Dimerization of an aptamer generated from Ligand-guided selection (LIGS) yields a high affinity scaffold against B-cells

Nucleic Acid Aptamers (NAAs) are a class of synthetic DNA or RNA molecules that bind specifically to their target. We recently introduced an aptamer termed R1.2 against membrane Immunoglobulin M (mIgM) expressing B-cell neoplasms using Ligand Guided Selection (LIGS). While LIGS-generated aptamers are highly specific, their lower affinity prevents aptamers from being used for translational applications. Highly specific aptamers with higher affinity can increase targetability, boosting the application of aptamers as diagnostic and therapeutic molecules. Herein, we report that dimerization of R1.2, an aptamer generated from LIGS, leads to high affinity variants without compromising the specificity. Three dimeric aptamer analogues with variable linker lengths were designed to evaluate the effect of linker length in affinity. The optimized dimeric R1.2 against cultured B-cell neoplasms, four donor B-cell samples and mIgM-positive Waldenström's Macroglobulinemia (WM) showed specificity. Furthermore, confocal imaging of dimeric aptamer and anti-IgM antibody in purified B-cells suggests co-localization. Binding assays against IgM knockout Burkitt's Lymphoma cells utilizing CRISPR/Cas9 further validated specificity of dimeric R1.2. Collectively, our findings show that LIGS-generated aptamers can be re-engineered into dimeric aptamers with high specificity and affinity, demonstrating wide-range of applicability of LIGS in developing clinically practical diagnostic and therapeutic aptamers.     

Effects of the Dopamine D2 Allosteric Modulator,PAOPA, on the Expression of GRK2, Arrestin-3, ERK1/2, and on Receptor Internalization

The activity of G protein-coupled receptors (GPCRs) is intricately regulated by a range of intracellular proteins, including G protein-coupled kinases (GRKs) and arrestins. Understanding the effects of ligands on these signaling pathways could provide insights into disease pathophysiologies and treatment. The dopamine D2 receptor is a GPCR strongly implicated in the pathophysiology of a range of neurological and neuropsychiatric disorders, particularly schizophrenia. Previous studies from our lab have shown the preclinical efficacy of a novel allosteric drug, 3(R)- [(2(S)-pyrrolidinylcarbonyl)amino]-2-oxo-1-pyrrolidineacetamide (PAOPA), in attenuating schizophrenia-like behavioural abnormalities in rodent models of the disease. As an allosteric modulator, PAOPA binds to a site on the D2 receptor, which is distinct from the endogenous ligand-binding site, in order to modulate the binding of the D2 receptor ligand, dopamine. The exact signaling pathways affected by this allosteric modulator are currently unknown. The objectives of this study were to decipher the in vivo effects, in rats, of chronic PAOPA administration on D2 receptor regulatory and downstream molecules, including GRK2, arrestin-3 and extracellular receptor kinase (ERK) 1/2. Additionally, an in vitro cellular model was also used to study PAOPA’s effects on D2 receptor internalization. Results from western immunoblots showed that chronic PAOPA treatment increased the striatal expression of GRK2 by 41%, arrestin-3 by 34%, phospho-ERK1 by 51% and phospho-ERK2 by 36%. Results also showed that the addition of PAOPA to agonist treatment in cells increased D2 receptor internalization by 33%. This study provides the foundational evidence of putative signaling pathways, and changes in receptor localization, affected by treatment with PAOPA. It improves our understanding on the diverse mechanisms of action of allosteric modulators, while advancing PAOPA’s development into a novel drug for the improved treatment of schizophrenia.     

HIV-1 Nef Down-Modulates C-C and C-X-C Chemokine Receptors via Ubiquitin and Ubiquitin-Independent Mechanism

Human and Simian Immunodeficiency virus (HIV-1, HIV-2, and SIV) encode an accessory protein, Nef, which is a pathogenesis and virulence factor. Nef is a multivalent adapter that dysregulates the trafficking of many immune cell receptors, including chemokine receptors (CKRs). Physiological endocytic itinerary of agonist occupied CXCR4 involves ubiquitinylation of the phosphorylated receptor at three critical lysine residues and dynamin-dependent trafficking through the ESCRT pathway into lysosomes for degradation. Likewise, Nef induced CXCR4 degradation was critically dependent on the three lysines in the C-terminal -SSLKILSKGK- motif. Nef directly recruits the HECT domain E3 ligases AIP4 or NEDD4 to CXCR4 in the resting state. This mechanism was confirmed by ternary interactions of Nef, CXCR4 and AIP4 or NEDD4; by reversal of Nef effect by expression of catalytically inactive AIP4-C830A mutant; and siRNA knockdown of AIP4, NEDD4 or some ESCRT-0 adapters. However, ubiquitinylation dependent lysosomal degradation was not the only mechanism by which Nef downregulated CKRs. Agonist and Nef mediated CXCR2 (and CXCR1) degradation was ubiquitinylation independent. Nef also profoundly downregulated the naturally truncated CXCR4 associated with WHIM syndrome and engineered variants of CXCR4 that resist CXCL12 induced internalization via an ubiquitinylation independent mechanism.     

Triterpene constituents of Caltha palustris

The structure of a new triterpene lactone, palustrolide, has been elucidated as 3β,23-dihydroxylupan-13β→28 lactone on the basis of physico-chemical studies. In addition, sitosterol, its glucoside, hederagenin, 16,17-dihydroxykauran-19-oic acid and hederagenic acid have been characterized.     

Effect of Different Gums on Features of 3D Printed Object Based on Vitamin-D Enriched Orange Concentrate

This study examined the effects of different gums viz. gum arabic (GA), guar gum (GG), k-carrageenan gum (KG), and xanthan gum (XG) on rheological and 3D printing characteristics of vitamin D (Vit D) enriched orange concentrate (OC) wheat starch (WS) blends. The textural and microstructural properties of printed objects from above mixture were evaluated and compared. The addition of gums induced an increase in apparent viscosity, storage modulus (G′), and loss modulus (G″) of the OC-WS mixtures, while GA decreased the apparent viscosity and G′. Nuclear magnetic resonance (NMR) analysis of 3D printed samples revealed that the movement of transverse time (T₂) toward closer to 0 ms indicated an increase in immobilized and bound water populations suggesting the gel formation. The slight shift toward shorter wavelength in FT-IR results for the broadband centered around 3400 cm^?1 after addition of gums possibly caused an increase of G′ and load bearing capacity of the blends. 3D printing characteristics revealed that the objects printed using KG containing blend possessed maximum fidelity to the target geometry and good loading bearing capacity, preventing collapsing over time due to the proper G′ value. At tanδ of 0.238, OC-WS-KG mixture achieved the best printing condition. Higher tanδ of GA (0.038) containing samples led to an unwanted collapse of the printed constructs. The objects printed using KG also exhibited the smoothest visible surface as well as microstructure and best mastication properties. Considering the studied features, vitamin D enriched OC with WS-KG was found to be the best match for orange fruit concentrate-based 3D food printing. This work demonstrates the novel ways to develop fortified 3D printed foods.     

Pseudomonas aeruginosa: A risk factor for fertility in male mice

The present study was designed to evaluate the effect of P. aeruginosa on reproductive potential of male mice via a series of in vitro and in vivo experiments. In vitro studies involved sperm parameters, Mg²⁺ATPase activity and acrosome status. In vivo study employed male mice which in the right vas deferens received 20?μl of either PBS (Group I) or 10⁴ cfu of P. aeruginosa (Group II). The animals were sacrificed on day 3, 7 and 14 and various parameters viz. body weight, TSI (%), bacterial load, spermiogram {i.e. sperm count, motility (%), viability (%) and morphology}, lipid peroxidation and tissue histopathology were evaluated. The results revealed that cell free supernatant of P. aeruginosa resulted in reduced motility, viability, Mg²⁺dependent ATPase activity and premature acrosomal loss of mouse spermatozoa in vitro. In vivo study showed that in comparison to group I, group II revealed significant alterations in all the parameters on all the days of sacrifice. Further, when reproductive organs of right and left side of mice in group II were compared, the right side demonstrated more devastating effects in terms of altered TSI (%) of testis and cauda epididymis, higher bacterial counts, azoospermia, increased malondialdehyde levels and severe inflammation in tissue histopathology in comparison to left side where bacteria disseminated in reduced numbers, thereby, resulting in insignificant changes in TSI (%), spermiogram, malondialdehyde levels and tissue histology. This study demonstrates that the colonization of P. aeruginosa in male genital tract could be a risk factor for fertility.     

Palaeoclimatic conditions in the upper Pleistocene and Holocene Bhimtal-Naukuchiatal lake basin in south-central Kumaun, North India

A 52 m thick upper Pleistocene and Holocene terrestrial succession in the Bhimtal-Naukuchiatal basin, south-central Kumaun Himalaya, India was studied using chronological, palaeontological, palynological and δ¹³C measurements. The section recorded evidence for climatic changes. At least two phases of arid climate and one phase of humid climate were recognised. Preliminary palaeomagnetic studies revealed a reversal of polarity, presumably correlatable with the Mono Lake excursion. Prior to this, no reversal event in the upper Pleistocene-Holocene terrestrial sediments of Indian subcontinent is known. A fossiliferous horizon, discovered in the lower part of the section, consisted of Sorex and Mus. This is the only report of a Late Pleistocene micromammalian assemblage in the Kumaun Himalaya.     

Somatic embryogenesis and regeneration of triploid plants in endosperm cultures of Acacia nilotica

Immature endosperm of Acacia nilotica formed a nodular callus on MS medium supplemented with 2,4-D, BAP and CH. In the third passage on this medium, in the dark, the callus differentiated somatic embryos. The embryos germinated on MS only after 15 d pre-treatment on modified MS medium in which major salts were replaced by those of major salts of B₅ medium and supplemented with glutamine, CH and CW. Triploid nature of the somatic embryos was confirmed by Feulgen cytophotometry.Abbreviations ABA abscisic acid - AC activated charcoal - BAP 6-benzylaminopurine - B₅ Gamborg et al. (1968) medium - CH casein hydrolysate - CW coconut water - d days - MS Murashige and Skoog (1962) medium - PEG 4000 polyethylene glycol - MW 3500–4000 - 2,4-D 2,4-dichlorophenoxyacetic acid