Comparison of Synergy Extrapolation and Static Optimization for Estimating Multiple Unmeasured Muscle Activations during Walking.

Background: Calibrated electromyography (EMG)-driven musculoskeletal models can provide great insight into internal quantities (e.g., muscle forces) that are difficult or impossible to measure experimentally. However, the need for EMG data from all involved muscles presents a significant barrier to the widespread application of EMG-driven modeling methods. Synergy extrapolation (SynX) is a computational method that can estimate a single missing EMG signal with reasonable accuracy during the EMG-driven model calibration process, yet its performance in estimating a larger number of missing EMG signals remains unclear. Methods: This study assessed the accuracy with which SynX can use eight measured EMG signals to estimate muscle activations and forces associated with eight missing EMG signals in the same leg during walking while simultaneously performing EMG-driven model calibration. Experimental gait data collected from two individuals post-stroke, including 16 channels of EMG data per leg, were used to calibrate an EMG-driven musculoskeletal model, providing "gold standard" muscle activations and forces for evaluation purposes. SynX was then used to predict the muscle activations and forces associated with the eight missing EMG signals while simultaneously calibrating EMG-driven model parameter values. Due to its widespread use, static optimization (SO) was also utilized to estimate the same muscle activations and forces. Estimation accuracy for SynX and SO was evaluated using root mean square errors (RMSE) to quantify amplitude errors and correlation coefficient r values to quantify shape similarity, each calculated with respect to "gold standard" muscle activations and forces. Results: On average, SynX produced significantly more accurate amplitude and shape estimates for unmeasured muscle activations (RMSE 0.08 vs. 0.15,r value 0.55 vs. 0.12) and forces (RMSE 101.3 N vs. 174.4 N,r value 0.53 vs. 0.07) compared to SO. SynX yielded calibrated Hill-type muscle-tendon model parameter values for all muscles and activation dynamics model parameter values for measured muscles that were similar to "gold standard" calibrated model parameter values. Conclusions: These findings suggest that SynX could make it possible to calibrate EMG-driven musculoskeletal models for all important lower-extremity muscles with as few as eight carefully chosen EMG signals and eventually contribute to the design of personalized rehabilitation and surgical interventions for mobility impairments.

A phosphoinositide switch mediates exocyst recruitment to multivesicular endosomes for exosome secretion.

Exosomes are secreted to the extracellular milieu when multivesicular endosomes (MVEs) dock and fuse with the plasma membrane. However, MVEs are also known to fuse with lysosomes for degradation. How MVEs are directed to the plasma membrane for exosome secretion rather than to lysosomes is unclear. Here we report that a conversion of phosphatidylinositol-3-phosphate (PI(3)P) to phosphatidylinositol-4-phosphate (PI(4)P) catalyzed sequentially by Myotubularin 1 (MTM1) and phosphatidylinositol 4-kinase type IIα (PI4KIIα) on the surface of MVEs mediates the recruitment of the exocyst complex. The exocyst then targets the MVEs to the plasma membrane for exosome secretion. We further demonstrate that disrupting PI(4)P generation or exocyst function blocked exosomal secretion of Programmed death-ligand 1 (PD-L1), a key immune checkpoint protein in tumor cells, and led to its accumulation in lysosomes. Together, our study suggests that the PI(3)P to PI(4)P conversion on MVEs and the recruitment of the exocyst direct the exocytic trafficking of MVEs for exosome secretion.

Stiff matrix induces exosome secretion to promote tumour growth.

Tissue fibrosis and extracellular matrix (ECM) stiffening promote tumour progression. The mechanisms by which ECM regulates its contacting cells have been extensively studied. However, how stiffness influences intercellular communications in the microenvironment for tumour progression remains unknown. Here we report that stiff ECM stimulates the release of exosomes from cancer cells. We delineate a molecular pathway that links stiff ECM to activation of Akt, which in turn promotes GTP loading to Rab8 that drives exosome secretion. We further show that exosomes generated from cells grown on stiff ECM effectively promote tumour growth. Proteomic analysis revealed that the Notch signalling pathway is activated in cells treated with exosomes derived from tumour cells grown on stiff ECM, consistent with our gene expression analysis of liver tissues from patients. Our study reveals a molecular mechanism that regulates exosome secretion and provides insight into how mechanical properties of the ECM control the tumour microenvironment for tumour growth.

Determine the Function of the Exocyst in Vesicle Tethering by Ectopic Targeting.

We describe an assay, in which ectopically targeting the exocyst subunit Sec3 to mitochondria is used to determine its role in tethering of post-Golgi vesicles to the plasma membrane. In the assay, we use a plasmid that encodes a fusion protein of the mitochondria protein Tom20 and Sec3 N-terminally tagged with the florescence protein mCherry, and coexpress the plasmid in yeast cells with CIT1-GFP, a marker protein of mitochondria. We then detect the colocalization between Sec3 and CIT1 and other exocyst subunits such as Sec5 on mitochondria using fluorescence microscopy. We further detect the colocalization between Sec3 and Sec4, a Rab protein and a marker of post-Golgi vesicles. Through this assay, we propose that the exocyst subunit Sec3 recruits the other exocyst subunits and secretory vesicles to a target membrane, suggesting that it plays a pivotal role in vesicle tethering. This approach is likely appropriate for studying other tethering complexes at their specific stages of trafficking and may also be used in other eukaryotic cells such as the cultured mammalian cells.

Chemical Characterization of Seasonal PM2.5 Samples and Their Cytotoxicity in Human Lung Epithelial Cells (A549).

In order to study the toxicity of fine particulate matter (PM2.5) sourced from different seasons on human health, we collected PM2.5 samples quarterly from March 2016 to February 2017 in Nanjing, China. The component analysis results showed that high proportions of water-soluble organic carbon (WSOC), SO42-, Ca2+ and Mg2+ were found in the summer samples, while high proportions of NO3-, NH4+ and heavy metals were observed in the spring and winter samples. Then human lung epithelial cells (A549) were exposed to the PM2.5 samples. The toxicological results indicated that reactive oxygen species (ROS) production in the spring and winter samples was higher than that in the summer and fall samples, which was related to the contribution of some heavy metals and inorganic ions (e.g., Pb and NO3-). However, the apoptosis rates of the cells showed the opposite seasonal changes as what the ROS did, which might be caused by the higher WSOC content in the summer. In addition, regression analysis also showed the importance of the PM2.5 components in ROS production and apoptosis. Particularly, Zn had the strongest correlation with ROS production (R = 0.863) and cell apoptosis (R = 0.675); thus, the specific toxicity of Zn in PM2.5 deserves further investigation. Our results could be beneficial for assessing the health risks and controlling the toxic components of PM2.5 in Nanjing.

Auto-ubiquitination of NEDD4-1 Recruits USP13 to Facilitate Autophagy through Deubiquitinating VPS34.

The class III phosphoinositide 3-kinase vacuolar protein sorting 34 (VPS34) is a core protein of autophagy initiation, yet the regulatory mechanisms responsible for its stringent control remain poorly understood. Here, we report that the E3 ubiquitin ligase NEDD4-1 promotes the autophagy flux by targeting VPS34. NEDD4-1 undergoes lysine 29 (K29)-linked auto-ubiquitination at K1279 and serves as a scaffold for recruiting the ubiquitin-specific protease 13 (USP13) to form an NEDD4-1-USP13 deubiquitination complex, which subsequently stabilizes VPS34 to promote autophagy through removing the K48-linked poly-ubiquitin chains from VPS34 at K419. Knockout of either NEDD4-1 or USP13 increased K48-linked ubiquitination and degradation of VPS34, thus attenuating the formation of the autophagosome. Our results identify an essential role for NEDD4-1 in regulating autophagy, which provides molecular insights into the mechanisms by which ubiquitination regulates autophagy flux.

A Patient-Derived Glioblastoma Organoid Model and Biobank Recapitulates Inter- and Intra-tumoral Heterogeneity.

Glioblastomas exhibit vast inter- and intra-tumoral heterogeneity, complicating the development of effective therapeutic strategies. Current in vitro models are limited in preserving the cellular and mutational diversity of parental tumors and require a prolonged generation time. Here, we report methods for generating and biobanking patient-derived glioblastoma organoids (GBOs) that recapitulate the histological features, cellular diversity, gene expression, and mutational profiles of their corresponding parental tumors. GBOs can be generated quickly with high reliability and exhibit rapid, aggressive infiltration when transplanted into adult rodent brains. We further demonstrate the utility of GBOs to test personalized therapies by correlating GBO mutational profiles with responses to specific drugs and by modeling chimeric antigen receptor T cell immunotherapy. Our studies show that GBOs maintain many key features of glioblastomas and can be rapidly deployed to investigate patient-specific treatment strategies. Additionally, our live biobank establishes a rich resource for basic and translational glioblastoma research.