The CRISPR/Cas9 Genome-editing system has revealed promising potential for generating gene mutation, deletion, and correction in human cells. Application of this powerful tool in Fabry disease (FD), however, still needs to be explored. Enzyme replacement therapy (ERT), a regular administration of recombinant human α Gal A (rhα-GLA), is a currently available and effective treatment to clear the accumulated Gb3 in FD patients. However, the short half-life of rhα-GLA in human body limits its application. Moreover, lack of an appropriate in vitro disease model restricted the high-throughput screening of drugs for improving ERT efficacy. Therefore, it is worth establishing a large-expanded in vitro FD model for screening potential candidates, which can enhance and prolong ERT potency. Using CRISPR/Cas9-mediated gene knockout of GLA in HEK-293T cells, we generated GLA-null cells to investigate rhα-GLA cellular pharmacokinetics. The half-life of administrated rhα-GLA was around 24 h in GLA-null cells; co-administration of proteasome inhibitor MG132 and rhα-GLA significantly restored the GLA enzyme activity by two-fold compared with rhα-GLA alone. Furthermore, co-treatment of rhα-GLA/MG132 in patient-derived fibroblasts increased Gb3 clearance by 30%, compared with rhα-GLA treatment alone. Collectively, the CRISPR/Cas9-mediated GLA-knockout HEK-293T cells provide an in vitro FD model for evaluating the intracellular pharmacokinetics of the rhα-GLA as well as for screening candidates to prolong rhα-GLA potency. Using this model, we demonstrated that MG132 prolongs rhα-GLA half-life and enhanced Gb3 clearance, shedding light on the direction of enhancing ERT efficacy in FD treatment.

Oncostatin M (OSM) is linked with multiple biological responses including growth and differentiation. Previous reports showed inhibitory effects of OSM in tumor progression while others showed promoting effects. The dual role of OSM in the development of various cancers is still unclear. We previously described OSM-mediated SLUG suppression, leading to repressed metastasis of lung adenocarcinoma (LAC) cells. However, the underlying mechanism remains elusive. Here, we showed that OSM suppresses SLUG express in LAC cells through a STAT1-dependent transcriptional inhibition. Knockdown of STAT1 reversed the OSM-suppressed SLUG expression and rescued the OSM-mediated inhibition of cell proliferation, migration, and invasion in vitro, as well as pulmonary metastasis in vivo. STAT1 suppressed SLUG transcription through binding to its promoter region in response to OSM. Furthermore, PIAS4, a co-repressor of STAT, and HDAC1 were able to bind to STAT1 on SLUG promoter region, resulting in reduced H3K9 acetylation and suppressed SLUG expression upon OSM treatment. In contrast, PIAS3 bound to activated STAT3, another effector of OSM, in response to OSM and blocked the binding of STAT3 to SLUG promoter region, preventing STAT3-dependent activation of SLUG transcription. Our findings suggested that OSM suppresses SLUG expression and tumor metastasis of LAC through inducing the inhibitory effect of the STAT1-dependent pathway and suppressing the activating effect of STAT3-dependent signaling. These results can serve as a scientific basis for the potential therapeutic intervention of OSM in cancer cells.

Clear cell (CCC), endometrioid (EC), mucinous (MC) and high-grade serous carcinoma (SC) are the four most common subtypes of epithelial ovarian carcinoma (EOC). The widely accepted dualistic model of ovarian carcinogenesis divided EOCs into type I and II categories based on the molecular features. However, this hypothesis has not been experimentally demonstrated. We carried out a gene set-based analysis by integrating the microarray gene expression profiles downloaded from the publicly available databases. These quantified biological functions of EOCs were defined by 1454 Gene Ontology (GO) term and 674 Reactome pathway gene sets. The pathogenesis of the four EOC subtypes was investigated by hierarchical clustering and exploratory factor analysis. The patterns of functional regulation among the four subtypes containing 1316 cases could be accurately classified by machine learning. The results revealed that the ERBB and PI3K-related pathways played important roles in the carcinogenesis of CCC, EC and MC; while deregulation of cell cycle was more predominant in SC. The study revealed that two different functional regulation patterns exist among the four EOC subtypes, which were compatible with the type I and II classifications proposed by the dualistic model of ovarian carcinogenesis.

Serous carcinoma (SC) is the most common subtype of epithelial ovarian carcinoma and is divided into four stages by the Federation of Gynecologists and Obstetrics (FIGO) staging system. Currently, the molecular functions and biological processes of SC at different FIGO stages have not been quantified. Here, we conducted a whole-genome integrative analysis to investigate the functions of SC at different stages. The function, as defined by the GO term or canonical pathway gene set, was quantified by measuring the changes in the gene expressional order between cancerous and normal control states. The quantified function, i.e., the gene set regularity (GSR) index, was utilized to investigate the pathogenesis and functional regulation of SC at different FIGO stages. We showed that the informativeness of the GSR indices was sufficient for accurate pattern recognition and classification for machine learning. The function regularity presented by the GSR indices showed stepwise deterioration during SC progression from FIGO stage I to stage IV. The pathogenesis of SC was centered on cell cycle deregulation and accompanied with multiple functional aberrations as well as their interactions.

The high invasiveness and frequent recurrence of lung adenocarcinoma (LAC) are major reasons for treatment failures and poor prognoses. Alterations in microRNAs (miRNAs) expression have been shown in lung cancers. Recent reports have demonstrated that tumors contain a small subpopulation of cancer stem cells (CSCs) that possesses self-renewing capacity and is responsible for tumor malignancy including metastasis, relapse, and chemoradioresistance. However, a miRNAs-based therapeutic approach in LAC-associated CSCs (LAC-CSCs) is still blurred. Using miRNA/mRNA-microarray and Quantitative RT-PCR, we found that the expression of miR145 is negatively correlated with the levels of Oct4/Sox2/Fascin1 in LAC patient specimens, and an Oct4(high)Sox2(high)Fascin1(high)miR145(low) phenotype predicted poor prognosis. We enriched LAC-CSCs by side population sorting or identification of CD133 markers and found that LAC-CSCs exhibited low miR145 and high Oct4/Sox2/Fascin1 expression, CSC-like properties, and chemoradioresistance. To clarify the role of miR145, we used a polyurethane-short branch-polyethylenimine (PU-PEI) as the vehicle to deliver miR145 into LAC-CSCs. PU-PEI-mediated miR145 delivery reduced CSC-like properties, and improved chemoradioresistance in LAC-CSCs by directly targeting Oct4/Sox2/Fascin1. Importantly, the repressive effect of miR145 on tumor metastasis was mediated by inhibiting the epithelial-mesenchymal transdifferentiation (EMT) and metastastic ability, partially by regulating Oct4/Sox2/Fascin1, Tcf4, and Wnt5a. Finally, in vivo study showed that PU-PEI-mediated miR145 delivery to xenograft tumors reduced tumor growth and metastasis, sensitized tumors to chemoradiotherapies, and prolonged the survival times of tumor-bearing mice. Our results demonstrated that miR145 acts as a switch regulating lung CSC-like and EMT properties, and provide insights into the clinical prospect of miR145-based therapies for malignant lung cancers.

Transient receptor potential vanilloid 1 (TRPV1), recognized as a hyperosmolarity sensor, is a crucial ion channel involved in the pathogenesis of neural and glial signaling. Recently, TRPV1 was determined to play a role in retinal physiology and visual transmission. In this study, we sought to clarify the role of TRPV1 and the downstream pathway in the osmotic stress-related retina ganglion cell (RGC) damage.

The coexistence of endometriosis (ES) with ovarian clear cell carcinoma (CCC) or endometrioid carcinoma (EC) suggested that malignant transformation of ES leads to endometriosis associated ovarian carcinoma (EAOC). However, there is still lack of an integrating data analysis of the accumulated experimental data to provide the evidence supporting the hypothesis of EAOC transformation. Herein we used a function-based analytic model with the publicly available microarray datasets to investigate the expression profiling between ES, CCC, and EC. We analyzed the functional regularity pattern of the three type of samples and hierarchically clustered the gene sets to identify key mechanisms regulating the malignant transformation of EAOC. We identified a list of 18 genes (NLRP3, AIM2, PYCARD, NAIP, Caspase-4, Caspase-7, Caspase-8, TLR1, TLR7, TOLLIP, NFKBIA, TNF, TNFAIP3, INFGR2, P2RX7, IL-1B, IL1RL1, IL-18) closely related to inflammasome complex, indicating an important role of inflammation/immunity in EAOC transformation. We next explore the association between these target genes and patient survival using Gene Expression Omnibus (GEO), and found significant correlation between the expression levels of the target genes and the progression-free survival. Interestingly, high expression levels of AIM2 and NLRP3, initiating proteins of inflammasomes, were significantly correlated with poor progression-free survival. Immunohistochemistry staining confirmed a correlation between high AIM2 and high Ki-67 in clinical EAOC samples, supporting its role in disease progression. Collectively, we established a bioinformatic platform of gene-set integrative molecular functionome to dissect the pathogenic pathways of EAOC, and demonstrated a key role of dysregulated inflammasome in modulating the malignant transformation of EAOC.

Inherited retinal dystrophies (IRDs) are rare but highly heterogeneous genetic disorders that affect individuals and families worldwide. However, given its wide variability, its analysis of the driver genes for over 50% of the cases remains unexplored. The present study aims to identify novel driver genes, disease-causing variants, and retinitis pigmentosa (RP)-associated pathways. Using family-based whole-exome sequencing (WES) to identify putative RP-causing rare variants, we identified a total of five potentially pathogenic variants located in genes OR56A5, OR52L1, CTSD, PRF1, KBTBD13, and ATP2B4. Of the variants present in all affected individuals, genes OR56A5, OR52L1, CTSD, KBTBD13, and ATP2B4 present as missense mutations, while PRF1 and CTSD present as frameshift variants. Sanger sequencing confirmed the presence of the novel pathogenic variant PRF1 (c.124_128del) that has not been reported previously. More causal-effect or evidence-based studies will be required to elucidate the precise roles of these SNPs in the RP pathogenesis. Taken together, our findings may allow us to explore the risk variants based on the sequencing data and upgrade the existing variant annotation database in Taiwan. It may help detect specific eye diseases such as retinitis pigmentosa in East Asia.

Retinal detachment (RD) is a sight-threatening ocular disease caused by separation of the neurosensory retina from the underlying retinal pigment epithelium layer. Its genetic basis is unclear because of a limited amount of data. In this study, we aimed to identify genetic risk loci associated with RD in participants without diabetes mellitus and to construct a polygenic risk score (PRS) to predict the risk of RD.

Our study presents a 319-gene panel targeting inherited retinal dystrophy (IRD) genes. Through a multi-center retrospective cohort study, we validated the assay's effectiveness and clinical utility and characterized the mutation spectrum of Taiwanese IRD patients. Between January 2018 and May 2022, 493 patients in 425 unrelated families, all initially suspected of having IRD without prior genetic diagnoses, underwent detailed ophthalmic and physical examinations (with extra-ocular features recorded) and genetic testing with our customized panel. Disease-causing variants were identified by segregation analysis and clinical interpretation, with validation via Sanger sequencing. We achieved a read depth of >200× for 94.2% of the targeted 1.2 Mb region. 68.5% (291/425) of the probands received molecular diagnoses, with 53.9% (229/425) resolved cases. Retinitis pigmentosa (RP) is the most prevalent initial clinical impression (64.2%), and 90.8% of the cohort have the five most prevalent phenotypes (RP, cone-rod syndrome, Usher's syndrome, Leber's congenital amaurosis, Bietti crystalline dystrophy). The most commonly mutated genes of probands that received molecular diagnosis are USH2A (13.7% of the cohort), EYS (11.3%), CYP4V2 (4.8%), ABCA4 (4.5%), RPGR (3.4%), and RP1 (3.1%), collectively accounted for 40.8% of diagnoses. We identify 87 unique unreported variants previously not associated with IRD and refine clinical diagnoses for 21 patients (7.22% of positive cases). We developed a customized gene panel and tested it on the largest Taiwanese cohort, showing that it provides excellent coverage for diverse IRD phenotypes.

The mitochondrial genetic disorder, Leber's hereditary optic neuropathy (LHON), is caused by a mutation in MT-ND4 gene, encoding NADH dehydrogenase subunit 4. It leads to the progressive death of retinal ganglion cells (RGCs) and causes visual impairment or even blindness. However, the precise mechanisms of LHON disease penetrance and progression are not completely elucidated. Human-induced pluripotent stem cells (hiPSCs) offer unique opportunities to investigate disease-relevant phenotypes and regulatory mechanisms underlying LHON pathogenesis at the cellular level. In this study, we successfully generated RGCs by differentiation of LHON patient-specific hiPSCs. We modified the protocol of differentiation to obtain a more enriched population of single-cell RGCs for LHON study. Based on assessing morphology, expression of specific markers and electrophysiological activity, we found that LHON-specific hiPSC-derived were more defective in comparison with normal wild-type RGCs. Based on our previous study, whereby by using microarray analysis we identified that the components of glutamatergic synapse signaling pathway were significantly downregulated in LHON-specific RGCs, we focused our study on glutamate-associated α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors. We found that the protein expression levels of the subunits of the AMPA receptor, GluR1 and GluR2, and their associated scaffold proteins were decreased in LHON-RGCs. By performing the co-immunoprecipitation assay, we found several differences in the efficiencies of interaction between AMPA subunits and scaffold proteins between normal and LHON-specific RGCs.

Glioblastoma multiform (GBM) is one of the most lethal human malignant brain tumors with high risks of recurrence and poor treatment outcomes. The RNA-binding protein Musashi-1 (MSI1) is a marker of neural stem/progenitor cells. Recent study showed that high expression level of MSI1 positively correlates with advanced grade of GBM, where MSI1 increases the growth of GBM. Herein, we explore the roles of MSI1 as well as the underlying mechanisms in the regulation of drug resistance and tumorigenesis of GBM cells. Our results demonstrated that overexpression of MSI1 effectively protected GBM cells from drug-induced apoptosis through down-regulating pro-apoptotic genes; whereas inhibition of AKT withdrew the MSI1-induced anti-apoptosis and cell survival. We further showed that MSI1 robustly promoted the secretion of the pro-inflammatory cytokine IL-6, which was governed by AKT activity. Autonomously, the secreted IL-6 enhanced AKT activity in an autocrine/paracrine manner, forming a positive feedback regulatory loop with the MSI1-AKT pathway. Our results conclusively demonstrated a novel drug resistance mechanism in GBM cells that MSI1 inhibits drug-induced apoptosis through AKT/IL6 regulatory circuit. MSI1 regulates both cellular signaling and tumor-microenvironmental cytokine secretion to create an intra- and intercellular niche for GBM to survive from chemo-drug attack.

Differentiation of human induced pluripotent stem cells (hiPSCs) into retinal lineages offers great potential for medical application. Therefore, it is of crucial importance to know the key intrinsic regulators of differentiation and the specific biomarker signatures of cell lineages.

MafA is a pancreatic transcriptional factor that controls β-cell-specific transcription of the insulin gene. However, the role of MafA in the regulation of pancreatic transdifferentiation and reprogramming in human stem cells is still unclear. In this study, we investigate the role of MafA in placenta-derived multipotent stem cells (PDMSCs) that constitutively expressed Oct-4 and Nanog. PDMSCs were isolated and transfected with MafA using a lentivector. Our results showed that overexpression of MafA in PDMSCs significantly up-regulated the expression of pancreatic development-related genes (Sox17, Foxa2, Pdx1 and Ngn3). Microarray analysis suggested that the gene expression profile of MafA-overexpressing PDMSCs was similar to that of pancreas and islet tissues. MafA increased the expression levels of the mRNAs of NKx2.2, Glut2, insulin, glucagons and somatostatin, and further facilitated the differentiation of PDMSCs into insulin(+) cells. The glucose-stimulated responses to insulin and c-peptide production in MafA-overexpressing PDMSCs were significantly higher than in PDMSCs with vector control. Our results indicated that MafA-overexpressing PDMSCs were more resistant to oxidative damage and oxidative damage-induced apoptosis than PDMSCs carrying the vector control were. Importantly, the expression of MafA in PDMSCs xenotransplanted into immunocompromised mice improved the restoration of blood insulin levels to control values and greatly prolonged the survival of graft cells in immunocompromised mice with STZ-induced diabetes. In summary, these data suggest that MafA plays a novel role in the reprogramming of stem cells into pancreatic β-progenitors, promotes the islet-like characteristics of PDMSCs, as well as functionally enhances insulin production to restore the regulation of blood glucose levels in transplanted grafts.

To evaluate the surgical outcomes of cryopreserved and dehydrated human amniotic membrane (hAM) graft transplantation for macular hole (MH) and macular hole retinal detachment (MHRD) repair.

Lung cancer (LC) causes the majority of cancer-related deaths. Circular RNAs (circRNAs) were reported to play roles in cancers by targeting pro- and anti-oncogenic miRNAs. However, the mechanisms of circRNAs in LC progression and their prognostic value of treatment response remain unclear. By using next generation sequencing (NGS) of LC cell lines' transcriptomes, we identified highly overexpressed hsa_circ_0000190 and hsa_circ_000164 as potential biomarkers. By using the highly sensitive RT-ddPCR method, these circRNAs were shown to be secreted by cell lines and were detected in human blood. Clinical validation by RT-ddPCR was carried out on 272 (231 LC patients and 41 controls) blood samples. Higher hsa_circ_0000190 levels were associated with larger tumor size (p < 0.0001), worse histological type of adenocarcinoma (p = 0.0028), later stage (p < 0.0001), more distant metastatic organs (p = 0.0039), extrathoracic metastasis (p = 0.0004), and poor survival (p = 0.047) and prognosis. Using liquid biopsy-based RT-ddPCR, we discovered the correlation between increased hsa_circ_0000190 plasma level (p < 0.0001) and higher programmed death-ligand 1 (PD-L1) level in tumor (p = 0.0283). Notably, long-term follow-up of the immunotherapy treated cases showed that upregulated plasma hsa_circ_0000190 level correlated with poor response to systemic therapy and immunotherapy (p = 0.0002, 0.0058, respectively). Secretory circRNAs are detectable in blood by LB-based RT-ddPCR and may serve as blood-based biomarkers to monitor disease progression and treatment efficacy.

Angiotensin-converting enzyme 2 (ACE2) was identified as the main host cell receptor for the entry of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its subsequent infection. In some coronavirus disease 2019 (COVID-19) patients, it has been reported that the nervous tissues and the eyes were also affected. However, evidence supporting that the retina is a target tissue for SARS-CoV-2 infection is still lacking. This present study aimed to investigate whether ACE2 expression plays a role in human retinal neurons during SARS-CoV-2 infection. Human induced pluripotent stem cell (hiPSC)-derived retinal organoids and monolayer cultures derived from dissociated retinal organoids were generated. To validate the potential entry of SARS-CoV-2 infection in the retina, we showed that hiPSC-derived retinal organoids and monolayer cultures endogenously express ACE2 and transmembrane serine protease 2 (TMPRSS2) on the mRNA level. Immunofluorescence staining confirmed the protein expression of ACE2 and TMPRSS2 in retinal organoids and monolayer cultures. Furthermore, using the SARS-CoV-2 pseudovirus spike protein with GFP expression system, we found that retinal organoids and monolayer cultures can potentially be infected by the SARS-CoV-2 pseudovirus. Collectively, our findings highlighted the potential of iPSC-derived retinal organoids as the models for ACE2 receptor-based SARS-CoV-2 infection.

Retinal vein occlusions (RVO) are associated with systemic risk factors. However, the ocular occlusive events might also influence a patient's systemic condition. This study tried to investigate serum biomarkers associated with oxidative stress, before and after intravitreal anti-vascular endothelial growth factor (aVEGF) therapy in patients with RVOs.

Glioblastomas (GBMs) are the most common primary brain tumors with poor prognosis. CD133 has been considered a putative marker of cancer stem cells (CSCs) in malignant cancers, including GBMs. MicroRNAs (miRNAs), highly conserved small RNA molecules, may target oncogenes and have potential as a therapeutic strategy against cancer. However, the role of miRNAs in GBM-associated CSCs remains mostly unclear. In this study, our miRNA/mRNA-microarray and RT-PCR analysis showed that the expression of miR145 (a tumor-suppressive miRNA) is inversely correlated with the levels of Oct4 and Sox2 in GBM-CD133(+) cells and malignant glioma specimens. We demonstrated that miR145 negatively regulates GBM tumorigenesis by targeting Oct4 and Sox2 in GBM-CD133(+). Using polyurethane-short branch polyethylenimine (PU-PEI) as a therapeutic-delivery vehicle, PU-PEI-mediated miR145 delivery to GBM-CD133(+) significantly inhibited their tumorigenic and CSC-like abilities and facilitated their differentiation into CD133(-)-non-CSCs. Furthermore, PU-PEI-miR145-treated GBM-CD133(+) effectively suppressed the expression of drug-resistance and anti-apoptotic genes and increased the sensitivity of the cells to radiation and temozolomide. Finally, the in vivo delivery of PU-PEI-miR145 alone significantly suppressed tumorigenesis with stemness, and synergistically improved the survival rate when used in combination with radiotherapy and temozolomide in orthotopic GBM-CD133(+)-transplanted immunocompromised mice. Therefore, PU-PEI-miR145 is a novel therapeutic approach for malignant brain tumors.

Lamotrigine (LTG) is generally considered as a voltage-gated sodium (Nav) channel blocker. However, recent studies suggest that LTG can also serve as a hyperpolarization-activated cyclic nucleotide-gated (HCN) channel enhancer and can increase the excitability of GABAergic interneurons (INs). Perisomatic inhibitory INs, predominantly fast-spiking basket cells (BCs), powerfully inhibit granule cells (GCs) in the hippocampal dentate gyrus. Notably, BCs express abundant Nav channels and HCN channels, both of which are able to support sustained action potential generation. Using whole-cell recording in rat hippocampal slices, we investigated the net LTG effect on BC output. We showed that bath application of LTG significantly decreased the amplitude of evoked compound inhibitory postsynaptic currents (IPSCs) in GCs. In contrast, simultaneous paired recordings from BCs to GCs showed that LTG had no effect on both the amplitude and the paired-pulse ratio of the unitary IPSCs, suggesting that LTG did not affect GABA release, though it suppressed cell excitability. In line with this, LTG decreased spontaneous IPSC (sIPSC) frequency, but not miniature IPSC frequency. When re-examining the LTG effect on GABAergic transmission in the cornus ammonis region 1 (CA1) area, we found that LTG markedly inhibits both the excitability of dendrite-targeting INs in the stratum oriens and the concurrent sIPSCs recorded on their targeting pyramidal cells (PCs) without significant hyperpolarization-activated current (Ih) enhancement. In summary, LTG has no effect on augmenting Ih in GABAergic INs and does not promote GABAergic inhibitory output. The antiepileptic effect of LTG is likely through Nav channel inhibition and the suppression of global neuronal network activity.