Desosomes and desimicelles - a novel vesicular and micellar system for enhanced oral delivery of poorly soluble drug: Optimization of in vitro characteristics and in vivo performance.

This study introduces two innovative nanocarrier systems to improve oral drug delivery. Desosomes and desimicelles combine Deep eutectic solvent (DES) with vesicular or micellar nanosystems, respectively. These novel nanosystems integrate the DES solubilization potency for administering drugs with low aqueous solubility and the vesicular and micellar systems to bypass physiological barriers and improve poor drug bioavailability. Lornoxicam (LRX) is a BCS class II anti-inflammatory with limited aqueous solubility and rapid clearance. Desosomes and desimicelles were prepared and successfully optimized. The optimization depended on particle size, zetapotential, entrapment efficiency, and solubility. The optimized desosomes (LRX-DES-V) and desimicelles (LRX-DES-M) were pictured by transmission electron microscope. Differential scanning calorimetry (DSC) and FTIR analysis indicated the successful inclusion of LRX inside each system. Invitro LRX release profiles revealed controlled release of LRX-DES-V and LRX-DES-M, with more sustained release by the later one. In-vivo study, inflammation was induced using a carrageenan rat model, and the anti-inflammatory effect of LRX-pure, marketed product, traditional niosomes, LRX-DES-V & LRX-DES-M were determined using inhibition %, serum inflammatory cytokines, and histopathology. After 4 h of induction, LRX-DES-M (68.05%) showed a significant inhibition compared to LRX-DES-V (63.57%). LRX-DES-M also showed a better reduction in COX2, PGE2, and TNF-α (1.25-fold, 1.24-fold, and 1.36-fold inhibition), respectively, compared to LRX-DES-V. We can conclude that LRX-DES-V and LRX-DES-M showed better effects than all other groups and that LRX-DES-M might be more effective than LRX-DES-V.

Myosin inhibitor reverses hypertrophic cardiomyopathy in genotypically diverse pediatric iPSC-cardiomyocytes to mirror variant correction.

Pathogenic variants in MYH7 and MYBPC3 account for the majority of hypertrophic cardiomyopathy (HCM). Targeted drugs like myosin ATPase inhibitors have not been evaluated in children. We generate patient and variant-corrected iPSC-cardiomyocytes (CMs) from pediatric HCM patients harboring single variants in MYH7 (V606M; R453C), MYBPC3 (G148R) or digenic variants (MYBPC3 P955fs, TNNI3 A157V). We also generate CMs harboring MYBPC3 mono- and biallelic variants using CRISPR editing of a healthy control. Compared with isogenic and healthy controls, variant-positive CMs show sarcomere disorganization, higher contractility, calcium transients, and ATPase activity. However, only MYH7 and biallelic MYBPC3 variant-positive CMs show stronger myosin-actin binding. Targeted myosin ATPase inhibitors show complete rescue of the phenotype in variant-positive CMs and in cardiac Biowires to mirror isogenic controls. The response is superior to verapamil or metoprolol. Myosin inhibitors can be effective in genotypically diverse HCM highlighting the need for myosin inhibitor drug trials in pediatric HCM.

Pseudomonas putida infection induces immune-antioxidant, hepato-renal, ethological, and histopathological/immunohistochemical disruptions in Oreochromis niloticus: the palliative role of titanium dioxide nanogel.

BACKGROUND: Pseudomonas putida is a pathogenic bacterium that induces great losses in fishes, including Nile tilapia (Oreochromis niloticus). Currently, the application of nanomaterials in aquaculture practices has gained more success as it endows promising results in therapies compared to traditional protocols. OBJECTIVE: Therefore, the current perspective is considered the first report to assess the anti-bacterial efficacy of titanium dioxide nanogel (TDNG) against Pseudomonas putida (P. putida) in Nile tilapia. METHODS: The fish (n = 200; average body weight: 47.50±1.32 g) were allocated into four random groups (control, TDNG, P. putida, and TDNG + P. putida), where 0.9 mg/L of TDNG was applied as bath treatment for ten days. RESULTS: Outcomes revealed that P. putida infection caused ethological alterations (surfacing, abnormal movement, and aggression) and depression of immune-antioxidant variables (complement 3, lysozyme activity, total antioxidant capacity, superoxide dismutase, and reduced glutathione content). Additionally, a substantial elevation in hepatorenal biomarkers (aspartate and alanine aminotransferases and creatinine) with clear histopathological changes and immuno-histochemical alterations (very weak BCL-2 and potent caspase-3 immuno-expressions) were seen. Surprisingly, treating P. putida-infected fish with TDNG improved these variables and obvious restoration of the tissue architectures. CONCLUSION: Overall, this report encompasses the key role of TDNG as an anti-bacterial agent for controlling P. putida infection and improving the health status of Nile tilapia.

Fexofenadine-loaded chitosan coated solid lipid nanoparticles (SLNs): A potential oral therapy for ulcerative colitis.

The targeting and mucoadhesive features of chitosan (CS)-linked solid lipid nanoparticles (SLNs) were exploited to efficiently deliver fexofenadine (FEX) into the colon, forming a novel and potential oral therapeutic option for ulcerative colitis (UC) treatment. Different FEX-CS-SLNs with varied molecular weights of CS were prepared and optimized. Optimized FEX-CS-SLNs exhibited 229 ± 6.08 nm nanometric size, 36.3 ± 3.18 mV zeta potential, 64.9 % EE, and a controlled release profile. FTIR, DSC, and TEM confirmed good drug entrapment and spherical particles. Mucoadhesive properties of FEX-CS-SLNs were investigated through mucin incubation and exhibited considerable mucoadhesion. The protective effect of FEX-pure, FEX-market, and FEX-CS-SLNs against acetic acid-induced ulcerative colitis in rats was examined. Oral administration of FEX-CS-SLNs for 14 days before ulcerative colitis induction reversed UC symptoms and almost restored the intestinal mucosa to normal integrity and inhibited Phosphatidylinositol-3 kinase (73.6 %), protein kinase B (73.28 %), and elevated nuclear factor erythroid 2-related factor 2 (185.9 %) in colonic tissue. Additionally, FEX-CS-SLNs inhibited tumor necrosis factor α (TNF-α) and interleukin 6 (IL-6) to (70.79 % & 72.99 %) in colonic tissue. The ameliorative potential of FEX-CS-SLNs outperformed that of FEX-pure and FEX-market. The exceptional protective effect of FEX-CS-SLNs makes it a potentially effective oral system for managing ulcerative colitis.

Systematic characterization of regulatory variants of blood pressure genes.

High blood pressure (BP) is the major risk factor for cardiovascular disease. Genome-wide association studies have identified genetic variants for BP, but functional insights into causality and related molecular mechanisms lag behind. We functionally characterize 4,608 genetic variants in linkage with 135 BP loci in vascular smooth muscle cells and cardiomyocytes by massively parallel reporter assays. High densities of regulatory variants at BP loci (i.e., ULK4, MAP4, CFDP1, PDE5A) indicate that multiple variants drive genetic association. Regulatory variants are enriched in repeats, alter cardiovascular-related transcription factor motifs, and spatially converge with genes controlling specific cardiovascular pathways. Using heuristic scoring, we define likely causal variants, and CRISPR prime editing finally determines causal variants for KCNK9, SFXN2, and PCGF6, which are candidates for developing high BP. Our systems-level approach provides a catalog of functionally relevant variants and their genomic architecture in two trait-relevant cell lines for a better understanding of BP gene regulation.

Regulatory functional role of NLRP3 inflammasome during Mycoplasma hyopneumoniae infection in swine.

Mycoplasma hyopneumoniae causes enzootic pneumonia, a highly contagious respiratory disease in swine that causes significant economic losses worldwide. It is unknown whether the nucleotide oligomerization domain-like receptor (NLR) family pyrin domain containing 3 (NLRP3) inflammasome regulates the immune response in swine during M. hyopneumoniae infection. The current study utilized an in vivo swine model of M. hyopneumoniae infection to investigate the regulatory functional role of the NLRP3 inflammasome during M. hyopneumoniae infection. Notable histopathological alterations were observed in M. hyopneumoniae-infected swine tissues, which were associated with an inflammatory response and disease progression. Swine M. hyopneumoniae infection was associated with an increase in the expression of the NLRP3 inflammasome, which stimulated pro-inflammatory cytokines such as tumor necrosis factor-alpha, interleukin 18, and interleukin 1 beta (IL-1ß). The impact of the NLRP3 inhibitor, MCC950 on NLRP3 and pro-inflammatory cytokines in M. hyopneumoniae-infected swine was examined to investigate the relationship between the NLRP3 inflammasome and M. hyopneumoniae infection. Taken together, our findings provide strong evidence that the NLRP3 inflammasome plays a critical regulatory functional role in M. hyopneumoniae infection in swine.

Our study highlights the importance of controlling the innate immune defense against respiratory mycoplasma invasion to suppress mycoplasma growth and minimize lung tissue damage. Using an in vivo swine model, we investigated the regulatory functional role of the NLR family pyrin domain containing 3 (NLRP3) inflammasome during acute Mycoplasma hyopneumoniae infection. Furthermore, we also found that NLRP3 expression levels have the potential to serve as a novel diagnostic marker for detecting M. hyopneumoniae infection in the respiratory tract of pigs. The NLRP3 inhibitor, MCC950, was used to investigate how NLRP3 inhibition affects the expression of inflammatory cytokines, and it was found that the NLRP3 inhibitor significantly reduced the mRNA and protein expression of NLRP3, indicating its specific targeting of the NLRP3 inflammasome during M. hyopneumoniae infection in swine. The findings suggest that MCC950 is a promising therapeutic option for treating NLRP3-related disorders, including porcine enzootic pneumonia.

Whole genome sequencing delineates regulatory, copy number, and cryptic splice variants in early onset cardiomyopathy.

Cardiomyopathy (CMP) is a heritable disorder. Over 50% of cases are gene-elusive on clinical gene panel testing. The contribution of variants in non-coding DNA elements that result in cryptic splicing and regulate gene expression has not been explored. We analyzed whole-genome sequencing (WGS) data in a discovery cohort of 209 pediatric CMP patients and 1953 independent replication genomes and exomes. We searched for protein-coding variants, and non-coding variants predicted to affect the function or expression of genes. Thirty-nine percent of cases harbored pathogenic coding variants in known CMP genes, and 5% harbored high-risk loss-of-function (LoF) variants in additional candidate CMP genes. Fifteen percent harbored high-risk regulatory variants in promoters and enhancers of CMP genes (odds ratio 2.25, p = 6.70 × 10-7 versus controls). Genes involved in α-dystroglycan glycosylation (FKTN, DTNA) and desmosomal signaling (DSC2, DSG2) were most highly enriched for regulatory variants (odds ratio 6.7-58.1). Functional effects were confirmed in patient myocardium and reporter assays in human cardiomyocytes, and in zebrafish CRISPR knockouts. We provide strong evidence for the genomic contribution of functionally active variants in new genes and in regulatory elements of known CMP genes to early onset CMP.

Nanostructured lipid carriers for enhanced in vitro and in vivo schistosomicidal activity of praziquantel: effect of charge.

WHO considers praziquantel (PZQ) as the drug of choice for treatment of Schistosoma mansoni infection but this requires high dose due to poor solubility and first pass metabolism. The aim of this work was to optimize nanostructured lipid carriers (NLCs) for enhanced PZQ oral delivery. The optimization involved testing the effect of surface charge of NLCs. NLCs comprised precirol ATO as solid lipid with oleic acid, Span 60 and Tween 80 as liquid components. Dicetyl phosphate and stearyl amine were the negative and positive charging agents, respectively. NLCs were prepared by microemulsification technique and were characterized. The schistosomicidal activity of PZQ loaded NLCs was monitored in vitro and in vivo using infected mice. PZQ showed high entrapment efficiency in all types of NLCs (ranged from 93.97 to 96.29%) with better PZQ loading in standard NLCs. This was clarified by thermal analysis which reflected displacement of PZQ by charging agents. In vitro schistosomicidal study revealed the superiority of PZQ loaded positively charged NLCs (LC50 and LC95 equal 0.147 and 0.193 µg/ml respectively) with traditional and negatively charged NLCs being inferior to simple PZQ solution after short incubation period. Scanning electron micrographs showed that PZQ loaded positively charged NLCs resulted in more intense ultrastructural changes in worms. The superiority of positively charged NLCs was confirmed by in vivo assessment as they showed better improvement in histopathological features of the liver of the infected mice compared with other formulations. The study introduced positively charged NLCs as promising carriers for oral delivery of PZQ.

Regions of bovine adenovirus-3 IVa2 involved in nuclear/nucleolar localization and interaction with pV.

Bovine adenovirus-3 (BAdV-3) is a non enveloped, icosahedral DNA virus containing a genome of 34446 bps. The intermediate region of BAdV-3 encodes pIX and IVa2 proteins. Here, we report the characterization of BAdV-3 IVa2. Anti-IVa2 serum detected a 50 kDa protein at 24-48 h post infection in BAdV-3 infected cells. The IVa2 localizes to nucleus and nucleolus of BAdV-3 infected cells. Analysis of mutant IVa2 demonstrated that amino acids 1-25 and 373-448 are required for nuclear and nucleolar localization of IVa2, respectively. The nuclear import of IVa2 utilize importin α -1 of importin nuclear import pathway. Although deletion/substitution of amino acids 4-18 is sufficient to abrogate the nuclear localization of IVa2, amino acids 1-25 are required for nuclear localization of a cytoplasmic protein. Furthermore, we demonstrate that amino acids 1-25 and 120-140 of IVa2 interact with importin α-1 and pV proteins, respectively in BAdV-3 infected cells.

Porcine Adenovirus Type 3 E3 Encodes a Structural Protein Essential for Capsid Stability and Production of Infectious Progeny Virions.

The adenovirus E3 region encodes proteins that are not essential for viral replication in vitro The porcine adenovirus type 3 (PAdV-3) E3 region encodes three proteins, including 13.7K. Here, we report that 13.7K is expressed as an early protein, which localizes to the nucleus of infected cells. The 13.7K protein is a structural protein, as it is incorporated in CsCl-purified virions. The 13.7K protein appears to be essential for PAdV-3 replication, as mutant PAV13.73A expressing a mutated 13.7K could be isolated only in VIDO AS2 cells expressing the 13.7K protein. Analysis of PAV13.73A suggested that even in the presence of reduced levels of some late viral proteins, there appeared to be no effect on virus assembly and production of mature virions. Further analysis of CsCl-purified PAV13.73A by transmission electron microscopy revealed the presence of disrupted/broken capsids, suggesting that inactivation of 13.7K protein expression may produce fragile capsids. Our results suggest that the PAdV-3 E3 region-encoded 13.7K protein is a capsid protein, which appears to be essential for the formation of stable capsids and production of infectious progeny virions.IMPORTANCE Although E3 region-encoded proteins are involved in the modulation of leukocyte functions (N. Arnberg, Proc Natl Acad Sci U S A 110:19976-19977, 2013) and inducing a lytic infection of lymphocytes (V. K. Murali, D. A. Ornelles, L. R. Gooding, H. T. Wilms, W. Huang, A. E. Tollefson, W. S. Wold, and C. Garnett-Benson, J Virol 88:903-912, 2014), none of the E3 proteins appear to be a component of virion capsid or required for replication of adenovirus. Here, we demonstrate that the 13.7K protein encoded by the E3 region of porcine adenovirus type 3 is a component of progeny virion capsids and appears to be essential for maintaining the integrity of virion capsid and production of infectious progeny virions. To our knowledge, this is the first report to suggest that an adenovirus E3-encoded protein is an essential structural protein.