Search Results (2,889)

Non-invasive temperature estimation during online operation is a critical challenge in enclosed micro-reaction systems, particularly when the thermal mass of the working fluid varies dynamically or is uncertain. Conventional model-based approaches typically rely on fixed thermal parameters, leading to significant estimation errors when the actual reagent volume deviates from nominal conditions. To address this limitation, this study proposes a volume-adaptive temperature estimation framework applied to an ultrafast quantitative polymerase chain reaction (qPCR) system. By modeling the heat-transfer pathways via a simplified resistance–capacitance (RC) network, a nonlinear least squares (NLS) algorithm within an output-error (OE) framework is employed to identify key thermal parameters online. The framework separates the estimation into an offline calibration stage—where a thermocouple-equipped chip provides ground-truth data—and an online deployment stage that relies solely on non-invasive external measurements. This approach allows the system to explicitly compensate for volume-induced variations in thermal inertia. Validation experiments on an ultrafast qPCR platform with reagent volumes ranging from 100 to 250 $\mathsf{\mu }\mathrm{L}$ and heating rates exceeding 20 °C/s demonstrate that the method achieves robust performance, maintaining a mean absolute error (MAE) of reagent temperature at 0.24 ℃ and restricting the average volume estimation error to within 1.37 $\mathsf{\mu }\mathrm{L}$. DNA gel electrophoresis results further confirm the biological reliability of the temperature prediction strategy by verifying amplification specificity. This work provides a generalised solution for precise thermal management in micro-systems subject to variable thermal loads. Full article

Accurate identification of cariogenic bacteria is crucial for understanding caries development in children. Classical culture methods often underestimate microbial diversity, while polymerase chain reaction (PCR) can detect species that are difficult to cultivate. The aim of this study was to compare culture-based and PCR-based methods for detecting key cariogenic microorganisms in the carious dentin of pediatric patients. Thirty dentin samples were collected from the permanent teeth of children aged 8–14 years. Parallel analyses were performed using standard culture techniques and PCR targeting the gtfB gene of S. mutans and the 16S rRNA gene of Lactobacillus spp. Culture results were quantified as colony-forming units, while PCR results were classified as negative, low-positive, or positive. The results show that culture-based methods identified S. mutans in 16.7% of the samples and Lactobacillus spp. in 3.3%, while PCR identified a signal for S. mutans in 43.3% and Lactobacillus spp. in 100% of the samples. PCR-based methods provide higher sensitivity for detecting key cariogenic bacteria, including S. mutans and Lactobacillus spp. However, PCR detects bacterial DNA and does not indicate bacterial viability or activity. Combining molecular and culture-based approaches allows a more comprehensive assessment of the cariogenic microbiota, supporting accurate microbiological evaluation in pediatric caries research. Full article

Background/Objectives: The treatment of gynecologic malignancies has moved towards a precision medicine model with an approach to prognostication and management based on biomarker testing. The objective of this review is to describe the current landscape of biomarker testing in gynecologic cancer including clinical implications and the approach to testing. Methods: A review of the literature was performed that included published clinical trials which utilized biomarker testing as part of inclusion/exclusion criteria, prospective trials that addressed the application and scoring of biomarkers utilized in gynecologic cancers, prospective clinical trials that utilized biomarker findings to determine management, and national or society guidelines for the scoring of biomarkers and treatment of gynecologic cancers. Findings: The use of biomarker testing as part of the management of gynecologic cancers is the standard of care for both treatment and prognostication. In endometrial cancer, biomarker testing has been incorporated into the staging system and impacts treatment in both the upfront and recurrent setting. Specific biomarkers of interest for endometrial cancer include estrogen receptor (ER), progesterone receptor (PR), DNA Polymerase Epsilon (POLE), mismatch repair proteins (MMR), and Human Epidermal growth factor Receptor-2 (HER2). In ovarian cancer, biomarker testing is primarily utilized in the recurrent setting to guide management of platinum-resistant ovarian cancer with a specific focus on targeted therapy utilizing antibody drug conjugates (ADCs) and immunotherapy. Immunotherapy has become an important component of therapy for cervical cancer and Programmed death-ligand 1 (PD-L1) testing is a key biomarker in determining treatment. Conclusions: The utilization of appropriate assays and processes to accurately assess the status of biomarkers in the pathology laboratory is crucial to the treatment of gynecologic malignancies in the precision medicine era. Full article

Background: Curcumin, a dietary polyphenol with anticancer potential, has been reported to enhance the efficacy of chemotherapeutic agents. Methods: The effects of combined curcumin and doxorubicin treatment on apoptosis and associated signaling pathways were investigated in human osteosarcoma HOS cells. Results: Combined treatment significantly reduced cell viability and induced apoptotic morphological changes, which were confirmed by increased sub-G1 population, enhanced DNA fragmentation, and elevated cleaved poly(ADP-ribose) polymerase (PARP) levels. Mechanistically, combined treatment markedly increased c-Jun N-terminal kinase (JNK) phosphorylation, whereas extracellular signal-regulated kinase (ERK) phosphorylation showed no appreciable change. Pharmacological inhibition revealed that JNK suppression attenuated PARP cleavage, while ERK inhibition also reduced apoptotic responses, suggesting a permissive role of basal ERK activity. In addition, combined treatment was associated with increased expression of the endoplasmic reticulum stress marker GRP78 and modulation of autophagy-associated markers. Although thioredoxin domain-containing protein 5 (TXNDC5) expression was reduced, TXNDC5 overexpression failed to attenuate apoptosis, indicating that apoptosis induction occurs independently of TXNDC5. Conclusions: These findings indicate that combined curcumin and doxorubicin induce apoptosis primarily through JNK-dependent MAPK signaling, accompanied by stress-associated cellular responses. Full article

Inflammatory joint diseases, including osteoarthritis, are multifactorial disorders in which dysregulated innate immune signaling and non-coding RNA (ncRNA)-mediated regulation of gene expression play essential roles. MicroRNA-155 (miR-155), its host gene MIR155HG, and Toll-like receptor 4 (TLR4) form a tightly linked inflammatory signaling axis, yet their combined genetic variability in chronic joint inflammation remains insufficiently characterized. The aim of this study was to investigate genetic variants in MIR155HG exon 3, mature miR-155, and TLR4 exon 3 and assess their potential synergistic role in chronic inflammatory joint disease. A case–control study was conducted with 100 cases (50 osteoarthritis patients and 50 matched healthy controls). Genomic DNA was analysed using polymerase chain reaction (PCR) and Sanger sequencing. Variant alleles and genotypes were identified, and their allele frequencies and genotypes were calculated using Mutation Surveyor. Detected variants were compared with public databases, and in silico tools were used to estimate the structural impact of TLR4 missense mutations. Sixteen heterozygous variants were identified in MIR155HG exon 3, most of them novel and population-specific. Interestingly, the highest variant frequencies for MIR155HG exon 3 were observed at positions 12448G>GC and 12481T>TA (both 64.3%), followed by 12442T>TC (57.1%). Additionally, two novel variants were detected in the miR-155 gene (chr21:29,694,314 G>A and chr21:29,646,351 T>C), each present at an allele frequency of 7.1% and absent from current external variant databases. Moreover, two rare TLR4 exon-3 variants were identified; a synonymous variant, c.147C>A (Pro49Pro; rs375037549), and a missense mutation, c.148G>A (Asp50Asn; rs776561489). Notably, in silico analyses and molecular dynamic simulations indicated that the Asp50Asn (D50N) substitution destabilizes the TLR4 protein. Conclusion: Concurrent variants in MIR155HG, miR-155, and TLR4 suggest a convergent regulatory molecular axis that may contribute to disease susceptibility and inflammatory progression. Full article

The insulin-like growth factor 1 (IGF-1) gene plays a key role in growth and production traits in livestock. Limited information is available regarding its genetic polymorphisms in Saudi camel breeds. This study aimed to investigate genetic variation in the IGF-1 gene among Saudi camel breeds to provide baseline genetic information for future association studies. A total of 176 camels representing six Saudi breeds were sampled. DNA was extracted and Polymerase chain reaction (PCR) amplification and Sanger sequencing were applied to detect IGF-1 polymorphisms. Genotype and allele frequencies were calculated across breeds, and statistical comparisons were performed based on proportional distributions to account for unequal sample sizes. Two single-nucleotide polymorphisms (SNPs) were identified: c.365G>A in exon 3 and c.435C>T in exon 5. The exon 3 variant resulted in a missense mutation (p. Arg122His) but was detected in heterozygous form in only one camel, and subsequent screening of 109 additional samples confirmed its rarity. The exon 5 variant was synonymous in isoform X1 and located in the 3′ untranslated region of isoform X2. Sequencing of 176 camels revealed that c.435C>T was highly polymorphic across the examined breeds. Significant differences in genotype frequencies were observed within and among breeds (p < 0.001). The CT genotype predominated in Waddah (60%), Shageh (48%), and Sofor (60%), significantly exceeding CC and TT frequencies (p < 0.001). In Majaheem and Saheli, CT (47%) and TT (45%) were nearly equal and both significantly higher than CC (p < 0.001). Shaele exhibited a distinct pattern, with TT being most frequent (57%), significantly higher than CC (7%, p < 0.001) and CT (36%, p < 0.01). These findings indicate directional selection favoring the C allele in the Waddah and Shageh breeds, whereas the T allele is favored in the remaining breeds. This study provides the first baseline characterization of IGF-1 polymorphisms among Saudi camel breeds. Although no phenotypic associations were assessed, the results offer a foundation for future research examining relationships between IGF-1 variants and economically important traits. Full article

Background/Objectives: Poly(ADP-ribose) polymerase 1 (PARP1) is an important therapeutic target in DNA repair-deficient cancers, but discovery of new inhibitors remains constrained by scaffold convergence, tolerability limits, and acquired resistance. This study aimed to develop an interpretable, reliability-stratified cheminformatics workflow for PARP1 potency prioritization and structure-based follow-up. Methods: A curated ChEMBL dataset of 3339 PARP1 inhibitors was encoded using RDKit 2D descriptors and Avalon fingerprints (1143 initial features), then reduced to 132 informative variables by Random Forest-based feature selection. Five regression models were optimized, including a stacked ensemble. Model interpretation was performed using permutation feature importance and SHAP. External near-domain corroboration was assessed using a stringent PubChem similarity expansion (Tanimoto > 0.90) around sub-10 nM seed compounds, followed by comparison with retrievable experimental PARP1 activity values. Top scaffold-diverse candidates were further evaluated by complementary docking against PARP1 (PDB: 4R6E) using AutoDock Vina and cavity-guided docking through the SwissDock platform. Results: The stacked ensemble achieved the best held-out performance (test R² = 0.723; RMSE = 0.610 pIC₅₀ units), with 83.7% of test predictions within ≤0.75 pIC₅₀ units and only 2.7% exceeding 1.5 pIC₅₀ units. PubChem similarity expansion retrieved approximately 32,450 analogs, of which 3349 were predicted to have IC₅₀ ≤ 10 nM. Among 366 compounds with retrievable experimental PARP1 activity values, predicted versus experimental pIC₅₀ showed a positive association (R² = 0.124; Pearson r = 0.479), with RMSE = 0.491 and MAE = 0.330 pIC₅₀ units. Three ligands—CID 168873053, CID 175154210, and CID 172894737—showed the strongest complementary docking support and pocket-consistent poses relative to niraparib. Conclusions: This workflow provides a transparent and practically useful framework for near-domain PARP1 inhibitor prioritization. The combined QSAR, explainability, external corroboration, and docking strategy supports shortlist generation for experimental follow-up. Full article

Background: Helicobacter pylori is a well-established risk factor for gastric carcinogenesis. Increasing evidence suggests that the oral cavity may serve as an extragastric reservoir for the bacterium, potentially contributing to persistent infection and reinfection. Orthodontic appliances can modify oral biofilm ecology and may facilitate bacterial colonization. This study aimed to investigate the association between oral H. pylori colonization and gastric cancer, while exploring the potential modifying role of fixed orthodontic appliances. Materials and Methods: In this cross-sectional observational study, 212 participants were recruited from gastroenterology and dental clinics between January 2023 and March 2025. Oral samples were collected and analyzed for H. pylori DNA using polymerase chain reaction (PCR). Gastric diagnoses were established through endoscopic examination and histopathological evaluation, classifying participants into gastric cancer, precancerous gastric lesions, non-atrophic gastritis, and control groups. Demographic, clinical, and oral health variables were recorded. Multivariable logistic regression models were used to evaluate the association between oral H. pylori detection and gastric cancer while adjusting for potential confounders, including age, sex, smoking status, oral hygiene indicators, and socioeconomic factors. Results: Oral Helicobacter pylori DNA was detected in 35/54 (64.8%) patients with gastric cancer, 30/56 (53.6%) with precancerous lesions, 21/52 (40.4%) with non-atrophic gastritis, and 15/50 (30.0%) controls. Gastric H. pylori infection was identified in 41/54 (75.9%) gastric cancer cases compared with 18/50 (36.0%) controls. Oral H. pylori positivity was more frequent among patients undergoing active orthodontic treatment (22/36, 61.1%) than among those without orthodontic appliances (79/188, 42.0%). In multivariable analysis, oral H. pylori positivity remained independently associated with gastric cancer (adjusted OR 3.02, 95% CI 1.51–6.03, p = 0.002). Conclusions: Our findings support an association between oral–gastric microbial interactions and H. pylori–associated disease, and suggest that the oral cavity may serve as a potential reservoir for gastric infection dynamics. The presence of orthodontic appliances may be associated with altered oral microbial ecology and could be linked to sustained H. pylori colonization. Full article

Antimicrobial resistance (AMR) poses a significant global health threat, with multidrug-resistant pathogens undermining the effectiveness of conventional antibiotics. Natural alkaloids, a diverse group of nitrogen-containing compounds mainly derived from plants, are gaining attention as potential antimicrobial agents due to their broad-spectrum activity, structural variety, and unique mechanisms of action. This review examines the antimicrobial properties of natural alkaloids, classifying them by chemical structure (e.g., quinoline, isoquinoline, pyridine, indole, and imidazole alkaloids). Their antibacterial, antifungal, and antiviral activities are discussed, along with the mechanisms by which they target pathogenic microorganisms, including disruption of cell walls and membranes, inhibition of protein synthesis, interference with DNA replication, and viral assembly. The review also explores the synergistic effects of alkaloids when combined with conventional antimicrobial agents. Alkaloids demonstrate potent antimicrobial activity against various pathogens. Quinoline alkaloids, such as quinine, inhibit DNA replication and damage cell membranes. Isoquinoline alkaloids like berberine and sanguinarine exhibit broad-spectrum antibacterial effects. Pyridine alkaloids, including nicotine, disrupt bacterial membranes. In fungi, alkaloids such as sanguinarine and indole derivatives prevent cell wall synthesis and spore germination. Antiviral alkaloids like lycorine target viral RNA polymerases. Additionally, alkaloids enhance the activity of traditional antibiotics by overcoming resistance. Natural alkaloids represent a promising source of antimicrobial agents with diverse mechanisms to combat AMR. Future research should focus on optimizing alkaloid structures, ensuring safety and efficacy, and exploring combination therapies to address the escalating AMR challenge. Full article

Targeted therapies are reshaping oncology by enabling treatment selection based on actionable molecular alterations, improving precision, and reducing unnecessary toxicity. This review provides an up-to-date overview of current targeted treatment modalities and the medicinal chemistry principles that support their discovery and optimization. We synthesize evidence on small-molecule and biologic strategies spanning receptor and non-receptor kinases and their major signaling axes (PI3K-AKT-mTOR and RAS-RAF-MEK-ERK), apoptosis regulation (BCL-2 family), DNA repair via poly(ADP-ribose) polymerase (PARP) inhibition, and epigenetic or metabolic targets including histone deacetylases (HDACs), bromodomain and extra-terminal proteins (BET), and mutant isocitrate dehydrogenases (IDH1/2). Across these areas, we summarize recurrent resistance mechanisms and the rationale for combination or sequential approaches. Biologic targeted therapy is discussed in parallel, including immune checkpoint blockade, antibody–drug conjugates, bispecific antibodies (BsAb), and cell therapies such as chimeric antigen receptor T cells, with emphasis on biomarker-guided patient stratification. Finally, we outline emerging directions beyond canonical nodes, including modulation of the p53-MDM2/MDM4 axis, ferroptosis control through AIFM2/FSP1, and innate immune pathways such as CD47-SIRPa and the stimulator of interferon genes (STING). Overall, the field is shifting from single-target inhibition toward integrated strategies that combine precise molecular targeting with an understanding of signaling network dynamics, resistance evolution, and therapeutic vulnerabilities. Full article

Singleplex and multiplex real-time PCR-HRM (polymerase chain reaction with high resolution melting), both with specific and non-specific amplicon detection, are used for a wide range of applications, from clinical diagnostics to food authentication. However, their results can be influenced by the quality of the template DNA and composition of the reaction mixture. The methods used for the analysis of these results then influence the conclusions drawn. In this work we present an example from our laboratory practice, where the results of singleplex and multiplex real-time PCR differed, despite using the same reaction conditions, primers and analyzed plant material. We show the influence of a singleplex and multiplex PCR setup on the results, as well as the influence of template contamination on the melting behaviour of amplicons. We also discuss the usefulness of cluster analysis for the clarification of real-time PCR-HRM results which appear unclear when only melting and difference curves or similarity scores are used for the analysis of these results. We provide a discussion of problems which we encountered during an analysis of commercial teas and which should be considered by researchers new to PCR-based analysis of plant material, especially if the studied material is rich in various contaminants. Full article

Toxoplasma gondii is a protozoan parasite with high zoonotic potential. Currently, no information is available on natural toxoplasmosis in domestic rabbits in Romania; therefore, the aim of the present study was to evaluate the seroprevalence of T. gondii IgG antibodies and to determine the prevalence of T. gondii DNA in domestic rabbits in our country. In total, 372 domestic rabbits were tested. Blood samples were obtained from 352 animals and tissue samples were obtained from 49 animals, of which 29 animals provided paired blood–tissue samples. Samples were collected from pet rabbits and from rabbits raised in household settings, hereafter referred to as farm rabbits. Sera samples were analyzed using a modified agglutination test (MAT), with a cut-off of 1:24, for anti-T. gondii antibody IgG-type detection, and the tissue specimens were tested by nested polymerase chain reaction (nested PCR) targeting the B1 gene for T. gondii DNA detection. A seropositivity of 16.5% (58/352) was obtained by MAT, while the prevalence of T. gondii DNA was 4.1% (2/49). The present study highlighted the presence of T. gondii in domestic rabbits in Romania, which suggests that rabbit meat consumption may represent a potential risk to human health and therefore warrants further attention. Moreover, to the best of our knowledge, this is the first study to report data on the prevalence of T. gondii in domestic rabbits from Romania. Full article

A comprehensive understanding of plant diversity is essential for ecological research, conservation planning, and sustainable resource management. Advances in genetic technologies have transformed the assessment of plant biodiversity, enabling more precise and efficient characterization of genetic variation. Early molecular markers, widely used in the late 2000s, have largely been replaced by polymerase chain reaction (PCR)-based tools that require less DNA, are easier to use, and are supported by accessible commercial kits. The 2020s have seen the emergence of new, more accessible tools driven by cost reduction and efficiency improvements. High-throughput sequencing (HTS) technologies have further revolutionized the field by providing genome-wide insights into allelic diversity, structural polymorphisms, and epigenetic modifications. These innovations enhance the detection of adaptive variation, improve understanding of spatial genetic structure, and support the evaluation of environmental impacts on plant populations. Marker-assisted selection, now common in modern breeding, leverages genomic data to develop cultivars with enhanced resistance and desirable agronomic traits. Emerging tools such as environmental DNA (eDNA) analysis, high-throughput phenotyping, and advanced bioinformatics workflows expand the capacity to monitor species, assess population viability, and identify key traits linked to adaptation. The present review aims to highlight these technological advancements and the more recent and useful tools available from Next-Generation Sequencing to genotyping-by-sequencing, discussing their role for conserving plant genetic resources, improving breeding programs, and deepening knowledge of plant biodiversity within changing ecosystems. Full article

Cytomegalovirus (CMV) infection of the anterior segment is increasingly recognized as an important cause of corneal endotheliitis and secondary glaucoma, even in immunocompetent individuals. CMV corneal endotheliitis typically presents with coin-shaped or linear keratic precipitates (KPs), corneal edema, mild anterior chamber inflammation, and recurrent intraocular pressure (IOP) elevation; persistent or episodic ocular hypertension may progress to glaucomatous optic neuropathy if inadequately treated. Definitive diagnosis relies on aqueous humor polymerase chain reaction (PCR) testing for CMV DNA, supported by adjunctive imaging including specular microscopy, anterior segment optical coherence tomography (AS-OCT), and in vivo confocal microscopy (IVCM). Management requires a comprehensive strategy integrating antiviral therapy, anti-inflammatory treatment, and appropriate IOP control. Topical or systemic ganciclovir remains the cornerstone, while refractory disease may necessitate surgical intervention. Older age and male sex, host immune status, prolonged or recurrent CMV infection, and pre-existing ocular conditions are major risk factors for progression and poor outcomes. The pathogenesis of secondary glaucoma is thought to involve both direct viral cytopathic effects and inflammation-mediated damage to the trabecular meshwork (TM), resulting in impaired aqueous outflow. Therefore, early recognition, accurate diagnosis, and effective treatment are essential to prevent corneal decompensation and permanent vision loss. Full article

The translation of nucleic acid amplification into practical point-of-care and biosensor-integrated diagnostics is still significantly impeded by the necessity for rapid sample preparation. For this reason, a broad comparison of seven commercially available kits for DNA/RNA extraction containing their temperature-related adjustments was performed. Extracts isolated from SARS-CoV-2-positive nasopharyngeal swabs, viral stocks, as well as laboratory-prepared suspensions of clinically relevant Gram-positive and Gram-negative bacteria were evaluated by recombinase polymerase amplification (RPA) and real-time PCR. In addition, the impact of transport media for SARS-CoV-2 samples was investigated. Extraction performance varied markedly according to the kit, pathogen, sample background. For SARS-CoV-2, rapid extraction was more effective for samples collected in viral transport medium than in inactivation buffer. Across bacterial targets, performance was species dependent, highlighting substantial differences in compatibility between simplified extraction workflows and downstream amplification. Among the rapid methods tested, a simplified QuickExtract protocol (95 °C, 5 min) provided the most consistent overall results, although it did not uniformly match the reference silica-based method for all targets. In conclusion, these results demonstrate that rapid nucleic acid extraction must be thoroughly evaluated as an essential element of the entire sample-to-answer workflow, rather than being chosen as a standalone preprocessing step for point-of-care molecular diagnostics. Full article