Single-cell sequencing on CD8+ TILs revealed the nature of exhausted T cells recognizing neoantigen and cancer/testis antigen in non-small cell lung cancer.

BACKGROUND: CD8+tumor infiltrating lymphocytes (TILs) are often observed in non-small cell lung cancers (NSCLC). However, the characteristics of CD8+ TILs, especially T-cell populations specific for tumor antigens, remain poorly understood. METHODS: High throughput single-cell RNA sequencing and single-cell T-cell receptor (TCR) sequencing were performed on CD8+ TILs from three surgically-resected lung cancer specimens. Dimensional reduction for clustering was performed using Uniform Manifold Approximation and Projection. CD8+ TIL TCR specific for the cancer/testis antigen KK-LC-1 and for predicted neoantigens were investigated. Differentially-expressed gene analysis, Gene Set Enrichment Analysis (GSEA) and single sample GSEA was performed to characterize antigen-specific T cells. RESULTS: A total of 6998 CD8+ T cells was analyzed, divided into 10 clusters according to their gene expression profile. An exhausted T-cell (exhausted T (Tex)) cluster characterized by the expression of ENTPD1 (CD39), TOX, PDCD1 (PD1), HAVCR2 (TIM3) and other genes, and by T-cell oligoclonality, was identified. The Tex TCR repertoire (Tex-TCRs) contained nine different TCR clonotypes recognizing five tumor antigens including a KK-LC-1 antigen and four neoantigens. By re-clustering the tumor antigen-specific T cells (n=140), it could be seen that the individual T-cell clonotypes were present on cells at different stages of differentiation and functional states even within the same Tex cluster. Stimulating these T cells with predicted cognate peptide indicated that TCR signal strength and subsequent T-cell proliferation and cytokine production was variable but always higher for neoantigens than KK-LC-1. CONCLUSIONS: Our approach focusing on T cells with an exhausted phenotype among CD8+ TILs may facilitate the identification of tumor antigens and clarify the nature of the antigen-specific T cells to specify the promising immunotherapeutic targets in patients with NSCLC.

Warm retrograde perfusion can remove more fat from lung grafts with fat embolism in a porcine model.

OBJECTIVE: In lung transplantation, unexpected pulmonary emboli, including thrombi and fat, have been observed with high probability and are associated with potential primary graft dysfunction. We evaluated a new perfusion method using warm retrograde flushing that removes more fat than conventional cold retrograde flushing. METHODS: We developed a novel porcine donor model for pulmonary fat embolism by administering autologous fat in the left pulmonary artery. The left pulmonary artery and the left superior and inferior pulmonary veins were cannulated for flushing and collecting these solutions. After flushing, the left lung was reperfused under observation for 3 h. Two groups underwent warm and cold additional retrograde flush (WS; warm solution group, CS; cold solution group). RESULTS: The fat removal rate in the antegrade flush was equal in both groups (3.0 ± 0.6% vs 3.0 ± 0.4%, p = 0.46); however, the rate was significantly greater in the WS group in retrograde flush (25.2 ± 3.2% vs 8.0 ± 1.4%, p = 0.01). Histology with Oil Red O staining and its software analysis showed more residual fat in the CS group (0.12 ± 0.01% vs 0.38 ± 0.07%, p = 0.01). There was no significant difference in the pulmonary function and hemodynamics during the 3-h period after reperfusion. CONCLUSION: Warm retrograde perfusion can remove more fat from lung grafts with fat embolism in a porcine donor model.

Group B streptococcal empyema necessitatis with pleural fistula after blunt trauma: A case report.

INTRODUCTION: We report the first case of empyema necessitatis (EN) with pleural fistula and septic arthritis caused by Streptococcus agalactiae following blunt trauma. PRESENTATION OF THE CASE: A 46-year-old man with diabetes mellitus and a history of recent right rib fracture and right knee bruising presented with dyspnea and right knee pain. He was diagnosed with EN and underwent chest drainage, followed by open-window thoracotomy. Septic arthritis occurred on day 8 after thoracotomy. The chest wall wound healed after 3 months. DISCUSSION: EN is a rare complication of empyema. In this patient, infection was invasive, causing necrotizing pneumonia with a pleural fistula. To our knowledge, there are no reports of group B streptococcal EN with a pleural fistula resulting from blunt chest trauma. CONCLUSION: Group B streptococcal infection might become invasive in immunocompromised patients, so careful follow-up for those patients is important.

Lung retransplantation in an adult 13 years after single lobar transplant in childhood.

Single living-donor lobar lung transplantation provides acceptable results for critically ill children; however, an additional lung transplantation may be required in the future as the recipient grows. We describe a case of successful lung retransplantation in a grown-up patient after single lobar lung transplantation in childhood. A 23-year-old man underwent bilateral cadaveric lung retransplantation for chronic lung allograft dysfunction 13 years after right single living-donor lobar transplantation for idiopathic pulmonary arterial hypertension performed at the age of 10 years. The postoperative course was uneventful. The patient had received growth hormone therapy at a local hospital for 3 years until the development of chronic lung allograft dysfunction after the initial transplantation. Pediatric recipients undergoing single living-donor lobar lung transplantation should be cautiously followed for potential retransplantation.

Balloon-expandable Metallic Stents for Airway Diseases.

Stent placement is an essential treatment for airway diseases. Although self-expandable metallic stents and silicone stents are commonly applied for the treatment of airway diseases, these stents are unsuitable for the treatment of small airway diseases encountered in pediatric patients and lung transplant recipients with airway complications. Currently, only vascular balloon-expandable metallic stents are available for the treatment of small airway diseases; however, little research has been conducted on the use of these stents in this field. We have launched a prospective feasibility study to clarify the safety and efficacy of balloon-expandable metallic stents for the treatment of airway diseases.

A case of miriplatin-induced lung injury.

A 69-year-old man with an 8-year history of hepatocellular carcinoma (HCC) was hospitalized for treatment of recurrent tumour. In 2010, the first transcatheter arterial chemoembolization (TACE) using miriplatin with agents (Lipiodol Ultra-Fluid) was performed and did not occur any adverse events. In 2014, since his HCC recurred, the TACE using miriplatin with agents was performed. Following this therapy, pyrexia occurred on day 3, followed by respiratory failure with cough and dyspnea on day 5. Chest radiography revealed scattered infiltration in the right upper lung fields, and chest computed tomography revealed ground grass attenuations, indicating fibrotic non-specific interstitial pneumonia. These findings progressively deteriorated, and a diagnosis of miriplatin-induced lung injury was made. His respiratory failure also progressively deteriorated. Treatment with pulse methylprednisolone therapy resulted in a dramatic improvement in both patient symptoms and radiological abnormalities.

Nano-imaging of the beating mouse heart in vivo: Importance of sarcomere dynamics, as opposed to sarcomere length per se, in the regulation of cardiac function.

Sarcomeric contraction in cardiomyocytes serves as the basis for the heart's pump functions in mammals. Although it plays a critical role in the circulatory system, myocardial sarcomere length (SL) change has not been directly measured in vivo under physiological conditions because of technical difficulties. In this study, we developed a high speed (100-frames per second), high resolution (20-nm) imaging system for myocardial sarcomeres in living mice. Using this system, we conducted three-dimensional analysis of sarcomere dynamics in left ventricular myocytes during the cardiac cycle, simultaneously with electrocardiogram and left ventricular pressure measurements. We found that (a) the working range of SL was on the shorter end of the resting distribution, and (b) the left ventricular-developed pressure was positively correlated with the SL change between diastole and systole. The present findings provide the first direct evidence for the tight coupling of sarcomere dynamics and ventricular pump functions in the physiology of the heart.

Directional bleb formation in spherical cells under temperature gradient.

Living cells sense absolute temperature and temporal changes in temperature using biological thermosensors such as ion channels. Here, we reveal, to our knowledge, a novel mechanism of sensing spatial temperature gradients within single cells. Spherical mitotic cells form directional membrane extensions (polar blebs) under sharp temperature gradients (≥∼0.065°C µm(-1); 1.3°C temperature difference within a cell), which are created by local heating with a focused 1455-nm laser beam under an optical microscope. On the other hand, multiple nondirectional blebs are formed under gradual temperature gradients or uniform heating. During heating, the distribution of actomyosin complexes becomes inhomogeneous due to a break in the symmetry of its contractile force, highlighting the role of the actomyosin complex as a sensor of local temperature gradients.

Endoscopic cell sheet transplantation device developed by using a 3-dimensional printer and its feasibility evaluation in a porcine model.

BACKGROUND: To prevent severe esophageal stenosis after aggressive endoscopic submucosal dissection (ESD), our group previously reported an efficient treatment using cell sheets that had been fabricated from patient cells. However, this transplantation procedure had not been easy for every endoscopist and needed to be improved to derive the full effect of epithelial cell sheets. OBJECTIVE: To develop an endoscopic device that enables easy and effective cell sheet transplantation and to evaluate its performance and clinical feasibility. DESIGN: Animal study. SETTING: Animal experimentation laboratory. INTERVENTION: Three pigs underwent circumferential esophageal ESD while under general anesthesia. A total of 12 cell sheets were endoscopically transplanted to the ESD site; 6 cell sheets were transplanted by using an endoscopic device that we developed, and 6 cell sheets were transplanted by using the conventional method. MAIN OUTCOME MEASUREMENTS: Procedure time, transplanted area on the ESD site, transplantation success rate, and monitoring of adverse events or incidents. RESULTS: The device allowed successful transplantation of all cell sheets with a shorter procedure time than with the conventional method (4.8 ± 0.8 minutes vs 13.3 ± 5.7 minutes, respectively) (P = .005) and onto a larger area (111.3 ± 56.3 mm(2) vs 41.8 ± 4.2 mm(2), respectively) (P = .023) with a higher success rate (100% vs 83%, respectively). No adverse incidents were monitored in each method. LIMITATIONS: Animal study, small sample. CONCLUSION: A newly designed endoscopic cell sheet transplantation device would be useful.

Cell-sized spherical confinement induces the spontaneous formation of contractile actomyosin rings in vitro.

During cell division, many animal cells transform into a spherical shape and assemble a contractile ring composed of actin filaments and myosin motors at the equator to separate the cell body into two. Although actomyosin regulatory proteins are spatio-temporally controlled during cytokinesis, the direct contribution of cell shape and actomyosin activity to the contractile ring assembly remains unclear. Here, we demonstrated in vitro that actin polymerization inside cell-sized spherical droplets induced the spontaneous formation of single ring-shaped actin bundles in the presence of bundling factors. Despite a lack of spatial regulatory signals, the rings always assembled at the equator to minimize the elastic energy of the bundles. Myosin promoted ring formation by the dynamic remodelling of actin networks, and an increase in the effective concentration of myosin triggered ring contraction. These results will help us understand how animal cells coordinate cell shape and actomyosin activities to direct cytokinesis.

Sarcomere length nanometry in rat neonatal cardiomyocytes expressed with α-actinin-AcGFP in Z discs.

Nanometry is widely used in biological sciences to analyze the movement of molecules or molecular assemblies in cells and in vivo. In cardiac muscle, a change in sarcomere length (SL) by a mere ∼100 nm causes a substantial change in contractility, indicating the need for the simultaneous measurement of SL and intracellular Ca(2+) concentration ([Ca(2+)]i) in cardiomyocytes at high spatial and temporal resolution. To accurately analyze the motion of individual sarcomeres with nanometer precision during excitation-contraction coupling, we applied nanometry techniques to primary-cultured rat neonatal cardiomyocytes. First, we developed an experimental system for simultaneous nanoscale analysis of single sarcomere dynamics and [Ca(2+)]i changes via the expression of AcGFP in Z discs. We found that the averaging of the lengths of sarcomeres along the myocyte, a method generally used in today's myocardial research, caused marked underestimation of sarcomere lengthening speed because of the superpositioning of different timings for lengthening between sequentially connected sarcomeres. Then, we found that after treatment with ionomycin, neonatal myocytes exhibited spontaneous sarcomeric oscillations (cell-SPOCs) at partial activation with blockage of sarcoplasmic reticulum functions, and the waveform properties were indistinguishable from those obtained in electric field stimulation. The myosin activator omecamtiv mecarbil markedly enhanced Z-disc displacement during cell-SPOC. Finally, we interpreted the present experimental findings in the framework of our mathematical model of SPOCs. The present experimental system has a broad range of application possibilities for unveiling single sarcomere dynamics during excitation-contraction coupling in cardiomyocytes under various settings.

[Clinical experience of the articulating needle-holder for completely video-assisted thoracoscopic surgery].

The autonomy Laparo-angle needle holder is a flexible device which has several articulating parts facilitating some traditionally difficult way of suture passage. This device is often used for laparoscopic surgery, and there have been few reports for video-assisted thoracic surgery (VATS). We used this device for complete VATS lobectomy and segmentectomy, and it enables us to suture a bronchus with the optimal direction even in the deep surgical field on complete VATS lobectomy and segmentectomy. Although some training may be needed to freely manipulate this device, it can be useful for minimallyinvasive video-assisted thoracic surgery.

Improvement of the yields of recombinant actin and myosin V-HMM in the insect cell/baculovirus system by the addition of nutrients to the high-density cell culture.

Baculovirus infection of Sf9 cells at high densities, such as during mid- and late exponential phase, often results in a significant reduction of protein yield per cell, compared to the early exponential phase. Nutrient depletion has been considered as a major cause for the decreased protein yield. In this study, we report that the addition of nutrients (glucose, yeastolate ultrafiltrate, and lactalbumin hydrolysate) and small fraction of fresh medium at time of infection restores the expression level of actin and myosin V-HMM at late exponential phase (11.3 × 10(6) cells/ml) to that at early exponential phase (1.0 × 10(6) cells/ml). The relative yields of actin and myosin V-HMM were approximately equal at both phases (typically 200 mg of actin and 5 mg of myosin V-HMM per 10(10) cells), i.e., the volumetric yield of proteins from the cell culture at late exponential phase was approximately tenfold higher than at early exponential phase. The functionality of the recombinant actin and myosin V-HMM was confirmed by measuring the rate of actin polymerization, actin-activated ATPase, and the gliding velocity of actin filaments in an in vitro motility assay.

Punch and spindle-shaped biopsies for collecting oral mucosal tissue for the fabrication of transplantable autologous epithelial cell sheets.

The oral mucosa is an easily accessible source of cells. Oral mucosal collection will be an essential surgical procedure for regenerative medicine and cell biological research. However, there is no current report that describes the details of the surgical procedure used for oral mucosal collection. Moreover, the number of cells that can be obtained has not been determined. Two different procedures, the punch biopsy and the spindle-shaped biopsy, were performed for the fabrication of transplantable autologous epithelial cell sheets. The mean values of the cells collected per square centimeter of tissue using the punch biopsy and the spindle-shaped biopsy were 76.8 ± 45 × 10(4) cells/cm(2) and 195.7 ± 120 × 10(4) cells/cm(2), respectively. There was no significant difference between the punch biopsy and the spindle-shaped biopsy. The coefficient of variation of the punch biopsy and the spindle-shaped biopsy was 58.9% and 69.8%, respectively. This result indicated that both procedures showed variations in the number of collected cells. Although the punch biopsy may be easier and simpler than the spindle-shaped biopsy, multiple punch biopsies may result in a more complicated procedure, and the spindle-shaped biopsy may be preferable when a large number of cells is necessary.

The bidirectional depolymerizer MCAK generates force by disassembling both microtubule ends.

During cell division the replicated chromosomes are segregated precisely towards the spindle poles. Although many cellular processes involving motility require ATP-fuelled force generation by motor proteins, most models of the chromosome movement invoke the release of energy stored at strained (owing to GTP hydrolysis) plus ends of microtubules. This energy is converted into chromosome movement through passive couplers, whereas the role of molecular motors is limited to the regulation of microtubule dynamics. Here we report, that the microtubule-depolymerizing activity of MCAK (mitotic centromere-associated kinesin), the founding member of the kinesin-13 family, is accompanied by the generation of significant tension-remarkably, at both microtubule ends. An MCAK-decorated bead strongly attaches to the microtubule side, but readily slides along it in either direction under weak external loads and tightly captures and disassembles both microtubule ends. We show that the depolymerization force increases with the number of interacting MCAK molecules and is ∼1 pN per motor. These results provide a simple model for the generation of driving force and the regulation of chromosome segregation by the activity of MCAK at both kinetochores and spindle poles through a 'side-sliding, end-catching' mechanism.

Modulation of the mechano-chemical properties of myosin V by drebrin-E.

The regulation of actin filament networks by various proteins has essential roles in the growth cone dynamics. In this study we focused on the actin-myosin interaction which has been suggested to be an important player in the neurite extension. We examined in vitro how the decoration of actin filaments with a side-binding protein, drebrin-E, affects the motile properties of an intracellular transporter myosin V. Single myosin V molecules landed on the drebrin-E-decorated actin filaments with a lower frequency and ran over shorter distances; however, their velocities were normal. Furthermore, the analysis of the movement of myosin V molecules in the optical trap revealed that the decoration of actin filaments with drebrin-E markedly increased the load-sensitivity of the myosin V stepping. These results are attributable to the delay in the attachment of the motor's leading head (ADP·P(i) state) to actin, induced by the competitive binding of drebrin-E to actin, whereas the rate of ADP release from the trailing head (the rate-limiting step in the ATPase cycle of myosin V) is unaffected. Our study indicates that, in addition to the regulation of binding affinity of myosin V, drebrin-E also modulates the chemo-mechanical coupling in the motile myosin V molecules, presumably affecting the movement of the growth cone.

Robust processivity of myosin V under off-axis loads.

The dimeric motor myosin V transports organelles along actin filament tracks over long distances in cells. Myosin V is a smart 'walker' that is able to swiftly adjust to variable 'road conditions' to continue its processive movement across dense cellular environments. Coordination between the two heads via intramolecular load modulates biochemical kinetics and ensures highly efficient unidirectional motion. However, little is known about how load-induced regulation of the processive stepping occurs in vivo, where myosin V experiences significant off-axis loads applied in various directions. To reveal how myosin V remains processive in cells, we measured the effect of the off-axis loads, applied to individual actomyosin V bonds in a range of angles, on the coordination between the two heads and myosin V processive stepping. We found that myosin V remains highly processive under diagonal loads owing to asymmetrical ADP affinities and that the native 6IQ lever optimizes the subunit coordination, which indicates that myosin V is designed to be an intracellular transporter.

Stimulatory effects of arachidonic acid on myosin ATPase activity and contraction of smooth muscle via myosin motor domain.

We have been searching for a mechanism to induce smooth muscle contraction that is not associated with phosphorylation of the regulatory light chain (RLC) of smooth muscle myosin (Nakamura A, Xie C, Zhang Y, Gao Y, Wang HH, Ye LH, Kishi H, Okagaki T, Yoshiyama S, Hayakawa K, Ishikawa R, Kohama K. Biochem Biophys Res Commun 369: 135-143, 2008). In this article, we report that arachidonic acid (AA) stimulates ATPase activity of unphosphorylated smooth muscle myosin with maximal stimulation (R(max)) of 6.84 +/- 0.51 relative to stimulation by the vehicle and with a half-maximal effective concentration (EC(50)) of 50.3 +/- 4.2 microM. In the presence of actin, R(max) was 1.72 +/- 0.08 and EC(50) was 26.3 +/- 2.3 microM. Our experiments with eicosanoids consisting of the AA cascade suggested that they neither stimulated nor inhibited the activity. Under conditions that did not allow RLC to be phosphorylated, AA stimulated contraction of smooth muscle tissue with an R(max) of 1.45 +/- 0.07 and an EC(50) of 27.0 +/- 4.4 microM. In addition to the ATPase activities of the myosin, AA stimulated those of heavy meromyosin, subfragment 1 (S1), S1 from which the RLC was removed, and a recombinant heavy chain consisting of the myosin head. The stimulatory effects of AA on these preparations were about twofold. The site of AA action was indicated to be the step-releasing inorganic phosphate (P(i)) from the reaction intermediate of the myosin-ADP-P(i) complex. The enhancement of P(i) release by AA was supported by computer simulation indicating that AA docked in the actin-binding cleft of the myosin motor domain. The stimulatory effect of AA was detectable with both unphosphorylated myosin and the myosin in which RLC was fully phosphorylated. The AA effect on both myosin forms was suggested to cause excess contraction such as vasospasm.

Purification of cytoplasmic actin by affinity chromatography using the C-terminal half of gelsolin.

A new rapid method of the cytoplasmic actin purification, not requiring the use of denaturants or high concentrations of salt, was developed, based on the affinity chromatography using the C-terminal half of gelsolin (G4-6), an actin filament severing and capping protein. When G4-6 expressed in Escherichia coli was added to the lysate of HeLa cells or insect cells infected with a baculovirus encoding the beta-actin gene, in the presence of Ca(2+) and incubated overnight at 4 degrees C, actin and G4-6 were both detected in the supernatant. Following the addition of Ni-Sepharose beads to the mixture, only actin was eluted from the Ni-NTA column by a Ca(2+)-chelating solution. The functionality of the cytoplasmic actins thus purified was confirmed by measuring the rate of actin polymerization, the gliding velocity of actin filaments in an in vitro motility assay on myosin V-HMM, and the ability to activate the ATPase activity of myosin V-S1.

Load-dependent ADP binding to myosins V and VI: implications for subunit coordination and function.

Dimeric myosins V and VI travel long distances in opposite directions along actin filaments in cells, taking multiple steps in a "hand-over-hand" fashion. The catalytic cycles of both myosins are limited by ADP dissociation, which is considered a key step in the walking mechanism of these motors. Here, we demonstrate that external loads applied to individual actomyosin V or VI bonds asymmetrically affect ADP affinity, such that ADP binds weaker under loads assisting motility. Model-based analysis reveals that forward and backward loads modulate the kinetics of ADP binding to both myosins, although the effect is less pronounced for myosin VI. ADP dissociation is modestly accelerated by forward loads and inhibited by backward loads. Loads applied in either direction slow ADP binding to myosin V but accelerate binding to myosin VI. We calculate that the intramolecular load generated during processive stepping is approximately 2 pN for both myosin V and myosin VI. The distinct load dependence of ADP binding allows these motors to perform different cellular functions.