV-A), duck adenovirus 3 (DAdV-3), or duck enteritis virus (DEV). The repeatability was excellent, with the coefficients of variation of repeated intragroup and intergroup tests ranging from 0.12–0.21% and 0.62–1.42%, respectively. Seventy-eight clinical samples collected from diseased or deceased ducklings were tested. The results showed that the DAAV positive rate was 21.79%, and a triple infection (DAAV+MDPV+GPV) was found. These data provide technical support for further molecular epidemiological surveillance and pathogenic mechanism studies of DAAV infection. [ABSTRACT FROM AUTHOR]

Biochemical methane potential (BMP) test is an important tool to evaluate the methane production biodegradability and toxicity of different wastes or wastewaters. This is a key parameter for assessing design and feasibility issues in the full-scale implementation of anaerobic digestion processes. A standardized and storable inoculum is the key to obtain reproducible results. In Uruguay, a local enterprise dedicated to design and install anaerobic digesters operated a lab-scale bioreactor as a source of biomass for BMP tests, using a protocol previously described. This reactor was controlled and fed with a mixture of varied organic compounds (lipids, cellulolytic wastes, proteins). Biomass was reintroduced into the reactor after BMP assays to maintain a constant volume and biomass concentration. The aim of this work was to evaluate how the microbial community evolved during this operation and the effect of storing biomass in the refrigerator. The composition of the microbial communities was analyzed by 16S rRNA amplicon sequencing using primers for Bacteria and Archaea. The methanogenic activity was determined, and the methanogens were quantified by mcrA qPCR. One sample was stored for a 5-month period in the refrigerator (4 °C); the activity and the microbial community composition were analyzed before and after storage. Results showed that applying the reported methodology, a reliable methanogenic sludge with an acceptable SMA was obtained even though the reactor suffered biomass alterations along the evaluated period. Refrigerating the acclimatized biomass for 5 months did not affect its activity nor its microbial composition according to the 16S rRNA gene sequence analysis, even though changes in the mcrA abundance were observed. Key points: • The applied methodology was successful to obtain biomass suitable to perform BMP assays. • The microbial community was resilient to external biomass addition. • Biomass storage at 4 °C for 5 months did not alter the methanogenic activity. [ABSTRACT FROM AUTHOR]

Efficient early pathogen detection, before symptom apparition, is crucial for optimizing disease management. In barley, the fungal pathogen Pyrenophora teres is the causative agent of net blotch disease, which exists in two forms: P. teres f. sp. teres (Ptt), causing net-form of net blotch (NTNB), and P. teres f. sp. maculata (Ptm), responsible for spot-form of net blotch (STNB). In this study, we developed primers and a TaqMan probe to detect both Ptt and Ptm. A comprehensive k-mer based analysis was performed across a collection of P. teres genomes to identify the conserved regions that had potential as universal genetic markers. These regions were then analyzed for their prevalence and copy number across diverse Moroccan P. teres strains, using both a k-mer analysis for sequence identification and a phylogenetic assessment to establish genetic relatedness. The designed primer-probe set was successfully validated through qPCR, and early disease detection, prior to symptom development, was achieved using ddPCR. The k-mer analysis performed across the available P. teres genomes suggests the potential for these sequences to serve as universal markers for P. teres, transcending environmental variations. [ABSTRACT FROM AUTHOR]

Background: Polycystic ovary syndrome (PCOS) is highly prevalent in women of reproductive age worldwide, exhibits highly heterogeneous clinical presentation and biochemical parameters, and has no cure. This study aimed to investigate the role of miR-34a-5p in PCOS, its effect on glycolysis in granulosa cells (GCs), and its potential contribution to follicular dysregulation. Methods: Herein, Follicular follicular fluid (FF) samples were collected from six patients with PCOS and six healthy controls undergoing in vitro fertilization-embryo transfer. The isolated extracellular vesicles were characterized by transmission electron microscopy, nanoparticle tracking analysis, and western blotting. Additionally, miRNA sequencing was performed to identify differentially expressed microRNAs, and their functions were analyzed through transcriptomics. The In vitro effects of miR-34a-5p on glycolysis, cell proliferation, and apoptosis were assessed in human ovarian granulosa tumour cell line (KGN cells). Targets of miR-34a-5p were identified by dual-luciferase reporter assays, and quantitative real-time polymerase chain reaction and western blotting were performed to determine gene and protein expression. Results: The level of miR-34a-5p in FF-derived extracellular vesicles derived from patients with PCOS was significantly higher than that of the control group. Transcriptomic analysis highlighted miR-34a-5p as a key regulator of glycolysis and apoptosis. Furthermore, in vitro analysis showed that miR-34a-5p targeted lactate dehydrogenase A (LDHA), inhibited glycolysis, reduced energy supply to GCs, and promoted apoptosis of KGN cells. Conversely, miR-34a-5p inhibition restored glycolysis function and cell viability. Conclusion: The findings of this study show that miR-34a-5p mediates GC apoptosis in PCOS by targeting LDHA and inhibiting glycolysis, suggesting its crucial role in PCOS pathophysiology, and offering potential therapeutic targets for improving follicular development and fertility outcomes in patients with PCOS. Further research is needed to explore the clinical implications of miR-34a-5p and its use as a biomarker for early diagnosis and prognosis of PCOS. [ABSTRACT FROM AUTHOR]

This study aimed to identify laboratory factors predicting leprosy relapse (LR) after multi‐drug therapy (MDT). A case–control study included 80 patients treated with MDT at a national reference center over 12 years. The Relapse Group had 40 patients who relapsed after an average of 89.2 months post‐MDT, while the Control Group had 40 patients who remained asymptomatic for an average of 113.1 months. Significant predictors of LR included neural/perineural lymphocytic infiltrate (OR = 4.67; p = 0.0076) and foamy granulomas (OR = 15.55; p = 0.0005), increasing odds by 4.7 and 15.6 times, respectively. The Relapse Group had a mean histological bacillary index (hBI) of 3.23+ compared to 1.8 in the Control Group (p = 0.004). An hBI ≥3+ had 72% sensitivity and 65% specificity for detecting LR (AUC = 0.72; p = 0.0002). Elevated anti‐phenolic glycolipid I (anti‐PGL‐I) IgM antibody levels (ELISA index, EI ≥1) were also associated with LR (OR = 4.67; p = 0.0031). An EI ≥3.6 had 71% sensitivity and 62% specificity (AUC = 0.70; p = 0.0012). Multivariate analysis indicated that neural/perineural infiltrate, foamy granulomas, hBI ≥ 1+, and EI ≥ 1 significantly predicted LR, with up to 94.32% probability. Conclusively, these factors can identify individuals at high probability of LR after MDT. [ABSTRACT FROM AUTHOR]

Salinity stress is a major concern in regions where irrigation relies on saline water. This study aimed to investigate the relative water content (RWC), electrolytic leakage (EL), total chlorophyll content, free amino acid content, and total soluble sugar content were analyzed in different groundnut species subjected to various salinity treatments. The results showed that salinity stress significantly reduced the RWC in groundnut leaves, with A. duranensis (wild type) exhibiting higher RWC values compared to the Arachis hypogaea species. RNA sequencing was performed to identify differentially expressed genes (DEGs) during salt stress. A total of 9079 DEGs were identified, with 1372 genes upregulated and 2509 genes downregulated. Genes belonging to transcription factor families, such as WRKY, MYB, bHLH, E2F, and Auxin efflux carrier proteins, were induced under salt stress in the tolerant genotype. Conversely, genes encoding NADH dehydrogenase, glutathione S-transferase, protein kinases, UDP-glycosyltransferase, and peroxidase were downregulated. Gene ontology and pathway analyses revealed several enriched categories and metabolic pathways associated with salt stress response, including catalytic activity, response to salt stress, ATP-dependent activity, and oxidative phosphorylation. The findings of this study provide insights into the physiological and molecular responses of groundnut to salinity stress. A. duranensis exhibited better salinity tolerance than Arachis hypogaea , as indicated by higher RWC values, lower electrolytic leakage, and differential gene expression patterns. These results contribute to our understanding of the mechanisms underlying salt stress tolerance in groundnut and may guide future efforts to develop salinity-tolerant groundnut species, ultimately improving crop yield in saline-affected regions. • Salinity stress decreases crop yield by affecting water content and leaf damage in groundnut plants. • A. duranensis variety shows higher tolerance to salinity stress compared to GG20. • Salinity stress reduces chlorophyll levels in groundnut leaves, with A. duranensis being more resilient. • Salinity stress triggers the accumulation of free amino acids and soluble sugars, especially in A. duranensis. • RNA-seq analysis reveals key genes involved in groundnut's response to salt stress, providing valuable insights. [ABSTRACT FROM AUTHOR]

Background/Objectives: Genetic newborn screening (NBS) has already entered the phase of common practice in many countries. In Germany, spinal muscular atrophy (SMA), severe combined immunodeficiency (SCID) and sickle cell disease (SCD) are currently a mandatory part of NBS. Here, we describe the experience of six years of genetic NBS including the prevalence of those three diseases in Germany. Methods: Samples and nucleic acids were extracted from dried blood spot cards, commonly used for NBS. A qPCR assay was used to detect disease-causing variants for SMA and SCD, and the detection of T-cell receptor excision circles (TRECs) was performed for SCID screening. Results: The results of the NBS of over 1 million newborns for SMA, approximately 770,000 for SCID and over 410,000 for SCD are discussed in detail. In these newborns, we have identified 121 cases of SMA, 15 cases of SCID and syndrome-based immunodeficiencies and 77 cases of SCD or β-thalassemia. Conclusions: The flexibility of multiplex qPCR is assessed as an effective tool for incorporating different molecular genetic markers for screening. The processing of dried blood spot (DBS) filter cards for molecular genetic assays and the assays are described in detail; turn-around times and cost estimations are included to give an insight into the processes and discuss further options for optimization. The identified cases are in the range expected for the total number of screened newborns, but present a more exact view on the actual prevalences for Germany. [ABSTRACT FROM AUTHOR]

Compared to single damming, the impact of cascade damming on nitrogen-related microorganisms in river ecosystems exhibits greater complexity. However, there is still a lack of research on the response of denitrifiers to the construction of cascade reservoirs. A study was conducted on 10 cascade reservoirs in the upper reaches of the Yellow River to investigate the impact of cascade reservoir construction on nirS-type denitrifying bacteria in sediments. Sediment samples were collected in May (dry season) and August (wet season) of 2023. The spatiotemporal characteristics of the nirS-type denitrifying bacterial community and gene abundance were analyzed using Illumina high-throughput sequencing and real-time fluorescence quantification PCR (qPCR). Redundancy analysis (RDA) and variation partitioning (VP) were utilized to assess the impact of environmental factors on these communities. The results showed the following: (1) Proteobacteria was the predominant phylum of nirS-type denitrifying bacteria in cascade reservoir sediments. At the genus level, unclassified Proteobacteria (69.51–95.64%) showed the highest relative abundance, followed by Paracoccus, Rhodanobacter, and Pseudomonas, indicating that unclassified Proteobacteria may dominate denitrification in these reservoir sediments. (2) The α and β diversity indices of nirS-type denitrifying bacteria were higher in the dry season than in the wet season, and also higher in young reservoirs compared to old reservoirs (p < 0.05). (3) Temporally, the abundance of the nirS gene was significantly higher in the wet season (12.71 × 107 copies/g dry sediment) compared to the dry season (66.35 × 105 copies/g dry sediment). Spatially, the abundance of the nirS gene was higher in the central region, while relatively lower at both ends. (4) RDA and VP analysis indicated that the community structure and abundance of nirS-type denitrifying bacteria were significantly influenced by the total nitrogen in sediments (19.31%) and water temperature (14.13%). Spearman correlation analysis showed that organic carbon significantly affected the diversity of nirS-type denitrifying bacteria (p < 0.05). The results contribute to a better understanding of the nitrogen-related microbial community in cascade reservoir sediments of the Yellow River, providing a scientific basis for reservoir management. [ABSTRACT FROM AUTHOR]

Lactopontin (LPN) is a highly phosphorylated O-glycosylated acidic protein closely associated with infant gut, brain, and immune development, and its recognition is urgent due to its rising application in fortified dairy products and infant formula. In this study, an ssDNA aptamer against LPN was obtained, among which two kinds of matrix-background-assisted systematic evolution of ligands via exponential enrichment (SELEX) approaches were performed and compared. The direct approach was to utilize the sample matrix as the mixing-incubation background between the ssDNA library and LPN that can theoretically increase screening pressure and simulate practical application scenarios. The indirect approach was to utilize a PBS buffer as a screening background and to include counter-screening steps that adopt the "sample matrix" as a whole as the counter-screening target. Their screening evolutions were monitored through qPCR assays from sequence diversity convergences of each sub-library based on the change in the proportion of hetero- and homo-duplexes from the dissociation curve and melting temperature, which were also verified from the sequence statistics of high-throughput sequencing. The common sequence of Seq.I1II3 from the two approaches was finally fished out as the aptamer through multiple analyses of combining the sequence frequency, secondary structures, homology, and binding assessments, which was demonstrated good specificity and low-nanomolar affinity by qPCR assay (KD, 5.9 nM). In addition, molecular docking and a dynamics simulation were performed for their binding site prediction and affinity confirmation. This study provides a potential identifying element and a basis for accelerating the development of methods for LPN detection in dairy products. [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]

• Developmental validation of novel qPCR multiplex for canine DNA quantification. • Canine DNA successfully recovered and quantified from attacked livestock. • Methods reveal limitations of current commercial canine STR kit. • Clear steps for improvements in canine forensic DNA pipeline identified and discussed. The absence of a standardised method to quantify canine DNA recovered from livestock attacks leaves forensic providers without an important quality control step to help support their decision making. Typically used to normalise the amount of DNA for STR amplification, modern forensic DNA quantification approaches use qPCR of target genes and can also include an Internal Positive Controls (IPC) to determine the presence of PCR inhibitors. The co-amplification of livestock DNA alongside canine DNA has meant that previously developed qPCR methods are not suitable for use so a standardised approach is needed. This research describes the development of a Taq-man multiplex qPCR assay that simultaneously quantifies the autosomal MC1R and Y-specific SRY gene to determine the concentration of canine DNA recovered from attacked livestock. Data suggests that the method is robust and reproducible with no significant difference in the standard curves produced from multiple runs or from different DNA standards derived from different canines. Assay sensitivity of between 15 and 31 pg is consistent with other forensic quantification assays and also in line with the sensitivity of the two tested canine STR kits, Canine Genotype 2.1 Kit and CaDNAP Panels 1 and 2. The assay is highly specific to canines when tested against 163 different dogs representing 33 different breeds and no cross-amplification of non-target species' DNA was observed even from livestock DNA tested at 31.25 ng/µl. This strongly suggests that any DNA detected on evidence collected from attacked livestock is canine. The assay also shows robust tolerance to common livestock inhibitors continuing to amplify when inhibitor-spiked DNA samples were tested. Both mixed and inhibited DNA samples underwent STR typing using two canine forensic STR kits with data showing the Canine Genotype 2.1 Kit displaying pronounced cross-amplification of livestock DNA and and/or extensive PCR inhibition leading to the complete loss of amplification when using this kit. Conversely the CaDNAP Panels 1 and 2 showed little cross-amplification of livestock DNA and improved inhibitor tolerance suggesting that this approach was better suited for the analysis of livestock attack samples. Findings are discussed and the impact of the observations on future work in this area are explored. [ABSTRACT FROM AUTHOR]

Hyperlipidemia is the root cause of numerous chronic conditions, leading to high mortality rates around the globe. Spirulina (Arthrospira platensis) microalgae serve as a promising reservoir of bioactive compounds with diverse pharmacological properties. The current study examined the nutritional profile of spirulina powder in relation to strict glycemic control, specifically focusing on its potential to lower lipid levels. In an in vivo investigation, normal healthy male Wistar albino rats (n = 60) were divided into two groups: a negative control group (NC) of ten rats and a high-fat diet group (n = 50) that were fed a cholesterol-rich diet until their cholesterol levels reached or exceeded 250 mg/dL. Subsequently, the hypercholesterolemic rats were then randomly allocated to several treatment groups: a positive control (PC); a standard treatment diet (STD) involving fenofibrate at a dose of 20 mg/kg body weight; and three experimental groups (T1, T2, and T3) that received spirulina powder supplementation at doses of 300, 600, and 900 mg per kg body weight, respectively, for the period of 12 weeks. Blood samples were analyzed for oxidative stress biomarkers, insulin levels, lipid profiles, liver function, and expression of gene levels in the diabetogenic pathway. The study utilized spectrophotometric colorimetric methods to identify oxidative stress biomarkers, serum kit methods to measure lipid profiles and liver enzymes, and the assessment of qPCR for mRNA quantity. According to the research findings, spirulina powder has certain noteworthy features. It had the greatest quantity of chlorogenic acid (4052.90 µg/g) among seven phenolics and two flavonoid compounds obtained by HPLC-UV analysis. Furthermore, the proximate analysis demonstrated that spirulina is high in protein (16.45 ± 0.8%) and has a significant energy yield of 269.51 K-calories per 100 g. A maximal spirulina dose of 900 mg/kg/wt significantly lowered oxidative stress, cholesterol, triglyceride, low-density lipoproteins (LDL), and insulin levels (p ≤ 0.05). In contrast, high-density lipoprotein (HDL) and total antioxidant capacity (TAC) levels increased significantly (p ≤ 0.05) compared to all other groups, except the NC group. The study provides remarkable proof about the pharmacological impact of spirulina powders. Significant reductions (p ≤ 0.05) in liver enzymes {alanine aminotransferase (ALT) and aspartate aminotransferase (AST)} were observed across all treatment groups, with the exception of the NC, compared to the positive control. The treatment groups had significantly greater gene expression levels of INS-1, PDX-1, IGF-1, and GLUT-2 than the positive control group (p ≤ 0.05). These findings highlight spirulina's potential as a long-term regulator of hyperglycemia in rat models with induced hyperlipidemia, owing to its phenolic bioactive components that serve as antioxidants. [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]

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]

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]

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]

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]

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]

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]

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: 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: 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: 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]

In January 2022, six gibbons (Nomascus spp.) in the same enclosure of a zoo successively showed symptoms of runny nose and cough. We separately collected six nasal swabs from the six gibbons, detected the pathogens in the samples by high-throughput sequencing technology. The results showed that two samples were positive for AIV. The sequence assembly was similar to H9N2 avian influenza virus. The two positive samples were tested by qPCR for H1 - H16 genes and N1 - N9 genes and the results showed that Ct values of H9 and N2 in sample 5 were 31. 35 and 36. 16 respectively, Ct values of H9 and N2 in sample 6 were 27. 46 and 29. 57. The gibbons were given comprehensive treatment, such as expectoration and cough relief, anti-inflammatory and secondary infection control, and disinfection of the feeding environment was strengthened. The sick gibbons recovered quickly. [ABSTRACT FROM AUTHOR]

Feline primary hypertrophic cardiomyopathy (HCM) is an intrinsic myocardial disease characterized by concentric hypertrophy of the left ventricle. In the present study, we investigated the microRNA‐mRNA regulatory network in feline myocardial tissue affected by primary (HCMI) and secondary HCM (HCMII). MRNA expression levels of sarcomeric genes, including, TNNT2, TNNI3, MYH7, MYBPC3, TPM1 and ACTC1 were assessed in the FFPE myocardial tissues. FFPE tissues from healthy cats were sequenced by the NGS, to explore, in the entire non‐deposited miRNome, the expression level of microRNAs targeting the complementary sequences of selected sarcomeric mRNAs. The sarcomeric genes TNNT2, MYH7, MYBPC3 and TPM1 showed a statistically significant upregulation in HCMI compared to HCMII (p <.01), except ACTC1 which was downregulated (p <.01); TNNI3 showed no statistically significant difference. In HCMII miR‐122‐5p, miR‐338‐3p, miR‐484, miR‐370‐3p, miR‐92b‐3p, miR‐375 and miR‐370‐3p showed a significant upregulation (p <.01) compared to control. The exception was miR‐30a‐5p which showed downregulation. Worthy of note is the 4‐fold higher expression of miR‐370‐3p, a key regulator of MYBPC3, in HMCI compared to HMCII. This research does not solve the aetiological mystery of HCM, but it may help to find a way to help diagnose and define the prognosis of HCM in cats. [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]

Using food ingredients and/or processed food products contaminated with pork, whether unintentionally or intentionally, has become a growing concern and issue. This condition encourages the development of an accurate method for specifically detecting the presence or absence of pork contamination. The research was carried out on two different samples: (1) fresh pork to provide an in-house positive control and (2) samples of pork-based processed meat products (floss, meatballs, corned beef, and sausages), tested using DNA markers. The use of samples originating from processed pork food is to determine the effect of the processing process on DNA fragments and the robustness of the extraction method for the detection process used. The research aimed to detect pork DNA fragments using the quantitative polymerase chain reaction (qPCR) method. The study stages were extracting fresh pork and processed products using an RNA extraction kit, DNA extraction kit, and salt extraction, as well as measuring the purity and concentration of DNA/RNA using a spectrophotometer. The RNA extract was converted into complementary DNA (cDNA), and the DNA extract was analyzed using qPCR with specific primers for pork DNA (Sus scrofa). The results showed that the concentration of RNA and DNA extracts was 71.1–296.025 ng/uL and of various purity. All processed meat product samples and in-house positive controls were amplified in the Ct range of 23–28 ng/uL. In this case, the meat processing had no effect on the DNA of the processed meat products analyzed, so DNA fragments could be detected. DNA qPCR was more time efficient than cDNA qPCR because it did not require an RNA reverse transcription step. [ABSTRACT FROM AUTHOR]

[Display omitted] • Toxoplasma gondii DNA was detected in bivalves from the Kerguelen and Galapagos archipelagos. • Its occurrence differed between sampling sites in Kerguelen but not in Galapagos. • In both archipelagos, quantitative PCR-positive bivalves were found on islands without cats. • Our finding demonstrated the waterborne transport of oocysts over at least 20 km. • Exposure to currents and giant kelp density affect T. gondii detection in bivalves. Oocysts of the protozoan Toxoplasma gondii are found in felid feces and can be washed into coastal waters, where they persist for months, attaching to algae and accumulating in invertebrates. We used wild bivalves to assess contamination of coastal waters of the Kerguelen and Galapagos archipelagos by this zoonotic parasite. Additionally, we leveraged the contrasting situations of these archipelagos to identify some potential drivers of contamination. In the Galapagos, with a cat density reaching 142 per km2, 15.38% of the sampled oysters (Saccostrea palmula) tested positive for T. gondii by quantitative real-time PCR (qPCR) (n = 260), and positive samples were found in all eight sampling sites. In Kerguelen, with 1-3 cats per km2, 40.83% of 120 tested mussels (Mytilus edulis platensis) were positive, and positive samples were found in four out of the five sampling sites. These findings provide evidence of T. gondii contamination in the coastal waters of these archipelagos. Furthermore, T. gondii -positive bivalves were found on islands located 20 km away (Galapagos) and 5 km away (Kerguelen) from the nearest cat population, indicating that T. gondii oocysts can disperse through waterborne mechanisms over several kilometers from their initial deposition site. In the Galapagos, where runoff is infrequent and all sites are exposed to currents, the prevalence of qPCR-positive bivalves did not show significant variations between sites (p = 0.107). In Kerguelen where runoff is frequent and site exposure variable, the prevalence varied significantly (p < 0.001). The detection of T. gondii in Kerguelen mussels was significantly correlated with the site exposure to currents (odds ratio (OR) 60.2, p < 0.001) and the on-site density of giant kelp forests (OR 2.624, p < 0.001). This suggests that bivalves can be contaminated not only by oocysts transported by currents but also by consuming marine aggregates containing oocysts that tend to form in kelp forests. [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]