α-Catenin and Piezo1 Mediate Cell Mechanical Communication via Cell Adhesions.

Cell-to-cell distant mechanical communication has been demonstrated using in vitro and in vivo models. However, the molecular mechanisms underlying long-range cell mechanoresponsive interactions remain to be fully elucidated. This study further examined the roles of α-Catenin and Piezo1 in traction force-induced rapid branch assembly of airway smooth muscle (ASM) cells on a Matrigel hydrogel containing type I collagen. Our findings demonstrated that siRNA-mediated downregulation of α-Catenin or Piezo1 expression or chemical inhibition of Piezo1 activity significantly reduced both directional cell movement and branch assembly. Regarding the role of N-cadherin in regulating branch assembly but not directional migration, our results further confirmed that siRNA-mediated downregulation of α-Catenin expression caused a marked reduction in focal adhesion formation, as assessed by focal Paxillin and Integrin α5 localization. These observations imply that mechanosensitive α-Catenin is involved in both cell-cell and cell-matrix adhesions. Additionally, Piezo1 partially localized in focal adhesions, which was inhibited by siRNA-mediated downregulation of α-Catenin expression. This result provides insights into the Piezo1-mediated mechanosensing of traction force on a hydrogel. Collectively, our findings highlight the significance of α-Catenin in the regulation of cell-matrix interactions and provide a possible interpretation of Piezo1-mediated mechanosensing activity at focal adhesions during cell-cell mechanical communication.

Dynamic analysis of circulating tumor DNA to predict the prognosis and monitor the treatment response of patients with metastatic triple-negative breast cancer: A prospective study.

Background: Limited data are available on applying circulating tumor DNA (ctDNA) in metastatic triple-negative breast cancer (mTNBC) patients. Here, we investigated the value of ctDNA for predicting the prognosis and monitoring the treatment response in mTNBC patients. Methods: We prospectively enrolled 70 Chinese patients with mTNBC who had progressed after ≤2 lines of chemotherapy and collected blood samples to extract ctDNA for 457-gene targeted panel sequencing. Results: Patients with ctDNA+, defined by 12 prognosis-relevant mutated genes, had a shorter progression-free survival (PFS) than ctDNA- patients (5.16 months vs. 9.05 months, p=0.001), and ctDNA +was independently associated with a shorter PFS (HR, 95% CI: 2.67, 1.2-5.96; p=0.016) by multivariable analyses. Patients with a higher mutant-allele tumor heterogeneity (MATH) score (≥6.316) or a higher ctDNA fraction (ctDNA%≥0.05) had a significantly shorter PFS than patients with a lower MATH score (5.67 months vs.11.27 months, p=0.007) and patients with a lower ctDNA% (5.45 months vs. 12.17 months, p<0.001), respectively. Positive correlations with treatment response were observed for MATH score (R=0.24, p=0.014) and ctDNA% (R=0.3, p=0.002), but not the CEA, CA125, or CA153. Moreover, patients who remained ctDNA +during dynamic monitoring tended to have a shorter PFS than those who did not (3.90 months vs. 6.10 months, p=0.135). Conclusions: ctDNA profiling provides insight into the mutational landscape of mTNBC and may reliably predict the prognosis and treatment response of mTNBC patients. Funding: This work was supported by the National Natural Science Foundation of China (Grant No. 81902713), Natural Science Foundation of Shandong Province (Grant No. ZR2019LZL018), Breast Disease Research Fund of Shandong Provincial Medical Association (Grant No. YXH2020ZX066), the Start-up Fund of Shandong Cancer Hospital (Grant No. 2020-PYB10), Beijing Science and Technology Innovation Fund (Grant No. KC2021-ZZ-0010-1).

Double-Transmembrane Domain of SNAREs Decelerates the Fusion by Increasing the Protein-Lipid Mismatch.

SNARE is the essential mediator of membrane fusion that highly relies on the molecular structure of SNAREs. For instance, the protein syntaxin-1 involved in neuronal SNAREs, has a single transmembrane domain (sTMD) leading to fast fusion, while the syntaxin 17 has a V-shape double TMDs (dTMDs), taking part in the autophagosome maturation. However, it is not clear how the TMD structure influences the fusion process. Here, we demonstrate that the dTMDs significantly reduce fusion rate compared with the sTMD by using an in vitro reconstitution system. Through theoretical analysis, we reveal that the V-shape dTMDs can significantly increase protein-lipid mismatch, thereby raising the energy barrier of the fusion, and that increasing the number of SNAREs can reduce the energy barrier or protein-lipid mismatch. This study provides a physicochemical mechanistic understanding of SNARE-regulated membrane fusion.

[A case of liver failure caused by fatal amanita poisoning was successfully treated by open cholecystostomy drainage].

The fatality rate of liver failure caused by fatal amanita poisoning is high, and there are no effective antidote drugs in China. On July 30, 2020, the department of infectious diseases and liver diseases of the First People's Hospital of Yunnan Province admitted a 67-year-old female patient with liver failure caused by fatal amanita poisoning. The patient went to the emergency department for treatment due to abdominal pain, vomiting and diarrhea after eating 350-400 g of amanita mushroom for 2 days, accompanied by fatigue for 1 day. There was no abnormality in physical examination. Laboratory indexes: alanine aminotransferase (ALT) 4 798 U/L, aspartate aminotransferase (AST) 10 030 U/L, activated partial thromboplastin time (APTT) 57.5 s, prothrombin time (PT) 72.1 s, international normalized ratio (INR) 8.66, prothrombinactivity (PA) 10%. Based on the patient's medical history, clinical manifestations and laboratory data, the diagnosis was amanita peptide mushroom poisoning and acute liver failure. According to the mechanism of amanita toxin poisoning as enterohepatic circulation, endoscopic retrograde cholangiopancreatography and ultrasound-guided gallbladder puncture and drainage for drainage of bile to discharge toxins were performed to interrupt the enterohepatic circulation of toxins. However, both methods failed, so open cholecystostomy was performed. Because the patient's coagulation function was very poor, artificial hepatic plasma exchange was given to improve coagulation function before open cholecystostomy, and eventually bile was drained successfully. After a total of 19 days of comprehensive medical treatment, the patient was cured and discharged from the hospital, and no sequelae was found after 1 year of follow-up. For such patients, early identification of the disease is required, and blocking the enterohepatic circulation of toxins as soon as possible according to the characteristics and toxicological mechanism of toxins may be the key treatment for rescuing patients with liver failure poisoned by amanita toxin, and it is necessary to combine comprehensive treatments such as active fluid replacement and blood purification to further improve the survival rate.

The Efficacy and Safety of Anlotinib Alone and in Combination with Other Drugs in Previously Treated Advanced Thymic Epithelia Tumors: A Retrospective Analysis.

BACKGROUND: Thymic epithelial tumors (TETs) are rare thoracic malignancies with no standard second-line treatment. Tumor angiogenesis is closely associated with the pathogenesis and invasiveness of TETs. Anlotinib is a small-molecule multitarget tyrosine kinase inhibitor (TKI) which inhibits tumor angiogenesis and tumor cell proliferation. Published studies have demonstrated the promising clinical effect of multitarget TKIs sunitinib and lenvatinib in previously treated TETs. However, TKIs have a high incidence of adverse events (AEs). OBJECTIVE: In this study, we investigated the clinical efficacy and safety of anlotinib in previously treated TET patients. METHODS: We collected clinical data of 22 patients from Shandong Cancer Hospital and Institute between October 2018 and March 2022. These patients were diagnosed with advanced TETs and received at least the first-line (1st-line) treatment. We analyzed the clinical effects between anlotinib monotherapy and anlotinib combination therapy in the second-line (2nd-line) or anlotinib treatment in different lines. RESULTS: These 22 patients included 18 cases of thymic carcinoma (TC) and 4 cases of thymoma (T). 68.2% of patients were males, and the median age was 53 years. Fourteen patients (63.6%) received anlotinib monotherapy and 8 patients (36.4%) received anlotinib combination therapy. The objective response rate (ORR) was 9.1% in the overall patients. The median progression-free survival (PFS) in the overall population was 12 months (14 months for T and 9 months for TC), and the median overall survival (OS) was 24 months (survival was not reached for T and was 24 months for TC). The incidence of AEs was 50%, most of them were grades I and II, and the incidence of grades III and IV AEs was 9%. CONCLUSION: This is the first study reporting the clinical effect of anlotinib in previously treated TETs patients. The survival data indicate that the efficacy of anlotinib is superior to sunitinib and lenvatinib. Our results suggest that anlotinib is a promising treatment option for previously treated TET patients and its toxicity is tolerable. More research and patents are needed in the future to explore better options for the diagnosis and treatment of TETs.

Mechanical communication-associated cell directional migration and branching connections mediated by calcium channels, integrin ß1, and N-cadherin.

Cell-cell mechanical communications at a large spatial scale (above hundreds of micrometers) have been increasingly recognized in recent decade, which shows importance in tissue-level assembly and morphodynamics. The involved mechanosensing mechanism and resulted physiological functions are still to be fully understood. Recent work showed that traction force sensation in the matrix induces cell communications for self-assembly. Here, based on the experimental model of cell directional migration on Matrigel hydrogel, containing 0.5 mg/ml type I collagen, we studied the mechano-responsive pathways for cell distant communications. Airway smooth muscle (ASM) cells assembled network structure on the hydrogel, whereas stayed isolated individually when cultured on glass without force transmission. Cell directional migration, or network assembly was significantly attenuated by inhibited actomyosin activity, or inhibition of inositol 1,4,5-trisphosphate receptor (IP3R) calcium channel or SERCA pump on endoplasmic reticulum (ER) membrane, or L-type calcium channel on the plasma membrane. Inhibition of integrin ß1 with siRNA knockdown reduced cell directional migration and branching assembly, whereas inhibition of cell junctional N-cadherin with siRNA had little effect on distant attractions but blocked branching assembly. Our work demonstrated that the endoplasmic reticulum calcium channels and integrin are mechanosensing signals for cell mechanical communications regulated by actomyosin activity, while N-cadherin is responsible for traction force-induced cell stable connections in the assembly.

Comparison of endocrine therapy and chemotherapy as different systemic treatment modes for metastatic luminal HER2-negative breast cancer patients -A retrospective study.

Purpose: The purpose of this study was to evaluate endocrine therapy and chemotherapy for first-line, maintenance, and second-line treatment of hormone receptor-positive HER-2-negative metastatic breast cancer (HR+HER-2-MBC) and the relationship between different treatment options and survival. Patients and methods: The patients included in this study were all diagnosed with metastatic breast cancer (MBC) at Shandong Cancer Hospital from January 2013 to June 2017. Of the 951 patients with MBC, 307 patients with HR+HER-2-MBC were included in the analysis. The progression-free survival (PFS) and overall survival (OS) of the various treatment modes were evaluated using Kaplan-Meier analysis and the log-rank test. Because of the imbalance in data, we used the synthetic minority oversampling technique (SMOTE) algorithm to oversample the data to increase the balanced amount of data. Results: This retrospective study included 307 patients with HR+HER-2-MBC; 246 patients (80.13%) and 61 patients (19.87%) were treated with first-line chemotherapy and first-line endocrine therapy, respectively. First-line endocrine therapy was better than first-line chemotherapy in terms of PFS and OS. After adjusting for known prognostic factors, patients receiving first-line chemotherapy had poorer PFS and OS outcomes than patients receiving first-line endocrine therapy. In terms of maintenance treatment, the endocrine therapy-endocrine therapy maintenance mode achieved the best prognosis, followed by the chemotherapy-endocrine therapy maintenance mode and chemotherapy-chemotherapy maintenance mode, and the no-maintenance mode has resulted in the worst prognosis. In terms of first-line/second-line treatment, the endocrine therapy/endocrine therapy mode achieved the best prognosis, while the chemotherapy/chemotherapy mode resulted in the worst prognosis. The chemotherapy/endocrine therapy mode achieved a better prognosis than the endocrine therapy/chemotherapy mode. There were no significant differences in the KI-67 index (<15%/15-30%/≥30%) among the patients receiving first-line treatment modes, maintenance treatment modes, and first-line/second-line treatment modes. There was no statistical evidence in this study to support that the KI-67 index affected survival. However, in the first-line/second-line model, after SMOTE, we could see that KI-67 ≥ 30% had a poor prognosis. Conclusions: Different treatment modes for HR+HER-2-MBC were analyzed. Endocrine therapy achieved better PFS and OS outcomes than chemotherapy. Endocrine therapy should be the first choice for first-line, maintenance, and second-line treatment of HR+HER-2-MBC.

Clinical Outcomes of BCMA CAR-T Cells in a Multiple Myeloma Patient With Central Nervous System Invasion.

Background: Multiple myeloma (MM) is the second most common hematological malignancy that still lacks effective clinical treatments. In particular, MM with central nervous system (CNS) invasion occurs rarely. Although B-cell maturation antigen (BCMA)-targeted chimeric antigen receptor-T (CAR-T) cell therapy has shown great promise for the treatment of relapsed/refractory MM, few studies have reported whether BCMA CAR-T could inhibit MM with CNS invasion. Case Presentation: In this study, we report a special case of a 63-year-old male patient who suffered MM with CNS invasion and presented rapid extramedullary disease (EMD) progression into multiple organs. Before CAR-T cell infusion, this patient received five cycles of bortezomib, Adriamycin, and dexamethasone (PAD) and an autologous transplant as the front-line treatment, followed by two cycles of bortezomib, lenalidomide, and dexamethasone (VRD) as the second-line regimen, and daratumumab, bortezomib, dexamethasone (DVD) as the third-line regimen. Since the patient still showed rapid progressive disease (PD), BCMA CAR-T cells were infused, and 1 month later, a stringent complete response (sCR) was achieved, and the response lasted for 4 months. Meanwhile, only grade 1 cytokine release syndrome (CRS) was observed. Conclusion: This case report demonstrated that BCMA CAR-T could effectively eradicate CNS-involved MM with low adverse events, suggesting that CAR-T cell therapy could be a feasible therapeutic option for this kind of refractory disease. Clinical Trial Registration: https://ClinicalTrials.gov, identifier: NCT04537442.a.

FRET Visualization of Cyclic Stretch-Activated ERK via Calcium Channels Mechanosensation While Not Integrin ß1 in Airway Smooth Muscle Cells.

Mechanical stretch is one type of common physiological activities such as during heart beating, lung breathing, blood flow through the vessels, and physical exercise. The mechanical stimulations regulate cellular functions and maintain body homeostasis. It still remains to further characterize the mechanical-biomechanical coupling mechanism. Here we applied fluorescence resonance energy transfer (FRET) technology to visualize ERK activity in airway smooth muscle (ASM) cells under cyclic stretch stimulation in airway smooth muscle (ASM) cells, and studied the mechanosensing pathway. FRET measurements showed apparent ERK activation by mechanical stretch, which was abolished by ERK inhibitor PD98059 pretreatment. Inhibition of extracellular Ca2+ influx reduced ERK activation, and selective inhibition of inositol 1,4,5-trisphosphate receptor (IP3R) Ca2+ channel or SERCA Ca2+ pump on endoplasmic reticulum (ER) blocked the activation. Chemical inhibition of the L-type or store-operated Ca2+ channels on plasma membrane, or inhibition of integrin ß1 with siRNA had little effect on ERK activation. Disruption of actin cytoskeleton but not microtubule one inhibited the stretch-induced ERK activation. Furthermore, the ER IP3R-dependent ERK activation was not dependent on phospholipase C-IP3 signal, indicating possibly more mechanical mechanism for IP3R activation. It is concluded from our study that the mechanical stretch activated intracellular ERK signal in ASM cells through membrane Ca2+ channels mechanosensation but not integrin ß1, which was mediated by actin cytoskeleton.

MircroRNA Let-7a-5p in Airway Smooth Muscle Cells is Most Responsive to High Stretch in Association With Cell Mechanics Modulation.

Objective: High stretch (strain >10%) can alter the biomechanical behaviors of airway smooth muscle cells which may play important roles in diverse lung diseases such as asthma and ventilator-induced lung injury. However, the underlying modulation mechanisms for high stretch-induced mechanobiological responses in ASMCs are not fully understood. Here, we hypothesize that ASMCs respond to high stretch with increased expression of specific microRNAs (miRNAs) that may in turn modulate the biomechanical behaviors of the cells. Thus, this study aimed to identify the miRNA in cultured ASMCs that is most responsive to high stretch, and subsequently investigate in these cells whether the miRNA expression level is associated with the modulation of cell biomechanics. Methods: MiRNAs related to inflammatory airway diseases were obtained via bioinformatics data mining, and then tested with cultured ASMCs for their expression variations in response to a cyclic high stretch (13% strain) simulating in vivo ventilator-imposed strain on airways. Subsequently, we transfected cultured ASMCs with mimics and inhibitors of the miRNA that is most responsive to the high stretch, followed by evaluation of the cells in terms of morphology, stiffness, traction force, and mRNA expression of cytoskeleton/focal adhesion-related molecules. Results: 29 miRNAs were identified to be related to inflammatory airway diseases, among which let-7a-5p was the most responsive to high stretch. Transfection of cultured human ASMCs with let-7a-5p mimics or inhibitors led to an increase or decrease in aspect ratio, stiffness, traction force, migration, stress fiber distribution, mRNA expression of α-smooth muscle actin (SMA), myosin light chain kinase, some subfamily members of integrin and talin. Direct binding between let-7a-5p and ItgαV was also verified in classical model cell line by using dual-luciferase assays. Conclusion: We demonstrated that high stretch indeed enhanced the expression of let-7a-5p in ASMCs, which in turn led to changes in the cells' morphology and biomechanical behaviors together with modulation of molecules associated with cytoskeletal structure and focal adhesion. These findings suggest that let-7a-5p regulation is an alternative mechanism for high stretch-induced effect on mechanobiology of ASMCs, which may contribute to understanding the pathogenesis of high stretch-related lung diseases.

A durable 4-1BB-based CD19 CAR-T cell for treatment of relapsed or refractory non-Hodgkin lymphoma.

Objective: Previous studies reported that 4-1BB-based CD19 chimeric antigen receptor (CAR)-T cells were more beneficial for the clinical outcomes than CD28-based CAR-T cells, especially the lower incidence rate of severe adverse events. However, the median progression-free survival (mPFS) of 4-1BB-based product Kymriah was shorter than that of CD28-based Yescarta (2.9 monthsvs. 5.9 months), suggesting that Kymriah was limited in the long-term efficacy. Thus, a safe and durable 4-1BB-based CD19 CAR-T needs to be developed. Methods: We designed a CD19-targeted CAR-T (named as IM19) which consisted of an FMC63 scFv, 4-1BB and CD3ζ intracellular domain and was manufactured into a memory T-enriched formulation. A phase I/II clinical trial was launched to evaluate the clinical outcomes of IM19 in relapsed or refractory (r/r) B cell non-Hodgkin lymphoma (B-NHL). Dose-escalation investigation (at a dose of 5×105/kg, 1×106/kg and 3×106/kg) was performed in 22 r/r B-NHL patients. All patients received a single infusion of IM19 after 3-day conditional regimen. Results: At month 3, the overall response rate (ORR) was 59.1%, the complete response rate (CRR) was 50.0%. The mPFS was 6 months and the 1-year overall survival rate was 77.8%. Cytokine release syndrome (CRS) occurred in 13 patients (59.1%), with 54.5% of grade 1-2 CRS. Only one patient (4.5%) experienced grade 3 CRS and grade 3 neurotoxicity. Conclusions: These results demonstrated the safety and durable efficacy of a 4-1BB-based CD19 CAR-T, IM19, which is promising for further development and clinical investigation.

Sensitive detection of cell-derived force and collagen matrix tension in microtissues undergoing large-scale densification.

Mechanical forces generated by cells and the tension of the extracellular matrix (ECM) play a decisive role in establishment, homeostasis maintenance, and repair of tissue morphology. However, the dynamic change of cell-derived force during large-scale remodeling of soft tissue is still unknown, mainly because the current techniques of force detection usually produce a nonnegligible and interfering feedback force on the cells during measurement. Here, we developed a method to fabricate highly stretchable polymer-based microstrings on which a microtissue of fibroblasts in collagen was cultured and allowed to contract to mimic the densification of soft tissue. Taking advantage of the low-spring constant and large deflection range of the microstrings, we detected a strain-induced contraction force as low as 5.2 µN without disturbing the irreversible densification. Meanwhile, the microtissues displayed extreme sensitivity to the mechanical boundary within a narrow range of tensile stress. More importantly, results indicated that the cell-derived force did not solely increase with increased ECM stiffness as previous studies suggested. Indeed, the cell-derived force and collagen tension exchanged dramatically in dominating the microtissue strain during the densification, and the proportion of cell-derived force decreased linearly as the microtissue densified, with stiffness increasing to ∼500 Pa. Thus, this study provides insights into the biomechanical cross-talk between the cells and ECM of extremely soft tissue during large-extent densification, which may be important to guide the construction of life-like tissue by applying appropriate mechanical boundary conditions.

Naringin as a plant-derived bitter tastant promotes proliferation of cultured human airway epithelial cells via activation of TAS2R signaling.

BACKGROUND: Bitter tastants can activate bitter taste receptors (TAS2Rs) and thus initiate relaxation of airway smooth muscle cells (ASMCs), which have great potential in the development of novel bronchodilator drugs for asthma therapy. However, the canonical bitter substance, denatonium is known to induce apoptosis of airway epithelial cells (AECs), indicating that other bitter tastants may also impair the epithelial integrity to prevent hazardous particulate matters such as coronaviruses. Therefore, any bitter tastants intended for treating airway disease should be carefully evaluated for potential toxicity to AECs. HYPOTHESIS/PURPOSE: Considering the vast diversity of bitter tastants in nature and different types of TAS2Rs expressed in airway cells, we hypothesized that there must be some natural bitter tastants to be not only potent in inducing relaxation of ASMCs but also unharmful to AECs. STUDY DESIGN AND METHODS: Here we evaluated a group of bitter flavonoids that are derived from fruits and commonly used in traditional herbal medicine, including apigenin, hesperetin, kaempferol, naringenin, quercetin, and naringin, for their effects on the proliferation of human airway epithelial-like (16HBE14o-, BEAS-2B, and A549) cells cultured in vitro. Cell proliferation and associated signaling pathways were assessed by cell counting, ATP assay, cell cycling assay, quantitative RT-PCR, Fluo-4 labeling, and fluorescence resonance energy transfer, respectively. RESULTS: The results show that five of the six tested bitter tastants inhibited, but only naringin promoted the proliferation of the 16HBE14o-, BEAS-2B, and A549 cells at the dose of a few hundred micromoles. Furthermore, the naringin-promoted proliferation of the 16HBE14o- cells was associated with enhanced cell cycle progression, mRNA expression of cyclin E, and evoked calcium signaling/ERK signaling, which were all attenuated by inhibition of the TAS2R signaling pathways with specific blockers. CONCLUSION: These findings indicate that although the majority of the bitter flavonoids may inhibit the proliferation of AECs, naringin emerged as one to promote the proliferation of AECs via cell cycle progression and TAS2R-activated intracellular signaling. It suggests that naringin and not a few other bitter tastants can be proven with nontoxicity to the airway epithelial structure and function, which provides further confidence in the development of safe and effective TAS2R-based bronchodilators for asthma therapy.

Membrane packing defects in synaptic vesicles recruit complexin and synuclein.

Complexin-1 (Cpx) and α-synuclein (α-Syn) are involved in neurotransmitter release through an interaction with synaptic vesicles (SVs). Recent studies demonstrated that Cpx and α-Syn preferentially associate with highly curved membranes, like SVs, to correctly position them for fusion. Here, based on recent experimental results, to further propose a possible explanation for this mechanism, we performed in silico simulations probing interactions between Cpx or α-Syn and membranes of varying curvature. We found that the preferential association is attributed to smaller, curved membranes containing more packing defects that expose hydrophobic acyl tails, which may favorably interact with hydrophobic residues of Cpx and α-Syn. The number of membrane defects is proportional to the curvature and the size can be regulated by cholesterol.

Sensing Traction Force on the Matrix Induces Cell-Cell Distant Mechanical Communications for Self-Assembly.

The long-range biomechanical force propagating across a large scale may reserve the capability to trigger coordinative responses within cell population such as during angiogenesis, epithelial tubulogenesis, and cancer metastasis. How cells communicate in a distant manner within the group for self-assembly remains largely unknown. Here, we found that airway smooth muscle cells (ASMCs) rapidly self-assembled into a well-constructed network on 3D Matrigel containing type I collagen (COL), which relied on long-range biomechanical force across the matrix to direct cell-cell distant interactions. Similar results happened by HUVEC cells to mimic angiogenesis. Interestingly, single ASMCs initiated multiple extended protrusions precisely pointing to neighboring cells in distance (100-300 µm away or 5-10 folds of the diameter of a round single cell), depending on traction force sensing. Individual ASMCs mechanosensed each other to move directionally on both nonfibrous Matrigel only and Matrigel containing fibrous COL but lost mutual sensing on the cross-linked gel or coated glass due to no long-range force transmission. The bead tracking assay demonstrated distant transmission of traction force (up to 400 µm) during the matrix deformation, and finite element method modeling confirmed the consistency between maximum strain distribution on the matrix and cell directional movements in experiments. Furthermore, ASMCs recruited COL from the hydrogel to build a fibrous network to mechanically stabilize the cell network. Our results revealed principally that cells can sense traction force transmitted through the matrix to initiate cell-cell distant mechanical communications, resulting in cell directional migration and coordinated cell and COL self-assembly with active matrix remodeling. As an interesting phenomenon, cells seem to be able to "make a phone call" via long-range biomechanics, which implicates physiological importance such as for tissue pattern formation.

Structure-guided engineering of Pseudomonas dacunhael-aspartate ß-decarboxylase for l-homophenylalanine synthesis.

Structure-guided engineering of Pseudomonas dacunhael-aspartate ß-decarboxylase (AspBDC) resulted in a double mutant (R37A/T382G) with remarkable 15 400-fold improvement in specific activity reaching 216 mU mg-1, towards the target substrate 3(R)-benzyl-l-aspartate. A novel strategy for enzymatic synthesis of l-homophenylalanine was developed by using the variant as a biocatalyst affording 75% product yield within 12 h. Our results underscore the potential of engineered AspBDC for the biocatalytic synthesis of pharmaceutically relevant and value added unnatural l-amino acids.

Single-vesicle imaging quantifies calcium's regulation of nanoscale vesicle clustering mediated by α-synuclein.

Although numerous studies have shown that the protein α-synuclein (α-Syn) plays a central role in Parkinson's disease, dementia with Lewy bodies, and other neurodegenerative diseases, the protein's physiological function remains poorly understood. Furthermore, despite recent reports suggesting that, under the influence of Ca2+, α-Syn can interact with synaptic vesicles, the mechanisms underlying that interaction are far from clear. Thus, we used single-vesicle imaging to quantify the extent to which Ca2+ regulates nanoscale vesicle clustering mediated by α-Syn. Our results revealed not only that vesicle clustering required α-Syn to bind to anionic lipid vesicles, but also that different concentrations of Ca2+ exerted different effects on how α-Syn induced vesicle clustering. In particular, low concentrations of Ca2+ inhibited vesicle clustering by blocking the electrostatic interaction between the lipid membrane and the N terminus of α-Syn, whereas high concentrations promoted vesicle clustering, possibly due to the electrostatic interaction between Ca2+ and the negatively charged lipids that is independent of α-Syn. Taken together, our results provide critical insights into α-Syn's physiological function, and how Ca2+ regulates vesicle clustering mediated by α-Syn.

Oxytocin involves in chronic stress-evoked melanoma metastasis via ß-arrestin 2-mediated ERK signaling pathway.

Stress is associated with an increased risk of lung metastasis in melanoma. However, the underlying mechanism is elusive. Oxytocin (OXT), a neurohormone produced by the hypothalamus, plays a vital role in laboring induction and lactation. Emerging evidence suggests that OXT also regulates human emotions, social cognition, social behaviors and stress-related disorders. Here, we reported that a significant up-regulation of oxytocin receptors (OXTRs) was observed in malignant melanoma. The activation of OXTRs dramatically promoted migration, invasion and angiogenesis but not the proliferation of melanoma cells in vitro and in vivo via ß-arrestin 2-dependent ERK-VEGF/MMP-2 pathway. Next, chronic restraint stress significantly elevated the plasma level of OXT. Notably, 21 days chronic restraint stress facilitated lung metastasis of melanoma and reduced overall survival in mice, which were largely abrogated by knocking down either OXTR or ß-arrestin 2. These findings provide evidence that chronic stress hormone-OXT promotes lung metastasis of melanoma via a ß-arrestin 2-dependent mechanism and suggest that OXT, a novel pro-metastasis factor, is a potential therapeutic target for melanoma.

Cholesterol suppresses membrane leakage by decreasing water penetrability.

Membrane fusion is a fundamental biological process that lies at the heart of enveloped virus infection, synaptic signaling, intracellular vesicle trafficking, gamete fertilization, and cell-cell fusion. Membrane fusion is initiated as two apposed membranes merge to a single bilayer called a hemifusion diaphragm. It is believed that the contents of the two fusing membranes are released through a fusion pore formed at the hemifusion diaphragm, and yet another possible pathway has been proposed in which an undefined pore may form outside the hemifusion diaphragm at the apposed membranes, leading to the so-called leaky fusion. Here, we performed all-atom molecular dynamics simulations to study the evolution of the hemifusion diaphragm structure with various lipid compositions. We found that the lipid cholesterol decreased water penetrability to inhibit leakage pore formation. Biochemical leakage experiments support these simulation results. This study may shed light on the underlying mechanism of the evolution pathways of the hemifusion structure, especially the understanding of content leakage during membrane fusion.

A missense variant in EZH2 is associated with colorectal cancer risk in a Chinese population.

Colorectal cancer (CRC) ranks the fifth leading cause of cancer death in China. EZH2 is a member of Polycomb-group (PcG) family and associated with transcriptional repression and cancer development. In this study, we report the association between a missense variant in EZH2 and risk of CRC. Through a systematic selection of variants in EZH2, we identified rs2302427 in the exon region of EZH2 and genotyped this variant in 852 CRC patients and 1,303 healthy controls using Taqman genotyping assay. The association between this variant and CRC risk was calculated using logistic regression with adjustment of sex, age, smoking status and drinking status. The result showed that rs2302427 was significantly associated with CRC susceptibility under an additive model (P=0.0068). Compared with CC genotype carriers, CG genotype and GG genotype carriers were associated with risk of CRC with odds ratio being 0.78 (95% CI: 0.63-0.96, P=0.0198) and 0.54 (95% CI: 0.24-1.18, P=0.1224), respectively. When stratified by sex, age, smoking status or drinking status, significant associations were observed only in younger individuals (OR=0.67, 95% CI: 0.50-0.89, P=0.0067) or smokers (OR=0.65, 95% CI: 0.48-0.88, P=0.0051). This study provides new insights into the personalized prevention of colorectal cancer.