Nucleic acid uptake occurs independent of lysosomal acidification but dependent on ATP consumption during RNautophagy/DNautophagy.

RNautophagy/DNautophagy (RDA) is an autophagic process that refers to the direct uptake of nucleic acids by lysosomes for degradation. Autophagy relies on lysosomes and lysosomal acidification is crucial for the degradation of intracellular components. However, whether lysosomal acidification interferes with nucleic acid uptake during RDA is unclear. In this study, we focused on vacuolar H+-ATPase (V-ATPase), the major proton pump responsible for maintaining an acidic pH in lysosomes. Our results show that lysosomes take up nucleic acids independently of the intralysosomal acidic pH during RDA. Isolated lysosomes treated with bafilomycin A1, a potent V-ATPase inhibitor, did not degrade, but took up RNA at similar levels as the control lysosomes. Similarly, the knockdown of Atp6v1a, the gene that encodes V-ATPase catalytic subunit A, did not affect the RNA uptake ability of isolated lysosomes. In addition, we demonstrated that nucleic acid uptake by isolated lysosomes necessitates ATP consumption, although V-ATPase is not required for the uptake process. These results broaden our understanding of the mechanisms underlying nucleic acid degradation via autophagy.

Optimal Timing of Serial 18F-Fluoro-2-Deoxyglucose Positron Emission Tomography after Prednisolone Treatment Introduction for Cardiac Sarcoidosis.

Immunosuppressive therapy with prednisolone (PSL) is the first-line treatment for cardiac sarcoidosis (CS), and 18F-fluoro-2-deoxyglucose positron emission tomography (FDG-PET) is used to evaluate its efficacy to guide treatment. However, the appropriate timing of FDG-PET in CS remains unknown. This single-center, retrospective, observational study included 15 consecutive CS patients who underwent 3 serial FDG-PET scans (at baseline, in the early phase [1-2 months after PSL introduction], and in the late phase [≥ 5 months after PSL introduction with a maintenance dose of PSL]). We adhered to the PSL tapering protocol by the Japanese Circulation Society even when early FDG-PET showed positive results (SUVmax ≥ 4.0). No patient died during the 908 (644-1600) days of observation. Negative results in the late phase were observed in 3 of 6 early-positive patients, and 3 of 9 early-negative patients showed positive results in the late phase. Changes in echocardiographic parameters from baseline to the late phase were significantly better in late-negative patients than in late-positive patients (left ventricular end-diastolic diameter: -0.7 (-9.3-[-0.5]) mm versus +3.5 (0.8-7.5) mm, P = 0.039; left ventricular end-systolic diameter: -4.2 (-6.9-[-0.1]) mm versus +5.1 (0.5-7.0) mm, P = 0.015; left ventricular ejection fraction: +4.7% (-1.0-9.0%) versus -1.5% (-11.3-1.5%), P = 0.045) ), although early FDG-PET did not predict those consequent changes. An interval of ≥ 5 months after introducing the PSL with a maintenance dose of PSL is long enough for FDG-PET to reflect consequent left ventricular functions, while an interval of 1-2 months can be too short.

Long-Term Outcomes in Patients with Not-Retrieval Inferior Vena Cava Filter Under Anticoagulation.

Since permanent inferior vena cava (IVC) filters increase deep vein thrombosis (DVT), filter retrieval should be performed as possible. Despite the guideline recommendation, IVC filters are not always retrieved in clinical practice. To date, many patients with not-retrieval IVC filters have been prescribed anticoagulant therapy, but the long-term prognosis, including venous thromboembolism (VTE) and bleeding events, remains unknown. In this study, 195 patients who underwent IVC filter implantation between 2006 and 2017 at 3 institutions in Niigata City have been investigated about their deaths, VTE recurrence, and bleeding events. After peaking 2009, the number of IVC filter implantation gradually decreased. During observational period, there were 158 patients with not-retrieval IVC filters (the overall retrieval rate of 19.0%). The not-retrieval group included significantly older and more patients with cancer compared to the retrieval group. Anticoagulation therapy was continued in 88% of the not-retrieval group. During a mean follow-up of 5.0 years, 6 symptomatic DVT events associated with inadequate control of anticoagulation and 13 bleeding events were observed. A total of 52 patients died and only the presence of cancer was prognostic risk factor. Although long-term anticoagulation therapy may be associated with bleeding events, there were few recurrent VTE under optimal anticoagulation. It is anticipated that even if the IVC filter cannot be retrieved, appropriate anticoagulation is useful for prevention of DVT recurrence despite the risk of bleeding.

The integral function of the endocytic recycling compartment is regulated by RFFL-mediated ubiquitylation of Rab11 effectors.

Endocytic trafficking is regulated by ubiquitylation (also known as ubiquitination) of cargoes and endocytic machineries. The role of ubiquitylation in lysosomal delivery has been well documented, but its role in the recycling pathway is largely unknown. Here, we report that the ubiquitin (Ub) ligase RFFL regulates ubiquitylation of endocytic recycling regulators. An RFFL dominant-negative (DN) mutant induced clustering of endocytic recycling compartments (ERCs) and delayed endocytic cargo recycling without affecting lysosomal traffic. A BioID RFFL interactome analysis revealed that RFFL interacts with the Rab11 effectors EHD1, MICALL1 and class I Rab11-FIPs. The RFFL DN mutant strongly captured these Rab11 effectors and inhibited their ubiquitylation. The prolonged interaction of RFFL with Rab11 effectors was sufficient to induce the clustered ERC phenotype and to delay cargo recycling. RFFL directly ubiquitylates these Rab11 effectors in vitro, but RFFL knockout (KO) only reduced the ubiquitylation of Rab11-FIP1. RFFL KO had a minimal effect on the ubiquitylation of EHD1, MICALL1, and Rab11-FIP2, and failed to delay transferrin recycling. These results suggest that multiple Ub ligases including RFFL regulate the ubiquitylation of Rab11 effectors, determining the integral function of the ERC.

Significance of Sarcopenia Evaluation in Acute Decompensated Heart Failure.

In patients with chronic heart failure (HF), the clinical importance of sarcopenia has been recognized in relation to disease severity, reduced exercise capacity, and adverse clinical outcome. Nevertheless, its impact on acute decompensated heart failure (ADHF) is still poorly understood. Dual-energy X-ray absorptiometry (DXA) is a technique for quantitatively analyzing muscle mass and the degree of sarcopenia. Fat-free mass index (FFMI) is a noninvasive and easily applicable marker of muscle mass.This was a prospective observational cohort study comprising 38 consecutive patients hospitalized for ADHF. Sarcopenia, derived from DXA, was defined as a skeletal muscle mass index (SMI) two standard deviations below the mean for healthy young subjects. FFMI (kg/m2) was calculated as 7.38 + 0.02908 × urinary creatinine (mg/day) divided by the square of height (m2).Sarcopenia was present in 52.6% of study patients. B-type natriuretic peptide (BNP) levels were significantly higher in ADHF patients with sarcopenia than in those without sarcopenia (1666 versus 429 pg/mL, P < 0.0001). Receiver operator curves were used to compare the predictive accuracy of SMI and FFMI for higher BNP levels. Areas under the curve for SMI and FFMI were 0.743 and 0.717, respectively. Multiple logistic regression analysis showed sarcopenia as a predictor of higher BNP level (OR = 18.4; 95% CI, 1.86-181.27; P = 0.013).Sarcopenia is associated with increased disease severity in ADHF. SMI based on DXA is potentially superior to FFMI in terms of predicting the degree of severity, but FFMI is also associated with ADHF severity.

Relationship Between Glucose Fluctuations and ST-Segment Resolution in Patients With ST-Elevation Acute Myocardial Infarction.

This study was conducted to assess whether any relationships exist between glucose fluctuations and electrocardiographic surrogate markers of reperfusion injury in patients with ST-elevation myocardial infarction (STEMI) undergoing primary percutaneous coronary intervention (PCI).We prospectively studied 63 consecutive patients with STEMI undergoing primary PCI. Patients had either diabetes (n = 30), impaired glucose tolerance (n = 26), impaired fasting glucose (n = 1), or normal glucose tolerance (n = 6). STsegment resolution (STR, %) was measured using electrocardiograms recorded 60 minutes after PCI. STR was categorized as ≥ 30% and < 30%. Glucose fluctuations were assessed by the following parameters obtained from a continuous glucose monitoring system: mean amplitude of glucose excursion (MAGE, mg/dL); and area under curve with reference to mean blood glucose (AUCMBG, mg/ dL/day).Both MAGE and AUCMBG were significantly higher in STR < 30%. In univariate analysis, MAGE ≥ 70 mg/dL (OR = 17.0; 95%CI, 1.93-150.12; P < 0.01), AUCMBG ≥ 20 mg/dL/day (OR = 10.9; 95%CI, 1.92-61.77; P < 0.01), and reperfusion arrhythmias (OR = 7.6; 95%CI, 1.32-44.29; P < 0.05) were significantly associated with suboptimal STR. Multiple logistic regression analysis showed only MAGE ≥ 70 mg/dL was predictive of suboptimal STR (OR = 22.5; 95%CI, 2.43-208.66, P < 0.01).Parameters of glucose fluctuations correlated with electrocardiographic surrogate markers of impaired myocardial salvage in STEMI after reperfusion therapy. Our results suggest that glucose fluctuations may represent a potential therapeutic target to reduce myocardial reperfusion injury in STEMI.

Evaluation of the four-dimensional motion of lung tumors during end-exhalation breath-hold conditions using volumetric cine computed tomography images.

BACKGROUND AND PURPOSE: This study was performed to evaluate the four-dimensional motion of lung tumors during end-exhalation (EE) breath-holding (BH) using cine computed tomography (CT) and investigate the correlation between tumor and surrogate marker motions. MATERIALS AND METHODS: This study included 28 patients who underwent stereotactic body radiation therapy at our institution and were capable of 15-20 s of EE BH within a ±1.5-mm gating window with external markers. During EE BH with cine CT, 21 s of continuous data were acquired using 320-row multislice CT. Displacements in the tumor position during EE BH were assessed in the left-right (LR), anterior-posterior (AP), and superior-inferior (SI) directions. Pearson's correlation coefficient (r) between tumor motions during EE BH and diaphragm/external marker motions was also determined. RESULTS: The mean absolute maximum displacements of the tumor position during EE BH were 1.3 (range: 0.2-4.0), 1.9 (range: 0.3-12.0), and 1.3 (range: 0.1-7.2) mm in the LR, AP, and SI directions, respectively. The displacement of the tumor position in the AP direction was weakly correlated (|r| < 0.4) with the external marker and diaphragm displacements in many cases (proportions of 50% and 46%, respectively). CONCLUSION: We found some cases showing substantial displacement in lung tumor positions during EE BH, especially in the AP direction. Because these tumor position displacements did not correlate with surrogate markers and were difficult to detect, we recommend pretreatment evaluation of the four-dimensional motions of tumors during BH using cine CT.

Baseline Blood Pressure and Left Ventricular Reverse Remodeling in Dilated Cardiomyopathy with Spontaneous Mechanical Alternans.

Objective Spontaneous mechanical alternans (MA), or pulsus alternans, has been observed in heart failure patients with hypertension or tachycardia for 150 years and is considered a sign of a poor prognosis. However, in some dilated cardiomyopathy (DCM) patients with MA, optimal medical therapy (OMT) brings left ventricular reverse remodeling (LVRR), a preferable prognostic indicator. This study examined the probability of LVRR in DCM patients with spontaneous MA and whether or not LVRR can be predicted by the baseline blood pressure or heart rate. Methods We conducted a single-center, retrospective observational study of newly diagnosed DCM patients from January 2017 to December 2020. Results Thirty-three newly diagnosed DCM patients were retrospectively examined. Spontaneous MA was observed during diagnostic cardiac catheterization in at least 1 of the pressure waveforms of the aorta, left ventricle, pulmonary artery, or right ventricle in 10 patients (30%) (MA-group). LVRR after OMT was achieved roughly equally in the MA group (6 of 10, 60%) and the non-MA group (12 of 23, 52%). In the MA group, those who achieved LVRR had a significantly higher baseline systolic aortic pressure (more than 120 mmHg in all 6 patients) than those who did not, although the baseline heart rate did not show a significant correlation with LVRR. In contrast, in the non-MA group, LVRR was unrelated to the baseline aortic pressure or heart rate. Conclusion The probability of LVRR in newly-diagnosed DCM patients with spontaneous MA was similar to that in those without spontaneous MA. Spontaneous MA may not necessarily be a sign of a poor prognosis if observed in patients with a preserved blood pressure.

A case of giant cell myocarditis mimicking cardiac sarcoidosis successfully maintained by prednisolone and tacrolimus.

A 45-year-old woman with no medical history underwent pacemaker implantation for a symptomatic complete atrioventricular block. On day 6, she noticed diplopia and then fever, general malaise, and elevation of serum creatinine kinase (CK). She was transferred to our hospital on day 21. Serum CK was elevated to 4543 IU/L, and echocardiography revealed a left ventricular ejection fraction of 43 %. We diagnosed her with giant cell myocarditis (GCM) via an emergent myocardial biopsy that revealed a proliferation of lymphocytes, eosinophils, and giant cells without granulomas. Initial treatment with high doses of intravenous methylprednisolone and immunoglobulin improved her symptoms in a few days, and prednisolone was given as follow-up treatment. CK was normalized in a week and a thinning of the interventricular septum mimicking cardiac sarcoidosis (CS) occurred. On day 38, we added a calcineurin inhibitor, tacrolimus, and maintained her with a combination of prednisolone and tacrolimus at a target dose of 10-15 ng/mL. Six months after the onset, there were no signs of relapse despite the persistent mild elevation of troponin I levels. We present a case of GCM mimicking CS successfully maintained by a combination of two immunosuppressive agents. Learning objective: Recommended treatment for giant cell myocarditis (GCM), a potentially fatal disease, is a combination of three immunosuppressive agents. However, GCM shares many characteristics with cardiac sarcoidosis (CS), which is treated using prednisolone alone in many cases. Recent studies on GCM and CS suggest they are different spectrums of a common entity. Although they can clinically overlap, they have different progressive speeds and severities. We present a case of GCM mimicking CS successfully treated with a combination of two immunosuppressive agents.

The Ubiquitin Ligase RNF34 Participates in the Peripheral Quality Control of CFTR (RNF34 Role in CFTR PeriQC).

The peripheral protein quality control (periQC) system eliminates the conformationally defective cystic fibrosis transmembrane conductance regulator (CFTR), including ∆F508-CFTR, from the plasma membrane (PM) and limits the efficacy of pharmacological therapy for cystic fibrosis (CF). The ubiquitin (Ub) ligase RFFL is responsible for the chaperone-independent ubiquitination and lysosomal degradation of CFTR in the periQC. Here, we report that the Ub ligase RNF34 participates in the CFTR periQC in parallel to RFFL. An in vitro study reveals that RNF34 directly recognizes the CFTR NBD1 and selectively promotes the ubiquitination of unfolded proteins. RNF34 was localized in the cytoplasm and endosomes, where RFFL was equally colocalized. RNF34 ablation increased the PM density as well as the mature form of ∆F508-CFTR rescued at low temperatures. RFFL ablation, with the exception of RNF34 ablation, increased the functional PM expression of ∆F508-CFTR upon a triple combination of CFTR modulators (Trikafta) treatment by inhibiting the K63-linked polyubiquitination. Interestingly, simultaneous ablation of RNF34 and RFFL dramatically increased the functional PM ∆F508-CFTR by inhibiting the ubiquitination in the post-Golgi compartments. The CFTR-NLuc assay demonstrates that simultaneous ablation of RNF34 and RFFL dramatically inhibits the degradation of mature ∆F508-CFTR after Trikafta treatment. Therefore, these results suggest that RNF34 plays a crucial role in the CFTR periQC, especially when there is insufficient RFFL. We propose that simultaneous inhibition of RFFL and RNF34 may improve the efficacy of CFTR modulators.

Pulmonary Artery Sarcoma Diagnosed Using an Endovascular Catheter Forceps Biopsy.

We herein report a case of pulmonary artery sarcoma (PAS) in a 64-year-old woman. She was admitted to our hospital because of massive genital bleeding from endometrial cancer. Contrast-enhanced computed tomography (CT) revealed a left pulmonary artery mass and deep vein thrombosis. She underwent anticoagulant therapy for one year. However, the mass lesion gradually expanded. 18F-Fluorodeoxyglucose (FDG) positron emission tomography (PET)/CT showed a positive uptake of FDG by the mass. An endovascular catheter biopsy was performed for the differentiation of endometrial cancer metastasis or primary sarcoma. The biopsy specimen tissue comprised spindle-shaped cells. Thus, the patient was diagnosed with PAS.

Cytosolic domain of SIDT2 carries an arginine-rich motif that binds to RNA/DNA and is important for the direct transport of nucleic acids into lysosomes.

RNautophagy and DNautophagy (RDA) are unconventional autophagic pathways where nucleic acids are directly transported through the lysosomal membrane, then degraded inside lysosomes. We have previously shown that bitopic protein LAMP2C and putative RNA transporter SIDT2, both lysosomal membrane proteins, mediate the direct transport of nucleic acids into lysosomes and that LAMP2C interacts with the nucleic acids and functions as a receptor during RDA. Because SIDT2-mediated RDA occurs in isolated lysosomes that lack LAMP2C, in this study, we tested the hypothesis that SIDT2 itself could also interact with the nucleic acids. Our results show that SIDT2 directly binds RNA and DNA through an arginine-rich motif (ARM) located within its main cytosolic domain, and disruption of this motif dramatically impairs SIDT2-mediated RNautophagic activity. We also found that SIDT2 interacts with exon 1 of HTT (huntingtin) transcript through the ARM in a CAG-dependent manner. Moreover, overexpression of SIDT2 promoted degradation of HTT mRNA and reduced the levels of polyglutamine-expanded HTT aggregates, hallmarks of Huntington disease. In addition, a comparative analysis of LAMP2C and SIDT2 functions at the cellular level revealed that the two proteins exert a synergistic effect on RNautophagic activity and that the ARMs which mediate the interactions of SIDT2 and LAMP2C with RNA are essential for the synergy. Together, our results point out the importance of nucleic acid-binding capacity of SIDT2 for its function in translocating nucleic acids through the lipid bilayer and suggests a potential application of RNautophagy activation to reduce the expression levels of disease-causing toxic proteins. Abbreviations: ACTB/ß-actin: actin beta; ARM: arginine-rich motif; CBB: Coomassie Brilliant Blue; CD: cytosolic domain; COX4I1/COX4: cytochrome c oxidase subunit 4I1; E. coli: Escherichia coli; EGFP: enhanced green fluorescent protein; EtBr: ethidium bromide; FITC: fluorescein isothiocyanate; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GOLGA2/GM130: golgin A2; GST: glutathione S-transferase; HRP: horseradish peroxidase; HSPA5/GRP78: heat shock protein family A (Hsp70) member 5; HTT: huntingtin; HTTex1: exon 1 of the HTT gene; LAMP2: lysosomal associated membrane protein 2; LMNA: lamin A/C; PAGE: polyacrylamide gel electrophoresis; PBS: phosphate-buffered saline; PEI: polyethyleneimine; polyQ: polyglutamine; qPCR: quantitative PCR; RAB5A: RAB5A, member RAS oncogene family; RDA: RNautophagy and DNautophagy; SCARB2/LIMP2: scavenger receptor class B member 2; SDS: sodium dodecyl sulfate; SID-1: systemic RNA interference deficient-1; SIDT2: SID1 transmembrane family member 2; WT: wild type.

Chaperone-Independent Peripheral Quality Control of CFTR by RFFL E3 Ligase.

The peripheral protein quality control (QC) system removes non-native membrane proteins, including ΔF508-CFTR, the most common CFTR mutant in cystic fibrosis (CF), from the plasma membrane (PM) for lysosomal degradation by ubiquitination. It remains unclear how unfolded membrane proteins are recognized and targeted for ubiquitination and how they are removed from the apical PM. Using comprehensive siRNA screens, we identified RFFL, an E3 ubiquitin (Ub) ligase that directly and selectively recognizes unfolded ΔF508-CFTR through its disordered regions. RFFL retrieves the unfolded CFTR from the PM for lysosomal degradation by chaperone-independent K63-linked poly-ubiquitination. RFFL ablation enhanced the functional expression of cell-surface ΔF508-CFTR in the presence of folding corrector molecules, and this effect was further improved by inhibiting the Hsc70-dependent ubiquitination machinery. We propose that multiple peripheral QC mechanisms evolved to dispose of non-native PM proteins and to preserve cellular proteostasis, even at the cost of eliminating partially functional polypeptides.

Photoelectrochemical and photocatalytic properties of biphasic organic p- and n-type semiconductor nanoparticles fabricated by a reprecipitation process.

The visible-light-responsive photoelectrochemical and photocatalytic properties of nanoparticles of C(60), partially hydrolyzed aluminum phthalocyanine chloride (denoted as AlPc), and a composite of the two are reported. The three types of nanoparticles were obtained through a reprecipitation method from N-methyl-2-pyrrolidone solutions of C(60), aluminum phthalocyanine chloride (AlPcCl), and their mixture, respectively. The nanoparticle composite's ultraviolet-visible absorption, diffuse-reflectance and Fourier transform IR spectra, X-ray diffraction pattern, and scanning electron microscopy image are all similar to the sum of those of the C(60) and AlPc particles, respectively. The nano-ordered composite exhibits p/n junctionlike photoelectrochemical characteristics, which were investigated in comparison with those of vapor-deposited C(60) (n-type), AlPcCl (p-type), C(60)/AlPcCl (n/p), and AlPcCl/C(60) (p/n) electrodes. The nanoparticle composite further shows photocatalytic activity for the decomposition of trimethylamine to carbon dioxide in a suspension system.