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Virtual reality (VR) is increasingly being used as a teaching and learning tool, however scaling this technology is difficult due to technological and cost considerations. An alternative approach that helps to address these problems is VR-by-proxy, where teaching takes place within a VR environment that is controlled by one lecturer and broadcast to students online. This allows the content to be accessed without specialist equipment while still offering an immersive and interactive experience. Taking advantage of the enforced move to online learning during the COVID-19 pandemic, this study evaluates the implementation of a novel VR-by-proxy disease diagnostic laboratory VR simulation within an undergraduate life sciences course in a higher education setting. Student participants were randomly allocated into two groups: the test group, who took part in a VR-by-proxy lesson; and a control group, who worked with interactive online lab manual material. We assessed improvement in learning and enjoyment through questionnaires before and after these tasks and collected qualitative data on student attitudes towards VR through focus groups. Our results indicate that although there is no observable difference in learning outcomes between the two groups, students in the test group reported an improved learning experience, confidence and enjoyment of learning. In our focus groups, confidence was understood in two ways by participants: firstly, as ‘understanding’ of the various steps involved in conducting a quantitative polymerase chain reaction experiment and secondly as a more general ‘familiarity’ with the laboratory setting. This study adds to the growing body of research into the effectiveness of VR for learning and teaching, highlighting that VR-by-proxy may provide many of the same benefits. [ABSTRACT FROM AUTHOR]

Neurogenesis is an active process of creating new neurons in the neurogenic zone. It is influenced by many factors, including the circadian system, which is synchronized by light. Neurogenesis in laboratory rodents peaks at night, and the rodents are nocturnal, contrary to humans that are active during the day. Here, we studied whether proliferation and apoptosis exhibit a daily rhythm in the brain of the diurnal songbird zebra finch (Taeniopygia guttata) and whether the cell proliferation peaks during the dark phase of the day, as in rodents. We injected the birds with the cell proliferation marker 5-ethynyl-2´-deoxyuridine (EdU; thymidine analog), quantified the number of dividing cells in the neurogenic ventricular zone (VZ), and measured mRNA expression of clock genes as well as genes indicating cell proliferation or apoptosis. First, we confirmed the daily rhythms of the clock genes. Next we found that proliferation along the whole VZ did not exhibit a daily rhythm. However, proliferation in the central ventral part of the VZ, i.e. "the hot-spot" area, showed a daily rhythm of proliferation. The highest number of newborn cells was detected in the dark phase of the day. The relative expression of the apoptotic genes caspase 3, Bcl-2, and Bax as well as the proliferating cell nuclear antigen (PCNA) did not show any rhythm. In summary, our results show that cell proliferation in the "hot-spot" region of the VZ in diurnal songbirds shows rhythmic activity over a period of 24 h and that the maximum cell proliferation occurs in the passive phase. This study may have implications for understanding the mechanisms underlying the daily regulation of brain cell proliferation in different species. [ABSTRACT FROM AUTHOR]

Background: Endometrial cancer is one of the most common malignancies and alternative successful biomarkers are needed in the diagnostic process. Objectives: This study aimed to investigate candidate genes' diagnostic potential in endometrial neoplasia. Material and Methods: The expression levels of USP28, JAG2, AURKA, PGK1, HRPT1, EZH2, YAP, and P53 genes were evaluated by qPCR and/or immunohistochemistry analyses on endometrioid-type adenocarcinomas including all three grades, as well as from non-atypical hyperplasia, and atypical hyperplasia. Results: We determined significant (p < 0.05) increases in USP28, PGK1, EZH2, JAG2, AURKA, and YAP mRNA expressions in endometrial cancer tissues. Significant differences in the expressions of USP28 and P53 genes were determined between tumor grade groups (p = 0.002, and p = 0.005, respectively). Immunohistochemically, significant differences were found between the study groups (p < 0.001) and tumor grades (p = 0.013) by the evaluation of USP28. Statistically significant differences were found between all study groups (p < 0.001) and tumor grades (p = 0.008) in terms of PGK1 immunohistochemical expressions. A positive correlation (p = 0.002, r = 0.356) was found between p53 and PGK1. Conclusions: Considering our qPCR and immunohistochemistry results together, it was concluded that USP28, PGK1, EZH2, JAG2, AURKA, HPRT1and YAP expressions could offer beneficial expression values in precancerous lesions and endometrial cancer. [ABSTRACT FROM AUTHOR]

This study aimed to optimize viral cleaning and indexing methods in sweet potato genotypes with potential for ethanol production. Two viral cleaning methods (apical meristem cultivation and thermotherapy) and three different PCR variations (conventional PCR, nested PCR, and real-time PCR) were evaluated to detect two sweet potato viruses, Sweet potato mosaic virus (SPMV) and Sweet potato symptomless mastrevirus 1 (SPSMV-1), in four sweet potato genotypes (Amanda, Beatriz, Duda and 520). The results revealed that all sweet potato genotypes were contaminated with SPMV and SPSMV-1 viruses. qPCR was the most accurate and reliable diagnostic technique in asymptomatic and symptomatic plants with a detection rate of 100% for plants infected with SPMV compared to 56.25% for PCR and 75% for nested PCR added PCR. However, for SP SMV-1, nested PCR increased detection sensitivity from 62.5% (PCR) to 93.75%, compared to 87.5% for qPCR. Virus-free materials were generated for all genotypes through meristem cultivation and thermotherapy. The cultivation of apical meristems was the most efficient treatment for viral cleaning, displaying an efficiency of 62.5%. On the other hand, the heat treatment, although less efficient (37.5%), was also capable of generating virus-free plants, with the advantage of being a faster alternative for viral cleaning. [ABSTRACT FROM AUTHOR]

Introduction: Pancreatic ductal adenocarcinoma (PDAC) is associated with poor prognosis. The roles of the transcription factor special AT-rich binding protein-2 (SATB2) and β-catenin in PDAC have been a subject of controversy. We aimed to assess the diagnostic and prognostic impact of SATB2 and β-catenin in PDAC. Methods: We analyzed 44 paraffin-embedded tissue blocks along with corresponding blood and pancreatic tissues. We evaluated SATB2 expression using immunohistochemistry (IHC) and enzyme-linked immunosorbent assay (ELISA). β-catenin was assessed using IHC and real-time polymerase chain reaction (qPCR). Results: High SATB2 expression and low β-catenin expression were associated with a poor prognosis in PDAC, including advanced pathological tumor stage (pT-stage), pathological lymph node stage (pN-stage), and TNM stage. We found a positive correlation between SATB2 expression assessed by IHC and the concentration of SATB2 in both serum and tissue samples measured by ELISA. We observed a positive correlation between β-catenin expression assessed by IHC and β-catenin levels measured by qPCR. Conclusions: SATB2 and β-catenin could provide valuable insights into the development of pancreatic cancer, and targeting them may be beneficial for the prevention and treatment of PDAC. The levels of SATB2 in serum show promise for the diagnosis and tumor invasion of pancreatic cancer. [ABSTRACT FROM AUTHOR]

Light sensitivity is important for marine benthic invertebrates, and it plays a vital role in the marine bivalves settling. Animal visual systems are enormously diverse; their development appears to be controlled by a set of conserved retinal determination genes (RDGs). Eyespots, as the simplest animal eyes, their appearance indicates the important effect on mussel larvae attachment. Nevertheless, the molecular mechanism of the eyespot’s development in Mytilus coruscus larvae is not clear. In this study, we identified 11 genes which play a regulatory role in the visual system (i.e. Pax1/9, Pax2/5/8, Pax6, Pax3/7, Six1/2, Six3/6, Six4/5, Dach, Eya, Brn and Tbx2) from transcriptome data and the whole genome sequence of M. coruscus. The results of chromosome localization showed that 11 genes were distributed on different chromosomes. Subcellular mapping revealed that all the proteins except Brn were located in the nucleus. Phylogeny and gene structure analyses revealed that the Pax members were divided into four subfamilies, the Six members were divided into three subfamilies and structures within the same subfamily were relatively conserved. Quantitative real-time PCR (qPCR) showed that Dach, Pax6, Pax3/7, Six1/2 and Six4/5 were expressed at high levels during the pediveliger stage. Moreover, Six1/2 and Six4/5 were highly expressed in mantle tissues. Subsequent overall in situ hybridization experiments in the planktonic larval stage revealed that Pax6, Six1/2 and Six4/5 detected signals in the region of the eyespot. Based on these analyses, we suggested that the development of vision in M. coruscus not only depended on the expression pattern of Pax6, but perhaps also related to Six1/2 and Six4/5 in the planktonic larval stage, while Six1/2 and Six4/5 were the dominant genes for visual function in the adult mussel. This study made a comprehensive analysis of the visual function of M. coruscus at the genome level, which helps us to understand the intrinsic mechanism of the visual system of marine bivalves, and also provides a molecular basis for improving the attachment and metamorphosis rate of M. coruscus larvae. [ABSTRACT FROM AUTHOR]

In Poland, red deer, roe deer and elk cause great damage to pine stands, and pine is the predominant forest-forming tree species in Polish forests. Pine seedlings planted adjacent to agricultural fields face heightened risk of damage from nocturnal foraging by game animals, which retreat to forested areas during the day. This damage creates openings for fungal entry, particularly by concerning plant pathogens, such as Fusarium species, which are prevalent in agricultural settings. This study aimed to analyze the mycobiome of pine seedlings damaged by forest animals, focusing on the presence of concerning pathogens in infested pine seedlings collected from ten forest plantations at various distances from rural areas. Samples of pine seedlings bitten by forest animals were taken from 10 forest plantations at different distances from rural areas. Selected diversity indices were determined to compare the fungal populations. Mycological analysis of the gnawed pine shoots using culture and qPCR methods revealed 746 fungal colonies. The most frequently isolated species were: Fusarium avenaceum, Alternaria alternata, Botrytis sp., Cladosporium cladosporioides, representatives of the genus Penicillium, Rhizopus nigricans and Fusarium oxysporum, which were found in samples from all study plots and are thus ubiquitous. Species that are typical pathogens of agricultural crops, such as Fusarium avenaceum, F. culmorum, F. poae, F. sporotrichioides and F. tricinctum, were also isolated from damaged seedling tissue in forest plantations neighbouring agricultural fields. Spearman's rank correlation analysis showed that the importance of secondary pathogens, represented by species of the genus Fusarium, decreased with increasing distance from agricultural fields, while the importance of primary pathogens increased. Younger pine seedlings exhibited lower species diversity and a higher prevalence of specific secondary pathogen species, coupled with heightened susceptibility to infection compared to older seedlings when subjected to tissue damage by forest animals. These findings underscore not only variations among plantations in proximity to agricultural fields but also suggest potential inter-land spread of fungi, emphasizing the need for integrated management strategies across agricultural and forestry domains. [ABSTRACT FROM AUTHOR]

Highlights: Canine parvovirus disease (CPVD) causes lymphopenia/immunosuppression, possibly through apoptosis of leukocytes possibly mediated by galectin-1 and Mincle. Blood samples were taken from confirmed CPV-positive and non-CPV dogs and used for (para)clinicoheamatological evaluation. Optimized qPCR was performed to analyze mRNA levels of caspases 3/7 and 9, galectin-1 and Mincle in blood leukocytes. Results revealed remarkable lymphopenia and changes in mRNA levels of Mincle and galectin-1, but of caspases 3/7 and 9. The lymphopenia and immunosuppression observed in CPVD is not due to lymphocyte apoptosis, and changes in Mincle and galectin-1 functions partially limit parvovirus pathogenecity.Canine parvovirus disease (CPVD) is one of the most common causes of viral diarrhea in dogs. The disease has a mortality rate of up to 90% if left untreated, and can cause gastroenteritis, vomiting, mucoid/hemorrhagic diarrhea, lymphopenia and even immunosuppression. Based on the effects of canine parvovirus type 2 (CPV-2) on the immune system, we investigated the effects of the CPV-2 on hematological indices and the expression of certain immune molecules in blood leukocytes of CPV-positive and non-CPV dogs. The dogs were (para)clinically evaluated, and their disease status was confirmed by antigen rapid detection kits and polymerase chain reaction (PCR). To elucidate the nature of the immunosuppression seen in CPVD, we investigated the expression of caspases 3/7 and 9, and some lectin family molecules such as galectin-1 (important in viral adhesion) and Mincle (macrophage‑inducible C‑type lectin receptor), in blood leukocytes using reverse transcription quantitative PCR (RT-qPCR). We observed remarkable lymphopenia, lower Hb concentration and higher red blood cell distribution width (RDW) value in CPV-positive dogs. No significant changes in expression of caspases 3/7 and 9 were detected, but galectin-1 and Mincle showed remarkable down and up-regulation, respectively. This proves that the immunosuppression seen in most CPVD is not caused by lymphocyte apoptosis in blood, and that Mincle is partially involved in the immune response to CPV-2. The observed changes in galectin-1 and Mincle may be a defense mechanism against parvovirus by potentially preventing the parvovirus from adhering to the cells. Further research is, nonetheless, needed to elucidate the possible roles of these molecules in CPV-2 pathogenesis and the immune system’s response to parvovirus. [ABSTRACT FROM AUTHOR]

Introduction: Ensuring accuracy in the diagnosis of leishmaniasis is crucial due to the myriad of potential differential diagnoses. Given the inherent limitations of serological techniques, real-time polymerase chain reaction (qPCR) emerges as a superior alternative. Furthermore, parasitological methods, conventionally regarded as the gold standard owing to their high specificity, encounter challenges concerning sensitivity and invasiveness for patients. In this context, the present study aims to assess, via meta-analysis, the performance of qPCR in diagnosing visceral and cutaneous leishmaniasis. Method: This meta-analysis encompassed studies published between January 2011 and December 2022, sourced from six databases (PubMed, LILACS, Scopus, Scielo, EMBASE, and Web of Science), utilizing the keywords “qPCR,” “molecular diagnosis,” and “leishmaniasis.” Epidemiological studies focusing on the efficacy of qPCR for leishmaniasis diagnosis were included. Data such as study demographics, geographic locations, sampling techniques, and the number of positive qPCR results were aggregated and analyzed to derive overall positivity rates, sensitivity, and specificity values associated with qPCR. Heterogeneity analysis was conducted on the data to select appropriate models, and the collective efficacy data of qPCR were illustrated in forest plots. Results: Fifty-four studies met all inclusion criteria. The positivity rates for human visceral and cutaneous leishmaniasis were 27.07% (95% CI: 17.81-36.33%) and 60.40% (95% CI: 30.23-90.57%), respectively. In cases of visceral leishmaniasis in dogs, cats, and wild animals, the positivity rates were 26.55% (95% CI: 21.40-31.70%), 0.92% (95% CI: 0.09-1.75%), and 28.98% (95% CI: 21.86-35.10%), respectively. Analysis of the selected studies revealed high overall sensitivity and specificity values achieved with qPCR, at 91.08% (95% CI: 81.77-100.39%) and 98.08% (95% CI: 97.13-99.03%), respectively. Conclusion: This study indicates that qPCR is a highly sensitive and specific tool, adequately suitable for the diagnosis of human visceral and cutaneous leishmaniasis, as well as visceral leishmaniasis in animals. [ABSTRACT FROM AUTHOR]

In this study, we measured human epidermal growth factor receptor (EGFR) mutations in both tissue and circulating tumor DNA (ctDNA) by using beads, emulsions, amplifications and magnetic polymerase chain reaction (BEAMing PCR). Noninvasive mutation detection by assessing circulating tumor DNA (ctDNA) offers many advantages over tumor biopsy. One hundred non-small cell lung cancer (NSCLC) patients were enrolled, and both preoperative plasma samples and formalin-fixed and paraffin-embedded (FFPE) samples were collected for the study. The EGFR mutation status was determined by BEAMing PCR in ctDNA. Real-time quantitative PCR (qPCR) data were collected from our hospital database (EMR-qPCR, Electronic Medical Records) for comparative analysis. Additionally, qPCR was also performed on FFPE tissues using a Diatech EGFR qPCR kit. The concordance rates were 98.8%, 98.9% and 95.5% for exons 19, 20 and 21, respectively, when the BEAMing data were compared with the EMR-qPCR data. Additionally, when the BEAMing and Diatech qPCR data were compared, 90%, 100%, 96% and 98% of the genes were obtained for exons 19, 20, 21 (L858R) and 21 (L861Q), respectively. For both comparisons, Cohen's kappa agreement was significant. The advantage of BEAMing is its ability to identify mutated DNA sequences in cancer cells in the background of normal cell DNA contamination. This could be useful for disease monitoring and progression. [ABSTRACT FROM AUTHOR]

Background and objectives: Radical prostatectomy is a standard treatment for prostate cancer, yet about 30% of patients experience rising biochemical markers within a decade post-surgery. Pelvic lymph node sampling during prostatectomy assesses potential lymph node metastases, but standard histological assessments, which typically examine only 2–3 tissue sections, often miss occult metastases. This study assesses the effectiveness of qPCR in detecting PSA coding KLK3 mRNA for identifying lymph node metastases post-prostatectomy and explores the correlation between PSA-mRNA and biochemical recurrence. Methods: A cohort of 157 patients who underwent radical prostatectomy with lymphadenectomy were examine. On average, 24.7 lymph nodes were removed per patient. Among them, 108 patients reached PSA value below 0.1 ng/ml without receiving additional therapy, and 106 were followed up over a duration of 5.4 years. This subgroup is of particular interest because it allows for the investigation of the correlation between the occurrence of PSA-mRNA in lymph nodes and later biochemical recurrence. Key findings and limitations qPCR of PSA-mRNA identified 47 out of 108 positive cases (43.5%), while histopathological examination only detected 16 out of 108 cases (14.8%). From the followed-up subgroup 37 out of 106 patients (34.9%) experienced biochemical recurrence. It is noteworthy that qPCR yields more positive findings, regardless of the presence of biochemical recurrence. Conclusion and clinical implications: The study findings illustrate that qPCR consistently outperforms conventional histology in detecting lymph node metastases, regardless of biochemical recurrence. The hypothesis that qPCR is better at predicting later biochemical recurrence than conventional histology has not been confirmed. [ABSTRACT FROM AUTHOR]

Industrial workplaces, particularly those involved in ore processing or smelting, pose a high risk of exposure to cadmium, a highly toxic metal. This study isolated and identified eight cadmium-resistant strains from industrial wastewater for their ability to resist cadmium. Enterobacter roggenkampii FACU2 demonstrated exceptional cadmium removal capabilities during our analysis, successfully eliminating 62% of the cadmium. Additionally, transmission electron microscopy (TEM) was utilized to examine the morphological change between the most and least efficient strains that responded to cadmium stress at the cellular level. Compared to the control bacteria, the treated bacteria exhibited notably higher levels of cadmium adsorption and accumulation within their cells. A complete genome analysis revealed that E. roggenkampii FACU2 has one chromosome and one plasmid with a size of 4,856,454 bp and 80,926 bp, respectively, in addition to harboring numerous heavy metal-resistant genes related to cadmium and other heavy metals. Moreover, the gene expression of four cadmium-resistant genes (czcA, cadA, czcC and czcD) showed that the high cadmium concentration led to a significant increase in czcA and cadA mRNA levels, thus indicating the activation of cadmium-resistant genes in the E. roggenkampii FACU2 compared to Enterobacter sp. strain FACU. Due to its ability to remove cadmium and other heavy metals, this strain holds promise as a source of genes for biological treatment methods. This application could contribute to environmental purification, ultimately benefiting human health. [ABSTRACT FROM AUTHOR]

Purpose: Fungal rhinosinusitis is a significant and growing health concern in arid regions, with an increasing incidence over recent decades. Without timely and appropriate management, it can lead to severe complications, including potential intracranial spread. This study aims to establish efficient and rapid diagnostics for non-invasive fungal rhinosinusitis (FRS), addressing the challenge of its difficult-to-culture diagnosis. Methods: Twenty-eight patients suspected of FRS were studied using endoscopic sinus surgery to obtain tissue samples for histopathology, direct microscopy, fungal culture, quantitative PCR (qPCR) and metagenomic next-generation sequencing (mNGS) detection. A patented qPCR targeting prevalent Aspergillus species was evaluated. Results: The patient cohort had a male-to-female ratio of 9:14, with disease duration up to 50 years. Histopathologically, 23 out of 28 cases were positive. Fungal culture exhibited a sensitivity of 21.74%, with one false positive. qPCR and mNGS showed 100% sensitivity and specificity, with a 100% consistency rate for identification at the species level (23/23), and potential detection of cases with co-infections. The most common pathogen was A. flavus, followed by A. fumigatus and A. niger. Two cases involved mixed infections of A. fumigatus and A. flavus. Conclusion: qPCR and mNGS proved effective in rapidly identifying fungi from fresh sinus tissue that are challenging to culture, surpassing conventional methods. However, further evaluation and optimization with a larger cohort of patients are necessary. Histopathology is still recommended to confirm the clinical significance of the detected fungal species. [ABSTRACT FROM AUTHOR]

The extent of viral infection significantly shapes disease susceptibility. Yellow mosaic disease induced by the begomovirus pathogen mungbean yellow mosaic India virus (MYMIV), revealed varying infection levels in both compatible and incompatible interactions across three distinct Vigna species such black gram, green gram, and rice bean. Differential gene expression analysis focused on MYMIV coat protein (AV1) and replication protein (AC1) highlighted elevated AV1 expression in the susceptible green gram genotype SML1082 compared to the black gram genotype KUG253. Conversely, AC1 showed higher expression in black gram than green gram, illustrating complex infection mechanisms among compatible MYMIV-Vigna interactions. A novel infection pathway, termed "Lack of Efficient Assembly (LEA)," has been hypothesized in MYMIV-Vigna interactions. Additionally, a whitefly-mediated artificial transmission model for begomoviruses, named Transparent Airflow Stress-free Container (TASC), has been designed and demonstrated for the efficient transmission of MYMIV. This study enhances the understanding of begomovirus infection dynamics in diverse Vigna species, offering insights into disease management strategies. [ABSTRACT FROM AUTHOR]

We present a quantitative polymerase chain reaction (qPCR)-based eDNA method to detect the presence of the endangered Asian arowana, Scleropages formosus (Müller and Schlegel, 1840) (Teleostei: Osteoglossidae), in Muda Lake where one of its last wild populations remains on the west side of Peninsular Malaysia. We first developed and tested a 123-base pair S. formosus-specific genetic marker using a new pair of primers. Then we applied our eDNA method at five sampling sites throughout Muda Lake and at five different months. The study observed a marked seasonal variation in detection rates, with significantly higher rates during the wet months of August, January, and December compared to the dry season spanning May and June. The Asian arowana is known to breed during the rainy season and we therefore hypothesize its reproductive biology influences the concentration of eDNA in the lake. Our results also highlight the possible impact of abiotic factors in tropical freshwater ecosystems (such as high water temperature) on eDNA persistence. In conclusion, future applications of qPCR-based eDNA methods in tropical environments would benefit from locally evaluating the effects of both biotic and abiotic factors on species detection. [ABSTRACT FROM AUTHOR]

Lumpy skin disease (LSD) is a highly contagious and fatal transboundary viral disease of cattle and water buffaloes caused by the Lumpy Skin Disease virus (LSDV) which belongs to the genus Capripoxvirus of the Poxviridae family. The disease causes huge economic impact and has been listed as a notifiable disease by World Organization for Animal Health. We present the molecular epidemiology of an LSD outbreak, the first-ever outbreak of the disease in the Union Territory (UT) of Jammu and Kashmir, India; a region lying in Northern Himalayas. During the outbreak, 1661 samples were collected from clinically affected, in-contact cattle as well as apparently healthy animals and screened for LSDV by qPCR. From these 1661 samples, 643 (38.7%) were found positive. Five LSDV-positive samples were randomly selected and the GPCR gene was amplified, cloned and sequenced. A 12-nucleotide deletion was observed in the GPCR gene when compared to the 2019 Indian LSDV isolate from Odhisa, India. Based on the data obtained by qPCR analysis, the occurrence of LSD was determined with respect to the age, sex and breed of the animals. Furthermore, ticks collected from the affected animals and flies on the farm were identified and were found positive for LSDV. [ABSTRACT FROM AUTHOR]

The Environmental DNA (eDNA) technology has attracted significant attention due to its convenience and high sensitivity. However, the variations of eDNA across diverse environments and biological species remain complex. Therefore, a detailed exploration of the release patterns of eDNA for specific species under different environments is crucial for the scientific utilization of eDNA detection techniques. This study conducted an experiment involving the aquaculture of Acanthopagrus schlegelii to explore the release and degradation mechanisms of eDNA. It also analyzed the influence of salinity and biomass on the concentration of eDNA in water. Through model simulations, the variation patterns of A. schlegelii eDNA were revealed. The study achieved three key findings: (1) The research on the release and degradation mechanisms of A. schlegelii eDNA indicated that the Generalized Additive Model (GAM) effectively fits the variation patterns of eDNA concentration. The peak concentration of eDNA released by A. schlegelii was observed at 42 h, and the degradation process exhibited two stages: rapid and slow degradation, with a negative correlation between eDNA concentration and time. (2) By investigating the relationship between the concentration of A. schlegelii eDNA and biomass, it was demonstrated that Linear Models (LM) effectively captured this relationship, indicating a correlation between eDNA concentration and biomass. (3) The detection of A. schlegelii eDNA concentration under different salinity conditions revealed that the GAM model better reflected the relationship between eDNA and salinity, exhibiting a negative correlation. As salinity increased, the concentration of eDNA decreased. This study lays a foundation for future assessments of the A. schlegelii biomass in natural waters using eDNA quantitative detection techniques, and provides relevant references for quantitative eDNA detection techniques in other marine fish species. [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]

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]

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]

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]

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 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]

Bakanae disease is widespread in rice cultivation areas globally, presenting a significant challenge to basmati rice farming in India. Chemical seed and seedling treatment methods are commonly employed for managing bakanae disease, however, very less information is available on spray treatments. Our study aimed to assess the effectiveness of various fungicidal spray in field conditions for managing bakanae disease and their impact on basmati rice yield and related characteristics. Seven fungicides, along with a pathogen-inoculated control, were evaluated as foliar treatments during the Kharif season of the year 2021 and 2022. A combined analysis of data from two years showed that T1 (Carbendazim 50% WP) had the lowest disease incidence at 44.68%, demonstrating a significantly high percentage disease inhibition of 44.08%. Following closely was T4 (Tebuconazole 50% + Trifloxystrobin 25% WG) with a disease incidence of 50.00% and a corresponding percentage disease inhibition of 37.37%, while, maximum disease incidence of 79.84% was recorded in control treatment (T8) followed by T5 (Copper oxychloride 50% WP). Further, significantly high grain yield was obtained in Carbendazim 50% WP treatments (219.37 g/m2) followed by Tebuconazole 50% + Trifloxystrobin 25% WG (212.00 g/m2). Additionally, a quantitative polymerase chain reaction assay on harvested grains with Fusarium fujikuroi specific marker revealed least pathogen colonization in harvested seeds of Carbendazim 50% WP treatment (T1), with a Ct value of 33.763 (copy no. 4.88E + 10), followed by Tebuconazole 50% + Trifloxystrobin 25% WG (Ct value 33.752, copy no. 4.94E + 10), conversely, Copper oxychloride (T5) demonstrated the least effectiveness with highest pathogen load (copy no. 9.44E + 13). Based on our findings, we recommend the use of Carbendazim 50% WP or Tebuconazole 50% + Trifloxystrobin 25% WG for the management of bakanae disease as foliar spray (2 sprays) treatments (First spray after 15 days of transplanting; second spray at maximum tillering stage (around 25–30 days post Ist spray)) showing low AUDPC and high yield compared to inoculated control. Further, fungicidal spray reduced the infected seed percentage and carried less inoculum in next season. Therefore, these investigations will be valuable for management of bakanae disease after symptom appearance in field conditions. Findings could be further helpful in developing and validating an integrated approach to manage bakanae disease effectively in near future. [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: 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]

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]

Wastewater-based epidemiology offers a complementary approach to clinical case-based surveillance of emergent diseases and can help identify regions with infected people to prioritize clinical surveillance strategies. However, tracking emergent diseases in wastewater requires reliance on novel testing assays with uncertain sensitivity and specificity. Limited pathogen shedding may cause detection to be below the limit of quantification or bordering the limit of detection. Here, we investigated how the definition of limit of detection for quantitative polymerase chain reaction (qPCR) impacts epidemiological insights during an mpox outbreak in Switzerland. 365 wastewater samples from three wastewater treatment plants in Switzerland from 9 March through 31 October 2022 were analyzed for mpox DNA using qPCR. We detected mpox DNA in 22% (79 of 365) wastewater samples based on a liberal definition of qPCR detection as any exponentially increasing fluorescence above the threshold. Based on a more restrictive definition as the lowest concentration at which there is 95% likelihood of detection, detection was 1% (5 of 365). The liberal definition shows high specificity (90%) and accuracy (78%), but moderate sensitivity (64%) when benchmarked against available clinical case reporting, which contrasts with higher specificity (98%) but lower sensitivity (10%) and accuracy (56%) of the 95% likelihood definition. Wastewater-based epidemiology applied to an emergent pathogen will require optimizing public health trade-offs between reporting data with high degrees of uncertainty and delaying communication and associated action. Information sharing with relevant public health stakeholders could couple early results with clear descriptions of uncertainty. Impact Statement: When a novel pathogen threatens to enter a community, wastewater-based epidemiology offers an opportunity to track its emergence and spread. However, rapid deployment of methods for to detect a novel pathogen may rely on assays with uncertain sensitivity and specificity. Benchmarking the detection of mpox DNA in Swiss wastewaters with reported clinical cases in 2022, we demonstrate how definitions of detection of a qPCR assay influence epidemiological insights from wastewater. The results highlight the need for information sharing between public health stakeholders that couple early insights from wastewater with descriptions of methodological uncertainty to optimize public health actions. [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]

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]

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]

Purpose: This study aimed to investigate the influence of bacterial vaginosis on time to pregnancy in subfertile couples. Methods: Couples attending a teaching hospital in the Netherlands having an initial fertility assessment (IFA) between July 2019 and June 2022 were included in this prospective study, with follow-up of pregnancies until June 2023. Vaginal samples at IFA were analyzed on pH, qPCR BV, and 16S rRNA gene microbiome analysis of V1-V2 region. Main outcome measures were time from initial fertility assessment to ongoing pregnancy at 12 weeks and live birth, analyzed by Kaplan–Meier and Cox regression with adjustment for potential confounders. Results: At IFA, 27% of 163 included participants tested positive for BV. BV status had no influence on time to ongoing pregnancy (HR 0.98, 0.60–1.61, aHR 0.97, 0.58–1.62). In persons with unexplained subfertility, positive BV status had a tendency of longer time to pregnancy. When persons had an indication for fertility treatment, positive BV status (HR 0.21, 0.05–0.88, aHR 0.19, 0.04–0.85) and microbiome community state type III and type IV had significant longer time to pregnancy. Conclusion: This study indicates that BV may have a potential negative impact on time to live birth pregnancy in subfertile persons with an indication for fertility treatment. This study did not find an association between BV and time to live birth pregnancy in a general group of subfertile couples or in unexplained subfertility. More research should be done in persons with unexplained subfertility and if treatment improves time to pregnancy. [ABSTRACT FROM AUTHOR]

Ramularia leaf spot of barley, caused by Ramularia collo-cygni has become a serious disease threat to barley production. By inducing necrotic lesions, the disease expiates the onset of leaf senescence, which can result in significant grain yield and quality losses. In addition to surviving between seasons in trash, stubble and volunteers, R. collo-cygni can be transmitted from seed to emerging seedlings. As a potential additional means of transmission it may also pose a risk for the spread of specific traits such as fungicide resistance. In the present study, we screened Irish barley seed stocks harvested in the years 2015–2017 for the presence of R. collo-cygni using qPCR, and subsequently using an amplicon sequencing assay screened for the presence of alleles in the SDHC gene of R. collo-cygni associated with SDHI resistance. Over the three year period > 60% of the samples screened had detectable levels of R. collo-cygni present. In a subsample of these five alleles associated with resistance (SDHC—N87S, G91R, H145R, R152M and H153R) were detected. The prevalence of the key alleles C-N87S and C-H146R remained stable over the three year period, and was similar to that detected amongst an isolate collection established in 2022. [ABSTRACT FROM AUTHOR]

Large and temperate Lake Peipsi is the fourth largest lake in Europe, where the massive cyanobacterial blooms are composed mostly of Microcystis spp., which have been common for several decades now. The seasonal dynamics of potentially toxic Microcystis were studied using microscopy and quantitative polymerase chain reaction (qPCR) by assessing the microcystin-encoding microcystin synthetase gene E (mcyE) abundances. Water samples were analyzed over the lake areas, varying in depth, trophic level, and cyanobacterial composition during the growing period of 2021. The Microcystis mcyE genes were detected through the growing period (May–October), forming peak abundances in September with decreasing temperatures (8.9–11.1 °C). Total phosphorus (TP) and nitrate (NO3−) were the most relevant environmental variables influencing the Microcystis biomass as well as mcyE abundances. Comparison with previous years (2011, 2012) indicated that the abundance and seasonal dynamics of toxigenic Microcystis can be highly variable between the years and lake areas, varying also in dominant Microcystis species. Contrary to expectations, based on mcyE abundances, the increased risk of toxin-producing Microcystis can occur in Peipsi through the growing period, independently of the water temperature and biomasses of Microcystis. [ABSTRACT FROM AUTHOR]

Pesticides like atrazine which are frequently present in everyday surroundings, have adverse impacts on human health and may contribute to male infertility. The work aimed to analyze the histological and biochemical effects of atrazine on the testis in adult albino rats and whether co-administration with resveratrol could reverse the effect of atrazine. Forty adult male albino rats in good health participated in this study. They were categorized at random into four groups: the Group Ӏ received water through a gastric tube for two months every day, the Group ӀӀ received resveratrol (20 mg/kg body weight (b.w.)) through a gastric tube for two months every day, the Group ӀӀӀ received atrazine (50 mg/kg bw) through a gastric tube for two months every day, the Group ӀV received concomitant doses of atrazine and resveratrol for two months every day. The testes of the animals were then carefully removed and prepared for biochemical, immunohistochemical, light, and electron microscopic studies. Atrazine exposure led to a significant decrease in serum testosterone hormone level, upregulation of caspase 3 and iNOS mRNA levels, destructed seminiferous tubules with few sperms in their lumens, many collagen fibres accumulation in the tunica albuginea and the interstitium, abnormal morphology of some sperms as well as many vacuolations, and damaged mitochondria in the cytoplasm of many germ cells. Concomitant administration of resveratrol can improve these adverse effects. It was concluded that atrazine exposure is toxic to the testis and impairs male fertility in adult rat and coadministration of resveratrol guards against this toxicity. [ABSTRACT FROM AUTHOR]

This study analyzed biogenic amine (BA) content in three varieties (types) of kimchi (Baechu kimchi, Baek kimchi, and Yeolmu kimchi), identified the causative bacteria, and evaluated the gene expression associated with the BA formation during kimchi fermentation at 4 °C. Histamine content exceeding the toxicity limit was detected in a single Baechu kimchi product. Tyramine content in most Baechu kimchi products was approximately half of the toxicity limit. Other varieties had relatively lower BA content. Most BA producers isolated from all kimchi varieties were identified as Levilactobacillus brevis, which prominently produced tyramine. To clarify the role of L. brevis in tyramine formation in Baechu kimchi, fermentation experiments were performed using L. brevis BC1M20. The results showed that tyramine content and tyrosine decarboxylase gene (tdc) expression were higher in the inoculated kimchi than in the control. In addition, in the inoculated kimchi, the content decreased while the expression level was almost constant. [ABSTRACT FROM AUTHOR]

In a three-year field study, we inoculated two potato varieties with a selection of four beneficial microbial strains (i.e. Rhizophagus irregularis MUCL41833, Trichoderma asperelloides A, Pseudomonas brassicacearum 3Re2-7 and Paraburkholderia phytofirmans PsJN), alone or in combination. Plants were grown under rainfed or irrigated conditions, and potato yield and development of several diseases were evaluated. The microbial inoculants were traced in the root system at different stages of crop development via molecular markers. Whatever the water supply, the inoculants had no effect on yield. Conversely, some of the inoculants were able to lower the incidence and/or severity of several blemish diseases, namely common scab-associated symptoms (CSAS) and silver scurf/black dot-associated symptoms (SSAS). Microbial consortia were more efficient in decreasing symptoms compared to single strain inoculations. The best control was obtained with the combination of R. irregularis and P. brassicacearum, which reduced the incidence of CSAS by 22% and severity of SSAS by 21%. Root tracking revealed that P. brassicacearum and P. phytofirmans PsJN were able to establish in the root system of the potato, while only P. brassicacearum was detected from emergence until flowering of the plants. [ABSTRACT FROM AUTHOR]

Fusarium graminearum is considered the main cause of Fusarium head blight (FHB) in Argentina. The development of this pathogen is dependent on weather conditions, and its occurrence results in economic losses associated with yield reduction, lower quality of grains, and frequent contamination with mycotoxins. The main inoculum source for FHB is F. graminearum perithecia in crop debris where the pathogen survives saprophytically. The adoption of conservationist tillage practices, which leave crop residues on the soil surface, impacts on the volume of inocula available which, in combination with the lack of FHB resistant wheat cultivars, increases the risk of disease development. Our objective was to analyze the long-term dynamics of F. graminearum inocula on different winter and summer crop residues. To do so, five winter cereal species (soft and durum wheat, barley, rye and tritordeum) were cultivated and harvested, and soybean was sown under no-till on the crop debris. The presence and viability of Fusarium graminearum propagules were later evaluated on the residues of the six crops in the 14 months following the harvest of soybean. A qPCR fungal quantification method was developed using previously available primers. The results obtained confirmed the presence and viability of the inoculum in all substrates throughout the time considered. Furthermore, a direct relationship was found between the amount of inoculum in the residues of the soybean sown under a zero-tillage system and the inoculum in the residues of the winter cereals previously sown. This research provides information that could help reduce the sources of F. graminearum inoculum and establish pre-harvest management strategies aimed at preventing mycotoxin contamination in the food chain. [ABSTRACT FROM AUTHOR]

Background: Karnal bunt of wheat is an important quarantine disease, incited by Tilletia indica. It limits India's trade in wheat export. The teliospores are major source of inoculum to initiate and spread the Karnal bunt disease. The study aimed to identify the germination-related genes in the teliospores of T. indica. Methods and results: The candidate genes in the teliospores germination were identified through the differential gene expression analysis with suitable bioinformatics analysis. Keeping in soil-borne nature of fungi, the teliospores of T. indica (2015 and 2018) were subjected to the qPCR analysis. 20 candidate genes were identified having role in germination of teliospores of T. indica. Twenty genes, viz. Ti9297 (9.31, 7.87-fold), Ti8696 (5.13, 6.54-fold), Ti7699 (8.9, 7.7-fold), Ti7858 (10.33, 6.21-fold), Ti7954 (7.46, 5.54-fold), Ti7739 (5.46, 6.46-fold), Ti9665 (10.74, 7.64-fold), Ti9335 (6.75, 4.36-fold), Ti8396 (9.35, 7.72-fold), Ti8126 (8.87, 11.31-fold), Ti7326 (6.04, 7.7-fold), Ti10208 (13.83, 5.81-fold), Ti12356 (7.83, 8.02-fold), Ti14271 (9.98, 6.32-fold), Ti9234 (11.2, 8.72-fold), Ti 8876 (6.47, 3.55-fold), Ti 10,606 (4.97, 2.35-fold), Ti7758 (10.33, 8.78-fold), Ti4692 (6.89, 9.88-fold), and Ti3932 (5.77, 4.5-fold) were found highly expressed in the germinating teliospores of 2015 and 2018, respectively. Eight genes (Ti508, Ti4152, Ti5346, Ti2375, Ti3739, Ti1134, Ti4399, and Ti4422) were downregulated in the germinating teliospores but these eight genes were showed higher expression in the dormant teliospores. Conclusions: Twenty candidate genes were upregulated in the germinating teliospores are supposed to be involved in the process of germination. Eight genes were downregulated which were related to the process of the dormancy of teliospores. The study will be helpful to devise the newer management strategies for Karnal bunt disease of wheat. [ABSTRACT FROM AUTHOR]

Phytoplasmas are bacterial pathogens located in the plant's phloem that are responsible for several plant diseases and are mainly transmitted by phloem-sucking insects. Apple proliferation (AP) is an economically important disease associated with the presence of 'Candidatus Phytoplasma mali' which is transmitted by two psyllid species. While Cacopsylla picta is a vector in different regions, the vector efficiency of C. melanoneura varies between different populations. This species is considered the main AP vector in Northwestern Italy but plays a minor role in Northeastern Italy and other European regions. To investigate whether the psyllid and/or the phytoplasma subtype drive the phytoplasma acquisition in C. melanoneura, a phytoplasma acquisition experiment was set up using single mating couples of overwintered individuals from different psyllid populations and phytoplasma subtypes. All analyzed insect populations acquired phytoplasma, but with different efficiencies and concentrations. The main factors driving the acquisition were the phytoplasma subtype and its concentration in the leaves of the infected trees together with the psyllid lineage. The phytoplasma concentration in the psyllids was again influenced by the phytoplasma subtype, the psyllid lineage and the region of origin, whereas the phytoplasma concentration in the leaves and the psyllid haplotype defined with the cytochrome oxidase I gene had only a minor impact on the phytoplasma concentration. This is the first study evaluating the roles of both the psyllid haplotype and the phytoplasma subtype on the acquisition process and highlights the importance of C. melanoneura as an additional AP vector. [ABSTRACT FROM AUTHOR]

The Lamiaceae family comprises herbaceous to woody plants that can thrive in a wide range of habitats and are cosmopolitan in distribution. In Turkey, there are 45 genera and 586 species belonging to the Lamiaceae family. Among these species, 239 taxa are endemic, making it the third-largest family in Turkey in terms of endemic individuals. In this study, seeds of three species from the Lamiaceae family, Izmir oregano (Origanum onites L.), French lavender (Lavandula stoechas L.), and basil (Ocimum basilicum L.), were used as test materials. The research aimed to overcome heterogeneous and low germination issues observed in these seeds, which were determined to be seed-related through preliminary studies, and to achieve the highest germination percentage and average germination time. Various pre-sowing treatments were applied to improve the germination performance and quality of Izmir oregano, French lavender, and basil seeds. For this purpose, the effects of treatments including control, KNO3, GA3, polymer coating, priming, priming + polymer coating, priming + polymer coating + KNO3, and priming + polymer coating + GA3 on seed germination rate and average germination days were investigated in comparison to the control. Prior to the treatments, molecular analyses were conducted on all seed samples to detect viral agents. As a result of the research, the highest germination percentage of 94% was found in Izmir oregano with the priming + polymer coating + KNO3 treatment. In contrast, for French lavender and basil seeds, the germination performance with a 92% and 94% germination rate, respectively, was achieved with the priming + polymer coating + GA3 treatment. Molecular analyses conducted on seed samples did not detect any viral agents. [ABSTRACT FROM AUTHOR]

The impact of invasive pulmonary aspergillosis (IPA) on non-neutropenic critically ill patients in intensive care units (ICU) has been demonstrated in recent decades. Furthermore, after the start of the COVID-19 pandemic, COVID-19 associated with pulmonary aspergillosis (CAPA) has become a major concern in ICUs. However, epidemiological data from different regions are scarce. We evaluated the prevalence and clinical-epidemiological data of IPA in patients with COVID-19 requiring mechanical ventilation (MV) in the ICU (“severe COVID-19”) and non-COVID ICU patients in MV of a tertiary hospital in the southern region of Brazil. Eighty-seven patients admitted between June 2020 and August 2022 were included; 31 with severe COVID-19. For the diagnosis of IPA or CAPA, algorithms including host factors and mycological criteria (positive culture for Aspergillus spp., immunoassay for galactomannan detection, and/or qPCR) were utilized. The overall incidence of IPA and CAPA in our ICU was 73 cases/1000 ICU hospitalizations. Aspergillosis occurred in 13% (4/31) of the COVID-19 patients, and in 16% (9/56) of the critically ill patients without COVID-19, with mortality rates of 75% (3/4) and 67% (6/9), respectively. Our results highlight the need for physicians enrolled in ICU care to be aware of aspergillosis and for more access of the patients to sensitive and robust diagnostic tests by biomarkers detection. [ABSTRACT FROM AUTHOR]

Toxoplasma gondii (T. gondii) is an obligate intracellular apicomplexan protozoan that causes toxoplasmosis. Approximately one-third of the world's population is currently T. gondii-seropositive. Although most infections are symptomless, a few can produce retinal lesions and, in immunocompromised persons or when congenitally contracted, can progress to life-threatening central nervous system disseminated infections. Therefore, quick, and precise diagnosis is a must. Molecular techniques nowadays play a crucial role in toxoplasmosis diagnosis, particularly in immunocompromised patients or congenital toxoplasmosis. This review aimed to detail recent advancements in molecular diagnostics of T. gondii infections. The terms "Toxoplasmosis," "Molecular diagnostics," "PCR," "qPCR," "B1," and "rep529" were used to search the English-language literature. In developed nations, conventional PCR (PCR) and nested PCR have been supplanted by quantitative PCR (qPCR), although they are still widely employed in poor nations. The diagnosis of toxoplasmosis has been revolutionized by the emergence of molecular diagnostics. Unfortunately, there is still substantial interlaboratory variability. There is an immediate need for standardization to increase the comparability of results between laboratories and clinical trials. A graphical abstract highlighting the summary of Toxoplasma molecular diagnostics, created using Biorender.com. [ABSTRACT FROM AUTHOR]

In recent years, the Mexican government has implemented several economic sanctions on producers and marketers of milk bovine cheese due to food fraud such as mislabeling or addition of vegetable fats. However, there have been no official reports on the authenticity of goat cheese. This research aimed to detect adulteration in goat cheese by terms of food substitution with bovine milk or soy protein. For which, bovine cytochrome b genes (274 bp) and lectin genes (118 bp) were amplified in 12 commercial brands of goat cheese by real-time PCR (qPCR) where the detection limit for each species was assigned to the lowest concentration detected with a confidence level ≥ 95% being 0.01 ng/µL for bovine milk, and 0.1 ng/µL for soy DNA. Research findings indicate the absence of soy protein; however, a high degree of adulteration with bovine milk (92.3%) was found. The above evidence suggests the need for regulatory agencies to improve verification and traceability mechanisms to ensure the authenticity of these products and protect consumers by providing reliable information. [ABSTRACT FROM AUTHOR]

Background: Silver nanoparticles (AgNPs) have been used in plant tissue culture as growth stimulants, promoting bud initiation, germination, and rooting. In prior studies, AgNPs were synthesized and characterized by green synthesis using extracts from Beta vulgaris var. cicla (BvAgNP), and their functionality as seed disinfectant and antimicrobial was verified. In this study, we evaluated the effect of BvAgNP on the growth and development of Mammillaria bombycina and Selenicereus undatus in vitro, as well as the expression of glyoxalase genes. Methods: Explants from M. bombycina and S. undatus in vitro were treated with 25, 50, and 100 mg/L of BvAgNP. After 90 days, morphological characteristics were evaluated, and the expression of glyoxalase genes was analyzed by qPCR. Results: All treatments inhibited rooting for M. bombycina and no bud initiation was observed. S. undatus, showed a maximum response in rooting and bud generation at 25 mg/L of BvAgNP. Scanning electron microscopy (SEM) results exhibited a higher number of vacuoles in stem cells treated with BvAgNP compared to the control for both species. Expression of glyoxalase genes in M. bombycina increased in all treatments, whereas it decreased for S. undatus, however, increasing in roots. Conclusions: This study presents the effects of BvAgNP on the growth and development of M. bombycina and S. undatus, with the aim of proposing treatments that promote in vitro rooting and bud initiation. [ABSTRACT FROM AUTHOR]

The high prevalence of antibiotic resistant bacteria (ARB) in several environments is a great concern threatening human health. Particularly, wastewater treatment plants (WWTP) become important contributors to the dissemination of ARB to receiving water bodies, due to the inefficient management or treatment of highly antibiotic-concentrated wastewaters. Hence, it is vital to develop molecular tools that allow proper monitoring of the genes encoding resistances to these important therapeutic compounds (antibiotic resistant genes, ARGs). For an accurate quantification of ARGs, there is a need for sensitive and robust qPCR assays supported by a good design of primers and validated protocols. In this study, eleven relevant ARGs were selected as targets, including aadA and aadB (conferring resistance to aminoglycosides); ampC, blaTEM, blaSHV, and mecA (resistance to beta-lactams); dfrA1 (resistance to trimethoprim); ermB (resistance to macrolides); fosA (resistance to fosfomycin); qnrS (resistance to quinolones); and tetA(A) (resistance to tetracyclines). The in silico design of the new primer sets was performed based on the alignment of all the sequences of the target ARGs (orthology grade > 70%) deposited in the Kyoto Encyclopedia of Genes and Genomes (KEGG) database, allowing higher coverages of the ARGs' biodiversity than those of several primers described to date. The adequate design and performance of the new molecular tools were validated in six samples, retrieved from both natural and engineered environments related to wastewater treatment. The hallmarks of the optimized qPCR assays were high amplification efficiency (> 90%), good linearity of the standard curve (R2 > 0.980), repeatability and reproducibility across experiments, and a wide linear dynamic range. The new primer sets and methodology described here are valuable tools to upgrade the monitorization of the abundance and emergence of the targeted ARGs by qPCR in WWTPs and related environments. [ABSTRACT FROM AUTHOR]