Pradeepraj Durairaj

Post-translational modifications, such as truncation facilitated by proteases and phosphorylation mediated by protein kinases, play pivotal roles in Tau protein function and cellular processes. The detection of distinct Tau forms in plasma has garnered significant interest in the scientific and translational communities. We discovered monoclonal antibodies (mAbs) using the hybridoma technique by immunizing mice with different Tau proteins. Enzyme-linked immunosorbent assay (ELISA), surface plasmon resonance, and Western blot experiments were used to evaluate these mAbs. Following that, we developed immunoassays on ELISA and single-molecule array (Simoa) platforms, respectively. The concentration of Tau fragments in human plasma was detected by Simoa assays to develop novel blood ratio biomarkers. Six mAbs were developed in this study, and on this basis, nine ELISA assays and three Simoa assays were established. The detection limits of Simoa-Assay 1-3 were 0.18, 0.12, and 2.70 pg/mL, respectively. The lower limit of quantification is 0.775, 0.647, and 3.630 pg/mL. The concentrations of three different Tau fragments in plasma were detected by Simoa-Assay 1-3, and the ratios of p-Tau217/t-Tau217 and Tau (1-22)-(187-208)/Tau (1-22)-(210-227) in human plasma are reported for the first time. Our three novel Tau Simoa-assays have the potential to be effective and efficient plasma-based screening tools for Alzheimer's disease (AD). Furthermore, the new ratios p-Tau217/t-Tau217 and Tau (1-22)-(187-208)/Tau (1-22)-(210-227) may be potential novel AD plasma biomarkers.

Extracellular vesicles (EVs) derived from human organoids are phospholipid bilayer-bound nanoparticles that carry therapeutic cargo. However, the low yield of EVs remains a critical bottleneck for clinical translation. Vertical-Wheel bioreactors (VWBRs), with unique design features, facilitate the scalable production of EVs secreted by human blood vessel organoids (BVOs) under controlled shear stress, using aggregate- and microcarrier-based culture systems. Human induced pluripotent stem cell-derived BVOs cultured as aggregates or on Synthemax II microcarriers within VWBRs (40 and 80 rpm) were compared to static controls. The organoids were characterized by metabolite profiling, flow cytometry, and gene expression of EV biogenesis markers. EVs were characterized by nanoparticle tracking analysis, electron microscopy, and Western blotting. Lipidomics provided insights into EV lipid composition, while functional assays assessed the impact of EVs in a D-galactose-induced senescence model. VWBR cultures showed more aerobic metabolism and higher expression of EV biogenesis genes compared to the static control. EVs from different conditions were comparable in size, but the yields were significantly higher for microcarrier and dynamic cultures than static aggregates. Lipidomic profiling revealed minimal variation (< 0.36%) in total lipid content; however, distinct differences were identified in lipid chain lengths and saturation levels, affecting key pathways such as sphingolipid and neurotrophin signaling. Human BVO EVs demonstrated the abilities of reducing oxidative stress and increasing cell proliferation in vitro. Human BVOs differentiated in VWBRs (in particular 40 rpm) produce 2–3 fold higher yield of EVs (per mL) than static control. The bio manufactured EVs from VWBRs have exosomal characteristics and therapeutic cargo, showing functional properties in in vitro assays. This innovative approach establishes VWBRs as a scalable platform for producing functional EVs with defined lipid profiles and therapeutic potential, paving the way for future in vivo studies.

Human cytochrome P450 (CYP) enzymes in the brain represent a crucial frontier in neuroscience, with far-reaching implications for drug detoxification, cellular metabolism, and the progression of neurodegenerative diseases. The brain’s complex architecture, composed of interconnected cell types and receptors, drives unique neuronal signaling pathways, modulates enzyme functions, and leads to distinct CYP gene expression and regulation patterns compared to the liver. Despite their relatively low levels of expression, brain CYPs exert significant influence on drug responses, neurotoxin susceptibility, behavior, and neurological disease risk. These enzymes are essential for maintaining brain homeostasis, mediating cholesterol turnover, and synthesizing and metabolizing neurochemicals, neurosteroids, and neurotransmitters. Moreover, they are key participants in oxidative stress responses, neuroprotection, and the regulation of inflammation. In addition to their roles in metabolizing psychotropic drugs, substances of abuse, and endogenous compounds, brain CYPs impact drug efficacy, safety, and resistance, underscoring their importance beyond traditional drug metabolism. Their involvement in critical physiological processes also links them to neuroprotection, with significant implications for the onset and progression of neurodegenerative diseases. Understanding the roles of cerebral CYP enzymes is vital for advancing neuroprotective strategies, personalizing treatments for brain disorders, and developing CNS-targeting therapeutics. This review explores the emerging roles of CYP enzymes, particularly those within the CYP1–3 and CYP46 families, highlighting their functional diversity and the pathological consequences of their dysregulation on neurological health. It also examines the potential of cerebral CYP-based biomarkers to improve the diagnosis and treatment of neurodegenerative disorders, offering new avenues for therapeutic innovation.

Quorum sensing (QS) inhibition has emerged as a promising target for directed drug design, providing an appealing strategy for developing antimicrobials, particularly against infections caused by drug‐resistant pathogens. In this study, we designed and synthesized a total of 33 β‐nitrostyrene derivatives using 1‐nitro‐2‐phenylethane (NPe) as the lead compound, to target the facultative anaerobic bacterial pathogen Serratia marcescens. The QS‐inhibitory effects of these compounds were evaluated using S. marcescens NJ01 and the reporter strain Chromobacterium violaceum CV026. Among the 33 new β‐nitrostyrene derivatives, (E)‐1‐methyl‐4‐(2‐nitrovinyl)benzene (m‐NPe, compound 28) was proven to be a potent inhibitor that reduced biofilm formation of S. marcescens NJ01 by 79%. Scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) results revealed that treatment with m‐NPe (50 μg/ml) not only enhanced the susceptibility of the formed biofilms but also disrupted the architecture of biofilms by 84%. m‐NPe (50 μg/ml) decreased virulence factors in S. marcescens NJ01, reducing the activity of protease, prodigiosin, and extracellular polysaccharide (EPS) by 36%, 72%, and 52%, respectively. In S. marcescens 4547, the activities of hemolysin and EPS were reduced by 28% and 40%, respectively, outperforming the positive control, vanillic acid (VAN). The study also found that the expression levels of QS‐ and biofilm‐related genes (flhD, fimA, fimC, sodB, bsmB, pigA, pigC, and shlA) were downregulated by 1.21‐ to 2.32‐fold. Molecular dynamics analysis showed that m‐NPe could bind stably to SmaR, RhlI, RhlR, LasR, and CviR proteins in a 0.1 M sodium chloride solution. Importantly, a microscale thermophoresis (MST) test revealed that SmaR could be a target protein for the screening of a quorum sensing inhibitor (QSI) against S. marcescens. Overall, this study highlights the efficacy of m‐NPe in suppressing the virulence factors of S. marcescens, identifying it as a new potential QSI and antibiofilm agent capable of restoring or improving antimicrobial drug sensitivity.

This work evaluated 3-(2-isocyanobenzyl)-1H-indole derivatives as quorum sensing inhibitors for the control of Pseudomonas aeruginosa infections in vitro.

Background: The calibrator in immunoassay plays an essential role in diagnosing Alzheimer’s disease (AD). Presently, the most well-studied biomarkers for AD diagnosis are three phosphorylated Tau (p-Tau): p-Tau231, p-Tau217, and p-Tau181. Glycogen synthase-3beta (GSK3β)-phosphorated Tau-441 is the most commonly used calibrator for p-Tau immunoassays. However, the batch-to-batch inconsistency issue of the commonly used GSK3β-phosphorylated Tau-441 limits its clinical application.Methods: We have successfully generated and characterized 61 Tau monoclonal antibodies (mAbs) with distinct epitopes by using the hybridoma technique and employed them as capture or detection antibodies for p-Tau immunoassays. Through chemical synthesis, we synthesized calibrators, which are three peptides including capture and detection antibody epitopes, for application in immunoassays that detect p-Tau231, p-Tau217, and p-Tau181. The novel calibrators were applied to Enzyme-linked immunosorbent assay (ELISA) and Single-molecule array (Simoa) platforms to validate their applicability and establish a range of p-Tau immunoassays.Results: By employing the hybridoma technique, 49 mAbs recognizing Tau (1–22), nine mAbs targeting p-Tau231, one mAb targeting p-Tau217, and two mAbs targeting p-Tau181 were developed. Peptides, including recognition epitopes of capture and detection antibodies, were synthesized. These peptides were used as calibrators to develop 60 immunoassays on the ELISA platform, of which six highly sensitive immunoassays were selected and applied to the ultra-sensitive Simoa platform. Remarkably, the LODs were 2.5, 2.4, 31.1, 32.9, 46.9, and 52.1 pg/ml, respectively.Conclusion: Three novel p-Tau calibrators were successfully generated and validated, which solved the batch-to-batch inconsistency issue of GSK3β-phosphorylated Tau-441. The novel calibrators exhibit the potential to promote the standardization of clinical AD diagnostic calibrators. Furthermore, we established a series of highly sensitive and specific immunoassays on the Simoa platform based on novel calibrators, which moved a steady step forward in p-Tau immunoassay application for AD diagnosis.

Quorum sensing (QS) inhibition is recognized as a novel antimicrobial target for infections caused by drug-resistant pathogens and is an attractive strategy for antipathogenic agent development. We designed and synthesized three parts of 3-(2-isocyanobenzyl)-1H-indole derivatives and tested their activity as novel quorum sensing inhibitors (QSIs). 3-(2-Isocyanobenzyl)-1H-indole derivatives demonstrated promising QS, biofilms, and prodigiosin inhibitory activities against Serratia marcescens at subminimum inhibitory concentrations (sub-MICs). In particular, 3-(2-isocyano-6-methylbenzyl)-1H-indole (IMBI, 32) was identified as the best candidate based on several screening assays, including biofilm and prodigiosin inhibition. Further studies demonstrated that exposure to IMBI at 1.56 mu g/mL to S. marcescens NJ01 significantly inhibited the formation of biofilms by 42%. The IMBI treatment on S. marcescens NJ01 notably enhanced the susceptibility of the formed biofilms, destroying the architecture of the biofilms by up to 40%, as evidenced by scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM). For interference of virulence factors in S. marcescens NJ01, IMBI at 3.12 mu g/mL inhibited the activity of protease and extracellular polysaccharides (EPS) by 17% and 51%, respectively, which were higher than that of the positive control vanillic acid (VAN). Furthermore, IMBI downregulated the expression of QS- and biofilm-related genes fimA, bsmA, pigP, flhC, rssB, fimC, and rsmA by 1.02- to 2.74-fold. To confirm these findings, molecular docking was performed, which indicated that the binding of IMBI to SmaR, RhlI, RhlR, LasR, and CviR could antagonize the expression of QS-linked traits. In addition, molecular dynamic simulations (MD) and energy calculations indicated that the binding of receptors with IMBI was extremely stable. The biofilms of S. marcescens NJ01 were markedly reduced by 50% when IMBI (0.39 mu g/mL) was combined with kanamycin (0.15 mu g/mL). In conclusion, this study highlights the potency of IMBI in inhibiting the virulence factors of S. marcescens. IMBI has all the potential to be developed as an effective and efficient QS inhibitor and antibiofilm agent in order to restore or improve antimicrobial drug sensitivity.

Human drug metabolites (HDMs) are important chemicals widely used in drug-related studies. However, acquiring these enzyme-derived and regio-/stereo-selectively modified compounds through chemical approaches is complicated. PikC is a biosynthetic P450 enzyme involved in pikromycin biosynthesis from the bacterium Streptomyces venezuelae. Here, we identify the mutant PikC_D50N as a potential biocatalyst, with a broad substrate scope, diversified product profile, and high catalytic efficiency, for preparation of HDMs. Remarkably, PikC_D50N can mediate the drug-metabolizing reactions using the low-cost H₂O₂ as a direct electron and oxygen donor.