Search Results (4,418)

Background/Objectives: Numerous studies have demonstrated the health benefits of polyphenols found in aronia fruits; however, little is known about how aronia leaf polyphenols impact colorectal cancer (CRC). This study aimed to evaluate the in vitro anti-metastatic and anti-invasive activity of crude aronia leaf extract (ACE) and purified phenolic-rich aronia leaf extract (APE) against two CRC cell lines (SW-480 and HT-29). Methods: Migration and invasion potential of ACE and APE were evaluated. Moreover, ELISA and gelatin zymography were performed to detect translational and activity changes in CRC cells after aronia extracts treatment. Results: We found that a 100 µg/mL concentration of ACE and APE almost entirely downregulated the migration and invasion of SW-480 cells, showing greater effectiveness than HT-29 cells. The observed inhibition was concentration-dependent and statistically significant. Additionally, extracts reduced the product of MMP-2 and MMP-9 gene expression at the protein level and simultaneously inhibited the activity of both MMPs. An APE at 300 µg/mL for SW-480 and 600 µg/mL for HT-29 resulted in a notable reduction in MMP-2 protein synthesis by 72% and 50%, respectively. In contrast, MMP-9 protein synthesis decreased by 48% and 59% in HT-29 cells treated with 300 µg/mL and 600 µg/mL of ACE, respectively. The levels of gelatinase activity were similar for both CRC lines, and the APE tested at a concentration of 300 µg/mL reached almost the IC₅₀ value after 48 h of incubation. Conclusions: Based on the presented results, we provided an experimental foundation for future in vitro and in vivo studies on the potential effects and activities of aronia leaves. Full article

Salmonella enterica serovar Infantis has emerged as a prevalent foodborne pathogen in poultry with significant global health implications. This study investigates the molecular characteristics influencing virulence in a S. Infantis rough variant collected from a poultry farm in the USA. In this study, whole genome sequencing and comparative genomics were performed on smooth and rough poultry S. Infantis isolates, while chicken embryo lethality assay was conducted to assess their virulence. Comparative genomics between isolates was analyzed using Mauve pairwise Locally Collinear Blocks to measure the genetic conservation. Embryo survival rates between the isolates were compared using the Kaplan–Meier curves. High genomic conservation was observed between the two isolates, but a frameshift mutation was detected in the Wzz(fepE) gene of the rough variant, resulting in early protein truncation. The chicken embryo lethality assay showed that the lethality rate of the smooth strain was higher than that of the rough strain (p < 0.05). This study identifies a frameshift mutation in the Wzz(fepE) gene, leading to protein truncation, which may reduce bacterial virulence by impacting O-antigen biosynthesis in the rough Salmonella Infantis variant. These findings deepen our understanding of S. Infantis pathogenesis and suggest that targeting the Wzz(fepE) gene or related pathways could be a promising strategy for developing effective vaccines and therapeutic interventions. Full article

Precocious sexual inducer (psi)-producing oxygenases (Ppos) participate in the production of C8 moldy volatile compounds (MVOCs), and these compounds could act as signal molecules modulating G protein signaling cascades, which participates in the growth and development, secondary metabolisms and pathogenicity of filamentous fungi. In this study, PePpoA and PePpoC proteins were identified in Penicillium expansum. The deletion of ppoA decreased C8 MVOC production in P. expansum, while they were not detected in the ΔppoC strain (p < 0.05). In addition, down-regulated cAMP/PKA and PKC/PLC signaling showed in the two mutants (p < 0.05). The two mutants showed slow colony growth and down-regulated expression of genes regulating spore development (abaA, wetA, brlA and vosA) with broken morphology of spore and hyphae. In addition, the two mutants had decreased pathogenicity on apple fruit and less patulin production in vitro and in vivo. Compared with ΔppoA strain, the deletion of ppoC inhibited G protein signaling pathways more, and the ΔppoC strain had more defective growth and development as well as reduced pathogenicity and patulin production (p < 0.05). Therefore, PePpoC proteins affect more growth and development, patulin biosynthesis and pathogenicity of P. expansum by regulating C8 MVOC-mediated G protein signaling transduction. Full article

A total of 104 samples of chicken meat acquired on the day of slaughter from two slaughterhouses in northwestern Spain were analyzed. These comprised 26 carcasses and 26 cuts from each of the two establishments. An average load of 5.39 ± 0.61 log₁₀ cfu/g (total aerobic counts) and 4.90 ± 0.40 log₁₀ cfu/g (psychrotrophic microorganisms) were obtained, with differences (p < 0.05) between types of samples and between slaughterhouses. Culturing methods involving isolation based on the UNE-EN-ISO 11290-1:2018 norm and identification of isolates by polymerase chain reaction (PCR) to detect the lmo1030 gene allowed the detection of Listeria monocytogenes in 75 samples (72.1% of the total; 50.0% of the carcasses and 94.2% of the cuts). The 75 isolates, one for each positive sample, were tested for resistance against a panel of 15 antibiotics of clinical interest by the disc diffusion method. All isolates belonged to the serogroup IIa (multiplex PCR assay) and showed resistance to between four and ten antibiotics, with an average value of 5.7 ± 2.0 resistances per isolate, this rising to 7.0 ± 2.1 when strains with resistance and reduced susceptibility were taken together. A high prevalence of resistance was observed for antibiotics belonging to the cephalosporin and quinolone families. However, the level of resistance was low for antibiotics commonly used to treat listeriosis (e.g., ampicillin or gentamicin). Nine different resistance patterns were noted. One isolate with each resistance pattern was tested for its ability to form biofilms on polystyrene during 72 h at 12 °C. The total biovolume of the biofilms registered through confocal laser scanning microscopy (CLSM) in the observation field of 16,078.24 μm² ranged between 13,967.7 ± 9065.0 μm³ and 33,478.0 ± 23,874.1 μm³, and the biovolume of inactivated bacteria between 0.5 ± 0.4 μm³ and 179.1 ± 327.6 μm³. A direct relationship between the level of resistance to antibiotics and the ability of L. monocytogenes strains to form biofilms is suggested. Full article

The ESX-1 secretion system is critical for the virulence of Mycobacterium tuberculosis as well as for conjugation in the saprophytic model Mycolicibacterium smegmatis. EsxB (CFP-10) and EsxA (ESAT-6) are secreted effectors required for the function of ESX-1 systems. While some transcription factors regulating the expression of esxB and esxA have been identified, little work has addressed their promoter structures or other determinants of their expression. Here, we defined two promoters, one located two genes upstream of esxB and one located immediately upstream, that contribute substantially to the expression of esxB and esxA. We also defined an mRNA cleavage site within the esxB 5′ untranslated region (UTR) and found that a single-nucleotide substitution reprogramed the position of this cleavage event without impacting esxB-esxA transcript abundance. We furthermore investigated the impact of a double stem-loop structure in the esxB 5′ UTR and found that it does not confer stability on a reporter gene transcript. Consistent with this, there was no detectable correlation between mRNA half-life and secondary structure near the 5′ ends of 5′ UTRs on a transcriptome-wide basis. Collectively, these data shed light on the determinants of esxB-esxA expression in M. smegmatis as well as provide broader insight into the determinants of mRNA cleavage in mycobacteria and the relationship between 5′ UTR secondary structure and mRNA stability. Full article

Background: Undifferentiated pleomorphic sarcoma (UPS) is a highly malignant mesenchymal tumor that ranks as one of the most common types of soft tissue sarcoma. Even though chemotherapy increases the 5-year survival rate in UPS, high tumor heterogeneity frequently leads to chemotherapy resistance and consequently to recurrences. In this study, we characterized the cell composition and the transcriptional profile of UPS with resistance to chemotherapy at the single cell resolution. Methods: A 58-year-old woman was diagnosed with a 13.6 × 9.3 × 6.0 cm multi-nodular tumor with heterogeneous cysto-solid structure at the level of the distal metadiaphysis of the left thigh during magnetic resonance tomography. Morphological and immunohistochemical analysis led to the diagnosis of high-grade (G3) UPS. Neoadjuvant chemotherapy, surgery (negative resection margins), and adjuvant chemotherapy were conducted, but tumor recurrence developed. The UPS sample was used to perform single-cell RNA sequencing by chromium-fixed RNA profiling. Results: Four subpopulations of tumor cells and seven subpopulations of tumor microenvironment (TME) have been identified in UPS. The expression of chemoresistance genes has been detected, including KLF4 (doxorubicin and ifosfamide), ULK1, LUM, GPNMB, and CAVIN1 (doxorubicin), and AHNAK2 (gemcitabine) in tumor cells and ETS1 (gemcitabine) in TME. Conclusions: This study provides the first description of the single-cell transcriptome of UPS with resistance to two lines of chemotherapy, showcasing the gene expression in subpopulations of tumor cells and TME, which may be potential markers for personalized cancer therapy. Full article

Fusarium avenaceum is an aggressive pathogen of pulse crops and a causal agent in root rot disease that negatively impacts Canadian agriculture. This study reports the results of a targeted metabolomics-based profiling of secondary metabolism in an 18-strain panel of Fusarium avenaceum cultured axenically in multiple media conditions, in addition to an in planta infection assay involving four strains inoculated on two pea cultivars. Multiple secondary metabolites with known roles as virulence factors were detected which have not been previously associated with F. avenaceum, including fungal decalin-containing diterpenoid pyrones (FDDPs), fusaoctaxins, sambutoxin and fusahexin, in addition to confirmation of previously reported secondary metabolites including enniatins, fusarins, chlamydosporols, JM-47 and others. Targeted genomic analysis of secondary metabolite biosynthetic gene clusters was used to confirm the presence/absence of the profiled secondary metabolites. The detection of secondary metabolites with diverse bioactivities is discussed in the context of virulence factor networks potentially coordinating the disruption of plant defenses during disease onset by this generalist plant pathogen. Full article

The adenomas in Cushing’s disease frequently exhibit mutations in exon 14, within a binding motif for the regulatory protein 14-3-3 located between the catalytic domain (DUB), responsible for ubiquitin hydrolysis, and the WW-like domain that mediates autoinhibition, resulting in constantly active USP8. The exact molecular mechanism of deubiquitinase activity disruption in Cushing’s disease remains unclear. To address this, Sanger sequencing of USP8 was performed to identify mutations in corticotropinomas. These mutations were subjected to computational screening, followed by molecular dynamics simulations to assess the structural alterations that might change the biological activity of USP8. Eight different variants of the USP8 gene were identified both within and outside the “hotspot” region. Six of these had previously been reported in Cushing’s disease, while two were detected for the first time in our patients with CD. One of the two new variants, initially classified as benign during screening, was found in the neighboring SH3 binding motif at a distance of 20 amino acids. This variant demonstrated pathogenicity patterns similar to those of known pathogenic variants. All USP8 variants identified in our patients caused conformational changes in the USP8 protein in a similar manner. The identified mutations, despite differences in annotation results—including evolutionary conservation assessments, automated predictor data, and variations in localization within exon 14—exhibit similar patterns of protein conformational change. This suggests a pathogenic effect that contributes to the development of CD. Full article

Background/Objectives: A modern classification distinguishes between two nosological entities posing an intermediate risk between differentiated and anaplastic carcinoma: poorly differentiated thyroid carcinoma and differentiated high-grade thyroid carcinoma. There are currently few studies searching for the preoperative molecular genetic markers of high-grade papillary thyroid carcinoma (PTC HG), primarily because of a recent WHO reclassification and singling out of a separate entity: high-grade follicular cell-derived nonanaplastic thyroid carcinoma. Therefore, this work was aimed at identifying PTC HG-specific microRNAs and mRNAs that reliably distinguish them from differentiated papillary thyroid carcinoma in preoperative cytology specimens (fine-needle aspiration biopsies). Methods: A molecular genetic profile (expression levels of 14 genes and eight microRNAs) was studied in 110 cytology specimens from patients with PTC: 13 PTCs HG and 97 PTCs without features of HG. Results: Of the examined eight microRNAs and 14 genes, significant differences in the expression levels between the PTC and PTC HG groups were revealed for genes SLC26A7, TFF3, and TPO. Only one gene (SLC26A7) proved to be crucial for detecting PTC HG. It showed the largest area under the ROC curve (0.816) in differentiation between the PTC and PTC HG groups and was the key element of the decision tree by ensuring 54% sensitivity and 87.6% specificity. Conclusions: Early preoperative diagnosis of PTC HG in patients with early stages of this cancer type will allow clinicians to modify a treatment strategy toward a larger surgery volume and lymph node dissection and may provide indications for subsequent radioactive iodine therapy. Full article

Sunflower Verticillium Wilt (SVW) caused by Verticillium dahliae is a significant threat to sunflower production in China. This soilborne disease is difficult to control. It has been observed that delayed sowing reduces the severity of SVW on different varieties and across various locations. Soil was collected from multiple locations with different sowing dates to understand the underlying biological mechanisms driving this phenomenon. The soil bacterial community was characterized through 16S rRNA gene amplicon sequencing performed on the Illumina MiSeq platform, followed by comprehensive bioinformatics analysis. Microsclerotia numbers in soil were detected using both NP-10 selective medium and quantitative polymerase chain reaction (qPCR). By delaying the sowing date, the number of microsclerotia in soil and the biomass of V. dahliae colonized inside sunflower roots were reduced during the early developmental stages (V2–V6) of sunflowers. Amplicon sequencing revealed an increased abundance of bacterial genera, such as Pseudomonas, Azoarcus, and Bacillus in soil samples collected from delayed sowing plots. Five bacterial strains isolated from the delayed sowing plot exhibited strong antagonistic effects against V. dahliae. The result of the pot experiments indicated that supplying two different synthetic communities (SynComs) in the pot did increase the control efficiencies on SVW by 19.08% and 37.82% separately. Additionally, soil temperature and humidity across different sowing dates were also monitored, and a significant correlation between disease severity and environmental factors was observed. In conclusion, delayed sowing appears to decrease microsclerotia levels by recruiting beneficial rhizosphere bacteria, thereby reducing the severity of SVW. Full article

Piglet diarrhea poses significant economic losses to the pig industry, posing a worldwide challenge that urgently needs to be addressed in pig breeding practices. Porcine rotavirus (PoRV) is an important viral diarrhea pathogen in piglets, with a high incidence rate and a tendency to cause growth retardation. To enhance the sensitivity and specificity of PoRV detection, we sequenced the NSP3 gene of G5 and G9 genotypes of rotavirus A (RVA), enabling simultaneous detection of the two serotypes. Subsequently, we developed a rapid PoRV detection method using a combination of recombinase-aided amplification (RAA) and CRISPR/Cas12a. In this method, Cas12a binds to RAA amplification products, guided by CRISPR-derived RNA (crRNA), which activates its cleavage activity and releases fluorescence by cutting FAM-BHQ-labeled single-stranded DNA (ssDNA). In the optimized reaction system, the recombinant plasmid PoRV can achieve a highly sensitive reaction within 30 min at 37 °C, with a detection limit as low as 2.43 copies/μL, which is ten times higher in sensitivity compared to the qPCR method. Results from specificity testing indicate that no cross-reactivity was observed between the RAA-CRISPR/Cas12a analysis of PoRV and other viral pathogens, including PoRV G3, PoRV G4, porcine epidemic diarrhea virus (PEDV), porcine epidemic diarrhea (PDCoV), and porcine reproductive and respiratory syndrome virus (PRRSV). In the clinical sample detection using the RAA-CRISPR/Cas12a method and qPCR, Cohen’s Kappa value reached as high as 0.952. Furthermore, this approach eliminates the need for large-scale instrumentation, offering a visual result under an ultraviolet lamp through fluorescence signal output. Full article

In the field of cattle medicine in Austria, to date, few studies have investigated the presence of methicillin-resistant Staphylococcus aureus and extended-spectrum β-lactamase-producing Escherichia coli in Austria. For this reason, milk and nasal samples were examined for the presence of methicillin-resistant Staphylococcus aureus as well as fecal samples for extended-spectrum cephalosporin-resistant Escherichia coli. The nasal and fecal swabs were collected during the veterinary treatment of calf pneumonia and calf diarrhea. For the milk samples, the first milk jets were milked into a pre-milking cup and then the teats were cleaned and disinfected before the samples were taken. The cows were selected during the veterinary visits to the farms when treatment was necessary due to mastitis. Depending on the severity of the mastitis (acute mastitis or subclinical mastitis), antibiotics and non-steroidal anti-inflammatory drugs were given immediately (acute disease) or after completion of the antibiogram (subclinical disease). Isolates were characterized by a polyphasic approach including susceptibility pheno- and genotyping and microarray-based assays. No methicillin-resistant Staphylococcus aureus was found in the milk samples, but one nasal swab was positive for methicillin-resistant Staphylococcus aureus. Twenty-two Escherichia coli isolates were detected among the fecal samples. All the Escherichia coli isolates were resistant to ceftazidime. In all the Escherichia coli isolates, genes from the bla_CTX family were detected with other bla genes or alone; the most frequently observed β-lactamase gene was bla_CTX-M-1/15 (n = 20). In total, 63.6% (n = 14) of the isolates exhibited a multidrug-resistant phenotype and one E. coli isolate (4.5%) harbored the AmpC gene. Precisely because the presence of data regarding extended-spectrum cephalosporin-resistant Escherichia coli and methicillin-resistant Staphylococcus aureus in calves and cows in Austria is rare, this study further expands our understanding of antimicrobial resistance in Austrian cattle, which is highly relevant for successful antibiotic therapy in sick cattle. Full article

Antibiotic resistance in cyanobacteria represents a global threat to public health. The widespread presence of cyanobacteria in aquatic environments exposes them to antibiotic contamination. Cyanobacteria are also in direct contact with pathogenic bacteria containing antibiotic-resistance genes (ARGs), which impart these characteristics to them. This study aims to examine the presence of some ARGs in locally isolated cyanobacteria species, Spirulina laxa, Chroococcus minutes, Oscillatoria princeps, Oscillatoria proteus, Oscillatoria terebriformis, and Lyngbya epiphytica, and compare the presence of these genes in two pathogenic bacteria, Escherichia coli and Klebsiella pneumoniae. Ampicillin (Ap) and erythromycin (Em) resistance genes were detected in five algal samples. Meanwhile, Chloramphenicol (Cm) and gentamicin (Gm) resistance genes were apparent in only two species. Genes encoding resistance towards kanamycin (Km) and spectinomycin (Sp) were recorded in three specimens. It was also found that E. coli possessed resistance genes for four antibiotics, ampicillin (Ap), erythromycin (Em), gentamicin (Gm), and kanamycin (Km), whereas K. pneumoniae was resistant towards three antibiotics, ampicillin (Ap), gentamicin (Gm), and kanamycin (Km). The results show that there is a match in antibiotic-resistance genes in both cyanobacteria and pathogenic bacteria. Suggesting the possibility that cyanobacteria could acquire ARGs from the environment through horizontal gene transfer. Thus, freshwater cyanobacteria may play a significant role in the prevalence of ARGs in their environment. Full article

Tick-borne pathogens are growing in importance for human and veterinary research worldwide. We developed, optimized, and validated a reliable quantitative PCR (qPCR; real-time PCR) assay to assess Borrelia burgdorferi infection by targeting two B. burgdorferi genes, ospA and flaB. When assessing previously tested tick samples, its performance surpassed the nested PCR in efficiency, sensitivity, and specificity. Since the detection of Borrelia is more difficult in mammalian samples, the qPCR assay was also assessed using wildlife tissues. For wildlife samples, the sensitivity and specificity of ospA primers, with the incorporation of a pre-amplification step, was equivalent or superior to the nested PCR. For human samples, no primer set was successful with human tissue without culture, but we detected Borrelia with ospA and flaB primers in 50% of the Lyme culture samples, corresponding to 60% of the participants with a Lyme disease diagnosis or suspicion. The specificity of amplification was confirmed by Sanger sequencing. The healthy participant culture samples were negative. This PCR-based direct detection assay performs well for the detection of Borrelia in different biological samples. Advancements in detection methods lead to a better surveillance of Borrelia in vectors and hosts, and, ultimately, enhance human and animal health. Full article

Background: Targeted next-generation sequencing (NGS) panels are increasingly being utilized to identify actionable gene amplifications (copy number > 4) among solid tumors. Methods: This study validated the analytical performance of two amplicon-based NGS assays, the Oncomine Comprehensive Panel (OCAv3) and the Oncomine Focus Assay (OFA), for detecting gene amplification in formalin-fixed paraffin-embedded (FFPE) tumors of varying cellularity. OCAv3 was assessed for amplification detection in 756 FFPE samples comprising various tumor types. Results: We demonstrated that with standardized quality control metrics, including median absolute pairwise difference score, these assays can achieve a near-perfect positive predictive value, although their sensitivity for detecting amplifications significantly decreased in tumors with cellularity below 30%. Stratifying tumor cellularity into 10–30%, 31–60%, and 61–95% groups revealed significantly higher gene amplification detection rates in the 31–60% and 61–95% groups versus the 10–30% group (20.6% and 26.7% vs. 9.2%, p < 0.0001). When considering all detected gene amplifications, the average amplification calling per sample was nearly five-fold lower in the 10–30% group versus the 61–95% group (0.11 vs. 0.52; p < 0.0001). To further investigate the analytic performance of OCAv3 in detecting ERBB2 amplification, we analyzed a cohort of 121 uterine carcinomas with confirmed ERBB2 status by HER2 IHC or FISH, in which a threshold incorporating amplifications and tumor cellularity achieved 79% sensitivity and 100% specificity, potentially eliminating the need for FISH analysis in 34% of equivocal cases. In a separate validation cohort, similar analytical performance was observed, with the threshold demonstrating consistent sensitivity and specificity. Conclusions: This study highlights the strengths and limitations of amplicon-based NGS assays in detecting amplifications using real-world data. Full article