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Impairment of pancreatic β cells is a principal driver of the development of diabetes. Restoring normal insulin release from the β cells depends on the ATP produced by the intracellular mitochondria. In maintaining mitochondrial function, the tumor suppressor p53 has emerged as a novel regulator of metabolic homeostasis and participates in adaptations to nutritional changes. In this study, we used orotic acid, an intermediate in the pathway for de novo synthesis of the pyrimidine nucleotide, to reduce genotoxicity. Administration of orotic acid reduced p53 activation of MIN6 β cells and subsequently reduced β cell death in the db/db mouse. Orotic acid intake helped to maintain the islet size, number of β cells, and protected insulin secretion in the db/db mouse. In conclusion, orotic acid treatment maintained β cell function and reduced cell death, and may therefore, be a future therapeutic strategy for the prevention and treatment of diabetes. more...

SARS-CoV-2 Omicron subvariants have evolved to evade receptor-binding site (RBS) antibodies that exist in diverse individuals as public antibody clones. We rationally selected RBS antibodies resilient to mutations in emerging Omicron subvariants. Y489 was identified as a site of virus vulnerability and a common footprint of broadly neutralizing antibodies against the subvariants. Multiple Y489-binding antibodies were encoded by public clonotypes and additionally recognized F486, potentially accounting for the emergence of Omicron subvariants harboring the F486V mutation. However, a subclass of antibodies broadly neutralized BA.4/BA.5 variants via hydrophobic binding sites of rare clonotypes along with extremely high mutation-resilience under escape mutation screening. The structural basis for mutation-resilience of this antibody group may inform the design of therapeutics resistant to viral escape. more...

The Omicron (B.1.1.529) SARS-CoV-2 variant contains an unusually high number of mutations in the spike protein, raising concerns of escape from vaccines, convalescent serum, and therapeutic drugs. Here, we analyzed the degree to which Omicron pseudo-virus evades neutralization by serum or therapeutic antibodies. Serum samples obtained 3 months after two doses of BNT162b2 vaccination exhibited 18-fold lower neutralization titers against Omicron than parental virus. Convalescent serum samples from individuals infected with the Alpha and Delta variants allowed similar frequencies of Omicron breakthrough infections. Domain-wise analysis using chimeric spike proteins revealed that this efficient evasion was primarily achieved by mutations clustered in the receptor binding domain but that multiple mutations in the N-terminal domain contributed as well. Omicron escaped a therapeutic cocktail of imdevimab and casirivimab, whereas sotrovimab, which targets a conserved region to avoid viral mutation, remains effective. Angiotensin-converting enzyme 2 (ACE2) decoys are another virus-neutralizing drug modality that are free, at least in theory, from complete escape. Deep mutational analysis demonstrated that an engineered ACE2 molecule prevented escape for each single-residue mutation in the receptor binding domain, similar to immunized serum. Engineered ACE2 neutralized Omicron comparably to the Wuhan strain and also showed a therapeutic effect against Omicron infection in hamsters and human ACE2 transgenic mice. Similar to previous SARS-CoV-2 variants, some sarbecoviruses showed high sensitivity against engineered ACE2, confirming the therapeutic value against diverse variants, including those that are yet to emerge. more...

Nonalcoholic steatohepatitis (NASH) is a progressive disorder with aberrant lipid accumulation and subsequent inflammatory and profibrotic response. Therapeutic efforts at lipid reduction via increasing cytoplasmic lipolysis unfortunately worsens hepatitis due to toxicity of liberated fatty acid. An alternative approach could be lipid reduction through autophagic disposal, i.e., lipophagy. We engineered a synthetic adaptor protein to induce lipophagy, combining a lipid droplet-targeting signal with optimized LC3-interacting domain. Activating hepatocyte lipophagy in vivo strongly mitigated both steatosis and hepatitis in a diet-induced mouse NASH model. Mechanistically, activated lipophagy promoted the excretion of lipid from hepatocytes via lysosomal exocytosis, thereby suppressing harmful intracellular accumulation of nonesterified fatty acid. A high-content compound screen identified alpelisib and digoxin, clinically-approved compounds, as effective activators of lipophagy. Administration of alpelisib or digoxin in vivo strongly inhibited the transition to steatohepatitis. These data thus identify lipophagy as a promising therapeutic approach to prevent NASH progression. more...

The novel SARS-CoV-2 variant, Omicron (B.1.1.529) contains an unusually high number of mutations (>30) in the spike protein, raising concerns of escape from vaccines, convalescent sera and therapeutic drugs. Here we analyze the alteration of neutralizing titer with Omicron pseudovirus. Sera obtained 3 months after double BNT162b2 vaccination exhibit approximately 18-fold lower neutralization titers against Omicron than parental virus. Convalescent sera from Alpha and Delta patients allow similar levels of breakthrough by Omicron. Domain-wise analysis using chimeric spike revealed that this efficient evasion was primarily achieved by mutations clustered in the receptor-binding domain, but that multiple mutations in the N-terminal domain contributed as well. Omicron escapes a therapeutic cocktail of imdevimab and casirivimab, whereas sotrovimab, which targets a conserved region to avoid viral mutation, remains effective. The ACE2 decoy is another virus-neutralizing drug modality that is free, at least in theory, from complete escape. Deep mutational analysis demonstrated that, indeed, engineered ACE2 prevented escape for each single-residue mutation in the receptor-binding domain, similar to immunized sera. Engineered ACE2 neutralized Omicron comparable to Wuhan and also showed a therapeutic effect against Omicron infection in hamsters and human ACE2 transgenic mice. Like previous SARS-CoV-2 variants, some sarbecoviruses showed high sensitivity against engineered ACE2, confirming the therapeutic value against diverse variants, including those that are yet to emerge.One Sentence SummaryOmicron, carrying ∼30 mutations in the spike, exhibits effective immune evasion but remains highly susceptible to blockade by engineered ACE2. more...

The novel SARS-CoV-2 variant, Omicron (B.1.1.529) contains about 30 mutations in the spike protein and the numerous mutations raise the concern of escape from vaccine, convalescent sera and therapeutic drugs. Here we analyze the alteration of their neutralizing titer with Omicron pseudovirus. Sera of 3 months after double BNT162b2 vaccination exhibite ∼27-fold lower neutralization titers against Omicron than D614G mutation. Neutralization titer is also reduced in convalescent sera from Alpha and Delta patients. However, some Delta patients have relatively preserved neutralization activity up to the level of 3-month double BNT162b2 vaccination. Omicron escapes from the cocktail of imdevimab and casirivimab, whereas sotrovimab that targets the conserved region to prevent viral escape is effective to Omicron similarly to the original SARS-CoV-2. The ACE2 decoy is another modality that neutralize the virus independently of mutational escape and Omicron is also sensitive to the engineered ACE2. more...

Cardiac fibrosis is the pathological feature of myocardial remodeling and clinically involved in various cardiovascular diseases including heart failure with preserved ejection fraction (HFpEF) and atrial fibrillation. Accumulating evidence revealed the molecular mechanism behind organ fibrosis, however, no effective anti-fibrotic therapy has been developed. To find novel anti-fibrotic molecular targets, we performed genome-wide CRISPR library screening with focus on myofibroblast activation. Since a feedforward autocrine loop accelerates myofibroblast differentiation, we employed connective tissue growth factor (CTGF), a center of profibrotic secretome, as a target factor for screening assay. We firstly generated a CTGF-GFP knock-in reporter cell in mesenchymal progenitor cells. The reporter cells were transduced with lentivirus CRISPR library and treated with TGF beta, then high and low GFP signal cells were sorted and sequenced. This screening showed that Keap1-NRF2 pathway was one of the top hits and a novel regulator for CTGF expression. Keap1 knockout attenuated TGF beta-mediated CTGF expression and additional NRF2 knockout cancelled it. Keap1-NRF2 pathway also regulated broadly other profibrotic factors, alpha SMA, periostin, and fibronectin. An activator of NRF2, CDDO-Im treatment had similar effect on TGF beta-mediated profibrotic response. CTGF knockout had little effect on these profibrotic factors, indicating that Keap1-NRF2 is better therapeutic target, as compared with CTGF. Keap1-NRF2 originally reported as a transcriptional regulator for antioxidant and detoxification genes. In silico analysis indicates CTFG promoter has NRF2 response element and ChiP-qPCR experiment confirmed that NRF2 directly regulated CTFG transcriptional expression, not indirectly through antioxidant gene expression. In conclusion, genome-wide screening identified Keap1-NRF2 pathway as a crucial regulator for myofibroblast activation and NRF2 activation might be novel therapy for fibrosis-related diseases. more...

Background: Although metabolic alterations were observed in heart failure (HF), only recently have the mechanisms underlying these changes been identified. Tumor suppressor p53 responds to metabolic changes thorough several mechanisms. One of the p53 targets, TIGAR (TP53-induced glycolysis and apoptosis regulator) reduces glycolysis and suppresses autophagy, which augments ischemic damage, however its role on HF is unclear. Method and Results: In order to investigate TIGAR’s function in HF, we compared myocardial metabolic and functional outcomes between TIGAR deficient (TIGAR–/–) mice and wild-type (TIGAR+/+) mice subjected to chronic thoracic transverse aortic constriction (TAC), a pressure-overload HF model. In wild-type mice hearts, p53 and TIGAR increased markedly during HF development. Eight weeks after TAC surgery, the left ventricular (LV) dysfunction, fibrosis, oxidative damage, and myocyte apoptosis were significantly advanced in wild-type than in TIGAR–/– mouse heart. Further, myocardial high-energy phosphates in wild-type hearts were significantly decreased compared to those of TIGAR–/– mouse heart. Glucose oxidation and glycolysis rates were also reduced in isolated perfused wild-type hearts following TAC than those in TIGAR–/– hearts, which suggest that the upregulation of TIGAR in HF causes impaired myocardial energetics and function. The effects of TIGAR knockout on LV function were also replicated in tamoxifen (TAM)-inducible cardiac-specific TIGAR knockout mice (TIGARflox/flox/ Tg(Myh6-cre/Esr1) mice). Conclusion: The ablation of TIGAR during pressure-overload HF preserves myocardial function and energetics. Thus, cardiac TIGAR targeted therapy to increase glucose metabolism will be a novel strategy for HF. more...

A 77-year-old Japanese woman underwent bioprosthetic aortic valve replacement (AVR) and the Maze procedure for severe aortic valve disease and paroxysmal atrial fibrillation (AF), and one year after the AVR, she also underwent a permanent pacemaker implantation for sick sinus syndrome. At two postoperative years, a large mural mass happened to be detected in her left atrium on routine trans-thoracic echocardiography. The cardiac rhythm records produced by the implanted pacemaker demonstrated the recurrence of AF. As anticoagulant therapy was not effective at reducing the size of the mass, surgery was performed and organized thrombus was detected on the ablation line made at the Maze procedure. more...

A 48-year-old man suffered from uncontrollable coronary vasospasms, even when taking the maximum dose of vasodilators. The patient had a history of hypereosinophilia, and as the eosinophilia worsened, more frequent and intense coronary spastic angina (CSA) attacks occurred. He was treated with 20 mg/day of oral prednisolone, and the chest symptoms of CSA completely resolved thereafter. We encountered a refractory CSA patient with an allergic predisposition for which the oral administration of corticosteroids was markedly effective. Although the priority of corticosteroid therapy is not clinically high in patients with CSA, it can be effective especially in patients with an allergic background. more...

Introduction Because of the unusual anatomy of an anomalous origin of the right coronary artery from the left sinus of Valsalva, selective cannulation of the guiding catheter in percutaneous coronary intervention for these cases is always challenging. Case presentation A 58-year-old Japanese man was admitted to our hospital complaining of worsening exertional chest pain. He was suspected of having unstable angina pectoris and underwent cardiac catheterization. We found a subtotal occlusive lesion in the mid-portion of his right coronary artery that originated from the left sinus of Valsalva. On the previous percutaneous coronary intervention, we failed to cannulate the guiding catheter to the anomalous orifice of the right coronary artery. Therefore, we decided to use the GuideLiner catheter for stable back-up support from the beginning. A 6Fr GuideLiner catheter was introduced into the right coronary artery by anchoring it coaxially with a semi-compliant balloon catheter. And we successfully deployed two drug-eluting stents by crossing over the posterior-descending artery. Final angiography demonstrated favorable dilatation of the target lesion, and native blood flow in the right coronary artery was completely recovered. Conclusion GuideLiner is a monorail-type “child” support catheter that facilitates coaxial guiding catheter engagement and an appropriate back-up force, achieving successful device delivery to target lesions in this kind of complex percutaneous coronary intervention. more...

Introduction Although several etiopathogenetic mechanisms have been proposed, the causes of left ventricular apical ballooning syndrome are still controversial. Case presentation A 51-year-old Japanese woman consulted the emergency room complaining of the sudden onset of anterior chest pain while shopping. We initially suspected her disease as left ventricular apical ballooning syndrome based on her clinical background and laboratory examinations. However, the initial coronary angiogram demonstrated diffuse lesions in her distal left anterior descending coronary artery, and she was finally diagnosed with apical myocardial infarction. The blood flow in her distal left anterior descending coronary artery had markedly improved in the chronic phase. If the reduced blood flow in her distal left anterior descending coronary artery was induced by coronary vasospasm and the vasospasm was relieved before the coronary angiogram was performed, this case must be diagnosed as left ventricular apical ballooning syndrome. Conclusion We think this case may promote discussion regarding the pathophysiology of left ventricular apical ballooning syndrome. more...