Quantification Approaches in Non-Target LC/ESI/HRMS Analysis: An Interlaboratory Comparison.

Nontargeted screening (NTS) utilizing liquid chromatography electrospray ionization high-resolution mass spectrometry (LC/ESI/HRMS) is increasingly used to identify environmental contaminants. Major differences in the ionization efficiency of compounds in ESI/HRMS result in widely varying responses and complicate quantitative analysis. Despite an increasing number of methods for quantification without authentic standards in NTS, the approaches are evaluated on limited and diverse data sets with varying chemical coverage collected on different instruments, complicating an unbiased comparison. In this interlaboratory comparison, organized by the NORMAN Network, we evaluated the accuracy and performance variability of five quantification approaches across 41 NTS methods from 37 laboratories. Three approaches are based on surrogate standard quantification (parent-transformation product, structurally similar or close eluting) and two on predicted ionization efficiencies (RandFor-IE and MLR-IE). Shortly, HPLC grade water, tap water, and surface water spiked with 45 compounds at 2 concentration levels were analyzed together with 41 calibrants at 6 known concentrations by the laboratories using in-house NTS workflows. The accuracy of the approaches was evaluated by comparing the estimated and spiked concentrations across quantification approaches, instrumentation, and laboratories. The RandFor-IE approach performed best with a reported mean prediction error of 15× and over 83% of compounds quantified within 10× error. Despite different instrumentation and workflows, the performance was stable across laboratories and did not depend on the complexity of water matrices.

High-quality haplotype-resolved chromosome assembly provides evolutionary insights and targeted steviol glycosides (SGs) biosynthesis in Stevia rebaudiana Bertoni.

Stevia rebaudiana Bertoni is popular source of plant-derived low/no-calorie natural sweeteners (LNCSs), collectively known as steviol glycosides (SGs). Nevertheless, genetic predisposition for targeted biosynthesis of SGs is complex due to multi-substrate functionality of key uridine diphosphate glycosyltransferases (UGTs). Here, we created a high-quality monoploid assembly of 1.34 Gb with N50 value of 110 Mb, 55 551 predicted protein-coding genes, and ~80% repetitive regions in Rebaudioside-A (Reb-A) enriched cultivar of S. rebaudiana. Additionally, a haplotype-based chromosome assembly consisting of haplotype A and haplotype B with an overall genome size of 2.33Gb was resolved, harbouring 639 634 variants including single nucleotide polymorphisms (SNPs), indels and structural variants (SVs). Furthermore, a lineage-specific whole genome duplication analysis revealed that gene families encoding UGTs and Cytochrome-P450 (CYPs) were tandemly duplicated. Additionally, expression analysis revealed five tandemly duplicated gene copies of UGT76G1 having significant correlations with Reb-A content, and identified key residue (leu200val) in the glycosylation of Reb-A. Furthermore, missense variations identified in the acceptor region of UGT76G1 in haplotype resolve genome, transcriptional and molecular docking analysis were confirmed with resequencing of 10 diverse stevia genotypes (~25X). Gene regulatory network analysis identified key transcription factors (MYB, bHLH, bZIP and AP2-ERF) as potential regulators of SG biosynthesis. Overall, this study provides haplotype-resolved chromosome-level genome assembly for genome editing and enhancing breeding efforts for targeted biosynthesis of SGs in S. rebaudiana.

Selenated NHC-Pd(II) Pincer Complex Catalyzed, Temperature-Dependent Selective Hydroamination and Oxidative Amination of Olefins: Formation of Enamine Esters and ß-Amino Esters under Solvent-Free and Aerobic Conditions.

NHC-Pd(II) pincer catalyzed oxidative amination and hydroamination of olefins is developed under solvent-free aerobic conditions. Reaction offered a temperature-controlled synthesis of (Z)-enamine and ß-amino esters to provide easy access and remarkable functional group tolerance for a variety of enamines. The developed approach renders an opportunity of scalability and flexibility, and besides this, the produced enamines can be transformed into many N-containing heterocycles, underscoring its potential usage in synthetic and pharmaceutical chemistry. Moreover, it is the first report for coupling of aniline with styrene.

Iron-Catalyzed Chemoselective Transfer Hydrogenation of α,ß-Unsaturated Ketones Using H2O as a Surrogate of Hydrogen.

Sustainable and highly economical iron-catalyzed chemoselective reduction of C═C of α,ß-unsaturated ketones has been established under mild reaction protocols. Water is used as a green and abundant surrogate of hydrogen and is scarcely used in organic synthetic transformations as a source of hydrogen. The developed protocol offers a broad spectrum for chemoselective transfer hydrogenation of α,ß-unsaturated ketones. Moreover, the method was found to be highly effective for aryl and ferrocenyl α,ß-unsaturated ketones consisting of one or two double bonds and with multiple functionalities. Moreover, the present method avoids prolonged reaction time, provides a wide range of substrates with excellent yield, and circumvents the tedious chromatographic workup.

Clinicopathological Pattern of Renal Biopsies in Children with Nephrotic Syndrome.

Background: A renal biopsy is essential for the identification and management of renal disorders. Although considered an invasive operation, it is necessary for a definitive diagnosis and treatment of many renal diseases. The primary goal of this study was to assess the clinicopathological aspect of renal diseases undergoing biopsy in children receiving tertiary care.Patients and Methods: Children (≤18 years) hospitalized with nephrotic syndrome were the subjects of this cross-sectional study, and comprehensive assessments confirmed the need for a kidney biopsy. Included were 277 children who met the inclusion and exclusion criteria. Data on patient outcomes, biopsy indications, complications, histopathologic results, and demographic information were documented.Results: Of the 277 patients who underwent renal biopsy, 63.2% were male, and 36.8% were female. Average age of the patients was 15 ± 2.9 years, with age distribution ranging from 3 to 18 years. The most frequent indication for renal biopsy was atypical age of <1 and >10-years-old (91.7%), steroid-resistant (5.1%), asymptomatic hematuria (21.3%), abnormal glomerular filtration rate (16.2%), and proteinuria (14.8%). The most common histopathological findings were focal segmental glomerulosclerosis (FSGS) (36.5%), followed by minimal change disease (MCD) (13.4%), membranoproliferative glomerulonephritis (MPGN) (10.5%), membranous glomerulonephritis (MGN) (7.94%), IgA nephropathy (IGAN) (7.58%), non-proliferative glomerulonephritis (NPGN) (7.58%), diffuse proliferative glomerulonephritis (DPGN) (6.9%), crescentic GN (5.8%), and systemic lupus erythematosus (SLE) (3.97%). The high frequency of positive samples was seen in SLE, followed by DPGN, MPGN, IGAN, and MGN. In contrast, MCD, crescentic GN, and NPGN showed negativity in all differential item functioning (DIF) parameters.Conclusion: Renal biopsy is a safe and effective procedure in the diagnosis and treatment of in children with nephrotic syndrome. FSGS had the highest frequency in examined biopsies.

Plant Cell-Engineered Gold Nanoparticles Conjugated to Quercetin Inhibit SARS-CoV-2 and HSV-1 Entry.

Recent studies have revealed considerable promise in the antiviral properties of metal nanomaterials, specifically when biologically prepared. This study demonstrates for the first time the antiviral roles of the plant cell-engineered gold nanoparticles (pAuNPs) alone and when conjugated with quercetin (pAuNPsQ). We show here that the quercetin conjugated nanoparticles (pAuNPsQ) preferentially inhibit the cell entry of two medically important viruses-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and herpes simplex virus type-1 (HSV-1) using different mechanisms. Interestingly, in the case of SARS-CoV-2, the pre-treatment of target cells with pAuNPsQ inhibited the viral entry, but the pre-treatment of the virus with pAuNPsQ did not affect viral entry into the host cell. In contrast, pAuNPsQ demonstrated effective blocking capabilities against HSV-1 entry, either during the pre-treatment of target cells or by inducing virus neutralization. In addition, pAuNPsQ also significantly affected HSV-1 replication, evidenced by the plaque-counting assay. In this study, we also tested the chemically synthesized gold nanoparticles (cAuNPs) of identical size and shape and observed comparable effects. The versatility of plant cell-based nanomaterial fabrication and its modification with bioactive compounds opens a new frontier in therapeutics, specifically in designing novel antiviral formulations.

Acute Methanol Toxicity: Clinical Correlation with Autopsy Findings, a Descriptive Study.

BACKGROUND: Methyl alcohol poisoning or deaths from drinking illegally brewed cheap alcohol which is often spiked with chemicals to increase its potency are frequent in India. Many outbreaks from different parts of the country have been reported from time to time. A total of 11,830 lives were lost between 2006 and 2015 due to the consumption of spurious liquor in the country. The symptoms can range from mild to severe depending upon factors like the amount of exposure and time of presentation. AIMS AND OBJECTIVES: The present study was designed to describe the clinical presentation, management, and outcome of the patients during a recent methanol outbreak that can form a basis for diagnosis and management. This study also highlights the salient autopsy findings and their correlation with clinical features. MATERIALS AND METHODS: It is a retrospective, descriptive study discussing clinical features of patients with methanol intoxication, their outcome, and the clinical correlation with autopsy findings of patients who succumbed to death. The study was conducted at King George's Medical University, Lucknow. The patients were enrolled from a methanol intoxication outbreak in Barabanki district on 28th May 2019 followed by a similar outbreak in Sitapur district two days later. RESULTS: A total of 33 patients were included in this study based on predefined clinical characteristics. The average amount of alcohol consumed was about 223 mL (range: 100-300 mL). The majority of patients had onset of symptoms between 12 and 24 hours. All patients had gastrointestinal symptoms, 97% of patients had visual disturbances, 91% of patients had central nervous system manifestation while frank coma was observed in 15% of patients. Decreased urine output was reported in 6% of patients. About 90% of patients had metabolic acidosis. Out of 33 patients included in this study, 30 patients were discharged in stable condition while two died and one absconded. Autopsy findings revealed marked cerebral edema and hyperemia, hyperemic heart, and congested lungs in all the patients. One patient showed putaminal necrosis which is characteristic of methanol poisoning. Kidneys in two cases were hyperemic and show parenchymal degeneration which co-relates with both patients being anuric. CONCLUSION: Methanol intoxication is a serious problem in developing countries like ours. Timely intervention is an important factor in reducing mortality among these patients. The study highlights the very important fact that methanol intoxication can be managed at the very ground level with minimal resources (as available) if intervened and recognized in time.

Non-IgE mediated mast cell activation.

Mast cells (MCs) are innate immune cells that are scattered in tissues throughout the organism being particularly abundant at sites exposed to the environment such as the skin and mucosal surfaces. Generally known for their role in IgE-mediated allergies, they have also important functions in the maintenance of tissue integrity by constantly sensing their microenvironment for signals by inflammatory triggers that can comprise infectious agents, toxins, hormones, alarmins, metabolic states, etc. When triggered their main function is to release a whole set of inflammatory mediators, cytokines, chemokines, and lipid products. This allows them to organize the ensuing innate immune and inflammatory response in tight coordination with resident tissue cells, other rapidly recruited immune effector cells as well as the endocrine and exocrine systems of the body. To complete these tasks, MCs are endowed with a large repertoire of receptors allowing them to respond to multiple stimuli or directly interact with other cells. Here we review some of the receptors expressed on MCs (ie, receptors for Immunoglobulins, pattern recognition receptors, nuclear receptors, receptors for alarmins, and a variety of other receptors) and discuss their functional implication in the immune and inflammatory response focusing on non-IgE-mediated activation mechanisms.

Efficacy of therapeutic plasma exchange in a patient with coagulation inhibitors (acquired haemophilia A) - A case report.

Acquired haemophilia A (AHA) is a rare disorder with mostly idiopathic aetiology that leads to factor VIII (FVIII) deficiency due to coagulation inhibitors formation. Treatment protocol includes immunosuppression and Factor VIII bypassing agents including activated Prothrombin Complex Concentrates (PCC). Nevertheless, the role of plasma exchange is not clear in the treatment of AHA. We report a case of 73 year old male who presented with haematuria, prolonged activated partial thromboplastin time (APTT) and a very high titres of Factor VIII inhibitors of 98 Bethesda units (BU) and was diagnosed with acquired haemophilia A. He failed to respond to multiple immunosuppressive therapies including rituximab. Therefore, therapeutic plasma exchange (TPE) therapy was planned due to persistence of haematuria despite immunosuppressive therapies. After five cycles of plasma exchange, APTT became normal, haematuria subsided and Factor VIII inhibitors became negative. Patient was discharged without any bleeding and in a stable condition. In this index case, plasma exchange played a very crucial role, resulting in recovery of the patient. These results advocate that therapeutic plasma exchange is an effective therapy for acquired haemophilia A.

Comparative Proteomic Analyses of Avirulent, Virulent, and Clinical Strains of Mycobacterium tuberculosis Identify Strain-specific Patterns.

Mycobacterium tuberculosis is an adaptable intracellular pathogen, existing in both dormant as well as active disease-causing states. Here, we report systematic proteomic analyses of four strains, H37Ra, H37Rv, and clinical isolates BND and JAL, to determine the differences in protein expression patterns that contribute to their virulence and drug resistance. Resolution of lysates of the four strains by liquid chromatography, coupled to mass spectrometry analysis, identified a total of 2161 protein groups covering ∼54% of the predicted M. tuberculosis proteome. Label-free quantification analysis of the data revealed 257 differentially expressed protein groups. The differentially expressed protein groups could be classified into seven K-means cluster bins, which broadly delineated strain-specific variations. Analysis of the data for possible mechanisms responsible for drug resistance phenotype of JAL suggested that it could be due to a combination of overexpression of proteins implicated in drug resistance and the other factors. Expression pattern analyses of transcription factors and their downstream targets demonstrated substantial differential modulation in JAL, suggesting a complex regulatory mechanism. Results showed distinct variations in the protein expression patterns of Esx and mce1 operon proteins in JAL and BND strains, respectively. Abrogating higher levels of ESAT6, an important Esx protein known to be critical for virulence, in the JAL strain diminished its virulence, although it had marginal impact on the other strains. Taken together, this study reveals that strain-specific variations in protein expression patterns have a meaningful impact on the biology of the pathogen.

Inhibition of CD40-induced N-Ras activation reduces leishmania major infection.

Leishmania major is a parasite that resides and replicates in macrophages. We previously showed that the parasite enhanced CD40-induced Raf-MEK-ERK signaling but inhibited PI3K-MKK-p38MAPK signaling to proleishmanial effects. As Raf and PI3K have a Ras-binding domain but exert opposite effects on Leishmania infection, we examined whether Ras isoforms had differential roles in Leishmania infection. We observed that L. major enhanced N-Ras and H-Ras expression but inhibited K-Ras expression in macrophages. L. major infection enhanced N-Ras activity but inhibited H-Ras and K-Ras activity. TLR2 short hairpin RNA or anti-TLR2 or anti-lipophosphoglycan Abs reversed the L. major-altered N-Ras and K-Ras expressions. Pam3CSK4, a TLR2 ligand, enhanced N-Ras expression but reduced K-Ras expression, indicating TLR2-regulated Ras expression in L. major infection. Whereas N-Ras silencing reduced L. major infection, K-Ras and H-Ras silencing enhanced the infection both in macrophages in vitro and in C57BL/6 mice. BALB/c-derived macrophages transduced with lentivirally expressed N-Ras short hairpin RNA and pulsed with L. major-expressed MAPK10 enhanced MAPK10-specific Th1-type response. CD40-deficient mice primed with these macrophages had reduced L. major infection, accompanied by higher IFN-γ but less IL-4 production. As N-Ras is activated by Sos, a guanine nucleotide exchange factor, we modeled the N-Ras-Sos interaction and designed two peptides from their interface. Both the cell-permeable peptides reduced L. major infection in BALB/c mice but not in CD40-deficient mice. These data reveal the L. major-enhanced CD40-induced N-Ras activation as a novel immune evasion strategy and the potential for Ras isoform-targeted antileishmanial immunotherapy and immunoprophylaxis.

Acute hypobaric hypoxia augments ROCK2 protein level and activity.

AIM: Rho kinase activation plays an important role in hypoxic pulmonary vasoconstriction and increased vascular resistance. The present study investigated changes in the level and activity of rho kinase isoform 2 (ROCK2) under acute hypobaric hypoxia exposure. For this, Wistar rats were taken as the model organism. MATERIALS AND METHODS: Fifteen male Wistar rats (4-6 week old, 250 grams) were normalized with the surrounding environment by providing a 12/12 hour day and night acclimatization cycle. The rats were divided into 3 groups: (a) control group (no exposure, n = 5), (b) Group 1 (12 hour hypobaric-hypoxia exposure, n = 5) and (c) Group 2 (12 hour hypobaric hypoxia and 12 hour normobaric normoxia exposure, n = 5). A change in behavior of the animals was noted before sacrifice. Blood was collected from the beating heart of the anesthetized animal. Lungs were dissected out and used to estimate the levels and activity of ROCK2 in different groups using commercially available kits. Lung histology was evaluated by immunohistochemistry. ROCK2 gene expression was studied in the blood and lungs using quantitative PCR. RESULTS AND CONCLUSIONS: Control and Group 2 animals had an active movement while the Group 1 animals were sluggish before the sacrifice. Formation of a large perivascular edema cuff and collagen deposition in lungs of Group 1 and a reduction in Group 2 was observed. The protein levels and activities of ROCK2 were increased in Group 1 (p < 0.05) and became normal in Group 2 which was akin to control group animals. ROCK2 expression in PBMCs was increased in Group 1 (10.7 fold, p = 0.005) and was decreased in Group 2 (5.5 fold, p = 0.02). The outcomes establish that acute hypobaric hypoxia augments ROCK2 protein level and activity.

Identification and interplay of sequence specific DNA binding proteins involved in regulation of human Pregnane and Xenobiotic Receptor gene.

Pregnane and Xenobiotic Receptor (PXR), a member of nuclear receptor superfamily, acts as a 'xenosensor' in our body and modulates a network of genes involved in xenobiotic metabolism and elimination. Expression levels of PXR in certain metabolic disorders including cancer are reported to be altered and its induced expression is associated with the development of resistance towards chemotherapy and adverse drug-drug interactions. Though the transcriptional regulation of PXR target genes have been elucidated in significant details, the structure and functional control of PXR promoter itself remains inadequately explored. In this work, we identify a Composite Element (CE) located within the proximal PXR promoter region that consists of multiple overlapping cis-elements and demonstrated that CE interacts specifically with some critical nuclear proteins. Subsequent DNA-protein interaction studies revealed mutually exclusive interactions on CE occurring between Sp1 and two unidentified DNA binding proteins with molecular masses of 50 and 54kDa. Here, we report the identification of 54kDa CE binding protein as a heterogeneous nuclear ribonucleoprotein K (hnRNPK) and demonstrate the effect of hnRNP K and Sp1 on PXR promoter transcriptional activity. Overall, the study indicates that PXR gene is tightly regulated to maintain a low receptor level.

Pregnane and Xenobiotic Receptor gene expression in liver cells is modulated by Ets-1 in synchrony with transcription factors Pax5, LEF-1 and c-Jun.

Nuclear receptor PXR is predominantly expressed in liver and intestine. Expression of PXR is observed to be dysregulated in various metabolic disorders indicating its involvement in disease development. However, information available on mechanisms of PXR self-regulation is fragmentary. The present investigation identifies some of the regulatory elements responsible for its tight regulation and low cellular expression. Here, we report that the PXR-promoter is a target for some key transcription factors like PU.1/Ets-1, Pax5, LEF-1 and c-Jun. Interestingly, we observed that PXR-promoter responsiveness to Pax5, LEF-1 and c-Jun, is considerably enhanced by Ets transcription factors (PU.1 and Ets-1). Co-transfection of cells with Ets-1, LEF-1 and c-Jun increased PXR-promoter activity by 5-fold and also induced expression of endogenous human PXR. Site-directed mutagenesis and transfection studies revealed that two Ets binding sites and two of the three LEF binding sites in the PXR-promoter are functional and have a positive effect on PXR transcription. Results suggest that expression of Ets family members, in conjunction with Pax5, LEF-1 and c-Jun, lead to coordinated up-regulation of PXR gene transcription. Insights obtained on the regulation of PXR gene have relevance in offering important cues towards normal functioning as well as development of several metabolic disorders via PXR signaling.

CCSfind: A tool for chemically informed LC-IM-MS database building.

In addition to providing critical knowledge of the accurate mass of ions, ion mobility-mass spectrometry (IM-MS) delivers complementary data relating to the conformation and size of ions in the form of an ion mobility spectrum and derived parameters, namely, the ion's mobility (K) and the IM-derived collision cross section (CCS). However, the maximum amount of information obtained in IM-MS measurements is not currently transferred into analytical databases including the full mobility spectra (CCS distributions) as well as capturing of additional ion species (e.g., adducts) into the same compound entry. We introduce CCSfind, a new tool for building comprehensive databases from experimental IM-MS measurements of small molecules. CCSfind allows predicted ion species to be chosen for input chemical formulae, which are then targeted by CCSfind after parsing open source mzML input files to provide a unified set of results within a single data processing step. CCSfind can handle both chromatographically separated isomers and IM separation of isomeric ions (e.g., "protomers" or conformers of the same ion species) with simple user control over the output for new database entries in SQL format. Files of up to 1 GB can be processed in less than 2 min on a desktop computer with 32 GB RAM with computational time scaling linearly with the size of the input mzML file or the number of input molecular formulae. Results are manually reviewed, annotated with experimental settings, before committing the database where the full dataset can be retrieved.

Ru-Catalyzed and Selenium-Directed Selective Formation of ortho- and Dialkenylated Selanes, Mixed Organoselenoethers, and Isoselenochromenes.

Herein, the Ru-catalyzed chemo- and regioselective formation of four novel organoselenium compounds is established. Mono- and dialkenylated selanes were formed by the Se-directed o-C-H activation of benzyl(phenyl)selanes with alkynes. Unprecedented debenzylative/dearylative hydroselenations of alkynes were performed by slightly varying the amount of catalyst and temperature. Catalyst-driven direct formation of novel isoselenochromenes is also recorded. Altogether, 45 new organoseleno compounds were synthesized in good amounts with varieties of alkynes and selanes. This is the first report of its kind to deal with the synthesis of novel, challenging, and unusual organoseleno compounds in one reaction.

Study of the Association of Semen Parameters With Embryo Quality in a Tertiary Care Center in Bihar: A Retrospective Study.

Introduction Semen quality, characterized by parameters such as sperm count, motility, and morphology, determines successful fertilization and subsequent embryo health. This study investigates the impact of sperm parameters on embryo quality in assisted reproductive technology (ART). Methods The study utilized a retrospective design with 194 male and 194 female participants who underwent fertility treatment from October 2020 to May 2024. Semen analysis included assessment of sperm count, motility, and morphology. Embryo quality was evaluated on days three and five post-fertilizations based on morphological criteria. Statistical analyses, including one-way ANOVA and chi-square tests, explored relationships between semen parameters and embryo quality. Results The study included 388 participants (males: 194 and females: 194). Female participants had a mean age of 31.0 ± 4.6 years and a mean BMI of 23.1 ± 5.3 kg/m², while males had a mean age of 36.6 ± 5.4 years and a mean BMI of 22.7 ± 2.8 kg/m². Paternal age and BMI showed no significant association (p > 0.05) with embryo quality. However, sperm quality parameters such as sperm count, motility, and morphology demonstrated significant associations (p < 0.05) with embryo quality on both day three and day five, indicating that abnormal sperm parameters were linked to poorer embryo quality. Factors such as alcohol consumption, smoking, tobacco use, living in industrial areas, and tea/coffee consumption showed no significant association with embryo quality. Conclusion The findings of the study emphasize the importance of comprehensive semen analysis in fertility assessments and highlight opportunities for improving ART outcomes through targeted interventions and further mechanistic research.

Necrotizing Fasciitis: When skin confuses - An autopsy case report.

Necrotizing Fasciitis (NF) is a severe life-threatening soft tissue infection characterized by the rapid destruction of muscle, fat and fascial layers. This report details an autopsy case report of a 40year old male, unclaimed body lacking the complete history except that given by the Police personnel accompanying in which there is no prior history of trauma. This person succumbed to septic shock secondary to NF, despite clinical interventions. This case emphasizes the importance of early diagnosis and the need for heightened clinical awareness to improve patient outcomes.

Mechanistic Insights into Cellular and Molecular Targets of Zinc Oxide Quantum Dots (ZnO QDs) in Fungal Pathogen, Candida albicans: One Drug Multi-Targeted Therapeutic Approach.

Rationally designed multitargeted drugs, known as network therapeutics/multimodal drugs, have emerged as versatile therapeutic solutions to combat drug-resistant microbes. Here, we report novel mechanistic insights into cellular and molecular targets of ZnO quantum dots (QDs) against Candida albicans, a representative of fungal pathogens. Stable, monodispersed 4-6 nm ZnO QDs were synthesized using a wet chemical route, which exhibited dose-dependent inhibition on the growth dynamics of Candida. Treatment with 200 µg/mL ZnO QDs revealed an aberrant morphology and a disrupted cellular ultrastructure in electron microscopy and led to a 23% reduction in ergosterol content and a 53% increase in intracellular reactive oxygen species. Significant increase in steady-state fluorescence polarization and fluorescence lifetime decay of membrane probe 1,6-diphenyl-1,3,5-hexatriene (DPH) in treated cells, respectively, implied reduction in membrane fluidity and enhanced microviscosity. The observed reduction in passive diffusion of fluorescent Rhodamine 6G across the membrane validated the intricate relationship between ergosterol, membrane fluidity, and microviscosity. An inverse relationship existing between ergosterol biosynthetic genes, ERG11 and ERG3 in treated cells, related well with displayed higher susceptibilities. Furthermore, treated cells exhibited impaired functionality and downregulation of ABC drug efflux pumps. Multiple cellular targets of ZnO QDs in Candida were validated by in silico molecular docking. Thus, targeting ERG11, ERG3, and ABC drug efflux pumps might emerge as a versatile, nano-ZnO-based strategy in fungal therapeutics to address the challenges of drug resistance.

Real-Time, High-Throughput Microscopic Quantification of Human Neutrophil Extracellular Trap Release and Assessing the Pharmacology of Antagonists.

Neutrophils play an important role in innate immune defense by using several strategies, including the release of neutrophil extracellular traps (NETs) in a process referred to as NETosis. However, in the past two decades, it has become clear that the accumulation of NETs in tissues contributes to the pathophysiology of multiple inflammatory and autoimmune diseases. Therefore, interest in the development of NETosis antagonists has risen. Variable and non-standardized methods to detect and analyze NETosis were developed concomitantly, each with its own advantages and limitations. Here, we describe a real-time microscopy method for the quantification of human NET release, allowing to study NETosis as well as NET inhibition in a high-throughput manner. The surface area-based semi-automated analysis recognizes NETs and distinguishes them from non-netting activated neutrophils. We demonstrate that the non-physiological NETosis inducers, calcium ionophore and phorbol-12-myristate-13-acetate (PMA), trigger the release of NETs with different characteristics and kinetics. Furthermore, we show that this approach allows studying NET release in response to disease-relevant stimuli, including immune complexes, N-Formylmethionine-leucyl-phenylalanine (fMLF), monosodium urate crystals, and calcium pyrophosphate crystals. To exemplify the utility of this method to study NETosis antagonists, we used CIT-013, a first-in-class monoclonal antibody inhibitor of NET release. CIT-013 targets citrullinated histone H2A and H4 and efficiently inhibits NET release with an IC50 of 4.6 nM. Other anti-histone antibodies tested lacked this NETosis-inhibitory capacity. Altogether, we demonstrate that this protocol enables specific, reliable, and reproducible high-throughput quantification of NETs, enhancing the study of NET release characteristics, kinetics, and pharmacology of NETosis antagonists.