Novel Programmed Death Ligand 1-AKT-engineered Mesenchymal Stem Cells Promote Neuroplasticity to Target Stroke Therapy.

Although tissue plasminogen activator (t-PA) and endovascular thrombectomy are well-established treatments for acute ischemic stroke, over half of patients with stroke remain disabled for a long time. Thus, a significant unmet need exists to develop an effective strategy for treating acute stroke. We developed a combination of programmed cell death-ligand 1 (PD-L1) and AKT-modified umbilical cord mesenchymal stem cells (UMSC-PD-L1-AKT) implanted through intravenous (IV) and intracarotid (IA) routes to enhance therapeutic efficacy in a murine stroke model for overcoming the hypoxic environment of the ischemic brain, to prolong stem cell survival, and to attenuate systemic inflammation to protect neuroglial cells from ischemic injury. Higher cellular proliferation and survival upon exposure to toxic agents were observed in UMSC-PD-L1-AKT cells than in UMSCs in vitro. Moreover, increased attenuation of CFSE+ cell proliferation and increased survival of primary cortical cells were verified by the interaction with UMSC-PD-L1-AKT. Consistently, dual-route administration (IV + IA) of UMSC-PD-L1-AKT resulted in a significant reduction in infarction volume and improvement of neurological dysfunction in a stroke model. Furthermore, enhancing CD8+CD122+IL-10+ T-regulatory (Treg) cells and reducing CD11b+CD80+ microglial/macrophages and CD3+CD8+TNF-α+ and CD3+CD8+ IFN-α+ cytotoxic T cells induced an anti-inflammatory microenvironment to protect neuroglial cells in the ischemic brain. Collectively, therapeutic intervention using UMSC-PD-L1-AKT could provide a niche for inducing neuroplastic regeneration in brains after stroke.

Ectopic ATP synthase stimulates the secretion of extracellular vesicles in cancer cells.

ABSTARCT: Ectopic ATP synthase on the plasma membrane (eATP synthase) has been found in various cancer types and is a potential target for cancer therapy. However, whether it provides a functional role in tumor progression remains unclear. Here, quantitative proteomics reveals that cancer cells under starvation stress express higher eATP synthase and enhance the production of extracellular vesicles (EVs), which are vital regulators within the tumor microenvironment. Further results show that eATP synthase generates extracellular ATP to stimulate EV secretion by enhancing P2X7 receptor-triggered Ca2+ influx. Surprisingly, eATP synthase is also located on the surface of tumor-secreted EVs. The EVs-surface eATP synthase increases the uptake of tumor-secreted EVs in Jurkat T-cells via association with Fyn, a plasma membrane protein found in immune cells. The eATP synthase-coated EVs uptake subsequently represses the proliferation and cytokine secretion of Jurkat T-cells. This study clarifies the role of eATP synthase on EV secretion and its influence on immune cells.

Spatial and temporal dynamics of ATP synthase from mitochondria toward the cell surface.

Ectopic ATP synthase complex (eATP synthase), located on cancer cell surface, has been reported to possess catalytic activity that facilitates the generation of ATP in the extracellular environment to establish a suitable microenvironment and to be a potential target for cancer therapy. However, the mechanism of intracellular ATP synthase complex transport remains unclear. Using a combination of spatial proteomics, interaction proteomics, and transcriptomics analyses, we find ATP synthase complex is first assembled in the mitochondria and subsequently delivered to the cell surface along the microtubule via the interplay of dynamin-related protein 1 (DRP1) and kinesin family member 5B (KIF5B). We further demonstrate that the mitochondrial membrane fuses to the plasma membrane in turn to anchor ATP syntheses on the cell surface using super-resolution imaging and real-time fusion assay in live cells. Our results provide a blueprint of eATP synthase trafficking and contribute to the understanding of the dynamics of tumor progression.

LncRNA SNHG1 regulates neuroblastoma cell fate via interactions with HDAC1/2.

The small nucleolar RNA host gene 1 (SNHG1) is a novel oncogenic long non-coding RNA (lncRNA) aberrantly expressed in different tumor types. We previously found highly expressed SNHG1 was associated with poor prognosis and MYCN status in neuroblastoma (NB). However, the molecular mechanisms of SNHG1 in NB are still unclear. Here, we disrupted endogenous SNHG1 in the MYCN-amplified NB cell line SK-N-BE(2)C using the CRISPR/Cas9 system and demonstrated the proliferation and colony formation ability of SNHG1-knowndown cells were suppressed. The transcriptome analysis and functional assays of SNHG1-knockdown cells revealed SNHG1 was involved in various biological processes including cell growth, migration, apoptosis, cell cycle, and reactive oxygen species (ROS). Interestingly, the expression of core regulatory circuitry (CRC) transcription factors in MYCN-amplified NB, including PHOX2B, HAND2, GATA3, ISL1, TBX1, and MYCN, were decreased in SNHG1-knockdown cells. The chromatin-immunoprecipitation sequencing (ChIP-seq) and transposase-accessible chromatin using sequencing (ATAC-seq) analyses showed that chromatin status of these CRC members was altered, which might stem from interactions between SNHG1 and HDAC1/2. These findings demonstrate that SNHG1 plays a crucial role in maintaining NB identity via chromatin regulation and reveal the function of the lncRNA SNHG1 in NB.

Quantitative phosphoproteomics reveals ectopic ATP synthase on mesenchymal stem cells to promote tumor progression via ERK/c-Fos pathway activation.

The tumor microenvironment (TME), which comprises cellular and noncellular components, is involved in the complex process of cancer development. Emerging evidence suggests that mesenchymal stem cells (MSCs), one of the vital regulators of the TME, foster tumor progression through paracrine secretion. However, the comprehensive phosphosignaling pathways that are mediated by MSC-secreting factors have not yet been fully established. In this study, we attempt to dissect the MSC-triggered mechanism in lung cancer using quantitative phosphoproteomics. A total of 1958 phosphorylation sites are identified in lung cancer cells stimulated with MSC-conditioned medium. Integrative analysis of the identified phosphoproteins and predicted kinases demonstrates that MSC-conditioned medium functionally promotes the proliferation and migration of lung cancer via the ERK/phospho-c-Fos-S374 pathway. Recent studies have reported that extracellular ATP accumulates in the TME and stimulates the P2X7R on the cancer cell membrane via purinergic signaling. We observe that ectopic ATP synthase is located on the surface of MSCs and excreted extracellular ATP into the lung cancer microenvironment to trigger the ERK/phospho-c-Fos-S374 pathway, which is consistent with these previous findings. Our results suggest that ectopic ATP synthase on the surface of MSCs releases extracellular ATP into the TME, which promotes cancer progression via activation of the ERK/phospho-c-Fos-S374 pathway.

Targeting protein interaction networks in mitochondrial dynamics for cancer therapy.

Mitochondria are crucial organelles that provide energy via oxidative phosphorylation in eukaryotic cells and also have critical roles in growth, division, and the cell cycle, as well as the rapid adaptation required to meet the metabolic needs of the cell. Mitochondrial processes are highly dynamic; fusion and fission can vary with cell type, cellular context, and stress levels. Accumulating evidence demonstrates that an imbalance in mitochondrial dynamics leads to death in numerous types of human cancer cells. Therefore, modulating mitochondrial dynamics could be a therapeutic target. In this review, we provide an overview of the protein interaction networks involved in mitochondrial dynamics as effective and feasible drug targets and discuss the related potential therapeutic strategies for cancer.

Loss of Fis1 impairs proteostasis during skeletal muscle aging in Drosophila.

Increased levels of dysfunctional mitochondria within skeletal muscle are correlated with numerous age-related physiopathological conditions. Improving our understanding of the links between mitochondrial function and muscle proteostasis, and the role played by individual genes and regulatory networks, is essential to develop treatments for these conditions. One potential player is the mitochondrial outer membrane protein Fis1, a crucial fission factor heavily involved in mitochondrial dynamics in yeast but with an unknown role in higher-order organisms. By using Drosophila melanogaster as a model, we explored the effect of Fis1 mutations generated by transposon Minos-mediated integration. Mutants exhibited a higher ratio of damaged mitochondria with age as well as elevated reactive oxygen species levels compared with controls. This caused an increase in oxidative stress, resulting in large accumulations of ubiquitinated proteins, accelerated muscle function decline, and mitochondrial myopathies in young mutant flies. Ectopic expression of Fis1 isoforms was sufficient to suppress this phenotype. Loss of Fis1 led to unbalanced mitochondrial proteostasis within fly muscle, decreasing both flight capabilities and lifespan. Fis1 thus clearly plays a role in fly mitochondrial dynamics. Further investigations into the detailed function of Fis1 are necessary for exploring how mitochondrial function correlates with muscle health during aging.

STAT3 phosphorylation at Ser727 and Tyr705 differentially regulates the EMT-MET switch and cancer metastasis.

Epithelial-mesenchymal transition (EMT)/mesenchymal-epithelial transition (MET) processes are proposed to be a driving force of cancer metastasis. By studying metastasis in bone marrow-derived mesenchymal stem cell (BM-MSC)-driven lung cancer models, microarray time-series data analysis by systems biology approaches revealed BM-MSC-induced signaling triggers early dissemination of CD133+/CD83+ cancer stem cells (CSCs) from primary sites shortly after STAT3 activation but promotes proliferation towards secondary sites. The switch from migration to proliferation was regulated by BM-MSC-secreted LIF and activated LIFR/p-ERK/pS727-STAT3 signaling to promote early disseminated cancer cells MET and premetastatic niche formation. Then, tumor-tropic BM-MSCs circulated to primary sites and triggered CD151+/CD38+ cells acquiring EMT-associated CSC properties through IL6R/pY705-STAT3 signaling to promote tumor initiation and were also attracted by and migrated towards the premetastatic niche. In summary, STAT3 phosphorylation at tyrosine 705 and serine 727 differentially regulates the EMT-MET switch within the distinct molecular subtypes of CSCs to complete the metastatic process.

Molecular Mechanisms of Laparoscopic Ovarian Drilling and Its Therapeutic Effects in Polycystic Ovary Syndrome.

Polycystic ovary syndrome (PCOS) is a common endocrinopathy, characterized by chronic anovulation, hyperandrogenism, and multiple small subcapsular cystic follicles in the ovary during ultrasonography, and affects 5-10% of women of reproductive age. PCOS is frequently associated with insulin resistance (IR) accompanied by compensatory hyperinsulinemia and, therefore, presents an increased risk of type 2 diabetes mellitus (DM). The pathophysiology of PCOS is unclear, and many hypotheses have been proposed. Among these hypotheses, IR and hyperandrogenism may be the two key factors. The first line of treatment in PCOS includes lifestyle changes and body weight reduction. Achieving a 5-15% body weight reduction may improve IR and PCOS-associated hormonal abnormalities. For women who desire pregnancy, clomiphene citrate (CC) is the front-line treatment for ovulation induction. Twenty five percent of women may fail to ovulate spontaneously after three cycles of CC treatment, which is called CC-resistant PCOS. For CC-resistant PCOS women, there are many strategies to improve ovulation rate, including medical treatment and surgical approaches. Among the various surgical approaches, one particular surgical method, called laparoscopic ovarian drilling (LOD), has been proposed as an alternative treatment. LOD results in an overall spontaneous ovulation rate of 30-90% and final pregnancy rates of 13-88%. These benefits are more significant for women with CC-resistant PCOS. Although the intra- and post-operative complications and sequelae are always important, we believe that a better understanding of the pathophysiological changes and/or molecular mechanisms after LOD may provide a rationale for this procedure. LOD, mediated mainly by thermal effects, produces a series of morphological and biochemical changes. These changes include the formation of artificial holes in the very thick cortical wall, loosening of the dense and hard cortical wall, destruction of ovarian follicles with a subsequently decreased amount of theca and/or granulosa cells, destruction of ovarian stromal tissue with the subsequent development of transient but purulent and acute inflammatory reactions to initiate the immune response, and the continuing leakage or drainage of "toxic" follicular fluid in these immature and growth-ceased pre-antral follicles. All these factors contribute to decreasing local and systemic androgen levels, the following apoptosis process with these pre-antral follicles to atresia; the re-starting of normal follicular recruitment, development, and maturation, and finally, the normalization of the "hypothalamus-pituitary-ovary" axis and subsequent spontaneous ovulation. The detailed local and systematic changes in PCOS women after LOD are comprehensively reviewed in the current article.

Multiomics Reveals Ectopic ATP Synthase Blockade Induces Cancer Cell Death via a lncRNA-mediated Phospho-signaling Network.

The EGFR tyrosine kinase inhibitor gefitinib is commonly used for lung cancer patients. However, some patients eventually become resistant to gefitinib and develop progressive disease. Here, we indicate that ecto-ATP synthase, which ectopically translocated from mitochondrial inner membrane to plasma membrane, is considered as a potential therapeutic target for drug-resistant cells. Quantitative multi-omics profiling reveals that ecto-ATP synthase inhibitor mediates CK2-dependent phosphorylation of DNA topoisomerase IIα (topo IIα) at serine 1106 and subsequently increases the expression of long noncoding RNA, GAS5. Additionally, we also determine that downstream of GAS5, p53 pathway, is activated by ecto-ATP synthase inhibitor for regulation of programed cell death. Interestingly, GAS5-proteins interactomic profiling elucidates that GAS5 associates with topo IIα and subsequently enhancing the phosphorylation level of topo IIα. Taken together, our findings suggest that ecto-ATP synthase blockade is an effective therapeutic strategy via regulation of CK2/phospho-topo IIα/GAS5 network in gefitinib-resistant lung cancer cells.

Phosphoproteome Analysis Reveals Dynamic Heat Shock Protein 27 Phosphorylation in Tanshinone IIA-Induced Cell Death.

Gastric cancer is one of the most common types of cancer worldwide. Nevertheless, effective therapeutic strategies have not yet been discovered. Several studies have shown that tanshinone IIA (TIIA), which is extracted from the traditional herbal medicine plant Danshen (Salvia miltiorrhiza), has potential activity against many kinds of cancer. Our previous research demonstrated that TIIA can induce cell death in gastric cancer. However, the exact signaling pathway response is still unclear. Post-translational modification (PTM) plays a significant role in a wide range of physiological processes in cancer, via regulation of both signal transduction cascades and many cellular pathways. Here, we integrated multilayer omics-transcriptomics and dynamic phosphoproteomics-to elucidate the regulatory networks triggered by TIIA in gastric cancer. We identified the phosphorylation of heat shock protein 27 (HSP27) at serine 82 in response to TIIA, which caused reactive oxygen species (ROS) production and unfolded protein response (UPR). Moreover, the accumulation of cellular stress increased the expression of heat shock factor 1 (HSF1). In addition, the downstream targets of HSF1, which were involved in heat shock stress and apoptosis, were also activated in TIIA-treated cells. In conclusion, this study performs a multiomic approach to clarify a comprehensive TIIA-responsive network leading to cell death in gastric cancer.

Effect of Loading Devices on Muscle Activation in Squat and Lunge.

CONTEXT: Squats and lunges are common exercises frequently applied in muscle-strengthening and therapeutic exercises. The loading devices are often used to increase the training intensity. OBJECTIVE: To determine the effect of loading devices on muscle activation in squat and lunge and to compare the differences in muscle activation between squat and lunge. DESIGN: Cross-sectional cohort. PARTICIPANTS: Nineteen healthy, male, recreationally active individuals without a history of lower limb injury. INTERVENTIONS: Each participant performed 10 repetitions of a squat under 5 conditions: unloaded, barbell, dumbbell, loaded vest, and kettlebell, and 10 repetitions of a lunge under 4 conditions: unloaded, barbell, dumbbell, and loaded vest. MAIN OUTCOME MEASURES: The electromyography signals of quadriceps, hamstrings, tibialis anterior, gastrocnemius lateralis and medialis were measured. One-way repeated-measure analysis of variance was used to compare the difference among different loading conditions. Paired t test was used to compare the difference between squat and lunge. RESULTS: The muscle activation in the loaded conditions was significantly higher than that in nonloaded conditions in squat and lunge. Compared with the barbell, dumbbell, and loaded vest conditions, the semitendinosus showed significantly higher activation, and the tibialis anterior showed significantly lower activation in kettlebell condition in squat. No significant difference in muscle activation was found among barbell, dumbbell, and kettlebell conditions in lunge. In addition, quadriceps and hamstring activities were significantly higher in lunge than in squat. CONCLUSIONS: Muscle activation was affected by the loading devices in squat but not affected in lunge. Kettlebell squat could be suggested for targeting in strengthening medial hamstring. Progressive strengthening exercise could be recommended from squat to lunge based on sequential activation level.

SFRPs Are Biphasic Modulators of Wnt-Signaling-Elicited Cancer Stem Cell Properties beyond Extracellular Control.

Secreted frizzled-related proteins (SFRPs) are mainly known for their role as extracellular modulators and tumor suppressors that downregulate Wnt signaling. Using the established (CRISPR/Cas9 targeting promoters of SFRPs and targeting SFRPs transcript) system, we find that nuclear SFRPs interact with ß-catenin and either promote or suppress TCF4 recruitment. SFRPs bind with ß-catenin on both their N and C termini, which the repressive effects caused by SFRP-ß-catenin-N-terminus binding overpower the promoting effects of their binding at the C terminus. By high Wnt activity, ß-catenin and SFRPs only bind with their C termini, which results in the upregulation of ß-catenin transcriptional activity and cancer stem cell (CSC)-related genes. Furthermore, we identify disulfide bonds of the cysteine-rich domain (CRD) and two threonine phosphorylation events of the netrin-related motif (NTR) domain of SFRPs that are essential for their role as biphasic modulators, suggesting that SFRPs are biphasic modulators of Wnt signaling-elicited CSC properties beyond extracellular control.

Cluster validity indices for mixture hazards regression models.

In the analysis of survival data, the problems of competing risks arise frequently in medical applications where individuals fail from multiple causes. Semiparametric mixture regression models have become a prominent approach in competing risks analysis due to their flexibility and easy interpretation of resultant estimates. The literature presents several semiparametric methods on the estimations for mixture Cox proportional hazards models, but fewer works appear on the determination of the number of model components and the estimation of baseline hazard functions using kernel approaches. These two issues are important because both incorrect number of components and inappropriate baseline functions can lead to insufficient estimates of mixture Cox hazard models. This research thus proposes four validity indices to select the optimal number of model components based on the posterior probabilities and residuals resulting from the application of an EM-based algorithm on a mixture Cox regression model. We also introduce a kernel approach to produce a smooth estimate of the baseline hazard function in a mixture model. The effectiveness and the preference of the proposed cluster indices are demonstrated through a simulation study. An analysis on a prostate cancer dataset illustrates the practical use of the proposed method.

RNA-Binding Proteomics Reveals MATR3 Interacting with lncRNA SNHG1 To Enhance Neuroblastoma Progression.

The interaction of long noncoding RNAs (lncRNAs) with one or more RNA-binding proteins (RBPs) is important to a plethora of cellular and physiological processes. The lncRNA SNHG1 was reported to be aberrantly expressed and associated with poor patient prognosis in several cancers including neuroblastoma. However, the interacting RBPs and biological functions associated with SNHG1 in neuroblastoma remain unknown. In this study, we identified 283, 31, and 164 SNHG1-interacting proteins in SK-N-BE(2)C, SK-N-DZ, and SK-N-AS neuroblastoma cells, respectively, using a RNA-protein pull-down assay coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Twenty-four SNHG1-interacting RBPs were identified in common from these three neuroblastoma cell lines. RBPs MATR3, YBX1, and HNRNPL have the binding sites for SNHG1 predicted by DeepBind motif analysis. Furthermore, the direct binding of MATR3 with SNHG1 was validated by Western blot and confirmed by RNA immunoprecipitation assay (RIP). Coexpression analysis revealed that the expression of SNHG1 is positively correlated with MATR3 ( P = 3.402 × 10-13). The high expression of MATR3 is associated with poor event-free survival ( P = 0.00711) and overall survival ( P = 0.00064). Biological functions such as ribonucleoprotein complex biogenesis, RNA processing, and RNA splicing are significantly enriched and in common between SNHG1 and MATR3. In conclusion, we identified MATR3 as binding to SNHG1 and the interaction might be involved in splicing events that enhance neuroblastoma progression.

Outcomes after major surgery in patients with myasthenia gravis: A nationwide matched cohort study.

OBJECTIVE: To validate the comprehensive features of adverse outcomes after surgery for patients with myasthenia gravis. METHODS: Using reimbursement claims from Taiwan's National Health Insurance Research Database, we analyzed 2290 patients who received major surgery between 2004 and 2010 and were diagnosed with myasthenia gravis preoperatively. Surgical patients without myasthenia gravis (n = 22,900) were randomly selected by matching procedure with propensity score for comparison. The adjusted odds ratios and 95% confidence intervals of postoperative adverse events associated with preoperative myasthenia gravis were calculated under the multiple logistic regressions. RESULTS: Compared with surgical patients without myasthenia gravis, surgical patients with myasthenia gravis had higher risks of postoperative pneumonia (OR = 2.09; 95% CI: 1.65-2.65), septicemia (OR = 1.31; 95% CI: 1.05-1.64), postoperative bleeding (OR = 1.71; 95% CI: 1.07-2.72), and overall complications (OR = 1.70; 95% CI: 1.44-2.00). The ORs of postoperative adverse events for patients with myasthenia gravis who had symptomatic therapy, chronic immunotherapy, and short-term immunotherapy were 1.76 (95% CI 1.50-2.08), 1.70 (95% CI 1.36-2.11), and 4.36 (95% CI 2.11-9.04), respectively. CONCLUSIONS: Patients with myasthenia gravis had increased risks of postoperative adverse events, particularly those experiencing emergency care, hospitalization, and thymectomy for care of myasthenia gravis. Our findings suggest the urgency of revising protocols for perioperative care for these populations.