Directed evolution of highly sensitive and stringent choline-induced gene expression controllers.

Gene expression controllers are useful tools for microbial production of recombinant proteins and valued bio-based chemicals. Despite its usefulness, they have rarely been applied to the practical industrial bioprocess, due to the lack of systems that meets the three requirements: low cost, safety, and tight control, to the inducer molecules. Previously, we have developed the high-spec gene induction system controlled by safe and cheap inducer choline. However, the system requires relatively high concentration (~100 mM) of choline to fully induce the gene under control. In this work, we attempted to drastically improve the sensitivity of this induction system to further reduce the induction costs. To this end, we devised a simple circuit which couple gene induction system with positive-feedback loop (P-loop) of choline importer protein BetT. After the tuning of translation level of BetT (strength of the P-loop) and deletion of endogenous betI (noise sources), highly active yet stringent control of gene expression was achieved using about 100 times less amount of inducer molecules. The choline induction system developed in this study has the lowest basal expression, the lowest choline needed to be activated, and the highest amplitude of induction as the highest available promoter such as those known as PT5 system. With this system, one can tightly control the expression level of genes of interest with negligible cost for inducer molecule, which has been the bottleneck for the application to the large-scale industrial processes.

Nonmagnetic framboid and associated iron nanoparticles with a space-weathered feature from asteroid Ryugu.

Extraterrestrial minerals on the surface of airless Solar System bodies undergo gradual alteration processes known as space weathering over long periods of time. The signatures of space weathering help us understand the phenomena occurring in the Solar System. However, meteorites rarely retain the signatures, making it impossible to study the space weathering processes precisely. Here, we examine samples retrieved from the asteroid Ryugu by the Hayabusa2 spacecraft and discover the presence of nonmagnetic framboids through electron holography measurements that can visualize magnetic flux. Magnetite particles, which normally provide a record of the nebular magnetic field, have lost their magnetic properties by reduction via a high-velocity (>5 km s-1) impact of a micrometeoroid with a diameter ranging from 2 to 20 µm after destruction of the parent body of Ryugu. Around these particles, thousands of metallic-iron nanoparticles with a vortex magnetic domain structure, which could have recorded a magnetic field in the impact event, are found. Through measuring the remanent magnetization of the iron nanoparticles, future studies are expected to elucidate the nature of the nebular/interplanetary magnetic fields after the termination of aqueous alteration in an asteroid.

Vagus nerve signal has an inhibitory influence on the development of peritoneal metastasis in murine gastric cancer.

The vagus nerve is the only pathway for transmitting parasympathetic signals between the brain and thoracoabdominal organs, thereby exhibiting anti-inflammatory functions through the cholinergic anti-inflammatory pathway. Despite often being resected during lymph node dissection in upper gastrointestinal cancer surgery, the impact of vagotomy on postoperative outcomes in gastric cancer patients remains unclear. Sub-diaphragmatic vagotomy was performed on C57BL/6 mice. Three weeks later, syngeneic murine gastric cancer cell line YTN16P was injected into the peritoneal cavity, and the number of peritoneal metastases (PM) on the mesentery and omentum compared with control mice. The phenotypes of immune cells in peritoneal lavage and omental milky spots one day after tumor inoculation were analyzed using flow cytometry and immunohistochemistry. Intraperitoneal transfer of 3 × 105 YTN16P significantly increased the number of metastatic nodules on the mesentery in the vagotomy group compared to the control group. The omental metastasis grade was also significantly higher in the vagotomy group. Phenotypic analysis of immune cells in peritoneal lavage did not reveal significant differences after vagotomy. However, vagotomized mice exhibited a notable increase in milky spot area, with a higher presence of cytokeratin(+) tumor cells, F4/80(+) macrophages, and CD3(+) T cells. Vagus nerve signaling appears to regulate the immune response dynamics within milky spots against disseminated tumor cells and inhibits the development of PM. Preserving the vagus nerve may offer advantages in advanced gastric cancer surgery to reduce peritoneal recurrence.

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.

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.

Splenectomy has opposite effects on the growth of primary compared with metastatic tumors in a murine colon cancer model.

The spleen is a key source of circulating and tumor-infiltrating immune cells. However, the effect of splenectomy on tumor growth remains unclear. At 3 weeks after splenectomy, we subcutaneously injected LuM1 cells into BALB/c mice and evaluated the growth of primary tumors and lung metastases at 4 weeks after tumor inoculation. In addition, we examined the phenotypes of immune cells in peripheral blood by using flow cytometry and in tumor tissue by using multiplex immunohistochemistry. The growth of primary tumors was reduced in splenectomized mice compared with the sham-operated group. Conversely, splenectomized mice had more lung metastases. Splenectomized mice had fewer CD11b+cells, especially monocytic MDSCs (CD11b+Gr-1neg-lowLy6chigh), and NK cells (CD49b+CD335+). The proportion of NK cells was inversely correlated with the number of lung metastases. In splenectomized mice, the density of CD3+ and granzyme B+ CD8+ T cells was increased, with fewer M2-type macrophages in primary tumors, but NK cells were decreased markedly in lung. Splenectomy concurrently enhances T cell-mediated acquired immunity by reducing the number of monocytic MDSCs and suppresses innate immunity by decreasing the number of NK cells. Splenectomy has opposite effects on primary and metastatic lesions through differential regulation on these two immune systems.

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.

MetJ-Based Mutually Interfering SAM-ON/SAM-OFF Biosensors.

SAM (S-adenosylmethionine) is an important metabolite that operates as a major donor of methyl groups and is a controller of various physiological processes. Its availability is also believed to be a major bottleneck in the biological production of numerous high-value metabolites. Here, we constructed SAM-sensing systems using MetJ, an SAM-dependent transcriptional regulator, as a core component. SAM is a corepressor of MetJ, which suppresses the MetJ promoter with an increasing cellular concentration of SAM (SAM-OFF sensor). The application of transcriptional interference and evolutionary tuning effectively inverted its response, yielding a SAM-ON sensor (signal increases with increasing SAM concentration). By linking two genes encoding fluorescent protein reporters in such a way that their transcription events interfere with each other's and by placing one of them under the control of MetJ, we could increase the effective signal-to-noise ratio of the SAM sensor while decreasing the batch-to-batch deviation in signal output, likely by canceling out the growth-associated fluctuation in translational resources. By taking the ratio of SAM-ON/SAM-OFF signals and by resetting the default pool size of SAM, we could rapidly identify SAM synthetase (MetK) mutants with increased cellular activity from a random library. The strategy described herein should be widely applicable for identifying activity mutants, which would be otherwise overlooked because of the strong homeostasis of metabolic networks.

Systematic promoter design for plasmid-encoded S-adenosylmethionine sensing systems.

S-adenosylmethionine (SAM) is an important biomolecule that mainly acts as a methyl donor and plays many roles in a variety of biological functions. SAM is also required for the biosynthesis of valuable methylated compounds, but its supply is a bottleneck for these biosynthetic pathways. To overcome this bottleneck and to reconfigure SAM homeostasis, a high-throughput sensing system for changes in intracellular SAM availability is required. We constructed a plasmid that can detect the factors that can alter SAM availability using minimal components. It does so by placing a fluorescent protein under a promoter controlled by endogenous MetJ, a transcription factor that represses its own regulons upon binding with SAM. Next, to validate SAM-responsive behavior, we systematically reconstructed 10 synthetic promoters with different positions and with different number of metbox sites, sequences of MetJ binding. We found that a position between the -35 box and the -10 box was the most effective for repression and that this setup was suitable for detecting the genetic or environmental factors that can deplete and recover the intracellular SAM availability. Overall, the response patterns of the synthetic MetJ-regulated promoters characterized in this study may be useful for the development of further SAM biosensing systems.

Chiral Spinodal-like Ordering of Homoimmiscible Water at Interface between Water and Chiral Ice III.

Diversity in structures of water endowed by a hydrogen-bonding network plays crucial roles in wide varieties of phenomena in nature. Chiral ordering of water molecules is an intriguing phenomenon from the viewpoint of bimolecular functions. However, experimental reports on chiral ordering have been limited to the water molecules interacting with biomolecules on the molecular scale. It remains unclear whether pure liquid water forms long-range chiral ordering without any interaction with biomolecules. Here, we show that chiral anisotropy can be observed in the macro/mesoscopic network pattern of an unknown water layer formed via spinodal phase separation-like dynamics at the interface between water and ice III with a chiral crystal structure. We named this unknown water homoimmiscible water. Our observations infer that the unknown water is a chiral liquid crystal. This possibility opens new avenues for a wide variety of research fields such as liquid polymorphism, biology, earth and planetary science, and so forth from the perspective of chirality.

Foods and beverages associated with smoking craving in heated tobacco product and cigarette smokers: A cross-sectional study.

INTRODUCTION: Certain foods and beverages are associated with smoking craving. However, only few studies have explored the relationship between food or beverage-related and taste-associated smoking craving. In this study, we aimed to identify the types of foods related to smoking craving in Japanese individuals who smoke cigarettes or heated tobacco products (HTPs). METHODS: A total of 657 individuals (HTP and cigarette smokers and never smokers) participated in this cross-sectional study. Participants were asked what foods/beverages, tastes, seasonings, cooking methods, and cuisine categories, made them want to smoke and what foods they consumed. RESULTS: Alcoholic beverages such as beer, coffee, and fat-rich foods were associated with a higher likelihood of smoking craving. Fruits, dairy products such as milk, and sweet and sour tastes, were associated with a lower likelihood of smoking craving. The daily intake of fruit and dairy products was significantly lower in cigarette and HTP smokers than in non-smokers (median fruit intake: non-smokers, 46.4 g/1000 kcal/day; cigarette smokers, 22.2 g/1000 kcal/day; HTP smokers, 31.4 g/1000 kcal/day; p<0.001; median dairy product intake: non-smokers, 76.3 g/day; cigarette smokers, 48.2 g/day; HTP smokers, 57.6 g/day; p<0.001) as assessed using a food frequency questionnaire (BDHQ). CONCLUSIONS: Specific foods and beverages such as alcohol, fruits, and dairy products are related to smoking craving, and their intake differs according to smoking status.

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.

Dissolution enables dolomite crystal growth near ambient conditions.

Crystals grow in supersaturated solutions. A mysterious counterexample is dolomite CaMg(CO3)2, a geologically abundant sedimentary mineral that does not readily grow at ambient conditions, not even under highly supersaturated solutions. Using atomistic simulations, we show that dolomite initially precipitates a cation-disordered surface, where high surface strains inhibit further crystal growth. However, mild undersaturation will preferentially dissolve these disordered regions, enabling increased order upon reprecipitation. Our simulations predict that frequent cycling of a solution between supersaturation and undersaturation can accelerate dolomite growth by up to seven orders of magnitude. We validated our theory with in situ liquid cell transmission electron microscopy, directly observing bulk dolomite growth after pulses of dissolution. This mechanism explains why modern dolomite is primarily found in natural environments with pH or salinity fluctuations. More generally, it reveals that the growth and ripening of defect-free crystals can be facilitated by deliberate periods of mild dissolution.

Anisotropy in spinodal-like dynamics of unknown water at ice V-water interface.

Experimentally demonstrating the existence of waters with local structures unlike that of common water is critical for understanding both the origin of the mysterious properties of water and liquid polymorphism in single component liquids. At the interfaces between water and ices Ih, III, and VI grown/melted under pressure, we previously discovered low- and high-density unknown waters, that are immiscible with the surrounding water. Here, we show, by in-situ optical microscopy, that an unknown water appears at the ice V-water interface via spinodal-like dynamics. The dewetting dynamics of the unknown water indicate that its characteristic velocity is ~ 90 m/s. The time evolution of the characteristic length of the spinodal-like undulation suggests that the dynamics may be described by a common model for spinodal decomposition of an immiscible liquid mixture. Spinodal-like dewetting dynamics of the unknown water transiently showed anisotropy, implying the property of a liquid crystal.

Unveiling the expansion of keratin genes in lungfishes: a possible link to terrestrial adaptation.

Keratins are intermediate filament proteins that are important for epidermal strength and protection from desiccation. Keratin genes are highly duplicated and have diversified by forming two major clusters in the genomes of terrestrial vertebrates. The keratin genes of lungfishes, the closest fish to tetrapods, have not been studied at the genomic level, despite the importance of lungfishes in terrestrial adaptation. Here, we identified keratin genes in the genomes of two lungfish species and performed syntenic and phylogenetic analyses. Additionally, we identified keratin genes from two gobies and two mudskippers, inhabiting underwater and terrestrial environments. We found that in lungfishes, keratin genes were duplicated and diversified within two major clusters, similar to but independent of terrestrial vertebrates. By contrast, keratin genes were not notably duplicated in mudskippers. The results indicate that keratin gene duplication occurred repeatedly in lineages close to tetrapods, but not in teleost fish, even in species adapted to terrestrial environments.

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.

Pancreas Ligation Device for Distal Pancreatectomy: An Ex Vivo Follow-Up Porcine Study.

Introduction Postoperative pancreatic fistula (POPF) is a critical complication occurring with a high incidence after distal pancreatectomy. To minimize the risk of POPF, we developed an innovative pancreas ligation device capable of closing the pancreatic stump without causing traumatic injury to the pancreatic duct and artery. We conducted an ex vivo follow-up study to compare the pressure resistance of the pancreas ligation device with that of a regular linear stapler. Materials and methods The pancreases were excised from 20 pigs and divided into two groups: ligation group (n = 10) and stapler group (n = 10). Distal pancreatectomy was performed, and the pancreatic stump was closed using either a pancreas ligation device or a regular linear stapler. The main pancreatic duct was cannulated with a 4-French catheter connected to a cannula and syringe filled with contrast medium. Using fluoroscopy detection, pressure resistance was defined as the maximum pressure without leakage from the pancreatic stump. Results No significant differences were found between the two groups regarding sex, age, body weight, or pancreatic thickness. In the ligation group, no leakage was observed at the stump in any pancreas. However, in the stapler group, six of 10 pancreases showed leakage at the staple line or into the parenchyma. Pressure resistance was significantly higher in the ligation group than in the stapler group (median: 42.8 vs. 34.3 mmHg, P = 0.023). Conclusions These findings suggest the effectiveness of a pancreas ligation device in reducing the incidence of POPF after distal pancreatectomy. Our ligation device is expected to be a useful alternative to a linear stapler for pancreatic stump closure.

Visualization of nanoscale magnetic domain states in the asteroid Ryugu.

In the samples collected from the asteroid Ryugu, magnetite displays natural remanent magnetization due to nebular magnetic field, whereas contemporaneously grown iron sulfide does not display stable remanent magnetization. To clarify this counterintuitive feature, we observed their nanoscale magnetic domain structures using electron holography and found that framboidal magnetites have an external magnetic field of 300 A m-1, similar to the bulk value, and its magnetic stability was enhanced by interactions with neighboring magnetites, permitting a disk magnetic field to be recorded. Micrometer-sized pyrrhotite showed a multidomain magnetic structure that was unable to retain natural remanent magnetization over a long time due to short relaxation time of magnetic-domain-wall movement, whereas submicron-sized sulfides formed a nonmagnetic phase. These results show that both magnetite and sulfide could have formed simultaneously during the aqueous alteration in the parent body of the asteroid Ryugu.

Cardiac sympathetic activity and relationship to cardiac events and left ventricular reverse remodeling in patients with non-ischemic dilated cardiomyopathy.

BACKGROUND: Delayed heart-to-mediastinum ratio (HMR) has been associated with catecholamine levels and contractile reserve in dilated cardiomyopathy (DCM); however, there is scant evidence regarding the association between cardiac sympathetic activity and left ventricular reverse remodeling (LV-RR). We calculated the 123I-metaiodobenzylguanidine (123I-mIBG) HMR and washout rate (WR) in patients with DCM and investigated their associations with LV-RR. METHODS: From April 2003 to January 2020, in 120 patients with DCM who underwent 123I-mIBG scintigraphy. 66 patients undergoing follow-up echo and taking a beta-blocker from baseline were examined the relationship between 123I-mIBG and LV-RR. After that, this prognostic value for composite cardiac events was evaluated in the entire 120 patients. RESULTS: In LV-RR analysis, patients were 50.4 ± 12.2 years, with a mean left ventricular ejection fraction of 28.6%. Of 66 patients, 28 (42.4%) achieved LV-RR. Multiple logistic regression analysis of LV-RR revealed that not delayed HMR but the WR (cutoff value: 13.5%) was an independent predictor of LV-RR (odds ratio 6.514, p = 0.002). In the analysis for composite cardiac events, even though WR itself does not have the prognostic capacity, Kaplan-Meier survival curves divided by the cutoff value (delayed HMR = 2.0, WR = 13.5) showed that delayed HMR and WR values enabled the stratification of high-risk patients (log-rank p < 0.001). CONCLUSIONS: The 123I-mIBG WR was associated with the prevalence of LV-RR in patients taking 100% of beta-blockers and 98.5% of renin-angiotensin system inhibitors. Reflecting the contractile reserve, the combined assessment of the delayed HMR and WR could be used to further precisely stratify the patients with DCM.

Dilated cardiomyopathy with anti-mitochondrial M2 antibody: A case series.

Patients with dilated cardiomyopathy (DCM) sometimes show anti-mitochondrial M2 antibody (AMA-M2) positivity. We aimed to compare the characteristics of DCM cases with and without AMA-M2, and to describe cases of DCM with AMA-M2 positivity.A total of 84 patients with DCM were analyzed. Six patients (7.1 %) were positive for AMA-M2. Of these six patients, five (83.3 %) had primary biliary cirrhosis (PBC) and four (66.7 %) had myositis. Patients with AMA-M2 positivity had more atrial fibrillation and more premature ventricular contractions than those without. Left and right atrial longitudinal dimensions were larger in patients with AMA positivity (left atrium, 65.9 mm vs. 54.7 mm, p = 0.02; right atrium, 57.0 mm vs. 46.1 mm, p = 0.02). Of the six patients with AMA-M2 positivity, three underwent cardiac resynchronization therapy with defibrillator implantation and three required catheter ablation treatment. Steroids were used in three patients. One patient died of unresolved lethal arrhythmia and another required re-hospitalization for heart failure; the remaining four patients did not have adverse events.Patients with DCM with AMA-M2 positivity had a higher affinity for PBC and myositis than those without, and are characterized by atrial enlargement and arrhythmias. Learning objective: Patients with dilated cardiomyopathy sometimes exhibit anti-mitochondrial M2 antibody positivity. These patients are at higher risk for primary biliary cirrhosis and inflammatory myositis, and their cardiac disorders are characterized by atrial enlargement and various arrhythmias. The course of the disease up to the time of diagnosis and after steroid use varies, and the prognosis is poor in advanced cases.