Microalgae are increasingly recognized as renewable biofactories for producing high-value bioactive molecules. However, their industrial exploitation is limited by their rigid cell walls, metabolite heterogeneity, and the energy-intensive nature of the extraction processes. Recent advances in process-intensification technologies, including microwave-assisted, ultrasound-assisted, enzymatic, pressurized liquid, and supercritical CO₂-based methods, have significantly improved extraction efficiency and selectivity, with reported lipid recoveries exceeding 40–50% in some microalgal systems and carotenoid recoveries approaching 90% under optimized conditions. NADES-assisted systems further enhance mass transfer and solubilization through tailored hydrogen-bonding interactions, enabling selective extraction of polar and semi-polar metabolites under mild conditions. However, limitations remain, including high viscosity, variability in extraction performance, and challenges in solvent recovery and scale-up. This review critically evaluates the extraction efficiency, mechanistic basis, and sustainability of NADES-assisted processes, highlighting key limitations and identifying research priorities for their integration into scalable microalgal biorefinery systems.

Introduction: Multidrug-resistant urinary tract pathogens, primarily Klebsiella pneumoniae and Pseudomonas aeruginosa , represent a significant and growing public health challenge. The overuse of antibiotics has accelerated the development of resistance, creating an urgent need for alternative antimicrobial strategies. This study aimed to evaluate the antibacterial efficacy of a peppermint oil nanoemulsion (PEONE) against clinical multidrug-resistant (MDR) isolates of K. pneumoniae and P. aeruginosa , with a specific focus on its potential for resistance modulation and its mechanism of action. Methods: Clinical isolates were obtained from patients with urinary tract infections and their antibiotic susceptibility profiles were determined. The PEONE was prepared using ultrasonic emulsification and characterized, revealing a droplet size of 190.21 ± 0.5 nm and a polydispersity index (PDI) of 0.15 ± 0.021. Antibacterial activity was assessed by determining the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). Membrane integrity was evaluated using DNA and protein leakage assays, and bacterial killing over time was measured with time-kill kinetics. Furthermore, molecular docking and 100 ns molecular dynamic simulations were performed against β-lactamase enzymes (PDB: 4EXY from K. pneumoniae , 6R73 from P. aeruginosa ) to identify key bioactive components within the nanoemulsion. Results: The bacterial isolates were resistant to Levofloxacin, Penicillin G, Ceftazidime, and amoxicillin-clavulanic acid (AMC). PEONE demonstrated potent antibacterial activity, with an MIC of 0.1% v/v and an MBC of 0.14% v/v. DNA and protein leakage increased significantly ( p < 0.05) with higher PEONE concentrations, indicating bacterial membrane disruption. Time-kill assays showed a sustained reduction in bacterial viability over 72 hours, with significant differences emerging after 12 hours of exposure. Molecular docking revealed that caryophyllene, a major component of peppermint oil, had the highest binding affinity against both β-lactamase targets (−9.2 kcal/mol for 4EXY; −7.1 kcal/mol for 6R73). The stability of this binding was confirmed through molecular dynamics simulations. Discussion: The findings indicate that PEONE is effective at inhibiting and killing MDR K. pneumoniae and P. aeruginosa . The observed leakage of DNA and proteins suggests that the mechanism of action likely involves disruption of the bacterial membrane, leading to the loss of intracellular contents. This is further supported by the computational data, which identified caryophyllene as a key component with stable binding to resistance-associated β-lactamase enzymes. These results position PEONE as a promising, plant-based alternative for combating antibiotic-resistant urinary tract infections. Further in vivo studies are warranted to explore its clinical applicability and safety profile.

Marburg Virus Disease (MVD) is a life-threatening hemorrhagic fever, caused by two viruses-Marburg virus (MARV) and Ravn virus (RAVV)-that belong to the family Filoviridae. Due to its high mortality rate and the lack of licensed vaccines, MVD remains a major global health concern. In this study, a comprehensive immunoinformatics workflow was used to design a universal chimeric multi-epitope subunit vaccine targeting conserved antigenic regions of both viruses to reduce MVD-related mortality. Antigenicity analysis identified glycoproteins (GP) of MARV and RAVV as the most immunogenic viral proteins, and multiple sequence alignment revealed conserved regions suitable for epitope selection. Three highly conserved, antigenic, non-allergenic, and non-toxic CTL, HTL, and B-cell epitopes from each virus were incorporated into the final vaccine construct. The designed vaccine exhibited high antigenicity, non-allergenicity, solubility, and acceptable physicochemical properties. Structural modeling generated a high-quality 3D structure, and molecular docking revealed stable binding to human TLR4 with a docking score of − 229.46 kcal/mol, supported by multiple hydrogen bonds and non-bonded interactions, suggesting robust immune activation potential. Codon optimization produced a CAI value of 0.96 and 52.2% GC content, indicating efficient expression in E. coli K-12, further validated through in silico cloning. Immune simulations showed robust primary and secondary immune responses, including high levels of IgM, IgG, IFN-γ, IL-2, and strong memory cell formation. Molecular dynamics simulation analyses confirmed the stability (RMSD), compactness (Rg), and consistent hydrogen-bonding interactions between the designed vaccine and TLR4. Overall, the computational analyses indicate that the proposed chimeric multi-epitope vaccine is stable, highly immunogenic, and capable of eliciting protective immune responses, providing a strong basis for subsequent in vitro and in vivo validation.

Peripheral nerve injuries (PNIs) remain a major cause of long-term disability, with standard treatments such as microsurgical repair and autologous grafting often yielding incomplete recovery due to slow axonal regeneration, fibrotic scarring, and limited reinnervation. Emerging therapies, including electrical stimulation (ES) and platelet-rich plasma (PRP), have shown promise but remain insufficient as standalone interventions. ES enhances axonal elongation, remyelination, and neuroplasticity by upregulating regeneration-associated genes and neurotrophins, while PRP delivers autologous growth factors that promote angiogenesis, Schwann cell activation, immunomodulation, and antioxidant defense. Both therapies converge on shared pathways by reducing inflammation, oxidative stress, and scar formation, thereby remodeling the microenvironment into a pro-regenerative niche. Preclinical evidence indicates that combining ES and PRP provides complementary benefits, with ES priming the injury site and PRP sustaining trophic support, resulting in superior axonal density, myelination, and functional recovery compared to monotherapies. Future directions emphasize personalized protocols, optimized ES parameters, standardized PRP formulations, and integration with biomaterials and closed-loop stimulation systems. Translation to clinical practice, however, requires standardized guidelines and rigorous randomized controlled trials to validate these multimodal strategies and enable patient-specific regenerative therapies. Graphical Abstract A visual summary of the nerve regeneration process, showcasing how electrical stimulation and platelet-rich plasma target shared pathways through complementary mechanisms. The graphic also explores future innovations like biomaterial integration and personalized medicine approaches. Peripheral nerve injury regeneration pathway

Neonatal sepsis is a significant cause of morbidity and mortality in infants, particularly those caused by Gram-negative bacteria. Antibiotic resistance ma

Background Methicillin-resistant Staphylococcus aureus (MRSA) represents a significant and growing public health challenge in Pakistan, due to antibiotic misuse, weak infection control measures and rapid bacterial evolution. Determining the overall prevalence of MRSA in the country is crucial for informing targeted treatment protocols and effective management strategies. This study conducted a systematic review and meta-analysis to establish the pooled prevalence of MRSA in Pakistan. Methods We searched electronic databases (Google Scholar, PubMed, Embase) for studies published between 2015 and 2025. The PRISMA guidelines were followed to conduct this systematic review. Meta-analyses were performed using R Studio software, applying both fixed- and random-effects models to calculate pooled prevalence. The heterogeneity was assessed using the I² statistics. Publication bias was assessed using Begg’s and Egger’s tests. Most studies used cefoxitin disk diffusion to characterize MRSA, while some studies used PCR to detect the mecA gene. Results Sixty-eight studies meeting the inclusion criteria were analyzed. The overall pooled MRSA prevalence was estimated at 50% (95% CI: 45–54%), with I² = 98.9). Year-wise subgroup analysis revealed significant variation from 2015 to 2025, with estimates ranging from 64% (95% CI: 26–90%), with I² = 96.68 in 2022 to a low of 34% (95% CI: 17–56%), with I² = 99.16 in 2020, which may be due to reduced healthcare access and infection control measures implemented during the COVID-19 lockdowns. The pooled estimate among general patients and healthcare personnel was 50% (95% CI: 44–57%), with I² = 98.5 and 44% (95% CI: 30–60%), with I² = 95.4, respectively. The subgroup differences test (p = 0.54) for population type revealed no statistically significant difference. The Begg’s test (p = 0.2378) and Egger’s test (p = 0.8893) showed no significant evidence of bias. The significant variation between the two diagnostic methods (p = 0.03) showed that the observed heterogeneity may be due to diagnostic methods used across the studies. Conclusion The high pooled prevalence of MRSA in Pakistan highlights an urgent need for strengthened antimicrobial stewardship, standardized surveillance systems, and integrated infection prevention strategies.