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Abstract Background Despite their low abundance in sperm, mitochondria have diverse functions in this cell type, including energy production, signalling and calcium regulation. In humans, sperm mitochondrial DNA content (mtDNAc) has been reported to be negatively linked to sperm function and fertility. Yet, the association between mtDNAc and sperm function in livestock remains unexplored. For this reason, this study aimed to shed some light on the link between mtDNAc and sperm function and fertilising potential in pigs. A qPCR method for mtDNAc quantification was optimised for pig sperm, and the association of this parameter with sperm motility, kinematics, mitochondrial activity, and fertility was subsequently interrogated. Results First, the qPCR method was found to be sensitive and efficient for mtDNAc quantification in pig sperm. By using this technique, mtDNAc was observed to be associated to sperm motility, mitochondrial activity and in vivo, but not in vitro, fertility outcomes. Specifically, sperm with low mtDNAc were seen to exhibit greater motility but decreased mitochondrial activity and intracellular reactive oxygen species. Interestingly, samples with lower mtDNAc showed higher conception and farrowing rates, but similar in vitro fertilisation rates and embryo development, when compared to those with greater mtDNAc. Conclusions These findings enrich our comprehension of the association of mtDNAc with sperm biology, and lay the foundation for future research into employing this parameter as a molecular predictor for sperm function and fertility in livestock.

Background: Soil-transmitted helminth (STH) infections are widespread in developing countries with inadequate sanitation facilities. These infections can lead to malnutrition, anemia, and other complications that can negatively impact both the mother's and fetus's health during pregnancy. This study aimed to estimate the burden of STH by Kato-Katz microscopy and quantitative polymerase chain reaction (qPCR) in pregnant women from rural India. The diagnostic accuracy of these two methods was also compared. Material and Methods: A community-based cross-sectional study was conducted in rural areas of Wardha district of Maharashtra state. We randomly selected and interviewed 688 pregnant women. A total of 534 (77.61%) participants gave stool samples. Two containers (60 mL) labeled with a unique identification number were given to pregnant women to collect stool samples for Kato-Katz microscopy and qPCR. Stool specimens were transported to an accredited lab and examined within 24 hours. Suitable examinations were conducted to compare sensitivity between two tests. Results: The results are presented for 534 participants who gave stool samples. The prevalence of any STH by Kato-Katz stool microscopy was 6.55% (95%CI 4.46–12.19), and that by qPCR was 20.41 (95%CI 17.53–29.40). The sensitivity of Kato-Katz microscopy was 29.17% compared to sensitivity of 90.83% by qPCR (P = 0.001). Pregnant women with STH infection have a significantly lower weight gain during pregnancy, and the majority were either mild or moderate anemic compared to those without STH. Conclusion: The study highlighted significant differences in prevalence of STH among pregnant women by Kato-Katz microscopy and qPCR. The sensitivity of qPCR for detecting STH was higher than that of Kato-Katz microscopy.

Abstract Objective Mitochondrial dysfunction and nuclear epigenetic alterations, two hallmarks of aging, are associated with aberrant development and complex disease risk. Here, we report a method for the simultaneous assessment of mitochondrial DNA copy number (mtDNA-CN) and DNA methylation age (DNAm age) from the same DNA extraction using quantitative polymerase chain reaction (qPCR) and array data, respectively. Result We present methods for the concurrent estimation of mtDNA-CN and DNAm age from the same DNA samples. This includes qPCR to estimate mtDNA-CN, representing the number of circular mitochondrial genomes in a cell, and DNA methylation microarray data to estimate the epigenetic age of an individual. Further, we provide a method for the combination of these metrics into a shared metric termed ‘mtEpiAge’. This approach provides a valuable tool for exploring the interplay between mitochondrial dysfunction and nuclear epigenetic alterations, and their associations with disease and aging.

Abstract Background Diabetic foot ulcers (DFU) are among the fastest-growing diseases worldwide. Recent evidence has emphasized the critical role of microRNA (miRNA)-mRNA networks in various chronic wounds, including DFU. In this study, we aimed to clarify the miRNA-mRNA axes associated with the occurrence of DFU. Methods Expression profiles of miRNAs and mRNAs were extracted from the Gene Expression Omnibus. Differentially expressed genes and differentially expressed miRNAs were identified, and miRNA-mRNA regulatory axes were constructed through integrated bioinformatics analyses. We validated the miRNA-mRNA axes using quantitative real-time PCR (qPCR) and dual-luciferase reporter assays. We conducted an immune infiltration analysis and confirmed the bioinformatics results using immunofluorescence staining. Single-sample gene set enrichment analysis (ssGSEA) was used to analyze the metabolic mechanisms. Results miR-182-5p-CHL1/MITF and miR-338-3p-NOVA1 interactions were identified using in silico analysis. The qPCR results showed apparent dysregulation of these miRNA-mRNA axes in DFU. The dual-luciferase reporter assay confirmed that miR-182-5p targeted CHL1 and MITF, and miR-338-3p targeted NOVA1. We conducted an immune infiltration analysis and observed that key genes correlated with decreased infiltration of M1 macrophages and resting mast cells in DFU. Immunofluorescence staining verified the co-localization of CHL1 and tryptase, while MITF and CD68 showed weak positive correlations. Metabolic pathways related to these three genes were identified using ssGSEA. Conclusions In summary, the miR-182-5p-CHL1/MITF and miR-338-3p-NOVA1 pathway interactions and decreased infiltration of M1 macrophages and resting mast cells may provide novel clues to the pathogenesis of DFU. Trial registration The clinical trial included in this study was registered in the Chinese Clinical Trial Registry ( ChiCTR2200066660 ) on December 13, 2022.

During the COVID-19 pandemic, extensive efforts focused on developing a better understanding of indirect transmission routes, environmental monitoring of fomites, and suitable surveillance strategies, providing new perspectives to also face other communicable diseases. Rapid methods for monitoring environmental contamination are strongly needed to support risk assessment, epidemiological surveillance and prevent infections from spreading. We optimized and automatized a protocol based on fomite detection by qPCR, using a microbial-signature approach based on marker genes belonging to the microbiota of droplets or different biological fluids. The procedure was implemented by exploiting the available tools developed for SARS-CoV-2 tracing, such as flocked swab sampling, real-time PCR equipment and automatic extraction of nucleic acids. This approach allowed scaling up, simplifying, and speeding up the extraction step of environmental swabs, processing at least 48 samples within 45 min vs. 90 min for about 24 samples by manual protocols. A comparison of microflora data by Next-Generation Sequencing (NGS) strongly supports the effectiveness of this semiautomated extraction procedure, providing good quality DNA with comparable representation of species as shown by biodiversity indexes. Today, equipment for qPCR is widely available and relatively inexpensive; therefore this approach may represent a promising tool for hospital hygiene in surveilling fomites associated with SARS-CoV-2 or other pathogen’s transmission.

This study aims to determine the expression of phenotypes and genotypes in Sapudi and Garut sheep, as well as differences between GDF9 exon 1 gene expression and phenotypic traits in Sapudi and Garut sheep. The research method used is a quantitative descriptive method. The material used was 48 rams kept at UPT Breeding Cattle and Forages in Jember and Seribu Pulau Children's Farm, Kangploso Farm, Malang. As for the observation of GDF9 gene expression by qPCR analysis using GDF9 gene primers with a sample of 8 tails. Data analysis used unpaired t-test analysis and continued with the BNT test. Observation of phenotypic traits included body length (PB), chest circumference (LD), and body height (TB). The genotype observed was GDF9 gene expression. The results showed very significant differences in the phenotypic characteristics of the Sapudi and Garut sheep (P

Satellite repeats are a significant component of the genome of Triticeae and play a crucial role in the speciation. They are a valuable tool for studying these processes. Pseudoroegneria species play a special role among grasses, as they are considered putative donors of the St-genome in many polyploid species. The aim of this study was to compare the copy number of satellite repeats in the genomes of Triticeae species. Quantitative real-time PCR was applied to determine the copy numbers of 22 newly discovered satellite repeats revealed in the whole-genome sequences of Pseudoroegneria species and one additional repeat previously identified in the genome of Aegilops crassa. The study focused on seven species of Pseudoroegneria, three species of Thinopyrum, Elymus pendulinus, Ae. tauschii, Secale cereale, and Triticum aestivum. Based on the copy number level and coefficients of variation, we identified three groups of repeats: those with low variability between species (medium-copy CL82), those with medium variability (low- and medium-copy CL67, CL3, CL185, CL119, CL192, CL89, CL115, CL95, CL168), and those with high coefficients of variation (CL190, CL184, CL300, CL128, CL207, CL69, CL220, CL101, CL262, CL186, CL134, CL251, CL244). CL69 exhibited a specific high copy number in all Pseudoroegneria species, while CL101 was found in both Pseudoroegneria and Th. junceum, CL244 in Th. bessarabicum, CL184 in P. cognata and S. cereale. CL95, CL128, CL168, CL186, CL207, and CL300 exhibited higher copy numbers in P. cognata compared to other species; CL3, CL95, CL115, CL119, CL190, CL220, CL207, and CL300 in P. kosaninii; CL89 in P. libanotica; CL134 in P. geniculata. Our assessment of the copy number of new satellite repeats in the St-genome and the analysis of their amplification specificity between species can contribute to the molecular-genetic and chromosome markers used for evolutionary, phylogenetic, and population studies of Triticeae species.

The zoonotic disease brucellosis is a serious public health and livestock industry concern. In the present study, we used bacteriological culture, RBT, and qPCR to determine the prevalence of brucellosis in the serum and milk samples of sheep with a history of abortion. Serum and milk samples were obtained from 100 sheep aged 3-5 years. In order to determine the prevalence of brucellosis, a modified RBT was performed on serum samples, Brucella was isolated from milk by bacteriological culture, and qPCR was applied to detect bacterial DNA in milk. The prevalence of brucellosis using modified RBT, bacteriological culture, and qPCR was 32%, 42%, and 44%, respectively. By considering qPCR as the standard, modified RBT showed a sensitivity of 95%, a specificity of 100%, an accuracy of 98%, a PV+ of 100%, and a PV- of 97%. The sensitivity, specificity, accuracy, PV+, and PV- for bacteriological culture were 77%, 100%, 90%, 100%, and 85%, respectively. The agreement between qPCR and modified RBT was 0.959 (95% CI: 0.896-1), between qPCR and bacteriological culture was 0.792 (95% CI: 0.667-0.897), and between modified RBT and bacteriological culture was 0.831 (95% CI: 0.709-0.38). Based on the results, bacterial isolation from sheep milk is not recommended except in specific cases due to its low sensitivity, as well as its time-consuming and hazardous nature. However, the modified RBT can be used as a routine method because of its cost-effectiveness, higher sensitivity, and higher accuracy compared to bacterial isolation. Moreover, qPCR is recommended as the gold standard test for detecting brucellosis in sheep milk, especially in those with a history of abortion.

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.

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

Brucellosis is a zoonosis caused by bacteria of the genus Brucella, which results in economic losses relating to livestock and threatens public health. A cross-sectional study was conducted to determine the molecular prevalence of Brucella species in smallholder dairy cattle in six regions of Tanzania from July 2019 to October 2020. Dairy cattle (n = 2048) were sampled from 1371 farms. DNA extracted from blood and vaginal swabs was tested for Brucella using qPCR targeting the IS711 gene and positives were tested for the alkB marker for B. abortus and BMEI1172 marker for B. melitensis. The molecular prevalence was 3.5% (95% CI: 2.8–4.4) with the highest prevalence 8.1% (95% CI: 4.6–13.0) in Njombe region. B. melitensis was the predominant species detected (66.2%). Further studies are recommended to understand the source of B. melitensis and its implications for veterinary public health. Livestock keepers should be informed of the risks and biosecurity practices to reduce the introduction and control of Brucella. Cattle and small ruminant vaccination programs could be implemented to control brucellosis in high-risk populations in the country.

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

Surface sampling for SARS-CoV-2 RNA detection has shown considerable promise to detect exposure of built environments to infected individuals shedding virus who would not otherwise be detected. Here, we compare two popular sampling media (VTM and SDS) and two popular workflows (Thermo and PerkinElmer) for implementation of a surface sampling program suitable for environmental monitoring in public schools. We find that the SDS/Thermo pipeline shows superior sensitivity and specificity, but that the VTM/PerkinElmer pipeline is still sufficient to support surface surveillance in any indoor setting with stable cohorts of occupants (e.g., schools, prisons, group homes, etc.) and may be used to leverage existing investments in infrastructure. IMPORTANCE The ongoing COVID-19 pandemic has claimed the lives of over 5 million people worldwide. Due to high density occupancy of indoor spaces for prolonged periods of time, schools are often of concern for transmission, leading to widespread school closings to combat pandemic spread when cases rise. Since pediatric clinical testing is expensive and difficult from a consent perspective, we have deployed surface sampling in SASEA (Safer at School Early Alert), which allows for detection of SARS-CoV-2 from surfaces within a classroom. In this previous work, we developed a high-throughput method which requires robotic automation and specific reagents that are often not available for public health laboratories such as the San Diego County Public Health Laboratory (SDPHL). Therefore, we benchmarked our method (Thermo pipeline) against SDPHL's (PerkinElmer) more widely used method for the detection and prediction of SARS-CoV-2 exposure. While our method shows superior sensitivity (false-negative rate of 9% versus 27% for SDPHL), the SDPHL pipeline is sufficient to support surface surveillance in indoor settings. These findings are important since they show that existing investments in infrastructure can be leveraged to slow the spread of SARS-CoV-2 not in just the classroom but also in prisons, nursing homes, and other high-risk, indoor settings.

A promising approach to help students safely return to in person learning is through the application of sentinel cards for accurate high resolution environmental monitoring of SARS-CoV-2 traces indoors. Because SARS-CoV-2 RNA can persist for up to a week on several indoor surface materials, there is a need for increased temporal resolution to determine whether consecutive surface positives arise from new infection events or continue to report past events. Cleaning sentinel cards after sampling would provide the needed resolution but might interfere with assay performance. We tested the effect of three cleaning solutions (BZK wipes, Wet Wipes, RNase Away) at three different viral loads: "high" (4 × 104 GE/mL), "medium" (1 × 104 GE/mL), and "low" (2.5 × 103 GE/mL). RNase Away, chosen as a positive control, was the most effective cleaning solution on all three viral loads. Wet Wipes were found to be more effective than BZK wipes in the medium viral load condition. The low viral load condition was easily reset with all three cleaning solutions. These findings will enable temporal SARS-CoV-2 monitoring in indoor environments where transmission risk of the virus is high and the need to avoid individual-level sampling for privacy or compliance reasons exists. IMPORTANCE Because SARS-CoV-2, the virus that causes COVID-19, persists on surfaces, testing swabs taken from surfaces is useful as a monitoring tool. This approach is especially valuable in school settings, where there are cost and privacy concerns that are eliminated by taking a single sample from a classroom. However, the virus persists for days to weeks on surface samples, so it is impossible to tell whether positive detection events on consecutive days are a persistent signal or new infectious cases and therefore whether the positive individuals have been successfully removed from the classroom. We compare several methods for cleaning "sentinel cards" to show that this approach can be used to identify new SARS-CoV-2 signals day to day. The results are important for determining how to monitor classrooms and other indoor environments for SARS-CoV-2 virus.

Viral infections have already been implicated with otitis media and sudden sensorineural hearing loss. However, the pathophysiology of COVID-19 as it relates to otologic disorders is not well-defined. With the spread of SARS-CoV-2, it is important to evaluate its colonization of middle ear mucosa. Middle ear and nasal tissue samples for quantitative RT-PCR and histologic evaluations were obtained from post-mortem COVID-19 patients and non-diseased control patients. Here we present evidence that SARS-CoV-2 colonizes the middle ear epithelium and co-localizes with the primary viral receptor, angiotensin-converting enzyme 2 (ACE2). Both middle ear and nasal epithelial cells show relatively high expression of ACE2, required for SARS-CoV-2 entry. The epithelial cell adhesion molecule (EpCAM) was use as a biomarker of epithelia. Furthermore, we found that the viral load in the middle ear is lower than that present in the nasal cavity.

Chameleons are well-known lizards with unique morphology and physiology, but their sex determination has remained poorly studied. Madagascan chameleons of the genus Furcifer have cytogenetically distinct Z and W sex chromosomes and occasionally Z1Z1Z2Z2/Z1Z2W multiple neo-sex chromosomes. To identify the gene content of their sex chromosomes, we microdissected and sequenced the sex chromosomes of F. oustaleti (ZZ/ZW) and F. pardalis (Z1Z1Z2Z2/Z1Z2W). In addition, we sequenced the genomes of a male and a female of F. lateralis (ZZ/ZW) and F. pardalis and performed a comparative coverage analysis between the sexes. Despite the notable heteromorphy and distinctiveness in heterochromatin content, the Z and W sex chromosomes share approximately 90% of their gene content. This finding demonstrates poor correlation of the degree of differentiation of sex chromosomes at the cytogenetic and gene level. The test of homology based on the comparison of gene copy number variation revealed that female heterogamety with differentiated sex chromosomes remained stable in the genus Furcifer for at least 20 million years. These chameleons co-opted for the role of sex chromosomes the same genomic region as viviparous mammals, lacertids and geckos of the genus Paroedura, which makes these groups excellent model for studies of convergent and divergent evolution of sex chromosomes. [ABSTRACT FROM AUTHOR]

Ophidiomycosis (snake fungal disease) is caused by the fungal pathogen Ophidiomyces ophidiicola, which causes dermal lesions, occasional systemic infections, and in some cases, mortality. To better understand potential conservation implications of ophidiomycosis (i.e., population-level effects), we investigated its impacts on individual fitness in a population of endangered eastern foxsnakes (Pantherophis vulpinus). We tracked 38 foxsnakes over 6 years and quantified body condition, movement patterns, oviposition rates, and survival. Body condition, distance travelled, and oviposition rates were similar between snakes with and without ophidiomycosis. Interestingly, snakes that tested positive for the pathogen travelled farther, suggesting that movement through a greater diversity of habitats increases risk of exposure. Ophidiomycosis did not negatively affect survival, and most apparently infected snakes persisted in a manner comparable to snakes without ophidiomycosis. Only one mortality was directly attributed to ophidiomycosis, although infected snakes were overrepresented in a sample of snakes killed by predators. Overall, our results suggest that ophidiomycosis may have sublethal effects on eastern foxsnakes, but do not suggest direct effects on survival, ovipositioning, or viability of the study population. [ABSTRACT FROM AUTHOR]

Long noncoding RNAs have been shown to be involved in a myriad of physiological and pathological pathways. To date, malignant pleural mesothelioma (MPM) is considered an extremely aggressive cancer. One reason for this is the late diagnosis of the disease, which can occur within 30–40 years of asbestos exposure. There is an immense need for the development of new, sensitive, inexpensive and easy methods for the early detection of this disease other than invasive methods such as biopsy. The aim of this study was to determine the expression of circulating lncRNAs in mesothelioma patient plasma to identify potential biomarkers. Ten previously identified lncRNAs that were shown to be aberrantly expressed in mesothelioma tissues were selected as candidates for subsequent validation. The expression of the ten selected candidate lncRNAs was verified via quantitative PCR (qPCR) in human plasma samples from mesothelioma patients versus healthy controls. The expression levels of circulating GAS5, SNHG8 and MALAT1 were significantly greater in plasma samples from patients than in those from controls. The ROC analysis of both MALAT1 and SNHG8 revealed 88.89% sensitivity and 66.67% specificity. The sensitivity of these markers was greater than that of GAS5 (sensitivity 72.22% and specificity 66.67%). The regression model for GAS5 was statistically significant, while that for SNHG8 and MALAT1 was not significant due to the small sample size. The area under the curve (AUC) of the three ROC curves was acceptable and significant: 0.7519 for GAS5, 0.7352 for SNHG8 and 0.7185 for MALAT1. This finding confirmed their ability to be used as markers. The three lncRNAs were not affected by age, sex or smoking status. The three lncRNAs showed great potential as independent predictive diagnostic biomarkers. Although the prediction model for MALAT1 did not significantly differ, MALAT1 was significantly expressed in patients more than in controls (p = 0.0266), and the recorded sensitivity and specificity were greater than those of GAS5. [ABSTRACT FROM AUTHOR]

Background: Frailty syndrome is a state of increased vulnerability to stressors, marked by lowered physical strength and increased dependence on others. The well-established changes in gut microbiota associated with old age suggest a probable relationship between gut microbiota and frailty. Methods and results: This study was aimed at finding the relationship between gut microbiota and frailty syndrome, by comparing the sociodemographic data and the gut microbiota profiles of 23 non-frail and 14 frail elderly individuals. We used the quantitative polymerase chain reaction method (qPCR) to determine the bacterial loads of Bifidobacteria, Lactobacillus, Bacteroidetes, Prevotella, and Escherichia coli in stool samples from test subjects. We discovered a significant increase in the bacterial load of Prevotella in frail elderly individuals aged 70 or above. Other bacterial loads and ratios were not significantly different between the two groups. Conclusions: More comprehensive studies with larger sample sizes and encompassing a wider range of inflammation-related bacteria need to be performed to discover the existence and exact nature of these relations. [ABSTRACT FROM AUTHOR]

Background: Bovine babesiosis caused by Babesia bovis is one of the most important tick-borne diseases of cattle in tropical and subtropical regions. Babesia bovis parasites have a complex lifecycle, including development within the mammalian host and tick vector. In the tick midgut, extracellular Babesia parasites transform into gametes that fuse to form zygotes. To date, little is known about genes and proteins expressed by male gametes. Methods and results: We developed a method to separate male gametes from in vitro induced B. bovis culture. Separation enabled the validation of sex-specific markers. Collected male gametocytes were observed by Giemsa-stained smear and live-cell fluorescence microscopy. Babesia male gametes were used to confirm sex-specific markers by quantitative real-time PCR. Some genes were found to be male gamete specific genes including pka, hap2, α-tubulin II and znfp2. However, α-tubulin I and ABC transporter, trap2-4 and ccp1-3 genes were found to be upregulated in culture depleted of male gametes (female-enriched). Live immunofluorescence analysis using polyclonal antibodies confirmed surface expression of HAP2 by male and TRAP2-4 by female gametes. These results revealed strong markers to distinguish between B. bovis male and female gametes. Conclusions: Herein, we describe the identification of sex-specific molecular markers essential for B. bovis sexual reproduction. These tools will enhance our understanding of the biology of sexual stages and, consequently, the development of additional strategies to control bovine babesiosis. [ABSTRACT FROM AUTHOR]

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[Display omitted] • A statistical relationship was created between quantitative PCR and microscopy to quantify Plasmodium relictum infection. • Infection detection probability differs between bird species due to differences in bird parasitemias. • Historical P. relictum (lineage GRW4) infection prevalence in Hawaii was likely much higher than previously thought. Plasmodium parasites infect thousands of species and provide an exceptional system for studying host-pathogen dynamics, especially for multi-host pathogens. However, understanding these interactions requires an accurate assay of infection. Assessing Plasmodium infections using microscopy on blood smears often misses infections with low parasitemias (the fractions of cells infected), and biases in malaria prevalence estimates will differ among hosts that differ in mean parasitemias. We examined Plasmodium relictum infection and parasitemia using both microscopy of blood smears and quantitative polymerase chain reaction (qPCR) on 299 samples from multiple bird species in Hawai'i and fit models to predict parasitemias from qPCR cycle threshold (Ct) values. We used these models to quantify the extent to which microscopy underestimated infection prevalence and to more accurately estimate infection patterns for each species for a large historical study done by microscopy. We found that most qPCR-positive wild-caught birds in Hawaii had low parasitemias (Ct scores ≥35), which were rarely detected by microscopy. The fraction of infections missed by microscopy differed substantially among eight species due to differences in species' parasitemia levels. Infection prevalence was likely 4–5-fold higher than previous microscopy estimates for three introduced species, including Zosterops japonicus , Hawaii's most abundant forest bird, which had low average parasitemias. In contrast, prevalence was likely only 1.5–2.3-fold higher than previous estimates for Himatione sanguinea and Chlorodrepanis virens , two native species with high average parasitemias. Our results indicate that relative patterns of infection among species differ substantially from those observed in previous microscopy studies, and that differences depend on variation in parasitemias among species. Although microscopy of blood smears is useful for estimating the frequency of different Plasmodium stages and host attributes, more sensitive quantitative methods, including qPCR, are needed to accurately estimate and compare infection prevalence among host species. [ABSTRACT FROM AUTHOR]

The objective of this study was to assess the occurrence and seasonal frequency of human adenovirus (HAdV), human polyomavirus (HPyV), and human papillomavirus (HPV) in urban sewage. The detection of these viruses was carried out by polymerase chain reaction (PCR), and then the viral concentrations in the positive samples were quantified by quantitative PCR (qPCR). Additionally, HAdV and HPyV genotyping was also performed by PCR. A total of 38/60 (63.3%) positive samples were found. HAdV was the most prevalent virus (26/60; 43.3%), followed by HPyV (21/60; 35%) and HPV (21/60; 35%). The viral concentrations ranged from 3.56× 10² to 7.55 × 107 genome copies/L. The most common dual viral agents was found between HAdV and HPyV, in eight samples (8/38, 21%). HAdV types 40 and 41 as well as HPyV types JC and BK were identified, with HAdV-40 and HPyV JC being the most prevalent types. Furthermore, the detection rates of HAdV, HPyV, and HPV were higher during the winter season than the other seasons. The high prevalence of HAdV and HPyV supports their suitability as viral indicators of sewage contamination. Furthermore, this study demonstrates the advantages of environmental surveillance as a tool to elucidate the community-circulating viruses. [ABSTRACT FROM AUTHOR]

Background: Despite their low abundance in sperm, mitochondria have diverse functions in this cell type, including energy production, signalling and calcium regulation. In humans, sperm mitochondrial DNA content (mtDNAc) has been reported to be negatively linked to sperm function and fertility. Yet, the association between mtDNAc and sperm function in livestock remains unexplored. For this reason, this study aimed to shed some light on the link between mtDNAc and sperm function and fertilising potential in pigs. A qPCR method for mtDNAc quantification was optimised for pig sperm, and the association of this parameter with sperm motility, kinematics, mitochondrial activity, and fertility was subsequently interrogated. Results: First, the qPCR method was found to be sensitive and efficient for mtDNAc quantification in pig sperm. By using this technique, mtDNAc was observed to be associated to sperm motility, mitochondrial activity and in vivo, but not in vitro, fertility outcomes. Specifically, sperm with low mtDNAc were seen to exhibit greater motility but decreased mitochondrial activity and intracellular reactive oxygen species. Interestingly, samples with lower mtDNAc showed higher conception and farrowing rates, but similar in vitro fertilisation rates and embryo development, when compared to those with greater mtDNAc. Conclusions: These findings enrich our comprehension of the association of mtDNAc with sperm biology, and lay the foundation for future research into employing this parameter as a molecular predictor for sperm function and fertility in livestock. [ABSTRACT FROM AUTHOR]

Potato mop top virus (PMTV) and Tobacco rattle virus (TRV) are significant soil borne pathogens of potato vectored by powdery scab and stubby-root nematodes, respectively. These viruses adversely impact tuber quality with infected tubers displaying brown streaks in the flesh and/or necrotic arcs on the surface contributing to the rejection of tubers in commercial settings. Currently, limited agricultural control methods for PMTV are available to farmers outside of planting resistant genotypes and avoiding fields with its vector; however, for TRV chemical control of the nematode vector is an option. Field screening for susceptibility to PMTV and TRV identified 'Castle Russet' to be resistant to both PMTV and TRV. In order to localize virus resistance genes for the development of marker assisted selection, a tetraploid mapping population (A15001) was developed by hybridizing 'Castle Russet' x A06084-1TE (susceptible to both viruses) and its progeny were subsequently trialed for two years in fields known to be infested with PMTV and TRV (two separate disease trial sites) near Larimore, ND. The population was phenotyped for PMTV and TRV incidence and severity of necrotic tubers at two time points post-harvest (approximately 19 days after harvest and 89 days after storage) with several genotypes in the population showing little or no virus induced necrosis over the years of evaluation, making them useful as parents in hybridizations by the potato breeding community. Tubers produced from the population were further assayed for PMTV and TRV infection by testing tuber core samples with a quantitative polymerase chain reaction (qPCR) assay. The A15001 population and the parents, 241 individuals, were genotyped with the Illumina Infinium SolCAP V2 12K Potato SNP Array yielding 6,704 quality-filtered, informative SNP markers. A genome wide association study (GWAS) analysis revealed SNP markers on multiple chromosomes (1, 2, 3, 5, and 11) that were significantly associated with PMTV incidence and negative qPCR suggesting polygenic inheritance. Conversely, GWAS revealed a significant QTL on chromosome 9 associated with all TRV phenotypes indicative of a major gene contributing to TRV resistance. These data can guide the development of molecular markers to select genotypes resistant to PMTV and TRV in potato breeding programs. [ABSTRACT FROM AUTHOR]

Migrating birds are often exposed to variable environments and face a multitude of stress exposures along their long-distance flights. During stopover refueling, migratory birds must balance the need to accumulate energy reserves to continue their migration with the need to respond to environmental and physiological stressors. We examined the gene expression patterns of different Heat Shock Proteins (HSPs) in migrating birds during stopover at different body condition states (lean vs. fat), to provide some first insights on the role of HSPs in bird migration and explore the concept of a trade-off between refueling and stress response. Our results showed upregulation of HSP expression at release that could be associated with muscle growth and increased cholesterol and lipid synthesis needed for birds to fuel their upcoming migration. On the other hand, during capture, upregulation of HSP5 could be attributed to physiological recovery from the non-stop endurance flight when crossing the Sahara Desert-Mediterranean Sea ecological barrier. All birds significantly increased their fuel loads up to 48% of lean body mass and we provide evidence for muscle rebuilding during stopover as flight muscle mass increased by 10%, highlighting the fact that stopover sites can play a major role in the physiological recovery of migrants. [ABSTRACT FROM AUTHOR]

Simple Summary: Contagious ovine digital dermatitis (CODD) and footrot (FR), a sub-acute or acute necrotic (decaying) infectious disease involving the hoof and underlying tissues, cause important economic losses all over the world. The aetiological agent for FR is Dichelobacter nodosus while CODD has been associated with pathogenic Treponema phylogroups. In the present work, we applied a set of quantitative polymerase chain reactions to detect in a set of 105 samples from diseased flocks and 15 from apparently healthy herds the following microbiological agents: Dichelobacter nodosus, Fusobacterium necrophorum, Treponema spp., and three pathogenic Treponema phylogroups (T. phagedenis, T. medium, and T. pedis). For Treponema spp., all the samples gave a positive result, so we focused our interest on the other agents. D. nodosus and F. necrophorum are the most frequently found bacteria (alone or in combination). Although F. necrophorum is nowadays known to be not the causative agent for FR, it is the only detected agent in 16.2% of the cases, and it is important to carry out analysis to identify it in order to allow veterinarians to apply the correct prevention and treatment procedures. Multiple serogroups of D. nodosus are often (35.1%) found in the pools. This report concludes with comprehensive proposals for diagnosing and preventing hoof ailments in ovine and caprine herds. Contagious ovine digital dermatitis (CODD) and footrot (FR), a sub-acute or acute necrotic (decaying) infectious disease involving the hoof and underlying tissues, pose economic challenges to herds in Spain and worldwide. The aetiological agent for FR is Dichelobacter nodosus, while CODD is caused by pathogenic Treponema phylogroups. We detail the findings derived from the analysis by qPCR of 105 pooled samples from 100 ovine and five caprine herds in Spain and Portugal, alongside 15 samples from healthy flocks in order to identify Dichelobacter nodosus, Fusobacterium necrophorum, Treponema spp., and three pathogenic Treponema phylogroups (T. phagedenis, T. medium, and T. pedis). Treponema spp. were detected in all 120 pools, including samples from the 15 healthy flocks where only one positive result for F. necrophorum was recorded. Mixed infections by agents different from Treponema spp. were identified in 68.57% of samples. Positive results for F. necrophorum and/or D. nodosus, were obtained for 91.4% of the pools, whereas the presence of the three pathogenic Treponema phylogroups was rare: each of them appeared in isolation in a single pool, while they were found in 18 pools in combination with other agents. While F. necrophorum was the sole finding in 16.2% of samples from affected herds, D. nodosus (the footrot causative agent) was only detected in 61% of affected farms. An improved qPCR protocol was implemented to determine the serogroups of D. nodosus in the samples and found all of them (except the G serogroup), often in combined infections (35.1%). This report concludes with comprehensive proposals for diagnosing, preventing, and treating hoof ailments, remarking the interest of the information about D. nodosus serogroups in order to improve the efficiency of immunization by choosing appropriate vaccine protocols. [ABSTRACT FROM AUTHOR]

Simple Summary: In this study, we examined how different water temperatures affect the severity of illness caused by the infectious spleen and kidney necrosis virus in juvenile tilapia and also analyzed the amount of the virus that remains in the fish at different temperatures. We found that the best conditions occurred at higher temperatures, specifically at 32 and 34 °C, where the fish showed no mortality. Furthermore, specific tests for the virus revealed a significant reduction in the viral load in fish tissues. On the other hand, when the fish were kept at lower temperatures, such as 26, 28, and 30 °C, they developed the disease and some died, indicating that the virus was more active in these conditions. These results are consistent with previous studies on aquatic animals. For example, research on shrimp showed that the white spot virus infection was inhibited at 31 °C, and carps infected with the herpes virus were found to be immune after being treated at 30 °C. Based on these results, we propose the use of preventive heat treatment against the infectious spleen and kidney necrosis virus in juvenile tilapia, taking into consideration similar findings in other aquatic animal species to control viral diseases. The infectious spleen and kidney necrosis virus (ISKNV) is one of the most important emerging viral pathogens for Nile tilapia (Oreochromis niloticus) farming. While prevalent worldwide, it has recently been detected in Brazil. However, despite the importance of the virus and the affected fish species, there are no scientific data on the effects of water temperature on disease pathogenesis in Nile tilapia. In the present study, we conducted two trials using juvenile Nile tilapia over a 15-day period. In trial 1, an experimental infection model was developed based on the intraperitoneal inoculation of active viral homogenates (4.3 × 104 virus fish−1), while control fish were similarly inoculated with inactivated viral homogenates. In trial 2, the fish were maintained at different water temperatures (26, 28, 30, 32, and 34 °C) and then infected with ISKNV. For virus detection, kidney and spleen samples were collected and analyzed by qPCR. Our results show that the disease was successfully reproduced in experimental conditions with active homogenates, with the first signs of the disease appearing on the third day after infection. In addition, a significant reduction in mortality was observed in the groups maintained at higher temperatures (>30 °C). This suggests that a treatment of the disease with non-lethal hyperthermia can be used to control the symptoms and mortality of ISKNV-infected Nile tilapia juveniles. [ABSTRACT FROM AUTHOR]

Harmful cyanobacterial blooms (HCBs) are of growing global concern due to their production of toxic compounds, which threaten ecosystems and human health. Saxitoxins (STXs), commonly known as paralytic shellfish poison, are a neurotoxic alkaloid produced by some cyanobacteria. Although many field studies indicate a widespread distribution of STX, it is understudied relative to other cyanotoxins such as microcystins (MCs). In this study, we assessed eleven U.S. urban lakes using qPCR, sxtA gene-targeting sequencing, and 16S rRNA gene sequencing to understand the spatio-temporal variations in cyanobacteria and their potential role in STX production. During the blooms, qPCR analysis confirmed the presence of the STX-encoding gene sxtA at all lakes. In particular, the abundance of the sxtA gene had a strong positive correlation with STX concentrations in Big 11 Lake in Kansas City, which was also the site with the highest quantified STX concentration. Sequencing analysis revealed that potential STX producers, such as Aphanizomenon, Dolichospermum, and Raphidiopsis, were present. Further analysis targeting amplicons of the sxtA gene identified that Aphanizomenon and/or Dolichospermum are the primary STX producer, showing a significant correlation with sxtA gene abundances and STX concentrations. In addition, Aphanizomenon was associated with environmental factors, such as conductivity, sulfate, and orthophosphate, whereas Dolichospermum was correlated with temperature and pH. Overall, the results herein enhance our understanding of the STX-producing cyanobacteria and aid in developing strategies to control HCBs. [ABSTRACT FROM AUTHOR]

Catalases (CATs) play crucial roles in scavenging H2O2 from reactive oxygen species, controlling the growth and development of plants. So far, genome-wide identification and characterization of CAT genes in oil palm have not been reported. In the present study, five EgCAT genes were obtained through a genome-wide identification approach. Phylogenetic analysis divided them into two subfamilies, with closer genes sharing similar structures. Gene structure and conserved motif analysis demonstrated the conserved nature of intron/exon organization and motifs among the EgCAT genes. Several cis-acting elements related to hormone, stress, and defense responses were identified in the promoter regions of EgCATs. Tissue-specific expression of EgCAT genes in five different tissues of oil palm was also revealed by heatmap analysis using the available transcriptome data. Stress-responsive expression analysis showed that five EgCAT genes were significantly expressed under cold, drought, and salinity stress conditions. Collectively, this study provided valuable information on the oil palm CAT gene family and the validated EgCAT genes can be used as potential candidates for improving abiotic stress tolerance in oil palm and other related crops. [ABSTRACT FROM AUTHOR]

Repetitive sequences form a substantial and still enigmatic part of the mammalian genome. We isolated repetitive DNA blocks of the X chromosomes of three species of the family Bovidae: Kobus defassa (KDEXr sequence), Bos taurus (BTAXr sequence) and Antilope cervicapra (ACEXr sequence). The copy numbers of the isolated sequences were assessed using qPCR, and their chromosomal localisations were analysed using FISH in ten bovid tribes and in outgroup species. Besides their localisation on the X chromosome, their presence was also revealed on the Y chromosome and autosomes in several species. The KDEXr sequence abundant in most Bovidae species also occurs in distant taxa (Perissodactyla and Carnivora) and seems to be evolutionarily older than BTAXr and ACEXr. The ACEXr sequence, visible only in several Antilopini species using FISH, is probably the youngest, and arised in an ancestor common to Bovidae and Cervidae. All three repetitive sequences analysed in this study are interspersed among gene-rich regions on the X chromosomes, apparently preventing the crossing-over in their close vicinity. This study demonstrates that repetitive sequences on the X chromosomes have undergone a fast evolution, and their variation among related species can be beneficial for evolutionary studies. [ABSTRACT FROM AUTHOR]

Changes in telomere length are increasingly used to indicate species' response to environmental stress across diverse taxa. Despite this broad use, few studies have explored telomere length in plants. Thus, evaluation of new approaches for measuring telomeres in plants is needed. Rapid advances in sequencing approaches and bioinformatic tools now allow estimation of telomere content from whole‐genome sequencing (WGS) data, a proxy for telomere length. While telomere content has been quantified extensively using quantitative polymerase chain reaction (qPCR) and WGS in humans, no study to date has compared the effectiveness of WGS in estimating telomere length in plants relative to qPCR approaches. In this study, we use 100 Populus clones re‐sequenced using short‐read Illumina sequencing to quantify telomere length comparing three different bioinformatic approaches (Computel, K‐seek and TRIP) in addition to qPCR. Overall, telomere length estimates varied across different bioinformatic approaches, but were highly correlated across methods for individual genotypes. A positive correlation was observed between WGS estimates and qPCR, however, Computel estimates exhibited the greatest correlation. Computel incorporates genome coverage into telomere length calculations, suggesting that genome coverage is likely important to telomere length quantification when using WGS data. Overall, telomere estimates from WGS provided greater precision and accuracy of telomere length estimates relative to qPCR. The findings suggest WGS is a promising approach for assessing telomere length and, as the field of telomere ecology evolves, may provide added value to assaying response to biotic and abiotic environments for plants needed to accelerate plant breeding and conservation management. [ABSTRACT FROM AUTHOR]

Root and crown rot incited by an oomycete, Phytophthora melonis, causes significant yield losses in commercial pumpkin (Cucurbita pepo) production worldwide. Currently, resistant cultivars and knowledge of molecular mechanism of C. pepo against P. melonis are scarce. Here, we analysed the quantitative gene expression changes of 10 candidate gene markers (bHLH87, ERF014, HSF, MYB, PR-1, WRKY21, CPI, POD, PSK, SGT) in pumpkin roots and leaves at three time points (h post-inoculation, hpi) following inoculation with P. melonis in two resistant (Ghelyani and Tanbal), and two susceptible (Marmari and Khoreshti) varieties of pumpkin. Gene expression using quantitative real time PCR along a time course revealed the strongest transcriptomic response at 48 and 72 hpi in resistant genotypes, 1.1-2.7-fold in roots and leaves, respectively, with a high significant correlation (r = 0.857**-0.974**). We also found that CPI, PSK, SGT1 and POD act as a dual regulator that similarly modulate immunity not only against P. melonis, but also against other diseases such as early blight (Alternaria cucumerina), powdery mildew (Podosphaera xanthii), downy mildews (Pseudoperonospora cubensis), and pathogenic plant nematodes (Meloidogyne javanica). Furthermore, significantly higher activities of the ROS scavenging defence enzymes, catalase (1.6-fold increase) and peroxidase (6-fold increase) were observed in the roots of resistant cultivars at different hpi compared with non-inoculated controls. In addition, the biomass growth parameters including leaf and root length, stem and root diameter, root fresh weight and volume were significantly different among studied genotypes. Cumulatively, the transcriptome data provide novel insights into the response of pumpkins for improving pumpkin breeding to P. melonis. [ABSTRACT FROM AUTHOR]

This article discusses the detection and confirmation of White Spot Syndrome Virus (WSSV) in crayfish in Alabama. The virus was detected using quantitative PCR (qPCR) and confirmed through whole genome sequencing. The genome sequence of the WSSV isolate from Alabama was found to be similar to shrimp and prawn isolates from China and Australia. The study suggests that further research is needed to understand the transmission and prevalence of WSSV in crayfish populations. The data from this study is available upon request from the corresponding author. [Extracted from the article]

We report here the structural and functional properties of an oxalate decarboxylase (OxDC)-like cupin domain-containing protein of Bacillus amyloliquefaciens MBNC and its role in imparting tolerance to acid stress conditions. Quantitative real-time PCR (qPCR) analysis revealed 32-fold and 20-fold upregulation of the target gene [(OxDC′)cupin] under acetic acid stress and hydrochloric acid stress, respectively, indicating its association with the acid stress response. Bacterial cells with targeted inactivation of the (OxDC′)cupin gene using the pMUTIN4 vector system showed decreased growth and survival rate in acidic pH, with drastically reduced exopolysaccharide production. In Silico protein–protein interaction studies revealed seven genes (viz. glmS, nagA, nagB, tuaF, tuaF, gcvT, and ykgA) related to cell wall biosynthesis and biofilm production to interact with OxDC-like cupin domain containing protein. While all these seven genes were upregulated in B. amyloliquefaciens MBNC after 6 h of exposure to pH 4.5, the mutant cells containing the inactivated (OxDC′)cupin gene displayed significantly lower expression (RQ: 0.001–0.02) (compared to the wild-type cells) in both neutral and acidic pH. Our results indicate that the OxDC-like cupin domain containing protein is necessary for cell wall biosynthesis and biofilm production in Bacillus amyloliquefaciens MBNC for survival in acid-stress conditions. [ABSTRACT FROM AUTHOR]

Quambalaria eucalypti is a fungal pathogen that causes leaf spot, shoot blight, and stem canker on Eucalyptus spp. Early diagnosis of the disease is difficult, although the symptoms are clear in its advanced phase. To enable a rapid and sensitive screening of asymptomatic or latently infected plant material for Q. eucalypti, a SYBR green-based real-time PCR assay targeting the partial histone-H3 region was developed. The assay demonstrated specificity for Q. eucalypti, not showing cross-reactivity with other Quambalaria species or the other eucalyptus fungal pathogens tested. The primers developed in this study ensured high analytical sensitivity, allowing the detection of Q. eucalypti DNA concentrations as low as 10 fg DNA from asymptomatic plants. The robustness and efficacy of the assay was demonstrated by interlaboratory comparisons with similar results. This newly developed quantitative PCR assay can be used for more comprehensive epidemiological investigations, testing the plant material in known Q. eucalypti distribution areas for early management strategies, or collecting data for resistance breeding programs. [ABSTRACT FROM AUTHOR]

Since DNA damage is of great importance in various biological processes, its rate is frequently assessed both in research studies and in medical diagnostics. The most precise methods of quantifying DNA damage are based on real-time PCR. However, in the conventional version, they require a large amount of genetic material and therefore their usefulness is limited to multicellular samples. Here, we present a novel approach to long-run real-time PCR-based DNA-damage quantification (L1-LORD-Q), which consists in amplification of long interspersed nuclear elements (L1) and allows for analysis of single-cell genomes. The L1-LORD-Q was compared with alternative methods of measuring DNA breaks (Bioanalyzer system, γ-H2AX foci staining), which confirmed its accuracy. Furthermore, it was demonstrated that the L1-LORD-Q is sensitive enough to distinguish between different levels of UV-induced DNA damage. The method was validated on mouse oocytes and fibroblasts, but the general idea is universal and can be applied to various types of cells and species. [ABSTRACT FROM AUTHOR]

Artemia nauplii are widely used as fish larvae feed due to its beneficial nutritional characteristics for larval development; however, efficient feeding strategies are needed to balance its high costs. Therefore, we evaluated the effects of different densities of Artemia nauplii (100, 250, 500, 750, and 1000 nauplii/post-larvae) on the growth, survival, water quality, and myogenic gene expression of tambaqui (Colossoma macropomum) post-larvae cultivated in a recirculating aquaculture system. After 2 weeks of trial, there was a significant decrease in dissolved oxygen concentration with the increase in nauplii density, but it did not interfere with larval performance and survival. In the first week, larvae fed with fewer than 500 nauplii/post-larvae presented slower growth, while in the second week, larvae fed with 1000 nauplii/post-larvae had the highest final weight and length. Regression analysis suggests that the optimum feeding density of Artemia nauplii during the first week is 411 nauplii/post-larvae, while for the second week, the growth increased proportionally to the feeding densities. The relative expression of the myod, myog, and mstn genes was higher in larvae fed with fewer than 500 nauplii/post-larvae. Although low-growing larvae showed increased expression of myod and myog genes, responsible for muscle hyperplasia and hypertrophy, respectively, mstn expression may have played a significant inhibitory role in larval development. Further research is needed to better determine the effects of the live food on the zootechnical performance and the expression of the myogenic genes in the initial phase of the life cycle of the tambaqui post-larvae. [ABSTRACT FROM AUTHOR]

Strigolactones (SLs), synthesized in plant roots, play a dual role in modulating plant growth and development, and in inducing the germination of parasitic plant seeds and arbuscular mycorrhizal fungi in the rhizosphere. As phytohormones, SLs are crucial in regulating branching and shaping plant architecture. Despite the significant impact of branching strategies on the yield performance of fruit crops, limited research has been conducted on SLs in these crops. In our study, we identified the transcript sequences of SL biosynthesis and signaling genes in olive (Olea europaea L.) using rapid amplification of cDNA ends. We predicted the corresponding protein sequences, analyzed their characteristics, and conducted molecular docking with bioinformatics tools. Furthermore, we quantified the expression levels of these genes in various tissues using quantitative real‐time PCR. Our findings demonstrate the predominant expression of SL biosynthesis and signaling genes (OeD27, OeMAX3, OeMAX4, OeMAX1, OeD14, and OeMAX2) in roots and lateral buds, highlighting their importance in branching. Treatment with rac‐GR24, an SL analog, enhanced the germination frequency of olive seeds in vitro compared with untreated embryos. Conversely, inhibition of SL biosynthesis with TIS108 increased lateral bud formation in a hard‐to‐root cultivar, underscoring the role of SLs as phytohormones in olives. These results suggest that modifying SL biosynthesis and signaling pathways could offer novel approaches for olive breeding, with potential applicability to other fruit crops. [ABSTRACT FROM AUTHOR]

Context: Accurate monitoring data on species presence and distribution are crucial for effective conservation management. Environmental DNA (eDNA)-based techniques, in which species are detected from trace amounts of DNA found throughout the environment, are promising tools that may complement traditional monitoring methods and improve detection. However, imperfect detection is a feature of all survey methods that should be properly assessed so that the probability of detecting a target species' DNA at a site where it is present (i.e. the sensitivity of the method) can be determined. The spot-tailed quoll (Dasyurus maculatus), a carnivorous marsupial found in eastern Australia, is a difficult species to detect as it is rare and has large home ranges, often in remote and difficult to access habitat. Aims: In this study, we aimed to evaluate the feasibility of using eDNA soil analysis as a viable alternative or complement to traditional monitoring techniques for detecting spot-tailed quoll. Methods: We developed a species-specific assay and validated it using synthetic oligos, tissue samples and soil collected from a captive quoll enclosure. We then assessed the assay on natural environment soil samples taken from the Snowy River region from communal quoll defecation sites (latrines) and from broader quoll habitat. We used amplification success data to model the concentration of quoll DNA in soil from different site types and calculate the sensitivity of our assay. Key results: Sensitivity was highest at latrine sites, but decreased sharply when sampling just 1 m away. In non-latrine habitat, the positive amplification rate was too low to allow for meaningful statistical analyses, suggesting that a prohibitively large number of samples would need to be analysed for detection probabilities to be adequate for routine monitoring programs. Conclusions: Overall, we found that low sensitivity was driven by the low concentration of spot-tailed quoll DNA at many of the surveyed sites. Implications: Given that quoll latrines can usually be identified from the accumulation of scats, and scats themselves can be sampled for DNA, we suggest that eDNA analysis of soil is unlikely to offer improvements over current spot-tailed quoll monitoring methods. The spot-tailed quoll is difficult to detect owing to its rarity, remote habitat and large home ranges. In this study, environmental DNA (eDNA) soil analysis was evaluated as an alternative to traditional monitoring methods for spot-tailed quoll detection. We designed an eDNA assay that showed high sensitivity at quoll latrine sites but performed poorly in non-latrine habitat due to low concentrations of quoll DNA in soil samples. We thus conclude that eDNA analysis of soil is unlikely to improve current monitoring methods for this species. Photograph by Jemma Cripps (ARI). [ABSTRACT FROM AUTHOR]

BACKROUND: Stored product protection from insect pests relies heavily on the use of phosphine. The most serious drawback of phosphine is the development of resistance in major stored product insects worldwide, including the red flour beetle, Tribolium castaneum (Herbst) and the lesser grain borer, Rhyzopertha dominica (F.). Two genetic loci are responsible for phosphine resistance: the rph1 (S349G mutation in the cyt‐b5‐r homolog) in T. castaneum and the rph2 (P45/49S mutation in the dihydrolipoamide dehydrogenase (dld) gene) in T. castaneum and R. dominica. RESULTS: In this study, we have developed and applied high‐throughput, practical and specific molecular diagnostics (TaqMan qPCR) for monitoring mutations S349G, P45S and P49S. In our pilot monitoring application, we have included phosphine‐resistant and susceptible populations from different parts of the world (USA, Australia, Brazil) and European strains from Greece and Serbia. Our results for the resistant T. castaneum showed a P45S mutant allele frequency (MAF) of 100% and 75.0% in the populations from Serbia and Brazil, respectively. Regarding the susceptible T. castaneum, P45S was detected in Greece (MAF = 62.5%) and was absent in Australia (MAF = 0.0%). Additionally, the S349G mutation was found to be fixed in all resistant populations, while it was also detected in susceptible ones (frequencies: 65.0% and 100.0%). The only case where both mutations were fixed (100%) was a resistant population from Serbia. In R. dominica, the P49S mutation was found only in the two resistant R. dominica populations from Serbia and Greece (50.0% and 100%) and was absent from the susceptible one from Greece; thus, P49S seems to be a satisfactory indicator for monitoring phosphine resistance. CONCLUSIONS: Our P49S detection assay in R. dominica seems to be a viable option in this direction, yet its utilization needs additional large‐scale confirmatory work. The identification of additional resistance markers also should be prioritized. © 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

Background: Quantitative real-time PCR (qPCR) is a highly reliable method for validating gene expression data in molecular studies due to its sensitivity, specificity, and efficiency. To ensure accurate qPCR results, it's essential to normalize the expression data using stable reference genes. Methods: This study aimed to identify suitable reference genes for qPCR studies in goats by evaluating 18 candidate reference genes (ACTB, BACH1, B2M, GAPDH, HMBS, HPRT1, PGK1, PPIA, PPIB, RPLP0, RPL19, RPS9, RPS15, RPS28, SDHA, TBP, UXT, and YWHAZ) in 10 different caprine tissues (heart, intestine, kidney, liver, lung, muscle, rumen, skin, spleen, and testis). An integrated tool called RefFinder, which incorporates various algorithms like NormFinder, GeNorm, BestKeeper, and ΔCt, was used to assess the stability of expression among these genes. Results: After thorough analysis, ACTB, PPIB, and B2M emerged as the most stable reference genes, while RPL19, RPS15, and RPS9 were found to be the least stable. The suitability of the selected internal control genes was further validated through target gene analysis, confirming their efficacy in ensuring accurate gene expression profiling in goats. Conclusion: The study determined that the geometric average of ACTB, PPIB, and B2M creates an appropriate normalization factor for gene expression studies in goat tissues. [ABSTRACT FROM AUTHOR]

The emerging use of qPCR and dPCR in regulated bioanalysis and absence of regulatory guidance on assay validations for these platforms has resulted in discussions on lack of harmonization on assay design and appropriate acceptance criteria for these assays. Both qPCR and dPCR are extensively used to answer bioanalytical questions for novel modalities such as cell and gene therapies. Following cross-industry conversations on the lack of information and guidelines for these assays, an American Association of Pharmaceutical Scientists working group was formed to address these gaps by bringing together 37 industry experts from 24 organizations to discuss best practices to gain a better understanding in the industry and facilitate filings to health authorities. Herein, this team provides considerations on assay design, development, and validation testing for PCR assays that are used in cell and gene therapies including (1) biodistribution; (2) transgene expression; (3) viral shedding; (4) and persistence or cellular kinetics of cell therapies. [ABSTRACT FROM AUTHOR]