Structural Insights into the Interaction between the C-Terminal-Deleted BH3-like Motif Peptide of Hepatitis B Virus X Protein and Bcl-xL.

Hepatitis B virus X protein (HBx) plays a crucial role in the development of hepatocellular carcinoma (HCC) associated with hepatitis B virus (HBV) infection. The full-length HBx protein interacts with Bcl-xL and is involved in the HBV replication and cell death processes. The three hydrophobic residues Trp120, Leu123, and Ile127 of the HBx BH3-like motif are essential for the Bcl-xL-binding. On the other hand, various lengths of C-terminal-truncated HBx mutants are frequently detected in HCC tissues, and these mutants, rather than the full-length HBx, appear to be responsible for HCC development. Notably, the region spanning residues 1-120 of HBx [HBx(1 and 120)] has been strongly associated with an increased risk of HCC development. However, the mode of interaction between HBx(1-120) and Bcl-xL remains unclear. HBx(1-120) possesses only Trp120 among the three hydrophobic residues essential for the Bcl-xL-binding. To elucidate this interaction mode, we employed a C-terminal-deleted HBx BH3-like motif peptide composed of residues 101-120. Here, we present the NMR complex structure of Bcl-xL and HBx(101-120). Our results demonstrate that HBx(101-120) binds to Bcl-xL in a weaker manner. Considering the high expression of Bcl-xL in HCC cells, this weak interaction, in conjunction with the overexpression of Bcl-xL in HCC cells, may potentially contribute to HCC development through the interaction between C-terminal-truncated HBx and Bcl-xL.

Structural basis for the unique multifaceted interaction of DPPA3 with the UHRF1 PHD finger.

Ubiquitin-like with PHD and RING finger domain-containing protein 1 (UHRF1)-dependent DNA methylation is essential for maintaining cell fate during cell proliferation. Developmental pluripotency-associated 3 (DPPA3) is an intrinsically disordered protein that specifically interacts with UHRF1 and promotes passive DNA demethylation by inhibiting UHRF1 chromatin localization. However, the molecular basis of how DPPA3 interacts with and inhibits UHRF1 remains unclear. We aimed to determine the structure of the mouse UHRF1 plant homeodomain (PHD) complexed with DPPA3 using nuclear magnetic resonance. Induced α-helices in DPPA3 upon binding of UHRF1 PHD contribute to stable complex formation with multifaceted interactions, unlike canonical ligand proteins of the PHD domain. Mutations in the binding interface and unfolding of the DPPA3 helical structure inhibited binding to UHRF1 and its chromatin localization. Our results provide structural insights into the mechanism and specificity underlying the inhibition of UHRF1 by DPPA3.

Practical methods to differentiate thymic malignancies by positron-emission tomography and tumor markers.

PURPOSE: An accurate diagnosis of thymic malignancies is important, but challenging due to the broad range of differential diagnoses. This study aims to evaluate the efficacy of PET/CT and tumor markers for diagnosing thymic malignancies. METHODS: Patients admitted to our department between January 2012 and December 2021 with primary anterior mediastinal tumors were retrospectively evaluated. We evaluated the relationship between the maximum standardized uptake value (SUVmax), tumor markers, and pathological diagnosis in four groups: thymic carcinoma, thymoma, lymphoma, and others. RESULTS: In total, 139 patients were included in this study. The SUVmax was significantly higher in lymphoma, thymic carcinoma, and thymoma, in that order. The cytokeratin 19 fragment (CYFRA 21-1) was significantly higher in thymic carcinoma than in the other groups. An ROC curve analysis indicated that the optimal cut-off values of SUVmax for thymic carcinoma plus lymphoma and CYFRA 21-1 for thymic carcinoma were 7.97 (AUC = 0.934) and 2.95 (AUC = 0.768), respectively. Using a combination of cut-off values (SUVmax = 8, CYFRA 21-1 = 3), the accuracy rate for diagnosing thymic carcinoma was 91.4%. CONCLUSIONS: The SUVmax and CYFRA 21-1 levels are significant indicators for the diagnosis of thymic carcinoma. Combining these indicators resulted in a more accurate diagnosis of thymic malignancies, which could facilitate the decision-making process for determining the optimal treatment strategies.

Relationship between changes in pulmonary function and patient-reported outcomes of lung cancer surgery.

PURPOSE: To investigate the relationship between changes in pulmonary function (PF) and patient-reported outcomes (PROs) of lung cancer surgery. METHODS: We recruited 262 patients who underwent lung resection for lung cancer, to evaluate the PROs, using the European Organization for Research and Treatment of Cancer Quality of Life Questionnaire (EORTC QLQ)-C30 and the Lung Cancer 13-question supplement (LC13). The patients underwent PF tests and PRO assessments preoperatively (Pre) and 1 year after surgery (Y1). Changes were calculated by subtracting the value at Pre from the value at Y1. We set two cohorts: patients under the ongoing protocol (Cohort 1) and patients who were eligible for lobectomy with clinical stage I lung cancer (Cohort 2). RESULTS: Cohorts 1 and 2 comprised 206 and 149 patients, respectively. In addition to dyspnea, changes in PF were also correlated with scores for global health status, physical and role function scores, fatigue, nausea and vomiting, pain, and financial difficulties. Absolute correlation coefficient values ranged from 0.149 to 0.311. Improvement of emotional and social function scores was independent of PF. Sublobar resection preserved PF more than lobectomy did. Wedge resection mitigated dyspnea in both cohorts. CONCLUSION: The correlation between PF and PROs was found to be weak; therefore, further studies are needed to improve the patient's postoperative experience.

Sex-specific emphysematous changes evaluated by a three-dimensional computed tomography volumetric analysis among patients with smoking histories who underwent resection for lung cancer.

PURPOSE: The present study evaluated the sex-specific susceptibility to the development of emphysema in patients with smoking histories who underwent lung cancer surgeries. METHODS: Lung cancer patients with smoking histories who underwent lung resection at the University of Tsukuba Hospital, Japan, were enrolled. Radiologic emphysematous changes were analyzed using three-dimensional computed tomography (3D-CT). The volume proportion of emphysematous lung per unit of smoking and the relationship between emphysematous change and clinicopathologic factors were evaluated. RESULTS: Radiologic emphysematous changes analyzed using 3D-CT per pack-year smoked, defined as the Smoking-Emphysema Index (SEI), were greater in females than males. The difference was more profound in adenocarcinoma patients than in non-adenocarcinoma patients (0.70 ± 2.30 vs. 0.21 ± 0.28, P = 0.037). CONCLUSION: Female lung cancer patients are more susceptible to smoking-induced emphysema than males. The SEI may be an effective indicator for evaluating smoking-induced emphysema.

Robust folding of a de novo designed ideal protein even with most of the core mutated to valine.

Protein design provides a stringent test for our understanding of protein folding. We previously described principles for designing ideal protein structures stabilized by consistent local and nonlocal interactions, based on a set of rules relating local backbone structures to tertiary packing motifs. The principles have made possible the design of protein structures having various topologies with high thermal stability. Whereas nonlocal interactions such as tight hydrophobic core packing have traditionally been considered to be crucial for protein folding and stability, the rules proposed by our previous studies suggest the importance of local backbone structures to protein folding. In this study, we investigated the robustness of folding of de novo designed proteins to the reduction of the hydrophobic core, by extensive mutation of large hydrophobic residues (Leu, Ile) to smaller ones (Val) for one of the designs. Surprisingly, even after 10 Leu and Ile residues were mutated to Val, this mutant with the core mostly filled with Val was found to not be in a molten globule state and fold into the same backbone structure as the original design, with high stability. These results indicate the importance of local backbone structures to the folding ability and high thermal stability of designed proteins and suggest a method for engineering thermally stabilized natural proteins.

Three-dimensional analysis reveals a high incidence of lung adenocarcinoma in the upper region.

PURPOSE: The lung is a unique organ with a ventilation-perfusion mismatch, which can cause inhomogeneous incidence rates of lung cancer depending on the location in the lung. We aimed to evaluate the incidence of lung adenocarcinoma in each lobe by analyzing the incidence per unit volume, to evaluate the incidence without being affected by differences in the size of each lobe or in the size of the lungs between individuals. METHODS: The number of adenocarcinomas in each lobe was counted. Lung volumes were measured using a three-dimensional computer workstation. The tumor incidence per unit volume was analyzed based on the number of tumors in each lobe. RESULTS: The number of tumors per unit volume was 0.467 in the right upper lobe (RUL), 0.182 in the right middle lobe, 0.209 in the right lower lobe, 0.306 in the left upper segment (LUS), 0.083 in the left lingular segment, and 0.169 in the left lower lobe. The tumor incidence rate of RUL + LUS was 2.269 times that of the other lobes, a value that was significantly higher when using the bootstrap method (p < 0.001). CONCLUSIONS: The incidence of adenocarcinoma per unit volume in both upper lobes was higher than that in other lobes.

Increase of Micro-vessels Beneath the Pleural Surface on CT Predicts Pleural Adhesions.

BACKGROUND: Pleural adhesions are often troublesome in lung surgeries. In some dense pleural adhesions, blood vessels between lung and chest wall (BVLC) are found during surgery. Theoretically, BVLC would increase the amount of blood flow just below the visceral pleura and could allow blood vessels beneath the pleural surface to be clearly visualized on CT. In this study, we investigated whether it was possible to identify the typical CT findings of cases with BVLC. METHODS: Medical records and imaging findings of 186 patients who underwent surgery for lung tumors in our institution were retrospectively reviewed. RESULTS: BVLC was found in 56 patients, of whom 44 (79%) had findings on preoperative CT that indicated increase of micro-vessels just below pleura. In the 21 patients with BVLC of ≥1 mm vessel diameter, the same CT findings were recognized in 19 cases (90%). CONCLUSION: Our results indicate that the CT finding of increased micro-vessels just below pleura has the potential to be used as a novel predictor of pleural adhesion.

Transcription Factor MAFB as a Prognostic Biomarker for the Lung Adenocarcinoma.

MAFB is a basic leucine zipper (bZIP) transcription factor specifically expressed in macrophages. We have previously identified MAFB as a candidate marker for tumor-associated macrophages (TAMs) in human and mouse models. Here, we analyzed single-cell sequencing data of patients with lung adenocarcinoma obtained from the GEO database (GSE131907). Analyzed data showed that general macrophage marker CD68 and macrophage scavenger receptor 1 (CD204) were expressed in TAM and lung tissue macrophage clusters, while transcription factor MAFB was expressed specifically in TAM clusters. Clinical records of 120 patients with lung adenocarcinoma stage I (n = 57), II (n = 21), and III (n = 42) were retrieved from Tsukuba Human Tissue Biobank Center (THB) in the University of Tsukuba Hospital, Japan. Tumor tissues from these patients were extracted and stained with anti-human MAFB antibody, and then MAFB-positive cells relative to the tissue area (MAFB+ cells/tissue area) were morphometrically quantified. Our results indicated that higher numbers of MAFB+ cells significantly correlated to increased local lymph node metastasis (nodal involvement), high recurrence rate, poor pathological stage, increased lymphatic permeation, higher vascular invasion, and pleural infiltration. Moreover, increased amounts of MAFB+ cells were related to poor overall survival and disease-free survival, especially in smokers. These data indicate that MAFB may be a suitable prognostic biomarker for smoker lung cancer patients.

Assessment of prediction methods for protein structures determined by NMR in CASP14: Impact of AlphaFold2.

NMR studies can provide unique information about protein conformations in solution. In CASP14, three reference structures provided by solution NMR methods were available (T1027, T1029, and T1055), as well as a fourth data set of NMR-derived contacts for an integral membrane protein (T1088). For the three targets with NMR-based structures, the best prediction results ranged from very good (GDT_TS = 0.90, for T1055) to poor (GDT_TS = 0.47, for T1029). We explored the basis of these results by comparing all CASP14 prediction models against experimental NMR data. For T1027, NMR data reveal extensive internal dynamics, presenting a unique challenge for protein structure prediction methods. The analysis of T1029 motivated exploration of a novel method of "inverse structure determination," in which an AlphaFold2 model was used to guide NMR data analysis. NMR data provided to CASP predictor groups for target T1088, a 238-residue integral membrane porin, was also used to assess several NMR-assisted prediction methods. Most groups involved in this exercise generated similar beta-barrel models, with good agreement with the experimental data. However, as was also observed in CASP13, some pure prediction groups that did not use any NMR data generated models for T1088 that better fit the NMR data than the models generated using these experimental data. These results demonstrate the remarkable power of modern methods to predict structures of proteins with accuracies rivaling solution NMR structures, and that it is now possible to reliably use prediction models to guide and complement experimental NMR data analysis.

Sensitivity-Enhanced Solid-State NMR Detection of Structural Differences and Unique Polymorphs in Pico- to Nanomolar Amounts of Brain-Derived and Synthetic 42-Residue Amyloid-ß Fibrils.

Amyloid-ß (Aß) fibrils in neuritic plaques are a hallmark of Alzheimer's disease (AD). Since the 42-residue Aß (Aß42) fibril is the most pathogenic among different Aß species, its structural characterization is crucial to our understanding of AD. While several polymorphs have been reported for Aß40, previous studies of Aß42 fibrils prepared at neutral pH detected essentially only one structure, with an S-shaped ß-sheet arrangement (e.g., Xiao et al. Nat. Struct. Mol. Biol. 2015, 22, 499). Herein, we demonstrate the feasibility of characterizing the structure of trace amounts of brain-derived and synthetic amyloid fibrils by sensitivity-enhanced 1H-detected solid-state NMR (SSNMR) under ultrafast magic angle spinning. By taking advantage of the high sensitivity of this technique, we first demonstrate its applicability for the high-throughput screening of trace amounts of selectively 13C- and 15N-labeled Aß42 fibril prepared with ∼0.01% patient-derived amyloid (ca. 4 pmol) as a seed. The comparison of 2D 13C/1H SSNMR data revealed marked structural differences between AD-derived Aß42 (∼40 nmol or ∼200 µg) and synthetic fibrils in less than 10 min, confirming the feasibility of assessing the fibril structure from ∼1 pmol of brain amyloid seed in ∼2.5 h. We also present the first structural characterization of synthetic fully protonated Aß42 fibril by 1H-detected 3D and 4D SSNMR. With procedures assisted by automated assignments, main-chain resonance assignments were completed for trace amounts (∼42 nmol) of a fully protonated amyloid fibril in the 1H-detection approach. The results suggest that this Aß42 fibril exhibits a novel fold or polymorph structure.

Consolidation volume and integration of computed tomography values on three-dimensional computed tomography may predict pathological invasiveness in early lung adenocarcinoma.

PURPOSE: To investigate the relationship between three-dimensional computed tomography (3D-CT) findings and pathological invasiveness in lung adenocarcinoma. METHODS: We retrospectively evaluated 95 patients who underwent surgical resection of lung adenocarcinoma of ≤ 20 mm. The diameters, volumes, and CT values of tumor consolidation were analyzed. We defined the modified CT value by setting air as 0 and water as 1000 and assumed a correlation with pathological invasiveness. Pre-invasive lesions and minimally invasive adenocarcinomas were classified as non-invasive adenocarcinoma. We compared the clinico-radiological features with pathological invasiveness. Receiver operator characteristic (ROC) curves and recurrence-free survival curves were constructed. RESULTS: Twenty-six non-invasive adenocarcinomas and 69 invasive adenocarcinomas were evaluated. The multivariate analysis revealed that the consolidation volume and the integration of modified CT values were the most important predictors of pathological invasion. The area under the ROC curve and the cut-off values of the consolidation volume were 0.868 and 75 mm3, respectively. The area under the ROC curve and the cut-off values of the integration of modified CT values were 0.871 and 80,000, respectively. There was no recurrence in cases with values below the cut-off across all parameters. CONCLUSION: The consolidation volume and integration of modified CT values were shown to be highly predictive of pathological invasiveness.

[Influence of Pleural Adhesions on Thoracoscopic Surgeries for Malignant Lung Tumors].

In the present study, influences of pleural adhesions on thoracoscopic lung surgeries were investigated. A total of 666 consecutive patients who had undergone thoracoscopic surgeries for lung malignant tumors were retrospectively analyzed. Pleural adhesions were present intraoperatively in 289 cases, of which 6 required conversion to thoracotomy due to the adhesions. The influences of pleural adhesions on the perioperative period were comparatively large under following conditions (level-A); the adhesion-type was tight which meant lung and pleural wall sticked closely even if lung collapse was encouraged, the strength was middle( required sharp-dissection) or strong( hard to dissect between visceral and parietal pleura), and the range was more than 10% of total pleural surface. Significant influences of the level-A of pleural adhesions were as follows;prolonged operation time in all procedures, frequent intraoperative lung fistula and prolonged pleural drainage period in wedge resections, and increased blood loss, intraoperative and postoperative lung fistula with prolonged pleural drainage time and postoperative hospitalization period in lobectomy. Other postoperative complications (pneumonia, empyema, exacerbation of interstitial pneumonitis, and arrhythmias) were not associated with pleural adhesions. Careful dissection procedure for pleural adhesions that minimize damage of visceral pleura would be the most important.

Factors related to physical and mental components of quality of life in the community-dwelling frail older persons.

[Purpose] The aim of this study was to investigate factors associated with changes in both the physical and mental components of quality of life (QOL) in of community-dwelling frail older persons in long-term care and to clarify which aspects are important to maintaining physical and mental components of QOL. [Participants and Methods] In this 1 year follow-up cohort study, participants were older persons from a single day care rehabilitation center in Japan. The Medical Outcome Study 8-Item Short-Form Health Survey (MOS-SF8), which gives both physical component summary (PCS) and mental component summary (MCS) scores, was used as the main QOL assessment. Participants were divided according to their level of QOL maintenance according to changes in PCS and MCS scores over the study period, and the variables were compared between the groups. [Results] PCS domain was significantly associated with forced vital capacity and the MCS domain was significantly associated with the Geriatric Depression Scale and Dysphagia Risk Assessment for the Community-Dwelling Elderly Test. [Conclusion] Depression, reduced pulmonary function, and reduced deglutition ability were independently related to low QOL. Assessment of these factors could be beneficial for maintaining the physical and mental components of QOL in community-dwelling frail older persons in long-term care.

Noise peak filtering in multi-dimensional NMR spectra using convolutional neural networks.

Motivation: Multi-dimensional NMR spectra are generally used for NMR signal assignment and structure analysis. There are several programs that can achieve highly automated NMR signal assignments and structure analysis. On the other hand, NMR spectra tend to have a large number of noise peaks even for data acquired with good sample and machine conditions, and it is still difficult to eliminate these noise peaks. Results: We have developed a method to eliminate noise peaks using convolutional neural networks, implemented in the program package Filt_Robot. The filtering accuracy of Filt_Robot was around 90-95% when applied to 2D and 3D NMR spectra, and the numbers of resulting non-noise peaks were close to those in corresponding manually prepared peaks lists. The filtering can strongly enhance automated NMR spectra analysis. Availability and implementation: The full package of the program, documents and example data are available from http://bmrbdep.pdbj.org/en/nmr_tool_box/Filt_Robot.html. Supplementary information: Supplementary data are available at Bioinformatics online.

Clickable gold nanoparticles for streamlining capture, enrichment and release of alkyne-labelled proteins.

Alkyne-labelled proteins are generated as key intermediates in the chemical probe-based approaches to proteomics analysis. Their efficient and selective detection and isolation is an important problem. We designed and synthesized azide-functionalized gold nanoparticles as new clickable capture reagents to streamline click chemistry-mediated capture, enrichment and release of the alkyne-labelled proteins in one-pot to expedite the post-labelling analysis. Because hydrophobic surface functionalities are known to render gold nanoparticles poorly water-dispersible, hydrophilic PEG linkers with two different lengths were explored to confer colloidal stability to the clickable capture reagents. We demonstrated the ability of the capture reagents to conjugate the alkyne containing proteins at a nanomolar concentration via click chemistry, which can be immediately followed by their enrichment and elution. Furthermore, a bifunctional clickable capture reagent bearing sulforhodamine and azide groups was shown to conveniently attach a fluorophore to the alkyne-labelled protein upon click capture, which facilitated their rapid detection in the gel analysis.

How Does the Recently Discovered Peptide MIP Exhibit Much Higher Binding Affinity than an Anticancer Protein p53 for an Oncoprotein MDM2?

An oncoprotein MDM2 binds to the extreme N-terminal peptide region of a tumor suppressor protein p53 (p53NTD) and inhibits its anticancer activity. We recently discovered a peptide named MIP which exhibits much higher binding affinity for MDM2 than p53NTD. Experiments showed that the binding free energy (BFE) of MDM2-MIP is lower than that of MDM2-p53NTD by approximately -4 kcal/mol. Here, we develop a theoretical method which is successful in reproducing this quantitative difference and elucidating its physical origins. It enables us to decompose the BFE into a variety of energetic and entropic components, evaluate their relative magnitudes, and identify the physical factors driving or opposing the binding. It should be applicable also to the assessment of differences among ligands in the binding affinity for a particular receptor, which is a central issue in modern chemistry. In the MDM2 case, the higher affinity of MIP is ascribed to a larger gain of translational, configurational entropy of water upon binding. This result is useful to the design of a peptide possessing even higher affinity for MDM2 as a reliable drug against a cancer.

nmrML: A Community Supported Open Data Standard for the Description, Storage, and Exchange of NMR Data.

NMR is a widely used analytical technique with a growing number of repositories available. As a result, demands for a vendor-agnostic, open data format for long-term archiving of NMR data have emerged with the aim to ease and encourage sharing, comparison, and reuse of NMR data. Here we present nmrML, an open XML-based exchange and storage format for NMR spectral data. The nmrML format is intended to be fully compatible with existing NMR data for chemical, biochemical, and metabolomics experiments. nmrML can capture raw NMR data, spectral data acquisition parameters, and where available spectral metadata, such as chemical structures associated with spectral assignments. The nmrML format is compatible with pure-compound NMR data for reference spectral libraries as well as NMR data from complex biomixtures, i.e., metabolomics experiments. To facilitate format conversions, we provide nmrML converters for Bruker, JEOL and Agilent/Varian vendor formats. In addition, easy-to-use Web-based spectral viewing, processing, and spectral assignment tools that read and write nmrML have been developed. Software libraries and Web services for data validation are available for tool developers and end-users. The nmrML format has already been adopted for capturing and disseminating NMR data for small molecules by several open source data processing tools and metabolomics reference spectral libraries, e.g., serving as storage format for the MetaboLights data repository. The nmrML open access data standard has been endorsed by the Metabolomics Standards Initiative (MSI), and we here encourage user participation and feedback to increase usability and make it a successful standard.

Synthesis of alkyne-tagged and biotin-tagged Sortin1 as novel photoaffinity probes.

Sortin1 is an inhibitor of vesicular biogenesis and transport, which is shared among eukaryotes and plants with an unknown mode of action. Toward exploration of its target proteins, we developed alkyne as well as biotin conjugated photoaffinity probes derived from Sortin1. Due to the presence of phenylketone moiety, Sortin1 was anticipated to serve as a photoreactive group in a similar manner to a commonly used photoreactive group, benzophenone. The core structure based on 5-oxo-1,4-dihydroindenopyridine was constructed in one step using three-component Hantzsch dihydropyridine synthesis. We demonstrated that Sortin1 displayed photocrosslinking reactivity against a model binding protein, which would be useful for capturing and detecting binding proteins.

Cyclophilin A expression and its prognostic significance in lung adenocarcinoma.

Cyclophilin A (CypA) has been reported to be upregulated in malignant tumors. CypA expression is thought to be associated with acquisition of tumor growth and anti-apoptotic function. Although upregulation of CypA has been reported in lung adenocarcinoma, its clinicopathological significance and roles in malignant progression remain unclear. Here we investigated the implications of CypA expression for outcome in patients with lung adenocarcinoma. Lung adenocarcinoma specimens from 198 cases were selected and reclassified according to the World Health Organization classification (4th edition) and the Noguchi classification. CypA expression was assessed by immunohistochemistry, and the H-score was calculated on the basis of intensity and proportion. The specificity of the antibody used was confirmed by Western blotting and the cut-off point was determined from the ROC curve. Sixty-seven cases (33.8%) had low CypA expression (CypA-L group) and 131 (66.2%) had high CypA expression (CypA-H group). Many cases of adenocarcinoma in situ were CypA-L, and advanced adenocarcinomas tended to be classified as CypA-H. Clinically, patients with CypA-H tumors showed a significantly poorer prognosis than those with CypA-L tumors. This is the first investigation of the implications of the CypA expression level in terms of the clinical characteristics of resected lung adenocarcinomas.