Plate Reader Research Articles

Toward point-of-care diagnostics: Running enzymatic assays on a photonic waveguide-based sensor chip with a portable, benchtop measurement system.

We demonstrate a portable, compact system to perform absorption-based enzymatic assays at a visible wavelength of 639 nm on a photonic waveguide-based sensor chip, suitable for lab-on-a-chip applications. The photonic design and fabrication of the sensor are described, and a detailed overview of the portable measurement system is presented. In this publication, we use an integrated photonic waveguide-based absorbance sensor to run a full enzymatic assay. An assay to detect creatinine in plasma is simultaneously performed on both the photonic sensor on the portable setup and on a commercial microplate reader for a clinically relevant creatinine concentration range. We observed a high correlation between the measured waveguide propagation loss and the optical density measurement from the plate reader and measured a limit-of-detection of 4.5 μM creatinine in the sensor well, covering the relevant clinical range for creatinine detection.

CccDNA-Targeted Drug Screen Reveals a Class of Antihistamines as Suppressors of HBV Genome Levels.

Chronic infection with hepatitis B virus (HBV) is incurable, as the current therapeutics cannot eliminate its persistent genomic material, cccDNA. Screening systems for cccDNA-targeting therapeutics are unavailable, as low copies of cccDNA in vitro complicate detection. To address this, cccDNA copies were massively increased to levels detectable via automated plate readers. This was achieved via continuous infection in a contact-free co-culture of an HBV generator (clone F881), which stably produced clinically relevant amounts of HBV, and HBV acceptors selected to carry high cccDNA loads. cccDNA-targeted therapeutics were then identified via reduced cccDNA-specific fluorescence, taking differences in the cell numbers and viability into account. Amongst the drugs tested, the H1 antihistamine Bilastine, HBVCP inhibitors and, surprisingly, current HBV therapeutics downregulated the cccDNA significantly, reflecting the assay's accuracy and sensitivity in identifying drugs that induce subtle changes in cccDNA levels, which take years to manifest in vivo. Bilastine was the only therapeutic that did not reduce HBV production from F881, indicating it to be a novel direct suppressor of cccDNA levels. When further assessed, only the structurally similar antihistamines Pitolisant and Nizatidine suppressed cccDNA levels when other H1 antihistamines could not. Taken together, our rapid fluorescence cccDNA-targeted drug screen successfully identified a class of molecules with the potential to treat hepatitis B.

Evaluation for endotoxin in intraocular materials used during phacoemulsification surgery using a recombinant factor C assay.

Cataract surgery remains the sole method to resolve blindness secondary to cataract formation. One complication includes fibrin web formation post-operatively. This study aimed to investigate the presence of endotoxin within materials used during cataract surgery as a possible cause of fibrin web phenomenon. Preservative-free epinephrine, heparin, viscoelastic devices, and intraocular lenses were collected for evaluation. Various manufacturers and manufacturing lot numbers were used when available. Viscosity of viscoelastics was reduced by incubating samples with human recombinant hyaluronidase. Intraocular product (IOL) packaging fluid was collected and stored for testing. The IOLs were then washed with a sterile balanced salt solution, incubated at 37°C for 48 h, and then fluid was collected for testing to mimic intraocular placement. Samples were tested using a commercially available rFC kit. Fluorescence was measured at time zero and after 1 h using a fluorescence microplate reader. The change in fluorescence was corrected for blank fluorescence and plotted to a standard curve. Endotoxin levels were below the limit of detection (0.05 EU/mL) in all samples. Incubation of IOLs at intraocular temperature did not increase extraction of endotoxin. Endotoxin was not identified in any tested sample, including those used in cases of fibrin web formation post-phacoemulsification. As fibrin webs are often observed episodically, it is possible that endotoxin levels may vary between batches, or that endotoxin is not related to fibrin formation.

Functional and Antioxidant Evaluation of Two Ecotypes of Control and Grafted Tree Tomato (Solanum betaceum) at Different Altitudes.

Tree tomato (Solanum betaceum) is susceptible to nematode attack; for this reason, grafting is used as an alternative to reduce this impact. In this study, the bioactive compounds of the fruit (shell, pulp, and seed jelly) of two tree tomato ecotypes ('giant orange' and 'giant purple') were evaluated in both control and grafted plants grown at different altitudes (2010-2250, 2260-2500, 2510-2750 and 2760-3000 masl). Commercial quality, vitamin C, organic acids, phenolics, carotenoids and antioxidant activity were determined by microextraction and quantified by liquid chromatography (RRLC) or spectrophotometry (microplate reader). The results showed high concentrations of vitamin C, organic acids and antioxidant activity in the seed jelly, organic acids in the pulp and phenolic compounds, carotenoids, and antioxidant activity in the shell. The main phenolics were ferulic acid, caffeic acid and luteolin, while the main carotenoids were lutein, B-cryptoxanthin and B-carotene. Multivariate analysis showed that tree tomato quality was mainly influenced by altitude and fruit part and that grafting positively affected soluble solids for both ecotypes and all altitudes.

The Use of Thioflavin T for the Estimation and Measurement of the Plasma Membrane Electric Potential Difference in Different Yeast Strains.

The use of the cationic, dye thioflavin T (ThT), to estimate the electric plasma membrane potential difference (PMP) via the fluorescence changes and to obtain its actual values from the accumulation of the dye, considering important correction factors by its binding to the internal components of the cell, was described previously for baker's yeast. However, it was considered important to explore whether the method developed could be applied to other yeast strains. Alternative ways to estimate the PMP by using flow cytometry and a multi-well plate reader are also presented here. The methods were tested with other strains of Saccharomyces cerevisiae (W303-1A and FY833), as well as with non-conventional yeasts: Debaryomyces hansenii, Candida albicans, Meyerozyma guilliermondii, and Rhodotorula mucilaginosa. Results of the estimation of the PMP via the fluorescence changes under different conditions were adequate with all strains. Consistent results were also obtained with several mutants of the main monovalent transporters, validating ThT as a monitor for PMP estimation.

Etiological features of urinary tract infections in pregnant women: current state of the problem

Background. Urinary tract infections in pregnant wo­men occupy a leading place in the structure of extragenital patho­logy. The purpose of the study is to determine the etiological features of urinary tract infections in pregnant women. Materials and ­methods. The study included 89 pregnant women with urinary tract infections. The material for microbiological research was urine, the samples of which were collected on the day of hospitalization of a pregnant woman before treatment. Identification of microorganisms was carried out with the use of ­MIKROLATEST® test systems (Erba Lachema, Czech Republic). Preparation of bacterial suspensions with a certain concentration of microbial cells was carried out using Densi-La-Meter electronic device (PLIVA-Lachema a.s., Czech Republic) according to the McFarland scale. Optical density was measured using a Multiskan EX microplate reader (type 355). Results. Escherichia coli, Proteus mirabilis, Proteus vulgaris, Streptococcus pyogenes, Enterococcus faecalis, Klebsiella pneumoniae, Pseudomonas aeruginosa are the leading pathogens of urinary tract infections in pregnant women of the Kharkiv region; Escherichia coli, Proteus mirabilis, Streptococcus pyogenes are predominant ones. The selected spectrum of etiological factors of urinary tract infections does not depend on the age of a pregnant woman. In the etiological structure of the urinary tract infections in pregnant women, Escherichia coli and Proteus mirabilis take the first and the second place during the first three trimesters of pregnancy. The third place in the first trimester belongs to Enterococcus faecalis, in the second trimester — to Proteus vulgaris and Streptococcus pyogenes, in the third trimester — to Streptococcus pyogenes. The etiological structure of urinary tract infections in pregnant women is determined by the topography of the inflammatory process. Thus, in women with asymptomatic bacteriuria, Proteus mirabilis takes the first place, Escherichia coli — the second place, Proteus vulgaris and Enterococcus faecalis — the third place; in acute pyelonephritis, the first place belongs to Escherichia coli, the second place to Streptococcus pyogenes, the third place to Proteus mirabilis, Enterococcus faecalis, Klebsiella pneumoniae; in urethritis, Escherichia coli takes the first place, Enterococcus faecalis, Klebsiella pneumoniae — the second place, Proteus mirabilis, Proteus vulgaris — the third place; in cystitis, the first place belongs to Escherichia coli, the second place to Proteus mirabilis, Proteus vulgaris, Enterococcus faecalis, Klebsiella pneumoniae, Pseudomonas aeruginosa. Conclusions. The etiological features of urinary tract infections with different topography of the inflammatory process of bacterial etiology determined by the authors must be taken into account in the treatment of this category of pregnant women.

Development of a rapid and sensitive fluorescent probe for high-throughput detecting SO2 in food samples

Sulfur dioxide (SO2), widely used as an antioxidant and preservative in food production, has been associated with detrimental cardiovascular and neurological effects when consumed excessively. This highlights the pressing need to develop a fast and sensitive probe capable of high-throughput screening for the quantitative determination of SO2 in food. Herein, we synthesized a new fluorescent probe, namely B3, specifically designed for high-throughput detection of SO2 in food. The vinyl chloride aldehyde within the B3 structure engages in a nucleophilic addition reaction with SO2, contributing to B3′s exceptional selectivity for SO2, and fast response time within 9 s. Furthermore, by integrating B3 with a microplate reader, we effectively achieved high-throughput detection of SO2 concentration up to 45 μM within a pH range of 5.5 to 8.0 in real food samples. This accomplishment serves as a significant contribution to ensuring consumer safety and facilitating health assessments.

Evaluation of in vitro antimalarial activity of <i>Citrus aurantifolia</i> (CHRISTM.) and <i>Ziziphus mauritiana</i> (LAM) using heme polymerization inhibition assay

Citrus aurantifolia and Ziziphus mauritiana (Lam) are plants that have numerous medicinal properties and are used extensively in traditional medicine for the treatment of several diseases. Malaria parasites metabolize haemoglobin and detoxify the resulting haem by polymerizing it to form haemozoin (malaria pigment). This study aimed at evaluating the antiplasmodial activity through in vitro cell-free heme polymerization inhibition assay (HPIA) of C. aurantifolia and Z. mauritiana. The analysis was conducted using a 96-well microtiter plate reader at an absorption of 400 nm, at different concentrations (0-2 mg/ml). The IC50 was determined by non-linear regression analysis using GraphPad Prism. The result indicated that both the root extracts of C. aurantifolia and Z. mauritiana showed significant difference (p˂0.0001) with IC50 of 0.81 mg/ml and 0.77 mg/ml respectively compared to positive control. Phytochemical screening of the ethanol root extracts showed the presence of saponins, alkaloids, terpenoids, glycosides, steroids, and anthraquinone, while tannins and reducing sugars were not detected in both samples used, flavonoid was detected in C. aurantifolia. Both the extracts exhibited potential for inhibiting heme polymerization in vitro.

Ucp4 Knockdown of Cerebellar Purkinje Cells Induces Bradykinesia

Although uncoupling protein 4 (UCP4) is the most abundant protein reported in the brain, the biological function of UCP4 in cerebellum and pathological outcome of UCP4 deficiency in cerebellum remain obscure. To evaluate the role of Ucp4 in the cerebellar Purkinje cells (PCs), we generated the conditional knockdown of Ucp4 in PCs (Pcp2cre;Ucp4fl/fl mice) by breeding Ucp4fl/fl mice with Pcp2cre mice. Series results by Western blot, immunofluorescent staining, and triple RNAscope in situ hybridization confirmed the specific ablation of Ucp4 in PCs in Pcp2cre;Ucp4fl/fl mice, but did not affect the expression of Ucp2, the analog of Ucp4. Combined behavioral tests showed that Pcp2cre;Ucp4fl/fl mice displayed a characteristic bradykinesia in the spontaneous movements. The electromyogram recordings detection excluded the possibility of hypotonia in Pcp2cre;Ucp4fl/fl mice. And the electrical patch clamp recordings showed the altered properties of PCs in Pcp2cre;Ucp4fl/fl mice. Moreover, transmission electron microscope (TEM) results showed the increased mitochondrial circularity in PCs; ROS probe imaging showed the increased ROS generation in molecular layer; and finally, microplate reader assay showed the significant changes of mitochondrial functions, including ROS, ATP, and MMP in the isolated cerebellum tissue. The results suggested that the specific knockdown of mitochondrial protein Ucp4 could damage PCs possibly by attacking their mitochondrial function. The present study is the first to report a close relationship between UCP4 deletion with PCs impairment, and suggests the importance of UCP4 in the substantial support of mitochondrial function homeostasis in bradykinesia. UCP4 might be a therapeutic target for the cerebellar-related movement disorder.Graphical

Development and Comparative Evaluation of Two Different Label-Free and Sensitive Fluorescence Platforms for Analysis of Olaparib: A Recently FDA-Approved Drug for the Treatment of Ovarian and Breast Cancer.

Olaparib (OLA) is a PARP inhibitor drug which has been recently approved by the Food and Drug Administration (FDA) for the treatment of ovarian and breast cancer. A convenient analytical tool for the quantitation of OLA in its dosage form and plasma samples was urgently needed. This study describes, for the first time, the development of two different label-free and sensitive fluorescence-based platforms for the pharmaceutical and bioanalysis of OLA. These platforms were microwell-assisted with a fluorescence microplate reader (MW-FLR) and high-performance liquid chromatography with fluorescence detection (HPLC-FD). Both MW-FLR and HPLC-FD employed the native fluorescence of OLA as an analytical signal. The MW-FLR involved measuring the fluorescence signals in 96-well white-opaque plates. The HPLC-FD involved chromatographic separation of OLA and duvelisib (DUV), as an internal standard on a Nucleosil-CN HPLC column (250 mm length × 4.6 mm i.d., 5 µm particle diameter) with a mobile phase composed of acetonitrile: water (25:75, v/v) pumped at a flow rate of 1.7 mL/min. Elution of OLA and DUV was detected using a fluorescence detector. The optimal conditions of both MW-FLR and HPLC-FD were established, and they were validated according to the guidelines of the International Council for Harmonization for the validation of analytical procedures. The linear ranges of MW-FLR and HPLC-FD were 25-1000 and 5-200 ng/mL, respectively, with limits of detection of 15 and 1.7 ng/mL, respectively. The accuracy and precision of both platforms were confirmed as the recovery values were ≥98.2% and the relative standard deviations (RSD) were ≤2.89%. Both methodologies were satisfactorily applied to the quantitation of OLA in its commercial dosage form (Lynparza® tablets) and plasma samples with high accuracy and precision. The greenness of both MW-FLR and HPLC-FD was assessed using two different multiple parameter-based metric tools, and the results proved their greenness and adherence to the requirements of green analytical approaches. Both platforms have simple procedures and acceptable levels of analytical throughput. In conclusion, the proposed MW-FLR and HPLC-FD are valuable tools for routine use in quality control and clinical laboratories for the quantitation of OLA for the purposes of pharmaceutical quality control, pharmacokinetic studies, and bioequivalence testing.

Preparation and Properties of Sodium Alginate/Gelatin Hydrogel-Microspheres

Calcium alginate gel microspheres exhibiting pH response release characteristics were prepared, and utilizing Bovine serum albumin (BSA) to evaluate drug release performance. Hydrogels composed of gelatin (GL) and sodium alginate oxide (NALO), with varying ratios (7:3; 4:1; 3:2) were prepared by controlling their composition. Scanning electron microscopy (SEM) and a microplate reader (ELIASA) were used to characterize the microspheres’ microstructure and in vitro BSA release behavior, respectively. The results demonstrated that, under acidic conditions, the BSA release was slower than in neutral conditions. The hydrogels’ physical and chemical properties were analyzed using optical microscopy, Fourier transform infrared spectroscopy (FTIR), swelling experiments and mechanical testing of the hydrogels and microsphere loaded hydrogels. The results showed that a GL to NALO ratio of 7:3 increased the swelling degree, while a 4:1 ratio optimized the mechanical characteristics. The study concluded that the microsphere-loaded hydrogels exhibited significant potential for human drug delivery applications.

Development of Novel Micellar-Enhanced High-Throughput Microwell Spectrofluorimetric Method for Quantification of Lorlatinib: Application to In Vitro Drug Release and Analysis of Urine Samples.

Lorlatinib (LOR) is a third-generation anaplastic lymphoma kinase (ALK) tyrosine kinase inhibitor drug. The Food and Drug Administration (FDA) has granted an approval for the use of LOR as a first therapeutic intervention for individuals diagnosed with ALK-positive metastatic and advanced non-small-cell lung cancer (NSCLC). The present study outlines, for the first time, the development and validation of an innovative microwell-based spectrofluorimetric (MW-SFL) method for the quantification of LOR. The proposed method involved the enhancement of the weak native fluorescence of LOR by its micellization into the sodium lauryl sulfate (SLS) micelles. The procedures of the method were conducted in white opaque plates with 96 microwells, and the enhanced fluorescence signals were measured by a fluorescence plate reader at 405 nm after excitation at 310 nm. The measured relative fluorescence intensity (RFI) had a linear relationship with LOR concentrations in the range of 60-1600 ng mL-1. The limit of detection (LOD) and the limit of quantification (LOQ) were found to be 19 and 56 ng mL-1, respectively. The method's accuracy and precision were assessed using a recovery study; the recovery values ranged from 99.98% to 101.40%, accompanied by relative standard deviation (RSD) values of 0.42% to 1.59%. The proposed MW-SFL method combined the advantages of the intrinsically high sensitivity of the spectrofluorimetric measurement and the excellent throughput of the microwell-based approach. The results proved the method is effective in the determination of LOR in its pharmaceutical tablets, tablet dissolution testing, as well as in spiked urine with a high degree of precision and accuracy. The MW-SFL method is notable for its simple procedures and utilization of water as a solvent, as well as minimal quantities of sample solutions. These features align with its ecofriendly approach to green chemistry principles. These advantages gave the proposed MW-SFL method a high potential value for the determination of LOR in clinical and quality control laboratories.

Ocular Pharmacology and Toxicology of TRPV1 Antagonist SAF312 (Libvatrep).

To evaluate the pharmacology and toxicology of SAF312, a transient receptor potential vanilloid 1 (TRPV1) antagonist. TRPV1 expression in human ocular tissues was evaluated with immunohistochemistry. Inhibition of calcium influx in Chinese hamster ovary (CHO) cells expressing human TRPV1 (hTRPV1) and selectivity of SAF312 were assessed by a fluorescent imaging plate reader assay. Ocular tissue and plasma pharmacokinetics (PK) were assessed following a single topical ocular dose of SAF312 (0.5%, 1.0%, 1.5%, 2.5%) in rabbits. Safety and tolerability of SAF312 were evaluated in rabbits and dogs. Effects of SAF312 on corneal wound healing after photorefractive keratectomy (PRK) surgery were assessed in rabbits. TRPV1 expression was noted in human cornea and conjunctiva. SAF312 inhibited calcium influx in CHO-hTRPV1 cells induced by pH 5.5 (2-[N-morpholino] ethanesulfonic acid), N-arachidonoylethanolamine, capsaicin, and N-arachidonoyl dopamine, with IC50 values of 5, 10, 12, and 27 nM, respectively, and inhibition appeared noncompetitive. SAF312 demonstrated high selectivity for TRPV1 (>149-fold) over other TRP channels. PK analysis showed highest concentrations of SAF312 in cornea and conjunctiva. SAF312 was found to be safe and well tolerated in rabbits and dogs up to the highest feasible concentration of 2.5%. No delay in wound healing after PRK was observed. SAF312 is a potent, selective, and noncompetitive antagonist of hTRPV1 with an acceptable preclinical safety profile for use in future clinical trials. SAF312, which was safe and well tolerated without causing delay in wound healing after PRK in rabbits, may be a potential therapeutic agent for ocular surface pain.

Antibiofilm Activity of Tapak Kuda Ipomoea pes-caprae against Pseudomonas aeruginosa ATCC 27853 and Methicillin-Resistant Staphylococcus aureus (MRSA) ATCC 43300: In-Vitro and In-Silico Evaluation

Ipomoea pes-caprae is one of the herbal plants that can treat various health problems such as skin infections, burns, boils, and various diseases caused by microbial infections. This study aims to identify ethanol extract compounds of I. pes-caprae leaf and evaluate their antibiofilm activity through in-vitro and in-silico assays. This study used two test bacteria, Pseudomonas aeruginosa and Methicilin Resistant Staphylococcus aureus. Antibacterial activity is carried out using the agar diffusion method and antibiofilm using a microplate reader. The biological activity was also evaluated through a computational approach using molecular docking. The results of preliminary test demonstrated the antibacterial activity. At a concentration of 100 mg/mL, I. pes-caprae extract produced a substantial inhibitory zone of 13.9 mm for P. aeruginosa and a moderate 8.5 mm zone for MRSA. The extract also showed high antibiofilm activity. It achieved impressive biofilm inhibition rates of 82.58% and 78.29%, respectively. Molecular docking shows the interaction between extract compounds and macromolecules that play a role in biofilm formation, namely SrtA and associated protein biofilms. 1,3,4,5-Tetrahydroxy-Cyclohexanecarboxylic Acid has the lowest binding energy of -7.5 Kcal/Mol and -5.9 Kcal/Mol at each target receptor. This study demonstrated the antibiofilm potential of I. pes-caprae extract, which was clarified through molecular docking studies.

Gβγ subunit inhibitor decreases DOM-induced head twitch response via the PLCβ/IP3/Ca2+/ERK and cAMP signaling pathways

Aims(−)-2,5-dimethoxy-4-methylamphetamine (DOM) induces the head-twitch response (HTR) primarily by activating the serotonin 5-hydroxytryptamine 2A receptor (5-HT2A receptor) in mice. However, the mechanisms underlying 5-HT2A receptor activation and the HTR remain elusive. Gβγ subunits are a potential treatment target in numerous diseases. The present study investigated the mechanism whereby Gβγ subunits influence DOM-induced HTR. Main methodsThe effects of the Gβγ inhibitor 3′,4′,5′,6′-tetrahydroxyspiro[2-benzofuran-3,9′-xanthene]-1-one (gallein) and antagonistic peptide βARKct (β-adrenergic receptor kinase C-terminal fragment) on DOM-induced HTR were studied via an HTR test. The activation of the phospholipase C β (PLCβ)/inositol triphosphate (IP3)/calcium (Ca2+) signaling pathway and extracellular signal-regulated kinase (ERK) following Gβγ subunit inhibition was detected by western blotting, Homogeneous Time-Resolved Fluorescence (HTRF) inositol phosphate (IP1) assay and Fluorometric Imaging Plate Reader (FLIPR) calcium 6 assay. The Gβγ subunit-mediated regulation of cyclic adenosine monophosphate (cAMP) was assessed via a GloSensor™ cAMP assay. Key findingsThe Gβγ subunit inhibitors gallein and βARKct reduced DOM-induced HTR in C57BL/6J mice. Like the 5-HT2A receptor-selective antagonist (R)-[2,3-di(methoxy)phenyl]-[1-[2-(4-fluorophenyl)ethyl]piperidin-4-yl]methanol (M100907), gallein inhibited PLCβ phosphorylation (pPLCβ), IP1 production, Ca2+ transients, ERK1/2 phosphorylation (pERK1/2) and cAMP accumulation induced by DOM in human embryonic kidney (HEK) 293T cells stably or transiently transfected with the human 5-HT2A receptor. Moreover, PLCβ protein inhibitor 1-[6-[[(8R,9S,13S,14S,17S)-3-methoxy-13-methyl-6,7,8,9,11,12,14,15,16,17-decahydrocyclopenta[a]phenanthren-17-yl]amino]hexyl]pyrrole-2,5-dione (U73122) (10 nmol/mouse), intracellular Ca2+ blocker 6-[6-[6-[5-acetamido-4,6-dihydroxy-2-(sulfooxymethyl)oxan-3-yl]oxy-2-carboxy-4-hydroxy-5-sulfooxyoxan-3-yl]oxy-2-(hydroxymethyl)-5-(sulfoamino)-4-sulfooxyoxan-3-yl]oxy-3,4-dihydroxy-5-sulfooxyoxane-2-carboxylic acid (heparin) (5 nmol/mouse), L-type Ca2+ channel blocker 3-O-(2-methoxyethyl) 5-O-propan-2-yl 2,6-dimethyl-4-(3-nitrophenyl)-1,4-dihydropyridine-3,5-dicarboxylate (nimodipine) (4 mg/kg), mitogen extracellular regulating kinase 1/2 (MEK1/2) inhibitor (Z)-3-amino-3-(4-aminophenyl)sulfanyl-2-[2-(trifluoromethyl)phenyl]prop-2-enenitrile (SL327) (30 mg/kg), and Gαs protein selective antagonist 4,4′,4″,4‴-(Carbonylbis-(imino-5,1,3-benzenetriylbis(carbonylimino)))tetrakisbenzene-1,3-disulfonic acid (NF449) (10 nmol/mouse) reduced DOM-induced HTR in C57BL/6J mice. SignificanceThe Gβγ subunits potentially mediate the HTR after 5-HT2A receptor activation via the PLCβ/IP3/Ca2+/ERK1/2 and cAMP signaling pathways. Inhibitors targeting the Gβγ subunits potentially inhibit the hallucinogenic effects of 5-HT2A receptor agonists.

Identification and characterization of a novel chromosomal aminoglycoside 3'-O-phosphotransferase, APH(3')-Id, from Kluyvera intermedia DW18 isolated from the sewage of an animal farm.

Aminoglycosides, as important clinical antimicrobials, are used as second-line drugs for treating multidrug-resistant tuberculosis or combined with β-lactam drugs for treating severe infections such as sepsis. Aminoglycoside-modifying enzyme (AME) is the most important mechanism of aminoglycoside resistance and deserves more attention. The bacterium Kluyvera intermedia DW18 was isolated from the sewage of an animal farm using the conventional method. The agar dilution method was used to determine the minimum inhibitory concentrations (MICs) of antimicrobials. A novel resistance gene was cloned, and the enzyme was expressed. The kinetic parameters were measured by a SpectraMax M5 multifunctional microplate reader. Bioinformatic analysis was performed to reveal the genetic context of the aph(3')-Id gene and its phylogenetic relationship with other AMEs. A novel aminoglycoside 3'-O-phosphotransferase gene designated aph(3')-Id was identified in K. intermedia DW18 and shared the highest amino acid identity of 77.49% with the functionally characterized aminoglycoside 3'-O-phosphotransferase APH(3')-Ia. The recombinant plasmid carrying the novel resistance gene (pMD19-aph(3')-Id/E. coli DH5α) showed 1,024-, 512-, 128- and 16-fold increased MIC levels for kanamycin, ribostamycin, paromomycin and neomycin, respectively, compared with the reference strain DH5α. APH(3')-Id showed the highest catalytic efficiency for ribostamycin [kcat/Km of (4.96 ± 1.63) × 105 M-1/s-1], followed by paromomycin [kcat/Km of (2.18 ± 0.21) × 105 M-1/s-1], neomycin [kcat/Km of (1.73 ± 0.20) × 105 M-1/s-1], and kanamycin [kcat/Km of (1.10 ± 0.18) × 105 M-1/s-1]. Three conserved functional domains of the aminoglycoside phosphotransferase family and ten amino acid residues responsible for the phosphorylation of kanamycin were found in the amino acid sequence of APH(3')-Id. No mobile genetic element (MGE) was discovered surrounding the aph(3')-Id gene. In this work, a novel aminoglycoside 3'-O-phosphotransferase gene designated aph(3')-Id encoded in the chromosome of the environmental isolate Kluyvera intermedia DW18 was identified and characterized. These findings will help clinicians select effective antimicrobials to treat infections caused by pathogens with this kind of resistance gene.

Paper-based analytical device integrated with bacteriophage tail fiber protein for bacteria detection and antimicrobial susceptibility test

Severe antimicrobial resistance calls for developing rapid, sensitive and affordable methodological platform for clinical diagnosis of bacterial infection. Herein, a paper-based analytical device (PAD) for fluorescent (FL) detection and antimicrobial susceptibility test (AST) of Pseudomonas aeruginosa (P. aeruginosa) was fabricated by defining 96 hydrophilic microzones on a filter paper using a handheld stamp dipped with liquid wax. The size of microzones was designed to be consistent with traditional 96-well microplate, thus the FL signals can be collected by a commercialized microplate reader. Streptavidin was immobilized into the microzones by chitosan-glutaraldehyde crosslinking reaction, and then biotinylated bacteriophage tail fiber protein (TFP) was conjugated through biotin-streptavidin affinity system. TFP and fluorescein isothiocyanate (FITC) labeled antimicrobial peptide were used as capture agent and signal probe, respectively, for FL detection of P. aeruginosa on the PAD. The linear range for quantifying P. aeruginosa is 1.0 × 103 CFU/mL to 1.0 × 107 CFU/mL, with a detection limit of 137 CFU/mL. The PAD was also applied to conduct AST of P. aeruginosa for imipenem, meropenem, cefepime, amikacin, and gentamicin, and the results are consistent with the traditional broth dilution method. The PAD provides an affordable diagnosis platform for bacterial infection, especially in resource-limited institutes and countries.

High-resolution neuraminidase inhibition profiling of Arnebia euchroma (Royle) I.M. Johnst. based on HR-MS and target isolation: An example study of anti-infectious constituents in traditional Chinese medicine

Ethnopharmacological relevanceTraditional Chinese Medicines (TCMs) are an important source to discover new anti-infectious drugs. Neuraminidases (NAs) not only play a key role on human health, but also are promising targets for anti-infectious drugs. Arnebia euchroma which is a widely used traditional Chinese medicine with the effect of cooling blood and detoxifying showed potential inhibitory activities on both bacterial NA and virus NA, suggesting that the material basis of A. euchroma deserves in-depth study. Aim of the studyTo investigate the anti-infectious constituents of A. euchroma based on NA inhibition. Material and methodsA HPLC-DAD system incorporated an auto-sampler was used for micro-fractionation. A nanoliter liquid handler and a high sensitive multimode plate reader system were used for high throughput NA inhibition screening. Thus a high-resolution NA inhibition profiling platform was constructed. The structures of potential active components in A. euchroma obtained by the high-resolution bioassay profiling were identified by DAD and MS in parallel. Then, a target and rapid isolation of NAIs from A. euchroma was achieved under the guidance of the spectrum-effect relationship obtained above. Finally, the isolated compounds were elucidated by extensive spectroscopic methods and their bioactivities were validated by in vitro assay and molecular docking. Results16 potential active ingredients in A. euchroma were isolated and identified, including a new mero-monoterpenoid. The in vitro bioassay results revealed that 12 out of the 16 isolated compounds showed potent inhibitory activities on bacterial NA (IC50s = 1–6 μM) and five of them exhibited potent anti-microbial activities on methicillin-resistant Staphylococccus aureus (MRSA) with MICs in the range of 0.5–4 μg/mL. Furthermore, some isolated compounds showed equal or even better inhibitory activities on oseltamivir resistant viral NA than oseltamivir sensitive NA. The mechanism study in silicon revealed that these natural compounds possessed absolutely different binding modes on the bacterial and viral NAs. ConclusionsOur study gave a clear spectrum-effect relationship of A. euchroma, providing a scientific evidence for future study of the multi-components synergistic effect of TCMs.

Sanggenon C alleviates palmitic acid-induced insulin resistance in HepG2 cells via AMPK pathway

Purpose: To investigate the potential role of sanggenon C alleviating in insulin resistance.Methods: HepG2 cell line was incubated with increasing concentrations of sanggenon C at 1, 5, 10, 15 or 20 μM for 4 h. to induce cytotoxicity, and then further incubated with 100 μM palmitic acid to induce insulin resistance. HepG2 cells without sanggenon C and palmitic acid treatment servered as control group. Glucose uptake was determined by measuring 2-NBDG (2-deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl)-amino]-D-glucose) fluorescence intensity using a microplate reader. Oil Red O staining was used to assess intracellular lipid accumulation, while oxidative stress was evaluated by enzyme-linked immunosorbent assay (ELISA).Results: Palmitic acid significantly decreased glucose uptake and increased intracellular lipid accumulation in HepG2 (p < 0.01), while sanggenon C enhanced t glucose uptake and lowered lipid accumulation in insulin-resistant HepG2 (p < 0.01). Sanggenon C significantly attenuated palmitic acidinduced increase in p-insulin receptor substrate 1 (p-IRS1), as well as decrease in p-AKT and p-FOXO1 (p < 0.01). Palmitic acid also induced oxidative stress in HepG2 through the up-regulation of reactive oxygen species (ROS) and malondialdehyde (MDA), as well as the down-regulation of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px). However, sanggenon C reduced ROS and MDA levels (p < 0.05), and enhanced SOD and GSH-Px in insulin-resistant HepG2 (p < 0.05). However, sanggenon C significantly increased p-AMP-activated protein kinase (p-AMPK) levels and p-ACC (acetyl-CoA carboxylase) in insulin-resistant HepG2 (p < 0.01).Conclusion: Sanggenon C lowers oxidative stress and ameliorates lipid accumulation thereby alleviating palmitic acid-induced insulin-resistant HepG2 cells via activation of AMPK pathway, thus suggesting that it is a potential strategy for overcoming insulin resistance.

Evaluation of the Inhibitory Activity of Syzygium aromaticum Extract -Chitosan Nanoparticles Against Biofilm Formation of Klebsiella pneumonia

The increasing resistance of Klebsiella pneumoniae to antibiotics has led to difficulties in treating infections due to its virulence factors. As one of its major pathogenic factors, this opportunistic pathogen may develop a thick biofilm coating, allowing the bacteria to attach to living or nonliving surfaces and promote drug resistance. Searching for therapeutic alternatives from a plant source that was safe and effective in treating this multi-drug-resistant bacteria was necessary. In this concept, Syzygium aromaticum extract (SAE) is used to combat K. pneumonia. The extract was confirmed by GC-MS and loaded onto chitosan nanoparticles (SACSNPs). The SACSNPs were prepared by the ionic gelation method with tripolyphosphate (TPP). And then characterized using UVvis, FTIR, AFM, SEM, and XRD techniques. The K. pneumonia isolates were obtained and identified using the VITEK-2 system. The MIC of SAE and SACSNPs were confirmed using a 96-well resazurin-aided microdilution method, which was 6.25 μg/ml for SACSNPs and 75.5 μg/ml for SAE. The inhibitory activity using sub-MIC of analytical substances was determined by measuring the optical density using a microplate reader with a 96-well plate and 0.1% crystal violet dye. The results show that the S. aromaticum extract loaded with chitosan nanoparticles has higher inhibitory activity against the biofilm formation of K. pneumonia than the S. aromaticum extract. Keywords: Chitosan nanoparticles, S. aromaticum, K. pneumonia, Biofilm, GCMS, resazurin, XRD.