Search Results (8,115)

Background: RT-qPCR is the gold standard for quantitative gene expression analysis, but it requires homogenized tissue and thus loses spatial information. RNA in situ hybridization (ISH) preserves tissue localization but is technically challenging, especially in calcified tissues such as bone and teeth, where decalcification can damage RNA. RNAscope, an advanced ISH method with high sensitivity and specificity, has been applied successfully to bone, but its use in dental pulp remains largely unexplored despite the pulp’s crucial role in tooth function and health. Our goal was to identify the optimal decalcification process of mouse tooth samples for RNAscope ISH, which preserves RNA integrity in mouse tooth pulp. Methods: We tested five different decalcification procedures (EDTA, Plank-Rychlo solution, 5% formic acid, ACD decalcification buffer and Morse solution) on tooth samples from 3-month-old male C57BL/6J mice. Micro-CT and hematoxylin-eosin (HE) staining was performed to evaluate the decalcification, the quality and the microstructure of the sections. RNAscope ISH was used to examine mRNA integrity by analyzing the expression patterns of three housekeeping genes with different expression levels (low, medium and high). Results: All five decalcification methods demonstrated well-preserved tissue structure based on HE staining, but RNA integrity was only preserved in the case of mouse dental pulp using the ACD decalcification buffer and Morse’s solution. Conclusions: We successfully identified the optimal decalcification procedures preserving RNA integrity in mouse tooth samples, which may be useful for any target RNA examinations by RNAscope ISH in the future. Full article

Soybean production is a cornerstone of Brazilian agriculture but is heavily threatened by insect pests such as the soybean looper Chrysodeixis includens, capable of reducing yields by up to 70% if uncontrolled. Reliance on chemical insecticides is increasingly unsustainable due to environmental impacts and resistance, highlighting the need for eco-friendly alternatives. The alphabaculovirus Chrysodeixis includens nucleopolyhedrovirus (ChinNPV) is an important biocontrol agent largely used in Brazilian fields because of its host specificity and safety, although its persistence is limited by ultraviolet (UV) sensitivity. Here, we characterize two ChinNPV isolates, CNPSo-168 (C168) and Tabatinga (Tb), using genomic and phenotypic analyses. Whole-genome sequencing revealed circular dsDNA genomes of 139,290 bp (154 ORFs) for C168 and 139,131 bp (153 ORFs) for Tb, both encoding the 38 baculovirus core genes and sharing >98.9% identity with reference genomes. Comparative genomics identified 431 SNPs, including 132 nonsynonymous changes in structural, regulatory, and infection-related genes. At low concentrations, C168 showed an approximately 2-fold lower LC₅₀ than Tb (higher potency), while both achieved near-complete mortality within 8 days at higher concentrations. This greater potency at lower concentrations reinforces the efficacy-based rationale for selecting isolate C168 for biocontrol applications. Infection reduced larval growth, pupation, and adult emergence, often with developmental impairments. Despite genetic differences, both isolates were highly UV-sensitive, and formulation tests indicated that titanium dioxide combined with kaolin conferred partial protection. These results provide insights into ChinNPV diversity and support its development as a sustainable tool for soybean pest management. Full article

In this study, a sliding microfluidic biosensor integrating RPA-SDA cascaded amplification was developed for the rapid, visual detection of Shigella. A novel RPA primer targeting the specific ipaH gene was designed to include a 5′-end G-quadruplex (G4) sequence and the complementary sequence of an Nt.BstNBI endonuclease recognition site. The RPA product templates a subsequent SDA reaction, generating abundant G4 structures that form peroxidase-mimicking DNAzymes with hemin, catalyzing a TMB reaction that produces a distinct blue color for visual readout (on-chip detection at OD₃₇₀, distinct from conventional tube assays at OD₄₅₀). The core on-chip detection process was completed within 13 min (10 min for SDA and 3 min for color development), achieving a limit of detection of 3.5 × 10⁻⁴ ng/μL for Shigella genomic DNA. This timing explicitly excludes the preceding, off-chip steps of nucleic acid extraction and RPA amplification. Validation using spiked lettuce samples confirmed the platform’s high specificity and sensitivity. This work establishes a proof-of-concept for a portable screening tool, highlighting its potential for on-site food safety applications. However, further validation in diverse food matrices and under real-world field conditions is required to fully establish its practical utility. Full article

This study developed a rapid and reliable SYBR-Green semiplex PCR assay for simulta-neous detection of major carbapenem resistance genes in Klebsiella pneumoniae and Acinetobacter baumannii. Two primer sets were used: one to detect blaKPC, blaNDM-1, and blaOXA-48 genes in K. pneumoniae and blaOXA-23 in A. baumannii, and another to amplify conserved 16S rRNA gene regions as internal controls. The intra- and inter-assay coeffi-cient of variation ranged from 0.03% to 3.8%. Standard curves exhibited excellent linearity across six logarithmic scales (10¹–10⁶ DNA copies/µL), with detection limits of 10–10² DNA copies/mL. Melting temperatures (Tm) were: 88.85 °C (KPIC), 90.65 °C (NDM-1), 89.45 °C (KPC), 84.23 °C (OXA-48), 87.81 °C (OXA-23), and 80.67 °C (ABIC). The SYBR-Green Semiplex PCR assay offers a rapid (65 min turnaround), cost-effective, and sensitive method for early detection of carbapenem-resistant pathogens, enabling timely targeted therapy and improved infection control by potentially reducing empirical antibiotic use before culture confirmation. Full article

Leishmaniasis and Chagas disease, caused by Leishmania spp. and Trypanosoma cruzi, are neglected tropical diseases with significant global health burden, particularly in resource-limited regions. Despite their impact, diagnosis and treatment remain challenging due to limited diagnostic tools and the toxicity of available therapies. Our objective is to propose the incorporation of markers for the diagnosis of leishmaniasis and Chagas disease using ncRNA. This narrative review evaluates studies published between 2010 and 2024 (PubMed, Scopus, Google Scholar) using the SANRA scale to assess the potential of non-coding RNAs (ncRNAs) as biomarkers for these infections. Both parasites release small RNAs via extracellular vesicles that modulate host–pathogen interactions and gene expression. Although RNA interference machinery is absent in T. cruzi and most Leishmania species, it persists in early-diverging lineages. In leishmaniasis, distinct miRNA expression profiles—including miR-155-5p, miR-5011-5p, miR-6785-5p, and miR-361-3p—demonstrate high diagnostic accuracy for detecting infection (AUC up to 1.0). Serum long ncRNAs such as MALAT1 and NUTM2A-AS1 show potential diagnostic value, though clinical validation remains pending. For Chagas disease, the available evidence on ncRNAs primarily addresses the diagnosis of clinical manifestations rather than initial infection. Host miRNAs, including miR-21, miR-145, miR-146a/b, and miR-19a-3p, correlate with cardiac involvement, immune dysregulation, and inflammation during chronic T. cruzi infection. Circulating miRNAs exhibit modest sensitivity (57–67%) and specificity (57–80%) for diagnosing chronic Chagas cardiomyopathy, indicating their utility in assessing disease progression and organ damage rather than detecting early infection. This review distinguishes between ncRNAs that diagnose infection and those that evaluate disease severity or organ involvement. Altered ncRNA expression profiles represent promising biomarkers for species differentiation, treatment monitoring, and assessing cardiac complications in Chagas disease, with broader diagnostic applications emerging for leishmaniasis. Full article

This study aimed to establish a multiplex PCR identification system capable of rapidly detecting adulteration in sheep and goat meat, while qualitatively identifying common adulterant meats (pork, chicken, and duck). Species-specific primers targeting mitochondrial DNA sequences were designed after screening for gene fragments with intraspecies conservation and interspecies specificity across five target species. The multiplex PCR conditions and system were systematically optimized and evaluated for specificity, reproducibility, sensitivity, and practical applicability using simulated mixed samples and heat-treated products. The results demonstrated that the system could successfully identify sheep meat, goat meat, and adulterant meat components in randomly combined target meat template DNAs with excellent reproducibility. The system maintained a high sensitivity, detecting target species even at low DNA template concentrations and in samples with low adulteration ratios. Moreover, target meat components remained detectable in heat-treated products, confirming the system’s robustness under realistic market conditions. This multiplex PCR identification system demonstrates strong specificity, good reproducibility, high sensitivity, and broad applicability. It provides an important tool for effectively monitoring sheep and goat meat adulteration and offers crucial technical support for ensuring the authenticity of sheep and goat meat. Full article

The increasing prevalence of the mobile tigecycline resistance gene tet(X) poses a severe global health threat, and the genus Acinetobacter is a major reservoir. This study aimed to develop a rapid and specific multiplex PCR assay for detecting the predominant tet(X)-positive Acinetobacter species. Through pan-genome analyses of 390 tet(X)-positive Acinetobacter genomes, a total of 20 tet(X) variants were identified in 24 Acinetobacter species, including 17 published lineages and seven taxonomically unresolved Taxa. Acinetobacter indicus (30.8%), Acinetobacter amyesii (17.2%), and Acinetobacter towneri (16.1%) were the top three hosts of diverse tet(X) variants. Species-specific signature genes were identified and used for primer design, yielding amplicons of 267 bp (tet(X)), 424 bp (A. indicus), 690 bp (A. amyesii), and 990 bp (A. towneri). The assay was rigorously adjusted for an optimal annealing temperature of 52.8 °C and a primer ratio of 1:1:1:1, demonstrating high sensitivity with a detection limit of 0.3 ng/μL DNA and excellent stability under −20 °C, 4 °C, 20 °C storage conditions. Validation experiments on 151 bacterial strains showed high accuracy for DNA templates (≥97.8%) and bacterial suspensions (≥93.5%) within two hours. This cost-effective and highly accurate multiplex PCR provides a powerful tool for proactive surveillance and control of the critical Acinetobacter sp. pathogens. Full article

Carum carvi, commonly known as caraway, is a medicinal and culinary plant recognized for its anti-inflammatory properties, primarily attributed to its essential oil components. However, the thermogenic potential of caraway—particularly the biological activity of its water-soluble extract—remains largely unexplored. In this study, we investigated the effects and underlying mechanisms of caraway on Ucp-1 mRNA expression in beige adipocytes and on inflammation-mediated suppression of thermogenesis, by treating C3H10T1/2 adipocytes with caraway water extract (CWE) or caraway hexane extract (CHE) during both the induction and maturation phases, followed by isoproterenol stimulation, and measurement of mRNA levels of Ucp-1 and differentiation-related genes. Additionally, RAW264.7 cells were treated with CWE prior to stimulation with lipopolysaccharides followed by evaluation of inflammatory marker expression. CWE increased Ucp-1 mRNA expression directly by enhancing adrenergic sensitivity and promoting beige adipocyte differentiation during the induction phase of differentiation. Further, CWE mediated an indirect effect on Ucp-1 expression by suppressing macrophage inflammation, thus restoring Ucp-1 expression otherwise inhibited under inflammatory conditions. These results suggest that caraway extracts—especially the water-soluble compounds—may serve as therapeutic candidates for obesity-related conditions by enhancing energy expenditure and mitigating chronic inflammation. Full article

African swine fever (ASF), induced by the African swine fever virus (ASFV), is an acute hemorrhagic disease characterized by high fever, systemic hemorrhages, and elevated mortality. Current diagnostic techniques including PCR and ELISA present limitations in field applications due to requirements for specialized equipment and prolonged processing duration. Therefore, rapid and accurate detection of ASFV has become a key link in ASF prevention and control. This study established a rapid and precise visual diagnostic approach by integrating the CRISPR/AapCas12b system with lateral flow strip (LFS) technology, specifically targeting the B646L gene encoding the major capsid protein p72. The CRISPR/AapCas12b-LFS platform achieved a sensitivity threshold of 6 copies/µL for B646L gene detection, completing analysis within an hour. Validation study confirmed exceptional specificity against common porcine pathogens including PRRSV, CSFV, PRV, PPV4, and PCV3. The developed assay demonstrated complete concordance with real-time PCR results when analyzing 34 clinical specimens including three heart samples, three liver samples, three spleen samples, three lung samples, three kidney samples, three lymph node samples, five serum samples, five blood samples, and five oral swab samples for ASFV detection. Overall, this method is sensitive, specific, and practicable onsite for ASFV detection, showing a great application potential for monitoring ASFV in the field. Full article

Paratuberculosis (Johne’s disease) is a chronic enteric infection of ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP), leading to significant economic losses in livestock production. While the solute carrier family 11 member 1 (SLC11A1) gene has been implicated in resistance to intracellular pathogens in several species, its role in ovine paratuberculosis remains largely uncharacterized. The present study investigated whether polymorphic variation in the SLC11A1 3′ untranslated region (3′UTR) (GT)_n microsatellite is associated with resistance or susceptibility to MAP infection in sheep. A total of 138 sheep from three breeds (Karagouniki, Boutsika, and Chios) were genotyped. Gene expression analysis was subsequently performed on a subset of 53 animals, which comprised rigorously phenotyped MAP-resistant (n = 18) and MAP-sensitive (n = 35) individuals from the Karagouniki breed. Four predominant alleles, (GT)₂₁, (GT)₂₂, (GT)₂₃, and (GT)₂₄, were identified. The (GT)₂₁ and (GT)₂₃ alleles were significantly enriched among resistant sheep, while (GT)₂₂ and (GT)₂₄ were more frequent in sensitive animals (χ² = 12.4, p = 0.006; Cramér’s V = 0.38). No significant differences in basal SLC11A1 mRNA expression were detected between phenotypic groups. These findings extend previous GWAS results in sheep by providing the first allele-level evidence linking SLC11A1 3′UTR microsatellite polymorphisms to paratuberculosis resistance in sheep. Although limited by sample size and single-breed representation, the results offer a foundation for future functional and genomic selection studies aimed at enhancing disease resilience in small ruminants. Full article

Macrolides are crucial for the management and treatment of bovine respiratory disease (BRD). However, antimicrobial resistance (AMR) threatens the efficacy of these and other antimicrobials. We developed real-time recombinase polymerase amplification (RPA) assays targeting three clinically relevant macrolide antimicrobial resistance genes (ARGs)—msrE-mphE and erm42—in ≤30 min using extracted DNA. A set of 199 deep nasopharyngeal swabs (DNPS) collected from feedlot calves near the time of arrival were selected based on bacterial culture (BC) results for Mannheimia haemolytica, Pasteurella multocida, and Histophilus somni and antimicrobial susceptibility testing (AST) for tulathromycin, tilmicosin, tildipirosin, or gamithromycin. Samples were also tested for the same targets using RPA and polymerase chain reaction (PCR). In samples that were culture-positive for one or more macrolide-resistant BRD-associated bacteria (n = 101), msrE-mphE and/or erm42 were detected in 95% of cases using RPA. The remaining 98 samples were either culture-negative, or the recovered bacteria were macrolide-susceptible: 43% of these were RPA-positive for at least one macrolide ARG. Together with BC-AST and PCR, Bayesian latent class modelling estimated the clinical sensitivity of RPA for macrolide ARGs to be 95% and specificity to be 58%, with moderate agreement between RPA and BC-AST (κ = 0.52) or PCR (κ = 0.55). The estimated sensitivity of the RPA multiplex assay for the targeted macrolide ARGs was very good, although estimated specificity was limited. However, Sanger sequencing confirmed RPA detection of msrE-mphE in BC-AST/PCR-negative samples (n = 23), reflecting the presence of this locus in non-target bacteria, as well as potential ARG variants among BRD bacteria. These findings support the potential of RPA for rapid ARG detection from extracted DNA. Continued assay optimization and evaluation for detection of respiratory bacteria and ARGs will further enhance its diagnostic utility. Full article

Peritubular capillaritis (ptc) is a hallmark lesion of antibody-mediated rejection (AMR), but the grading of its extent is historically based on arbitrary defined cut-offs. Molecular AMR diagnosis via intragraft gene expression measurements may provide evidence to challenge established ptc categories. We retrospectively included 38 renal allograft biopsies from clinical routine, performed because of suspicion of AMR. Biopsies were re-assessed by an experienced nephropathologist and intragraft gene expression was measured using the NanoString nCounter^® platform. Ptc categories were correlated with AMR gene expression to identify a ptc extent cut-off with optimal prediction of molecular diagnosis of AMR [gene expression levels above first quartile (AMR^Q>1)]. Finally, an independent validation cohort (n = 25, Erasmus MC, Rotterdam, The Netherlands) was included to reproduce the results. Re-assessment of biopsies revealed AMR in 26/68.4%, mixed rejection in 5/13.2%, and T-cell-mediated rejection in 3/7.9%. Biopsies with diffuse ptc had significantly higher AMR gene expression compared to biopsies with focal ptc and biopsies with no ptc (64.0/53.3–84.0 vs. 31.5/27.0–49.5, p = 0.023 and 27.0/14.3–31.8, p = 0.003, median/IQR). Sensitivity analysis revealed that a ≥35% ptc cut-off resulted in higher AUCs for predicting AMR^Q>1 compared to ptc^50% (AUC 0.78, 95% CI: 0.63–0.93, p = 0.009 versus AUC: 0.74, CI: 0.56–0.90, p = 0.03). In the validation cohort, only the ptc^35–, but not the ptc^50%, cut-off significantly predicted AMR^Q>1 (AUC 0.75, 95% CI: 0.54–0.96 p = 0.04 vs. AUC 0.69, CI: 0.46–0.93, p = 0.13). Using intragraft gene expression measurement, we identified a new ptc extent threshold with better prediction of molecular AMR. The newly proposed cut-off of ≥35% could potentially improve diagnostic evaluation and prognostication in cases with suspected or diagnosed AMR. Full article

Wheat blast, caused by Pyricularia oryzae Triticum lineage (PoTl), is one of the most destructive and significant fungal diseases affecting wheat crops. The stability of the G143A mutation in the cytB gene, which confers resistance to Quinone outside inhibitor fungicides (QoIs) in PoTl isolates, has not been extensively studied. This study was conducted to evaluate the stability of fungicide resistance, fitness, and competitive ability of the QoI-resistant (R) PoTl isolates group over nine and five consecutive transfer cycles in vitro and in vivo, respectively, without fungicide exposure. No changes in azoxystrobin sensitivity were observed in either the QoI-resistant or sensitive (S) PoTl isolate groups after the successive transfer cycles in vitro and in vivo. The mycelial growth of the QoI-R PoTl isolate group remained stable, while the conidial germination capacity increased over time. For the QoI-resistant isolates, leaf and head disease, conidial production, and the latent period on wheat leaves did not change between the first and fifth infection cycles. In each transfer cycle, the highest levels of leaf and head disease, as well as the largest quantities of conidia collected from wheat leaves, were observed in isolate mixtures. Also, the G143A mutation responsible for QoI resistance remained stable after five transfer cycles of the QoI-resistant (0S:100R) isolate on wheat leaves. Our findings indicate that the G143A mutation remains stable, and there are adaptive benefits in QoI-R PoTl isolates. We discuss the ecological implications of the wheat blast population’s adaptation and PoTl QoIs resistance stability in wheat-cropping areas in Brazil. Full article

A two-year field study was performed to evaluate the cadmium (Cd) phytoremediation potential of two hyperaccumulators, Sedum alfredii (S.A.) and Sedum plumbizincicola (S.P.), in contaminated farmland. Biomass and Cd uptake in both species followed logistic growth models. S.A. reached maturity about 20 days earlier than S.P., with optimal harvest timing at the early late-flowering stage (early–mid May), compared to the full late-flowering stage (early June) for S.P. The primary Cd-accumulating organs were stems and flowers in S.A. and leaves and stems in S.P. Under identical conditions, S.P. exhibited higher theoretical biomass, Cd content, bioconcentration factor (BCF), and Cd uptake, supported by transcriptomic data showing upregulation of metal transporter and stress-related genes under Cd exposure. However, S.P. demonstrated greater environmental sensitivity and lower stress resistance, resulting in more variable real-world remediation efficiency than S.A. It is recommended to harvest at flowering stages, enhance biomass in key Cd-accumulating tissues, and select species based on local conditions. Future work should aim to breed Sedum varieties with greater biomass, Cd accumulation capacity, and stress tolerance. This study provides actionable insights for optimizing the timing and species selection in Cd phytoremediation. Full article

Variability in blood RNA-Seq data can obscure transcriptional changes that reflect tumor responses, and conventional normalization methods such as RLE/DESeq2 or TMM/edgeR often fail to capture these changes consistently. To address this challenge, we applied DiRT (DEG-by-index Ratio Transformation), a normalization and analysis strategy previously used in insect models, to 111 blood RNA-Seq datasets from mouse tumorigenesis models. DiRT achieved clearer separation between tumor and control samples as early as three days after tumor induction and maintained consistent marker signals across all stages of disease progression. In contrast, standard methods typically revealed differences only at later or scattered time points. KEGG pathway analysis further showed that DiRT-derived differentially expressed genes (DEGs) were enriched in the platelet activation signaling pathway, a pathway not identified using RLE/DESeq2 or TMM/edgeR. These findings demonstrate that DiRT enhances both sensitivity and reproducibility, enabling earlier and more consistent detection of transcriptional changes in blood during tumor development. Full article