Structural Biochemistry of Muscle Contraction.

Muscles are essential for movement and heart function. Contraction and relaxation of muscles rely on the sliding of two types of filaments-the thin filament and the thick myosin filament. The thin filament is composed mainly of filamentous actin (F-actin), tropomyosin, and troponin. Additionally, several other proteins are involved in the contraction mechanism, and their malfunction can lead to diverse muscle diseases, such as cardiomyopathies. We review recent high-resolution structural data that explain the mechanism of action of muscle proteins at an unprecedented level of molecular detail. We focus on the molecular structures of the components of the thin and thick filaments and highlight the mechanisms underlying force generation through actin-myosin interactions, as well as Ca2+-dependent regulation via the dihydropyridine receptor, the ryanodine receptor, and troponin. We particularly emphasize the impact of cryo-electron microscopy and cryo-electron tomography in leading muscle research into a new era.

The molecular basis for sarcomere organization in vertebrate skeletal muscle.

Sarcomeres are force-generating and load-bearing devices of muscles. A precise molecular picture of how sarcomeres are built underpins understanding their role in health and disease. Here, we determine the molecular architecture of native vertebrate skeletal sarcomeres by electron cryo-tomography. Our reconstruction reveals molecular details of the three-dimensional organization and interaction of actin and myosin in the A-band, I-band, and Z-disc and demonstrates that α-actinin cross-links antiparallel actin filaments by forming doublets with 6-nm spacing. Structures of myosin, tropomyosin, and actin at ~10 Å further reveal two conformations of the "double-head" myosin, where the flexible orientation of the lever arm and light chains enable myosin not only to interact with the same actin filament, but also to split between two actin filaments. Our results provide unexpected insights into the fundamental organization of vertebrate skeletal muscle and serve as a strong foundation for future investigations of muscle diseases.

Structure of the native myosin filament in the relaxed cardiac sarcomere.

The thick filament is a key component of sarcomeres, the basic units of striated muscle1. Alterations in thick filament proteins are associated with familial hypertrophic cardiomyopathy and other heart and muscle diseases2. Despite the central importance of the thick filament, its molecular organization remains unclear. Here we present the molecular architecture of native cardiac sarcomeres in the relaxed state, determined by cryo-electron tomography. Our reconstruction of the thick filament reveals the three-dimensional organization of myosin, titin and myosin-binding protein C (MyBP-C). The arrangement of myosin molecules is dependent on their position along the filament, suggesting specialized capacities in terms of strain susceptibility and force generation. Three pairs of titin-α and titin-ß chains run axially along the filament, intertwining with myosin tails and probably orchestrating the length-dependent activation of the sarcomere. Notably, whereas the three titin-α chains run along the entire length of the thick filament, titin-ß chains do not. The structure also demonstrates that MyBP-C bridges thin and thick filaments, with its carboxy-terminal region binding to the myosin tails and directly stabilizing the OFF state of the myosin heads in an unforeseen manner. These results provide a foundation for future research investigating muscle disorders involving sarcomeric components.

Impact of Lymph Node Dissection on Survival After Neoadjuvant Chemoradiotherapy for Locally Advanced Esophageal Squamous Cell Carcinoma: From the Results of NEOCRTEC5010, a Randomized Multicenter Study.

OBJECTIVE: To clarify whether systemic LND influences the safety of surgery and the survival of patients with locally advanced esophageal squamous cell carcinoma (ESCC) after neoadjuvant chemoradiotherapy (nCRT). SUMMARY OF BACKGROUND DATA: Prognostic impact of systemic lymphadenectomy during surgery after nCRT for ESCC is still uncertain and requires clarification. METHODS: This is a secondary analysis of NEOCRTEC5010 trial which compared nCRT followed by surgery versus surgery alone for locally advanced ESCC. Relationship between number of LND and perioperative, recurrence, and survival outcomes were analyzed in the nCRT group. RESULTS: Three-year overall survival was significantly better in the nCRT group than the S group (75.2% vs 61.5%; P = 0.011). In the nCRT group, greater number of LND was associated with significantly better overall survival (hazard ratio, 0.358; P < 0.001) and disease-free survival (hazard ratio, 0.415; P = 0.001), but without any negative impact on postoperative complications. Less LND (<20 vs ≥20) was significantly associated with increased local recurrence (18.8% vs 5.2%, P = 0.004) and total recurrence rates (41.2% vs 25.8%, P = 0.027). Compared to patients with persistent nodal disease, significantly better survival was seen in patients with complete response and with LND ≥20, but not in those with LND <20. CONCLUSIONS: Systemic LND does not increase surgical risks after nCRT in ESCC patients. And it is associated with better survival and local diseasecontrol. Therefore, systemic lymphadenectomy should still be considered as an integrated part of surgery after nCRT for ESCC.

Application of a Mixed-Ligand Metal-Organic Framework in Photocatalytic CO2 Reduction, Antibacterial Activity and Dye Adsorption.

In this paper, a known mixed-ligand MOF {[Co2(TZMB)2(1,4-bib)0.5(H2O)2]·(H2O)2}n (compound 1) was reproduced, and its potential application potential was explored. It was found that compound 1 had high photocatalytic activity for CO2 reduction. After 12 h of illumination, the formation rate of CO, which is the product of CO2 reduction by compound 1, reached 3012.5 µmol/g/h. At the same time, compound 1 has a good antibacterial effect on Staphylococcus aureus (S. aureus), Escherichia coli (E. coli) and Candida albicans (C. albicans), which has potential research value in the medical field. In addition, compound 1 can effectively remove Congo Red from aqueous solutions and achieve the separation of Congo red from mixed dye solutions.

A streamlined workflow for automated cryo focused ion beam milling.

Cryo-electron tomography (cryo-ET) is an emerging technique to study the cellular architecture and the structure of proteins at high resolution in situ. Most biological specimens are too thick to be directly investigated and are therefore thinned by milling with a focused ion beam under cryogenic conditions (cryo-FIB). This procedure is prone to contaminations, which makes it a tedious process, often leading to suboptimal results. Here, we present new hardware that overcomes the current limitations. We developed a new glove box and a high vacuum cryo transfer system and installed a stage heater, a cryo-shield and a cryo-shutter in the FIB milling microscope. This reduces the ice contamination during the transfer and milling process and simplifies the handling of the sample. In addition, we tested a new software application that automates the key milling steps. Together, these improvements allow for high-quality, high-throughput cryo-FIB milling. This paves the way for new types of experiments, which have been previously considered infeasible.

Full shut-off of Escherichia coli RNA-polymerase by T7 phage requires a small phage-encoded DNA-binding protein.

Infection of Escherichia coli by the T7 phage leads to rapid and selective inhibition of the bacterial RNA polymerase (RNAP) by the 7 kDa T7 protein Gp2. We describe the identification and functional and structural characterisation of a novel 7 kDa T7 protein, Gp5.7, which adopts a winged helix-turn-helix-like structure and specifically represses transcription initiation from host RNAP-dependent promoters on the phage genome via a mechanism that involves interaction with DNA and the bacterial RNAP. Whereas Gp2 is indispensable for T7 growth in E. coli, we show that Gp5.7 is required for optimal infection outcome. Our findings provide novel insights into how phages fine-tune the activity of the host transcription machinery to ensure both successful and efficient phage progeny development.

Yersinia entomophaga Tc toxin is released by T10SS-dependent lysis of specialized cell subpopulations.

Disease-causing bacteria secrete numerous toxins to invade and subjugate their hosts. Unlike many smaller toxins, the secretion machinery of most large toxins remains enigmatic. By combining genomic editing, proteomic profiling and cryo-electron tomography of the insect pathogen Yersinia entomophaga, we demonstrate that a specialized subset of these cells produces a complex toxin cocktail, including the nearly ribosome-sized Tc toxin YenTc, which is subsequently exported by controlled cell lysis using a transcriptionally coupled, pH-dependent type 10 secretion system (T10SS). Our results dissect the Tc toxin export process by a T10SS, identifying that T10SSs operate via a previously unknown lytic mode of action and establishing them as crucial players in the size-insensitive release of cytoplasmically folded toxins. With T10SSs directly embedded in Tc toxin operons of major pathogens, we anticipate that our findings may model an important aspect of pathogenesis in bacteria with substantial impact on agriculture and healthcare.

Improvement of Whole-body Bone Planar Images on a Bone-dedicated Single-photon Emission Computed Tomography Scanner by Blind Deconvolution Algorithm.

Purpose: We have developed a bone-dedicated collimator with higher sensitivity but slightly degraded resolution on single-photon emission computed tomography (SPECT) for planar bone scintigraphy, compared with conventional low-energy high-resolution collimator. In this work, we investigated the feasibility of using the blind deconvolution algorithm to improve the resolution of planar images on bone scintigraphy. Materials and Methods: Monte Carlo simulation was performed with the NCAT phantom for modeling bone scintigraphy on the clinical dual-head SPECT scanner (Imagine NET 632, Beijing Novel Medical Equipment Ltd.) equipped with the bone-dedicated collimator. Maximum likelihood estimation method was used for the blind deconvolution algorithm. The initial estimation of point spread function (PSF) and iteration number for the method were determined by comparing the deblurred images obtained from different input parameters. We simulated different tumors in five different locations and with five different diameters to evaluate the robustness of the initial inputs. Furthermore, we performed chest phantom studies on the clinical SPECT scanner. The quantified increased contrast ratio (CR) between the tumor and the background was evaluated. Results: The 2 mm PSF kernel and 10 iterations provided a practical and robust deblurred image on our system. Those two inputs can generate robust deblurred images in terms of the tumor location and size with an average increased CR of 21.6%. The phantom studies also demonstrated the ability of blind deconvolution, using those two inputs, with increased CRs of 17%, 17%, 22%, 20%, and 13% for lesions with diameters of 1 cm, 2 cm, 3 cm, 4 cm, and 5 cm, respectively. Conclusions: It is feasible to use the blind deconvolution algorithm to deblur the planar images for SPECT bone scintigraphy. The appropriate values of the PSF kernel and the iteration number for the blind deconvolution can be determined using simulation studies.

Tyrosine hydroxylase inhibits HCC progression by downregulating TGFß/Smad signaling.

The alteration of metabolic processes has been found to have significant impacts on the development of hepatocellular carcinoma (HCC). Nevertheless, the effects of dysfunction of tyrosine metabolism on the development of HCC remains to be discovered. This research demonstrated that tyrosine hydroxylase (TH), which responsible for the initial and limiting step in the bio-generation of the neuro-transmitters dopamine and adrenaline, et al. was shown to be reduced in HCC. Increased expression of TH was found facilitates the survival of HCC patients. In addition, decreased TH indicated larger tumor size, much more numbers of tumor, higher level of AFP, and the presence of cirrhosis. TH effectively impairs the growth and metastasis of HCC cells, a process dependent on the phosphorylation of serine residues (S19/S40). TH directly binds to Smad2 and hinders the cascade activation of TGFß/Smad signaling with the treatment of TGFß1. In summary, our study uncovered the non-metabolic functions of TH in the development of HCC and proposes that TH might be a promising biomarker for diagnosis as well as an innovative target for metastatic HCC.

Trustworthy learning with (un)sure annotation for lung nodule diagnosis with CT.

Recent evolution in deep learning has proven its value for CT-based lung nodule classification. Most current techniques are intrinsically black-box systems, suffering from two generalizability issues in clinical practice. First, benign-malignant discrimination is often assessed by human observers without pathologic diagnoses at the nodule level. We termed these data as "unsure-annotation data". Second, a classifier does not necessarily acquire reliable nodule features for stable learning and robust prediction with patch-level labels during learning. In this study, we construct a sure-annotation dataset with pathologically-confirmed labels and propose a collaborative learning framework to facilitate sure nodule classification by integrating unsure-annotation data knowledge through nodule segmentation and malignancy score regression. A loss function is designed to learn reliable features by introducing interpretability constraints regulated with nodule segmentation maps. Furthermore, based on model inference results that reflect the understanding from both machine and experts, we explore a new nodule analysis method for similar historical nodule retrieval and interpretable diagnosis. Detailed experimental results demonstrate that our approach is beneficial for achieving improved performance coupled with trustworthy model reasoning for lung cancer prediction with limited data. Extensive cross-evaluation results further illustrate the effect of unsure-annotation data for deep-learning based methods in lung nodule classification.

Establishment and validation of a novel immune-related prognostic signature in malignant pleural mesothelioma.

Background: Immune-related genes (IRGs) play an important role in the tumor immune microenvironment and affect tumor prognosis. This study aimed to establish a prognostic signature for malignant pleural mesothelioma (MPM) patients. Methods: We obtained the relevant data of MPM patients in The Cancer Genome Atlas (TCGA), and univariate and multivariate Cox regression were used to construct the prediction signature and verify it with the external validation dataset GSE2549. A nomogram was then constructed, and its predictive ability was evaluated and analyzed the level of immune cell infiltration in different groups in the signature. Results: An IRG-related prognostic signature composed of INHBA, CAT, SORT1, TNFSF13B, and BIRC5 was constructed, with patients divided into high-risk and low-risk groups according to the risk score. The survival time of overall survival (OS), progression-free survival (PFS), disease-free interval (DFI), and relapse-free survival (RFS) in low-risk groups was longer than in high-risk groups. Furthermore, the signature had high predictive performance, and the receiver operating characteristic (ROC) of 1, 2, and 3 years could reach 0.853, 0.881, and 0.914, respectively. The predictive accuracy of the signature was verified by using the independent GSE2549 dataset. The levels of activated CD4 T cells, immature dendritic cells, and type 2 T helper cells were higher in high-risk patients. The gene set enrichment analysis (GSEA) analysis showed that a high concentration and P53 signal pathways were found in high-risk groups. Conclusions: This research developed and verified a new type of immune prognostic signature based on five IRGs, which can predict the prognosis of tumor patients and provide new ideas for individualized treatment.

Limited airway resection and reconstruction for paediatric tracheobronchial inflammatory myofibroblastic tumour.

OBJECTIVES: The paediatric tracheobronchial inflammatory myofibroblastic tumour (IMT) is a rare disease. Whether limited surgical resection is a feasible surgical approach for these patients remains controversial. The objectives of this study were to report the long-term prognosis after limited surgical resections on paediatric tracheobronchial IMT and provide a surgical management strategy for this rare disease. METHODS: Paediatric tracheobronchial IMT patients who underwent limited surgical resection from 2012 to 2020 were enrolled in this study. The clinical characteristics, course of treatment and long-term outcomes of all participants were collated. We presented the accumulated data and analysed the feasibility of limited surgical resection on the paediatric tracheobronchial IMT. RESULTS: A total of 9 children with tracheobronchial IMTs were enrolled in our study. Cough and shortness of breath were the most common symptoms. All 9 participants underwent surgical treatment, including 2 tracheal reconstructions, 4 carinal reconstructions and 3 bronchial sleeve resections. Among the participants, 6/9 (66%) were positive for the anaplastic lymphoma receptor tyrosine kinase gene in terms of immunohistochemistry. None of the participants died of short-term complications. The follow-up period was 5.4 (range, 1.1-9.3) years, during which all participants remained well. CONCLUSIONS: Limited surgical resection is preferred for paediatrics with tracheobronchial IMTs. Meanwhile, patients with complete resection have an excellent long-term prognosis.

Primary pulmonary choriocarcinoma in male: report a case with genetic testing and review of the literature.

Background: Primary pulmonary choriocarcinoma is an extremely rare malignant trophoblastic tumor with a poor prognosis. Most choriocarcinomas originated from gonads, such as the ovaries and testes. Review the previous literature, only 41 cases were reported. Case Description: We reported that a 65-year-old man found shadows in the lungs when undergoing the X-ray examination. Positron emission tomography (PET) was performed to exclude metastatic disease before surgery. The patient underwent three-dimension uniportal thoracoscopic left upper lung resection and lymph node dissection. The operation was uneventful, and he was discharged on the fourth day postoperatively. Postoperative pathology: malignant trophoblastic tumors (choriocarcinoma). After the operation, the patient has genetically tested, the mutations in tumor protein p53 (TP53), NRAS proto-oncogene (NRAS), and fibroblast growth factor receptor 1 (FGFR1) were found. Conclusions: Primary pulmonary choriocarcinoma is an extremely rare and highly malignant tumor difficult to detect in the early stage. By analyzing the previous literature, the patients with active treatment have more extended survival periods than the patients without treatment (P=0.0051). Patients, including surgery, had better survival than patients without surgery (P=0.027) depending on the different treatment regimens. Hence, once the diagnosis was confirmed, the comprehensive treatment of surgical resection combined with chemotherapy and radiotherapy is of great significance to improve the prognosis of patients.

Structures from intact myofibrils reveal mechanism of thin filament regulation through nebulin.

In skeletal muscle, nebulin stabilizes and regulates the length of thin filaments, but the underlying mechanism remains nebulous. In this work, we used cryo-electron tomography and subtomogram averaging to reveal structures of native nebulin bound to thin filaments within intact sarcomeres. This in situ reconstruction provided high-resolution details of the interaction between nebulin and actin, demonstrating the stabilizing role of nebulin. Myosin bound to the thin filaments exhibited different conformations of the neck domain, highlighting its inherent structural variability in muscle. Unexpectedly, nebulin did not interact with myosin or tropomyosin, but it did interact with a troponin T linker through two potential binding motifs on nebulin, explaining its regulatory role. Our structures support the role of nebulin as a thin filament "molecular ruler" and provide a molecular basis for studying nemaline myopathies.

Indocyanine green fluorescence-navigated thoracoscopy versus traditional inflation-deflation approach in precise uniportal segmentectomy: a short-term outcome comparative study.

Background: Uniportal video-assisted thoracoscopic surgery (VATS) segmentectomy is widely used in the field of thoracic surgery. However, anatomical variations in the bronchi and lung vessels may be critical obstacles during precise pulmonary segmentectomy. Thus, it is necessary to optimize uniportal VATS segmentectomy and to accurately identify the plane between lung segments by precisely transecting the bronchi and blood vessels of the lung segments. The indocyanine green fluorescence (ICGF)-based method has the potential to be a feasible and effective technique to facilitate the uniportal VATS segmentectomy. The present study aims at comparing the short-term outcomes of ICGF versus the traditional inflation-deflation method for uniportal VATS segmentectomy. Methods: The perioperative clinical data in 200 consecutive patients undergoing uniportal VATS segmentectomy from December 2018 to August 2020 at Shanghai Chest Hospital were analyzed retrospectively. The targeted segment structures were identified and dissected precisely by using ICGF-based (N=100) or the traditional inflation-deflation (N=100) methods. The parameters of intraoperative blood loss and operation time, postoperative drainage volume, air leakage time, drainage tube retention time, length of hospital stay, and complications in the ICGF group were collected. Further, the operation time between the ICGF and the inflation-deflation groups was compared. The data summary and statistical analysis were performed by SPSS 19.0. P value <0.05 was considered statistically significant. Results: No massive hemorrhage, hypoxemia, allergy, conversion to lobectomy, or wedge resection was noted during the surgery. ICGF groups resulted in a shorter operative time (90±11.46 vs. 118±10.59 min, P<0.001). No postoperative complications were observed, e.g., bronchopleural fistula, hemoptysis, or atelectasis. All patients were discharged as routinely scheduled. No disease recurrence or metastasis was found during the follow-ups. Conclusions: Our study indicated that the ICGF-based navigation approach is a simple, effective, and reliable technique that can greatly facilitate the uniportal VATS segmentectomy.

Case report: Complete resection of invasive thymoma invading the superior vena cava and right atrium under cardiopulmonary bypass support.

Here we describe an uncommon case of a 48-year-old male patient with an invasive thymoma invading the superior vena cava, bilateral innominate veins, right internal jugular vein, right subclavian vein, right atrium, azygos vein, and part of the lung tissues. The tumor was resected entirely under cardiopulmonary bypass support, and the venous bypass using a vascular graft was successfully established between the left innominate vein and the right atrium. The postoperative course was uneventful, and the patient was discharged 15 days after surgery without complications.

Multi-scale characterization of tumor-draining lymph nodes in resectable lung cancer treated with neoadjuvant immune checkpoint inhibitors.

BACKGROUND: Regional lymph node (LN) acts as a pivotal organ for antitumor immunity. Paradoxically, tumor-draining LNs (TDLNs) are usually the first site of tumor metastasis in lung cancer. It is largely unknown about the association between the status of TDLNs and the response of primary tumor beds to immune checkpoint inhibitors (ICIs) in lung cancer patients. Also, studies characterizing the TDLNs in response to ICIs are scarce. METHODS: We characterized and compared the radiological, metabolic (18F-FDG) and pathologic responses between primary tumor beds and paired TDLNs (invaded/non-invaded) from 68 lung cancer patients who underwent neoadjuvant ICIs plus surgery. Additionally, we performed the spatial profiling of immune and non-immune cells within TDLNs using multiplexed immunofluorescence. Therapy responses (e.g., pathologic complete (pCR) or major response (MPR)) of primary lung tumor beds and paired TDLNs were investigated separately. FINDINGS: We observed that responses of TDLNs to ICIs markedly differ from their paired primary lung tumors regarding the radiological, metabolic (18F-FDG uptake), and pathologic alterations. Neoadjuvant ICIs therapy specifically decreased 18F-FDG-reflected metabolic activity in the primary tumor beds with pCR/MPR but not their TDLNs counterparts. Furthermore, the presence of invaded TDLNs was associated with poor pathologic responses in the matched primary tumor beds and predictive of rapid post-treatment tumor relapse. Spatial profiling demonstrated exclusion of T cell infiltrates within the metastatic lesions of invaded TDLNs, and diminished multiple immune and non-immune compositions in non-involved regions surrounding the metastatic lesions. INTERPRETATION: These results provide the first clinically-relevant evidence demonstrating unique response patterns of TDLNs under ICIs treatment and revealing the underappreciated association of TDLNs status with the response of their paired primary tumors to ICIs in lung cancer. FUNDING: This work was supported by the National Natural Science Foundation of China (82072570 to F. Yao; 82002941 to B. Sun), the excellent talent program of Shanghai Chest Hospital (to F.Y), the Basic Foundation Program for Youth of Shanghai Chest Hospital (2021YNJCQ2 to H.Yang), and the Innovative Research Team of High-level Local Universities in Shanghai (SHSMU-ZLCX20212302 to F. Yao).

The improved success rate and reduced complications of a novel localization device vs. hookwire for thoracoscopic resection of small pulmonary nodules: a single-center, open-label, randomized clinical trial.

Background: In our previous study, we developed a 4-hook claw-suture localization device for pulmonary nodule resection, which acheived satifisfactory results. Following this, we conducted this single-center, open-label, randomized clinical trial to compare the success rate and complication rate of this novel localization device and currently widely-used hookwire. Methods: Patients with small pulmonary nodules (0.4-1 cm) who received preoperative localization and thoracoscopic resection at Shanghai Chest Hospital were randomly assigned (1:2 ratio, via computer-generated randomized numbers) to undergo localization using either a novel claw-suture system (claw group) or classical (hookwire group) localization device. The primary endpoint of this study was localization success rate, and the secondary endpoints included complications, localization-related time, and pain. Results: A total of 411 patients were randomly assigned to the claw group (n=136) or the hookwire group (n=275) before thoracoscopic resection of small pulmonary nodules and analyzed. Compared with the hookwire group, the claw group had a significantly higher success rate (133/136, 97.8% vs. 254/275, 92.4%, P=0.027), less asymptomatic hemorrhage (16.9% vs. 37.5%, P=0.003) and pleural reaction (0% vs. 5.1%, P=0.017), as well as better pain alleviation 10 min after localization (measured using the difference between two visual analog scale scores, 0.84±0.98 vs. 0.35±0.79, P<0.001). In contrast, the hookwire group was associated with a shorter localization procedure duration than the claw group (7.2±2.9 vs. 14.4±6.6 min, P<0.001). In the multiple localization subgroup, the claw group compared to the hookwire group also achieved higher success (32/33, 97.0% vs. 70/86, 81.4%) and less pleural reaction (0% vs. 16.3%). Conclusions: The new claw-suture localization device is superior to traditional hookwire, with a higher success rate, fewer complications, and better patient tolerance for preoperative localization of small pulmonary nodules. Trial Registration: Chinese Clinical Trial Registry ChiCTR1900027346.