Identification of CRKL as an oncogenic biomarker for prognosis and immunotherapy in melanoma, and its potential molecular mechanism.

CRKL (CRK Like Proto-Oncogene) belongs to the Crk family and is a 39-kDa adapter protein that encodes SH2 and SH3 (src homologs) domains. To identify its oncogenic role in malignant melanoma, we investigated the association between CRKL and mutation, prognosis, tumor mutation burden, immune cell infiltration of melanoma, and explored the associations between CRKL and immunotherapy response. Our results showed that abnormal CRKL expression is associated with poor prognosis in melanoma and is significantly correlated with immune-activated pathways and processes, immune cell infiltrations, and expression of immunoregulators. Importantly, we found that CRKL expression is a predictive biomarker for anti-PD1 therapy response in melanoma patients. Furthermore, inhibiting CRKL expression in melanoma cell lines suppressed their proliferation and metastasis, as well as activated the pyroptosis-related pathway. Our study provides potential mechanisms of melanoma pathogenesis, which may suggest new avenues for targeted therapy in this disease.

Developmental Xist induction is mediated by enhanced splicing.

X-inactive-specific transcript (Xist) is a long noncoding RNA (lncRNA) essential for inactivating one of the two X chromosomes in mammalian females. Random X chromosome inactivation is mediated by Xist RNA expressed from the inactive X chromosome. We found that Xist RNA is unspliced in naïve embryonic stem (ES) cells. Upon differentiation, Xist splicing becomes efficient across all exons independent of transcription, suggesting interdependent or coordinated removal of Xist introns. In female cells with mutated polypyrimidine tract binding protein 1 (Ptbp1), differentiation fails to substantially upregulate mature Xist RNA because of a defect in Xist splicing. We further found both Xist129 and XistCAS RNA are unspliced in Mus musculus 129SvJ/Mus castaneous (CAS) hybrid female ES cells. Upon differentiation, Xist129 exhibits a higher splicing efficiency than XistCAS, likely contributing to preferential inhibition of the X129 chromosome. Single cell analysis shows that the allelic choice of Xist splicing is linked to the inactive X chromosome. We conclude post-transcriptional control of Xist RNA splicing is an essential regulatory step of Xist induction. Our studies shed light on the developmental roles of splicing for nuclear-retained Xist lncRNA and suggest inefficient Xist splicing is an additional fail-safe mechanism to prevent Xist activity in ES cells.

Inhibition of nonsense-mediated RNA decay by ER stress.

Nonsense-mediated RNA decay (NMD) selectively degrades mutated and aberrantly processed transcripts that contain premature termination codons (PTC). Cellular NMD activity is typically assessed using exogenous PTC-containing reporters. We overcame some inherently problematic aspects of assaying endogenous targets and developed a broadly applicable strategy to reliably and easily monitor changes in cellular NMD activity. Our new method was genetically validated for distinguishing NMD regulation from transcriptional control and alternative splicing regulation, and unexpectedly disclosed a different sensitivity of NMD targets to NMD inhibition. Applying this robust method for screening, we identified NMD-inhibiting stressors but also found that NMD inactivation was not universal to cellular stresses. The high sensitivity and broad dynamic range of our method revealed a strong correlation between NMD inhibition, endoplasmic reticulum (ER) stress, and polysome disassembly upon thapsigargin treatment in a temporal and dose-dependent manner. We found little evidence of calcium signaling mediating thapsigargin-induced NMD inhibition. Instead, we discovered that of the three unfolded protein response (UPR) pathways activated by thapsigargin, mainly protein kinase RNA-like endoplasmic reticulum kinase (PERK) was required for NMD inhibition. Finally, we showed that ER stress compounded TDP-43 depletion in the up-regulation of NMD isoforms that had been implicated in the pathogenic mechanisms of amyotrophic lateral sclerosis and frontotemporal dementia, and that the additive effect of ER stress was completely blocked by PERK deficiency.

Enhancing cellular behavior in repaired tissue via silk fibroin-integrated triboelectric nanogenerators.

Triboelectric nanogenerators (TENGs) have emerged as a promising approach for generating electricity and providing electrical stimuli in medical electronic devices. Despite their potential benefits, the clinical implementation of TENGs faces challenges such as skin compliance and a lack of comprehensive assessment regarding their biosafety and efficacy. Therefore, further research is imperative to overcome these limitations and unlock the full potential of TENGs in various biomedical applications. In this study, we present a flexible silk fibroin-based triboelectric nanogenerator (SFB-TENG) that features an on-skin substrate and is characterized by excellent skin compliance and air/water permeability. The range of electrical output generated by the SFB-TENG was shown to facilitate the migration and proliferation of Hy926, NIH-3T3 and RSC96 cells. However, apoptosis of fibroblast NIH-3T3 cells was observed when the output voltage increased to more than 20 V at a frequency of 2 Hz. In addition, the moderate electrical stimulation provided by the SFB-TENG promoted the cell proliferation cycle in Hy926 cells. This research highlights the efficacy of a TENG system featuring a flexible and skin-friendly design, as well as its safe operating conditions for use in biomedical applications. These findings position TENGs as highly promising candidates for practical applications in the field of tissue regeneration.

Epistatic interactions between NMD and TRP53 control progenitor cell maintenance and brain size.

Mutations in human nonsense-mediated mRNA decay (NMD) factors are enriched in neurodevelopmental disorders. We show that deletion of key NMD factor Upf2 in mouse embryonic neural progenitor cells causes perinatal microcephaly but deletion in immature neurons does not, indicating NMD's critical roles in progenitors. Upf2 knockout (KO) prolongs the cell cycle of radial glia progenitor cells, promotes their transition into intermediate progenitors, and leads to reduced upper-layer neurons. CRISPRi screening identified Trp53 knockdown rescuing Upf2KO progenitors without globally reversing NMD inhibition, implying marginal contributions of most NMD targets to the cell cycle defect. Integrated functional genomics shows that NMD degrades selective TRP53 downstream targets, including Cdkn1a, which, without NMD suppression, slow the cell cycle. Trp53KO restores the progenitor cell pool and rescues the microcephaly of Upf2KO mice. Therefore, one physiological role of NMD in the developing brain is to degrade selective TRP53 targets to control progenitor cell cycle and brain size.

Quantitative Measurement of Alternatively Spliced RNA Isoform Levels.

Conventional approaches to quantify alternative splicing are exon-centric and derive a ratio based on relative levels of the isoforms (or isoform groups) that include versus exclude a particular alternative RNA segment. The ratio measurement to study alternative splicing regulation can be confounded when alternative isoforms undergo differential RNA decay, for example, nonsense-mediated mRNA decay (NMD). Isoform-centric quantification is more informative for functional studies of alternative splicing, but challenges remain in distinguishing specific isoforms. Here, we provide a practical guide on addressing the specificity of isoform quantification and describe a simple sensitive method. Quantitative measurement of alternatively spliced RNA isoforms can be used to differentiate splicing regulation from transcriptional control and isoform-specific RNA decay regulation.

Molecular profiling of individual FDA-approved clinical drugs identifies modulators of nonsense-mediated mRNA decay.

Nonsense-mediated mRNA decay (NMD) degrades transcripts with premature stop codons. Given the prevalence of nonsense single nucleotide polymorphisms (SNPs) in the general population, it is urgent to catalog the effects of clinically approved drugs on NMD activity: any interference could alter the expression of nonsense SNPs, inadvertently inducing adverse effects. This risk is higher for patients with disease-causing nonsense mutations or an illness linked to dysregulated nonsense transcripts. On the other hand, hundreds of disorders are affected by cellular NMD efficiency and may benefit from NMD-modulatory drugs. Here, we profiled individual FDA-approved drugs for their impact on cellular NMD efficiency using a sensitive method that directly probes multiple endogenous NMD targets for a robust readout of NMD modulation. We found most FDA-approved drugs cause unremarkable effects on NMD, while many elicit clear transcriptional responses. Besides several potential mild NMD modulators, the anticancer drug homoharringtonine (HHT or omacetaxine mepesuccinate) consistently upregulates various endogenous NMD substrates in a dose-dependent manner in multiple cell types. We further showed translation inhibition mediates HHT's NMD effect. In summary, many FDA drugs induce transcriptional changes, and a few impact global NMD, and direct measurement of endogenous NMD substrate expression is robust to monitor cellular NMD.

Establishment of open web platform based on 3D head model for product adaptability analysis and evaluation.

With improved living conditions, the demand for wearable products has increased, particularly for headwear. Traditionally, the dimensions of headwear products are designed according to 1D anthropometric data, such as head length, width, and eye width. However, this design method, applied to fit the 3D human head, results in differences in adaptability, which affect the wearing comfort of the user. Hence, this study aimed to develop an aided design platform based on a three-dimensional standard head shape to evaluate the virtual wearing of headwear products and adaptability testing. Specifically, a WebGL-based service, named the Chinese Headwear Adaptability Testing (CHAT) platform, was established. Validation and user studies were then conducted using an eye massager device. After the product was redesigned according to evaluation by the proposed system, the results show that the comfort and adaptability of the redesigned eye massager were significantly improved. The findings suggest that using the CHAT platform to design headwear products can help achieve a better wearing fit for the products.

Knockdown of enhancer of rudimentary homolog expression attenuates proliferation, cell cycle and apoptosis of melanoma cells.

Early stage or localized melanoma can be surgically resected with satisfactory outcome, whereas advanced malignant melanoma responds to treatment poorly and has a negative prognosis even after surgery, radiotherapy and other comprehensive treatments. Gene therapy targeting various biological signaling pathways has become an increasingly popular area in melanoma research. However, for gene therapy success, it is important to reveal the molecular mechanisms of melanoma tumorigenesis and development. The present study examined the effects of downregulating enhancer of rudimentary homolog (ERH) expression on the proliferation, metastasis and cell cycle of melanoma cells. ERH expression levels in melanoma tissues and cells were determined. Then, ERH gene expression in melanoma cell lines was downregulated or overexpressed by the lentiviral RNA interference technique. Furthermore, we performed cell counting kit-8, clone formation, scratch, transwell migration, subcutaneous tumorigenesis and venous metastasis assays as well as carried out flow cytometry analysis to explore the effects of ERH expression on cell proliferation, cell cycle, apoptosis and metastasis. We found that ERH expression in melanoma tissues and cells was markedly higher than in normal melanin nevus. Suppressing ERH expression by RNA interference in melanoma A375, WM35 and SK28 cell lines inhibited their proliferation and induced cell apoptosis. The cell cycle was also found to be blocked in the G1 phase. However, the metastatic properties of melanoma cells in vitro and in vivo remained largely unaltered by ERH knockdown. Our results show that ERH expression is increased in melanoma. Meanwhile, the proliferation and cell cycle transformation abilities are impaired potentially by downregulating the ERH expression in melanoma cells. Therefore, targeting ERH might serve as a novel therapeutic approach for malignant melanoma.