Assay Protocol Research Articles

An improved MTT colorimetric method for rapid viable bacteria counting

The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay has been employed in the analysis of bacterial growth. In comparison to experiments conducted on mammalian cells, the MTT bacterial assay encounters a greater number of interfering factors and obstacles that impact the accuracy of results. In this study, we have elucidated an improved MTT assay protocol and put forth an equation that establishes a correlation between colony-forming units (CFU) and the amount of formazan converted by the bacteria, drawing upon the fundamental principle of the MTT assay. This equation is represented as CFU=kF. Furthermore, we have explicated a methodology to determine the scale factor “k” by employing S. aureus and E. coli as illustrative examples. The findings indicate that S. aureus and E. coli reduce MTT by a cyclic process, from which the optimal reduction time at room temperature was determined to be approximately 30 mins. Furthermore, individual E. coli exhibits an MTT reduction capacity approximately four times greater than that of S. aureus. HPLC analysis proves to be the most accurate method for mitigating interferences during the dissolution and quantification of formazan. Additionally, this study has identified a new constraint related to the narrow linear range (0–125 μg/mL) of formazan concentration-absorbance and has presented strategies to circumvent this limitation.

In-Cell Arrestin-Receptor Interaction Assays.

G protein-coupled receptors (GPCRs) represent ∼30% of current drug targets. Ligand binding to these receptors activates G proteins and arrestins, which function in different signaling pathways. Given that functionally selective or biased ligands preferentially activate one of these two groups of pathways, they may be superior medications for certain disease states. The identification of such ligands requires robust drug screening assays for both G protein and arrestin activity. This unit describes protocols for assays that monitor reversible arrestin recruitment to GPCRs in living cells using either bioluminescence resonance energy transfer (BRET) or nanoluciferase complementation (NanoLuc). Two types of assays can be used: one configuration directly measures arrestin recruitment to a GPCR fused to a protein tag at its intracellular C-terminus, whereas the other configuration detects arrestin translocation to the plasma membrane in response to activation of an unmodified GPCR. Together, these assays are powerful tools for studying dynamic interactions between GPCRs and arrestins. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Receptor-arrestin BRET assay to measure ligand-induced recruitment of arrestin to receptors Basic Protocol 2: Receptor-arrestin NANOBIT assay to measure ligand-induced recruitment of arrestin to receptors Alternative Protocol 1: BRET assay to measure ligand-induced recruitment of arrestin to the plasma membrane Alternative Protocol 2: NANOBIT assay to measure ligand-induced recruitment of arrestin to the plasma membrane Support Protocol 1: Optimization of polyethylenimine (PEI) concentration for transfection.

Development of Polymerase Chain Reaction assay protocol for assessment Salmonella sp. in cow raw milk

Salmonella sp. is a pathogenic bacterium that may associated with acute diarrhoea in human. These bacteria may be transmitted in a variety of ways, including consumption of contaminated cow raw milk. Salmonella sp. is troublesome to assessment due to methodological restrictions. The aim of this study was to development the protocol of Polymerase Chain reaction (PCR) assay for Assessment Salmonella sp. in cow raw milk. This method is comprised of (1) biochemical assay and (2) bacterial DNA purification from Selenite Cystine Broth and XLD Xylose Lysine Deoxycholate culture using Wizard® Genomic DNA Purification Kit followed by PCR detection. The biochemical assay is divided into several stages, namely bacterial isolation, Gram staining and conventional biochemical tests. The PCR optimization was done with Salmonella sp. The oligonucleotide primer invasion protein (invA) gene (F: 5’-TCGTCATTCCATTACCTACC-3’; R: 5’-AAACGTTGAAAAACTGAGGA-3’) were used for targeting the diagnosis of Salmonella at the genus level. In biochemical tests, Salmonella sp. results showed posit if result of catalase, oxidase, citrate, TSIA, lactose-sucrose-mannitol fermentation, urea, and methyl-red. Conversely, negative result for Voges-Proskauer. PCR protocol consist of 30 PCR cycles with initial denaturation at 94°C for 45 seconds, denaturation at 94°C for 20 seconds, annealing at 57°C for 15 seconds, extension at 72°C for 15 seconds and finally at 72°C for 2 minutes. The conventional method detection results obtained as many as 9 positive samples Salmonella sp. and the PCR method obtained 7 positive samples Salmonella sp. In conclusion, our results indicate that the developed protocol would be utilized as a routine analysis for monitoring cow raw milk contamination and the protocol of the PCR technique provides results at a handful of time required by the biochemical assay (24 hours compared to 2–3 days).

Four Decades of the Comet Assay: pH Optimum of Lysis Buffer Still Needs to be Elucidated.

The proper course and reproducibility of diagnostic techniques depend on narrowly defined reaction conditions, including the reaction pH. Nevertheless, numerous assays are affected by an inaccurately defined reaction pH. Buffers are sometimes suggested for use outside their useful pH ranges, which complicates the reproducibility of results because the buffering capacity is insufficient to retain the disclosed pH. Here, we focus on the comet assay lysis buffer. Comet assay is broadly used for quantifying DNA breaks in eukaryotic cells. The most widespread comet assay protocols employ lysis of the cells before electrophoresis in a buffer containing Triton X-100, a high concentration of NaCl, sodium sarcosinate, EDTA, and Tris, with some modifications. However, nearly all researchers report that they use Tris buffer at pH 10, and some report the pH of the Tris additive alone. Alternatively, others report the pH of the final lysis buffer. However, the lysis solution used in the comet assay is buffered at a pH outside the useful range of Tris. Tris-based buffers have a useful pH range of 7.0 - 9.0. The buffer composed of 10 mM Tris has pKa 8.10 at 25°C and 8.69 at 4°C. The cell lysis conditions used in nearly all modifications of comet assay protocols remain imprecise and uncritically employed. Despite the pH of the lysis buffer likely has negligible effect on the detection of DNA breaks, precise lysis conditions are highly important for the use of comet assay in the detection of base modifications, which are often unstable and sensitive to pH.

Integrated High-Throughput Screening and Large-Scale Isobolographic Analysis to Accelerate the Discovery of Radiosensitizers With Greater Selectivity for Cancer Cells

High-throughput screening (HTS) platforms have been widely used to identify candidate anticancer drugs and drug-drug combinations; however, HTS-based identification of new drug-ionizing radiation (IR) combinations has rarely been reported. Herein, we developed an integrated approach including cell-based HTS and computational large-scale isobolographic analysis to accelerate the identification of radiosensitizing compounds acting strongly and more specifically on cancer cells. In a 384-well plate format, 160 compounds likely to interfere with the cell response to radiation were screened on human glioblastoma (U251-MG) and cervix carcinoma (ME-180) cell lines, as well as on normal fibroblasts (CCD-19Lu). After drug exposure, cells were irradiated or not and short-term cell survival was assessed by high-throughput cell microscopy. Computational large-scale dose-response and isobolographic approach were used to identify promising synergistic drugs radiosensitizing cancer cells rather than normal cells. Synergy of a promising compound was confirmed on ME-180 cells by an independent 96-well assay protocol, and finally, by the gold-standard colony forming assay. We retained 4 compounds synergistic at 2 isoeffects in U251-MG and ME-180 cell lines and 11 compounds synergistically effective in only one cancer cell line. Among these 15 promising radiosensitizers, 5 compounds showed limited toxicity combined or not with IR on normal fibroblasts. Overall, this study demonstrated that HTS chemoradiation screening together with large-scale computational analysis is an efficient tool to identify synergistic drug-IR combinations, with concomitant assessment of unwanted toxicity on normal fibroblasts. It sparks expectations to accelerate the discovery of highly desired agents improving the therapeutic index of radiation therapy.

Novel biogenic preparation of gold nanoparticles decorated on sepiolite clay and evaluation of anti-cancer effect on gastric cancer cell and electrochemical sensing of nitrite

An environmentally friendly strategy was used in this study to synthesize gold nanoparticles decorated on sepiolite clay (GNPs-SC) using Heracleum persicum grass extract. The physicochemical characters of the prepared composite were characterized using transmission electron microscopy (TEM), scanning electron microscopy (SEM), and X-ray diffraction (XRD). A GNPs-SC modified carbon pate electrode (CPE) was used to study the electrochemical oxidation of nitrite. The proposed nitrite sensor exhibits excellent performance, including a broad linear range (1.0–150 μM), a low limit of detection (0.4 μM), and acceptable reproducibility (RSD = 2.6%). As well, the prepared GNPs-SC was tested for its effectiveness against human gastric adenocarcinoma (AGS) cell line. The MTT assay protocol revealed that the bio-synthesized product displayed significant cytotoxic activity against gastric cancer in human subjects. The findings of this study indicate that GNPs-SC, synthesized using environmentally friendly protocol, exhibit great potential for use in electrochemical sensing and treatment of human cancer.

B-212 Application of Annexin A5Coated Beads for Collection of Extracellular Vesicles and Their Content RNA as Liquid Biopsy for Lung Cancer

Abstract Background Extracellular vesicles (EVs) carry molecules derived from cells, such as RNA. Measurement of tumor-derived extracellular RNA (exRNA) in EVs from plasma can be a promising approach for the development of blood-based cancer detection. However, the lack of an easy method for EVs separation limits its application. The aim of this study is to establish a procedure for EV collection and exRNA detection for non-small-cell lung cancer (NSCLC) diagnosis and monitoring. Methods Annexin A5-coated magnetic beads (ANX beads) were developed for EVs isolation. Next, we established a procedure for EV separation and exRNA detection from fluidic specimens using the ANX beads. Then blood samples from 22 NSCLC patients and 20 healthy controls were collected to evaluate the procedure and we detected different RNAs in the samples. Results We evaluated the ability of ANX beads in EVs separation and found that the beads could capture large and small EVs and could increase RNA concentration. Then we detected different RNAs in the samples and found that CK19 mRNA, MALAT1 lncRNA, and miR155 microRNA had good performance to differentiate NSCLC from healthy controls. Conclusion In conclusion, this assay protocol could separate EVs to enrich RNA concentration. The EVs separation employed was captured by magnetic beads which could be adopted into an automatic nucleic acid extract system to conveniently concentrate and extract nucleic acid from EVs.

Establishment and application of multiplex droplet digital polymerase chain reaction assay for bovine enterovirus, bovine coronavirus, and bovine rotavirus.

Bovine enterovirus (BEV), bovine coronavirus (BCoV), and bovine rotavirus (BRV) are still the major worldwide concerns in the health care of cattle, causing serious economic losses in the livestock industry. It is urgent to establish specific and sensitive methods to detect viruses for the early control of diseases. Droplet digital PCR (ddPCR) has been proposed to effectively detect viral particles, and it does not involve Ct values or standard curves. In this study, we designed specific primers and probes, based on conserved regions of viral genomes, to optimize protocols for a dual ddPCR assay for detecting BCoV and BRV and a multiplex ddPCR assay for BEV, BCoV, and BRV. Sensitivity assays revealed that the lower limit of detection for qPCR was 1,000 copies/μL and for ddPCR for BEV, BCoV, and BRV, 2.7 copies/μL, 1 copy/μL and 2.4 copies/μL, respectively. Studying 82 samples collected from diarrheal calves on a farm, our dual ddPCR method detected BCoV, BRV, and co-infection at rates of 18.29%, 14.63%, and 6.1%, respectively. In contrast, conventional qPCR methods detected BCoV, BRV, and co-infection at rates of 10.98%, 12.2%, and 3.66%, respectively. On the other hand, studying 68 samples from another farm, qPCR detected BCoV, BRV, BEV, and co-infection of BCoV and BEV at rates of 14.49%, 1.45%, 5.80%, and 1.45%, respectively. Our multiplex ddPCR method detected BCoV, BRV, BEV, co-infection of BCoV and BEV, and co-infection of BRV and BEV. at rates of 14.49%, 2.9%, 8.7%, 2.9%, and 1.45%, respectively. Studying 93 samples from another farm, qPCR detected BCoV, BRV, BEV, and co-infection of BCoV and BEV was detected at rates of 5.38%, 1.08%, 18.28%, and 1.08%, respectively. Co-infection of BCoV, BRV, BEV, BCoV, and BEV, and co-infection of BRV and BEV, were detected by multiplex ddPCR methods at rates of 5.38%, 2.15%, 20.45%, 1.08%, and 1.08%, respectively. These results indicated that our optimized dual and multiplex ddPCR methods were more effective than conventional qPCR assays to detect these viral infections.

Development and validation of a real-time PCR assay protocol for the specific detection and quantification of pelagiphages in seawater samples

Earth is inhabited by numerous adaptations of cellular forms shaped by the persistent scrutiny of natural selection. Thus, as natural selection has fixed beneficial adaptations of functional traits, cellular life has conquered almost all environmental niches on our planet. However, cellular life succumbs in number and genetic diversity to viruses. Among all viruses, phages are highly prevalent in diverse environments, and due to their vast genetic diversity and abundance, their relevant role as significant players in several ecological processes is now fully recognized. Pelagiphages, bacteriophages infecting bacteria of the SAR11 clade, are the most abundant viruses in the oceans. However, the ecological contribution of pelagiphages on populations of Pelagibacterales remains largely underestimated. An essential aspect of estimating the impact of bacteriophages is their absolute and precise quantification, which provides relevant information about the host-virus interactions and the structure of viral assemblages. Consequently, due to its abundance and claimed influence in the biogeochemical cycling of elements, the accurate quantification of pelagiphages results in an essential task. This study describes the development and validation of a sensitive, specific, accurate and reproducible qPCR platform targeting pelagiphages. Moreover, this method allowed the detection and quantification of pelagiphages in the Gulf of California for the first time.

Novel biosynthesis of gold nanoparticles for multifunctional applications: Electrochemical detection of hydrazine and treatment of gastric cancer

In this work, an environmentally friendly strategy was used to synthesize gold nanoparticles (Au NPs) using Olea europaea (olive) fruit. Transmission electron microscopy (TEM), UV–Vis spectroscopy, X-ray diffraction (XRD) and energy-dispersive X-ray (EDX) were used to characterize the physicochemical properties of the synthesized NPs. An Au NPs modified glassy carbon electrode was used to investigate the direct electrochemical oxidation of hydrazine. The suggested hydrazine sensor has good performance, such as a wide linear range (2.5–275 μM), low limit of detection (0.09 μM), notable selectivity and excellent reproducibility (RSD = 2.2%). The in-vitro cytotoxicity of three human cancer cell lines (KATOIII, NCI-N87, and SNU-16) was also explored with various concentrations of Au NPs prepared from olive fruit extract. Bio-synthesized Au NPs were found to have cytotoxic properties against gastric cancer in humans based on MTT assay protocol. The obtained results show that green synthesized Au NPs can be successfully employed in electrochemical sensing and cancer treatment applications.

Quantifying assays: inhibition of signalling pathways of cancer.

Inhibiting a signalling pathway concerns controlling the cellular processes of a cancer cell's viability, cell division and death. Assay protocols created to see if the molecular structures of the drugs being tested have the desired inhibition qualities often show great variability across experiments, and it is imperative to diminish the effects of such variability while inferences are drawn. In this paper, we propose the study of experimental data through the lenses of a mathematical model depicting the inhibition mechanism and the activation-inhibition dynamics. The method is exemplified through assay data obtained from an experimental study of the inhibition of the chemokine receptor 4 (CXCR4) and chemokine ligand 12 (CXCL12) signalling pathway of melanoma cells. The quantitative analysis is conducted as a two step process: (i) deriving theoretically from the model the cell viability as a function of time depending on several parameters; (ii) estimating the values of the parameters by using the experimental data. The cell viability is obtained as a function of concentration of the inhibitor and time, thus providing a comprehensive characterization of the potential therapeutic effect of the considered inhibitor, e.g. $IC_{50}$ can be computed for any time point.

Design, Synthesis, and Assay of New N-acyl-4-(4-aminoalkoxy- phenyl)- thiazole-2-amine Derivatives as Acetylcholinesterase Inhibitors

Background: Thiazoles are an important class of heterocyclic compounds with many biological effects, including anticholinesterase activity. Objective: The purpose of this work was to synthesize new thiazole derivatives and evaluate as acetylcholinesterase inhibitors (AChEIs) for Alzheimer’s disease. Methods: A series of new N-acyl-4-(4-aminoalkoxy-phenyl)-thiazole-2-amine derivatives was designed and synthesized. Ellman assay protocol was used for the AchE and BuChE inhibitory activity. To correlate better the drug-like property, the theoretical prediction was calculated using Mol inspiration software 2015 online. The potential binding mode of compounds with AChE and BuChE was investigated by the molecular docking simulation. Results: All synthesized compounds exhibited a certain inhibitory activity on AChE and 5p had the most effective selective inhibitory effect on AChE. The inhibitory form of 5p on AChE was shown to be a combination of competitive and noncompetitive inhibition, according to enzyme kinetic tests. Docking simulation studies revealed that the binding energy of 5p with AChE was lower than that of it with BuChE, which also explained the selective inhibitory activity of 5p on AChE. Conclusion: These results provided valuable information for the design of potent AChEIs, and it was believed that 5p could be a promising lead structure for its further development for the treatment of AD.

A spectroscopic response factor-based toluidine blue assay towards a universal assay protocol for sulfated polysaccharides: Application to fucoidan content in crude extract

Sulfated polysaccharides (SPS) have attracted a lot of interest because of their diverse pharmacological functions. Numerous scientific studies have shown that SPS exhibit better biological activity than those that are not sulfated, such as immunomodulatory, anti-viral, and antioxidant activities. A crucial step to a better understanding of the mechanism of action and health effects is the production of high purity SPS. This calls for the development of selective assay techniques that can identify SPS preferentially without being influenced by other substances or the co-extracted polysaccharides. A universal modified toluidine blue (TB) assay was developed in this study to detect SPS. The assay procedures were conducted using different SPS standards including fucoidans from different biogenic sources, in addition to heparin and dextran sulfate. Spectroscopic response factor was calculated for each SPS which showed very good correlation (R2 = 0.998) with the corresponding sulfation degree. The proposed method was applied for determination of SPS content of crude fucoidan product using five different SPS standards. The method was cross validated by conducting ANOVA test to the obtained % recovery revealing that there is no significant difference between the results obtained by identical reference standard and four nonidentical natural SPS standards. This is the first report of a selective universal assay of SPS that enables the selective determination of SPS using a nonidentical reference standard.

Green synthesis of silver nanoparticles using Ocimum sanctum Linn. and its antibacterial activity against multidrug resistant Acinetobacter baumannii.

The biosynthesis of nanoparticles using the green route is an effective strategy in nanotechnology that provides a cost-effective and environmentally friendly alternative to physical and chemical methods. This study aims to prepare an aqueous extract of Ocimum sanctum (O. sanctum)-based silver nanoparticles (AgNPs) through the green route and test their antibacterial activity. The biosynthesized silver nanoparticles were characterised by colour change, UV spectrometric analysis, FTIR, and particle shape and size morphology by SEM and TEM images. The nanoparticles are almost spherical to oval or rod-shaped with smooth surfaces and have a mean particle size in the range of 55 nm with a zeta potential of -2.7 mV. The antibacterial activities of AgNPs evaluated against clinically isolated multidrug-resistant Acinetobacter baumannii (A. baumannii) showed that the AgNPs from O. sanctum are effective in inhibiting A. baumannii growth with a zone of inhibition of 15 mm in the agar well diffusion method and MIC and MBC of 32 µg/mL and 64 µg/mL, respectively. The SEM images of A. baumannii treated with AgNPs revealed damage and rupture in bacterial cells. The time-killing assay by spectrophotometry revealed the time- and dose-dependent killing action of AgNPs against A. baumannii, and the assay at various concentrations and time intervals indicated a statistically significant result in comparison with the positive control colistin at 2 µg/mL (P<0.05). The cytotoxicity test using the MTT assay protocol showed that prepared nanoparticles of O. sanctum are less toxic against human cell A549. This study opens up a ray of hope to explore the further research in this area and to improve the antimicrobial activities against multidrug resistant bacteria.

Freeze-drying of few microliters of antibody formulations to implement 384-wells homogeneous instant assays

Enzyme-linked immunosorbent assay protocols have traditionally complex workflows with several intensive wash steps. Analytical tools with both shorter time-to-result and hands-on-time using smaller sample and assays reagents volumes are now investigated. In this context, fluorescence resonance energy transfer (FRET)-based assays are emerging as one of the most promising analytical tools in high-throughput screening (HTS). These immunoassays allow fast quantification of antigens at the nano-gram level in a final assay volume of only a few μL. We used a homogeneous time-resolved FRET (called HTRF) assay to develop a freeze-dried screening and ready-to-use format with only one rehydration step called “instant assay”.To assure optimal performance of the developed homogeneous instant assay, we investigated the critical quality attributes by studying the functionality and stability of the critical reagents and fluorophores. The cyclic adenosine 3′-5′-monophosphate (cAMP) was selected as the antigen target. We tested various formulations (with different buffers, sugars, bulking reagents, surfactants and co-solvants) combined with a slow freezing and the use of an aluminium plate holder during the freeze-drying of few microliter of bioreagents.The optimized freeze-drying procedure permits to preserve more than 70% of Ab recognition properties. The developed off-the-shelf homogeneous FRET immunoassay allows direct and fast quantification of cAMP at a nanogram level.

P-063 Microfluidic technology is highly effective in selecting a sperm population with high progressive motility and low DNA fragmentation index

Abstract Study question Which sperm preparation procedure is able to select the best population of sperm? Summary answer Microfluidic sperm sorting using Fertile Plus device isolates sperm with the highest motility and lowest levels of DNA fragmentation. What is known already The sperm selection in Medically Assisted Reproduction should lead to fully functional spermatozoa able to reach the fertilization site and allow optimal fertilization and embryo development. These abilities are dependent on specific sperm parameters such as, but not limited to, motility, morphology, Acrosome Index (AI), and DNA fragmentation Index (DFI). At present, the routine sperm selection is suboptimal, does not replicates the physiological processes observed in nature and consequently may induce sperm damage. The new Microfluidic Sperm Sorting technology allows a physiological selection of sperm. However, no comparison with other selection procedures regarding all the above-mentioned parameters was reported. Study design, size, duration Prospective-observational study performed at an University Hospital-IVF Centre between April-November 2022 comparing the effect of four different sperm selection procedures on various semen parameters. Fifty-two samples with minimum 20x106 sperm/ml and progressive motility ≥30% were included. Descriptive statistics reported the mean±SD [minimum-maximum] values for each variable. Interclass correlation coefficient (ICC) was calculated for the absolute agreement in between the four different procedures, where DGC was considered the standard-of-care. An ICC-value &amp;lt;0.4 indicates a low agreement. Participants/materials, setting, methods Unused raw semen after routine diagnostic analysis were individually split in 4 fractions and processed immediately by different sperm preparation methods: 1) sperm wash (SW), 2) Density Gradient Centrifugation (DGC), 3) Magnetic Activated Cell Sorting (MACS, (Miltenyi Biotec)), and 4) Microfluidic Sperm Sorting (MSS) using Fertile Plus Device (Koek Biotechnology). Each fraction was analyzed for progressive motility, morphology, Acrosome Index (AI) and DNA fragmentation Index (DFI; TUNEL assay protocol). Main results and the role of chance The mean age of the patients was 35.9 years (SD ± 6.8). The mean value of days of abstinence was 4.1 (SD ± 6.8). Mean progressive motility rates were: 54.3±10.6% [23-86%], 74.3±11.8% [38-90%], 77.2±12.5% [37-92%] and 88.6±4.2% [73-96%] for SW, DGC, MACS, and MSS, respectively. Mean percentages of normal morphology were: 3.3±2.9% [0-13%], 4.1±3.1% [0-13%], 4.2±3.7% [0-18%], and 5.1±3.9% [0-16%] for SW, DGC, MACS, and MSS, respectively. Mean AI were: 8.5±4.9% [1-20%], 9.7±6.0% [1-30%], 8.7±4.9% [0-19%], and 10.8±6.8% [1-30%] for SW, DGC, MACS, and MSS, respectively. Mean DFI were: 6.2±4.6% [0.8-26.1%], 2.7±3.2% [ 0.2-14%], 2.1±4.3% [0.9-20.8%], and 0.2±0.4% [0.0-2.3%] for SW, DGC, MACS, and MSS, respectively. Values referring to ICC absolute agreement between the DGC and the other methods indicate MSS as the only procedure in strong disagreement regarding progressive motility [0.29, 95%CI (-0.2-0.58)] and DFI [0.17; 95%CI (-0.19-0.45)]. No disagreement regarding morphology and AI was observed between all techniques. A further paired comparison confirmed the strong disagreement between MSS and MACS [0.2; 95%CI (-0.17-0.49) and 0.21; 95%CI (-0.25-0.52)] and between MSS and SW [0.06; 95%CI (-0.04-0.25) and 0.06; 95%CI (-0.15-0.28)] regarding progressive motility and DFI, respectively. Limitations, reasons for caution Differences in DNA-damage may have been smaller between techniques if abstinence period was shorter. Due to the design of the experiment, data on clinical outcomes are not available. Wider implications of the findings The selection of a population of highly motile spermatozoa with undamaged DNA from unprocessed semen is ideally performed with MSS using Fertile Plus. Question remains how these results compare with a shorter abstinence period and whether this improves the embryological outcomes in the IVF laboratory. Trial registration number NCT05383599

Design, synthesis, antiviral evaluation, and In silico studies of acrylamides targeting nsP2 from Chikungunya virus

The Togaviridae family comprises several New- and Old-World Alphaviruses that have been responsible for thousands of human illnesses, including the RNA arbovirus Chikungunya virus (CHIKV). Firstly, it was reported in Tanzania in 1952 but rapidly it spread to several countries from Europe, Asia, and the Americas. Since then, CHIKV has been circulating in diverse countries around the world, leading to increased morbidity rates. Currently, there are no FDA-approved drugs or licensed vaccines to specifically treat CHIKV infections. Thus, there is a lack of alternatives to fight against this viral disease, making it an unmet need. Structurally, CHIKV is composed of five structural proteins (E3, E2, E1, C, and 6k) and four non-structural proteins (nsP1-4), in which nsP2 represents an attractive antiviral target for designing novel inhibitors since it has an essential role in the virus replication and transcription. Herein, we used a rational drug design strategy to select some acrylamide derivatives to be synthesized and evaluated against CHIKV nsP2 and also screened on CHIKV-infected cells. Thus, two regions of modifications were considered for these types of inhibitors, based on a previous study of our group, generating 1560 possible inhibitors. Then, the 24 most promising ones were synthesized and screened by using a FRET-based enzymatic assay protocol targeting CHIKV nsP2, identifying LQM330, 333, 336, and 338 as the most potent inhibitors, with Ki values of 48.6 ± 2.8, 92.3 ± 1.4, 2.3 ± 1.5, and 181.8 ± 2.5 μM, respectively. Still, their Km and Vmax kinetic parameters were also determined, along with their competitive binding modes of CHIKV nsP2 inhibition. Then, ITC analyses revealed KD values of 127, 159, 198, and 218 μM for LQM330, 333, 336, and 338, respectively. Also, their ΔH, ΔS, and ΔG physicochemical parameters were determined. MD simulations demonstrated that these inhibitors present a stable binding mode with nsP2, interacting with important residues of this protease, according to docking analyzes. Moreover, MM/PBSA calculations displayed that van der Waals interactions are mainly responsible for stabilizing the inhibitor-nsP2 complex, and their binding energies corroborated with their Ki values, having −198.7 ± 15.68, −124.8 ± 17.27, −247.4 ± 23.78, and −100.6 ± 19.21 kcal/mol for LQM330, 333, 336, and 338, respectively. Since Sindbis (SINV) nsP2 is similar to CHIKV nsP2, these best inhibitors were screened against SINV-infected cells, and it was verified that LQM330 presented the best result, with an EC50 value of 0.95 ± 0.09 μM. Even at 50 μM concentration, LQM338 was found to be cytotoxic on Vero cells after 48 h. Then, LQM330, 333, and 336 were evaluated against CHIKV-infected cells in antiviral assays, in which LQM330 was found to be the most promising antiviral candidate in this study, exhibiting an EC50 value of 5.2 ± 0.52 μM and SI of 31.78. The intracellular flow cytometry demonstrated that LQM330 is able to reduce the CHIKV cytopathogenic effect on cells, and also reduce the percentage of CHIKV-positive cells from 66.1% ± 7.05 to 35.8% ± 5.78 at 50 μM concentration. Finally, qPCR studies demonstrated that LQM330 was capable of reducing the number of viral RNA copies/μL, suggesting that CHIKV nsP2 is targeted by this inhibitor as its mechanism of action.

Detection and Quantification of Calcitonin Gene-Related Peptide (CGRP) in Human Plasma Using a Modified Enzyme-Linked Immunosorbent Assay

Calcitonin gene-related peptide (CGRP) is a vasoactive neuropeptide that plays a putative role in the pathophysiology of migraine headaches and may be a candidate for biomarker status. CGRP is released from neuronal fibers upon activation and induces sterile neurogenic inflammation and arterial vasodilation in the vasculature that receives trigeminal efferent innervation. The presence of CGRP in the peripheral vasculature has spurred investigations to detect and quantify this neuropeptide in human plasma using proteomic assays, such as the enzyme-linked immunosorbent assay (ELISA). However, its half-life of 6.9 min and the variability in technical details of assay protocols, which are often not fully described, have yielded inconsistent CGRP ELISA data in the literature. Here, a modified ELISA protocol for the purification and quantification of CGRP in human plasma is presented. The procedural steps involve sample collection and preparation, extraction using a polar sorbent as a means of purification, additional steps to block non-specific binding, and quantification via ELISA. Further, the protocol has been validated with spike and recovery and linearity of dilution experiments. This validated protocol can theoretically be used to quantify CGRP concentrations in the plasma of individuals not only with migraine, but also with other diseases in which CGRP may play a role.

Use of automated capillary immunoassays for quantification of antibodies in chicken sera against recombinant Salmonella enterica serotype Heidelberg proteins

The classic immunoblot technique is an important tool for identification and characterization of target proteins. However, a standard protocol for this classic immunoblot assay involves many steps that may cause experimental variations in each step and make quantification of antibodies in sera difficult. A capillary electrophoresis-based immunoblot system was developed to reduce potential problems in variations during the experimental process, enable protein identification in an automatic manner and quantitate various isotypes of antibodies in sera. In the present study, we used this system to examine the purity of the recombinant proteins and measure amounts of various isotypes of immunoglobins in chicken sera after immunization with two recombinant Salmonella FliD and FimA proteins. A single band of each protein was detected in the gel like images by this system after purification by nickel-chelated affinity chromatography. A good linear range of the protein concentrations was also obtained for each recombinant protein. This automated capillary immunoblot system was successfully used for detection and quantification of various immunoglobin isotypes against two recombinant Salmonella proteins from the immunized chicken sera, but not the un-immunized chicken sera. The chicken immunoglobin G (IgG) antibody response to the FliD protein from the immunized group was 1110- and 51,400-fold higher than that from the un-immunized chickens both two- and three-weeks post-vaccination, respectively. It was also observed that IgM antibody against the FliD protein from the immunized chickens was 1030-fold higher than that from the un-immunized chickens two weeks post-vaccination, but the IgM response declined to 120-fold between two groups from two weeks to three weeks after immunization. The IgM antibody response to the FimA protein from the immunized group was 1.84- and 1.12-fold higher than that from the un-immunized group, respectively, both two- and three-weeks post-vaccination, while the IgG antibody response from the immunized group was 8.07- and 27.6-fold higher than that from the un-immunized group, respectively, during the same period. These results suggest that this capillary-based immunoblot assay can be an alternative method for analyses and quantitation of chicken humoral immune response before and after immunization with any antigens and/or for investigation in Salmonella outbreaks.

Antiproliferative Activity of Novel Imatinib Analogue as Potential Anticancer Agents, Synthesis and in vitro Screening

Abstract: Background: A series of N-(2,5-dimethylphenyl)-4-pyridin-3-ylpyrimidin-2-amine derivatives were synthesized as Imatinib derivatives, bearing 2-chloroquinoline as a heteroaryl motif. Materials and Methods: The compounds were synthesized by reducing in situ prepared azomethine intermediate using NaBH4 as a reducing agent in methanol as a solvent. Fourier transformation-IR, proton-NMR along with mass spectrometry were used to determine the structures of the compounds. The antiproliferative activity of the compounds against cell lines A549 and MCF7 was evaluated in vitro using the MTT assay protocol. Results: The compounds showed moderate cell growth inhibition at a concentration of 10 μM. Among the test compounds, the compound with a dimethoxy group at the 6 and 8 positions of the 2-chloroquinoline ring displayed the highest antiproliferative activity. Conclusion: The synthesized Imatinib derivatives exhibited moderate antiproliferative activity against A549 and MCF7 cell lines, and the compound with a dimethoxy group at the 6 and 8 positions of the 2-chloroquinoline ring showed the highest activity. These findings suggest that further studies can be performed to optimize the antiproliferative activity of these compounds. Keywords: Imatinib derivatives, Quinoline, Reducing amination, Antiproliferative activity.