Metabolites of Cannabis Induce Cardiac Toxicity and Morphological Alterations in Cardiac Myocytes.

Cannabis is one of the most commonly used recreational drugs worldwide. Rrecent epidemiology studies have linked increased cardiac complications to cannabis use. However, this literature is predominantly based on case incidents and post-mortem investigations. This study elucidates the molecular mechanism of Δ9-tetrahydrocannabinol (THC), and its primary metabolites 11-Hydroxy-Δ9-THC (THC-OH) and 11-nor-9-carboxy-Δ9-tetrahydrocannabinol (THC-COOH). Treatment of cardiac myocytes with THC-OH and THC-COOH increased cell migration and proliferation (p < 0.05), with no effect on cell adhesion, with higher doses (250-100 ng/mL) resulting in increased cell death and significant deterioration in cellular architecture. Conversely, no changes in cell morphology or viability were observed in response to THC. Expression of key ECM proteins α-SMA and collagen were up-regulated in response to THC-OH and THC-COOH treatments with concomitant modulation of PI3K and MAPK signalling. Investigations in the planarian animal model Polycelis nigra demonstrated that treatments with cannabinoid metabolites resulted in increased protein deposition at transection sites while higher doses resulted in significant lethality and decline in regeneration. These results highlight that the key metabolites of cannabis elicit toxic effects independent of the parent and psychoactive compound, with implications for cardiotoxicity relating to hypertrophy and fibrogenesis.

Reinforced Aortic Root Reconstruction in Type A Aortic Dissection: A Prospective Study.

BACKGROUND: Type A aortic dissection is a challenging surgical emergency associated with high morbidity and mortality. Many techniques have evolved to repair the dissected sinus segments and restore aortic valve dynamics. Herein, we evaluate the early outcome of a novel technique for reconstruction of dissected aortic root. METHODS: A prospective study was conducted on 300 patients to evaluate the early results of repair of dissected root in type A aortic dissection. The mean age was 59.65±8.52 years, and 76% of patients were males. All patients had four standard steps for aortic reconstruction: 1) commissural resuspension; 2) right coronary sinus reinforcement with pericardial and Dacron bands; 3) non-coronary sinus reinforcement using external Dacron patch; 4) circumferential inversion of adventitial layer of the root. Patients were followed up clinically, echocardiographically, and by CT scan. RESULTS: The in-hospital mortality was 8%. The mean cross-clamp time was 120±30 minutes, and circulatory arrest time was 25+10 minutes. Twenty-seven patients (9%) experienced postoperative complications, including bleeding and acute kidney injury. During a mean follow-up time of 48±12 months, there were no recurrent aortic dissection, aortic dilatation, pseudoaneurysm, or progression of aortic regurgitation during the entire study period. CONCLUSIONS: This reconstructive technique technically is undemanding, feasible, safe, and durable with good early results. A larger cohort of patients with longer period of follow up should generate a more powerful evaluation of this technique.

Formulation and characterization of propolis and tea tree oil nanoemulsion loaded with clindamycin hydrochloride for wound healing: In-vitro and in-vivo wound healing assessment.

This study aimed to develop propolis and tea tree oil nanoemulsion loaded with clindamycin hydrochloride to heal wound effectively. Nanoemulsion formulae were prepared and characterized by droplet size analysis, zeta potential, viscosity, ex-vivo permeation, and skin deposition. The optimal formula was evaluated in terms of morphology, cytotoxicity, and in-vitro wound healing assay. Also, the efficacy of the optimal formula was evaluated by in-vivo wound healing and histopathological studies. The optimal formula (F3) was composed of 9% tea tree oil and 0.4% propolis extracts with mean droplet size 19.42 ± 1.7 nm, zeta potential value -24.5 ± 0.2 mV, and viscosity 69.4 ± 1.8 mP. Furthermore, the optimal formula showed the highest skin deposition value 550.00 ± 4.9 µg/cm2 compared to other formulae. The TEM micrograph of the optimal formula showed that the nanoemulsion droplet has an almost spherical shape. Also, the optimal formula did not show noticeable toxicity to the human skin fibroblast cells. The in-vitro and in-vivo wound healing assay showed unexpected results that the un-loaded drug nanoemulsion formula had a comparable wound healing efficacy to the drug-loaded nanoemulsion formula. These results were confirmed with histopathological studies. Our results showed that the propolis and tea tree oil nanoemulsion, whether loaded or unloaded with an antibiotic, is an efficient local therapy for wound healing.

Marine Carotenoid Fucoxanthin Possesses Anti-Metastasis Activity: Molecular Evidence.

Fucoxanthin is commonly found in marine organisms; however, to date, it has been one of the scarcely explored natural compounds. We investigated its activities in human cancer cell culture-based viability, migration, and molecular assays, and found that it possesses strong anticancer and anti-metastatic activities that work irrespective of the p53 status of cancer cells. In our experiments, fucoxanthin caused the transcriptional suppression of mortalin. Cell phenotype-driven molecular analyses on control and treated cells demonstrated that fucoxanthin caused a decrease in hallmark proteins associated with cell proliferation, survival, and the metastatic spread of cancer cells at doses that were relatively safe to the normal cells. The data suggested that the cancer therapy regimen may benefit from the recruitment of fucoxanthin; hence, it warrants further attention for basic mechanistic studies as well as drug development.

Unlocking Dendritic Cell-Based Vaccine Efficacy through Genetic Modulation-How Soon Is Now?

The dendritic cell (DC) vaccine anti-cancer strategy involves tumour-associated antigen loading and maturation of autologous ex vivo cultured DCs, followed by infusion into the cancer patient. This strategy stemmed from the idea that to induce a robust anti-tumour immune response, it was necessary to bypass the fundamental immunosuppressive mechanisms of the tumour microenvironment that dampen down endogenous innate immune cell activation and enable tumours to evade immune attack. Even though the feasibility and safety of DC vaccines have long been confirmed, clinical response rates remain disappointing. Hence, the full potential of DC vaccines has yet to be reached. Whether this cellular-based vaccination approach will fully realise its position in the immunotherapy arsenal is yet to be determined. Attempts to increase DC vaccine immunogenicity will depend on increasing our understanding of DC biology and the signalling pathways involved in antigen uptake, maturation, migration, and T lymphocyte priming to identify amenable molecular targets to improve DC vaccine performance. This review evaluates various genetic engineering strategies that have been employed to optimise and boost the efficacy of DC vaccines.

Abrogating the Interaction Between p53 and Mortalin (Grp75/HSPA9/mtHsp70) for Cancer Therapy: The Story so far.

p53 is a transcription factor that activates the expression of a set of genes that serve as a critical barrier to oncogenesis. Inactivation of p53 is the most common characteristic in sporadic human cancers. Mortalin is a differentially sub-cellularly localized member of the heat shock protein 70 family of chaperones that has essential mitochondrial and extra-mitochondrial functions. Elevated mortalin levels in multiple cancerous tissues and tumor-derived cell lines emphasized its key role in oncogenesis. One of mortalin's major oncogenic roles is the inactivation of p53. Mortalin binds to p53 sequestering it in the cytoplasm. Hence, p53 cannot freely shuttle to the nucleus to perform its tumor suppressor functions as a transcription factor. This protein-protein interaction was reported to be cancer-specific, hence, a selective druggable target for a rationalistic cancer therapeutic strategy. In this review article, the chronological identification of mortalin-p53 interactions is summarized, the challenges and general strategies for targeting protein-protein interactions are briefly discussed, and information about compounds that have been reported to abrogate mortalin-p53 interaction is provided. Finally, the reasons why the disruption of this druggable interaction has not yet been applied clinically are discussed.

A Low Dose Combination of Withaferin A and Caffeic Acid Phenethyl Ester Possesses Anti-Metastatic Potential In Vitro: Molecular Targets and Mechanisms.

Withaferin A (Wi-A) and Caffeic Acid Phenethyl Ester (CAPE) are the bioactive ingredients of Ashwagandha (Withania somnifera) and propolis, respectively. Both of these natural compounds have been shown to possess anticancer activity. In the present study, we recruited a low dose of each of these compounds and developed a combination that exhibited remarkably potent anti-migratory and anti-angiogenic activities. Extensive molecular analyses including a cDNA array and expression analyses of the specific gene targets demonstrated that such activities are mediated through their effect on cell adhesion/tight junction proteins (Claudins, E-cadherin), inhibition of canonical Wnt/ß-catenin signaling pathways and the consequent downregulation of EMT-signaling proteins (Vimentin, MMPs, VEGF and VEGFR) that play a critical role in cancer metastasis. The data supported that this novel combination of Wi-A and CAPE (Wi-ACAPE, containing 0.5 µM of Wi-A and 10 µM of CAPE) may be recruited for the treatment of metastatic and aggressive cancers and, hence, warrant further evaluation by recruiting a variety of experimental and clinical metastatic models.

Identification and Characterization of MortaparibPlus-A Novel Triazole Derivative That Targets Mortalin-p53 Interaction and Inhibits Cancer-Cell Proliferation by Wild-Type p53-Dependent and -Independent Mechanisms.

p53 has an essential role in suppressing the carcinogenesis process by inducing cell cycle arrest/apoptosis/senescence. Mortalin/GRP75 is a member of the Hsp70 protein family that binds to p53 causing its sequestration in the cell cytoplasm. Hence, p53 cannot translocate to the nucleus to execute its canonical tumour suppression function as a transcription factor. Abrogation of mortalin-p53 interaction and subsequent reactivation of p53's tumour suppression function has been anticipated as a possible approach in developing a novel cancer therapeutic drug candidate. A chemical library was screened in a high-content screening system to identify potential mortalin-p53 interaction disruptors. By four rounds of visual assays for mortalin and p53, we identified a novel synthetic small-molecule triazole derivative (4-[(1E)-2-(2-phenylindol-3-yl)-1-azavinyl]-1,2,4-triazole, henceforth named MortaparibPlus). Its activities were validated using multiple bioinformatics and experimental approaches in colorectal cancer cells possessing either wild-type (HCT116) or mutant (DLD-1) p53. Bioinformatics and computational analyses predicted the ability of MortaparibPlus to competitively prevent the interaction of mortalin with p53 as it interacted with the p53 binding site of mortalin. Immunoprecipitation analyses demonstrated the abrogation of mortalin-p53 complex formation in MortaparibPlus-treated cells that showed growth arrest and apoptosis mediated by activation of p21WAF1, or BAX and PUMA signalling, respectively. Furthermore, we demonstrate that MortaparibPlus-induced cytotoxicity to cancer cells is mediated by multiple mechanisms that included the inhibition of PARP1, up-regulation of p73, and also the down-regulation of mortalin and CARF proteins that play critical roles in carcinogenesis. MortaparibPlus is a novel multimodal candidate anticancer drug that warrants further experimental and clinical attention.

Mutant p53L194F Harboring Luminal-A Breast Cancer Cells Are Refractory to Apoptosis and Cell Cycle Arrest in Response to MortaparibPlus, a Multimodal Small Molecule Inhibitor.

We previously performed a drug screening to identify a potential inhibitor of mortalin-p53 interaction. In four rounds of screenings based on the shift in mortalin immunostaining pattern from perinuclear to pan-cytoplasmic and nuclear enrichment of p53, we had identified MortaparibPlus (4-[(1E)-2-(2-phenylindol-3-yl)-1-azavinyl]-1,2,4-triazole) as a novel synthetic small molecule. In order to validate its activity and mechanism of action, we recruited Luminal-A breast cancer cells, MCF-7 (p53wild type) and T47D (p53L194F) and performed extensive biochemical and immunocytochemical analyses. Molecular analyses revealed that MortaparibPlus is capable of abrogating mortalin-p53 interaction in both MCF-7 and T47D cells. Intriguingly, upregulation of transcriptional activation function of p53 (as marked by upregulation of the p53 effector gene-p21WAF1-responsible for cell cycle arrest and apoptosis) was recorded only in MortaparibPlus-treated MCF-7 cells. On the other hand, MortaparibPlus-treated T47D cells exhibited hyperactivation of PARP1 (accumulation of PAR polymer and decrease in ATP levels) as a possible non-p53 tumor suppression program. However, these cells did not show full signs of either apoptosis or PAR-Thanatos. Molecular analyses attributed such a response to the inability of MortaparibPlus to disrupt the AIF-mortalin complexes; hence, AIF did not translocate to the nucleus to induce chromatinolysis and DNA degradation. These data suggested that the cancer cells possessing enriched levels of such complexes may not respond to MortaparibPlus. Taken together, we report the multimodal anticancer potential of MortaparibPlus that warrants further attention in laboratory and clinical studies.

Differentiation of Capripox Viruses by Nanopore Sequencing.

The genus capripoxvirus (CaPV), family Poxviridae, includes three virus species: goatpox virus (GPV), sheeppox virus (SPV) and lumpy skin disease virus (LSDV). CaPV causes disease outbreaks with consequent economic losses in Africa and the Middle East. LSDV has recently spread to Southeast Europe. As CaPVs share 96-97% genetic similarity along the length of the entire genome and are difficult to distinguish using serological assays, simple, reliable and fast methods for diagnosis and species differentiation are crucial in cases of disease outbreak. The present study aimed to develop a field-applicable CaPV differentiation method. Nanopore technology was used for whole genome sequencing. A local database of complete CaPV genomes and partial sequences of three genes (RPO30, P32 and GPCR) was established for offline Basic Local Alignment Search Tool (BLAST). Specificities of 98.04% in whole genome and 97.86% in RPO30 gene runs were obtained among the three virus species, while other databases were less specific. The total run time was shortened to approximately 2 h. Functionality of the developed procedure was proved by samples with high host background sequences. Reliable differentiation options for the quality and capacity of hardware, and sample quality of suspected cases, were derived from these findings. The whole workflow can be performed rapidly with a mobile suitcase laboratory and mini-computer, allowing application at the point-of-need with limited resource settings.

Soyasapogenol-A targets CARF and results in suppression of tumor growth and metastasis in p53 compromised cancer cells.

We screened some phytochemicals for cytotoxic activity to human cancer cells and identified Soyasapogenol-A (Snol-A) as a potent candidate anti-cancer compound. Interestingly, Soyasapogenin-I (Snin-I) was ineffective. Viability assays endorsed toxicity of Snol-A to a wide variety of cancer cells. Of note, wild type p53 deficient cancer cells (SKOV-3 and Saos-2) also showed potent growth inhibitory effect. Molecular analyses demonstrated that it targets CARF yielding transcriptional upregulation of p21WAF1 (an inhibitor of cyclin-dependent kinases) and downregulation of its effector proteins, CDK2, CDK-4, Cyclin A and Cyclin D1. Targeting of CARF by Snol-A also caused (i) downregulation of pATR-Chk1 signaling leading to caspase-mediated apoptosis and (ii) inactivation of ß-catenin/Vimentin/hnRNPK-mediated EMT signaling resulting in decrease in migration and invasion of cancer cells. In in vivo assays, Snol-A caused suppression of tumor growth in subcutaneous xenograft model and inhibited lung metastasis in tail vein injection model. Taken together, we demonstrate that Snol-A is a natural inhibitor of CARF and may be recruited as a potent anti-tumor and anti-metastasis compound for treatment of p53-deficient aggressive malignancies.