A current challenge regarding microfluidic paper-based analytical devices (µPAD) for blood plasma separation (BPS) and electrochemical immunodetection of protein biomarkers is how to achieve a µPAD that yields enough plasma to retain the biomarker for affinity biosensing in a functionalized electrode system. This paper describes the development of a BPS µPAD to detect and quantify the S100B biomarker from peripheral whole blood. The device uses NaCl functionalized VF2 filter paper as a sample collection pad, an MF1 filter paper for plasma retention, and an optimized microfluidic channel geometry. An inverted light microscope, scanning electron microscope (SEM), and image processing software were used for visualizing BPS efficiency. A design of experiments (DOE) assessed the device’s efficacy using an S100B ELISA Kit to measure clinically relevant S100B concentrations in plasma. The BPS device obtained 50 μL of plasma from 300 μL of whole blood after 3.5 min. The statistical correlation of S100B concentrations obtained using plasma from standard centrifugation and the BPS device was 0.98. The BPS device provides a simple manufacturing protocol, short fabrication time, and is capable of S100B detection using ELISA, making one step towards the integration of technologies aimed at low-cost POC testing of clinically relevant biomarkers.

DNA is packaged in an octamer of histones, forming chromatin, a complex of DNA and proteins. The structural matrix of a chromosome, chromatin and its changes are now regarded as important factors in controlling gene expression, which has sparked a lot of interest in understanding genetic pathways governing various diseases, including cancer. DNA methylation in the CpG dinucleotide as a transcriptional silencing mechanism, post-translational histone modifications such as acetylation, methylation, and others that affect chromatin structure, ATP-dependent chromatin remodelling, and miRNA-mediated gene silencing are all found to be important in various types of cancer. In this review, we analyze the main alterations in gene expression, epigenetic modification patterns in cancer cells, the main modulators and inhibitors of each epigenetic mechanism, and the molecular evolution of the most representative inhibitors, all of which point to a promising future for HAT, HDAC, non-glycoside DNMT inhibitors, and domain inhibitors.

Respiratory syncytial virus (RSV) is the most frequent cause of bronchiolitis in children under five years of age. No vaccines against this virus are currently available. RSV infection of a cell is initiated by fusion between the virion membrane and a cellular membrane, but it is not clear if the fusion process takes place at the plasma membrane or within an endosome. Most such experiments have been initiated at the traditional synchronization temperature of 4°C, an abnormal temperature for animal cells and one at which cellular homeostasis may be negatively affected. We have compared two synchronization temperatures (4°C and 22°C) to determine the kinetics of RSV entry into human bronchial epithelial cells. Following inoculation, virus entry was halted at different times by the addition of neutralizing antibody or temperature reduction to 4°C. We engineered a virus that encodes an extra viral gene, beta-lactamase fused to the viral phosphoprotein (P), to enable rapid detection after infection initiation. We found that the synchronization temperature used during inoculation determines the site of fusion. Transition from 4°C to 37°C resulted in RSV entry via the endosomal pathway but also induced F-actin disruption and plasma membrane blebbing, whether the cells were inoculated with RSV or not. Transition from 22°C to 37°C resulted in RSV entry by fusion at the plasma membrane and without the F-actin and plasma membrane disruptions. These results suggest that RSV normally enters cells by fusion at the plasma membrane and that the induction of endocytosis by infection synchronization at 4°C may be an artefact caused by distortion of the plasma membrane-supporting cytoskeleton. Author Summary In order to understand the overall mechanism driving infection, it is important to determine how the virus enters cells. The pathway that RSV uses to infect cells is unclear. It is a common practice to attach the virions at 4°C to synchronize the viral infection. In this report, we found that warming up primary cultures of undifferentiated normal human bronchial epithelial cells to 37°C from 4°C triggered dramatic changes in their cell membrane and cytoskeleton totally unrelated to the presence of the virions. The assessment of viral content delivery to the cytoplasm using RSV engineered to express BlaM allowed us to find that the virions attached at 4°C or 22°C fused their envelope with endosome or plasma membrane, respectively. Consequently, the entry via endosome after attachment at 4°C is an experimental artefact and RSV infects by fusing its envelope with the plasma membrane. The implications go beyond RSV since the entry of several virus species have been explored by synchronizing the infection after attachment at 4°C.

The diagnosis of pediatric meningoencephalitis has traditionally relied on the detection of inflammatory markers in cerebrospinal fluid (CSF), particularly pleocytosis, as an indirect indicator of infection. This assumption presumes that microbial invasion of the central nervous system consistently triggers a measurable immune response. In this prospective observational study conducted in Cartagena, Colombia, we enrolled 166 pediatric patients with suspected meningoencephalitis. All patients underwent testing with the FilmArray Meningitis/Encephalitis (FA-M/E) panel for direct pathogen detection in CSF, and results were compared to conventional CSF cytochemical analysis and culture. The FA-M/E panel identified a pathogen in 21 of 166 patients (12.6%), whereas CSF cultures were negative in all cases. Detected pathogens included 16 viruses (76.2%), most frequently Enterovirus and Human Herpesvirus 6 and five bacteria (23.8%), including Escherichia coli K1 and Streptococcus agalactiae . Remarkably, 100% (21/21) of the FA-M/E-positive samples exhibited complete absence of pleocytosis (0 white blood cells/µL). Additionally, 33.3% (7/21) showed entirely normal CSF biochemistry. Among these cases with a normal CSF profile, the panel identified two clinically significant bacterial infections and five viral infections that would have otherwise gone undetected. These findings demonstrate that traditional CSF parameters are poor predictors of infection in patients with a positive molecular result. Routine reliance on cytochemical analysis alone may therefore delay diagnosis and treatment. Our results support the use of molecular diagnostics as a frontline tool, even in the absence of classical inflammatory CSF markers. IMPORTANCE: This study challenges the long-standing paradigm that abnormal cerebrospinal fluid (CSF) findings are required to justify molecular testing in pediatric meningoencephalitis. We show that critical central nervous system (CNS) infections can present with entirely normal CSF profiles, including cases caused by high-risk pathogens such as E. coli K1 and Streptococcus agalactiae . By highlighting the limitations of pleocytosis and biochemical markers in detecting early CNS infection, our data underscore the importance of incorporating molecular diagnostics into routine clinical evaluation, particularly for high-risk pediatric populations. Failure to do so may result in missed or delayed diagnoses and suboptimal treatment.

Background: Antiretroviral resistance-associated mutations, within the broader context of HIV-1 genetic variability, represent a growing challenge for HIV-1 control, highlighting the need for continuous molecular surveillance and mechanistic understanding of drug resistance. This study aimed to characterize mutations in the pol gene associated with resistance to protease inhibitors and to explore their structural implications. Methods: Viral RNA was extracted from plasma samples of HIV-positive patients, and a 266 bp fragment of the HIV-1 pol gene was amplified by RT-PCR and sequenced using the Sanger method. Sequences showing ≥98% homology were aligned and analyzed using MEGA v11 and the Stanford HIV Drug Resistance Database to identify resistance-associated mutations, while viral subtypes were determined using COMET, jpHMM-HIV, and STAR tools. Amino acid sequences were used for structural modeling with AlphaFold, followed by molecular docking with Nelfinavir using the CB-Dock2 server. Results: Four samples exhibited resistance-associated profiles, including high-level, intermediate, and low-level resistance, with one isolate showing high-level resistance to multiple protease inhibitors. Structural analyses revealed that Nelfinavir preferentially binds to alternative hydrophobic cavities rather than the canonical catalytic site, lacking direct interactions with the Asp25/Asp25′ dyad. Conclusions: These findings suggest a structural mechanism of resistance based on non-canonical ligand binding that may impair effective protease inhibition.