Microfluidic device-fabricated spiky nano-burflower shape gold nanomaterials facilitate large biomolecule delivery into cells using infrared light pulses.

Photothermal nanoparticle-sensitised photoporation is an emerging approach, which is considered an efficient tool for the intracellular delivery of biomolecules. Nevertheless, using this method to achieve high transfection efficiency generally compromises cell viability and uneven distribution of nanoparticles results in non-uniform delivery. Here, we show that high aspect ratio gold nano-burflowers, synthesised in a microfluidic device, facilitate highly efficient small to very-large cargo delivery uniformly using infrared light pulses without sacrificing cell viability. By precisely controlling the flow rates of shaping reagent and reducing agent, high-density (24 numbers) sharply branched spikes (∼80 nm tip-to-tip length) of higher aspect ratios (∼6.5) with a small core diameter (∼45 nm) were synthesised. As produced gold burflower-shape nanoparticles are biocompatible, colloidally stable (large surface zeta potential value), and uniform in morphology with a higher plasmonic peak (max. 890 nm). Theoretical analysis revealed that spikes on the nanoparticles generate a higher electromagnetic field enhancement upon interaction with light pulses. It induces plasmonic nanobubbles in the vicinity of the cells, followed by pore formation on the membrane leading to diverse biomolecular delivery into cells. Our platform has been successfully implemented for uniform delivery of small to very large biomolecules, including siRNA (20-24 bp), plasmid DNA expressing green fluorescent protein (6.2 kbp), Cas-9 plasmid (9.3 kbp), and ß-galactosidase enzyme (465 kDa) into diverse mammalian cells with high transfection efficiency and cell viability. For very large biomolecules such as enzymes, the best results were achieved as ∼100% transfection efficiency and ∼100% cell viability in SiHa cells. Together, our findings demonstrate that the spiky gold nano-burflower shape nanoparticles manufactured in a microfluidic system exhibited excellent plasmonic behaviour and could serve as an effective tool in manipulating cell physiology.

Chromogranin A and its derived peptides: potential regulators of cholesterol homeostasis.

Chromogranin A (CHGA), a member of the granin family of proteins, has been an attractive therapeutic target and candidate biomarker for several cardiovascular, neurological, and inflammatory disorders. The prominence of CHGA stems from the pleiotropic roles of several bioactive peptides (e.g., catestatin, pancreastatin, vasostatins) generated by its proteolytic cleavage and by their wide anatomical distribution. These peptides are emerging as novel modulators of cardiometabolic diseases that are often linked to high blood cholesterol levels. However, their impact on cholesterol homeostasis is poorly understood. The dynamic nature of cholesterol and its multitudinous roles in almost every aspect of normal body function makes it an integral component of metabolic physiology. A tightly regulated coordination of cholesterol homeostasis is imperative for proper functioning of cellular and metabolic processes. The deregulation of cholesterol levels can result in several pathophysiological states. Although studies till date suggest regulatory roles for CHGA and its derived peptides on cholesterol levels, the mechanisms by which this is achieved still remain unclear. This review aims to aggregate and consolidate the available evidence linking CHGA with cholesterol homeostasis in health and disease. In addition, we also look at common molecular regulatory factors (viz., transcription factors and microRNAs) which could govern the expression of CHGA and genes involved in cholesterol homeostasis under basal and pathological conditions. In order to gain further insights into the pathways mediating cholesterol regulation by CHGA/its derived peptides, a few prospective signaling pathways are explored, which could act as primers for future studies.

Cyclic-AMP response element binding protein (CREB) and microRNA miR-29b regulate renalase gene expression under catecholamine excess conditions.

AIMS: Renalase, a key mediator of cross-talk between kidneys and sympathetic nervous system, exerts protective roles in various cardiovascular/renal disease states. However, molecular mechanisms underpinning renalase gene expression remain incompletely understood. Here, we sought to identify the key molecular regulators of renalase under basal/catecholamine-excess conditions. MATERIALS AND METHODS: Identification of the core promoter domain of renalase was carried out by promoter-reporter assays in N2a/HEK-293/H9c2 cells. Computational analysis of the renalase core promoter domain, over-expression of cyclic-AMP-response-element-binding-protein (CREB)/dominant negative mutant of CREB, ChIP assays were performed to determine the role of CREB in transcription regulation. Role of the miR-29b-mediated-suppression of renalase was validated in-vivo by using locked-nucleic-acid-inhibitors of miR-29. qRT-PCR and Western-blot analyses measured the expression of renalase, CREB, miR-29b and normalization controls in cell lysates/ tissue samples under basal/epinephrine-treated conditions. KEY FINDINGS: CREB, a downstream effector in epinephrine signaling, activated renalase expression via its binding to the renalase-promoter. Physiological doses of epinephrine and isoproterenol enhanced renalase-promoter activity and endogenous renalase protein level while propranolol diminished the promoter activity and endogenous renalase protein level indicating a potential role of beta-adrenergic receptor in renalase gene regulation. Multiple animal models (acute exercise, genetically hypertensive/stroke-prone mice/rat) displayed directionally-concordant expression of CREB and renalase. Administration of miR-29b inhibitor in mice upregulated endogenous renalase expression. Moreover, epinephrine treatment down-regulated miR-29b promoter-activity/transcript levels. SIGNIFICANCE: This study provides evidence for renalase gene regulation by concomitant transcriptional activation via CREB and post-transcriptional attenuation via miR-29b under excess epinephrine conditions. These findings have implications for disease states with dysregulated catecholamines.

A common Matrix metalloproteinase 8 promoter haplotype enhances the risk for hypertension via diminished interactions with nuclear factor kappa B.

OBJECTIVES: Matrix metalloproteinase 8 (MMP8) has a prominent role in collagen turnover in blood vessels and vascular remodeling. The contribution of regulatory single nucleotide polymorphisms in MMP8 to cardiovascular diseases is unclear. We aimed to delineate the influence of MMP8 promoter variations on hypertension. METHODS: A case-control study in unrelated individuals ( n  = 2565) was carried out. Resequencing of the MMP8 proximal promoter, linkage disequilibrium analysis, genotyping of variants and regression analyses were performed. MMP8 promoter-reporter constructs were generated and expressed in human vascular endothelial cells under various conditions. RESULTS: We identified four single nucleotide polymorphisms (SNPs) in the promoter region of MMP8 : -1089A/G (rs17099452), -815G/T (rs17099451), -795C/T (rs11225395), -763A/T (rs35308160); these SNPs form three major haplotypes. Hap3 (viz., GTTT haplotype) carriers showed significant associations with hypertension in two geographically distinct human populations (e.g., Chennai: odds ratio [OR] = 1.47, 95% confidence interval [CI] = 1.16-1.86, P  = 2 × 10 -3 ; Chandigarh: OR = 1.85, 95% CI = 1.21-2.81, P  = 4 × 10 -3 ). Hap3 carriers also displayed elevated systolic blood pressure, diastolic blood pressure and mean arterial pressure levels. Hap3 promoter-reporter construct showed lower promoter activity than the wild-type (Hap1) construct. In silico analysis and molecular dynamics studies predicted diminished binding of the transcription factor nuclear factor kappa B (NF-κB) to the functional -815T allele of Hap3 compared to the -815G wild-type allele; this prediction was validated by in-vitro experiments. Hap3 displayed impaired response to tumor necrosis factor-alpha treatment, possibly due to weaker binding of NF-κB. Notably, MMP8 promoter haplotypes were identified as independent predictors of plasma MMP8 and endothelial dysfunction markers (von Willebrand factor and endothelin-1) levels. CONCLUSION: MMP8 promoter GTTT haplotype has a functional role in reducing MMP8 expression during inflammation via diminished interaction with NF-κB and in enhancing the risk of hypertension.

Functional Gly297Ser Variant of the Physiological Dysglycemic Peptide Pancreastatin Is a Novel Risk Factor for Cardiometabolic Disorders.

Pancreastatin (PST), a chromogranin A-derived potent physiological dysglycemic peptide, regulates glucose/insulin homeostasis. We have identified a nonsynonymous functional PST variant (p.Gly297Ser; rs9658664) that occurs in a large section of human populations. Association analysis of this single nucleotide polymorphism with cardiovascular/metabolic disease states in Indian populations (n = 4,300 subjects) displays elevated plasma glucose, glycosylated hemoglobin, diastolic blood pressure, and catecholamines in Gly/Ser subjects as compared with wild-type individuals (Gly/Gly). Consistently, the 297Ser allele confers an increased risk (∼1.3-1.6-fold) for type 2 diabetes/hypertension/coronary artery disease/metabolic syndrome. In corroboration, the variant peptide (PST-297S) displays gain-of-potency in several cellular events relevant for cardiometabolic disorders (e.g., increased expression of gluconeogenic genes, increased catecholamine secretion, and greater inhibition of insulin-stimulated glucose uptake) than the wild-type peptide. Computational docking analysis and molecular dynamics simulations show higher affinity binding of PST-297S peptide with glucose-regulated protein 78 (GRP78) and insulin receptor than the wild-type peptide, providing a mechanistic basis for the enhanced activity of the variant peptide. In vitro binding assays validate these in silico predictions of PST peptides binding to GRP78 and insulin receptor. In conclusion, the PST 297Ser allele influences cardiovascular/metabolic phenotypes and emerges as a novel risk factor for type 2 diabetes/hypertension/coronary artery disease in human populations.