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

Abstract Background and Objective Genetic and epigenetic changes characterize the multi-step process of breast carcinogenesis. It is believed that abnormal microRNA (miRNA) expression has a role in the onset and progression of breast cancer. This study aimed to examine the link between miRNA-127 and miRNA-138 and metastasis, tumor invasion, and apoptosis in Egyptian women with breast cancer, as well as their correlation with its molecular types. Methodology A total of 150 participants were included in this study, including 75 women with breast cancer and 75 supposedly healthy women who were age and gender-matched. Every patient underwent a thorough physical examination, a general clinical examination, a mammogram, and lab tests, such as the determination of the levels of miRNA-127 and miRNA-138 expression by real-time PCR and the measurement of blood carcinoembryonic antigen (CEA) and carcinoma antigen 15–3 (CA15-3) and CA15-3 and CEA levels. Results There was a significant low expression of miRNA-127 in favor of high TNM stage (Classification of Malignant Tumors), left-sided tumor, metastasis, high-grade disease, increased axillary nodal involvement, absence of estrogen and progesterone receptors, and low antigen Kiel 67 (Ki67) expression. Also, a significant expression of miRNA 127 in triple-negative breast cancer was found, followed by human epidermal growth factor receptor 2 (HER2/neu) overexpression, then luminal B, and the highest expression was with the Luminal A molecular subtype. A significant negative correlation existed between miRNA 127 and miRNA 138 with CEA and CA15.3 levels. Conclusion The miRNA-127 and miRNA-138 suppression may promote metastasis. Consequently, the restoration of miRNA-127 and miRNA-138 in breast cancer may have therapeutic potential; so, the miRNA-127 and miRNA-138 may play a role in breast cancer development.

Abstract Background Nebulized administration of dexamethasone on cytokine regulation in horses with moderate asthma has not been investigated. Objective To investigate the changes in expression of inflammatory cytokine mRNA after nebulized administration of dexamethasone treatment of horses with moderate asthma. Animals Horses with naturally occurring moderate asthma (n = 16) and healthy control horses (n = 4). All horses were kept in a dusty environment during the study. Methods Prospective, parallel, randomized, controlled, blinded clinical trial. Blood endogenous cortisol, tracheal mucus, and bronchoalveolar lavage (BAL) were sampled before and after 13 days treatment with either nebulized administration of dexamethasone (15 mg once daily) or 0.9% saline (3 mL). Treatment groups were randomly allocated via randomization function (Microsoft Excel). Amplification of target mRNA in BAL fluid (IL‐1β, IL‐4, IL‐5, IL‐6, IL‐8, IL‐10, IL‐12, IL‐17, IL‐23, IFN‐γ, Eotaxin‐2, and TNF‐α) was achieved by qPCR, and the relative expression software tool was used to analyze BAL inflammatory cytokine mRNA. Results Horses treated with nebulized administration of dexamethasone had increased relative expression of IL‐5 (1.70‐fold), IL‐6 (1.71‐fold), IL‐17 (3.25‐fold), IL‐12 (1.66‐fold), and TNF‐α (1.94‐fold), and decreased relative expression of IL‐23 (1.76‐fold; P = .04) in samples collected on Day 14, in comparison to samples collected on Day 0 (all P .05). Conclusions and Clinical Importance Nebulized administration of dexamethasone was associated with increased expression of inflammatory cytokine mRNA. There was no improvement in inflammatory airway cytology associated with either dexamethasone or saline treatment.

Improving the nutritional value of grain sorghum, a drought- and heat-tolerant grain crop, is an important task in the context of global warming. One of the reasons for the low nutritional value of sorghum grain is the resistance of its storage proteins (kafirins) to proteolytic digestion, which is due, among other things, to the structural organization of protein bodies, in which γ-kafirin, the most resistant to proteases, is located on the periphery, encapsulating more easily digested α-kafirins. The introduction of genetic constructs capable of inducing RNA silencing of the γ-kafirin (gKAF1) gene opens up prospects for solving this problem. Using Agrobacterium-mediated genetic transformation of immature embryos of the grain sorghum cv. Avans we have obtained a mutant with improved digestibility of endosperm proteins (up to 92 %) carrying a genetic construct for RNA silencing of the gKAF1 gene. The goal of this work was to study the stability of inheritance of the introduced genetic construct in T2–T4 generations, to identify the number of its copies, as well as to trace the manifestation of agronomically valuable traits in the offspring of the mutant. The mutant lines were grown in experimental plots in three randomized blocks. The studied lines were characterized by improved digestibility of kafirins, a modified type of endosperm, completely or partially devoid of the vitreous layer, an increased percentage of lysine (by 75 %), reduced plant height, peduncle length, 1000-grains weight, and grain yield from the panicle. In T2, a line with monogenic control of GA resistance was selected. qPCR analysis showed that in different T3 and T4 plants, the genetic construct was present in 2–4 copies. In T3, a line with a high digestibility of endosperm proteins (81 %) and a minimal decrease in agronomically valuable traits (by 5–7 %) was selected.

Total joint arthroplasty is a commonly used surgical procedure in orthopedics. Revision surgeries are required in >10% of patients mainly because of prosthetic joint infection caused by bacteria or aseptic implant loosening caused by chronic inflammation. Encephalitozoon cuniculi is a microsporidium, an obligate intracellular parasite, capable of exploiting migrating proinflammatory immune cells for dissemination within the host. We used molecular detection methods to evaluate the incidence of E. cuniculi among patients who had total hip or knee arthroplasty revision. Out of 49 patients, E. cuniculi genotypes I, II, or III were confirmed in joint samples from 3 men and 2 women who had implant loosening. Understanding the risks associated with the presence of microsporidia in periprosthetic joint infections is essential for proper management of arthroplasty. Furthermore, E. cuniculi should be considered a potential contributing cause of joint inflammation and arthrosis.

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. HIGHLIGHTS This study aimed to investigate the occurrence and seasonality variation of HAdV, HPyV, and HPV in raw sewage collected regularly over 12 months from sewage pump stations.; This study represents the first data about the occurrence of HAdV, HPyV, and HPV in Zagazig city, Al Sharqia Governorate, Egypt.; This study supports using both HAdV and HPyV as indicators than either of them as a single fecal indicator.;

Abstract 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. Graphical Abstract

Abstract 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 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.

We detected 24 Encephalitozoon cuniculi positive Ixodes ricinus ticks of 284 collected in the Czech Republic. Since the route of transmission of microsporidia is not fully understood, the presence of microsporidia in ticks raises the question of whether they may be involved in the transmission of these pathogens.

Black tea is highly favored by consumers worldwide, with enzymatic reactions being recognized as a pivotal factor influencing tea quality. The role of microorganisms in shaping the composition of black tea has emerged as a focus of research due to their involvement in enzyme catalysis and metabolic processes. In this study, full-length amplicon sequencing combined with qPCR more accurately reflected microbial profile, and Pantoea, Pseudomonas, Paucibacter, and Cladosporium were identified as the main microbial genera. Moreover, by comprehensively analyzing color, aroma, and taste components over time in black tea samples, correlations were established between the dominant genus and various quality factors. Notably, peroxidase activity levels, total soluble sugar content, and tea pigments concentration exhibited significant associations with the dominant genus. Consequently, this microbiological perspective facilitated the exploration of driving factors for improving black tea quality while establishing a theoretical foundation for quality control in industrial production.

Helicases, motor proteins present in both prokaryotes and eukaryotes, play a direct role in various steps of RNA metabolism. Specifically, SF2 RNA helicases, a subset of the DEAD-box family, are essential players in plant developmental processes and responses to biotic and abiotic stresses. Despite this, information on this family in the physic nut (Jatropha curcas L.) remains limited, spanning from structural patterns to stress responses. We identified 79 genes encoding DEAD-box RNA helicases (JcDHX) in the J. curcas genome. These genes were further categorized into three subfamilies: DEAD (42 genes), DEAH (30 genes), and DExH/D (seven genes). Characterization of the encoded proteins revealed a remarkable diversity, with observed patterns in domains, motifs, and exon–intron structures suggesting that the DEAH and DExH/D subfamilies in J. curcas likely contribute to the overall versatility of the family. Three-dimensional modeling of the candidates showed characteristic hallmarks, highlighting the expected functional performance of these enzymes. The promoter regions of the JcDHX genes revealed potential cis-elements such as Dof-type, BBR-BPC, and AP2-ERF, indicating their potential involvement in the response to abiotic stresses. Analysis of RNA-Seq data from the roots of physic nut accessions exposed to 150 mM of NaCl for 3 h showed most of the JcDHX candidates repressed. The protein–protein interaction network indicated that JcDHX proteins occupy central positions, connecting events associated with RNA metabolism. Quantitative PCR analysis validated the expression of nine DEAD-box RNA helicase transcripts, showing significant associations with key components of the stress response, including RNA turnover, ribosome biogenesis, DNA repair, clathrin-mediated vesicular transport, phosphatidyl 3,5-inositol synthesis, and mitochondrial translation. Furthermore, the induced expression of one transcript (JcDHX44) was confirmed, suggesting that it is a potential candidate for future functional analyses to better understand its role in salinity stress tolerance. This study represents the first global report on the DEAD-box family of RNA helicases in physic nuts and displays structural characteristics compatible with their functions, likely serving as a critical component of the plant's response pathways. [ABSTRACT FROM AUTHOR]

Background: Gene expression profiling via qPCR is an essential tool for unraveling the intricate molecular mechanisms underlying growth and development. Identifying and validating the most appropriate reference genes is essential for qPCR experiments. Nevertheless, there exists a deficiency in a thorough assessment of reference genes concerning the expression of the genes in the research in the context of the growth and development of the Black Tiger Shrimp, P. monodon. This popular marine crustacean is extensively raised for human consumption. In this study, we assessed the expression stability of seven reference genes (ACTB, 18S, EF-1α, AK, PK, cox1, and CLTC) in adult tissues (hepatopancreas, gills, and stomach) of small and large polymorphs of P. monodon. Methods and results: The stability of gene expressions was assessed utilizing NormFinder, BestKeeper, and geNorm, and a comprehensive ranking of these genes was conducted through the online tool RefFinder. In the overall ranking, 18S and CLTC emerged as the most stable genes in the hepatopancreas and stomach, while CLTC and AK exhibited significant statistical reliability in the gills of adult P. monodon. The validation of these identified stable genes was carried out using a growth-associated gene, insr-1. Conclusion: The results indicated that 18S and CLTC stand out as the most versatile reference genes for conducting qPCR analysis focused on the growth of P. monodon. This study represents the first comprehensive exploration that identifies and assesses reference genes for qPCR analysis in P. monodon, providing valuable tools for research involving similar crustaceans. [ABSTRACT FROM AUTHOR]

Background and Objective: Genetic and epigenetic changes characterize the multi-step process of breast carcinogenesis. It is believed that abnormal microRNA (miRNA) expression has a role in the onset and progression of breast cancer. This study aimed to examine the link between miRNA-127 and miRNA-138 and metastasis, tumor invasion, and apoptosis in Egyptian women with breast cancer, as well as their correlation with its molecular types. Methodology: A total of 150 participants were included in this study, including 75 women with breast cancer and 75 supposedly healthy women who were age and gender-matched. Every patient underwent a thorough physical examination, a general clinical examination, a mammogram, and lab tests, such as the determination of the levels of miRNA-127 and miRNA-138 expression by real-time PCR and the measurement of blood carcinoembryonic antigen (CEA) and carcinoma antigen 15–3 (CA15-3) and CA15-3 and CEA levels. Results: There was a significant low expression of miRNA-127 in favor of high TNM stage (Classification of Malignant Tumors), left-sided tumor, metastasis, high-grade disease, increased axillary nodal involvement, absence of estrogen and progesterone receptors, and low antigen Kiel 67 (Ki67) expression. Also, a significant expression of miRNA 127 in triple-negative breast cancer was found, followed by human epidermal growth factor receptor 2 (HER2/neu) overexpression, then luminal B, and the highest expression was with the Luminal A molecular subtype. A significant negative correlation existed between miRNA 127 and miRNA 138 with CEA and CA15.3 levels. Conclusion: The miRNA-127 and miRNA-138 suppression may promote metastasis. Consequently, the restoration of miRNA-127 and miRNA-138 in breast cancer may have therapeutic potential; so, the miRNA-127 and miRNA-138 may play a role in breast cancer development. [ABSTRACT FROM AUTHOR]

Background: Nebulized administration of dexamethasone on cytokine regulation in horses with moderate asthma has not been investigated. Objective: To investigate the changes in expression of inflammatory cytokine mRNA after nebulized administration of dexamethasone treatment of horses with moderate asthma. Animals: Horses with naturally occurring moderate asthma (n = 16) and healthy control horses (n = 4). All horses were kept in a dusty environment during the study. Methods: Prospective, parallel, randomized, controlled, blinded clinical trial. Blood endogenous cortisol, tracheal mucus, and bronchoalveolar lavage (BAL) were sampled before and after 13 days treatment with either nebulized administration of dexamethasone (15 mg once daily) or 0.9% saline (3 mL). Treatment groups were randomly allocated via randomization function (Microsoft Excel). Amplification of target mRNA in BAL fluid (IL‐1β, IL‐4, IL‐5, IL‐6, IL‐8, IL‐10, IL‐12, IL‐17, IL‐23, IFN‐γ, Eotaxin‐2, and TNF‐α) was achieved by qPCR, and the relative expression software tool was used to analyze BAL inflammatory cytokine mRNA. Results: Horses treated with nebulized administration of dexamethasone had increased relative expression of IL‐5 (1.70‐fold), IL‐6 (1.71‐fold), IL‐17 (3.25‐fold), IL‐12 (1.66‐fold), and TNF‐α (1.94‐fold), and decreased relative expression of IL‐23 (1.76‐fold; P =.04) in samples collected on Day 14, in comparison to samples collected on Day 0 (all P <.05). Horses treated with nebulized administration of saline had no significant difference in the relative expression of any gene (all P >.05). Conclusions and Clinical Importance: Nebulized administration of dexamethasone was associated with increased expression of inflammatory cytokine mRNA. There was no improvement in inflammatory airway cytology associated with either dexamethasone or saline treatment. [ABSTRACT FROM AUTHOR]

Fusarium Oxysporum f. sp. Ciceris (FOC) is the causal agent of Fusarium wilt, a destructive and widespread disease of chickpea. Rapid and accurate identification and detection of plant pathogens are essential for timely Disease Management (DM) strategies with appropriate measures. This study aimed to quantitatively determine FOC by using Quantitative Real-Time Polymerase Chain Reaction (qPCR) technique with specific primer pairs [Histone (H3) and Ribosomal (J5)] in seed, root, and root collar, and to discriminate it from other pathogenic fungi [Fusarium Oxysporum formae speciales (FO f. sp.) and Ascocyhta rabiei]. Total RNAs were isolated, converted to cDNAs (limit of 5 ng/rxn.-0.05 pg/rxn.) and used as template for qPCR studies. The FOC was detected in plant samples starting from the first day after inoculation. The FOC was detected in root, root collar and seed samples and was differentiated by qPCR assay from other pathogenic fungi. Melting curves, in which no primer dimers and non-specific complementation were observed, presented a single peak. Quantification was successfully performed using specific H3 and J5 primer pairs (P< 0.05), and the FOC was distinguished from other pathogenic fungi with J5 primer (P< 0.05). The results of these studies may support the development of new biochemical and molecular methods that allow direct, faster and more accurate determination of pathogens. Thus, it will also enable us to reduce the losses caused by diseases and the costs of DM. [ABSTRACT FROM AUTHOR]

Monitoring of bioinoculants once released into the field remains largely unexplored; thus, more information is required about their survival and interactions after root colonization. Therefore, specific primers were used to perform a long-term tracking to elucidate the effect of Hartmannibacter diazotrophicus on wheat and barley production at two experimental organic agriculture field stations. Three factors were evaluated: organic fertilizer application (with and without), row spacing (15 and 50 cm), and bacterial inoculation (H. diazotrophicus and control without bacteria). Hartmannibacter diazotrophicus was detected by quantitative polymerase chain reaction on the roots (up to 5 × 105 copies g−1 dry weight) until advanced developmental stages under field conditions during two seasons, and mostly in one farm. Correlation analysis showed a significant effect of H. diazotrophicus copy numbers on the yield parameters straw yield (increase of 453 kg ha−1 in wheat compared to the mean) and crude grain protein concentration (increase of 0.30% in wheat and 0.80% in barley compared to the mean). Our findings showed an apparently constant presence of H. diazotrophicus on both wheat and barley roots until 273 and 119 days after seeding, respectively, and its addition and concentration in the roots are associated with higher yields in one crop. [ABSTRACT FROM AUTHOR]

Versatility, sensitivity, and accuracy have made the real-time polymerase chain reaction (qPCR) a crucial tool for research, as well as diagnostic applications. However, for point-of-care (PoC) use, traditional qPCR faces two main challenges: long run times mean results are not available for half an hour or more, and the requisite high-temperature denaturation requires more robust and power-demanding instrumentation. This study addresses both issues and revises primer and probe designs, modified buffers, and low ∆T protocols which, together, speed up qPCR on conventional qPCR instruments and will allow for the development of robust, point-of-care devices. Our approach, called "FlashPCR", uses a protocol involving a 15-second denaturation at 79 °C, followed by repeated cycling for 1 s at 79 °C and 71 °C, together with high Tm primers and specific but simple buffers. It also allows for efficient reverse transcription as part of a one-step RT-qPCR protocol, making it universally applicable for both rapid research and diagnostic applications. [ABSTRACT FROM AUTHOR]

【Objective】In mammals, Toll-like receptors and antimicrobial peptide genes are important components of the innate immune system, which play a crucial role in fighting against pathogen attacks. The study was conducted to explore the expression differences of Toll-like receptor and antimicrobial peptide genes between different immune organs or tissues in Tibetan pigs and Duroc-Landrace-Yorkshire pigs, with an aim to reveal the potential contribution of these genes to disease resistance and immune response and provide theoretical support for the screening of molecular markers for disease resistance. 【Method】The mRNA abundance of Toll-like receptors genes (TLR1-TLR9) and two types of antimicrobial peptide genes (PBD-1 and PR-39) in lungs, mesenteric lymph nodes, inguinal lymph nodes, submandibular lymph nodes and spleens of 6-month-old Tibetan and Duroc-Landrace-Yorkshire pigs were detected by qPCR.【Result】The mRNA expression of Tolllike receptors and antimicrobial peptide genes in most of the immune organs or tissues of Tibetan pigs was significantly higher than that of Duroc-Landrace-Yorkshire pigs. Among them, the mRNA expression of TLR1 and TLR2 in lungs was increased by about 50%, and PR-39 was increased by 2.6 times; the expression of TLR4 in mesenteric lymph nodes was increased by 40%, and the expression of TLR1 and PR-39 was increased by 88% and 3 times, respectively. In the inguinal lymph nodes, the expression of TLR1 and TLR2 was increased by about 2 times, and the expression of TLR9 and PR-39 was increased by 70%, especially, the expression of PR-39 increasing by 7 times; The expression of TLR1, TLR2, TLR4 and TLR7 in submandibular lymph nodes was increased by more than 2 times, and the expression of PR-39 was increased by nearly 7 times, which was similar to that in inguinal lymph nodes; the expression of TLR1 in the spleen rose by 3.5 times, which was similar to that in submandibular lymph nodes. The expression of TLR4 and TLR9 increased by about 50%, and the expression of PR-39 increased by 2.5 times.【Conclusion】Tibetan pigs show higher expression levels of Toll-like receptors and antimicrobial peptide genes in multiple immune organs or tissues compared to Duroc-Landrace-Yorkshire pigs. It is implied that Tibetan pigs may possess stronger innate immunity and be able to generate more effective local or systemic immune responses against pathogenic microbial infections. The results of this study provide important theoretical support for the identification of disease resistance molecular markers and are expected to provide a scientific basis for further improvement of disease resistance in Tibetan pigs and other pig breeds. [ABSTRACT FROM AUTHOR]

Grass carp (Ctenopharyngodon idella), an invasive cyprinid within the Laurentian Great Lakes, is naturally reproducing in several Lake Erie tributaries, which has raised concerns of the species' spread throughout Lake Erie and the other Great Lakes. Knowledge of the recent invasion extent outside of the western basin of Lake Erie, particularly in eastern tributaries and nearshore waters, is limited. Understanding the invasion extent would improve the efficacy of ongoing coordinated multi-agency control efforts. Molecular tools, such as environmental DNA (eDNA), have shown promise for early detection of aquatic invasive species. In this study, water samples (N = 476) were collected for grass carp eDNA monthly between May and November in 2018 and 2019, at three sites in the Michigan waters of Lake Erie and the Detroit River. We fit Bayesian multi-scale occupancy models to determine differences in eDNA capture and detection probability among grass carp qPCR assays, sampling sites, and across time. To determine whether grass carp were physically present, and to validate eDNA samples, we quantified recent grass carp presence in sampled areas using an existing acoustic telemetry and field sampling framework. Our results indicate that grass carp eDNA capture probability differed among sites, but there was no difference among months. Positive grass carp eDNA detections were observed across multiple months at each site, with 69% of sitespecific sampling events testing positive for grass carp eDNA on at least one assay and replicate. The majority (65%) of weeks where positive eDNA sampling detections occurred also concurrently had one or more grass carp detected via acoustic telemetry 1-6 days prior. Our results highlight the potential utility of using eDNA to monitor the invasion extent of grass carp within the nearshore waters of Lake Erie. However, further evaluation of the factors that influence grass carp eDNA characteristics among sites within Lake Erie are needed to determine its efficacy for surveillance protocols by natural resource management agencies. [ABSTRACT FROM AUTHOR]

The dairy fermentation industry relies on the activity of lactic acid bacteria in robust starter cultures to accomplish milk acidification. Maintenance of the composition of these starter cultures, whether defined or undefined, is essential to ensure consistent and high-quality fermentation end products. To date, limited information exists regarding the microbial composition of undefined starter culture systems. Here, we describe a culture-based analysis combined with a metagenomics approach to evaluate the composition of two undefined mesophilic starter cultures. In addition, we describe a qPCR-based genotype detection assay, which is capable of discerning nine distinct lactococcal genotypes to characterize these undefined starter cultures, and which can be applied to monitor compositional changes in an undefined starter culture during a fermentation. IMPORTANCE This study reports on the development of a combined culture-based analysis and metagenomics approach to evaluate the composition of two undefined mesophilic starter cultures. In addition, a novel qPCR-based genotype detection assay, capable of discerning nine distinct lactococcal genotypes (based on lactococcal cell wall polysaccharide biosynthesis gene clusters), was used to monitor compositional changes in an undefined starter culture following phage attack. These analytical approaches facilitate a multifaceted assessment of starter culture compositional stability during milk fermentation, which has become an important QC aspect due to the increasing demand for consistent and high-quality dairy products. [ABSTRACT FROM AUTHOR]

Introduction and Objectives: The relation linking type1 diabetes (T1DM) to Alzheimer's disease (AD) was recorded. Recently, widespread attention for gene modification of stem cells (SCs) was established. The present work aimed at investigating possible ameliorating impact of gene modified SCs on cerebral cortex and hippocampus in Alzheimer's disease (AD) complicating T1DM. Methods and Results: 40 female rats were classified into: In Vitro Study Group: 4 animals used to perform SCs culture and Sarco-Endoplasmic Reticulum Ca2+ATPase (SERCA) 2b preparation. Control Group (A): 6 animals. Diabetic Group (B): 10 animals received IP injection 50 mg/kg streptozotocin (STZ). Non-transfected SCs Group (C): 10 rats, received 1×106 rat SCs intravenous. SERCA2b transfected SCs Group (D): 10 rats received 1x106 transfected SCs. 52 days after proving incidence of diabetes, neurological, serum glucose, morphological, morphometric, gene quantitation, western blot and biochemical studies were performed. Neurological study expressed a decrease in group B and in group C. Group B clarified degenerated neurons in external pyramidal (EP) layer and dentate gyrus (DG). Group D revealed more obvious regression of degenerative changes than group C. An increase in area of deformed nerve cells and Congo red +ve neurons, mean area% of immunoexpression (IE) were found in groups B and C. Mean Ca ion concentration, catalase, SERCA2b gene and protein values expressed a significant (sig) decrease in groups B and C. Mean MDA and beta amyloid protein proved a sig increase in groups B and C. Conclusions: Stem cells transfected with sarcoplasmic reticulum calcium ATPase2b gene proved pronounced therapeutic impact suggesting its potency and regenerative capacity in remarkable amelioration of Alzheimer's disease complicating diabetes I. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Herpesviruses are associated with disease in many penguin species. Herpesvirus-associated lesions can cause significant morbidity and mortality in penguins and have been identified in African penguins (Spheniscus demersus), Humboldt penguins (Spheniscus humboldti), and a little blue penguin (Eudyptula minor) infected with spheniscid alphaherpesvirus 1 (SpAHV1). Further investigation is necessary to understand the impact of herpesviruses on penguin health, but there are no rapid, sensitive, and specific methods for detecting and quantifying herpesviral load. We therefore developed a quantitative real-time PCR (qPCR) assay for the detection of SpAHV1 in penguins. TaqMan primer-probes targeting the DNA polymerase gene were designed using a commercial software program. Inter- and intra-assay variability, dynamic range, limit of detection, and analytical specificity were assessed. We used our assay to analyze previously collected field samples from Punta San Juan, Peru, in which conventional consensus PCR had detected one SpAHV1-positive penguin sample. Our qPCR assay was highly specific for SpAHV1. It had a dynamic range of 107–101 target copies per reaction and performed with high efficiency and low intra- and inter-assay variability. Reaction efficiency was not impacted by penguin DNA from SpAHV1-negative tracheal swabs. We detected an additional field sample as positive with our newly developed qPCR assay, and although this likely represents detection of another infected penguin, the true disease status of this population is currently uncharacterized given that no gold-standard test exists for SpAHV1. Our qPCR assay may provide a valuable tool in the surveillance and characterization of SpAHV1 in penguins. [ABSTRACT FROM AUTHOR]

Circular economy recycling-derived fertilizers (RDF) have the potential to replace linear economy fertilizers such as unsustainable superphosphates. Here, effects of ash RDF treatments in Irish grassland cultivation were investigated in a simulated second growing season. Soil fertilized in a preceding pot trial with superphosphate (SP), poultry-litter ash (PLA) and sewage-sludge ash (SSA) at P concentration of 60 kg P ha−1 and a P-free control (SP0) was reused in a microcosm trial. Lolium perenne was cultivated for 54 days in six replicates with a full complement of micro- and macro-nutrients other than P. PLA treatments provided higher dry weight shoot yields than SP0, while SSA and SP overlapped with SP0 and PLA. Most probable number (MPN) analysis showed that phosphonate- and phytate-utilizing bacterial abundance was significantly increased in PLA. Alkaline (phoD) phosphomonoesterase gene fragments were significantly more abundant (qPCR) in the ashes than the superphosphate or P-free control. Bacterial communities were significantly affected by the P application. Similarly, a significant separation of treatments was confirmed in a canonical correspondence analysis of the phoD-harboring community. The genera Streptomyces and Xanthomonas were significantly higher in abundance in the ash RDFs. These results demonstrated the potential benefits of ash RDF treatments as an alternative P source. [ABSTRACT FROM AUTHOR]

Globally, many pond turtles (Family Emydidae) are of conservation concern due to contracting ranges, increasingly fragmented habitats, and declining populations. One of these turtles, the diamondback terrapin (Malaclemys terrapin), is of concern within the United States, but surveying potential estuarine habitat for the presence of the species requires high levels of time and effort. Here a species-specific, probe-based qPCR assay for identifying terrapin DNA from environmental DNA (eDNA) is described. To expand the utility of this tool, the assay was designed to allow for potential detection of at least 19 other pond turtles taxa and the utility was tested by designing a second species-specific probe for red-eared slider (Trachemys scripta elegans) with a different fluorophore, which can be used simultaneously with the terrapin probe to detect either species. Probes were found to be species-specific and effective at detecting low levels of DNA for the target species, the diamondback terrapin (10 fg/µL) and the red-eared slider (100 fg/µL) in laboratory conditions. The assay also showed species-specific detection in environmental samples taken from field locations where one species or the other were known to reside. Through citizen science approaches, this assay could be used for discovering novel terrapin habitats, monitoring invasive red-eared slider populations or surveying other emydids, with species-specific probes, at a fraction of the cost of traditional surveys. [ABSTRACT FROM AUTHOR]

The early identification of aggressive forms of cancer is of high importance in treating papillary thyroid cancer (PTC). Disease dissemination is a major factor influencing patient survival. Mutation status of BRAF oncogene, BRAF V600E, is proposed to be an indicator of disease recurrence; however, its influence on PTC dissemination has not been deciphered. This study aimed to explore the association of the frequency of BRAF V600E alleles in PTC with disease dissemination. In this study, 173 PTC samples were analyzed, measuring the proportion of BRAF V600E alleles by qPCR, which was then normalized against the proportion of tumor cells. Semiquantitative analysis of BRAF V600E mutant protein was performed by immunohistochemistry. The BRAF V600E mutation was present in 60% of samples, while the normalized frequency of mutated BRAF alleles ranged from 1.55% to 92.06%. There was no significant association between the presence and/or proportion of the BRAF V600E mutation with the degree of PTC dissemination. However, the presence of the BRAF mutation was significantly linked with angioinvasion. This study's results suggest that there is a heterogeneous distribution of the BRAF mutation and the presence of oligoclonal forms of PTC. It is likely that the BRAF mutation alone does not significantly contribute to PTC aggressiveness. [ABSTRACT FROM AUTHOR]

Microbial proton-pump rhodopsin (PPR)-based phototrophy, a light-harvesting mechanism different from chlorophyll-based photosystems, may contribute significantly to solar energy entry into the marine ecosystem. PPR transforms solar energy into cellular energy that is used for various metabolic processes in the cells or flagellar movement. Although rhodopsins or their encoding genes have been documented in a wide phylogenetic range of cultured dinoflagellates, information is limited about how widespread and how spatiotemporally dynamical dinoflagellate PPR (DiPPR) are in natural marine ecosystems. In this study, we investigated DiPPR in Long Island Sound (LIS), a temperate estuary of the Atlantic Ocean between Connecticut and Long Island, New York, USA. We isolated six novel full-length dinoflagellate proton-pump rhodopsin cDNAs, expanding the DiPPR database that is crucial to PPR research. Based on these new sequences and existing sequences of DiPPR, we designed primers and conducted quantitative PCR and sequencing to determine the abundance and diversity of DiPPR genes spatially and temporally throughout a year in the water samples collected from LIS. DiPPR genes were found year-round and throughout LIS but with higher abundances in the eutrophic Western Sound and in April and July. The gene diversity data suggest that there are at least five distinct rhodopsin-harboring groups of dinoflagellates throughout the year. The abundance of DiPPR genes, measured as copy number per mL of seawater, appeared not to be influenced by water temperature or nitrogen nutrient concentration but exhibited weak negative correlations with orthophosphate concentration and salinity and a positive correlation with the abundance of DiPPR-harboring dinoflagellates. This first quantitative profiling of PPR in natural plankton reveals the prevalence and dynamics of this plastid-independent photoenergy harvesting mechanism in a temperate estuary and provides efficient DiPPR primers potentially useful for future research. Furthermore, this study shed light on the potential role of DiPPR in phosphor nutrition and dinoflagellate population, which warrants further studies. [ABSTRACT FROM AUTHOR]

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]