Recognition of Amino Acid Salts by Temperature-Dependent Allosteric Binding with Stereodynamic Urea Receptors.

Positive homotropic artificial allosteric systems are important for the regulation of cooperativity, selectivity and nonlinear amplification. Stereodynamic homotropic allosteric receptors can transmit and amplify induced chirality by the first ligand binding to axial chirality between two chromophores. We herein report stereodynamic allosteric urea receptors consisting of a rotational shaft as the axial chirality unit, terphenyl units as structural transmission sites and four urea units as binding sites. NMR titration experiments revealed that the receptor can bind two carboxylate guests in a positive homotropic allosteric manner attributed to the inactivation by intramolecular hydrogen-bonding between urea units within the receptor. In addition, the VT-CD spectra observed upon binding of the urea receptor with l- or D-amino acid salts in MeCN showed interesting temperature-dependent Cotton effects, based on the differences of the receptor shaft unit and the guest structure. The successful discrimination of hydrocarbon-based side chains of amino acid salts indicated that the input of chiral and steric information for the guest was amplified as outputs of the Cotton effect and the temperature-dependence of VT-CD spectra through cooperativity of positive allosteric binding.

The Balance of CD8-Positive T Cells and PD-L1 Expression in the Myocardium Predicts Prognosis in Lymphocytic Fulminant Myocarditis.

INTRODUCTION: The clinical significance and prognostic value of T cell involvement and programmed cell death-1 (PD-1)/programmed cell death ligand-1 (PD-L1) have not been established in lymphocytic fulminant myocarditis (FM). We investigated the prognostic impact of the number of CD4+, CD8+, FoxP3+, and PD-1+ T cells, as well as PD-L1 expression, in cardiomyocytes in lymphocytic FM. METHODS: This is a single-center observational cohort study. Myocardial tissue was obtained from 16 consecutive patients at lymphocytic FM onset. The median follow-up was 140 days. Cardiac events were defined as a composite of cardiac death and left ventricular-assist device implantation. CD4, CD8, FoxP3, PD-1, and PD-L1 immunostaining were performed on myocardial specimens. RESULTS: The median age of the patients was 52 years (seven men and nine women). There was no significant difference in the number of CD4+ cells. The number of CD8+ cells and the CD8+/CD4+ T cell ratio were higher in the cardiac event group (Event+) than in the group without cardiac events (Event-) (p = 0.048 and p = 0.022, respectively). The number of FoxP3+ T cells was higher in the Event+ group (p = 0.049). Although there was no difference in the number of PD-1+ cells, cardiomyocyte PD-L1 expression was higher in the Event+ group (p = 0.112). Event-free survival was worse in the group with a high CD8+ cell count (p = 0.012) and high PD-L1 expression (p = 0.049). When divided into three groups based on the number of CD8+ cells and PD-L1 expression (CD8highPD-L1high [n = 8], CD8lowPD-L1high [n = 1], and CD8lowPD-L1low [n = 7]), the CD8highPD-L1high group demonstrated the worst event-free survival, while the CD8lowPD-L1high group had a favorable prognosis without cardiac events (p = 0.041). CONCLUSION: High myocardial expression of CD8+ T cells and PD-L1 may predict a poor prognosis in lymphocytic FM.

Efficacy of endoscopic surveillance for pharyngeal mucosa during endoscopic resection for pharyngeal carcinoma: a multicenter prospective study.

INTRODUCTION: Since patients with pharyngeal squamous cell carcinoma (SCC) often have multiple pharyngeal lesions, evaluation of pharyngeal lesions before endoscopic resection (ER) is important. However, detailed endoscopic observation of the entire pharyngeal mucosa under conscious sedation is difficult. We examined the usefulness of endoscopic surveillance with narrow band imaging (NBI) and lugol staining for detection of pharyngeal sublesions during ER for pharyngeal SCC under general anesthesia (endoscopic surveillance during treatment; ESDT). METHODS: From January 2021 through June 2022, we examined 78 patients who were diagnosed with superficial pharyngeal SCC and underwent ER. They underwent the ESDT and for patients who were diagnosed with new lesions of pharyngeal SCC or high-grade dysplasia (HGD) that were not detected in the endoscopic examination before treatment, ER were performed simultaneously for new lesions and the main lesions. The primary endpoint of this study was the detection rate of new lesions of pharyngeal SCC or HGD in the ESDT. RESULTS: Fifteen of the 78 patients were diagnosed as having undetected new pharyngeal lesions in the ESDT and 10 (12.8%) (95% CI 6.9-22.2%) were histopathologically confirmed to have new lesions of pharyngeal SCC or HGD. Among the 13 lesions of SCC or HGD, 8 were found by NBI observation; however, 5 were undetectable using NBI but detectable by lugol staining. All of the 13 lesions had endoscopic findings of pink color sign on lugol staining. CONCLUSIONS: Endoscopic surveillance for pharyngeal sublesions during ER for pharyngeal SCC is feasible and useful.

Machine Learning Refinement of In Situ Images Acquired by Low Electron Dose LC-TEM.

We have studied a machine learning (ML) technique for refining images acquired during in situ observation using liquid-cell transmission electron microscopy. Our model is constructed using a U-Net architecture and a ResNet encoder. For training our ML model, we prepared an original image dataset that contained pairs of images of samples acquired with and without a solution present. The former images were used as noisy images, and the latter images were used as corresponding ground truth images. The number of pairs of image sets was 1,204, and the image sets included images acquired at several different magnifications and electron doses. The trained model converted a noisy image into a clear image. The time necessary for the conversion was on the order of 10 ms, and we applied the model to in situ observations using the software Gatan DigitalMicrograph (DM). Even if a nanoparticle was not visible in a view window in the DM software because of the low electron dose, it was visible in a successive refined image generated by our ML model.

Intraperitoneal transfer of microRNA-29b-containing small extracellular vesicles can suppress peritoneal metastases of gastric cancer.

Small extracellular vesicles (sEV) contain various microRNAs (miRNAs) and play crucial roles in the tumor metastatic process. Although miR-29b levels in peritoneal exosomes were markedly reduced in patients with peritoneal metastases (PM), their role has not been fully clarified. In this study, we asked whether the replacement of miR-29b can affect the development of PM in a murine model. UE6E7T-12, human bone marrow-derived mesenchymal stem cells (BMSCs), were transfected with miR-29b-integrating recombinant lentiviral vector and sEV were isolated from culture supernatants using ultracentrifugation. The sEV contained markedly increased amounts of miR-29b compared with negative controls. Treatment with transforming growth factor-ß1 decreased the expression of E-cadherin and calretinin with increased expression of vimentin and fibronectin on human omental tissue-derived mesothelial cells (HPMCs). However, the effects were totally abrogated by adding miR-29b-rich sEV. The sEV inhibited proliferation and migration of HPMCs by 15% (p < 0.005, n = 6) and 70% (p < 0.005, n = 6), respectively, and inhibited adhesion of NUGC-4 and MKN45 to HPMCs by 90% (p < 0.0001, n = 5) and 77% (p < 0.0001, n = 5), respectively. MicroRNA-29b-rich murine sEV were similarly obtained using mouse BMSCs and examined for in vivo effects with a syngeneic murine model using YTN16P, a highly metastatic clone of gastric cancer cell. Intraperitoneal (IP) transfer of the sEV every 3 days markedly reduced the number of PM from YTN16P in the mesentery (p < 0.05, n = 6) and the omentum (p < 0.05, n = 6). Bone marrow mesenchymal stem cell-derived sEV are a useful carrier for IP administration of miR-29b, which can suppress the development of PM of gastric cancer.

Increased risk of purge system malfunction after Impella 5.0 replacement: a case series.

The Impella 5.0 is an axial-flow percutaneous ventricular assist device used in patients with cardiogenic shock. Although the recommended period of use is 10 days or less, weaning can be delayed because of ongoing hemodynamic instability. In clinical practice, this device sometimes malfunctions during long-term management with heparin and must be replaced; however, the relationship between the duration of support with the initial and replacement Impella 5.0 and the changes in value of the purge system has not been fully elucidated. From July 2018 to May 2021, Impella 5.0 was implanted and used for more than 10 days in 11 patients at our institution. Four patients required Impella replacement because of device malfunction and the second Impella had purge system malfunction in all cases. The second Impella was used for a significantly shorter time than the first Impella (p = 0016). We calculated the ratio of purge pressure to purge flow rate and found that the ratio exceeded 50 mm Hg/mL/h in all cases with purge system malfunction. In conclusion, it is important to construct a treatment strategy considering the duration of use, because the risk of purge system malfunction is high after replaced Impella 5.0.

In Situ Transmission Electron Microscopy Study of Bubble Behavior Near the Surface of Ice Crystals by Using a Liquid Cell With a Peltier Cooling Holder.

Liquid cell transmission electron microscopy (LC-TEM) is a unique technique that permits in situ observations of various phenomena in liquids with high spatial and temporal resolutions. One difficulty with this technique is the control of the environmental conditions in the observation area. Control of the temperature ranging from room temperature to minus several tens of degrees Celsius, is desirable for controlling the supersaturation in various materials and for observing crystallization more easily. We have developed a cooling transmission electron microscopy specimen holder that uses Peltier devices, and we have combined it with a liquid cell to realize accurate temperature control in LC-TEM. We evaluated this system by using water as a specimen. Motionless bubbles, shown to be voids containing pressurized gas, formed in the specimen sometime after the temperature had reached -12°C. An electron diffraction pattern showed that the specimen turned into ice Ih after the formation of these bubbles, confirming that our system works properly and can induce crystallization. In addition, we analyzed the behavior of bubbles formed in the ice Ih, and we discussed the formation of these bubbles and their internal pressure.

[Intraperitoneal Transfer of miR-29b Containing Exosome Suppresses the Development of Peritoneal Metastases from Gastric Cancer].

Although miR-29b levels in peritoneal exosomes was markedly reduced in patients with peritoneal metastases(PM), their role has not been fully clarified. Bone marrow derived mesenchymal stem cells(BMSC)were transfected with miR-29b- integrating lentivirus and exosomes isolated from culture supernatants using ultracentrifugation. The effects of the exosomes on human peritoneal mesothelial cells(HPMC)were examined in vitro. The in vivo effect of murine BMSC-derived exosomes was examined with a syngeneic PM model. Culture of HPMC with TGF-ß1 decreased expression of E-cadherin and calretinin with increased expression of vimentin, totally restored by adding miR-29b-rich exosomes. The exosomes inhibited proliferation and migration of HPMC, and inhibited adhesion of gastric cancer cells to HPMC. Intraperitoneal(IP)transfer of miR- 29b-rich exosomes every 3 days markedly reduced the number of PM of a murine gastric cancer cell, YTN16P, on the mesentery of C57/BL6 mice. IP administration of miR-29b-containing exosome suppresses the development of PM of gastric cancer.

Diacylglycerol kinase δ functions as a proliferation suppressor in pancreatic ß-cells.

Although an aberrant reduction in pancreatic ß-cell mass contributes to the pathogenesis of diabetes, the mechanism underlying the regulation of ß-cell mass is poorly understood. Here, we show that diacylglycerol kinase δ (DGKδ) is a key enzyme in the regulation of ß-cell mass. DGKδ expression was detected in the nucleus of ß-cells. We developed ß-cell-specific DGKδ knockout (ßDGKδ KO) mice, which showed lower blood glucose, higher plasma insulin levels, and better glucose tolerance compared to control mice. Moreover, an increased number of small islets and Ki-67-positive islet cells, as well as elevated cyclin B1 expression in the islets, were detected in the pancreas of ßDGKδ KO mice. DGKδ knockdown in the ß-cell line MIN6 induced significant increases in bromodeoxyuridine (BrdU) incorporation and cyclin B1 expression. Finally, we confirmed that streptozotocin-induced hyperglycemia and ß-cell loss were alleviated in ßDGKδ KO mice. Thus, suppressing the expression or enzymatic activity of DGKδ that functions as a suppressor of ß-cell proliferation could be a novel therapeutic approach to increase ß-cell mass for the treatment of diabetes.

IL-33 induces orofacial neuropathic pain through Fyn-dependent phosphorylation of GluN2B in the trigeminal spinal subnucleus caudalis.

Orofacial neuropathic pain can cause considerable disruptions in patients' daily lives, especially because of a lack of effective medications as its underlying causative mechanisms are not fully understood. Here, we found neuron-specific expression of the interleukin (IL)-33 receptor in the trigeminal spinal subnucleus caudalis (Vc), distinct from the spinal dorsal horn. Reduction in head withdrawal threshold in response to von Frey filament stimulation of the whisker pad skin was inversely correlated with the upregulation of IL-33 in the Vc after infraorbital nerve injury (IONI). Neutralization of IL-33 in the Vc alleviated mechanical allodynia in the whisker pad skin after IONI; conversely, intracisternal administration of IL-33 elicited mechanical allodynia in the whisker pad skin, which was relieved by GluN2B antagonism. Moreover, IL-33 triggered the potentiation of GluN2B-containing N-methyl-D-aspartate receptor-mediated synaptic currents and phosphorylation of synaptosomal GluN2B in the Vc, whereas IONI-induced GluN2B phosphorylation was inhibited by neutralization of IL-33 in the Vc. IL-33-induced GluN2B phosphorylation was mediated by phosphorylation of Fyn kinase, and inhibition of the Fyn kinase pathway prevented the development of IL-33-induced mechanical allodynia. Our findings provide insights into a new mechanism by which IL-33 directly regulates synaptic transmission and suggest that IL-33 signaling could be a candidate target for therapeutic interventions for orofacial neuropathic pain.

Possible embryos and precursors of crystalline nuclei of calcium carbonate observed by liquid-cell transmission electron microscopy.

Several different building blocks or precursors play an important role in the early stages of the crystallization of calcium carbonate (CaCO3). Many studies have been conducted over a long period to elucidate the fundamental processes involved in this crystallization. Here, we report the role of an amorphous phase and embryo at the beginning of the nucleation of CaCO3 from solutions of relatively low supersaturation. Prenucleation crystals formed in amorphous calcium carbonate (ACC) at a significantly large formation rate of 2 × 1022 m-3 s-1, suggesting that a low interfacial energy exists between the ACC and crystals. Only one calcite crystal exceeded the size for a critical nucleus (∼104 molecules) in 150 pre-nucleation crystals. Each pre-nucleation crystal might consist of a different polymorph, and ACCs have a similar composition and structure. A particle-detection algorithm, used in conjunction with machine learning, suggested that an embryo with a characteristic structure exists in solution and might play a crucial role in nucleation. No similar embryonic structure could be observed immediately after the dissolution of pre-nucleation particles, implying that their dissolution process is not simply the reverse process of their growth. This method should provide a new approach to understanding nucleation processes.

Trend in Age at the Initial Pacemaker Implantation in Patients With Bradyarrhythmia　- A 50-Year Analysis (1970-2019) in Japan.

BACKGROUND: This study investigated whether the age of patients undergoing pacemaker implantation is increasing.Methods and Results: This study retrospectively reviewed the consecutive cases of 3,582 patients who underwent an initial pacemaker implantation at our hospitals because of symptomatic bradyarrhythmias between 1970 and 2019. The exclusion criteria were: patients with AV block due to cardiac surgery or AV junction ablation, and patients aged <20 years. The patients were divided into 5×10-year groups: those treated in the 1970s (1970-1979), 1980s (1980-1989), 1990s (1990-1999), 2000s (2000-2009), and 2010s (2010-2019). A total of 3,395 patients satisfied the study criteria. The average age at which the patients underwent a first pacemaker implantation increased across the 10-year periods: 63.7±13.2 years in the 1970s, 66.2±12.6 years (1980s), 69.1±12.4 years (1990s), 72.0±11.1 years (2000s), and 75.8±10.0 years (2010s) and advanced significantly in the 1990s, 2000s, and 2010s compared to the 1970s (all P<0.001). The ratio of patients aged ≥80 and ≥90 years increased from 10.6% and 0% in the 1970 s to 38.2% (P<0.001) and 5.2% (P= 0.017) in the 2010s, respectively. CONCLUSIONS: The average age at initial pacemaker implantation increased by 12.1 years over the last 50 years in Japan. In particular, the ratios of ≥80 and ≥90 years as the patients age increased significantly.

Clinical Effect of Pulmonary Artery Diameter/Ascending Aorta Diameter Ratio on Left Ventricular Reverse Remodeling in Patients With Dilated Cardiomyopathy.

BACKGROUND: Many patients with dilated cardiomyopathy (DCM) progress to heart failure (HF), although some demonstrate left ventricular (LV) reverse remodeling (LVRR), which is associated with better outcomes. The pulmonary artery diameter (PAD) to ascending aortic diameter (AoD) ratio has been used as a prognostic predictor in patients with HF, although this tool's usefulness in predicting LVRR remains unknown.Methods and Results: Data from a prospective observational study of 211 patients diagnosed in 2000-2020 with DCM were retrospectively analyzed. Sixty-nine patients with New York Heart Association class I or II HF were included. LVRR was observed in 23 patients (33.3%). The mean LV ejection fraction (29%) and LV end-diastolic dimension (64.5 mm) were similar in patients with and without LVRR. The PAD/AoD ratio was significantly lower in patients with LVRR than those without (81.4% vs. 92.4%, respectively; P=0.003). The optimal PAD/AoD cut-off value for detecting LVRR was 0.9 according to the receiver operating characteristic curve analysis. Multivariate analysis identified a PAD/AoD ratio ≥0.9 as an independent predictor of presence/absence of LVRR. Cardiac events were significantly more common in patients with a PAD/AoD ratio ≥0.9 than those with a ratio <0.9, after a median follow up of 2.5 years (log-rank, P=0.007). CONCLUSIONS: The PAD/AoD ratio can predict LVRR in patients with DCM.

Programmed cell death ligand 1 expression on monocytes is inversely correlated with tumour response to preoperative chemoradiotherapy for locally advanced rectal cancer.

AIM: The clinical efficacy of chemoradiotherapy (CRT) is largely dependent on host immune status. The aim of this study was to identify possible markers expressed on circulating mononuclear cells to predict tumour response in patients with locally advanced rectal cancer (LARC). METHODS: Peripheral blood samples were obtained from 47 patients diagnosed with LARC before and after CRT. The numbers of lymphocytes and monocyte subsets were analysed using flow cytometry. Based on clinical and pathological findings, patients were classified as high or low responders. RESULTS: Lymphocyte counts were markedly decreased after CRT. Total numbers of lymphocytes (p = 0.030) and CD4(+) T cells (p = 0.041) in post-CRT samples were significantly lower in low responders than in high responders. In contrast, monocyte counts were not reduced and the number of CD14dim (+) CD16(+) nonclassical (patrolling) monocytes were somewhat increased after CRT (p = 0.050). Moreover, the ratios of programmed cell death ligand 1 (PD-L1) (+) cells on patrolling monocytes before and after CRT were significantly higher in low responders than in high responders (p = 0.0046, p = 0.0006). The same trend was observed for classical and intermediate monocytes. The expression of PD-L1 on patrolling monocytes before CRT correlated inversely with the number of T cells and natural killer (NK) cells after CRT. PD-L1(+) ratio in patrolling monocytes was an independent predictor for response to CRT. CONCLUSION: Programmed cell death ligand 1 (PD-L1) expression on patrolling monocytes suppresses cell-mediated immunity in patients receiving CRT which could be related to tumour response, and may be a useful biomarker for decision-making in the management of patients with LARC.

Prognostic value of malnutrition evaluated using the Global Leadership Initiative on Malnutrition criteria and its association with psoas muscle volume in non-ischemic dilated cardiomyopathy.

Heart failure (HF) is a systemic inflammatory disease that causes hypotrophy and skeletal muscle loss. The Global Leadership Initiative on Malnutrition (GLIM) criteria have been developed as a novel evaluation index for malnutrition, with reported usefulness in HF caused by ischemic heart disease. However, reports on the usefulness of malnutrition evaluated by the GLIM criteria in non-ischemic dilated cardiomyopathy (NIDCM) and its relationship with psoas muscle volume are lacking. We investigated the prognostic value of malnutrition evaluated using the GLIM criteria and its association with psoas muscle volume in patients with NIDCM. We enrolled 139 consecutive patients with NIDCM between December 2000 and June 2020. Malnutrition was evaluated using the GLIM criteria on admission. The median follow-up period was 4.7 years. Cardiac events were defined as a composite of cardiac death, hospitalization for worsening HF, and lethal arrhythmia. Furthermore, we measured the psoas muscle volume using computed tomography volumetry in 48 patients. At baseline, the median age was 50 years, and 132 patients (95.0%) had New York Heart Association functional class I or II HF. The median psoas muscle volume was 460.8 cm3. A total of 26 patients (18.7%) were malnourished according to the GLIM criteria. The Kaplan-Meier survival analysis showed that malnourished patients had more cardiac events than non-malnourished patients (log-rank, P < 0.001). The multivariate Cox proportional hazards regression analysis revealed that GLIM criteria-based malnutrition was an independent determinant of cardiac events (hazard ratio, 2.065; 95% confidence interval, 1.166-3.656; P = 0.014). Psoas muscle volume, which was assessed in a total of 48 patients, was lower in malnourished than in non-malnourished patients (median, 369.0 vs. 502.3 cm3; P = 0.035) and correlated with body mass index (r = 0.441; P = 0.002). Nutritional screening using the GLIM criteria may be useful in predicting future cardiac events in patients with NIDCM, reflecting a potential relationship between malnutrition and a low psoas muscle volume.

Splenic size as an indicator of hemodynamics and prognosis in patients with heart failure.

The spleen is an important immune organ that releases erythrocytes and monocytes and destroys aged platelets. It also reserves 20-30% of the total blood volume, and its size decreases in hypovolemic shock. However, the clinical significance of splenic size in patients with heart failure (HF) remains unclear. We retrospectively analyzed the data of 206 patients with clinically stable HF gathered between January 2001 and August 2020 and recorded in a single-center registry. All patients underwent right heart catheterization and computed tomography (CT). Splenic size was measured using CT volumetry. The primary outcomes were composite cardiac events occurring for the first time during follow-up, namely, cardiac death and hospitalization for worsening HF. The median splenic volume and splenic volume index (SVI) were 118.0 mL and 68.9 mL/m2, respectively. SVI was positively correlated with cardiac output (r = 0.269, P < 0.001) and stroke volume (r = 0.228, P = 0.002), and negatively correlated with systemic vascular resistance (r = - 0.302, P < 0.001). Seventy cardiac events occurred, and the optimal receiver operating characteristic curve SVI cutoff value for predicting cardiac events was 68.9 mL/m2. The median blood adrenaline concentration was higher in the low-SVI group than the high-SVI group (0.039 ng/mL vs. 0.026 ng/mL, respectively; P = 0.004), and the low-SVI group experienced more cardiac events (log-rank test, P < 0.001). Multivariate Cox proportional hazards regression revealed that a low SVI was an independent predictor of cardiac events, even when adjusted for the validated HF risk score, blood-brain natriuretic peptide concentration, blood catecholamine concentrations, and hemodynamic parameters. Splenic size reflects hemodynamics, including systemic circulating blood volume status and sympathetic nerve activity, and is associated with HF prognosis.

Impact of sleep-disordered breathing on ventricular tachyarrhythmias after left ventricular assist device implantation.

Sleep-disordered breathing (SDB) is associated with an increased risk of adverse events in patients with heart failure (HF); however, its impact in patients implanted with a left ventricular assist device (LVAD) remains unclear. We aimed to investigate the prevalence of SDB in patients with LVAD and its impact on their clinical outcomes. Fifty consecutive patients with LVAD who underwent portable sleep monitoring between September 2017 and April 2018 were prospectively enrolled, and they were followed up for 170 ± 36 days. According to their respiratory disturbance indexes (RDIs), they were categorized into the SDB group (RDI ≥ 15, n = 12) and the non-SDB group (RDI < 15, n = 38). The incidence of adverse events during the follow-up period was investigated after enrollment. Multivariate logistic regression analysis revealed significant differences in SDB in LVAD-implanted patients in terms of the logarithmic transformation brain natriuretic peptide (BNP) values (p = 0.005). The optimal BNP cut-off value for SDB prediction in LVAD-implanted patients was 300 pg/mL (sensitivity: 58.3%, specificity: 94.7%). During follow-up, ventricular tachyarrhythmias (VTas) occurred significantly more frequently in the SDB group (4 [33%] vs. 2 [5%] patients, p = 0.02); Atrial tachyarrhythmia (ATa) also tended to occur more frequently in the SDB group (2 [25%] vs. 2 [2%] patients, p = 0.07). SBD was prevalent in 24% of the LVAD-implanted patients with advanced HF. Furthermore, SDB was significantly associated with high BNP levels and was also potentially associated with subsequent incidence of VTa in patients with LVAD.

Fast Improvement of TEM Images with Low-Dose Electrons by Deep Learning.

Low electron dose observation is indispensable for observing various samples using a transmission electron microscope; consequently, image processing has been used to improve transmission electron microscopy (TEM) images. To apply such image processing to in situ observations, we here apply a convolutional neural network to TEM imaging. Using a dataset that includes short-exposure images and long-exposure images, we develop a pipeline for processed short-exposure images, based on end-to-end training. The quality of images acquired with a total dose of approximately $5$$e^{-}$ per pixel becomes comparable to that of images acquired with a total dose of approximately $1{,}000$$e^{-}$ per pixel. Because the conversion time is approximately 8 ms, in situ observation at 125 fps is possible. This imaging technique enables in situ observation of electron-beam-sensitive specimens.

Conserved keratin gene clusters in ancient fish: An evolutionary seed for terrestrial adaptation.

Type I and type II keratins are subgroups of intermediate filament proteins that provide toughness to the epidermis and protect it from water loss. In terrestrial vertebrates, the keratin genes form two major clusters, clusters 1 and 2, each of which is dominated by type I and II keratin genes. By contrast, such clusters are not observed in teleost fish. Although the diversification of keratins is believed to have made a substantial contribution to terrestrial adaptation, its evolutionary process has not been clarified. Here, we performed a comprehensive genomic survey of the keratin genes of a broad range of vertebrates. As a result, we found that ancient fish lineages such as elephant shark, reedfish, spotted gar, and coelacanth share both keratin gene clusters. We also discovered an expansion of keratin genes that form a novel subcluster in reedfish. Syntenic and phylogenetic analyses revealed that two pairs of krt18/krt8 keratin genes were shared among all vertebrates, thus implying that they encode ancestral type I and II keratin protein sets. We further revealed that distinct keratin gene subclusters, which show specific expressions in the epidermis of adult amphibians, stemmed from canonical keratin genes in non-terrestrial ancestors. Molecular evolutionary analyses suggested that the selective constraints were relaxed in the adult epidermal subclusters of amphibians as well as the novel subcluster of reedfish. The results of the present study represent the process of diversification of keratins through a series of gene duplications that could have facilitated the terrestrial adaptation of vertebrates.

Impella5.0 "ILIPELLA" Approach for a Fulminant Myocarditis Patient With a Small Peripheral Artery.

Cardiogenic shock with fulminant myocarditis is a life-threatening diagnosis. Extracorporeal membrane oxygenation (ECMO) with an Impella for left ventricle unloading is often required to maintain the haemodynamics. However, the small peripheral vascularity in small-bodied patients interrupts the upgrade from ECMO to Impella5.0, which usually requires grafting to a femoral artery or subclavian artery of at least 7 mm in size. This report outlines the external iliac artery approach, named the "ILIPELLA" technique, which uses a reconstructed external iliac artery to introduce Impella5.0 in patients with small peripheral vascularity.