Modification of the gut microbiota by beneficial microbes can enhance an organism’s lifespan, giving rise to the concept of probiotics. Probiotics are live microorganisms that provide health benefits when taken in sufficient amounts. Owing to their outstanding health benefits, probiotics have experienced rapid expansion and gained interest for the development of new applications. The exploration of microbial applications via genetic modification is currently of great interest to researchers. Genetic engineering using the clustered regularly interspaced short palindromic repeat (CRISPR)-Cas system has received considerable attention and has established applications. Owing to these enhanced properties, the CRISPR-Cas system is currently used in medicine, agriculture, food, and biotechnology. Considering the adaptive immune system in bacteria, this genetic tool is used to alter the microbial genome. Lactic acid bacteria (LAB) are widely recognized for their probiotic potential, and over 40% of LAB species contain the CRISPR-Cas system. The rising demand for probiotics and their expanding applications necessitate the enhancement of their existing characteristics. The CRISPR-Cas system, recognized for its precision, accuracy, and speed, has enabled researchers to modify the genomes of probiotics, thereby enhancing their beneficial attributes. This system can enhance probiotic properties through additive, subtractive, or modulatory mechanisms. Various approaches have been developed to improve probiotic functionalities using the CRISPR-Cas system, such as substituting slow promoters with efficient alternatives, eliminating undesirable components, boosting metabolism, and increasing tolerance levels. Furthermore, CRISPR-engineered probiotics have emerged as next-generation probiotics with enhanced properties and advanced applications across diverse fields, including the food, medicine, agriculture, and pharmaceutical sectors.

Glycerophosphodiester phosphodiesterases (GDPDs) are biocatalysts and highly conserved enzymes in both prokaryotic and eukaryotic organisms. GDPD plays an important role in cellular metabolism by hydrolyzing glycerophosphodiester substrates into smaller but useful molecules, such as glycerol-3-phosphate and its corresponding alcohol. GDPD directly affects the pathogenicity of infectious bacteria, including Staphylococcus aureus, and consequently represents a potential therapeutic drug target. In the current study, the GDPD enzyme from vancomycin-resistant S. aureus (VRSA) was heterologously expressed in BL21(DE3)pLysS and purified. Its catalytic activity was assessed on bis(p-nitrophenyl) phosphate (BpNPP), a commonly used non-physiological substrate for phosphodiesterases. 24 compounds based on pharmacologically important aminoquinolines were evaluated for their inhibitory potential against GDPD. Among these, 15 compounds exhibited significant to moderate inhibitory potential with IC50 values ranging from 79.3 ± 4.8 to 943.5 ± 17.3 µM. Notably, compound 6 exhibited the most potent inhibition (IC50 = 79.3 ± 4.8 µM), as compared to the standard ethylenediaminetetraacetic acid EDTA (IC50 = 468.5 ± 1.09 µM). Circular dichroism (CD) spectroscopy indicates that GDPD has a properly folded secondary structure, which is essential for enzyme activity. CD spectra showed a red shift upon inhibitor addition, from 211 to 237 nm, reflecting alterations in the enzyme's secondary structure. The thermal shift assay was performed to observe the thermal stability and structural integrity of the enzyme in the presence of inhibitors. Saturation transfer difference (STD) NMR spectroscopy provided limited but key insights into the binding interactions of inhibitors with the target enzyme. Binding interactions were further explored using extensive molecular docking studies.

The rapid rise of multidrug-resistant (MDR) pathogens poses a major challenge to global healthcare, reducing the effectiveness of conventional antimicrobial therapies and necessitating innovative treatment strategies. Probiotics have emerged as promising biotherapeutic agents due to their ability to modulate the gut microbiome, inhibit pathogen colonization, produce antimicrobial compounds, and enhance host immunity. Recent advances suggest that integrating probiotics with nanotechnology and artificial intelligence (AI) may provide a powerful approach to combat MDR infections. Nanotechnology-based delivery systems improve probiotic stability, gastrointestinal survival, controlled release, and targeted delivery, thereby enhancing therapeutic efficacy. Meanwhile, AI-driven tools facilitate microbial profiling, strain selection, resistance surveillance, predictive modeling, and formulation optimization. The convergence of probiotics, nanotechnology, and AI offers a personalized and adaptive strategy for preventing and managing MDR infections while minimizing the selective pressures that drive antimicrobial resistance. This review highlights the potential of this emerging tri-modal approach as a next-generation solution against MDR pathogens.

The probiogenomic approach has gained considerable attention for elucidating the genetic basis of probiotic functionality. In this study, the probiotic potential of Enterococcus faecium F25 was comprehensively evaluated using an integrated strategy combining traditional biochemical characterization, in vitro functional assays, and whole-genome-based analyses. The in vitro assessments were conducted using plate-based assays, while genomic investigations were performed using publicly available bioinformatic tools and platforms. Both approaches consistently confirmed the strain’s typical enterococcal characteristics and its promising probiotic potential. Comprehensive genomic profiling revealed the absence of transmissible virulence-associated determinants and transferable antibiotic resistance genes, together with a comparatively low abundance of mobile genetic elements, supporting the strain’s biosafety for potential industrial and health-related applications. Functional genome annotation further identified multiple probiotic-associated determinants involved in stress tolerance, metabolic adaptability, antimicrobial activity, and bioactive compound production. In particular, genes encoding enterocins A, B, and enterolysin A, together with stress response elements and enhanced metabolic capabilities, may contribute to improved environmental resilience, gastrointestinal survival, and antagonistic activity against pathogens. Collectively, these findings indicate that E. faecium F25 possesses a promising combination of safety, functional, and metabolic traits, supporting its potential as a versatile probiotic candidate. Further in vivo investigations are required to validate its probiotic efficacy and functional performance under host-associated conditions.

Enteromorpha intestinalis is a green seaweed enriched with diverse bioactive compounds that possess substantial pharmacological and biotechnological properties. Despite the focus on their therapeutic applications, research on the development of nano-formulations is limited. Therefore, this study aimed to develop an E. intestinalis (EI) extract-based nanoemulsion for topical use. Olive oil, Tween-80 (surfactant), and PEG-400 (co-surfactant) were selected for the formulation of the nanoemulsion. The Smix ratio was set to 1:1 using a pseudo-ternary phase diagram. Moreover, a design experiment ascertained the composition of the formulation, followed by physicochemical characterization. The optimal formulation, based on droplet size, was selected for further analysis. Stability studies, antioxidant and anti-inflammatory properties of selected nanoemulsion formulation were determined and acute dermal toxicity assay was also performed. The best formulation (F6) displayed a droplet size of 183.27 ± 20.04 nm, PDI of 0.6, and viscosity of 290 ± 5.77 m-Pa.S. The developed nanoemulsion exhibited good skin compatibility and a slightly acidic pH. Both the extract and nanoemulsion formulation exhibited concentration-dependent antioxidant activity. The nanoemulsion had a lower IC50 value of 163.19 μg/mL, showing greater efficacy than the seaweed extract alone. Formulation (F6) also significantly (p < 0.05) inhibited paw volume (8-31%) compared to the control, while diclofenac sodium achieved a maximum inhibition of 41%. The designed formulation was stable, effective, and non-irritating demonstrating its potential topical application. This study presents, for the first time, a nanoemulsion formulation that incorporates E. intestinalis extract. This advancement paves the way for further in vivo studies to assess the efficacy and safety of this formulation for clinical applications.

Senescence or aging is often associated with onset of morbidities, which have been attributed to the harmful phenomenon of glycation. To date, no drug is available to combat this deleterious process. The drug repurposing is a robust and cost-effective approach to identify potential candidate molecules for drug discovery programs. In present study, Meloxicam (MEL) was evaluated for its capacity to be repurposed against glycation. Using the Fructose-BSA model, the anti-glycation effect was evaluated by measuring intrinsic fluorescence of Advanced Glycation End Products (AGEs). The fructoseamine load and free lysine availability was estimated using NBT and TNBSA assays respectively. The BSA secondary structure was assessed by Thioflavin-T, Congo red and Circular Dichroism tests. Finally, lysine blockade and carbonyl entrapment was evaluated as a possible mode of anti-glycation action. Our data showed that MEL (0.5, 1, and 2mM) has significantly reduced the AGEs formation (IC50 = 0.25mM). The load of fructosamine adducts along with free lysine availability was found to be reduced. The secondary structure of BSA was preserved. Regarding mode of action, MEL did not block lysine residues, which was also supported by computational data. However, it was found to entrap carbonyl intermediates. In conclusion, the present study demonstrate that MEL possess anti-glycation potential, which can be attributed to entrapment of carbonyl intermediates. Hence, MEL, a clinically used NSAIDs, present itself as promising candidate to be repurposed against deleterious phenomenon of glycation.

Aims and Objective: Presence of Enterococcus species in the Microbiota of humans, animals and environment has been established. E. faecium and E. faecalis are the major species involved in nosocomial infections due to their ability to resist clinically important antibiotics. Soil, one of the most important environmental niches is considered as a vector for the dissemination of pathogenic bacteria to the food chain and back to the environment. This study was conducted to evaluate the presence of specific antibiotic resistance genes among Enterococcus species previously isolated and showed resistance to clinically important antibiotics from bulk soil (BS). Methodology: Multiplex PCR strategy using specific primers and thermal conditions were used for the amplification of genes responsible for antibiotic resistance. Result: The most prevalent resistance gene among the isolates was gyrA (18%) followed by parC (15%) gene. 12% and 9% isolates carried aph (3)-IIIa and aac6-aph2 genes, respectively. While TetM, TetL, aac(3)-IV were found in different frequencies. The emergence of resistance in enterococci from BS poses a substantial risk to public health, mainly owing to the heightened likelihood of treatment failures. Conclusion: This study provides a baseline data regarding presence of antibiotic-resistant genes among enterococci in BS environment that can serve as a source for further dissemination to humans and animals. Thus, continuous identification and monitoring of their incidence and emerging antibiotic resistance is important to prevent the risk of public health.

Biomolecular functionalization offers a powerful way to tailor protein-nanoparticle interactions. In this study, bromelain, a proteolytic enzyme, has been employed as a surface modifier to investigate its effect on corona formation on selenium nanoparticles (SeNPs) and bromelain-functionalized SeNPs (BR-SeNPs) of similar hydrodynamic diameters (D-h similar to 50 nm). The dynamic light scattering (DLS) and spectroscopic techniques (UV-vis, FTIR, fluorescence, and circular dichroism) have been systematically used as qualitative and quantitative tools to comparatively monitor adsorption kinetics and structural investigations of bromelain corona in both systems. The rate of bromelain adsorption was approximately twice as fast for SeNPs compared to BR-SeNPs, reflecting the steric and kinetic effects of pre-adsorbed protein layers. BR-SeNPs formed thinner coronas, with reduced saturation levels (204 nm for soft, 178 nm for hard corona) and demonstrated higher binding (k similar to 2.0) in soft corona states. The adsorption reached a plateau at 10 g/L for SeNPs (q(e) = 0.0857) and at 8 g/L for BR-SeNPs (q(e) = 0.0675), confirming the earlier saturation of BR-SeNPs. CD analysis confirmed that bromelain largely retained its native conformation upon adsorption, particularly on SeNPs-BR. The adsorption behavior was best described by the Freundlich isotherm, indicating multilayer and heterogeneous binding, while the kinetic modeling followed a pseudo-second-order mechanism. Thermodynamic analysis confirmed that binding was spontaneous, enthalpy-entropy co-driven, and predominantly mediated by hydrogen bonding and electrostatic interactions. Notably, in serum stability assays, only bare SeNPs showed a significant increase in size, while SeNPs-BR maintained their colloidal stability. This study offers a robust strategy for designing next-generation biocompatible therapeutic and diagnostic nanocarriers.

This review explores the innovative approaches in localized drug delivery systems specifically designed to manage periodontal diseases (PD). The primary focus is on how these systems enhance the concentration of therapeutic agents at the site of infection, thereby minimizing systemic side effects and improving treatment outcomes. It emphasizes the role of biodegradable and bioadhesive polymers as carriers for sustained drug release. The chapter reviews different local drug delivery systems, such as fibers, films, chips, microparticles, and gels, highlighting their mechanisms of action, advantages, and limitations. It also discusses commonly used drugs, including chlorhexidine, doxycycline, metronidazole, and tetracycline, which are encapsulated within these systems to improve therapeutic efficacy. Additionally, the chapter delves into the polymers used in drug delivery, categorizing them into natural and synthetic biodegradable polymers, each offering unique benefits regarding biocompatibility and drug release kinetics. Polymeric therapeutics for controlled and sustained release further support the development of localized treatments. Overall, this chapter underscores the advancements in drug delivery technologies that promise to revolutionize the treatment of PD by reducing systemic side effects and optimizing therapeutic outcomes.

The oxadiazole derivatives are part of the azole family and have drawn significant interest in medicinal chemistry due to their wide range of biological activities and promising pharmacological profiles. This patent spotlight highlights the developments of the oxadiazole-based compounds reported between 2020 and 2024, with applications in treating diseases such as cancer, bacterial infections, metabolic disorders, and neurodegenerative conditions. These compounds act through various mechanisms, including enzyme inhibition, receptor modulation, and disruption of microbial and cancer cell pathways. Their structural flexibility allows for the design of novel molecules targeting specific therapeutic areas. The spotlight on these recent patents underscores oxadiazole derivatives' growing importance in drug discovery, offering potential advancements in efficacy and safety for future therapeutic agents.

OBJECTIVES:The primary aim of this study was to develop an effective treatment strategy for periodontal diseases that maximizes therapeutic effects while minimizing systemic adverse effects. Specifically, the study focused on creating a xerogel-based localized drug delivery system for the slow release of doxycycline hyclate (DH) to treat periodontal disease. METHODS:Xerogels were prepared using the solvent casting method, with the solvent being evaporated slowly at ambient conditions. The prepared DH xerogels underwent comprehensive characterization to assess their in-silico compatibility, pharmacokinetics, and physicochemical properties. The properties studied included drying time and rate, thickness, moisture content, swelling index, organoleptic properties, scanning electron microscopy, FTIR spectroscopy, differential scanning calorimetry, drug release and kinetics, and antibacterial activity. RESULTS:In-silico studies demonstrated compatibility between the ingredients, indicating minimal adverse effects on the body. The analysis revealed hydrogen bonding between the drug and polymers, changing the drug's crystallization characteristics to an amorphous form. The release profiles of DH from the xerogels indicated a slow release, ranging from 29.42% to 66.30% over 10 hours, following the Hopfenberg model. CONCLUSION:The findings of this study suggest that the formulated xerogels are well-suited for periodontal applications. The slow-release profile of DH from the xerogels offers a promising approach for localized treatment of periodontal disease, reducing the risk of systemic adverse effects. This data is valuable for dental practitioners and pharmaceutical formulators, providing a new avenue for enhancing periodontal disease treatment.

Objectives:Glycation is one of the primary underlying processes attributed to senescence and related diseases. No medicine currently targets this harmful manifestation. Drug repurposing is an efficient and cost-effective way of developing drugs. The present study evaluated meloxicam, a clinically used NSAID, for its ability to offer protection against glycative stress. Materials and Methods:Methylglyoxal (MGO; 17.25 mg/kg) was administered for two weeks to create a rat model of glycative stress. Aminoguanidine (AG; 50 mg/kg) and Meloxicam (MEL; 0.15, 0.3, and 0.6 mg/kg) were used as standard and test agents, respectively. Afterward, the cognitive (Morris Water Maze), liver (LFT), and kidney (Creatinine) functioning were evaluated. The expression of genes of interest (TNF-α, RAGE, BACE, Glyoxalase, and VEGF) were estimated (qPCR) in the liver, brain, and kidney along with histopathology (H&E staining). Carboxymethyllysine (CML) levels in rat plasma were evaluated via ELISA. Results:MEL treatment has significantly (P<0.05) protected the MGO-induced cognitive (duration in target quadrant, time taken to get to target quadrant, and the frequency of crossings via platform location), hepatic, and renal impairment. The qPCR data revealed that MEL prevented MGO-induced enhancement in the expression of genes of interest. Additionally, the CML levels were significantly (P<0.005) normalized by concomitant administration of MEL. Histopathological examination did not reveal any remarkable outcomes. Conclusion:MEL has significantly mitigated the rats' MGO-induced cognitive, liver, and kidney impairments. Hence, it appears to be a potential molecule for repurposing as an antiglycation agent.

It is essential to adopt effective therapy strategies for periodontal diseases to achieve optimal results while avoiding adverse effects on the system. This study has developed various PEGylated chitosan-based biodegradable xerogels for localized release of doxycycline hyclate (DH) to treat periodontal infectious diseases. The xerogels were formulated using the solvent casting method, and the solvent (0.25 M HCl) was slowly evaporated at ambient conditions. Two different molecular weights were employed for chitosan and polyethylene glycol, and twelve combinations, including the placebos and controls, were prepared for the formulation of xerogels. Different physical and chemical characteristics of the prepared DH xerogels were studied, such as drying time and rate, thickness, moisture content, swelling index, organoleptic characteristics, scanning electron microscopy, FTIR spectrometry, differential scanning calorimetry, drug release and kinetics, and antibacterial activity. The results revealed that the drug transforms from a crystalline to an amorphous state, thus rapidly releasing the drug (> 60% in 30 min. in all xerogels), followed by a sustained release up to 10 h. The release kinetics results revealed that the drug followed the Korsmeyer-Peppas model. It is concluded that the formulated DH-loaded xerogels showed promising results for use in the periodontal pockets to treat various infectious diseases.

The emergence and applications of probiotic species across industries are growing rapidly, requiring the isolation, identification, and robust characterization of new strains. Enterococcus faecium, a dominant species of the genus Enterococcus, is widely distributed and has a prominent role in biotechnological applications. The probiotic potential of E. faecium is well established, and various strains have been commercially available. In this study, we aimed to provide a strategic road map to explore the probiotic potential, postbiotic production, antioxidant activities, aggregation properties, and functional characterization of the selected E. faecium strains (n = 6) isolated locally. All selected strains demonstrated significant probiotic potential, with stress tolerance, aggregation, and postbiotic production. They were free from biogenic amines while exhibiting notable free radical scavenging and reducing activities. Additionally, their ability to adhere to fibrinogen and mucin indicates enhanced potential for mucosal colonization, competitive exclusion of pathogens, and improved host interaction. All strains tolerated digestive stress, two strains (E. faecium Se142 and E. faecium F25) produced slime, and all exhibited antioxidant activity. The influence of digestive enzymes on enterocins, the production of arginine hydrolases, and the impact of glycine, arginine, and glucose on their growth performance reflected positive attributes. These attributes indicate their potential as ideal candidates for developing new probiotic formulations, with intended food and biotechnological applications. In the future, genomic and in vivo validation studies are warranted.

Klebsiella pneumoniae is a nosocomial pathogen that poses a serious concern due to the high prevalence of extended-spectrum beta-lactamases (ESBL) and carbapenem-resistant K. pneumoniae (CRKP) strains. However, the data on the prevalence of contributing virulence determinants are limited in the Pakistani population. The study aims to characterize clinical isolates of K. pneumoniae to understand clonal relationships and determine the relationship of antibiotic resistance with different virulence factors (i.e., biofilm, capsular polysaccharide, hemolysis, efflux pump, and outer membrane porins). The clinical strains were collected from the diagnostic facility of two public sector hospitals in Karachi. The resistance and virulence profile of the isolates were evaluated via antibiotic susceptibility test, double disk synergy test (DDST), ChromAgar, string test, blood hemolysis, and biofilm assay. Genotypically, the isolates were identified by 16S rRNA and rpoB gene, and further characterized for the presence of ESBL, CRKP, biofilm, efflux pump, and outer membrane porins genes. The clonal lineage among isolates was established by Enterobacterial Repetitive Intergenic Consensus Polymerase Chain Reaction (ERIC PCR). The antibiotic susceptibility test was analyzed through the Multiple Antibiotic Resistance (MAR) index, which revealed 90.2 % (n = 102) strains were MDR. Whereas, the genetic diversity was revealed through ERIC PCR and clade wise data revealed genetic variations due to ESBL (85 %), carbapenem resistance (73 %), biofilm (97 %), efflux pump (40-53 %), outer membrane porins (38-49 %), and hypermucoidity (6 %). The Pearson correlation analysis revealed a strong relationship of MDR strains with biofilm (r = 0.99), efflux pump (r = 0.92), and outer membrane porins (r = 0.88). The study highlighted the prevalence of MDR K. pneumonia with the plethora of virulence factors in the local clinical setting, necessitating stringent screening to develop national policies to tackle further antimicrobial resistance development and outbreaks.

Probiotics are live microorganisms that, when administered in adequate amounts, confer a health benefit to the host. There are selection criteria that are followed while claiming the probiotic potential of a test strain. Besides other health benefits of probiotics, their anti-aging properties have recently grasped the attention of researchers and are considered of high significance. Aging is a natural, biological, and irreversible process through which every individual passes. The world's elderly population is increasing, and it is anticipated that there will be 1.6 billion elderly people in 2050. Efforts, both individually and globally, are made to reduce or slow down the aging process. Presently, the role of probiotics, majorly from the genera lactobacilli and Bifidobacterium, is widely investigated for their anti-aging potential. The exact mechanisms by which gerobiotics (anti-aging probiotics) can attenuate aging are still under explanation, but evidence shows that gerobiotics can target transcription factors, act via oxidative stress mechanisms, and reduce the physiological process of aging. C. elegans, mice, and rats are used as experimental models in aging studies. Several gerobiotics were tailored in an experimental model and obtained promising results. Although the current data is not sufficient for human trials, there is a need for more experiments that correctly define the mode of action, pathways, and association of the host with gerobiotics. Considering the importance of the field, the increase in experimental observations, and the availability of less literature, this review article aims to provide recent, updated, and cumulative literature about the anti-aging properties of probiotics.

Collagenases are enzymes that break down collagen and are used in wound healing and treating various disorders. Currently, collagenase is commercially available in only ointment and injectable forms and is sensitive to various environmental factors. In the present study, different hydrogel formulations of collagenase have been prepared at pH 6.5 using carboxymethylcellulose sodium and zinc acetate with and without humectants such as propylene glycol (PG) and glycerin (GL) in varying concentrations. The formulated gels were stored at room temperature (25±2°C, 60±5% RH) and refrigerator temperature (5±3°C) for six months to evaluate their physical and up to six years for chemical stability. The gels were subjected to various tests, including organoleptic studies, spreadability, moisture content, swelling index, swelling/de-swelling, syneresis, viscosity, gelation time, and weight variation. The purity and molecular weight of collagenase have been determined using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). At the same time, its activity during the storage period was evaluated by gelatin zymography. Casein zymography was also performed to detect any caseinase contamination in the formulations. The release of the enzyme from different gel formulations was assessed using the Franz diffusion apparatus and analyzed by gelatin zymography. The results showed some physical changes that were more prominent in gels stored at room temperature than those kept refrigerated. The difference in humectant concentration was also found to affect the stability of gels. PG was found to be a better humectant than GL, particularly in a concentration of 25%. The zymography results indicated that collagenase was stable in all formulations kept in the refrigerator. In contrast, its complete degradation was noted in the preparations stored at room temperature within a month. The data generated in this study will help the formulators to commercialize a relatively economical gel formulation of collagenase that is highly stable for up to six years at refrigerator temperature (5±3°C).