Development of Apoptotic-Cell-Inspired Antibody-Drug Conjugate for Effective Immune Modulation.

BACKGROUND: Apoptotic cells' phosphoserine (PS) groups have a significant immunosuppressive effect. They inhibit proinflammatory signals by interacting with various immune cells, including macrophages, dendritic cells, and CD4+ cells. Previously, we synthesized PS-group-immobilized polymers and verified their immunomodulatory effects. Despite its confirmed immunomodulatory potential, the PS group has not been considered as a payload for antibody-drug conjugates (ADCs) in a targeted anti-inflammatory approach. AIM: We conducted this research to introduce an apoptotic-cell-inspired antibody-drug conjugate for effective immunomodulation. METHOD: Poly(2-hydroxyethyl methacrylate-co-2-methacryloyloxyethyl phosphorylserine) (p(HEMA-co-MPS)) was synthesized as a payload using RAFT polymerization, and goat anti-mouse IgG was selected as a model antibody, which was conjugated with the synthesized p(HEMA-co-MPS) via 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide/N-Hydroxysuccinimide (EDC/NHS) reaction. The antibody-binding affinity, anti-inflammatory potential, and cytotoxicity measurements were evaluated. RESULTS: We successfully synthesized ADCs with a significant anti-inflammatory effect and optimized the antibody-polymer ratio to achieve the highest antibody-binding affinity. CONCLUSION: We successfully introduced p(HEMA-co-MPS) to IgG without decreasing the anti-inflammatory potential of the polymer while maintaining its targeting ability. We suggest that the antibody-polymer ratio be appropriately adjusted for effective therapy. In the future, this technology can be applied to therapeutic antibodies, such as Tocilizumab or Abatacept.

Drainage pattern of the splenic flexure vein and its accompanying arteries using three-dimensional computed tomography angiography: a single-centre study of 600 patients.

AIM: The splenic flexure has variable vascular anatomy, and the details of the venous forms are not known. In this study, we report the flow pattern of the splenic flexure vein (SFV) and the positional relationship between the SFV and arteries such as the accessory middle colic artery (AMCA). METHODS: This was a single-centre study using preoperative enhanced CT colonography images of 600 colorectal surgery patients. CT images were reconstructed into 3D angiography. SFV was defined as a vein flowing centrally from the marginal vein of the splenic flexure visible on CT. AMCA was defined as the artery feeding the left side of the transverse colon, separate from the left branch of the middle colic artery. RESULTS: The SFV returned to the inferior mesenteric vein (IMV) in 494 cases (82.3%), the superior mesenteric vein in 51 cases (8.5%) and the splenic vein in seven cases (1.2%). The AMCA was present in 244 cases (40.7%). The AMCA branched from the superior mesenteric artery or its branches in 227 cases (93.0% of cases with existing AMCA). In the 552 cases in which the SFV returned to the IMV, superior mesenteric vein or splenic vein, the left colic artery was the most frequent artery accompanying the SFV (42.2%), followed by the AMCA (38.1%) and the left branch of the middle colic artery (14.3%). CONCLUSIONS: The most common flow pattern of the vein in the splenic flexure is from the SFV to IMV. The SFV is frequently accompanied by the left colic artery or AMCA.

Defective import of mitochondrial metabolic enzyme elicits ectopic metabolic stress.

Deficiencies in mitochondrial protein import are associated with a number of diseases. However, although nonimported mitochondrial proteins are at great risk of aggregation, it remains largely unclear how their accumulation causes cell dysfunction. Here, we show that nonimported citrate synthase is targeted for proteasomal degradation by the ubiquitin ligase SCFUcc1. Unexpectedly, our structural and genetic analyses revealed that nonimported citrate synthase appears to form an enzymatically active conformation in the cytosol. Its excess accumulation caused ectopic citrate synthesis, which, in turn, led to an imbalance in carbon flux of sugar, a reduction of the pool of amino acids and nucleotides, and a growth defect. Under these conditions, translation repression is induced and acts as a protective mechanism that mitigates the growth defect. We propose that the consequence of mitochondrial import failure is not limited to proteotoxic insults, but that the accumulation of a nonimported metabolic enzyme elicits ectopic metabolic stress.

Msp1-mediated proofreading mechanism for localization of tail-anchored membrane proteins.

Protein targeting to organelles has been thought to be a very precise process, and proteins that fail to localize correctly are rapidly degraded. Tail-anchored proteins are posttranslationally targeted to the endoplasmic reticulum membrane via guided entry of tail-anchored (TA) proteins pathway. However, these proteins can be mislocalized to the mitochondrial outer membrane. We found that the AAA-ATPase Msp1 on the mitochondrial outer membrane extracts mislocalized TA proteins to the cytosol, passing them to the guided entry of the TA proteins pathway to facilitate their transfer to the endoplasmic reticulum membrane. After the transfer to the endoplasmic reticulum, such TA proteins are directed to degradation if they are recognized by the quality control system on the endoplasmic reticulum. If not recognized, they are retargeted to their original destination along the secretory pathway. Thus, we have identified an intracellular proofreading system that corrects the localization of TA proteins.

Proofreading of protein localization mediated by a mitochondrial AAA-ATPase Msp1.

Normal cellular functions rely on correct protein localization within cells. Protein targeting had been thought to be a precise process, and even if it fails, the mistargeted proteins were supposed to be quickly degraded. However, this view is rapidly changing. Tail-anchored (TA) proteins are a class of membrane proteins that possess a single transmembrane domain (TMD) near the C-terminus and are posttranslationally targeted to the endoplasmic reticulum (ER) membrane, mitochondrial outer membrane (OM), and peroxisomal membrane, yet they can be mistargeted to the mitochondrial OM. The mistargeted TA proteins can be extracted from the OM by a mitochondrial AAA-ATPase Msp1/ATAD1 and transferred to the ER. If they are regarded as aberrant by the ER protein quality control system, they are extracted from the ER membrane for proteasomal degradation in the cytosol. If they are not regarded as aberrant, they are further transported to downstream organelles or original destinations along the secretory pathway. Thus, Msp1 contributes to not only degradation but also "proofreading" of the targeting of mislocalized TA proteins.

GET pathway mediates transfer of mislocalized tail-anchored proteins from mitochondria to the ER.

Tail-anchored (TA) membrane proteins have a potential risk to be mistargeted to the mitochondrial outer membrane (OM). Such mislocalized TA proteins can be extracted by the mitochondrial AAA-ATPase Msp1 from the OM and transferred to the ER for ER protein quality control involving ubiquitination by the ER-resident Doa10 complex. Yet it remains unclear how the extracted TA proteins can move to the ER crossing the aqueous cytosol and whether this transfer to the ER is essential for the clearance of mislocalized TA proteins. Here we show by time-lapse microscopy that mislocalized TA proteins, including an authentic ER-TA protein, indeed move from mitochondria to the ER in a manner strictly dependent on Msp1 expression. The Msp1-dependent mitochondria-to-ER transfer of TA proteins is blocked by defects in the GET system, and this block is not due to impaired Doa10 functions. Thus, the GET pathway facilitates the transfer of mislocalized TA proteins from mitochondria to the ER.

Radiological changes in the formation of Brodie's abscess by sequential magnetic resonance imaging: a case report.

Brodie's abscess is a relatively rare subacute form of osteomyelitis. Early diagnosis is challenging because of its insidious onset and vague symptoms. Magnetic resonance imaging is helpful in the diagnosis of Brodie's abscess; however, to date, no study has described the imaging findings of this disease in the early stage. Here, we present the case of a 14 year-old boy with Brodie's abscess in the proximal tibia. The lesion initially presented as a bone marrow edema in the proximal metaphysis of the left tibia on MRI and was misinterpreted as a bone bruise. Further radiological examination was performed 1 month later; this revealed the formation of an abscess cavity, which suggested Brodie's abscess. The patient was referred to our hospital and underwent curettage and debridement, which led to the definitive diagnosis of Brodie's abscess on histopathological findings and bacterial culture. On careful retrospective evaluation, the initial radiological findings suggested a microabscess on the metaphyseal side of the growth plate and bone marrow edema spreading from the lesion to the epiphysis. These radiological changes could be reliable evidence proving that the metaphyseal side of the growth plate is the origin of Brodie's abscess. Moreover, bone marrow edema with suspected microabscess in the metaphysis of the long bones can be the initial stage of the formation of Brodie's abscess and should be carefully followed up.

Difference in the airway luminal area between the standing and supine positions using upright and conventional computed tomography.

No clinical studies to date have compared the airway luminal area between supine and standing positions. Our aim was therefore to compare the airway luminal area between these two positions on computed tomography (CT) and to determine its correlation with forced expiratory volume in 1 s (FEV1). Thirty-two asymptomatic volunteers underwent both conventional (supine position) and upright (standing position) CT during deep inspiration breath-holding. Pulmonary function tests were conducted on the same day. We measured the airway luminal area on CT in each position. Paired t-tests and Pearson's correlation coefficients were used for statistical analysis. The average luminal areas of the trachea, right and left main bronchi, and average third-generation airway were greater in the standing than the supine position by 3.4%, 6.1%, 5.5%, and 5.2%, respectively. The correlation coefficients between airway luminal areas and FEV1 tended to be higher in the standing than the supine position; this correlation was highest for the average third-generation airway (r = 0.70, P < 0.0001). The airway luminal areas of the trachea, bilateral main bronchi, and average third-generation airway were greater in the standing than the supine position. The average third-generation airway area in the standing position had the highest correlation with FEV1.

Treatment effect prediction using CT after balloon pulmonary angioplasty in chronic thromboembolic pulmonary hypertension.

OBJECTIVE: To evaluate whether the change in computed tomography pulmonary angiography (CTPA) metrics after balloon pulmonary angioplasty (BPA) can predict treatment effect in chronic thromboembolic pulmonary hypertension (CTEPH) patients. METHODS: This study included 82 CTEPH patients who underwent both CTPA and right heart catheterization (RHC) before and at the scheduled time of 6 months after BPA. The diameters of the main pulmonary artery (dPA), ascending aorta (dAA), right atrium (dRA), right ventricular free wall thickness (dRVW), and right and left ventricles (dRV, dLV) were measured on CTPA. The correlation of the New York Heart Association functional class (NYHA FC), 6-minute walking distance (6MWD), brain natriuretic peptide (BNP) level, and calculated CT metrics with a decrease in mean pulmonary artery pressure (ΔmPAP) using RHC (used as the reference for BPA effect) was investigated. Using multiple regression analysis, independent variables were also identified. RESULTS: In univariate analysis, clinical indicators (NYHA FC, 6MWD, and BNP level) improved significantly after BPA and were significantly correlated with ΔmPAP (p < 0.01). In the univariate analysis of CTPA parameters, dPA, dRA, dPA/dAA ratio, dRVW, and dRV/dLV ratio decreased significantly and were significantly correlated with ΔmPAP (p < 0.01). Multivariate analysis demonstrated that decreased dPA (p = 0.001) and decreased dRA (p = 0.039) on CTPA were independent predictive factors of ΔmPAP. CONCLUSIONS: Decreased dPA and dRA on CTPA could predict a decrease in mPAP after BPA, thus potentially eliminating unnecessary invasive catheterization. KEY POINTS: • The reduction in mean pulmonary artery pressure after balloon pulmonary angioplasty in CTEPH patients was significantly correlated with the clinical indices improvement and CTPA parameter decrease. • The decreased diameter of the main pulmonary artery and the decreased diameter of the right atrium on CTPA were independent predictors of mean pulmonary artery pressure reduction.

CT screening for COVID-19 in asymptomatic patients before hospital admission.

INTRODUCTION: In the novel coronavirus disease (COVID-19) pandemic era, it is essential to rule out COVID-19 effectively to prevent transmission in both communities and medical facilities. According to previous reports in high prevalence areas, CT screening may be useful in the diagnosis of COVID-19. However, the value of CT screening in low prevalence areas has scarcely been reported. METHODS: This report examines the diagnostic efficacy of CT screening before admission to a hospital in Tokyo. We conducted a retrospective analysis at Keio University Hospital from April 6, 2020, through May 29, 2020. We set up an outpatient screening clinic on April 6 for COVID-19, administering both PCR with nasopharyngeal swabs and chest CT for all patients scheduled for surgery under general anesthesia. RESULTS: A total of 292 asymptomatic patients were included in this study. There were three PCR-positive patients, and they all had negative CT findings, which revealed that both the sensitivity and positive predictive value of CT (PPV) were 0%. There were nine CT-positive patients; the specificity and the negative predictive value (NPV) were 96.9% and 98.9%, respectively. CONCLUSION: CT screening was not useful in low prevalence areas at this time in Tokyo, even with the inclusion of the most prevalent phase. Given that the utility of CT screening depends on disease prevalence, the criteria for performing CT screening based on the prevalence of COVID-19 should be established.

Msp1 Clears Mistargeted Proteins by Facilitating Their Transfer from Mitochondria to the ER.

Normal mitochondrial functions rely on optimized composition of their resident proteins, and proteins mistargeted to mitochondria need to be efficiently removed. Msp1, an AAA-ATPase in the mitochondrial outer membrane (OM), facilitates degradation of tail-anchored (TA) proteins mistargeted to the OM, yet how Msp1 cooperates with other factors to conduct this process was unclear. Here, we show that Msp1 recognizes substrate TA proteins and facilitates their transfer to the endoplasmic reticulum (ER). Doa10 in the ER membrane then ubiquitinates them with Ubc6 and Ubc7. Ubiquitinated substrates are extracted from the ER membrane by another AAA-ATPase in the cytosol, Cdc48, with Ufd1 and Npl4 for proteasomal degradation in the cytosol. Thus, Msp1 functions as an extractase that mediates clearance of mistargeted TA proteins by facilitating their transfer to the ER for protein quality control.

Myocardial T1 values in healthy volunteers measured with saturation method using adaptive recovery times for T1 mapping (SMART1Map) at 1.5 T and 3 T.

Myocardial T1 mapping is clinically valuable for assessing the myocardium, and modified look-locker inversion-recovery (MOLLI) approaches have been commonly used for measuring myocardial T1 values. To date, several other sequences have been developed for measuring myocardial T1 values, and saturation-recovery-based sequences have been shown to be less dependent on various factors, such as T2 times and magnetization transfer, than inversion-recovery techniques. Systematic differences in T1 values between different sequences have been reported; therefore, definition of the normal range of native T1 values is required before clinical usage can begin. The purpose of this study was to evaluate the reference range and sex dependency of native T1 values in the myocardium measured using one such saturation-recovery sequence, i.e., saturation method using adaptive recovery times for cardiac T1 mapping (SMART1Map). Myocardial T1 values were compared between SMART1Map and MOLLI in 24 young healthy volunteers at 1.5 T and 3 T, and differences in the T1 values between the sexes were assessed. The mean native T1 values in the myocardium were significantly longer with SMART1Map than MOLLI [1530.4 ± 49.2 vs 1222.1 ± 48.9 ms at 3 T (p < 0.001) and 1227.3 ± 41.9 ms vs 1014.8 ± 49.4 ms at 1.5 T (p < 0.001)]. A significant difference between the sexes was observed in the T1 values obtained using each sequence, excluding SMART1Map at 3 T. The SMART1Map has a potential advantage to overcome the shortcoming of MOLLI, which underestimates T1 values; however, the sex-dependent difference remains obscure using SMART1Map.

Myristoyl group-aided protein import into the mitochondrial intermembrane space.

The MICOS complex mediates formation of the crista junctions in mitochondria. Here we analyzed the mitochondrial import pathways for the six yeast MICOS subunits as a step toward understanding of the assembly mechanisms of the MICOS complex. Mic10, Mic12, Mic26, Mic27, and Mic60 used the presequence pathway to reach the intermembrane space (IMS). In contrast, Mic19 took the TIM40/MIA pathway, through its CHCH domain, to reach the IMS. Unlike canonical TIM40/MIA substrates, presence of the N-terminal unfolded DUF domain impaired the import efficiency of Mic19, yet N-terminal myristoylation of Mic19 circumvented this effect. The myristoyl group of Mic19 binds to Tom20 of the TOM complex as well as the outer membrane, which may lead to "entropy pushing" of the DUF domain followed by the CHCH domain of Mic19 into the import channel, thereby achieving efficient import.

Deubiquitylases USP5 and USP13 are recruited to and regulate heat-induced stress granules through their deubiquitylating activities.

Stress granules are transient cytoplasmic foci induced by various stresses that contain translation-stalled mRNAs and RNA-binding proteins. They are proposed to modulate mRNA translation and stress responses. Here, we show that the deubiquitylases USP5 and USP13 are recruited to heat-induced stress granules. Heat-induced stress granules also contained K48- and K63-linked ubiquitin chains. Depletion of USP5 or USP13 resulted in elevated ubiquitin chain levels and accelerated assembly of heat-induced stress granules, suggesting that these enzymes regulate the stability of the stress granules through their ubiquitin isopeptidase activity. Moreover, disassembly of heat-induced stress granules after returning the cells to normal temperatures was markedly repressed by individual depletion of USP5 or USP13. Finally, overexpression of a ubiquitin mutant lacking the C-terminal diglycine motif caused the accumulation of unanchored ubiquitin chains and the repression of the disassembly of heat-induced stress granules. As unanchored ubiquitin chains are preferred substrates for USP5, we suggest that USP5 regulates the assembly and disassembly of heat-induced stress granules by mediating the hydrolysis of unanchored ubiquitin chains while USP13 regulates stress granules through deubiquitylating protein-conjugated ubiquitin chains.This article has an associated First Person interview with the first author of the paper.

Giant Cell Tumors of the Bone: Changes in Image Features after Denosumab Administration.

PURPOSE: To assess the clinical importance in the feature change in giant cell tumors of the bone (GCTB) after denosumab treatment, detected by MRI. METHODS: In 12 patients, MRI and CT of GCTB obtained before and after the treatment retrospectively compared. The tumor size, the signal intensity (SI) ratio between the solid part of the GCTB and muscle, cystic part size, gadolinium enhancement and apparent diffusion coefficient (ADC) value were measured on MRI. The bone formation in the tumor was observed on CT and X-ray. RESULTS: The mean number of denosumab injections was 19 ± 10. The follow-up period was up to 2 years. One case showed partial remission, while the other 11 cases were stable. A mean SI ratio on T2-weighted image statistically significantly decreased from 3.9 to 1.9 after the treatment. A cystic component in the tumor was observed in five cases before the treatment, and the diameter of the cystic part decreased after the treatment in 80% of cases (4/5). All the tumors showed contrast enhancement on T1-weighted image pre- and post-treatment (11/11). The averaged ADC values were 1.52 × 10-3 mm2/s before and 1.44 × 10-3 mm2/s after the treatment (P = 0.63). Bone formation in the tumor was observed in 58% of cases (7/12). CONCLUSION: The decrease of SI ratio on T2-weighted image, shrinkage of cystic part and bone formation should be regarded as the effectiveness of denosumab treatment despite of no substantial change in the tumor size.

Preoperatively determining the margins of subcutaneous malignant soft tissue tumours using contrast-enhanced ultrasonography.

The purpose of this technical report is to demonstrate the feasibility of contrast-enhanced ultrasonography for preoperative evaluation of the lateral extent of subcutaneous malignant soft tissue tumours. Patients with subcutaneous malignant soft tissue tumours who underwent surgery between March 2016 and May 2016 were selected for inclusion. Contrast-enhanced ultrasonography using Sonazoid® was performed 1 day before the operation. The tumour margin was determined based on contrast-enhanced ultrasonography and marked on the skin using a permanent marker. After surgery, the extent of the tumour area was determined based on microscopic findings. The distance between the edge of the tumour and the skin markings was histologically measured. The mean distance between the extent of infiltrative tumour growth based on the microscopic findings and the skin marking was 1.3 ± 1.6 mm. The greatest distance between microscopically determined tumour invasion and the skin marking was 4 mm. Contrast-enhanced ultrasonography of subcutaneous malignant soft tissue tumours was not only feasible, but also highly accurate in estimating tumour margins as well as infiltrative tumour growth compared with macroscopic and microscopic analysis, respectively.

Pre-treatment interleukin-6 levels strongly affect bone erosion progression and repair detected by magnetic resonance imaging in rheumatoid arthritis patients.

Objective: To examine the relationship between MRI structural damage and repair and plasma inflammatory cytokines in patients with RA. Methods: A total of 88 newly diagnosed, untreated RA patients were enrolled. Contrast MRI of the dominant hand and X-rays of the hands and feet were performed at baseline and 1 year later. MR images were evaluated using RA MRI scoring, and X-ray. Results: Progression of bone erosion and repair were observed more frequently in MRI than in X-rays (erosion, 52% vs 26%, P < 0.001; repair, 26% vs 15%, P = 0.003, respectively). Baseline IL-6 levels and seropositivity were independent relevant factors for MRI erosion progression, with IL-6 having stronger effect than seropositivity. A receiver operating characteristic curve identified the baseline IL-6 level of 7.6 pg/ml for predicting erosion progression during 1 year, with an area under the curve of 0.82; higher IL-6 levels resulted in more erosion progression. Baseline low IL-6 was also an independent predictor for MRI erosion repair. Conclusion: In newly diagnosed, untreated RA patients, baseline plasma IL-6 levels are responsible for 1-year MRI bone erosion progression and repair.

CT imaging before transcatheter aortic valve implantation (TAVI) using variable helical pitch scanning and its diagnostic performance for coronary artery disease.

OBJECTIVES: To evaluate the effectiveness of CT before TAVI using variable helical pitch (VHP) scanning and its diagnostic performance for coronary artery disease (CAD). METHODS: Sixty patients (84.4 ± 4.6 years) scheduled for TAVI underwent CT using VHP scanning with the contrast material (CM) volume calculated as scanning time × weight [kg] × 0.06 mL. Retrospective electrocardiography (ECG)-gated scanning was utilized to examine the thorax, and non-ECG-gated scanning of the abdomen immediately followed. We analyzed CT attenuation values of the coronary arteries, aorta, iliac and femoral arteries. The coronary CT angiography images were evaluated for the presence of stenosis (≥50 %); invasive coronary angiography served as a reference standard. RESULTS: The average attenuations of all of the arteries were greater than 400 HU. We could evaluate the peripheral access vessels and dimensions of the ascending aorta, aortic root, and aortic annulus in all patients. The average volume of CM was 38.7 ± 8.5 mL. On per-patient and vessel analysis, CT showed 91.7 % and 89.5 % sensitivity, and 91.3 % and 97.4 % negative predictive value (NPV). CONCLUSIONS: CT using VHP scanning with an average CM volume of 38.7 mL is useful before TAVI and had a high sensitivity and NPV in excluding obstructive CAD. KEY POINTS: • TAVI-planning CT using variable helical pitch (VHP) scanning is useful. • The average volume of contrast material was 38.7 ± 8.5 mL. • The average attenuations of all the arteries were >400 HU. • This CT had a high sensitivity and NPV for excluding obstructive CAD.

Tethering an N-Glycosylation Sequon-Containing Peptide Creates a Catalytically Competent Oligosaccharyltransferase Complex.

Oligosaccharyltransferase (OST) transfers an oligosaccharide chain to the Asn residue in the Asn-X-Ser/Thr sequon in proteins, where X is not proline. A sequon was tethered to an archaeal OST enzyme via a disulfide bond. The positions of the cysteine residues in the OST protein and the sequon-containing acceptor peptide were selected by reference to the eubacterial OST structure in a noncovalent complex with an acceptor peptide. We determined the crystal structure of the cross-linked OST-sequon complex. The Ser/Thr-binding pocket recognizes the Thr residue in the sequon, and the catalytic structure termed the "carboxylate dyad" interacted with the Asn residue. Thus, the recognition and the catalytic mechanism of the sequon are conserved between the archaeal and eubacterial OSTs. We found that the tethered peptides in the complex were efficiently glycosylated in the presence of the oligosaccharide donor. The stringent requirements are greatly relaxed in the cross-linked state. The two conserved acidic residues in the catalytic structure were each dispensable, although the double mutation abolished the activity. A Gln residue at the Asn position in the sequon functioned as an acceptor, and the hydroxy group at position +2 was not required. In the standard assay using short free peptides, strong amino acid preferences were observed at the X position, but the preferences, except for Pro, completely disappeared in the cross-linked state. By skipping the initial binding process and stabilizing the complex state, the catalytically competent cross-linked complex offers a unique system for studying the oligosaccharyl transfer reaction.