RCC2 promotes prostate cancer cell proliferation and migration through Hh/GLI1 signaling pathway and cancer stem-like cells.

BACKGROUND: Regulator of chromosome condensation 2 (RCC2) was a telophase disk-binding protein on mitosis, and functions as an oncogene in many human cancers. However, its role on prostate cancer (PCa) was unknown. The goal of this study is to explore the function of RCC 2 on PCa development. METHODS: The expression of RCC2 and its methylation level, its correlation with lymph node metastasis or disease-free survival (DFS) was analyzed using TCGA database. The effect of RCC2 on PCa cell proliferation, migration and invasion were detected using CCK-8, cell colony formation, Transwell and wood healing assays. RNA-seq and GSEA analysis were used to search the downstream genes and pathways of RCC2 in mediated PCa progression. Western blot was used to detect the proteins in PCa cells transfected with indicated siRNAs or plasmids. RESULTS: RCC2 had high expression and low promoter methylation level in PCa, and its expression was correlated with regional node metastasis and disease-free survival. Cell proliferation, migration, invasion and EMT of PCa cells in vitro were greatly enhanced after RCC2 overexpression, while the RCC2 knockdown suppressed these processes. RNA-seq and GSEA results showed the Hedgehog signaling regulator Gli1 and Gli3 were involved in RCC2 knockdown DU145 cells. Gli1 was also a marker of cancer stem-like cells (CSCs). Mechanistically, RCC2 induced cell growth, EMT, CSCs markers through Gli1; inhibiting Gli1 expression using siGli1 or GLI inhibitor suppressed cell progression in vitro and tumor growth in vivo. CONCLUSION: In summary, RCC2 promoted PCa development through Hh/Gli1 signaling pathway via regulating EMT and CSCs.

Model for predicting prognosis and immunotherapy based on CD+8 T cells infiltration in neuroblastoma.

BACKGROUND: Neuroblastoma (NBL) is an extracranial malignant tumor in children deriving from the neural crest in the sympathetic nervous system. Although various immunotherapy interventions have made significant breakthroughs in many adult cancers, the efficacy of these immunotherapies was still limited in NBL. NBL has low immunogenicity which results in a lack of tumor-infiltrating T lymphocytes in the tumor microenvironment (TME). Moreover, tumor cells can wield many immune evasion strategies both in the TME and systemically to impede lymphocyte infiltration and activation. All these factors hamper the anti-tumor effects of CD8+ T cells during immunotherapy and the levels of infiltrating CD8+ T cells correlate with therapy response. MATERIALS AND METHODS: In this study, we utilized multidimensional bioinformatic methods to establish a risk model based on CD8+ T cells -related genes (CD8+ TRGs). RESULTS: We obtained 33 CD8+ TRGs with well-predictive ability for prognosis in both GSE49711 and E-MTAB-8248 cohorts. Then, 12 CD8+ TRGs including HK2, RP2, HPSE, ELL2, GFI1, SLC22A16, FCGR3A, CTSS, SH2D1A, RBP5, ATF5, and ADAM9 were finally identified for risk model construction and validation. This model revealed a stable performance in prognostic prediction of the overall survival (OS) and event-free survival (EFS) in patients with NBL. Additionally, our research indicated that the immune and stromal scores, immune-related pathways, immune cell infiltration, the expression of major histocompatibility complex (MHC) and immune checkpoint molecules, immunotherapy response, and drug susceptibility revealed significant differences between high and low-risk groups. CONCLUSIONS: According to our analyses, the constructed CD8+ TRGs-based risk model may be promising for the clinical prediction of anti-tumor therapy responses and prognoses in NBL.

PI(4,5)P2-dependent regulation of endothelial tip cell specification contributes to angiogenesis.

Dynamic positioning of endothelial tip and stalk cells, via the interplay between VEGFR2 and NOTCH signaling, is essential for angiogenesis. VEGFR2 activates PI3K, which phosphorylates PI(4,5)P2 to PI(3,4,5)P3, activating AKT; however, PI3K/AKT does not direct tip cell specification. We report that PI(4,5)P2 hydrolysis by the phosphoinositide-5-phosphatase, INPP5K, contributes to angiogenesis. INPP5K ablation disrupted tip cell specification and impaired embryonic angiogenesis associated with enhanced DLL4/NOTCH signaling. INPP5K degraded a pool of PI(4,5)P2 generated by PIP5K1C phosphorylation of PI(4)P in endothelial cells. INPP5K ablation increased PI(4,5)P2, thereby releasing ß-catenin from the plasma membrane, and concurrently increased PI(3,4,5)P3-dependent AKT activation, conditions that licensed DLL4/NOTCH transcription. Suppression of PI(4,5)P2 in INPP5K-siRNA cells by PIP5K1C-siRNA, restored ß-catenin membrane localization and normalized AKT signaling. Pharmacological NOTCH or AKT inhibition in vivo or genetic ß-catenin attenuation rescued angiogenesis defects in INPP5K-null mice. Therefore, PI(4,5)P2 is critical for ß-catenin/DLL4/NOTCH signaling, which governs tip cell specification during angiogenesis.

Inferences of carboplatin response-related signature by integrating multiomics data in ovarian serous cystadenocarcinoma.

Platinum-based chemotherapy, especially carboplatin, is the primary measure to treat patients with ovarian cancer (OC). However, OC patients still have an adverse prognosis due to emergency of chemotherapy resistance. Ovarian serous cystadenocarcinoma (OSC) is the most common histological subtype of OC. Therefore, identifying the key factors that affect chemotherapy resistance and searching novel treatments had become a top priority. In this study, we analyzed carboplatin response-related mRNA, miRNA, DNA methylation, and alternative splicing (AS) and established a drug-resistant signature for carboplatin in OSC. This drug-resistant signature was obviously higher in resistant group than in non-resistant group and had accuracy predictive performance, which demonstrated that this signature could be considered as a superior indicator for OSC patients with carboplatin resistance. Furthermore, we selected three potential small molecule drugs including liranaftate, siguazodan, and tramiprostate to inhibit carboplatin resistance of OSC. In addition, we also identified ZINC00000205417, ZINC00000140928, and ZINC00021908260 were potential small molecule compounds for SLC17A7 based on Molecular Operating Environment (MOE) virtual screening. Finally, we confirmed the drug-like properties of these small molecule drugs via evaluating absorption, distribution, metabolism, elimination, and toxicity (ADMET) property. In summary, the signature could be used as biomarker for carboplatin resistance and small molecule drugs targeting these genes could improve clinical treatment for OSC in the future.

Subcutaneous etonogestrel implant combined with endometrial ablation for the treatment of adenomyosis: two case reports.

Adenomyosis is a common disease that affects many premenopausal women. Two patients with adenomyosis, aged 51 and 42 years, presented with dysmenorrhea and increased menstrual volume. They refused laparoscopy or laparotomy surgery and were not eligible for the levonorgestrel-releasing intrauterine system (LNG-IUS). The first patient underwent endometrial ablation and subcutaneous etonogestrel (ENG)-releasing implant placement at the same time. Her symptoms of dysmenorrhea and heavy menstruation improved significantly. When serum follicle-stimulating hormone (FSH) and estradiol (E2) levels suggested menopause, the ENG-releasing implant was removed. However, her abdominal pain recurred and was relieved by medication. For the second patient, an ENG-releasing implant was placed first, and her dysmenorrhea and heavy menstrual volume were relieved. However, the bleeding pattern changed from regular bleeding to prolonged bleeding, which troubled the patient. Endometrial ablation was performed 4 months later to solve the problem. Both patients had improved symptoms and were satisfied with the treatment. For patients with adenomyosis who refuse surgery and are not candidates for the use of LNG-IUS, an ENG-releasing implant combined with endometrial ablation may be an effective alternative.

Single-cell RNA sequencing reveals intratumoral heterogeneity and potential mechanisms of malignant progression in prostate cancer with perineural invasion.

Background: Prostate cancer (PCa) is the second most common cancer among men worldwide. Perineural invasion (PNI) was a prominent characteristic of PCa, which was recognized as a key factor in promoting PCa progression. As a complex and heterogeneous disease, its true condition is difficult to explain thoroughly with conventional bulk RNA sequencing. Thus, an improved understanding of PNI-PCa progression at the single-cell level is needed. Methods: In this study, we performed scRNAseq on tumor tissues of three PNI-PCa patients. Principal component analysis (PCA) and Uniform manifold approximation and projection (UMAP) were used to reduce dimensionality and visualize the cellular composition of tumor tissues. The differently expressed genes among each cluster were identified by EdgeR. GO enrichment analysis was used to understand the roles of genes within the clusters. Pseudotime cell trajectory was used to reveal the molecular pathways underlying cell fate decisions and identify genes whose expression changed as the cells underwent transition. We applied CellPhoneDB to identify cell-cell interactions among the epithelial and neural cells in PNI-PCa. Results: Analysis of the ∼17,000 single-cell transcriptomes in three PNI prostate cancer tissues, we identified 12 major cell clusters, including neural cells and two epithelial subtypes with different expression profiles. We found that basal/intermediate epithelial cell subtypes highly expressed PCa progression-related genes, including PIGR, MMP7, and AGR2. Pseudotime trajectory analysis showed that luminal epithelial cells could be the initiating cells and transition to based/intermediate cells. Gene ontology (GO) enrichment analysis showed that pathways related to cancer progressions, such as lipid catabolic and fatty acid metabolic processes, were significantly enriched in basal/intermediate cells. Our analysis also suggested that basal/intermediate cells communicate closely with neural cells played a potential role in PNI-PCa progression. Conclusion: These results provide our understanding of PNI-PCa cellular heterogeneity and characterize the potential role of basal/intermediate cells in the PNI-PCa progression.

N-Butylphthalide vs. Human Urinary Kallidinogenase for the Treatment of Acute Ischemic Stroke: Functional Outcome and Impact on Serum VEGF and TNF-α Expressions.

OBJECTIVE: To compare the e!cacy and functional outcomes of dl-3-n-Butylphthalide (NBP) and human urinary kallidinogenase (HUK) on ischemic stroke patients and to determine their effects on serum tumor necrosis factor-alpha (TNF-α) and vascular endothelial growth factor (VEGF). METHODS: A prospective study was conducted on 57 ischemic stroke patients. Functional outcomes were assessed by the National Institute Health Stroke Scale (NIHSS), the modified Rankin Scale (mRS), and the activities of daily living score (ADL), whereas TNF-α and VEGF expressions were measured by enzyme-linked immunosorbent assay (ELISA). RESULTS: TNF-α was significantly down-regulated in the NBP group and upregulated in the control group two weeks after treatment (p=0.017 and p=0.047, respectively). A significant difference in VEGF expressions was observed between the two groups (330.25±120.64 vs. 437.15±137.68, p=0.041) two weeks after treatment. Both groups showed significant improvement in NIHSS and ADL scores three months after treatment (p<0.001), with the NBP group exhibiting improvement in NIHSS scores as early as two weeks after treatment (p=0.008). The three-month NIHSS scores of the two groups were significantly lower than those of the control group (p=0.010 and p=0.008, respectively). Both the NBP and HUK groups showed a significant decline in mRS scores two weeks and three months after treatment (p<0.05). CONCLUSIONS: Both treatments are effective and can significantly promote recovery in stroke patients. Additionally, both options have similar effects in promoting long-term recovery, with NBP exerting a greater impact on serum VEGF and TNF-α expressions.

Development of alternative splicing signature in lung squamous cell carcinoma.

Increasing evidence demonstrated that alternative splicing (AS) plays a vital role in tumorigenesis and clinical outcome of patient. However, systematical analysis of AS in lung squamous cell carcinoma (LUSC) is lacking and greatly necessary. Thus, this study was to systematically estimate the function of AS events served as prognostic indicators in LUSC. Among 31,345 mRNA AS events in 9633 genes, we detected 1996 AS in 1409 genes which have significant connection with overall survival (OS) of LUSC patients. Then, prognostic model based on seven types of AS events was established and we further constructed a combined prognostic model. The Kaplan-Meier curve results suggested that seven types of AS signatures and the combined prognostic model could exhibit robust performance in predicting prognosis. Patients in the high-risk group had significantly shorter OS than those in the low-risk group. The ROC showed all prognostic models had high accuracy and powerful predictive performance with different AUC ranging from 0.837 to 0.978. Moreover, the combined prognostic model had highest performance in risk stratification and predictive accuracy than single prognostic models and had higher accuracy than other mRNA model. Finally, a significant correlation network between survival-related AS genes and prognostic splicing factors (SFs) was established. In conclusion, our study provided several potential prognostic AS models and constructed splicing network between AS and SFs in LUSC, which could be used as potential indicators and treatment targets for LUSC patients.

The performance of the alarmin HMGB1 in pediatric diseases: From lab to clinic.

INTRODUCTION: The ubiquitously expressed nonhistone nuclear protein high-mobility group box protein 1 (HMGB1) has different functions related to posttranslational modifications and cellular localization. In the nucleus, HMGB1 modulates gene transcription, replication and DNA repair as well as determines chromosomal architecture. When the post-transcriptional modified HMGB1 is released into the extracellular space, it triggers several physiological and pathological responses and initiates innate immunity through interacting with its reciprocal receptors (i.e., TLR4/2 and RAGE). The effect of HMGB1-mediated inflammatory activation on different systems has received increasing attention. HMGB1 is now considered to be an alarmin and participates in multiple inflammation-related diseases. In addition, HMGB1 also affects the occurrence and progression of tumors. However, most studies involving HMGB1 have been focused on adults or mature animals. Due to differences in disease characteristics between children and adults, it is necessary to clarify the role of HMGB1 in pediatric diseases. METHODS AND RESULTS: Through systematic database retrieval, this review aimed to first elaborate the characteristics of HMGB1 under physiological and pathological conditions and then discuss the clinical significance of HMGB1 in the pediatric diseases according to different systems. CONCLUSIONS: HMGB1 plays an important role in a variety of pediatric diseases and may be used as a diagnostic biomarker and therapeutic target for new strategies for the prevention and treatment of pediatric diseases.

Polypyrrole-coated Fe2O3 nanotubes constructed from nanoneedles as high-performance anodes for aqueous asymmetric supercapacitors.

Asymmetric supercapacitors (ASCs) show promising potential for electrochemical energy storage applications. However, the energy density of ASCs is limited by the poor electrochemical performance of anodes. To achieve high-performance ASCs, herein, Fe2O3 nanotubes constructed from Fe2O3 nanoneedles were fabricated by employing MnO2 nanotubes as a self-sacrificing template, and then a layer of polypyrrole (PPy) was coated through an in situ chemical oxidative polymerization method to enhance their performance. The electrochemical tests indicate that the resultant PPy-coated Fe2O3 nanotubes (Fe2O3@PPy) exhibit a high areal capacitance of 530 mF cm-2 at 1 mA cm-2 and good cycling stability, which are superior to those of the Fe2O3 nanotubes. The superior performance of the Fe2O3@PPy nanotubes can be attributed to the synergistic effect between the PPy shell and Fe2O3 core, in which the conducting PPy shell not only works as a superhighway for charge transport, but also stabilizes the Fe2O3 nanotubes during charge-discharge processes. When the Fe2O3@PPy nanotubes were assembled with MnO2 nanotubes, the as-assembled ASCs possess a high cell voltage of 2.0 V and deliver a high energy density of up to 51.2 Wh kg-1 at a power density of 285.4 W kg-1 with an excellent cycling stability (83.5% capacitance retention over 5000 cycles).

cis-Silicon phthalocyanine conformation endows J-aggregated nanosphere with unique near-infrared absorbance and fluorescence enhancement: a tumor sensitive phototheranostic agent with deep tissue penetrating ability.

Organic phototheranostic nanomedicines with an optimized near-infrared (NIR) biological transparent window (700-900 nm) are highly desirable for the diagnosis and treatment of deep-seated tumors in clinic. As excellent organic photosensitizers for photodynamic therapy (PDT) with outstanding photo- and thermo-stability, phthalocyanines (Pcs) have been used as the building blocks of single-component nanomedicines. However, to the best of our knowledge, all the Pc-based single-component self-assemblies reported to date are of an H-aggregate nature. This results in the simultaneous self-quenching of fluorescence emission and photodynamic activity as well as greatly reduced tissue penetration due to blue-shifted absorption. In the present work, intramolecular hydrogen bonding was formed between the two long and flexible axial NH2-terminated diethylene glycol ligands of the amphiphilic SiPc molecule (SiPc-NH2) in solution, leading to the employment of a cis-conformation of this molecule according to the 1H-NMR spectroscopy result, which as a building block then further self-assembled into monodisperse nanospheres (SiPcNano) with a J-aggregation nature on the basis of electronic absorption spectroscopic results. As a result, SiPcNano exhibited significantly enhanced red-shifted absorption in the NIR range of 750-850 nm and fluorescence emission. This in combination with the increased photodynamic effect for SiPcNano triggered by the protonation of amine groups due to the acidic nature of tumors endowed effective synergistic NIR photodynamic and photothermal effects in different cancer cells and thus effective inhibition of tumor growth in A549 tumor-bearing mice on the basis of a series of in vitro and in vivo evaluations. The present result provides a new approach for constructing novel single-component NIR organic nanomedicines for multifunctional cancer therapy.

Overexpression of Mesothelin in Pancreatic Ductal Adenocarcinoma (PDAC).

Purpose: Pancreatic ductal adenocarcinoma (PDAC) with difficulty in early diagnosis does not respond well to conventional treatments and has not occurred significant improvement in the overall 5-year survival rates. Mesothelin (MSLN) is a tumor differentiation antigen expressed in several solid neoplasms and a limited number of healthy tissues. Its selective expression on malignant cells makes it an interesting candidate for investigation as a diagnostic and prognostic biomarker and as a therapeutic target. In this study, we detected the expression of MSLN in PDAC and analyzed the correlation between the expression of MSLN and clinicopathological data, so as to provide more theoretical basis for the role of MSLN in the diagnosis and treatment of PDAC. Patients and methods: Cancer and para-cancer tissues of 24 cases with PDAC were assessed by standardized immunohistochemical (IHC) detection with two kinds of anti-MSLN antibodies (EPR4509 and EPR19025-42) to detect their positive expression rates and study the correlation between the expression of MSLN and the clinicopathological data. Results: The two anti-MSLN antibodies of cancer tissues showed positive expression with tan yellow or tan brown granules diffusely distributed on the cell membrane in 22 of 24 cases with PDAC (positive rate of 91.67%), and the positive expression of the two antibodies EPR4509 and EPR19025-42 was completely consistent in all tissue samples. No expression of the two anti-MSLN antibodies was found in para-cancer tissues and the difference was statistically significant (χ2=40.615, p=0.000, p<0.05) when compared with PDAC tissues. There was no significant correlation between MSLN expression and clinicopathological data, such as gender, tumor size, location, pathological stage, differentiation degree and lymph node metastasis (p>0.05). Conclusion: MSLN was highly expressed in PDAC tissues, but not in paracancerous tissues. There was no significant correlation between MSLN expression and clinicopathological factors. The overexpression of MSLN may have promising prospects in diagnosis, targeted therapy and immunotherapy of PDAC.

Platelet-rich fibrin prevents postoperative intestinal adhesion.

Platelet-rich fibrin (PRF) was prepared from the blood of BALB/C inbred mice to explore potential effects on postoperative intestinal adhesion. A murine model of intestinal adhesion characterized by abdominal wall defect/and cecum damage was established by scraping caecum serosa and cutting peritoneum and muscles in the abdominal wall. The wound was covered with PRF (group A), sodium hyaluronate (group B), or left alone (blank control; group C). All animals were monitored for 28 days. The incidence of adhesion was 35.0, 66.7, and 73.7% in groups A, B, and C, respectively. The incidence of adhesion in group A was significantly lower than that in group C (p < .05). Histopathologically, severity of fibrosis and the number of fibroblasts or inflammatory cells in group A were lower than those in groups B and C (p < .05), whereas the number of mesothelial cells was higher (p = .001). Furthermore, the severity of fibrosis and number of fibroblasts or inflammatory cells were lower in low grade than those in high grade of adhesion (p < .05), whereas the number of mesothelial cells was higher (p < .05). Collectively, PRF applied to abdominal surgery may reduce the incidence of intestinal adhesion by promoting proliferation of mesothelial cells whereas inhibiting proliferation of fibroblasts and infiltration of inflammatory cells.

Hypoxia-inducible factor-prolyl hydroxylase inhibitor ameliorates myopathy in a mouse model of chronic kidney disease.

Muscle wasting and diminished physical performance contribute to the morbidity and mortality of chronic kidney disease (CKD), for which no curative therapy exists. Accumulating evidence indicates that impaired angiogenesis occurs in the muscles of CKD models. Therefore, proangiogenesis therapy is considered a potentially effective strategy for limiting CKD-associated myopathy. Hypoxia-inducible factor (HIF)-prolyl hydroxylase inhibitor (HIF-PHI) stabilizes HIF and enhances muscle angiogenesis during acute ischemia; however, little evidence was available from CKD models. Here, we assessed whether pharmacological activation of HIF by MK-8617 (MK), a novel orally active HIF-PHI, improves CKD-associated myopathy. Mice were divided into sham or CKD groups, and CKD mice were subdivided into CKD + vehicle or MK treatment groups (1.5, 5, or 12.5 mg/kg for 12 wk). In CKD mice, skeletal muscle mass, mitochondrial amount, and exercise capacity decreased compared with sham mice. Compared with the CKD + vehicle group, low (1.5 mg/kg) and medium (5 mg/kg) doses of MK, but not the high dose (12.5 mg/kg), significantly restored these changes and was accompanied by incremental increases in HIF-1α. Furthermore, increased capillary density and area were observed in a MK dose-dependent manner, which is likely related to an improved VEGF response in the skeletal muscle of CKD mice. In addition, macrophage and proinflammatory cytokines, including monocyte chemoattractant protein 1, TNF-α, and IL-6, significantly increased in the high-dose MK group. These results indicate that HIF-PHI provides a potential therapeutic strategy to improve CKD-associated myopathy.

Effective angiogenesis requires regulation of phosphoinositide signaling.

Phosphoinositide signaling regulates numerous downstream effectors that mediate cellular processes which influence cell cycle progression, migration, proliferation, growth, survival, metabolism and vesicular trafficking. A prominent role for phosphoinositide 3-kinase, which generates phosphatidylinositol 3,4,5-trisphosphate, a phospholipid that activates a plethora of effectors including AKT and FOXO during embryonic and postnatal angiogenesis, has been described. In addition, phosphatidylinositol 3-phosphate signaling is required for endosomal trafficking, which contributes to vascular remodeling. This review will examine the role phosphoinositide signaling plays in the endothelium and its contribution to sprouting angiogenesis.

Aggressive Resection of Congenital Lumbosacral Lipomas in Adults: Indications, Techniques, and Outcomes in 122 Patients.

OBJECTIVE: The authors reviewed the treatment of adult patients with congenital intraspinal lipomas with total/near-total resection and discussed their preoperative characteristics, prognostic factors, and surgical outcomes. METHODS: Medical records of 122 adult patients with congenital lumbosacral lipomas undergoing total/near-total resection were systematically analyzed. The cohort was subdivided into 3 groups depending on symptom onset age: group 1 (≤5 years, n = 40), group 2 (>5 years but <18 years, n = 33), and group 3 (>18 years, n = 49). Preoperative and postoperative neurologic status were compared between groups and analyzed as a whole. RESULTS: The most common symptom was bladder dysfunction (82.0%), followed by constipation (76.2%). At the 3-month follow-up, improvement was noted in most patients presenting with pain (87.2%) and neuropathic ulcers (70.0%). Overall, neurologic status was improved in 73.0% of patients and stabilized in 19.7% of patients. A binary logistic regression model identified shorter preoperative duration (P = 0.013) and preoperative pain (P = 0.005) as independent predictors of postoperative improvement. Neurosurgical complications developed in 16 patients, and wound complications occurred in 2 patients. Two of 3 patients who had recurred symptoms underwent repeated detethering surgery during long-term follow-up. CONCLUSIONS: Despite longer preoperative duration than the pediatric population, adult patients with lumbosacral lipomas can still benefit from total/near-total resection especially regarding pain and foot ulcers, with low surgery-related morbidity. The long-term advantage of resecting additional lipoma in adults remains a point of discussion.

Alternative splicing as a biomarker and potential target for drug discovery.

Alternative splicing is a key process of multi-exonic gene expression during pre-mRNA maturation. In this process, particular exons of a gene will be included within or excluded from the final matured mRNA, and the resulting transcripts generate diverse protein isoforms. Recent evidence demonstrates that approximately 95% of human genes with multiple exons undergo alternative splicing during pre-mRNA maturation. Thus, alternative splicing plays a critical role in physiological processes and cell development programs, and.dysregulation of alternative splicing is highly associated with human diseases, such as cancer, diabetes and neurodegenerative diseases. In this review, we discuss the regulation of alternative splicing, examine the relationship between alternative splicing and human diseases, and describe several approaches that modify alternative splicing, which could aid in human disease diagnosis and therapy.