Towards CRISPR powered electrochemical sensing for smart diagnostics

Even though global health has been steadily improved, the global disease burden associated with communicable and non-communicable diseases extensively increased healthcare expenditure. The present COVID-19 pandemic scenario has again ascertained the importance of clinical diagnostics as a basis to make life-saving decisions. In this context, there is a need for developing next-generation integrated smart real-time responsive biosensors with high selectivity and sensitivity. The emergence of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas biosensing systems has shown remarkable potential for developing next-generation biosensors. CRISPR/Cas integrated electrochemical biosensors (E-CRISPR) stands out with excellent properties. In this opinionated review, we illustrate the rapidly evolving applications for E-CRISPR-integrated detection systems towards biosensing and the future scope associated with E-CRISPR based diagnostics.     

Estimation of Treatment Time for Microbial Preprocessing of Biomass

Biochemical conversion of lignocellulosic biomass to ethanol involves size reduction, preprocessing, pretreatment, enzyme hydrolysis, and fermentation. In recent years, microbial preprocessing has been gaining attention as a means to produce labile biomass for lessening the requirement of pretreatment severity. However, loss of sugars due to microbial consumption is a major consequence, suggesting its minimization through optimization of nutrients, temperature, and preprocessing time. In this work, we emphasized estimation of fungal preprocessing time, at which higher sugar yields can be achieved after preprocessing and enzyme hydrolysis. The estimation is based on the enzymatic activity profile obtained by treating switchgrass with Phanerochaete chrysosporium for 28 days. Enzyme assays were conducted once in every 7 days for 28 days, for activities of phenol oxidase, peroxidase, β-glucosidase, β-xylosidase, and cellobiohydrolase. We found no activity for phenol oxidase and peroxidase, but the greatest activities for cellulases on the seventh day. We then treated switchgrass for 7 days with P. chrysosporium and observed that the preprocessed switchgrass had higher glucan (39%), xylan (17.5%), and total sugar yields (25.5%) than the unpreprocessed switchgrass (34%, 37.5%, and 20.5%, respectively, p < 0.05). This verifies the utility of using enzyme assays for initial estimation of preprocessing time to enhance sugar yields.     

A rapid and highly efficient Michael addition of methoxybenzenes and indoles to α,β-unsaturated ketones using BF3·OEt2 as a catalyst

An efficient and general protocol is described for the Michael addition of α,β-unsaturated ketones with electron-rich arenes/indoles to give alkylated arenes/indoles under mild reaction condition at room temperature. Shorter reaction time, convenient and good isolated yields are the significant features of this protocol. Moreover, the procedure is environmentally benign in nature and applicable to variety of arenes/indoles as well as α,β-unsaturated ketones.     

Determination of GABA, glutamate and carbamathione in brain microdialysis samples by capillary electrophoresis with fluorescence detection

Disulfiram has been used as a deterrent in the treatment of alcohol abuse for almost 60 years. Our laboratory has shown that a disulfiram metabolite, S‐(N,N‐diethylcarbamoyl) glutathione (carbamathione), is formed from disulfiram and appears in the brain after the administration of disulfiram. Carbamathione does not inhibit aldehyde dehydrogenase but has been shown to be a partial non‐competitive inhibitor of the N‐methyl‐D ‐aspartic acid glutamate (Glu) receptor. In light of disulfiram's apparent clinical effectiveness in cocaine dependence, and carbamathione's effect on the N‐methyl‐D ‐aspartic acid receptor, the effect of carbamathione on brain Glu and γ‐aminobutyric acid (GABA) needs to be further examined. A CE‐LIF method based on derivatization with napthalene‐2,3‐dicarboxyaldehyde to simultaneously detect both neurotransmitter amino acids and carbamathione in brain microdialysis samples is described. The separation of Glu, GABA and carbamathione was carried out using a 50 mmol/L boric acid buffer (pH 9.6) on a 75 cm×50 μm id fused‐silica capillary (60 cm effective) at +27.5 kV voltage with a run time of 11 min. The detection limits for Glu, GABA and carbamathione were 6, 10 and 15 nmol/L, respectively. This method was used to monitor carbamathione and the amino acid neurotransmitters in brain microdialysis samples from the nucleus accumbens after the administration of an intravenous dose of the drug (200 mg/kg) and revealed a carbamathione‐induced change in GABA and Glu levels. This method demonstrates a simple, rapid and accurate measurement of two amino acid neurotransmitters and carbamathione for in vivo monitoring in the brain using microdialysis sampling.     

Calcium Alginate Bead-mediated Enhancement of the Selective Recovery of a Lead Novel Antifungal Bacillomycin Variant

In the pursuit of new antifungal compounds, five coproduced lipopeptide variants (AF₁ to AF₅) from wild-type Bacillus subtilis RLID 12.1 were identified in our previous study. Out of five, AF₄ was identified as a novel lead molecule belonging to the bacillomycin family showing less cytotoxicity at its respective minimum inhibitory concentrations (MIC) evaluated against 81 strains of Candida and Cryptococcus species (including clinical isolates); besides this, AF₄ purified in the present study exhibited encouraging MIC values against 10 clinical mycelial fungi. Aiming for a selective production augmentation of AF₄ lipopeptide variant, a new fermentation media comprising malt extract (1.01%), dextrose (0.55%), peptone (1.79%), MnSO₄ (2 mM), and NaCl (0.5%) was formulated. Maximum production of 954.8?±?10.8 mg/L was achieved with 44% selectivity at 30 °C compared to unoptimized conditions (186.4?±?6.1 mg/L). Use of calcium alginate beads in the formulated media during the onset of lipopeptide production resulted in an augmentation in the selectivity of the most efficacious AF₄ variant to about 72% presumably due to attenuation of other coproduced lipopeptide variants AF₁ and AF₂. Difference in yield of lipopeptides varied with bead size, bead preparation ratios, and sodium alginate concentrations. Use of Ca-alginate beads in the upstream production process of the lead AF₄ variant may be considered as a novel strategy to address the potential challenge that may arise during the scale-up and downstream processing steps. Another significant finding derived from the study is that the proportion of bacillomycin variants of B. subtilis RLID 12.1 could be controlled by temperature and metal ions under static and shaking conditions.     

LC–MS/MS and NMR characterization of forced degradation products of mirabegron

A rapid, precise, and reliable liquid chromatography tandem mass spectrometry (LC–MS/MS) method has been developed for the characterization of stressed degradation products of mirabegron. It is used in the treatment of overactive bladder and administered to treat urinary symptoms such as urgency or frequency and incontinence. It also works by relaxing the muscles around bladder.

Mirabegron was subjected to hydrolysis (acidic, alkaline, and neutral) and peroxidation, as per ICH-specified conditions. The drug showed degradation under stress conditions. However, it was stable to neutral conditions. A total of seven degradation products were observed and the chromatographic separation of the drug and its degradation products was achieved on X-TerraRP-8 (250 mm × 4.6 mm, i.d., 5 µm) column using 0.01 M ammonium acetate as mobile phase-A and 60:40 ratio of acetonitrile (ACN):water as mobile phase-B. The degradation products were characterized by LC–MS/MS and its fragmentation pathways were proposed. Probable possible structures were drawn based on parent and daughter molecular ions. One peroxide degradant impurity was isolated using preparative LC and characterized using liquid chromatography–mass spectrometry and NMR data.     

Combined ligand-based and structure-based design of PDE 9A inhibitors against Alzheimer’s disease

Molecular Diversity - PDE9 enzyme hydrolyzes cGMP, which is involved in the regulation of synaptic plasticity through the NMDA pathway (a well-known excitotoxic target for AD) via activation of...     

Transition Metal-Free Regio- and Stereo-Selective trans Hydroboration of 1,3-Diynes: A Phosphine-Catalyzed Access to (E)-1-Boryl-1,3-Enynes

We report a transition metal-free, regio- and stereo-selective, phosphine-catalyzed method for the trans hydroboration of 1,3-diynes with pinacolborane that affords (E)-1-boryl-1,3-enynes. The reaction proceeds with excellent selectivity for boron addition to the external carbon of the 1,3-diyne framework as unambiguously established by NMR and X-ray crystallographic studies. The reaction displays a broad substrate scope including unsymmetrical diynes to generate products in high yield (up to 95 %). Experimental and theoretical studies suggest that phosphine attack on the alkyne is a key process in the catalytic cycle.     

Identification of Crucial Intermediates in the Formation of Humins from Cellulose-Derived Platform Chemicals Under Brønsted Acid Catalyzed Reaction Conditions

Humins are one of the undesirable products formed during the dehydration of sugars as well as the conversion of 5-hydroxymethylfurfural (HMF) to value-added products. Thus, reducing the formation of humins is an important strategy for improving the yield of the aforementioned reactions. Even after a plethora of studies, the mechanism of formation and the structure of humins are still elusive. In this regard, we have employed density functional theory-based mechanistic studies and microkinetic analysis to identify crucial intermediates formed from glucose, fructose, and HMF that can initiate the polymerization reactions resulting in humins under Brønsted acid-catalyzed reaction conditions. This study brings light into crucial elementary reaction steps that can be targeted for controlling humins formation. Moreover, this work provides a rationale for the experimentally observed aliphatic chains and HMF condensation products in the humins structure. Different possible polymerization routes that could contribute to the structure of humins are also suggested based on the results. Importantly, the findings of this work indicate that increasing the rate of isomerization of glucose to fructose and reducing the rate of reaction between HMF molecules could be an efficient strategy for reducing humins formation.