Microvascular Experimentation in the Chick Chorioallantoic Membrane as a Model for Screening Angiogenic Agents including from Gene-Modified Cells

The chick chorioallantoic membrane (CAM) assay model of angiogenesis has been highlighted as a relatively quick, low cost and effective model for the study of pro-angiogenic and anti-angiogenic factors. The chick CAM is a highly vascularised extraembryonic membrane which functions for gas exchange, nutrient exchange and waste removal for the growing chick embryo. It is beneficial as it can function as a treatment screening tool, which bridges the gap between cell based in vitro studies and in vivo animal experimentation. In this review, we explore the benefits and drawbacks of the CAM assay to study microcirculation, by the investigation of each distinct stage of the CAM assay procedure, including cultivation techniques, treatment applications and methods of determining an angiogenic response using this assay. We detail the angiogenic effect of treatments, including drugs, metabolites, genes and cells used in conjunction with the CAM assay, while also highlighting the testing of genetically modified cells. We also present a detailed exploration of the advantages and limitations of different CAM analysis techniques, including visual assessment, histological and molecular analysis along with vascular casting methods and live blood flow observations.

EFFECTS OF DIFFERENT PROCESSING METHODS ON THE RESISTANT STARCH CONTENT OF SOME LEGUMES

This work describes the effects of different processing methods on resistant starch (RS) contents of Canavalia ensiformis, Detarium microcarpum, Jatropha curcas and Glycine max. meals. The legume seeds were subjected to different processing methods (Raw, soaked, Boiled, Toasted and Fermented). Resistant Starch was determined by Megazyme Resistant Starch Assay procedure (A.O.A.C, 2002). In the results, the highest resistant starch contents were recorded in the toasted method for the three legume meals (Canavalia ensiformis 11.69 %, Detarium microcarpum 10.49 %, Jatropha curcas 13.06 %, while in Glycine max. 12.0 % was recorded in the boiled method). The lowest resistant starch contents were recorded in the raw processing method for the three legume meals (Canavalia ensiformis 8.47 %, Detarium microcarpum 7.25 %, Jatropha curcas 9.13 %, while in Glycine max. 7.51 % was recorded in the soaked method). The results of this research have proven the type 3 (RS3) resistant starch, which is retrograded starch made by cooking/cooling processes on starchy materials. Data were analyzed using one-way ANOVA and significant differences (p<0.05) were recorded among the different processing methods

Efficiency Improvements and Discovery of New Substrates for a SARS-CoV-2 Main Protease FRET Assay

The COVID-19 pandemic, caused by the SARS-CoV-2 virus, has a huge impact on the world. Although several vaccines have recently reached the market, the development of specific antiviral drugs against SARS-CoV-2 is an important additional strategy in fighting the pandemic. One of the most promising pharmacological targets is the viral main protease (Mpro). Here, we present an optimized biochemical assay procedure for SARS-CoV-2 Mpro. We have comprehensively investigated the influence of different buffer components and conditions on the assay performance and characterized Förster resonance energy transfer (FRET) substrates with a preference for 2-Abz/Tyr(3-NO2) FRET pairs. The substrates 2-AbzSAVLQSGTyr(3-NO2)R-OH, a truncated version of the established DABCYL/EDANS FRET substrate, and 2-AbzVVTLQSGTyr(3-NO2)R-OH are promising candidates for screening and inhibitor characterization. In the latter substrate, the incorporation of Val at position P5 improved the catalytic efficiency. Based on the obtained results, we present here a reproducible, reliable assay protocol using highly affordable buffer components.

An efficient and label-free LC-MS/MS method for assessing drug’s activity at dopamine and serotonin transporters using transporter-transfected HEK293T cells

Background: Dopamine transporter (DAT) and serotonin transporter (SERT) are targets for many psychoactive substances. Functional assays including uptake inhibition and release assays often involve radiolabeled compounds like [3H]-dopamine and [3H]-serotonin to assess drug activity at transporters, which have high requirements on handling radioactive samples. Aims: The aim of this study was to establish a label-free method to assess drug activity at DAT and SERT. Methods: A liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) method was established using transporter-transfected human embryonic kidney 293T (HEK293T) cells. This method was evaluated by testing the effects of amphetamine and cocaine in the assay procedure. Results: The limits of detection of this method were 0.2 nM for both dopamine (DA) and serotonin (5-HT), with good linearities in the range of 0.5–160 nM. Amphetamine and cocaine’s IC50 and EC50 on DAT and SERT determined by this method were consistent with previous reports. Conclusions: A rapid, reliable and label-free LC-MS/MS method for assessing drug activity was established, which affords an attractive alternative for those laboratories that do not have a radiation license or capabilities.

Efficiency Improvements and Discovery of New Substrates for a SARS-CoV-2 Main Protease FRET Assay

ABSTRACTThe COVID-19 pandemic, caused by the SARS-CoV-2 virus, has a huge impact on the world. Although several vaccines have recently reached the market, the development of specific antiviral drugs against SARS-CoV-2 is an important additional strategy in fighting the pandemic. One of the most promising pharmacological targets is the viral main protease (Mpro). Here, we present an optimized biochemical assay procedure for SARS-CoV-2 Mpro. We have comprehensively investigated the influence of different buffer components and conditions on the assay performance, and characterized six FRET substrates with a 2-Abz/Tyr(3-NO2) FRET pair. The substrates 2-AbzSAVLQSGTyr(3-NO2)R-OH, a truncated version of the established DABCYL/EDANS FRET substrate, and a new substrate 2-AbzVVTLQSGTyr(3-NO2)R-OH are promising candidates for screening and inhibitor characterization. In the latter substrate, the incorporation of Val at the position P5 improved the catalytic efficacy. Based on the obtained results, we present here a reproducible, reliable assay protocol using highly affordable buffer components.

Third generation radioimmunoassay (RIA) for TSH receptor autoantibodies (TRAb) – one step less, similar results?

Aim TSH-receptor (TSHR)-autoantibody (TRAb) is the serological hallmark of Graves’ disease (GD). Recently, 3rd-generation radioimmunoassays (RIA) employing monoclonal TRAb such as M22 or T7 instead of TSH for the inhibition of human TRAb binding with solid-phase TSHR (coated tubes) have been introduced into laboratory routine. Methods As current assays typically employ a consecutive incubation of patient serum and labelled monoclonal TRAb, automation of TRAb RIA is a challenge. Thus, the assay procedure using human TSHR-coated tubes and the mouse monoclonal TRAb T7 was modified by combining both steps. The novel one-step method was compared with its corresponding consecutive 3rd-generation RIA by investigating 304 individuals encompassing 102 patients with active GD (GDa), 43 patients with GD after successful therapy (GDt), 31 with Hashimoto’s disease (HD), 28 with non-autoimmune thyroid diseases (NAITD) and 100 healthy subjects (HS). Results With the new method, the incubation time was shortened by approximately one hour. Both 3rd-generation RIAs did not reveal a significantly different assay performance by comparing areas under the curve (AUC) with receiver operating characteristics curve analysis (AUC one-step: 0.94, AUC two-step: 0.96, p > 0.05, respectively). The two-step TRAb RIA demonstrated sensitivity and specificity values of 87.5 % and 96.2 %, respectively, whereas the one-step revealed 84.6 % and 96.2 %, respectively. Conclusion One-step 3rd-generation RIA may be used for the reliable detection of TRAb. The shorter and easier assay design may improve its use and enable automation in routine nuclear medicine laboratories.

A new grade-capping approach based on coarse duplicate data correlation

SYNOPSIS In most exploration or mining grade data-sets, the presence of outliers or extreme values represents a significant challenge to mineral resource estimators. The most common practice is to cap the extreme values at a predefined level. A new capping approach is presented that uses QA/QC coarse duplicate data correlation to predict the real data coefficient of variation (i.e., error-free CV). The cap grade is determined such that the capped data has a CV equal to the predicted CV. The robustness of the approach with regard to original core assay length decisions, departure from lognormality, and capping before or after compositing is assessed using simulated data-sets. Real case studies of gold and nickel deposits are used to compare the proposed approach to the methods most widely used in industry. The new approach is simple and objective. It provides a cap grade that is determined automatically, based on predicted CV, and takes into account the quality of the assay procedure as determined by coarse duplicates correlation. Keywords: geostatistics, outliers, capping, duplicates, QA/QC, lognormal distribution.

Long-Chain Base (LCB)-Targeted Lipidomics Study Uncovering the Presence of a Variety of LCBs in Mammalian Blood

Globotriaosylsphingosine (LysoGb3) is a biomarker for Fabry disease (OMIM 301500) that contains long-chain bases (LCBs) as a building block. There have been several studies proposing that LysoGb3 forms with distinct LCBs could be putative disease subtype-related biomarkers for this congenital disorder; however, there have been no detailed multiple reaction monitoring-based studies examining the LCB distribution in this lysosphingolipid. To achieve this, we established an assay procedure that aimed at elucidating the LCB-targeted lipidome using liquid chromatography–tandem mass spectrometry. Consistent with previous studies, we found d18:1 to be the major LCB species of the LysoGb3 in pooled human plasma, while some atypical LCBs, such as d18:2, d18:0, t18:1, d16:1, and d17:1, were detected as minor fractions. When the same methodology was applied to fetal bovine serum (FBS) as a positive control, we identified additional unique LCB species, such as t18:0, d20:1, t19:1, and t21:1, in herbivore LysoGb3. Furthermore, we found an elevation of sphingosine and LysoGb3, which are N-deacylated forms of ceramide and Gb3, respectively, in FBS, suggesting that ceramidase activity may be involved in this process. Thus, our LCB-targeted lipidomics data revealed that mammalian LCBs in glycosphingolipids have a greater variety of molecular species than previously expected.

Synthesis and Study of Some 17a-aza-D-homo Steroids as 5α-Reductase Inhibitors

Background and Objective: Tremendous advances have been made in the development of new pharmacotherapuetic agents and less invasive techniques to help men with lower urinary tract symptoms. The use of 5α-reductase inhibitor (5-ARI) is restricted to the patients with large prostate volumes, whose symptoms are refractory to antiandrogens or α– adrenergic blockers Out of the various synthesized5-reductase inhibitors with different substituents on steroidal nucleus, esters have been found to exhibited high anti-androgenic activity. Methods: In our attempt to find new, safer and potent 5-ARI and our continued interest in azasteorids, esters of 17a-Aza-Dhomo-5-androsten-3β-ol with synergistic effect were synthesized and characterized using different analytical techniques. The compounds were evaluated for their 5α-reductase inhibitory activity in-vivo by their effect on serum androgen level by ELISA assay procedure. The interaction with receptors was studied using advanced docking programme to predict the correlation of the synthesized compounds with actual biological activity. Results: The target compounds (6-12) showed increased anti-androgenic activity as compared to finasteride and control, which imply that the target compounds are effective in inhibiting 5α-reductase. Particularly, compound 6 showing highest inhibitory activity and greater affinity for 5-AR receptor with highest dock score. Results of these studies when compared with Finasteride showed increased solubility and dissolution of target compound 6. Conclusion: Compound6 showed immense potential with improved efficacy and better bioavailability, thus makes it a suitable candidate for further studies and optimal formulation.