Search Results (925)

:Solvents represent the quiet majority in biomolecular systems, yet modeling their influence with both speed and ri:gor remains a central challenge. This study maps the state of the art in implicit solvent theory and practice, spanning classical continuum electrostatics (PB/GB; DelPhi, APBS), modern nonpolar and cavity/dispersion treatments, and quantum–continuum models (PCM, COSMO/COSMO-RS, SMx/SMD). We highlight where these methods excel and where they falter, namely, around ion specificity, heterogeneous interfaces, entropic effects, and parameter sensitivity. We then spotlight two fast-moving frontiers that raise both accuracy and throughput: machine learning-augmented approaches that serve as PB-accurate surrogates, learn solvent-averaged potentials for MD, or supply residual corrections to GB/PB baselines, and quantum-centric workflows that couple continuum solvation methods, such as IEF-PCM, to sampling on real quantum hardware, pointing toward realistic solution-phase electronic structures at emerging scales. Applications across protein–ligand binding, nucleic acids, and intrinsically disordered proteins illustrate how implicit models enable rapid hypothesis testing, large design sweeps, and long-time sampling. Our perspective argues for hybridization as a best practice, meaning continuum cores refined by improved physics, such as multipolar water, ML correctors with uncertainty quantification and active learning, and quantum–continuum modules for chemically demanding steps. Full article

This study investigates the spatiotemporal optimization of small-scale emergency infrastructure in high-density urban environments, using nucleic acid testing sites in Macao as a case study. The objective is to enhance emergency responsiveness during future public health crises by aligning infrastructure deployment with dynamic patterns of population influx. A behaviorally informed spatial decision-making framework is developed through the integration of kernel density estimation, point-of-interest (POI) distribution, and origin–destination (OD) path simulation based on an Ant Colony Optimization (ACO) algorithm. The results reveal pronounced temporal fluctuations in testing demand—most notably with crowd peaks occurring around 12:00 and 18:00—and highlight spatial mismatches between existing facility locations and key residential or functional clusters. The proposed approach illustrates the feasibility of coupling infrastructure layout with real-time mobility behavior and offers transferable insights for emergency planning in compact urban settings. Full article

Background: Circulating miRNAs have been identified as potential biomarkers for the early diagnosis and monitoring of cancers. However, limitations of polymerase chain reaction (PCR)-based methods are currently delaying the transition of miRNA research into clinical practice. These include labour-intensive workflows, exposure to errors and difficulties in detecting and quantifying low-abundance miRNAs. Objectives: This review emphasizes the need to develop amplification-free (“PCR-free”) technologies to improve the reliability, scalability and practicality of miRNA diagnostics in clinical settings. Methods: This review explores recent advances in PCR-free technologies developed over the past five years. It focuses on innovative methods, such as bead-based assays and sensor detection platforms, which serve as valuable alternatives to conventional PCR-based approaches. These emerging technologies have the potential to overcome the key limitations of PCR by offering streamlined workflows, reduced error rates and enhanced compatibility with a variety of clinical sample types. Crucially, they enable absolute quantification without the need for pre-nucleic acid extraction, reverse transcription or amplification, as well as the simultaneous detection of multiple miRNAs within a single assay. These provide cost-effective and scalable solutions for comprehensive biomarker profiling. The transition from PCR-based to PCR-free technologies is a significant step forward in miRNA diagnostics, overcoming long-standing technical barriers and paving the way for broader adoption of miRNA analysis in routine clinical settings. This shift supports the advancement of precision medicine and holds promises for improving early cancer detection. Full article

From the corona virus pandemic in Japan that started with the “Diamond Princess” accident, it became clear that rapid detection, a high sensitivity, multiple diagnostic items, one-step one-base point mutation detection, a fast speed of system development, portability (small size and light weight), full automation, random access, and other conditions are required for future point-of-care testing systems. The Eprimer-SmartAmp technology that was developed possesses characteristics fully aligned with these requirements. Building upon this platform, the “GenPad” system was subsequently established. The GenPad system is widely applicable not only to emerging foreign infectious diseases, but also to cancer, lifestyle-related diseases, and other areas of healthcare through telemedicine and intraoperative nucleic acid diagnoses. In collaboration with telecommunication systems, GenPad is expected to contribute to the establishment of a smart medical city with a countermeasure against emerging foreign infectious diseases, where individuals can check their own health conditions in all healthcare areas. Full article

Origanum vulgare L. is an important aromatic plant traditionally used in folk medicine since ancient times. Its growing interest for the scientific community is mainly attributed to its distinctive chemical profile, which includes bioactive compounds, such as polyphenols (phenolic acids and flavonoids) and volatile compounds (essential oil). These components collectively contribute to oregano’s wide spectrum of biological activities. In this study, the volatile components of the essential oil (WEO_OR) and the polyphenolic fraction of the methanolic extract (ME_OR) obtained from leaves and inflorescences of wild Origanum vulgare collected in central Italy were characterized using GC-MS and UHPLC-DAD, respectively. Carvacrol was identified as the major compound in the essential oil, while rosmarinic acid was predominant in the methanolic extract. A comparative analysis was also carried out with a commercially available essential oil (CEO_OR), aiming to evaluate potential differences in chemical composition and antioxidant activity (DPPH, ABTS, and FRAP assays). ME_OR showed the strongest antioxidant activity (DPPH IC₅₀ = 0.052 mg mL⁻¹; ABTS = 3.94 mg TE mL⁻¹; FRAP = 30.58 mg TE g⁻¹), followed by CEO_OR (DPPH IC₅₀ = 0.45 mg mL⁻¹; ABTS = 9.57 mg TE mL⁻¹; FRAP = 7.33 mg TE g⁻¹), while WEO_OR displayed the lowest values (DPPH IC₅₀ = 1.54 mg mL⁻¹; ABTS = 0.10 mg TE mL⁻¹). Furthermore, ME_OR and WEO_OR were tested in vitro using the human hepatoblastoma cell line HepG2 to assess their potential biological activities related to cell survival and oxidative stress. The results indicated that at the tested doses, neither the ME nor the EO showed significant toxicity, as evidenced by the unchanged proliferation rate of HepG2 cells. However, the ME at low doses (50 and 100 μg mL⁻¹) and the EO (0.005%), administered as a pre-treatment, exhibited a protective effect against oxidative stress, as inferred from the reduction in 8-OHdG levels, a marker of oxidative damage to nucleic acids. Full article

Background: COVID-19 vaccination has been a key tool in protecting healthcare workers (HCWs), but breakthrough infections have occurred. The durability of vaccine-induced immunity and its impact on HCWs remain critical for public health strategies. Methods: In this small cohort study (n = 32), we assessed antibody levels and breakthrough infection rates in HCWs over 12 months post-vaccination, providing insights for booster strategies and infection control. A cohort of 32 HCWs was screened for SARS-CoV-2 infection using weekly self-administered swabs and blood samples collected at baseline, 6 months, and 12 months. SARS-CoV-2 antibodies (IgG, IgM) targeting spike proteins and nucleocapsids were analyzed using a multi-antigen serology panel. Pooled nucleic acid testing was employed for infection detection. Results: Nine participants showed breakthrough infections, with nucleocapsid antibodies indicating prior infection. Eight of these cases occurred after the third vaccine dose during the Omicron-dominant period. Anti-spike antibody levels declined significantly in participants without prior infection, while those with breakthrough infections exhibited increased levels. The half-life of S1 and S1 receptor-binding domain (RDB) vaccine-induced antibodies was 144 and 166 days, respectively, which aligns with CDC data. These findings provide valuable insights for determining the optimal timing of booster doses. Conclusions: Our findings highlight the waning antibody levels over time and the occurrence of breakthrough infections. Although based on a small sample, these data support the need for ongoing monitoring and timely boosters. Full article

Neuroinflammation is the major contributor to the pathology of neonatal hypoxic–ischemic (HI) brain injury. Our previous studies have demonstrated that microRNA210 (miR210) inhibition with antisense locked nucleic acid (LNA) inhibitor mitigates neuroinflammation and provides neuroprotection after neonatal HI insult. However, the underlying mechanisms remain elusive. In the present study, using miR210 knockout (KO) mice and microglial cultures, we tested the hypothesis that miR210 promotes microglial activation and neuroinflammation through suppressing mitochondrial function in microglia after HI. Neonatal HI brain injury was conducted on postnatal day 9 (P9) wild-type (WT) and miR210 knockout (KO) mouse pups. We found that miR210 KO significantly reduced brain infarct size at 48 h and improved long-term locomotor functions assessed by an open field test three weeks after HI. Moreover, miR210 KO mice exhibited reduced IL1β levels, microglia activation and immune cell infiltration after HI. In addition, in vitro studies of microglia exposed to oxygen–glucose deprivation (OGD) revealed that miR210 inhibition with LNA reduced OGD-induced expression of Il1b and rescued OGD-mediated downregulation of mitochondrial iron–sulfur cluster assembly enzyme (ISCU) and mitochondrial oxidative phosphorylation activity. To validate the link between miR210 and microglia activation, isolated primary murine microglia were transfected with miR210 mimic or negative control. The results showed that miR210 mimic downregulated the expression of mitochondrial ISCU protein abundance and induced the expression of proinflammatory cytokines similar to the effect observed with ISCU silencing RNA. In summary, our results suggest that miR210 is a key regulator of microglial proinflammatory activation through reprogramming mitochondrial function in neonatal HI brain injury. Full article

Honey bees are essential pollinators for the ecosystem and food crops. However, their health and survival face threats from both biotic and abiotic stresses. Fungi, microsporidia, and bacteria might significantly contribute to colony losses. Therefore, rapid and sensitive diagnostic tools are crucial for effective disease management. In this study, molecular assays were developed to quickly and efficiently detect the main honey bee pathogens: Nosema ceranae, Aspergillus flavus, Paenibacillus larvae, and Black queen cell virus. In this context, new primer pairs were designed for use in quantitative Real-time PCR (qPCR) reactions. Various protocols for extracting total nucleic acids from bee tissues were tested, indicating a CTAB-based protocol as the most efficient and cost-effective. Furthermore, excluding the head of the bee from the extraction, better results were obtained in terms of quantity and purity of extracted nucleic acids. These assays showed high specificity and sensitivity, detecting up to 250 fg of N. ceranae, 25 fg of P. larvae, and 2.5 pg of A. flavus DNA, and 5 pg of BQCV cDNA, without interference from bee DNA. These qPCR assays allowed pathogen detection within 3 h and at early stages of infection, supporting timely and efficient management interventions. Full article

Background: Influenza viruses are major pathogens responsible for respiratory infections and pose significant risks to densely populated urban areas. RT-qPCR has made substantial contributions in controlling virus transmission during previous COVID-19 epidemics, but it faces challenges in terms of detection time for large sample sizes and susceptibility to nucleic acid contamination. Methods: Our study designed loop-mediated isothermal amplification primers for three common influenza viruses: A/H3N2, A/H1N1, and B/Victoria, and utilized a 4-channel microfluidic chip to achieve simultaneous detection. The chip initiates amplification by centrifugation and allows testing of up to eight samples at a time. Results: By creating a closed amplification system in the microfluidic chip, aerosol-induced nucleic acid contamination can be prevented through physically isolating the reaction from the operating environment. The chip can specifically detect A/H1N1, A/H3N2, and B/Victoria and has no signal for other common respiratory viruses. The testing process can be completed within 1 h and can be sensitive to viral RNA at concentrations as low as 10⁻³ ng/μL for A/H1N1 and A/H3N2 and 10⁻¹ ng/μL for B/Victori. A total of 296 virus swab samples were further analyzed using the microfluidic chip method and compared with the classical qPCR method, which resulted in high consistency. Conclusions: Our chip enables faster detection of influenza virus and avoids nucleic acid contamination, which is beneficial for POCT establishment and has lower requirements for the operating environment. Full article

Background/Objectives. Efficient nucleic acid delivery into target cells remains a critical challenge in gene therapy. Due to its advantages in biocompatibility and safety, recent research has increasingly focused on non-viral gene delivery. Methods. A series of copolymers—synthesized by integrating thermally sensitive poly(N-isopropylacrylamide) (PNIPAm), hydrophilic poly(ethylene glycol) (PEG) grafts, and a polycationic poly(L-lysine) (PLL) block of varying lengths ((PNIPAm)₇₇-graft-(PEG)₉-block-(PLL)_z, z = 10–65)—were investigated. Plasmid DNA complexation with the copolymers was achieved through temperature-modulated methods. The resulting polyplexes were characterized by evaluating complex strength, particle size, zeta potential, plasmid DNA loading capacity, resistance to anionic stress, stability in serum, and lysosomal membrane destabilization assay. The copolymers’ potential for plasmid DNA delivery was assessed through cytotoxicity and transfection studies in cancer cell lines. Results. Across all complexation methods, the copolymers effectively condensed plasmid DNA into stable polyplexes. Particle sizes (60–90 nm) ranged with no apparent correlation to copolymer type, complexation method, or N/P ratio, whereas zeta potentials (+10–+20 mV) and resistance to polyanionic stress were dependent on the PLL length and N/P ratio. Cytotoxicity analysis revealed a direct correlation between PLL chain length and cell viability, with all copolymers demonstrating minimal cytotoxicity at concentrations required for efficient transfection. PNL-20 ((PNIPAm)₇₇-graft-(PEG)₉-block-(PLL)₂₀) exhibited the highest transfection efficiency among the tested formulations while maintaining low cytotoxicity. Conclusions. The study highlights the promising potential of (PNIPAm)₇₇-graft-(PEG)₉-block-(PLL)_z copolymers for effective plasmid DNA delivery to cancer cells. It reveals the importance of attaining the right balance between polyplex tightness and plasmid release to achieve improved biocompatibility and transfection efficiency. Full article

A virome survey of banana plantations and their surrounding plants was carried out at nation-wide level in Malawi using virion associated nucleic acids (VANA) high throughput sequencing (HTS) on pooled samples and appropriate alien controls. In total, 366 plants were sequenced, and 23 plant virus species were detected, three species on banana (275 plants) and 20 species in surrounding plants (91 plants). Two putative novel virus species; ginger tymo-like virus and pepper derived totivirus were detected and confirmed by RT-PCR on ginger and pepper. Nine known virus species and detected a host plant was identified for two of them. No viral exchange between banana and surrounding plants was observed. Results from the VANA protocol, applied to pooled banana samples, were compared with previous targeted PCR results obtained from individual banana samples. HTS test detected better BanMMV than IC-(RT)-PCR on individual samples (better inclusivity) but detected with much lower sensitivity BBTV and BSV species, often with less than 10 reads per sample. Detection of novel and known viruses and new host plants calls for strengthened sanitory and phytosanitory measures within and beyond banana production systems. Our research confirms that HTS sensitivity depends on sampling, pooling protocol and targeted virus species. Full article

Extracellular vesicles (EVs), nanoscale membrane-enclosed particles, are natural carriers of proteins and nucleic acids. Microalgae are widely used as a source of bioactive substances in the food and cosmetic industries and definitely have a potential to be used as the producers of EVs for biomedical applications. In this study, the extracellular vesicles isolated from the culture medium of two unicellular microalgae, Chlamydomonas reinhardtii (Chlamy-EVs) and Parachlorella kessleri (Chlore-EVs), were characterized by atomic force microscopy (AFM), cryo-electronic microscopy (cryo-EM), and nanoparticle tracking analysis (NTA). The biocompatibility with human cells in vitro (HEK-293T, DF-2 and A172) and biodistribution in mouse organs and tissues in vivo were tested for both microalgal EVs. An exogenous therapeutic protein, human heat shock protein 70 (HSP70), was successfully loaded to Chlamy- and Chlore-EVs, and its efficient delivery to human glioma and colon carcinoma cell lines has been confirmed. Additionally, in order to search for potential therapeutic biomolecules within the EVs, their proteomes have been characterized. A total of 105 proteins were identified for Chlamy-EVs and 33 for Chlore-EVs. The presence of superoxide dismutase and catalase in the Chlamy-EV constituents allows for considering them as antioxidant agents. The effective delivery of exogenous cargo to human cells and the possibility of the particle yield optimization by varying the microalgae growth conditions make them favorable producers of EVs for biotechnology and biomedical application. Full article

Irradiation with ultraviolet light-emitting diodes (UV-LEDs) represents a promising method for viral inactivation, but a detailed understanding of the wavelength-dependent action spectra remains limited, particularly across different viral components. In this study, we established standardized UV action spectra for infectivity reduction in pathogenic viruses using a system equipped with interchangeable LEDs at 13 different peak wavelengths (250–365 nm). The reduction in viral infectivity induced by UV-LED exposure was strongly related to viral genome damage, whereas no significant degradation of viral structural proteins was detected. Peak virucidal efficiency was observed at 267–270 nm across all tested viruses, representing a slight shift from the traditionally expected 260 nm nucleic acid absorption peak. Enveloped RNA viruses, including influenza A virus, respiratory syncytial virus, and coronavirus, exhibited greater UV sensitivity than nonenveloped viruses such as feline calicivirus and adenovirus. These observations indicate that structural characteristics, such as the presence of an envelope and genome organization, influence UV susceptibility. The wavelength-specific action spectra established in this study provide critical data for optimizing UV-LED disinfection systems to achieve efficient viral inactivation while minimizing energy consumption in healthcare, food safety, and environmental sanitation. Full article

In recent years, the persistent emergence of novel infectious pathogens (epitomized by the global coronavirus disease-2019 (COVID-2019) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)) has propelled nucleic acid testing (NAT) into an unprecedented phase of rapid development. As a key technology in modern molecular diagnostics, NAT achieves precise pathogen identification through specific nucleic acid sequence recognition, establishing itself as an indispensable diagnostic tool across diverse scenarios, including public health surveillance, clinical decision-making, and food safety control. The reliability of NAT systems fundamentally depends on reference materials (RMs) that authentically mimic the biological characteristics of natural viruses. This critical requirement reveals significant limitations of current RMs in the NAT area: naked nucleic acids lack the structural authenticity of viral particles and exhibit restricted applicability due to stability deficiencies, while inactivated viruses have biosafety risks and inter-batch heterogeneity. Notably, pseudovirus has emerged as a novel RM that integrates non-replicative viral vectors with target nucleic acid sequences. Demonstrating superior performance in mimicking authentic viral structure, biosafety, and stability compared to conventional RMs, the pseudovirus has garnered substantial attention. In this comprehensive review, we critically summarize the engineering strategies of pseudovirus platforms and their emerging role in ensuring the reliability of NAT systems. We also discuss future prospects for standardized pseudovirus RMs, addressing key challenges in scalability, stability, and clinical validation, aiming to provide guidance for optimizing pseudovirus design and practical implementation, thereby facilitating the continuous improvement and innovation of NAT technologies. Full article

Swine infectious diseases, often caused by multiple co-infecting agents, pose severe global threats to pig health and industry economics. Conventional single-plex testing assays, whether relying on pathogen antigens or nucleic acids, exhibit limited efficacy in the face of co-infection events. The modern nucleic acid-based multiplex testing (NAMT) methods demonstrate substantial strengths in the simultaneous detection of multiple pathogens involving co-infections owing to their remarkable sensitivity, exceptional specificity, high-throughput, and short turnaround time. The development, commercialization, and application of NAMT assays in swine infectious disease surveillance would be advantageous for early detection and control of pathogens at the onset of an epidemic, prior to community transmission. Such approaches not only contribute to saving the lives of pigs but also aid pig farmers in mitigating or preventing substantial economic losses resulting from infectious disease outbreaks, thereby alleviating unwanted pressure on animal and human health systems. The current literature review provides an overview of some modern NAMT methods, such as multiplex quantitative real-time PCR, multiplex digital PCR, microarrays, microfluidics, next-generation sequencing, and their applications in the diagnosis of swine infectious diseases. Furthermore, the strengths and weaknesses of these methods were discussed, as well as their future development and application trends in swine disease diagnosis. Full article