Skin rejuvenation and photoaging protection using adipose-derived stem cell extracellular vesicles loaded with exogenous cargos.

BACKGROUND: Small extracellular vesicles from adipose-derived stem cells (ASC-sEVs) have gained remarkable attention for their regenerative and protective properties against skin aging. However, the use of ASC-sEVs to further encapsulate certain natural anti-aging compounds for synergistic effects has not been actively explored. For large-scale production in skincare industry, it is also crucial to standardize cost-effective methods to produce highly pure ASC-sEVs. METHODS: Human ASCs were expanded in serum-free media with different compositions to first optimize the sEV production. ASC-sEVs from different batches were then purified using tangential flow filtration and sucrose cushion ultracentrifugation, followed by extensive characterization for identity and content profiling including proteomics, lipidomics and miRNA sequencing. ASC-sEVs were further loaded with nicotinamide riboside (NR) and resveratrol by sonication-incubation method. The therapeutic effect of ASC-sEVs and loaded ASC-sEVs was tested on human keratinocyte cell line HaCaT exposed to UVB by measuring reactive oxygen species (ROS). The loaded ASC-sEVs were later applied on the hand skin of three volunteers once a day for 8 weeks and skin analysis was performed every 2 weeks. RESULTS: Our standardized workflow produced ASC-sEVs with high yield, high purity and with stable characteristics and consistent biocargo among different batches. The most abundant subpopulations in ASC-sEVs were CD63+ (∼30%) and CD81+ -CD63+ (∼35%). Purified ASC-sEVs could be loaded with NR and resveratrol at the optimized loading efficiency of ∼20%. In UVB-exposed HaCaT cells, loaded ASC-sEVs could reduce ROS by 38.3%, higher than the sEVs (13.3%) or compounds (18.5%) individually. In human trial, application of loaded ASC-sEVs after 8 weeks substantially improved skin texture, increased skin hydration and elasticity by 104% and reduced mean pore volume by 51%. CONCLUSIONS: This study demonstrated a robust protocol to produce ASC-sEVs and exogenously load them with natural compounds. The loaded ASC-sEVs exhibited synergistic effects of both sEVs and anti-aging compounds in photoaging protection and skin rejuvenation.

Denatured collagen inhibits neuroblastoma tumor-sphere migration and growth via the LOX/LOXL2 - FAK signaling pathway.

A complex interplay exists within the tumor microenvironment and extracellular matrix, which could contribute to solid tumor progression. Collagen, a major component of the extracellular matrix, may correlate with cancer prognosis. While thermal ablation has shown promise as a minimally invasive treatment of solid tumors, its impact on collagen is still unknown. In this study, we demonstrate that thermal ablation, but not cryo-ablation, induces irreversible collagen denaturation in a neuroblastoma sphere model. Prolonged collagen denaturation resulted in a significant reduction in sphere stiffness, migration, and proliferation, and an increase in apoptosis. Mechanistic analysis revealed that collagen denaturation inhibited collagen cross-linking, reduced extracellular LOX/LOXL2 expression, and resulted in decreased phosphorylation of FAK. Downstream of FAK, we observed reduced epithelial to mesenchymal transition, attenuated CDC42 expression, and decreased migration. Collectively, these results suggest that denatured collagen presents a novel target for modulating the tumor microenvironment and treating solid cancers via the LOX1/LOXL2-FAK signaling pathway.

ARID1A-SIN3A drives retinoic acid-induced neuroblastoma differentiation by transcriptional repression of TERT.

Aggressive, high-risk neuroblastoma (NB) exhibits an immature differentiation state, profound epigenetic dysregulation and high telomerase activity. It has been suggested that aggressive NB may be treatable by inducing differentiation whereas therapeutic targeting of telomerase is under investigation for multiple cancer types. While epigenetic regulation of the telomerase reverse transcriptase (TERT) promoter has been described in high-risk NB, the exact molecular mechanisms are still not completely understood. Here we used quantitative real-time polymerase chain reaction (PCR), chromatin immunoprecipitation qPCR, quantitative telomeric repeat amplification protocol, and immunoblot techniques to investigate epigenetic regulation of TERT in wild-type and genetically modified NB cell lines. We demonstrated that TERT expression is reduced during 13-cis retinoic acid-induced NB differentiation and that this inversely correlated with increased expression of AT-rich interaction domain 1A (ARID1A), a subunit of the SWItch/sucrose nonfermentable chromatin remodeling complex. We showed that ARID1A directly caused suppression of TERT and was reliant on DNA binding and co-occupancy of the TERT promoter by the SIN3 transcription regulator family member A (SIN3A) repressor complex allowing NB differentiation to proceed. Finally, using data from NB patient cohorts, we reported a significant correlation between low ARID1A expression, elevated expression of TERT, and poorly differentiated, high-risk NB. These results provide insights into a key epigenetic pathway responsible for modulating TERT-driven NB progression, which could represent a target for therapeutic intervention.

Cellular TRIM33 restrains HIV-1 infection by targeting viral integrase for proteasomal degradation.

Productive HIV-1 replication requires viral integrase (IN), which catalyzes integration of the viral genome into the host cell DNA. IN, however, is short lived and is rapidly degraded by the host ubiquitin-proteasome system. To identify the cellular factors responsible for HIV-1 IN degradation, we performed a targeted RNAi screen using a library of siRNAs against all components of the ubiquitin-conjugation machinery using high-content microscopy. Here we report that the E3 RING ligase TRIM33 is a major determinant of HIV-1 IN stability. CD4-positive cells with TRIM33 knock down show increased HIV-1 replication and proviral DNA formation, while those overexpressing the factor display opposite effects. Knock down of TRIM33 reverts the phenotype of an HIV-1 molecular clone carrying substitution of IN serine 57 to alanine, a mutation known to impair viral DNA integration. Thus, TRIM33 acts as a cellular factor restricting HIV-1 infection by preventing provirus formation.

SCN1A Gene Mutation and Adaptive Functioning in 18 Vietnamese Children with Dravet Syndrome.

BACKGROUND AND PURPOSE: Dravet syndrome is a rare and severe type of epilepsy in infants. The heterogeneity in the overall intellectual disability that these patients suffer from has been attributed to differences in genetic background and epilepsy severity. METHODS: Eighteen Vietnamese children diagnosed with Dravet syndrome were included in this study. SCN1A variants were screened by direct sequencing and multiplex ligation-dependent probe amplification. Adaptive functioning was assessed in all patients using the Vietnamese version of the Vineland Adaptive Behavior Scales, and the results were analyzed relative to the SCN1A variants and epilepsy severity. RESULTS: We identified 13 pathogenic or likely pathogenic variants, including 6 that have not been reported previously. We found no correlations between the presence or type of SCN1A variants and the level of adaptive functioning impairment or severity of epilepsy. Only two of nine patients aged at least 5 years had an adaptive functioning score higher than 50. Both of these patients had a low frequency of convulsive seizures and no history of status epilepticus or prolonged seizures. The remaining seven had very low adaptive functioning scores (39 or less) despite the variability in the severity of their epilepsy confirming the involvement of factors other than the severity of epilepsy in determining the developmental outcome. CONCLUSIONS: Our study expands the spectrum of known SCN1A variants and confirms the current understanding of the role of the genetic background and epilepsy severity in determining the developmental outcome of Dravet syndrome patients.