Search Results (158)

Background/Objectives: The gene pool of the Apennine wolf is affected by admixture with domestic variants due to anthropogenic hybridisation with dogs. Genetic monitoring at the population level involves assessing the extent of admixture in single individuals, ranging from pure wolves to recent hybrids or wolf backcrosses, through the analysis of nuclear and mitochondrial DNA (mtDNA) markers. Although individually non-diagnostic, mtDNA is nevertheless essential for completing the final diagnosis of genetic admixture. Typically, the identification of wolf mtDNA haplotypes is carried out via sequencing of coding genes and non-coding DNA stretches. Our objective was to develop a fast real-time PCR assay to detect the mtDNA haplotypes that occur exclusively in the Apennine wolf population, as a valuable alternative to the demanding sequence-based typing. Methods: We validated a qualitative duplex real-time PCR that exploits the combined presence of diagnostic point mutations in two mtDNA segments, the NDH-4 gene and the control region, and is performed in a single-tube step through TaqMan-MGB chemistry. The aim was to detect mtDNA multi-fragment haplotypes that are exclusive to the Apennine wolf, bypassing sequencing. Results: Basic validation of 149 field samples, consisting of pure Apennine wolves, dogs, wolf × dog hybrids, and Dinaric wolves, showed that the assay is highly specific and sensitive, with genomic DNA amounts as low as 10⁻⁵ ng still producing positive results. It also proved high repeatability and reproducibility, thereby enabling reliable high-throughput testing. Conclusions: The results indicate that the assay presented here provides a valuable alternative method to the time- and cost-consuming sequencing procedure to reliably diagnose the maternal lineage of the still-threatened Apennine wolf, and it covers a wide range of applications, from scientific research to conservation, diagnostics, and forensics. Full article

Antiphospholipid syndrome (APS) is an autoimmune disorder characterized by venous and arterial thrombosis and obstetric complications, driven by antiphospholipid antibodies (APLAs). This review synthesizes the latest advancements and current understanding, diagnosis, and treatment of APS. APLAs, including lupus anticoagulant (LAC), anticardiolipin (aCL), and anti-β2-glycoprotein I (aβ2-GPI), interfere with coagulation and endothelial function, as well as with placental health. APS can be primary or secondary; it is often associated with systemic autoimmune diseases like lupus. The pathogenesis of APS remains only partially understood. APLAs promote thrombosis through endothelial damage, platelet activation, and inflammatory signaling pathways. Laboratory diagnosis relies on persistent positivity for APLAs and LAC through tests like ELISA and clotting assays, following a three-step confirmation process. New integrated test systems have been introduced to improve standardization. Classification criteria have evolved, with the 2023 EULAR-ACR criteria providing a weighted, domain-based scoring system, enhancing diagnostic precision. Catastrophic APS (CAPS) is a severe, rare manifestation of APS, characterized by multi-organ failure due to rapid, widespread microthrombosis and systemic inflammation, which requires urgent anticoagulation. Seronegative APS is proposed for patients with clinical features of APS but negative standard antibody tests, possibly due to non-criteria antibodies or transient immunosuppression. Treatment primarily involves long-term anticoagulation with vitamin K antagonists; direct oral anticoagulants are generally not recommended. APS diagnosis and management remain complex due to clinical heterogeneity and laboratory challenges. Continued refinement of diagnostic tools and criteria is essential for improving outcomes in this life-threatening condition. Full article

Innovative functional materials integrating host–guest complexes in cryogels offer promising applications in topical drug delivery, enhancing drug solubility and stability. In this study, we designed and developed a cryogel-based patch for acne treatment by polymerizing an acrylate-functionalized γ-cyclodextrin (γ-CD) and trimethoprim (TMP) inclusion complex with [2-(acryloyloxy)ethyl]trimethylammonium chloride (AETMA) at low temperatures. A multistep workflow was applied to synthesize the inclusion complex via mortar-assisted kneading, followed by cryogel formulation through radical cryopolymerization. The resulting hybrid system leverages the cationic nature of AETMA to promote adhesion and electrostatic interactions with the skin surface. At the same time, γ-CD serves as a drug reservoir, facilitating sustained release of the drug. The system was characterized by FT-IR, TGA, and SEM analyses. In vitro release studies demonstrated a sustained TMP release profile, best described by the Korsmeyer–Peppas diffusion model. Antibacterial assays confirmed the system’s effectiveness against Staphylococcus aureus, supporting its potential for localized and prolonged acne treatment. Moreover, cytocompatibility tests demonstrated that the formulation is biocompatible, further validating its suitability for topical application. Full article

Background/Objectives: Angiogenesis is the multistep process of the formation of new blood vessels. It is beneficial in scenarios that require tissue repair and regeneration, such as wound healing, bone fracture repair, and recovery from ischemic injuries like stroke, where new blood vessel formation restores oxygen and nutrient supply to damaged areas. Extremely low-frequency electromagnetic stimulation (ELF-EMS), which involves electromagnetic fields in the frequency range of 0–300 Hz, have been shown to reduce ischemic stroke volume by improving cerebral blood flow and recovery effects that are dependent on eNOS. Based on previous results, we herein explore the effects of ELF-EMS treatment (13.5 mT/10 and 60 Hz) on the activation of angiogenic processes in vitro in homeostatic conditions. Methods: Using human microvascular endothelial cells (HMEC-1), we studied cell proliferation, migration, and tube formation in vitro, as well as nitric oxide production and the effect of calcium and nitric oxide (NO) on these processes. Moreover, blood vessel formation was studied using a chicken chorioallantoic membrane (CAM) assay. Results: Our results showed that ELF-EMS increases proliferation, tube formation, and both the migration and transmigration of these cells, the latter of which was mediated via NO. In turn, calcium inhibition decreased ELF-EMF-induced NO production. Furthermore, ELF-EMS significantly increased blood vessel formation in the CAM assay. Conclusions: Our results indicated that ELF-EMS exposure (13.5 mT/10 and 60 Hz) significantly induces angiogenesis in vitro and in ovo, underscoring its potential application in the treatment of conditions characterized by insufficient blood supply. Full article

Background/Objectives: Neurotensin receptors (NTSRs), members of the G protein-coupled receptor (GPCR) family, have been found to be overexpressed in several types of human cancers, including breast, colon, lung, liver, prostate, and pancreatic cancer. In particular, NTSR1 is overexpressed in at least 75% of pancreatic ductal adenocarcinomas. The aim of the present study was the development and evaluation of new ^99mTc-labeled nonpeptide NTSR1-antagonists for SPECT imaging of NTSR-positive tumors. Methods: Multistep syntheses of NTSR1 antagonist derivatives were performed following our previously described procedure. Two different chelating strategies were applied for ^99mTc radiolabeling to provide the [^99mTc]Tc-HYNIC complex [^99mTc]1 and the [^99mTc]Tc-tricarbonyl complex [^99mTc]2. Receptor binding assays were performed using hNTSR1-expressing CHO cells. Radiochemical yields (RCYs) were determined by radio-HPLC. For [^99mTc]1 and [^99mTc]2, log D_7.4, plasma protein binding, stability in human plasma and serum, and cellular uptake in HT-29 cells were determined. Biodistribution studies and small animal SPECT studies were performed in HT-29 tumor-bearing nude mice. Results: The radiosynthesis of [^99mTc]1 (log D_7.4 = −0.27) and [^99mTc]2 (log D_7.4 = 1.00) was successfully performed with RCYs of 94–96% (decay-corrected). Both radioligands were stable in human serum and plasma, showed plasma protein binding of 72% ([^99mTc]1) and 82% ([^99mTc]2), and exhibited high and specific uptake in HT-29 cells. Biodistribution studies in HT-29 tumor-bearing mice showed a higher tumor accumulation of [^99mTc]1 compared to [^99mTc]2 (8.8 ± 3.4 %ID/g vs. 2.7 ± 0.2 %ID/g at 2 h p.i.). [^99mTc]2 showed exceptionally high intestinal accumulation (49 ± 22 %ID/g at 1 h p.i.) and was therefore considered unfavorable. In the SPECT/CT imaging of HT-29 tumor xenografts, [^99mTc]1 showed a higher NTSR1-specific tumor uptake than [^99mTc]2 at all time points after tracer injection, with 12 ± 2.8 %ID/g for [^99mTc]1 vs. 3.1 ± 1.1 %ID/g for [^99mTc]2 at 4 h p.i. and adequate tumor-to-background ratios. Conclusions: In particular, the [^99mTc]Tc-HYNIC ligand ([^99mTc]1) showed promising preclinical results, being a potential candidate for SPECT imaging and, therefore, appropriate for translation into the clinic. Full article

Cleft palate is one of the most common congenital abnormalities and one of the main symptoms of Stickler syndrome. Secondary palate development is a complex multi-step process that involves raising the palatal frame from a vertical to a horizontal position. Lysyl oxidase-like 3 (LOXL3), a member of the lysyl oxidase family responsible for the crosslinking in collagen, is also one of the mutated genes detected in Stickler syndrome. Loss of Loxl3 causes delayed palatal shelf elevation, which in turn resulted in cleft palate. However, the precise mechanisms of palatal shelf delayed elevation remain unclear. In this study, we deeply investigated the mechanism of Loxl3 induced delayed elevation in palatal shelves. We found that Loxl3 deficiency caused reduced cell proliferation in both medial and posterior palatal mesenchyme through BrdU labeling and Western blot analysis (p < 0.05, p < 0.01), decreased migration of palatal mesenchymal cells through cell scratch assay (p < 0.05), and decreased expression of genes associated with proliferation through Western blot analysis (p < 0.05, p < 0.01) at E14. We found that the specific deletion of Loxl3 in the palatal mesenchyme resulted in delayed elevation but normal fusion of palatal shelves, also reduced cell proliferation and collagen fibers deposition in medial palatal mesenchyme through BrdU labeling and histological analysis (p < 0.05, p < 0.01). Thus, our data suggest that Loxl3 regulates cell proliferation and collagen fibers deposition in the palatal mesenchyme, thus controlling palatal shelf elevation. Full article

Prostate cancer (PCa) is one of the most common malignancies in men, where early and accurate detection is crucial. While PSA testing has been the diagnostic standard, its limited specificity leads to unnecessary biopsies and missed significant cancers. Urinary biomarkers such as PCA3 and TMPRSS2-ERG and multi-marker assays (MyProstateScore, SelectMDx, and ExoDx) offer a promising alternative. This narrative review examines their diagnostic performance and clinical utility with the aim of understanding whether they can be integrated with the established tests and exams already in use. A literature search of PubMed, Scopus, and Medline identified some relevant recent studies (2010–2025). The findings show that PCA3 and TMPRSS2-ERG improve specificity over PSA, while multi-marker tests enhance risk stratification and reduce unnecessary procedures. MPS integrates urinary biomarkers with PSA, achieving over 95% sensitivity and negative predictive value for clinically significant cancers. SelectMDx demonstrates ~90% negative predictive value, and ExoDx assesses urinary exosomes to predict aggressive disease. Despite their advantages, challenges persist, including variability in performance, cost, and accessibility. Urinary biomarkers represent a major step toward more precise, less invasive diagnostics, with future research needed to optimize clinical integration and cost-effectiveness. Full article

African swine fever virus (ASFV) is a highly virulent DNA virus that has spread globally since its introduction into Georgia in 2007, causing substantial economic losses in the swine industry. In the absence of an effective vaccine, chemical disinfection remains a key strategy for disease control. However, in cell-based disinfectant efficacy testing, distinguishing between disinfectant-induced cytotoxicity and virus-induced cytopathic effects (CPEs) remains a major challenge, leading to the potential misinterpretation of results. To address this, we developed a multi-step analytical framework to differentiate CPEs from cytotoxicity using a Vero cell-adapted ASFV strain. Virkon^® S was tested at three dilutions—375×, 275× (manufacturer-recommended), and 175×—and evaluated through CPE observation, lactate dehydrogenase (LDH) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays, and antigen detection via lateral flow immunoassay (p30) and immunofluorescence (p54). Notably, the 375× dilution achieved effective viral inactivation with significantly lower cytotoxicity, demonstrating that this framework can facilitate a more refined determination of disinfectant working dilutions. Furthermore, increased p30 signals after disinfection and the observation of lower cytotoxicity in virus-plus-disinfectant groups compared to disinfectant-only groups highlight the complexity of virus-disinfectant interactions and the potential for misinterpretation. This study provides a standardized and interpretable strategy for assessing ASFV disinfectant efficacy and offers a practical basis for evaluating other enveloped viruses in future disinfection studies. Full article

Lateral flow tests (LFTs) had a pivotal role in combating the spread of the SARS-CoV-2 virus throughout the COVID-19 pandemic thanks to their affordability and ease of use. Most of LFT devices were based on nitrocellulose membrane strips whose industrial upscaling to billions of devices has already been extensively demonstrated. Nevertheless, the assay option in an LFT format is largely restricted to qualitative detection of the target antigens. In this research, we surveyed the potential of UV nanoimprint lithography (UV-NIL) and extrusion coating (EC) for the high-throughput production of disposable capillary-driven, foil-based tests that allow multistep assays to be implemented for quantitative readout to address the inherent lack of on-demand fluid control and sensitivity of paper-based devices. Both manufacturing technologies operate on the principle of imprinting that enables high-volume, continuous structuring of microfluidic patterns in a roll-to-roll (R2R) production scheme. To demonstrate the feasibility of R2R-fabricated foil chips in a point-of-care biosensing application, we adapted a commercial chemiluminescence multiplex test for COVID-19 antibody detection originally developed for a capillary-driven microfluidic chip manufactured with injection molding (IM). In an effort to build a complete ecosystem for the R2R manufacturing of foil chips, we also recruited additional processes to streamline chip production: R2R biofunctionalization and R2R lamination. Compared to conventional fabrication techniques for microfluidic devices, the R2R techniques highlighted in this work offer unparalleled advantages concerning improved scalability, dexterity of seamless handling, and significant cost reduction. Our preliminary evaluation indicated that the foil chips exhibited comparable performance characteristics to the original IM-fabricated devices. This early success in assay translation highlights the promise of implementing biochemical assays on R2R-manufactured foil chips. Most importantly, it underscores the potential utilization of UV-NIL and EC as an alternative to conventional technologies for the future development in vitro diagnostics (IVD) in response to emerging point-of-care testing demands. Full article

Insulin-like growth factor-1 (IGF-1) is a regulatory factor closely associated with diabetes, obesity, and breast cancer, and it also acts as one of the most abundant growth factors in bovine colostrum. Current methods generally have the problem of low sensitivity, a time-consuming nature, and low stability, which makes it difficult to crack down on the false advertising of IGF-1 content in dairy products. In this work, an ultrasensitive fluorescent enzyme-linked immunosorbent assay (ELISA) is proposed, where the antibody and the target are combined in the form of a “sandwich” to ensure the accuracy and specificity of the assay. IGF-1 is quantified based on an effective hydrogen peroxide (H₂O₂) probe with 10-acetyl-3,7-dihydroxyphenoxazine (ADHP) as the fluorogenic substrate. The proposed fluorescent sandwich ELISA has a low limit of detection (LOD) of 77.29 pg/mL, fast experimental process within 1 h, and stable signal of 1 h. Furthermore, multi-step pretreatment methods for bovine colostrum powders are established to remove interfering substances, including fat, casein, and binding proteins, achieving the accurate and specific detection of IGF-1. IGF-1 recovery studies on treated bovine colostrum powders exhibit good recovery rates ranging from 91.71% to 102.32%, which proves the feasibility of detecting IGF-1 in real bovine colostrum. Full article

Reactive oxygen species play a significant role in various pathological conditions, driving the need for novel, potent antioxidants. While polyphenols are known for their antioxidant properties, their limited stability and bioavailability present challenges for therapeutic applications. To address these limitations, a series of novel thiazolyl-polyphenolic compounds was synthesized via a multi-step synthetic route incorporating Hantzsch heterocyclization in the final step. The synthesized compounds 7a–k were structurally characterized using spectroscopic techniques, including NMR, MS, and IR. In silico thermodynamic calculations, including HOMO–LUMO gap and bond dissociation enthalpy (BDE) calculations, revealed a promising antioxidant profile for these compounds and indicated that the substitution in position 2 of the thiazole ring does not substantially influence the antioxidant activity conferred by the catechol moiety in position 4. The antioxidant capacity of the synthesized compounds was experimentally validated using a panel of six distinct assays: two radical scavenging assays (ABTS and DPPH) and four electron transfer-based assays (RP, TAC, FRAP, and CUPRAC). The in vitro evaluation demonstrated that compounds 7j and 7k exhibited significantly enhanced antioxidant activity compared to the established antioxidant standards, ascorbic acid and Trolox. These findings suggest that the strategic modifications in position 2 of the thiazole scaffold represent a promising direction for future research aimed at developing novel therapeutic agents with enhanced antioxidant properties. The present study is limited to the in vitro evaluation of compounds based on the N-methyl substituted thiazole scaffold, but future studies can include modifications such as changing the substituent on the thiazole nitrogen, the hydrazone linker or possible insertion of substituents in position 5 of thiazole ring of substituents with various electronic or physico-chemical properties. Full article

Point-of-care (POC) antigen detection plays a crucial role in curbing the spread of viruses. Paper-based fluorescence aptasensors are expected to offer a low-cost tool to meet the needs of decentralized POC diagnosis. Herein, we report on a fluorescent paper-based sensing system for detecting the SARS-CoV-2 spike protein. The sensing system was constructed by loading multi-layer Nb₂C MXene nano-quenchers and carbon-dot-labeled aptamer (G-CDs@Apt) probes onto a mixed cellulose ester (MCE) paper substrate. On the Nb₂C MXene/G-CDs@Apt sensing paper, abundant G-CDs@Apt probes were attached to the multilayer MXene nano-quenchers and kept in a fluorescence-off state, while recognition of the target detached the G-CDs@Apt probes formed the nano--quenchers, resulting in fluorescence recovery of the sensing paper. The developed paper-based sensor performed well in the one-step detection of the SARS-CoV-2 S1 protein with a detection limit of 0.067 ng/mL (0.335 pg/test). The assay exhibited good selectivity and anti-interference in the detection of the SARS-CoV-2 S1 protein in artificial saliva. Moreover, the paper-based aptasensor was successfully used to detect the SARS-CoV-2 S1 protein in actual environmental samples with recoveries of 90.87–100.55% and relative standard deviations of 1.52–3.41%. The proposed technology provides a cost-effective alternative to traditional antibody test strips for a wide range of POC diagnostic applications. Full article

Background: Host cell proteins (HCPs) from primary hamster kidney cells (PHKCs) used to produce rabies vaccines may cause an allergic reaction in humans, so these residual HCPs must be controlled. Establishing a national standard for PHKC HCP is very important to ensure the consistency of HCPs between batches of the vaccine and to standardize the control of HCPs. Objectives: We aimed to establish a novel national standard substance to determine the HCP residue in rabies vaccines produced with PHKCs. Methods: A two-step multi-laboratory collaborative collaboration was undertaken. In the first step, the protein concentration of the standard substance stock solution was determined using Lowry’s method. In the second step, the concentration of the candidate standard substance was determined with an enzyme-linked immunosorbent assay. Results: The concentration of the PHKC protein standard was 4.0 μg/mL (95% confidence interval: 3.5–4.4 μg/mL). Conclusions: The PHKC protein standard was approved by the Chinese National Committee on Standards for the quality control of PHKC-based rabies vaccines for human use, and it plays an important role in controlling the quality of these human vaccines. Full article

Hyaluronic acid (HA)-based delivery systems for doxorubicin (DOX) have been developed to selectively target cancer cells and enhance their therapeutic effects while reducing systemic side effects. However, conventional methods for preparing HA-based drug delivery systems are often limited by multistep synthetic processes, time-consuming purification, and the use of crosslinkers or surfactants, which can cause undesired toxicities. To resolve these issues, we developed a facile one-pot method to prepare self-assembled sodium hyaluronate/doxorubicin (HA/DOX) nanoaggregates by mixing HA and DOX. The self-assembled HA/DOX nanoaggregates were formed via cation–π interactions between the aromatic moiety of DOX and Na⁺ ions in HA as well as electrostatic interactions between HA and DOX. The optimized HA/DOX nanoaggregates with a [DOX]/[HA] molar ratio of 5 had an average particle size of approximately 250 nm and a sphere-like shape. In vitro studies revealed that HA/DOX nanoaggregates effectively targeted CD44-overexpressing cancer cells, selectively delivering DOX into the cell nuclei more efficiently than free DOX and resulting in enhanced cytotoxic effects. Annexin V and transferase dUTP nick-end labeling assays confirmed that HA/DOX nanoaggregates induced apoptosis via DNA fragmentation more effectively than free DOX. Full article

Lipase from Pseudomonas fluorescens (PFL) and laccase from Trametes versicolor were co-immobilized onto nanocellulose (NC), using a layer-by-layer approach. Initially, PFL was adsorbed onto NC through ionic and hydrophobic interactions. To achieve higher PFL immobilization yield and activity, NC was functionalized with aldehyde groups through periodate oxidation (NCox) or glutaraldehyde activation (NC-GA). FTIR analysis confirmed these chemical modifications. Among the functionalized NCs, NCox showed the best capacity to retain higher amounts of PFL (maximum load: 20 mg/g), and this support was selected to proceed with the co-immobilization experiments. In this process, NCox-250-PFL (NCox activated with 250 µmol/g of aldehyde groups) was covered with polyethyleneimine (PEI), laccase was co-immobilized, and a crosslinking step using glutaraldehyde was used to covalently attach the enzymes to the support, producing the biocatalyst NCox-250-PFL-PEI-Lac-GA. Co-immobilized enzymes presented higher thermal stability (50 °C) than soluble enzymes; co-immobilized laccase retained 61.1% of its activity after 24 h, and PFL retained about 90% after 48 h of deactivation at 50 °C. In operational stability assays, the heterogeneous biocatalysts maintained more than 45% of their activity after five cycles of pNPB hydrolysis and ABTS oxidation. This co-immobilized biocatalyst, with its high stability and activity retention, is a promising multi-active heterogeneous biocatalyst for use in cascade reactions of industrial interest. Full article