High throughput screening for SARS-CoV-2 helicase inhibitors.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for nearly 7 million deaths worldwide since its outbreak in late 2019. Even with the rapid development and production of vaccines and intensive research, there is still a huge need for specific anti-viral drugs that address the rapidly arising new variants. To address this concern, the National Institute of Allergy and Infectious Diseases (NIAID) established nine Antiviral Drug Discovery (AViDD) Centers, tasked with exploring approaches to target pathogens with pandemic potential, including SARS-CoV-2. In this study, we sought inhibitors of SARS-CoV2 non-structural protein 13 (nsP13) as potential antivirals, first developing a HTS-compatible assay to measure SARS-CoV2 nsP13 helicase activity. Here we present our effort in implementing the assay in a 1,536 well-plate format and in identifying nsP13 inhibitor hit compounds from a ∼650,000 compound library. The primary screen was robust (average Z' = 0.86 ± 0.05) and resulted in 7,009 primary hits. 1,763 of these compounds upon repeated retests were further confirmed, showing consistent inhibition. Following in-silico analysis, an additional orthogonal assay and titration assays, we identified 674 compounds with IC50 <10 µM. We confirmed activity of independent compound batches from de novo powders while also incorporating multiple counterscreen assays. Our study highlights the potential of this assay for use on HTS platforms to discover novel compounds inhibiting SARS-CoV2 nsP13, which merit further development as an effective SARS-CoV2 antiviral.

Pyrazine-based small molecule kinase inhibitors: clinical applications and patent review (2019-2023).

Protein kinases play a key role in cellular signaling pathways including proliferation, apoptosis, inflammation and immune regulation. Therefore, targeting kinases with small molecules has emerged as a therapeutic potential in cancers and other diseases including inflammatory and autoimmune disorders. The main chemical motifs of the available small molecule kinase inhibitors are heterocyclic, nitrogen-containing and six-membered rings including pyrazine. Several potent and selective pyrazine-based kinase inhibitors have been developed and progressed into clinical trials. The data of clinical application of kinase inhibitors demonstrate good clinical activity with manageable toxicity in several relapse-resistant malignancies and severe to moderate immunological disorders. All pyrazine-based kinase inhibitors are orally active. This paper reviews the most recent kinase literature (2019-2023) related to pyrazine-based small molecule inhibitors. This review includes the FDA (Food and Drug Administration)-approved and patent agents along with their targeted kinase, scaffold, potency, selectivity profile, assignee and biological results in clinical and preclinical studies.

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Effects of gamma-radiation on microbial, nutritional, and functional properties of Katimon mango peels: A combined biochemical and in silico studies.

Gamma radiation has notable impacts on the flesh of mangoes. In this research, Katimon mangoes were subjected to different levels of irradiation (0.5, 1.0, 1.5, and 2.0 kGy) using a60Co irradiator. The results showed that irradiation significantly reduced the microbial population in the mango peels, with the 1.5 kGy dose showing the most significant reduction. Irradiation also delayed ripening and extended the shelf life of the mango peels. The total fat, protein, ash, moisture, and sugar content of the mango peels were all affected by irradiation. The total protein content, ash content and moisture content increased after irradiation, while the fat content remained relatively unchanged. The sugar content increased in all samples after storage, but the non-irradiated samples had higher sugar levels than the irradiated ones. The dietary fiber content of the mango peels was not significantly affected by irradiation. The vitamin C content decreased in all samples after storage. The titratable acidity and total soluble solids content of the mango peels increased after storage, but there were no significant differences between the irradiated and non-irradiated samples. Antioxidant activity and cytotoxicity assessment highlighted the antioxidant potential and reduced toxicity of irradiated samples. Additionally, the antimicrobial effectiveness of irradiated mango peels was evaluated. The most substantial inhibitory zones (measuring 16.90 ± 0.35) against Pseudomonas sp. were observed at a radiation dose of 1.5 kGy with 150 µg/disc. To identify potential antimicrobial agents, the volatile components of mangoes irradiated with 1.5 kGy were analyzed through GC-MS. Subsequently, these compounds were subjected to in silico studies against a viable protein, TgpA, of Pseudomonas sp. (PDB ID: 6G49). Based on molecular dynamic simulations and ADMET properties, (-)-Carvone (-6.2), p-Cymene (-6.1), and Acetic acid phenylmethyl ester (-6.1) were identified as promising compounds for controlling Pseudomonas sp.

Rapid, high throughput, automated detection of SARS-CoV-2 neutralizing antibodies against Wuhan-WT, delta and omicron BA1, BA2 spike trimers.

Traditional cellular and live-virus methods for detection of SARS-CoV-2 neutralizing antibodies (nAbs) are labor- and time-intensive, and thus not suited for routine use in the clinical lab to predict vaccine efficacy and natural immune protection. Here, we report the development and validation of a rapid, high throughput method for measuring SARS-CoV-2 nAbs against native-like trimeric spike proteins. This assay uses a blockade of human angiotensin converting enzyme 2 (hACE-2) binding (BoAb) approach in an automated digital immunoassay on the Quanterix HD-X platform. BoAb assays using Wuhan-WT (vaccine strain), delta (B.1.167.2), omicron BA1 and BA2 variant viral strains showed strong correlation with cell-based pseudovirus neutralization activity (PNA) and live-virus neutralization activity. Importantly, we were able to detect similar patterns of delta and omicron variant resistance to neutralization in samples with paired vaccine strain and delta variant BoAb measurements. Finally, we screened clinical samples from patients with or without evidence of SARS-CoV-2 exposure by a single-dilution screening version of our assays, finding significant nAb activity only in exposed individuals. Importantly, this completely automated assay can be performed in 4 h to measure neutralizing antibody titers for 16 samples over 8 serial dilutions or, 128 samples at a single dilution with replicates. In principle, these assays offer a rapid, robust, and scalable alternative to time-, skill-, and cost-intensive standard methods for measuring SARS-CoV-2 nAb levels.

Metagenomic sequencing for identifying pathogen-specific circulating DNAs and development of diagnostic methods for schistosomiasis.

Timely diagnosis of Schistosoma infection, particularly in the early stage is crucial for identifying infected hosts and then taking effective control strategies. Here, metagenomic next-generation sequencing was used to identify pathogen-specific circulating DNAs (cDNAs) in the sera/plasma of New Zealand rabbits infected with S. japonicum, and the identified cDNAs were validated by PCR and qPCR. Loop-mediated isothermal amplification (LAMP)-based CRISPR-Cas12a and recombinase polymerase amplification-based lateral flow strip (RPA-LF) methods combined with the newly identified cDNA were developed to evaluate the potentials for diagnosing murine and human schistosomiasis. The results indicated that twenty-two cDNAs were identified. The developed LAMP-based CRISPR/Cas12a and RPA-LF methods showed a good potential for diagnosing murine or human schistosomiasis as early as 5 days of post-infection with 5 cercariae infection. In a word, S. japonicum specific cDNAs in circulation of infected hosts could be effective biomarkers for detecting Schistosoma infection particularly for early stages.

Structural and biochemical insights into the bacteriophage PlyGRCS endolysin targeting methicillin-resistant Staphylococcus aureus (MRSA) and serendipitous discovery of its interaction with a cold shock protein C (CspC).

Methicillin-resistant Staphylococcus aureus (MRSA) causes life-threatening human infections. Bacteriophage-encoded endolysins degrade the cell walls of Gram-positive bacteria by selectively hydrolyzing the peptidoglycan layer and thus are promising candidates to combat bacterial infections. PlyGRCS, the S. aureus-specific bacteriophage endolysin, contains a catalytic CHAP domain and a cell-wall binding SH3_5 domain connected by a linker. Here, we show the crystal structure of full-length PlyGRCS refined to 2.1 Å resolution. In addition, a serendipitous finding revealed that PlyGRCS binds to cold-shock protein C (CspC) by interacting with its CHAP and SH3_5 domains. CspC is an RNA chaperone that plays regulatory roles by conferring bacterial adaptability to various stress conditions. PlyGRCS has substantial lytic activity against S. aureus and showed only minimal change in its lytic activity in the presence of CspC. Whereas the PlyGRCS-CspC complex greatly reduced CspC-nucleic acid binding, the aforesaid complex may downregulate the CspC function during bacterial infection. Overall, the crystal structure and biochemical results of PlyGRCS provide a molecular basis for the bacteriolytic activity of PlyGRCS against S. aureus.

Coumarins as factor XIIa inhibitors: Potency and selectivity improvements using a fragment-based strategy.

Previously, we described weak coumarin inhibitors of factor XIIa, a promising target for artificial surface-induced thrombosis and various inflammatory diseases. In this work, we used fragment-based drug discovery approach to improve our coumarin series. First, we screened about 200 fragments for the S1 pocket. The S1 pocket of trypsin-like serine proteases, such as factor XIIa, is highly conserved and is known to drive a major part of the association energy. From the screening, we selected fragments displaying a micromolar activity and studied their selectivity on other serine proteases. Then, these fragments were merged to our coumarin templates, leading to the generation of nanomolar inhibitors. The mechanism of inhibition was further studied by mass spectrometry demonstrating the covalent binding through the formation of an acyl enzyme complex. The most potent compound was tested in plasma to evaluate its stability and efficacy on coagulation assays. It exhibited a plasmatic half-life of 1.9 h and a good selectivity for the intrinsic coagulation pathway over the extrinsic one.

Implementing best practices on data generation and reporting of Mycobacterium tuberculosis in vitro assays within the ERA4TB consortium.

Tuberculosis (TB) is the historical leading cause of death by a single infectious agent. The European Regimen Accelerator for Tuberculosis (ERA4TB) is a public-private partnership of 30+ institutions with the objective to progress new anti-TB regimens into the clinic. Thus, robust and replicable results across independent laboratories are essential for reliable interpretation of treatment efficacy. A standardization workgroup unified in vitro protocols and data reporting templates. Time-kill assays provide essential input data for pharmacometric model-informed translation of single agents and regimens activity from in vitro to in vivo and the clinic. Five conditions were assessed by time-kill assays in six independent laboratories using four bacterial plating methods. Baseline bacterial burden varied between laboratories but variability was limited in net drug effect, confirming 2.5 µL equally robust as 100 µL plating. This exercise establishes the foundations of collaborative data generation, reporting, and integration within the overarching Antimicrobial Resistance Accelerator program.

Global profiling of AMG510 modified proteins identified tumor suppressor KEAP1 as an off-target.

KRAS inhibitor AMG510 covalently modifies the G12C residue and inactivates the KRAS/G12C function. Because there are many reactive cysteines in the proteome, it is important to characterize AMG510 on-target modification and off-targets. Here, we presented a streamlined workflow to measure abundant AMG510 modified peptides including that of KRAS/G12C by direct profiling, and a pan-AMG510 antibody peptide IP workflow to profile less abundant AMG510 off-targets. We identified over 300 off-target sites with three distinct kinetic patterns, expanding the AMG510 modified proteome involved in the nucleocytoplasmic transport, response to oxidative stress, adaptive immune system, and glycolysis. We found that AMG510 covalently modified cys339 of ALDOA and inhibited its enzyme activity. Moreover, AMG510 modified KEAP1 cys288 and induced NRF2 accumulation in the nuclear of NSCLC cells independent of KRAS/G12C mutation. Our study provides a comprehensive resource of protein off-targets of AMG510 and elucidates potential toxicological sideeffects for this covalent KRASG12C inhibitor.

Caenorhabditis elegans: a model organism in the toxicity assessment of environmental pollutants.

The unfavorable effects of environmental pollutants are becoming increasingly evident. In recent years, Caenorhabditis elegans (C. elegans) has been used as a powerful terrestrial model organism for environmental toxicity studies owing to its various advantages, including ease of culture, short lifespan, small size, transparent body, and well-characterized genome. In vivo bioassays and field studies can analyze and evaluate various toxic effects of the toxicants on the model organism, while emerging technologies allow profound insights into molecular disturbances underlying the observed phenotypes. In this review, we discuss the applications of C. elegans as a model organism in environmental toxicity studies and delineate apical assays such as lifespan, growth rate, reproduction, and locomotion, which are widely used in toxicity evaluation. In addition to phenotype assays, a comprehensive understanding of the toxic mode of action and mechanism can be achieved through a highly sensitive multi-omics approach, including the expression levels of genes and endogenous metabolites. Recent studies on environmental toxicity using these approaches have been summarized. This review highlights the practicality and advantages of C. elegans in evaluating the toxicity of environmental pollutants and presents the findings of recent toxicity studies performed using this model organism. Finally, we propose crucial technical considerations to escalate the appropriate use of C. elegans in examining the toxic effects of environmental pollutants.

Bioactivity Profiles on 15 Different Effect Mechanisms for 15 Golden Root Products via High-Performance Thin-Layer Chromatography, Planar Assays, and High-Resolution Mass Spectrometry.

Planar chromatography has recently been combined with six different effect-directed assays for three golden root (Rhodiola rosea L.) samples. However, the profiles obtained showed an intense tailing, making zone differentiation impossible. The profiling was therefore improved to allow for the detection of individual bioactive compounds, and the range of samples was extended to 15 commercial golden root products. Further effect-directed assays were studied providing information on 15 different effect mechanisms, i.e., (1) tyrosinase, (2) acetylcholinesterase, (3) butyrylcholinesterase, (4) ß-glucuronidase, and (5) α-amylase inhibition, as well as endocrine activity via the triplex planar yeast antagonist-verified (6-8) estrogen or (9-11) androgen screen, (12) genotoxicity via the planar SOS-Umu-C bioassay, antimicrobial activity against (13) Gram-negative Aliivibrio fischeri and (14) Gram-positive Bacillus subtilis bacteria, and (15) antioxidative activity (DPPH• radical scavengers). Most of the golden root profiles obtained were characteristic, but some samples differed substantially. The United States Pharmacopeia reference product showed medium activity in most of the assays. The six most active compound zones were further characterized using high-resolution mass spectrometry, and the mass signals obtained were tentatively assigned to molecular formulae. In addition to confirming the known activities, this study is the first to report that golden root constituents inhibit butyrylcholinesterase (rosin was tentatively assigned), ß-glucuronidase (rosavin, rosarin, rosiridin, viridoside, and salidroside were tentatively assigned), and α-amylase (stearic acid and palmitic acid were tentatively assigned) and that they are genotoxic (hydroquinone was tentatively assigned) and are both agonistic and antagonistic endocrine active.

Comparative kinase and cancer cell panel profiling of kinase inhibitors approved for clinical use from 2018 to 2020.

During the last two decades, kinase inhibitors have become the major drug class for targeted cancer therapy. Although the number of approved kinase inhibitors increases rapidly, comprehensive in vitro profiling and comparison of inhibitor activities is often lacking in the public domain. Here we report the extensive profiling and comparison of 21 kinase inhibitors approved by the FDA for oncology indications since June 2018 and 13 previously approved comparators on panels of 255 biochemical kinase assays and 134 cancer cell line viability assays. Comparison of the cellular inhibition profiles of the EGFR inhibitors gefitinib, dacomitinib, and osimertinib identified the uncommon EGFR p.G719S mutation as a common response marker for EGFR inhibitors. Additionally, the FGFR inhibitors erdafitinib, infigratinib, and pemigatinib potently inhibited the viability of cell lines which harbored oncogenic alterations in FGFR1-3, irrespective of the specific clinical indications of the FGFR inhibitors. These results underscore the utility of in vitro kinase inhibitor profiling in cells for identifying new potential stratification markers for patient selection. Furthermore, comparison of the in vitro inhibition profiles of the RET inhibitors pralsetinib and selpercatinib revealed they had very similar biochemical and cellular selectivity. As an exception, an NTRK3 fusion-positive cell line was potently inhibited by pralsetinib but not by selpercatinib, which could be explained by the targeting of TRK kinases in biochemical assays by pralsetinib but not selpercatinib. This illustrates that unexpected differences in cellular activities between inhibitors that act through the same primary target can be explained by subtle differences in biochemical targeting. Lastly, FLT3-mutant cell lines were responsive to both FLT3 inhibitors gilteritinib and midostaurin, and the PI3K inhibitor duvelisib. Biochemical profiling revealed that the FLT3 and PI3K inhibitors targeted distinct kinases, indicating that unique dependencies can be identified by combined biochemical and cellular profiling of kinase inhibitors. This study provides the first large scale kinase assay or cell panel profiling study for newly approved kinase inhibitors, and shows that comprehensive in vitro profiling of kinase inhibitors can provide rationales for therapy selection and indication expansion of approved kinase inhibitors.

Cotton flower metabolites inhibit SARS-CoV-2 main protease.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been spreading globally for over 2 years, causing serious contagious disease and incalculable damage. The introduction of vaccines has slowed the spread of SARS-CoV-2 to some extent, but there remains a need for specific and effective treatment. The high chemical diversity and safety profiles of natural products make them a potential source of effective anti-SARS-CoV-2 drugs. Cotton plant is one of the most important economic and medical crops and is the source of a large number of antiviral phytochemicals. In this work, we used SARS-CoV-2 main protein (Mpro ) as the target to identify potential anti-SARS-CoV-2 natural products in cotton. An in vitro assay showed that of all cotton tissues examined, cotton flower extracts (CFs) exhibited optimal inhibitory effects against Mpro . We proceeded to use the CF metabolite database to screen natural Mpro inhibitors by combining virtual screening and biochemical assays. We identified that several CF natural products, including astragalin, myricitrin, and astilbin, significantly inhibited Mpro with half-maximal inhibitory concentrations (IC50s) of 0.13, 10.73, and 7.92 µm, respectively. These findings may serve as a basis for further studies into the suitability of cotton as a source of potential therapeutics for SARS-CoV-2.

Toxic effects of excess exposure to boric acid on serum biochemical aspect, hematology and histological alterations and ameliorative potential role of melatonin in rats.

The current work clarifies the negative effects of excess exposure to boric acid (H3BO3) as a boron-containing compound on rats and the possible ameliorative effect of melatonin (MEL). Forty rats were equally divided into 5 groups as follows: group 1 was treated as control while groups 2, 3, 4 and 5 were orally administered corn oil (0.5 ml), H3BO3 (1330 mg/kg BW), MEL (10 mg/kg BW) and H3BO3 + MEL for 28 consecutive days, respectively. At the end of the experiment, blood was sampled for biochemical and hematological analysis and tissues were collected for histopathological examination. The obtained results demonstrated that the exposure to H3BO3 induced hepatorenal dysfunctions, alterations in bone-related minerals and hormones levels, prostaglandin E2 as inflammatory mediator and hematological indices. H3BO3 induced histological alterations in the liver, kidneys, bone and skin. The co-administration of MEL with H3BO3 resulted in a significant improvement in most of the measured parameters and restoration of morpho-functional state of different organs compared to the H3BO3 group. In conclusion, the study clearly demonstrated that H3BO3- induced various adverse effects and that melatonin may be beneficial in a partial mitigating the H3BO3 and may represent a novel approach in the counteracting its toxicity.

Clinical Biochemical Indicators and Intestinal Microbiota Testing Reveal the Influence of Reproductive Age Extending from the Mother to the Offspring.

Age is an important factor that determines the physiological functions of the human body, but the changes in maternal physiology, biochemistry, and intestinal flora related to reproductive age and their impact on offspring are not clear. Here, we tested and analyzed the clinical physiological and biochemical indicators and/or intestinal flora, matching the data of 252 parturients and their newborns. We found that 4 clinical indicators, including the white blood cell count and the absolute value of monocytes, were significantly related to the reproductive age (P < 0.05). The composition of the intestinal flora also varied with age, and the intestinal flora of advanced-age women (≥35 years old) was different from that of middle-aged women (>25 and <35 years old). We also found that changes in maternal clinical physiological and biochemical indicators related to reproductive age could reflect changes in the abundance of bacteria, such as Peptococcus and Vibrio, and changes in the intestinal flora spread to offspring. These results provide new evidence to explain the increased adverse pregnancy outcomes of mothers of inappropriate age, describe the increased health risks of newborns, help us examine the importance of age-appropriate birth from a broader perspective, and contribute to the discovery of mother-child bonds for a better understanding of healthy reproduction. IMPORTANCE In this study, we demonstrated that physiological indicators and the gut microbiome fluctuate drastically among parturients of different reproductive ages and that there is a significant correlation between the two changes. Mothers of different ages had different gut microbes, and the gut microbiota varied as the childbearing age became too high. Changes in the gut microbiome with maternal reproductive age affected the offspring, and the influence of reproductive age on the intestinal flora had a synergistic effect between mother and child that was revealed for the first time. The maternal childbearing age might affect the colonization of the offspring's initial flora. The results provide new evidence to explain the increased adverse pregnancy outcomes of mothers of inappropriate age, describe the increased health risks of newborns, and contribute to the discovery of mother-child bonds for a better understanding of healthy reproduction.

Subacute dermal toxicity study of bensulfuron-methyl in Sprague-Dawley rats.

Background: Bensulfuron-methyl has recently attracted attention given its widespread use as an herbicide in crops, especially its transdermal safety. However, no dermal toxicity study of this pesticide to mammals has been reported. The present study aims to investigate subacute dermal toxicity of bensulfuron-methyl following repeated doses exposure.Methods: Forty-eight Sprague-Dawley rats were randomly divided into four groups: a normal control group and bensulfuron-methyl groups of different concentrations (250, 500, 1000 mg/kg.bw/day). The rats were topically applied with the substance dermally for 6 h per day for 28 days. At the end of the experiment, all rats were monitored for any changes in their hematological, biochemical parameters, and pathological and histological sections.Results: There were no statistically significant differences (P ≥ 0.05) in the hematological parameters and biochemical parameters. The pathological histological results of rats in the control and the highest concentration group showed no significant abnormalities. The NOAEL of subacute dermal toxicity study was found to be 1000 mg/kg.bw/day in both female and male rats.Conclusion: The result indicated that bensulfuron-methyl is probably safe for humans as a pesticide.

A novel KLF13 mutation underlying congenital patent ductus arteriosus and ventricular septal defect, as well as bicuspid aortic valve.

Recently, mutations in the Kruppel-like factor 13 (KLF13) gene encoding a Kruppel-like transcription factor have been reported to cause congenital heart disease (CHD). However, due to pronounced genetic heterogeneity, the mutational spectrum of KLF13 in other cohorts of cases suffering from distinct types of CHD remain to be ascertained. In the present investigation, by Sanger sequencing of KLF13 in 316 unrelated cases affected by different forms of CHD, a new mutation in heterozygous status, NM_015995.3: c.430G>T; p.(Glu144*), was detected in an index patient affected with patent ductus arteriosus (PDA) and ventricular septal defect (VSD), as well as bicuspid aortic valve (BAV), with a mutation frequency of ~0.32%. Genetic investigation of the available family members of the proband demonstrated that the truncating mutation co-segregated with CHD. The nonsense mutation was not observed in 400 unrelated volunteers without CHD who were enrolled as control subjects. Quantitative biological measurements with dual luciferase reporters revealed that Glu144*-mutant KLF13 did not transactivate the downstream genes vascular endothelial growth factor A and natriuretic peptide A. In addition, the mutation abrogated the synergistic transcriptional activation between KLF13 and T-box transcription factor 5, a well-established CHD-causing gene. In conclusion, the present study indicates that genetically defective KLF13 contributes to familial PDA and VSD, as well as BAV, which expands the phenotypic spectrum linked to KLF13, and reveals a novel molecular pathogenesis of the disease, providing a new molecular target for the early prophylaxis and individualized treatment of CHD.

Comparison of Three Expanded-Spectrum Cephalosporin Hydrolysis Assays and the NG-Test CTX-M Multi Assay That Detects All CTX-M-Like Enzymes.

Rapid detection of expanded-spectrum cephalosporins (ESC) hydrolysing enzymes is crucial to implement infection control measures and antibiotic stewardship. Here, we have evaluated three biochemical ESC hydrolysis assays (ESBL NDP test, ß-LACTA™ test, LFIA-CTX assay) and the NG-Test® CTX-M MULTI that detects CTX-M enzymes, on 93 well-characterized Gram-negative isolates, including 60 Enterobacterales, 21 Pseudomonas spp. and 12 Acinetobacter spp. The performances were good for all three hydrolysis assays, with the LFIA-CTX being slightly more sensitive and specific on the tested panel of isolates especially with Enterobacterales, without ambiguous results. This study showed that LFIA-CTX may be used for the detection of ESC hydrolysis as a competitive alternative to already available assays (ß-LACTA™ test and ESBL NDP test) without any specific equipment and reduced hands-on-time. The lateral flow immunoassay NG-Test® CTX-M MULTI has proven to be a useful, easy, rapid, and reliable confirmatory test in Enterobacterales for detection of CTX-M-type ESBLs, which account for most of the resistance mechanisms leading to ESC resistance in Enterobacterales, but it misses rare ESC hydrolysing ß-lactamases (AmpC, minor ESBLs, and carbapenemases). Combining it with the LFIA-CTX assay would yield an assay detecting the most frequently-encountered ESBLs (CTX-M-like ß-lactamases) together with ESC hydrolysis.

Evaluating Hedgehog Acyltransferase Activity and Inhibition Using the Acylation-coupled Lipophilic Induction of Polarization (Acyl-cLIP) Assay.

Palmitoylation of the Hedgehog family of proteins is a critical step in the Hedgehog signaling pathway and is performed by the membrane-bound O-acyltransferase enzyme Hedgehog acyltransferase (HHAT). Measurement of HHAT activity has traditionally relied on radiolabeled fatty acid substrates, which imposes considerable constraints on throughput, cost, and safety, consequently hindering the efficient identification and development of small-molecule HHAT inhibitors. The Acylation-coupled Lipophilic Induction of Polarisation (Acyl-cLIP) assay was recently developed in our lab as a novel platform to evaluate lipidation of peptides in real time and high throughput. In this chapter, we describe the isolation of active HHAT from HEK293a cells and application of the Acyl-cLIP assay to characterize HHAT inhibitors. Our methodology uses standard chemical biology lab equipment and yields high-quality kinetic data from minimal sample volumes. The assay uses standard 384-well plates and is easily adapted to medium- or high-throughput screening formats.

Osmotically Enabled Wearable Patch for Sweat Harvesting and Lactate Quantification.

Lactate is an essential biomarker for determining the health of the muscles and oxidative stress levels in the human body. However, most of the currently available sweat lactate monitoring devices require external power, cannot measure lactate under low sweat rates (such as in humans at rest), and do not provide adequate information about the relationship between sweat and blood lactate levels. Here, we discuss the on-skin operation of our recently developed wearable sweat sampling patch. The patch combines osmosis (using hydrogel discs) and capillary action (using paper microfluidic channel) for long-term sweat withdrawal and management. When subjects are at rest, the hydrogel disc can withdraw fluid from the skin via osmosis and deliver it to the paper. The lactate amount in the fluid is determined using a colorimetric assay. During active sweating (e.g., exercise), the paper can harvest sweat even in the absence of the hydrogel patch. The captured fluid contains lactate, which we quantify using a colorimetric assay. The measurements show the that the total number of moles of lactate in sweat is correlated to sweat rate. Lactate concentrations in sweat and blood correlate well only during high-intensity exercise. Hence, sweat appears to be a suitable biofluid for lactate quantification. Overall, this wearable patch holds the potential of providing a comprehensive analysis of sweat lactate trends in the human body.