Search Results (204)

Background: Peyronie’s disease (PD) is a chronic inflammatory condition that affects the penile albuginea. Oxidative stress (OS) plays a crucial role in the development of the disease, prompting us to investigate OS levels at the site of the disease and in peripheral blood. This article presents our second study in which the OS was evaluated by calculating the OS index (OSI) in blood samples taken directly from the penile corpora cavernosa of patients with PD. Our innovative diagnostic method, which focuses on the analysis of oxidative stress (OS) in the corpora cavernosa of the penis, allows us to accurately identify the “chemical” signals (OS levels) of the pathology in the area where it is present. Methods: Our study included 102 PD patients from our Peyronie’s care center and 100 control cases. To conduct a comprehensive OS analysis, we measured both the total oxidant status (TOS) and total antioxidant status (TAS) and calculated the oxidative stress index (OSI) as OSI = TOS/TAS × 100. Blood samples were collected from the penis and a vein in the upper extremity, and OS was measured using d-ROMs and PATs (FRAS kit). Results: Pearson’s analyses revealed a significant statistical correlation between penile OSI values and PD plaque volumes (p = 0.003), while no correlation was found between systemic OSI values and plaque volumes (p = 0.356). Penile OSI values decreased significantly after PD plaque removal (p < 0.0001). A comparison of penile OSI values in PD patients (post plaque removal) and the control group showed no significant differences (p = 0.418). Conclusions: The lack of correlation between systemic OSI values and Peyronie’s plaque volume suggests that direct sampling from the site of the disease is preferable for OS studies. Conducting a penile OSI study could provide a precise oxidative marker dependent on plaque volume. In addition, the penile OSI study can biochemically monitor the therapeutic result, alongside penile ultrasound imaging. Full article

Background: Cancer vaccines represent a groundbreaking advancement in cancer immunotherapy, utilizing tumor antigens to induce tumor-specific immune responses. However, challenges like tumor-induced immune resistance and technical barriers limit the widespread application of predefined antigen vaccines. Here, we investigated the potential of viral protein R (Vpr) peptides as effective candidates for constructing anonymous antigen vaccines in situ by directly injecting at the tumor site and releasing whole-tumor antigens, inducing robust anti-tumor immune responses to overcome the limitations of predefined antigen vaccines. Methods: The cytotoxic effects of Vpr peptides were evaluated using the CCK8 reagent kit. Membrane penetration ability of Vpr peptides was observed using a confocal laser scanning microscope and quantitatively analyzed using flow cytometry. EGFR levels in the cell culture supernatants of cells treated with Vpr peptides were evaluated using an ELISA. Surface exposure of CRT on the tumor cell surface was observed using a confocal laser scanning microscope and quantitatively analyzed using flow cytometry. The secretion levels of ATP from tumor cells were evaluated using an ATP assay kit. HMGB1 release was evaluated using an ELISA. Mouse (Male C57BL/6 mice aged 4 weeks) MC38 and LLC bilateral subcutaneous tumor models were established to evaluate the therapeutic effects of Vpr peptides through in situ vaccination. Proteomic analysis was performed to explore the mechanism of anti-tumor activity of Vpr peptides. Results: Four Vpr peptides were designed and synthesized, with P1 and P4 exhibiting cytotoxic effects on tumor cells, inducing apoptosis and immunogenic cell death. In mouse tumor models, in situ vaccination with Vpr peptide significantly inhibited tumor growth and activated various immune cells. High-dose P1 monotherapy demonstrated potent anti-tumor effects, activating DCs, T cells, and macrophages. Combining ISV of P1 with a CD47 inhibitor SIRPαFc fusion protein showed potent distant tumor suppression effects. Proteomic analysis suggested that Vpr peptides exerted anti-tumor effects by disrupting tumor cell morphology, movement, and adhesion, and promoting immune cell infiltration. Conclusions: The designed Vpr peptides show promise as candidates for in situ vaccination, with significant anti-tumor effects, immune activation, and favorable safety profiles observed in mouse models. In situ vaccination with Vpr-derived peptides represents a potential approach for cancer immunotherapy. Full article

DNA nanoball sequencing (DNBSEQ) is one of the most rapidly developing sequencing technologies and is widely applied in genomic and transcriptomic investigations. Recently, a new PE300 sequencing option primarily recommended for amplicon analysis was released for DNBSEQ-G99 and G400 devices. Given their unprecedentedly high data yield per flow cell, the new PE300 kits could be a great choice for various sequencing tasks, but we found that combining different types of DNA libraries in a single run could lead to undesired artifacts in the data. In this study, we investigate the occasional read cross-contamination that we first observed in our DNBSEQ PE300 run. The phenomenon, which we refer to as “software contamination”, is not actual contamination but primarily manifests as improper forward/reverse read pairing, improper demultiplexing, or as “digital chimeric” reads. Although rare, these artifacts were found in all runs we have analyzed, including several MGI demo datasets (both PE100 and PE150). In this study, we demonstrate that these artifacts arise primarily from the incorrect resolution of sequencing signals produced by neighboring DNA nanoballs, leading to mixing out forward and reverse reads or improper demultiplexing. The artifacts occur most frequently with read pairs where the length of insert sequence is shorter than the read length. Based on a few external NA12878 human exome sequencing data, we conclude that the total improper pairing rate in DNBSEQ data is comparable to Illumina ones. Overall, the problem only affects the analysis results when simultaneously sequenced libraries have markedly different insert size distribution or flow cell loading. Additionally, we demonstrate here that raw DNBSEQ data might contain ~2% optical duplicates, resulting from the same effect of close neighboring of DNB-sites in the flow cell. Full article

Heavy metal and cyanide contamination in soil presents serious environmental and ecological concerns due to their persistence, bioavailability, and toxicity to soil biota. In this study, a novel solid-phase direct contact bioassay kit was developed using immobilized Chlorella vulgaris spheres to evaluate the toxicity of soils contaminated with mercury (Hg²⁺), silver (Ag⁺), copper (Cu²⁺), and cyanide (CN⁻). The assay was designed using 25 mL glass vials in which algal spheres were directly exposed to spiked soils for 72 h without the need for pollutant extraction. Oxygen evolution in the headspace was measured as the primary endpoint, alongside optical density and chlorophyll a fluorescence (OJIP) to assess photosynthetic inhibition. The assay demonstrated high sensitivity and reproducibility, with strong correlations (R² > 0.93) between oxygen evolution and optical density. EC₅₀ values based on oxygen evolution were 4.43, 4.18, 3.10, and 61.3 mg/kg for Hg²⁺, Ag⁺, CN⁻, and Cu²⁺, respectively, and 7.8, 7.4, 2.9, and 29.7 mg/kg based on optical density. The relatively higher EC₅₀ for copper was attributed to its biological role as an essential micronutrient. OJIP transient profiles supported the observed photosynthetic inhibition, particularly under Hg²⁺, Ag⁺, and CN⁻ exposure. The present study overcomes the limitations of conventional chemical analyses by providing a rapid, low-cost, and ecologically relevant tool for direct soil toxicity assessment, with potential applications in environmental monitoring and contaminated site evaluation. Full article

Changes in land use and agricultural practices have altered the resilience of plant communities and can lead to the emergence of invasive species. One of these is the perennial grass species Bothriochloa ischaemum (L.) Kleng., whose diversity-reducing effects are known from several studies. Our exploratory questions were as follows: How does the presence of B. ischaemum affect the diversity and ratio of the species of sandy grasslands? To what extent does this diversity change depend on site characteristics? The supporting studies were carried out in five low-lying sand dune slacks and six relatively higher areas in the upper-intermediate part of the dunes and on an abandoned old field located in the Hungarian Great Plain in the Carpathian Basin. The cover of vascular plant species was recorded in all sampling sites in twelve 2 by 2 m plots, and the dataset was analysed using agglomerative cluster analyses and a non-parametric Kruskal–Wallis test. Five significantly different groups were identified, separating the vegetation types of the sides of the sand dunes, the vegetation types of the dune slack and the old field, and a Stipa borysthenica Kolkov ex Prokudin-dominated vegetation type. Our results suggest that B. ischaemum is only present as small tussocks on the drier, more exposed sides of dunes, with 3.9–24.2% average coverage; is less able to outcompete Festuca vaginata Waldst. et Kit. ex Willd. and S. borysthenica; and is only able to form large tussocks mainly in the lower dune slacks, with 45.6–79.5% average coverage. Here, in the wetter areas, it achieves high cover with a considerable accumulation of litter, and it becomes a dominant species in this association. The diversity-reducing effect of B. ischaemum on old-field grasslands depends on the age of the site and on the stability of the vegetation. Full article

Central nervous system (CNS) tumors are the most common solid malignancy in the pediatric population. These lesions are the result of the aberrant cell signaling step proteins, which normally regulate cell proliferation. Mitogen-activated protein kinase (MAPK) pathways and tyrosine kinase receptors are involved in tumorigenesis of low-grade gliomas. High-grade gliomas may carry similar mutations, but loss of epigenetic control is the dominant molecular event; it can occur either due to histone mutations or inappropriate binding or unbinding of DNA on histones. Therefore, despite the absence of genetic alteration in the classic oncogenes or tumor suppressor genes, uncontrolled transcription results in tumorigenesis. Isocitric dehydrogenase (IDH) mutations do not predominate compared to their adult counterpart. Embryonic tumors include medulloblastomas, which bear mutations of transcription-regulating pathways, such as wingless-related integration sites or sonic hedgehog pathways. They may also relate to high expression of Myc family genes. Atypical teratoid rhabdoid tumors harbor alterations of molecules that contribute to ATP hydrolysis of chromatin. Embryonic tumors with multilayered rosettes are associated with microRNA mutations and impaired translation. Ependymomas exhibit great variability. As far as supratentorial lesions are concerned, the major events are mutations either of NFkB or Hippo pathways. Posterior fossa tumors are further divided into two types with different prognoses. Type A group is associated with mutations of DNA damage repair molecules. Lastly, germ cell tumors are a heterogeneous group. Among them, germinomas manifest KIT receptor mutations, a subgroup of the tyrosine kinase receptor family. Full article

Background/Objectives: Gastrointestinal stromal tumors (GISTs) are rare mesenchymal tumors of the gastrointestinal (GI) tract, predominantly driven by KIT or PDGFRα oncogene mutations. Nonspecific symptoms contribute to diagnostic delays, with general practitioners (GPs) playing a pivotal role in early detection. However, studies on GIST-specific primary care pathways are limited. This study examines GP contacts, diagnoses, and prescribed drugs in primary care during the 12 months preceding GIST diagnosis. Methods: This case-control study utilized data from the Netherlands Cancer Registry and Nivel Primary Care Database. It included 294 GIST patients diagnosed between 2010 and 2020 and 576 matched cancer-free controls. GP contacts, diagnoses, and newly prescribed drugs were analyzed across two time intervals: 0–4 and 5–12 months prediagnosis. Statistical comparisons were conducted using the Wilcoxon rank-sum test and descriptive analyses. Results: GIST cases had a median of six GP contacts (IQR 4–11) in the 12 months prediagnosis versus three (IQR 2–6) for controls (p < 0.05). Contacts increased 4 months before diagnosis, peaking 1 month prior. Common diagnoses in the 4-month interval included malignant neoplasms of the stomach (27.9%) and other digestive sites (27.6% and 11.2%), abdominal pain (9.5%), and iron deficiency anemia (9.5%). Newly prescribed drugs included proton pump inhibitors (13.9%) and osmotically acting laxatives (15.0%). Conclusions: This study highlights increased GP visits and specific reasons for these visits before GIST diagnosis. Future research should further examine GP records, not only through coded data but also unstructured data, and incorporate patient and GP perspectives to explore potential improvements in the diagnostic process. Full article

A data-driven 3D simulation for the robotic automation of the most labor-intensive packaging process in meal kit production was developed using Tecnomatix plant simulation software. The workflow and environments of the existing manual process were analyzed. An existing production site was scanned using a 3D Lidar scanner to create 3D models and design the initial assembly layout. Two types of 3D simulation models, implemented with a single or double delta robot, were designed to determine the optimal robot-automated packaging process. Key performance indicators for simulation models of a manual and two robot automation systems were analyzed. The throughputs of the manual, single delta robot and double delta robot models were 2112, 1510, and 2568 ea/h, respectively. The single robot system achieved only 68.3% of the throughput of the manual process, which is attributed to a cycle time of 2.36 s for picking and placing all components. On the other hand, the cycle time of the double robot system was 1.66 times faster, and the throughput was 1.7 times greater compared to the single robot system. The developed 3D simulation model for the meal kit packaging system demonstrates the potential of robotic automation in addressing the labor shortage issue as well as improving production efficiency. Full article

Background: The alarming increase in carbapenemase-producing Enterobacterales is a matter of grave public health concern. The most ubiquitous carbapenemases, Klebsiella pneumoniae carbapenemase (KPC)-, New Delhi metallo-β-lactamase (NDM)-, and oxacillinase (OXA-48)-like enzymes, belong to the Ambler molecular classes A, B, and D, respectively. KPC- and OXA-48-like enzymes have a serine-based hydrolytic mechanism, while NDMs are metallo-β-lactamases that contain zinc in the active site. For the judicious use of reserve drugs and promoting antimicrobial stewardship, timely detection of carbapenemases is essential. While molecular tools are the gold standard for the detection of these enzymes, many laboratories have limited access to them. This study focused on evaluating in-house tools and commercial phenotypic tests for the detection of OXA-48-, KPC-, and NDM-like enzymes in K. pneumoniae, the predominant extremely drug-resistant pathogen in Oman. Methods: In total, 80 GeneXpert/PCR-confirmed (40 OXA-48 and 20 KPC and NDM each) and 37 whole-genome-sequenced (25 OXA-232 and 6 KPC-2, plus NDM-1 and NDM-5) K. pneumoniae were subjected to screening by temocillin (30 μg disk) (MAST Diagnostica, Germany) and D71C (MASTDISCS^®). Isolates resistant to temocillin (<11 mm) and D71C were subjected to four tests: an in-house tool (OXA-48 disk test) and three commercial phenotypic tests: (i) the MASTDISCS^® Combi (D72C) (MAST Group Ltd., Bootle, UK); (ii) the MASTDISCS^® Combi (D73C) (MAST Group Ltd., UK); and (iii) an immunochromatographic assay (ICT), which is the KPC/IMP/NDM/VIM/OXA-48 Combo test kit (Medomics, China), for the detection of OXA-48-, KPC-, and NDM-like carbapenemases. Results: Temocillin exhibited good sensitivity and specificity (100% and 97.50%) compared to D71C (70% and 100%). Among the confirmatory tests, the in-house OXA-48 disk test had 92.50% sensitivity and 100% specificity, while the commercial MAST DISC tests D72C, D73C, and ICT had 97.50%, 95.00%, and 100% sensitivity and 100%, 91.67%, and 95% specificity, respectively. Conclusions: The temocillin disk test is a good screening tool. With high sensitivity and specificity, ease of performance, short turnaround time, and low cost, we recommend the ICT format for routine diagnostic use. In resource-constrained centers, the OXA-48 disk test is an excellent alternative with high sensitivity and specificity. Full article

Introduction: Pancreatic ductal adenocarcinoma (PDAC) is insidious, with only 15–20% of those diagnosed suitable for surgical resection as it is either too advanced and has invaded local structures or has already spread to distant sites. The associated tumor microenvironment provides a protective shield which limits the efficacy of chemotherapeutic agents, but also impairs the delivery of nutrients required for the PDAC cells. To compensate for this, metabolic adaptions occur to provide alternative sources of fuel. The aim of this study is to explore metabolomic differences between participants with resectable PDAC compared to healthy volunteers (HV). The objectives were to use nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS) to determine if resectable PDAC induces sufficient metabolic adaptations and variations which could be used to discriminate between the two groups. Methods: Plasma samples were collected from fasted individuals with resectable PDAC (n = 23, median age 68 [IQR 56–75], 69.6% male) and HV (n = 24, median age 63 [IQR 58–71], 54.2% male). Samples were analyzed using NMR and the Biocrates MxP Quant 500 kit at University Hospital Southampton. Results: NMR spectroscopy identified six independent metabolites that significantly discriminated between the PDAC and HV groups, including elevated plasma concentrations of 3-hydroxybutyrate and citrate, with decreased amounts of glutamine and histidine. MS analysis identified 84 metabolites with a significant difference between the PDAC and HV cohorts. The metabolites with a fold change (FC) > 1.5 in the PDAC population were conjugated bile acids (taurocholic acid, glycocholic acid, and glycochenodexoycholic acid). Discussion: In conclusion, using metabolomics, biochemical differences between resectable PDAC and HV were detected. These differences indicate metabolic plasticity and utilization of alternative fuel sources. Full article

Objective: This in vitro study aimed to investigate the impact of folic acid on DNA methylation and gene expression in adipocytes from subcutaneous adipose tissue of patients with systemic lupus erythematosus (SLE), with a focus on the influence of obesity on these epigenetic changes. Methods: Tissue biopsies were collected from patients with normal weight (NW) and obesity (OBS). Adipocytes were isolated via enzymatic digestion and density separation. Each group was divided into control (standard medium) and folic acid treatment (2 mg/24 h for 48 h) conditions. After treatment, DNA methylation levels were analyzed using the Infinium Methylation EPIC v2.0 Kit, and gene expression analyses were performed by RT-qPCR. A pathway enrichment analysis was conducted using the KEGG database for functional insight. Results: Folic acid induced differential methylation at 755 CpG sites in NW adipocytes, which were associated with immune regulation, including MAPK signaling. Also, OBS adipocytes showed methylation changes at 92 CpG sites, affecting pathways related to metabolic regulation, such as cAMP signaling. LEP gene expression was upregulated (5.2-fold) in OBS adipocytes, while CREM2 expression was increased (2.8-fold) in NW adipocytes after treatment. These gene expression differences underscore weight-dependent responses to folic acid, with LEP upregulation in OBS cells suggesting links to metabolic dysregulation and CREM2 upregulation in NW cells potentially contributing to immune modulation. Conclusions: Folic acid treatment exerts distinct epigenetic and gene expression effects in adipocytes of SLE patients, modulated by obesity status. This weight-dependent response, marked by changes in pathways relevant to immune and metabolic function, highlights the need for further investigation into how nutrient-based interventions might support SLE management. From a clinical perspective, this study underscores the potential of targeted nutrient-based interventions to address immunometabolic dysfunctions in SLE patients. Further research could explore folic acid supplementation as a complementary approach to personalized treatment strategies, particularly for patients with obesity. Full article

Although the Universal Salt Iodization (USI) program has been highly successful, it remains relevant due to the continued risk of Iodine Deficiency Disorders (IDDs) in vulnerable groups, such as children and pregnant women. This program empowers the relevant authority to continuously monitor iodine levels in iodized salt. Our study reports on the use of a Salt Iodate Micro-Method Reagent (SIMR) detection kit for this purpose. The kit was validated, with a linearity of 5.0–60.0 mg/Kg, at a detection limit of 6.8 mg/Kg, with excellent recovery ranging from 93.0 to 108.3%, whereas the repeatability, intermediate precision, and reproducibility achieved a mean coefficient of variation (CV) of 5.3%, 6.8%, and 5.9%, respectively. The stability of the reagents used in the kit was tested using freshly prepared iodine standard quality control (QC) samples of 20.0 mg/Kg and 40.0 mg/Kg, all of which were observed to be stable, within the range of the mean ± 2 × (standard deviation, SD), for 10 days. The suitability of the kit was proven when no difference was found in the mean results of 70 salt samples, using a paired t-test and the Bland–Altman plot, compared to the reference method, at a 95% confidence interval (CI). Thus, the SIMR detection kit is a highly feasible alternative method for iodine monitoring, with a fast analysis time, as well as being cost effective, and environmentally friendly. Full article

Trigonella foenum-graecum is a widely cultivated legume in Mediterranean regions, and it is used for human and animal consumption, as well as for medical purposes. High temperatures and abundant rainfall during the spring season in Sicily favor the formation of an environment suitable for the growth and proliferation of fungi with the production of mycotoxins. In this study, ochratoxin A, aflatoxin, deoxynivalenol, zearalenone, fumonisin, and T-2 toxin concentrations in Trigonella foenum-graecum were determined in feed administered to ruminants and also in blood samples from cattle and sheep in order to evaluate the toxicity correlated to the possible presence of these mycotoxins based on the clinical signs observed in the animals. Analyses of mycotoxins in fenugreek and blood samples were conducted using the enzyme immunoassay KIT. Five extensive farms sited in the northwest of the Sicily region, with a total of 90 intoxicated animals, reported a concomitant unusual outbreak of neurological disorders. Decreased spinal reflex responses, postural abnormalities associated with weakness or recumbency, and hyperesthesia of the limbs suggested a problem regarding the peripheral nervous system. The mortality rate recorded was very high, even reaching 100% of the intoxicated animals. OTA intoxication in Sicilian ruminants represents an important warning on the vulnerability of farms to mycotoxin contamination and underlines the importance of preventive measures and monitoring in animal health management. Full article

SNAP-tag and Halo-tag have been employed to achieve targeted RNA editing by directing the deaminase domain of human ADAR to specific sites in the transcriptome. This targeting is facilitated by short guide RNAs (gRNAs) complementary to the target transcript, which are chemically modified with benzylguanine or chloroalkane moieties to enable covalent binding to the respective self-labeling enzymes. However, broad application of this approach has been limited by challenges such as low scalability, the requirement for specialized chemical expertise and equipment, and labor-intensive protocols. In this study, we introduce streamlined, efficient protocols for the synthesis and purification of these linkers, suitable for SNAP-tag and Halo-tag applications, without the need for advanced chemical equipment. Our methods enable linker coupling in a kit-like manner and support the high-yield production of modified gRNAs. We demonstrate that the newly synthesized linkers and gRNA designs perform similarly to previously published constructs with regard to RNA editing efficiency. Moreover, large-scale production of modified gRNAs facilitates their use in studies involving cellular uptake and in vivo experiments. Full article

Background/Objectives: The study of DNA transfer and persistence has become increasingly significant, driven by advancements in DNA detection sensitivity and the need for reliable forensic evidence. In forensic investigations, saliva and saliva-stained materials are recognised as valuable DNA sources, particularly in cases of homicide, sexual assault, and burglary, where saliva can be transferred between individuals during the criminal act. The time between the crime and sample collection is a critical factor that can influence the success of the analysis. The value of the specimens collected from the victim’s skin or mouth (perilabial and labial sites, teeth and tongue) after the crime has not been investigated with currently used highly sensitive and specific molecular methods. Methods: On the assumption that a significant loss of DNA occurred, in our study, 10 voluntary pairs were tested at different time points after intense kissing and samples were taken from the above-mentioned sites to assess the presence of the donor’s DNA. Extracted DNA was quantified using the Plexor HY System kit (Promega), and both autosomal STRs and Y-STRs were analysed. Results: The results reveal a greater persistence of male DNA on the female partner, particularly in the labial and perilabial regions, even up to 120 min after contact, in terms of both concentration and duration. Conclusions: This study emphasises the forensic importance of salivary DNA as a solid source of evidence, particularly in investigations involving mixed DNA profiles. Full article