Functional characterization of novel variants in SMPD1 in Indian patients with acid sphingomyelinase deficiency.

Pathogenic variations in SMPD1 lead to acid sphingomyelinase deficiency (ASMD), that is, Niemann-Pick disease (NPD) type A and B (NPA, NPB), which is a recessive lysosomal storage disease. The knowledge of variant spectrum in Indian patients is crucial for early and accurate NPD diagnosis and genetic counseling of families. In this study, we recruited 40 unrelated pediatric patients manifesting symptoms of ASMD and subnormal ASM enzyme activity. Variations in SMPD1 were studied using Sanger sequencing for all exons, followed by interpretation of variants based on American College of Medical Genetics and Genomics & Association for Molecular Pathology (ACMG/AMP) criteria. We identified 18 previously unreported variants and 21 known variants, including missense, nonsense, deletions, duplications, and splice site variations with disease-causing potential. Eight missense variants were functionally characterized using in silico molecular dynamic simulation and in vitro transient transfection in HEK293T cells, followed by ASM enzyme assay, immunoblot, and immunofluorescence studies. All the variants showed reduced ASM activity in transfected cells confirming their disease-causing potential. The study provides data for efficient prenatal diagnosis and genetic counseling of families with NPD type A and B.

Novel splice-site variant of UCHL1 in an Indian family with autosomal recessive spastic paraplegia-79.

Spastic Paraplegia-79 (SPG79) is an autosomal recessive type of childhood onset complicated by hereditary spastic paraplegia. SPG79 is characterized by spasticity, paraplegia, optic atrophy, cerebellar signs, and other variable clinical features. Recessive, disease causing variants in Ubiquitin C-terminal hydrolase-L1 (UCHL1) gene have been implicated as a cause for SPG79 in two families till now. In this study, we report on a third family of SPG79 with two similarly affected siblings, harboring a novel homozygous splice-site variant in the UCHL1 gene (NM_004181.4: c.459+2T>C). The variant was identified by whole-exome sequencing and validated by Sanger sequencing in the family.

Familial choreoathetosis due to novel heterozygous mutation in PDE10A.

PDE10A encodes a dual cAMP-cGMP phosphodiesterase that is enriched in the medium spiny neurons of the corpus striatum in the brain and plays an important role in basal ganglia circuitry. Three unrelated patients with childhood onset chorea and striatal abnormalities on MRI brain with heterozygous de novo variants in PDE10A have been described previously. Two families with eight affected individuals with biallelic mutations in PDE10A have also been described previously. We report a family with multiple affected individuals with childhood onset chorea, striatal abnormalities, and a novel heterozygous mutation, c.1001T>G(p.F334C) in PDE10A which was identified by exome sequencing.

Engineering of an ω-3 polyunsaturated fatty acid-containing nanoemulsion system for combination C6-ceramide and 17ß-estradiol delivery and bioactivity in human vascular endothelial and smooth muscle cells.

Delayed endothelial cell (EC) regeneration and the medial vascular smooth muscle cells (VSMCs) proliferation contribute to arterial restenosis. Although ω-3-polyunsaturated fatty acids (PUFAs), 17ß-estradiol (17-ßE) and C6-ceramide (CER) have shown therapeutic promise in addressing restenosis, extensive protein binding and lipophilicity complicate their (co-)delivery to cellular targets. We report engineering of an ω-3-PUFA-rich oil-in-water nanoemulsion formulation that effectively delivers 17-ßE and CER cargo to cultured vascular cells. The cargo-free, ω-3-PUFA-rich nanoemulsion itself typically reduced growth factor-stimulated cellular proliferation, as did nanoemulsion-delivered CER alone, through enhanced pro-apoptotic caspase 3/7 activity. 17-ßE loaded nanoemulsion inhibited VSMC proliferation and supported EC proliferation, responses associated with the mitogen-activated-protein-kinase (MAPK) signaling. Co-administration of 17-ßE and CER loaded nanoemulsions exerted an anti-proliferative effect more pronounced on VSMCs than ECs. These therapeutically beneficial responses to ω-3-PUFA, CER, and/or 17-ßE in our nanoemulsion formulation invite evaluation of this novel approach in animal models of restenosis and other occlusive vasculopathies. FROM THE CLINICAL EDITOR: This team of investigators report the engineering of an ω-3-PUFA-rich oil-in-water nanoemulsion formulation that effectively delivers 17-ßE and C6-ceramide cargo to cultured vascular cells in an effort to address vascular restenosis. Further preclinical studies will be needed in animal models before this approach could be considered for clinical trials.

Genomic surveillance reveals early detection and transition of delta to omicron lineages of SARS-CoV-2 variants in wastewater treatment plants of Pune, India.

The COVID-19 pandemic has emphasized the urgency for rapid public health surveillance methods to detect and monitor the transmission of infectious diseases. The wastewater-based epidemiology (WBE) has emerged as a promising tool for proactive analysis and quantification of infectious pathogens within a population before clinical cases emerge. In the present study, we aimed to assess the trend and dynamics of SARS-CoV-2 variants using a longitudinal approach. Our objective included early detection and monitoring of these variants to enhance our understanding of their prevalence and potential impact. To achieve our goals, we conducted real-time quantitative polymerase chain reaction (RT-qPCR) and Illumina sequencing on 442 wastewater (WW) samples collected from 10 sewage treatment plants (STPs) in Pune city, India, spanning from November 2021 to April 2022. Our comprehensive analysis identified 426 distinct lineages representing 17 highly transmissible variants of SARS-CoV-2. Notably, fragments of Omicron variant were detected in WW samples prior to its first clinical detection in Botswana. Furthermore, we observed highly contagious sub-lineages of the Omicron variant, including BA.1 (~28%), BA.1.X (1.0-72%), BA.2 (1.0-18%), BA.2.X (1.0-97.4%) BA.2.12 (0.8-0.25%), BA.2.38 (0.8-1.0%), BA.2.75 (0.01-0.02%), BA.3 (0.09-6.3%), BA.4 (0.24-0.29%), and XBB (0.01-21.83%), with varying prevalence rates. Overall, the present study demonstrated the practicality of WBE in the early detection of SARS-CoV-2 variants, which could help track future outbreaks of SARS-CoV-2. Such approaches could be implicated in monitoring infectious agents before they appear in clinical cases.

A review of multifunctional nanoemulsion systems to overcome oral and CNS drug delivery barriers.

The oral and central nervous systems (CNS) present a unique set of barriers to the delivery of important diagnostic and therapeutic agents. Extensive research over the past few years has enabled a better understanding of these physical and biological barriers based on tight cellular junctions and expression of active transporters and metabolizing enzymes at the luminal surfaces of the gastrointestinal (GI) tract and the blood-brain barrier (BBB). This review focuses on the recent understanding of transport across the GI tract and BBB and the development of nanotechnology-based delivery strategies that can enhance bioavailability of drugs. Multifunctional lipid nanosystems, such as oil-in-water nanoemulsions, that integrate enhancement in permeability, tissue and cell targeting, imaging, and therapeutic functions are especially promising. Based on strategic choice of edible oils, surfactants and additional surface modifiers, and different types of payloads, rationale design of multifunctional nanoemulsions can serve as a safe and effective delivery vehicle across oral and CNS barriers.

Multifunctional Effects of a Small-Molecule STAT3 Inhibitor on NASH and Hepatocellular Carcinoma in Mice.

Purpose: The incidence of hepatocellular carcinoma is increasing in the United States, and liver cancer is the second leading cause of cancer-related mortality worldwide. Nonalcoholic steatohepatitis (NASH) is becoming an important risk for hepatocellular carcinoma, and most patients with hepatocellular carcinoma have underlying liver cirrhosis and compromised liver function, which limit treatment options. Thus, novel therapeutic strategies to prevent or treat hepatocellular carcinoma in the context of NASH and cirrhosis are urgently needed.Experimental Design: Constitutive activation of STAT3 is frequently detected in hepatocellular carcinoma tumors. STAT3 signaling plays a pivotal role in hepatocellular carcinoma survival, growth, angiogenesis, and metastasis. We identified C188-9, a novel small-molecule STAT3 inhibitor using computer-aided rational drug design. In this study, we evaluated the therapeutic potential of C188-9 for hepatocellular carcinoma treatment and prevention.Results: C188-9 showed antitumor activity in vitro in three hepatocellular carcinoma cell lines. In mice with hepatocyte-specific deletion of Pten (HepPten- mice), C188-9 treatment blocked hepatocellular carcinoma tumor growth, reduced tumor development, and reduced liver steatosis, inflammation, and bile ductular reactions, resulting in improvement of the pathological lesions of NASH. Remarkably, C188-9 also greatly reduced liver injury in these mice as measured by serum aspartate aminotransferase and alanine transaminase levels. Analysis of gene expression showed that C188-9 treatment of HepPten- mice resulted in inhibition of signaling pathways downstream of STAT3, STAT1, TREM-1, and Toll-like receptors. In contrast, C188-9 treatment increased liver specification and differentiation gene pathways.Conclusions: Our results suggest that C188-9 should be evaluated further for the treatment and/or prevention of hepatocellular carcinoma. Clin Cancer Res; 23(18); 5537-46. ©2017 AACR.

Therapeutic Efficacy of an ω-3-Fatty Acid-Containing 17-ß Estradiol Nano-Delivery System against Experimental Atherosclerosis.

Atherosclerosis and its consequences remain prevalent clinical challenges throughout the world. Initiation and progression of atherosclerosis involves a complex, dynamic interplay among inflammation, hyperlipidemia, and endothelial dysfunction. A multicomponent treatment approach targeted for delivery within diseased vessels could prove beneficial in treating atherosclerosis. This study was undertaken to evaluate the multimodal effects of a novel ω-3-fatty acid-rich, 17-ß-estradiol (17-ßE)-loaded, CREKA-peptide-modified nanoemulsion system on experimental atherosclerosis. In vitro treatment of cultured human aortic endothelial cells (ECs) with the 17-ßE-loaded, CREKA-peptide-modified nanoemulsion system increased cellular nitrate/nitrite, indicating improved nitric oxide formation. In vivo, systemic administration of this nanoemulsion system to apolipoprotein-E knock out (ApoE-/-) mice fed a high-fat diet significantly improved multiple parameters related to the etiology and development of occlusive atherosclerotic vasculopathy: lesion area, circulating plasma lipid levels, and expression of aortic-wall inflammatory markers. These salutary effects were attributed selectively to the 17-ßE and/or ω-3 polyunsaturated fatty acid components of the nano-delivery system. At therapeutic doses, the 17-ßE-loaded, CREKA-peptide modified nanoemulsion system appeared to be biocompatible in that it elicited no apparent adverse/toxic effects, as indexed by body weight, plasma alanine aminotransferase/aspartate aminotransferase levels, and liver and kidney histopathology. The study demonstrates the therapeutic potential of a novel, 17-ßE-loaded, CREKA-peptide-modified nanoemulsion system against atherosclerosis in a multimodal fashion by reducing lesion size, lowering the levels of circulating plasma lipids and decreasing the gene expression of inflammatory markers associated with the disease.

Nucleic Acid Delivery for Endothelial Dysfunction in Cardiovascular Diseases.

Endothelial dysfunction has been implicated in the pathophysiology of multiple cardiovascular diseases and involves components of both innate and acquired immune mechanisms. Identifying signature patterns and targets associated with endothelial dysfunction can help in the development of novel nanotherapeutic platforms for treatment of vascular diseases. This review discusses nucleic acid-based regulation of endothelial function and the different nucleic acid-based nanotherapeutic approaches designed to target endothelial dysfunction in cardiovascular disorders.

Comparative pharmacokinetics and tissue distribution analysis of systemically administered 17-ß-estradiol and its metabolites in vivo delivered using a cationic nanoemulsion or a peptide-modified nanoemulsion system for targeting atherosclerosis.

The primary objective of this study was to compare the biodistribution and pharmacokinetic profile of 17-ß-estradiol (17-ßE) on systemic delivery using either the cationic or the CREKA-peptide-modified (Cysteine-Arginine-Glutamic-acid-Lysine-Alanine) omega-3-fatty acid oil containing nanoemulsion system in vivo in the wild type C57BL/6 mice. Higher blood concentrations of 17-ßE, higher accumulation in the tissues of interest - heart and aorta, and higher accumulation within the other tissues - liver and kidney was observed on delivering 17-ßE using the CREKA-peptide-modified nanoemulsion system (AUClast in plasma - 263.89±21.81min*%/injected dose/ml) as compared to the cationic nanoemulsion (AUClast in plasma - 20.2±1.86min*%/injected dose/ml) and solution form (AUClast in plasma - 44.9±1.24min*%/injected dose/ml) respectively. Both, the cationic nanoemulsion and the CREKA-peptide-modified nanoemulsion showed a higher relative targeting efficiency of 4.57 and 4.86 respectively for 17-ßE than the relative targeting efficiency of 1.78 observed with the solution form. In conclusion, since the maximum exposure (highest AUClast for plasma and tissues) for 17-ßE was observed with the CREKA-peptide-modified nanoemulsion system, the study shows that CREKA-peptide-modified nanoemulsion system was the most suitable vehicle for systemic delivery of 17-ßE in the wild type C57BL/6 mice.

Therapeutic strategies for endothelial dysfunction.

INTRODUCTION: A functionally compromised vascular endothelium is associated with tissue-damaging responses including inflammation, immune stimulation, oxidative stress and platelet activation/aggregation and can lead to severe end-organ damage, as implicated in the pathology of several cardiac, cerebral and renal disorders. Multiple noninvasive techniques are available for assessing endothelial dysfunction in clinical settings. Diverse interventions have been identified as having therapeutic potential for treating endothelial dysfunction and preventing its pathophysiological sequellae. AREAS COVERED: Evaluation techniques and interventional treatment approaches for endothelial dysfunction, with particular reference to prevalent cardiovascular and metabolic disorders such as coronary artery disease and diabetes. Limitations of the current treatments and avenues for improved endothelium-targeted therapies. EXPERT OPINION: Beneficial pleiotropic effects of various agents (cardiovascular medicines, antioxidants, nutritional supplements) on vascular endothelial function in humans notwithstanding, a growing body of preclinical data suggests that protein-, cell- and gene-based approaches hold promise for selective therapeutic targeting of the dysfunctional vascular endothelium. Additional efficacy data in appropriate animal models of vascular injury and cardiometabolic disease, further refinement of delivery modalities and continued investigation of the mechanisms underlying endothelial repair and regeneration should help identify the most promising therapeutic approaches for improving endothelial function that merit evaluation in human trials.

Enhancement in anti-proliferative effects of paclitaxel in aortic smooth muscle cells upon co-administration with ceramide using biodegradable polymeric nanoparticles.

PURPOSE: Using a combination of paclitaxel (PTX), and the apoptotic signaling molecule, C6-ceramide (CER), the enhancement in anti-proliferative effect of human aortic smooth muscle cells (SMC) was examined by administering in polymeric nanoparticles. METHODS: PTX- and CER-loaded poly(ethylene oxide)-modified poly(epsilon caprolactone) (PEO-PCL) nanoparticles were formulated by solvent displacement and characterized. The uptake and intracellular localization of the nanoparticle in SMC was examined using Z-stack fluorescent confocal microscopy. Anti-proliferative and pro-apoptotic effects of SMC were determined upon administration of PTX and CER, either as single agent or in combination, in aqueous solution and in PEO-PCL nanoparticle formulations. RESULTS: High encapsulation efficiencies (i.e., >95%) of PTX and CER at 10% (w/w) loading were attained in the PEO-PCL nanoparticles of around 270 nm in diameter. Fluorescence confocal analysis showed that nanoparticle delivery did facilitate cellular uptake and internalization. Additionally, combination of PTX and CER delivery in PEO-PCL nanoparticles was significantly more effective in decreasing the proliferation of SMC, probably by enhancing the apoptotic response. CONCLUSIONS: The results of this study show that combination of PTX and CER when administered in PEO-PCL nanoparticles can significantly augment the anti-proliferative effect in SMC. This strategy may potentially be useful in the treatment of coronary restenosis.