Nomogram for predicting survival of patients with diffuse large B-cell lymphoma.

The international prognostic index (IPI) system has been widely used to predict prognosis in diffuse large B-cell lymphoma (DLBCL). However, this system categorizes DLBCL patients into four risk groups, and cannot optimize individualized prognosis. In addition, other clinicopathological factors, such as molecular aberrations, are not incorporated into the system. To partly overcome these weak points, we developed nomograms to predict individual patient survival. We also incorporated MYD88L265P and CD79BY196 mutations into the nomograms since these mutations are associated with a worse prognosis and their signaling pathways have been highlighted as a therapeutic target. We analyzed 302 DLBCL cases for which multivariate analysis by Cox proportional hazard regression was performed. Nomograms for progression-free survival (PFS) and overall survival (OS) were constructed and assessed by a concordance index (C-index). The nomograms were also evaluated using an open external dataset (n = 187). The MYD88L265P and/or CD79BY196 (MYD88/CD79B) mutation was detected in 62/302 patients. The nomograms incorporating IPI factors exhibited a C-index of 0.738 for PFS and a C-index of 0.765 for OS. The nomograms incorporating IPI factors and the MYD88/CD79B mutation showed a C-index of 0.745 for PFS and a C-index of 0.769 for OS. The nomograms we created were evaluated using an external dataset and were well validated. The present nomograms incorporating IPI factors and the MYD88/CD79B mutation have sufficient discrimination ability, and may effectively predict prognosis in DLBCL patients. The prognostic models we have presented here may help clinicians personalize prognostic assessments and clinical decisions.

Autopsy of Drug-induced Lung Injury with Atypical Diffuse Alveolar Disorder due to Amiodarone: A Case Report.

Amiodarone, a prominent antiarrhythmic drug, may cause lung injury. We herein report the case of an 87-year-old man who had been taking amiodarone for 5 years and was subsequently referred due to respiratory failure. Chest computed tomography revealed multiple consolidations with air bronchograms in both lungs. Despite administering steroid pulse therapy, his respiratory failure worsened, and he died 3 days later. Autopsy revealed hyaline membrane formation and organic formation with fibrin deposition. Drug-induced lung injury caused by amiodarone was confirmed by autopsy. Caution is therefore required when fibrin deposition in the alveolar spaces is observed in such cases, which are prone to suffer a rapid deterioration.

Dissection of the IDA promoter identifies WRKY transcription factors as abscission regulators in Arabidopsis.

Plants shed organs such as leaves, petals, or fruits through the process of abscission. Monitoring cues such as age, resource availability, and biotic and abiotic stresses allow plants to abscise organs in a timely manner. How these signals are integrated into the molecular pathways that drive abscission is largely unknown. The INFLORESCENCE DEFICIENT IN ABSCISSION (IDA) gene is one of the main drivers of floral organ abscission in Arabidopsis and is known to transcriptionally respond to most abscission-regulating cues. By interrogating the IDA promoter in silico and in vitro, we identified transcription factors that could potentially modulate IDA expression. We probed the importance of ERF- and WRKY-binding sites for IDA expression during floral organ abscission, with WRKYs being of special relevance to mediate IDA up-regulation in response to biotic stress in tissues destined for separation. We further characterized WRKY57 as a positive regulator of IDA and IDA-like gene expression in abscission zones. Our findings highlight the promise of promoter element-targeted approaches to modulate the responsiveness of the IDA signaling pathway to harness controlled abscission timing for improved crop productivity.

Pathogenicity of enterotoxigenic Escherichia coli in Caenorhabditis elegans as an alternative model host.

Enterotoxigenic Escherichia coli (ETEC), one of the diarrheagenic E. coli, is the most common cause of diarrhea in developing country and in travelers to those areas. In this study, Caenorhabditis elegans was used as an alternative model host to evaluate ETEC infections. The ETEC strain ETEC1, which was isolated from a patient with diarrhea, possessed enterotoxins STh, LT1, and EAST1 and colonization factors CS2 and CS3. Live ETEC1 shortened the life span and body size of C. elegans in association with increased expression of enterotoxin genes and intestinal colonization. In contrast, heat-killed ETEC1 did not affect the life span of C. elegans. Caenorhabditis elegans infected with ETEC1 showed upregulated expression of genes related to insulin-like peptides and host defense responses. These results suggest that ETEC1 exhibits pathogenicity through intestinal colonization and enterotoxin production in C. elegans. This system is useful as an ETEC infection model.

Comparing Supervised Learning and Rigorous Approach for Predicting Protein Stability upon Point Mutations in Difficult Targets.

Accurate prediction of protein stability upon a point mutation has important applications in drug discovery and personalized medicine. It remains a challenging issue in computational biology. Existing computational prediction methods, which range from mechanistic to supervised learning approaches, have experienced limited progress over the last few decades. This stagnation is largely due to their heavy reliance on both the quantity and quality of the training data. This is evident in recent state-of-the-art methods that continue to yield substantial errors on two challenging blind test sets: frataxin and p53, with average root-mean-square errors exceeding 3 and 1.5 kcal/mol, respectively, which is still above the theoretical 1 kcal/mol prediction barrier. Rigorous approaches, on the other hand, offer greater potential for accuracy without relying on training data but are computationally demanding and require both wild-type and mutant structure information. Although they showed high accuracy for conserving mutations, their performance is still limited for charge-changing mutation cases. This might be due to the lack of an available mutant structure, often represented by a simplified capped peptide. The recent advances in protein structure prediction methods now make it possible to obtain structures comparable to experimental ones, including complete mutant structure information. In this work, we compare the performance of supervised learning-based methods and rigorous approaches for predicting protein stability on point mutations in difficult targets: frataxin and p53. The rigorous alchemical method significantly surpasses state-of-the-art techniques in terms of both the root-mean-squared error and Pearson correlation coefficient in these two challenging blind test sets. Additionally, we propose an improved alchemical method that employs the pmx double-system/single-box approach to accurately predict the folding free energy change upon both conserving and charge-changing mutations. The enhanced protocol can accurately predict both types of mutations, thereby outperforming existing state-of-the-art methods in overall performance.

Chromatin Profiling of CBFA2T3-GLIS2 AMLs Identifies Key Transcription Factor Dependencies and BRG1 Inhibition as a Novel Therapeutic Strategy.

Oncogenic fusions involving transcription factors are present in the majority of pediatric leukemias; however, the context-specific mechanisms they employ to drive cancer remain poorly understood. CBFA2T3-GLIS2 (C/G) fusions occur in treatment-refractory acute myeloid leukemias and are restricted to young children. To understand how the C/G fusion drives oncogenesis we applied CUT&RUN chromatin profiling to an umbilical cord blood/endothelial cell (EC) co-culture model of C/G AML that recapitulates the biology of this malignancy. We find C/G fusion binding is mediated by its zinc finger domains. Integration of fusion binding sites in C/G- transduced cells with Polycomb Repressive Complex 2 (PRC2) sites in control cord blood cells identifies MYCN, ZFPM1, ZBTB16 and LMO2 as direct C/G targets. Transcriptomic analysis of a large pediatric AML cohort shows that these genes are upregulated in C/G patient samples. Single cell RNA-sequencing of umbilical cord blood identifies a population of megakaryocyte precursors that already express many of these genes despite lacking the fusion. By integrating CUT&RUN data with CRISPR dependency screens we identify BRG1/SMARCA4 as a vulnerability in C/G AML. BRG1 profiling in C/G patient-derived cell lines shows that the CBFA2T3 locus is a binding site, and treatment with clinically-available BRG1 inhibitors reduces fusion levels and downstream C/G targets including N-MYC, resulting in C/G leukemia cell death and extending survival in a murine xenograft model.

Effects of anesthetics on nociceptive sensory evoked potentials by intraepidermal noxious electrical stimulation of A-δ fibers.

PURPOSE: Generation of nociceptive sensory evoked potentials (NEPs) by selective stimulation of nociceptive intraepidermal nerve fibers is a simple technique which could be used as intraoperative nociception monitor. We evaluated the effects of remifentanil, propofol and sevoflurane on NEPs by this technique. METHODS: Patients undergoing general anesthesia were assigned to groups in two studies. A-δ fiber selective NEPs were recorded. Study 1: NEPs were recorded at control, under anesthetics administration: remifentanil at an effect-site concentration (Ce) of 1.0 ng/mL (n = 10), propofol at Ce of 0.5 µg/mL (n = 10), or sevoflurane at 0.2 minimum alveolar concentration (MAC) (n = 10), and recovery from the anesthetics. Study 2: NEPs were recorded at control and under administration of higher dose anesthetics: propofol at Ce of 0.5 and 1.0 µg/mL (n = 10) or sevoflurane at 0.2 and 0.5 MAC (n = 10). A P-value < 0.016 was considered statistically significant in multiple analyses. RESULTS: Study 1: Remifentanil at Ce of 1.0 ng/mL significantly suppressed the amplitude of NEPs (mean amplitude (standard deviation) of control vs. remifentanil administration: 16.8 µV (3.8) vs. 10.1 µV (2.5), P < 0.001). Propofol and sevoflurane did not suppress the amplitude significantly. Study 2: Propofol at Ce of 0.5 and 1.0 µg/mL and sevoflurane at 0.2 and 0.5 MAC did not suppress the amplitude significantly. CONCLUSION: The amplitude of A-δ fiber selective NEPs was suppressed by remifentanil but not propofol or sevoflurane. NEPs with intraepidermal electrical stimulation can assess the analgesic effect of anesthetics. CLINICAL TRIAL NUMBER: UMIN000038214 REGISTRY URL: https://center6.umin.ac.jp/cgi-open-bin/ctr_e/ctr_view.cgi?recptno=R000043328.

Ninety-day mortality of extremely elderly patients undergoing hip fracture surgery and its association with preoperative cardiac function: a single-center retrospective study.

PURPOSE: We investigated the 90-day mortality rate in elderly patients who underwent hip fracture surgery and the association of preoperative cardiac function with mortality. METHODS: We retrospectively enrolled 133 consecutive patients aged 80 years or older who underwent hip fracture surgery. We obtained information for patient sex, age, comorbidities, medications, anesthesia method, left ventricular systolic and diastolic functions assessed by echocardiography, and preoperative brain natriuretic peptide (BNP) levels. Multivariate logistic regression analysis was performed. RESULTS: The 90-day mortality rate in patients with a mean age of 88.9 years was 7.5% (10/133). More than half of the patients had diastolic dysfunction of the left ventricle. There were no significant differences in preoperative cardiac systolic and diastolic functions between the mortality group and non-mortality group. The preoperative BNP level in the mortality group was significantly higher than that in the non-mortality group (p = 0.038). Preoperative BNP level was not an independent risk factor for 90-day mortality (p = 0.081) in the primary multivariate logistic regression analysis but was an independent risk factor (p = 0.039) with an odds ratio of 1.004 (95% CI 1.000-1.008) in the sensitivity analysis with different explanatory variables. CONCLUSION: The 90-day mortality rate in patients over 80 years old after hip fracture surgery was 7.5%. There were no significant differences in preoperative cardiac function assessed by echocardiography between the mortality and non-mortality groups. Our results suggest that there is no association or only a weak association of high BNP level with 90-day mortality in this age population.

Discovery of a Hidden Trypanosoma cruzi Spermidine Synthase Binding Site and Inhibitors through In Silico, In Vitro, and X-ray Crystallography.

In drug discovery research, the selection of promising binding sites and understanding the binding mode of compounds are crucial fundamental studies. The current understanding of the proteins-ligand binding model extends beyond the simple lock and key model to include the induced-fit model, which alters the conformation to match the shape of the ligand, and the pre-existing equilibrium model, selectively binding structures with high binding affinity from a diverse ensemble of proteins. Although methods for detecting target protein binding sites and virtual screening techniques using docking simulation are well-established, with numerous studies reported, they only consider a very limited number of structures in the diverse ensemble of proteins, as these methods are applied to a single structure. Molecular dynamics (MD) simulation is a method for predicting protein dynamics and can detect potential ensembles of protein binding sites and hidden sites unobservable in a single-point structure. In this study, to demonstrate the utility of virtual screening with protein dynamics, MD simulations were performed on Trypanosoma cruzi spermidine synthase to obtain an ensemble of dominant binding sites with a high probability of existence. The structure of the binding site obtained through MD simulation revealed pockets in addition to the active site that was present in the initial structure. Using the obtained binding site structures, virtual screening of 4.8 million compounds by docking simulation, in vitro assays, and X-ray analysis was conducted, successfully identifying two hit compounds.

Root-knot nematode modulates plant CLE3-CLV1 signaling as a long-distance signal for successful infection.

Plants use many long-distance and systemic signals to modulate growth and development, as well as respond to biotic and abiotic stresses. Parasitic nematodes infect host plant roots and cause severe damage to crop plants. However, the molecular mechanisms that regulate parasitic nematode infections are still unknown. Here, we show that plant parasitic root-knot nematodes (RKNs), Meloidogyne incognita, modulate the host CLAVATA3 (CLV3)/EMBRYO SURROUNDING REGION (CLE)-CLV1 signaling module to promote the infection progression. Plants deficient in the CLE signaling pathway show enhanced RKN resistance, whereas CLE overexpression leads to increased susceptibility toward RKN. Grafting analysis shows that CLV1 expression in the shoot alone is sufficient to positively regulate RKN infection. Together with results from the split-root culture system, infection assays, and CLE3-CLV1 binding assays, we conclude that mobile root-derived CLE signals are perceived by CLV1 in the shoot, which subsequently produce systemic signals to promote gall formation and RKN reproduction.

Helix encoder: a compound-protein interaction prediction model specifically designed for class A GPCRs.

Class A G protein-coupled receptors (GPCRs) represent the largest class of GPCRs. They are essential targets of drug discovery and thus various computational approaches have been applied to predict their ligands. However, there are a large number of orphan receptors in class A GPCRs and it is difficult to use a general protein-specific supervised prediction scheme. Therefore, the compound-protein interaction (CPI) prediction approach has been considered one of the most suitable for class A GPCRs. However, the accuracy of CPI prediction is still insufficient. The current CPI prediction model generally employs the whole protein sequence as the input because it is difficult to identify the important regions in general proteins. In contrast, it is well-known that only a few transmembrane helices of class A GPCRs play a critical role in ligand binding. Therefore, using such domain knowledge, the CPI prediction performance could be improved by developing an encoding method that is specifically designed for this family. In this study, we developed a protein sequence encoder called the Helix encoder, which takes only a protein sequence of transmembrane regions of class A GPCRs as input. The performance evaluation showed that the proposed model achieved a higher prediction accuracy compared to a prediction model using the entire protein sequence. Additionally, our analysis indicated that several extracellular loops are also important for the prediction as mentioned in several biological researches.

Differentiation latency and dormancy signatures define fetal liver HSCs at single cell resolution.

Decoding the gene regulatory mechanisms mediating self-renewal of hematopoietic stem cells (HSCs) during their amplification in the fetal liver (FL) is relevant for advancing therapeutic applications aiming to expand transplantable HSCs, a long-standing challenge. Here, to explore intrinsic and extrinsic regulation of self-renewal in FL-HSCs at the single cell level, we engineered a culture platform designed to recapitulate the FL endothelial niche, which supports the amplification of serially engraftable HSCs ex vivo. Leveraging this platform in combination with single cell index flow cytometry, serial transplantation assays, and single cell RNA-sequencing, we elucidated previously unrecognized heterogeneity in immunophenotypically defined FL-HSCs and demonstrated that differentiation latency and transcriptional signatures of biosynthetic dormancy are distinguishing properties of self-renewing FL-HSCs with capacity for serial, long-term multilineage hematopoietic reconstitution. Altogether, our findings provide key insights into HSC expansion and generate a novel resource for future exploration of the intrinsic and niche-derived signaling pathways that support FL-HSC self-renewal.

The hepatic niche leads to aggressive natural killer cell leukemia proliferation through the transferrin-transferrin receptor 1 axis.

Aggressive natural killer cell leukemia (ANKL) is a rare lymphoid neoplasm frequently associated with Epstein-Barr virus, with a disastrously poor prognosis. Owing to the lack of samples from patients with ANKL and relevant murine models, comprehensive investigation of its pathogenesis including the tumor microenvironment (TME) has been hindered. Here we established 3 xenograft mice derived from patients with ANKL (PDXs), which enabled extensive analysis of tumor cells and their TME. ANKL cells primarily engrafted and proliferated in the hepatic sinusoid. Hepatic ANKL cells were characterized by an enriched Myc-pathway and proliferated faster than those in other organs. Interactome analyses and in vivo CRISPR-Cas9 analyses revealed transferrin (Tf)-transferrin receptor 1 (TfR1) axis as a potential molecular interaction between the liver and ANKL. ANKL cells were rather vulnerable to iron deprivation. PPMX-T003, a humanized anti-TfR1 monoclonal antibody, showed remarkable therapeutic efficacy in a preclinical setting using ANKL-PDXs. These findings indicate that the liver, a noncanonical hematopoietic organ in adults, serves as a principal niche for ANKL and the inhibition of the Tf-TfR1 axis is a promising therapeutic strategy for ANKL.

Inflammation and subsequent nociceptor sensitization in the bone marrow are involved in an animal model of osteoarthritis pain.

AIMS: This study aimed to determine whether pathological changes in the bone marrow cause Osteoarthritis (OA) pain based on magnetic resonance imaging (MRI), immunohistochemistry, and electrophysiology. MAIN METHODS: Adjuvant-induced arthritis (AIA) was achieved by injecting 150 µL of complete Freund's adjuvant into the right knee joints of male Sprague-Dawley rats. AIA rats were compared with saline-injected rats. KEY FINDINGS: AIA significantly induced mechanical hyperalgesia and spontaneous pain in the right hind paw 1-14 days after induction. Intratibial injection of 50 µL of 1 % lidocaine significantly suppressed AIA-induced mechanical hyperalgesia (p = 0.0001) and spontaneous pain (p = 0.0006) 3 days after induction. In T2-weighted MRI, AIA induced high-signal intensity within the proximal tibial metaphysis, and the mean T2 values in this area significantly increased on days 3 (p = 0.0043) and 14 (p = 0.0012) after induction. AIA induced intraosseous edema and significantly increased the number of intraosseous granulocytes on days 3 (p < 0.0001) and 14 (p < 0.0001) after induction. The electrophysiological study on days 3-7 after induction showed significantly increased spontaneous firing rates (p = 0.0166) and evoked responses to cutaneous stimuli (brush, p < 0.0001; pinching, p = 0.0359) in the right hind paw plantar surface and intratibial stimuli (p = 0.0002) in wide-dynamic-range neurons of the spinal dorsal horn. SIGNIFICANCE: Intraosseous changes caused by OA induce hypersensitivity in the sensory afferents innervating bone marrow may be involved in OA pain. Novel bone marrow-targeted therapies could be beneficial for treating OA pain.

The Efficacy and Safety of Immune Checkpoint Inhibitor and Tyrosine Kinase Inhibitor Combination Therapy for Advanced or Metastatic Renal Cell Carcinoma: A Multicenter Retrospective Real-World Cohort Study.

We evaluated the efficacy and safety of combination therapy with immune checkpoint inhibitors (ICIs) and tyrosine kinase inhibitors (TKI) as first-line therapy for patients diagnosed as having advanced or metastatic renal cell carcinoma (mRCC). We enrolled 51 patients to receive ICI+TKI therapy for mRCC at 9 Japanese institutions. The overall survival (OS) of the patients treated with ICI+TKI was the primary endpoint., and the secondary endpoints were progression-free survival (PFS), objective response rate (ORR), and disease control rate (DCR). Furthermore, we analyzed the clinical prognostic and predictive factors in patients with mRCC treated with ICI+TKI therapy. Seven months was the median follow-up period. The OS rates at 6, 12, and 18 months were 93.1, 82.5, and 68.8%, respectively. The median PFS for patients who received ICI+TKI was 19.0 months, ORR was 68.6%, and DCR was 88.2%. ICI+TKI-related adverse events occurred in 43 patients (84.3%) with any grade and in 22 patients (43.1%) with grade ≥3. Treatment selection with poor prognostic factors may be prudent, even though ICI+TKI is an efficacious and safe first-line treatment in patients with mRCC.

Refractory Eosinophilic Granulomatosis with Polyangiitis Complicated with IgG4-related Disease Showing Different Treatment Responses for Each Organ.

We herein report a 75-year-old woman who presented with dyspnea and purpura. She was diagnosed with eosinophilic granulomatosis with polyangiitis (EGPA) based on axonal damage observed in the left tibial nerve and skin and lung pathologies. Lung pathology showed IgG4-positive plasma cells, considered a complication of IgG4-related disease (IgG4-RD). Computed tomography revealed thickening of the abdominal aorta and a poor contrast area in the left kidney, which was indicative of IgG4-RD. Steroid administration improved the IgG4-RD. However, the EGPA resisted treatment; therefore, immunosuppressive drugs and mepolizumab were administered. Refractory EGPA complicated with IgG4-RD showed different treatment responses for each organ.

CLE3 and its homologs share overlapping functions in the modulation of lateral root formation through CLV1 and BAM1 in Arabidopsis thaliana.

Lateral roots are important for a wide range of processes, including uptake of water and nutrients. The CLAVATA3 (CLV3)/EMBRYO SURROUNDING REGION-RELATED (CLE) 1 ~ 7 peptide family and their cognate receptor CLV1 have been shown to negatively regulate lateral root formation under low-nitrate conditions. However, little is known about how CLE signaling regulates lateral root formation. A persistent obstacle in CLE peptide research is their functional redundancies, which makes functional analyses difficult. To address this problem, we generate the cle1 ~ 7 septuple mutant (cle1 ~ 7-cr1, cr stands for mutant allele generated with CRISPR/Cas9). cle1 ~ 7-cr1 exhibits longer lateral roots under normal conditions. Specifically, in cle1 ~ 7-cr1, the lateral root density is increased, and lateral root primordia initiation is found to be accelerated. Further analysis shows that cle3 single mutant exhibits slightly longer lateral roots. On the other hand, plants that overexpress CLE2 and CLE3 exhibit decreased lateral root lengths. To explore cognate receptor(s) of CLE2 and CLE3, we analyze lateral root lengths in clv1 barely any meristem 1(bam1) double mutant. Mutating both the CLV1 and BAM1 causes longer lateral roots, but not in each single mutant. In addition, genetic analysis reveals that CLV1 and BAM1 are epistatic to CLE2 and CLE3. Furthermore, gene expression analysis shows that the LATERAL ORGAN BOUNDARIES DOMAIN/ASYMMETRIC LEAVES2-LIKE (LBD/ASL) genes, which promote lateral root formation, are upregulated in cle1 ~ 7-cr1 and clv1 bam1. We therefore propose that CLE2 and CLE3 peptides are perceived by CLV1 and BAM1 to mediate lateral root formation through LBDs regulation.

The impact of preclinical clerkship in general surgery on medical students' attitude to a surgical career.

PURPOSE: With the advent of a new program for postgraduate medical students in 2004, the number of applicants choosing surgical careers in Japan has been declining. We conducted this study to evaluate the impact of preclinical clerkship and how it affects students' attitudes toward a surgical career. METHODS: The subjects of our study were fifth-year medical students who participated in a clinical clerkship in general surgery in our department between April 2021 and March 2022. We conducted pre- and post-preclinical clerkship surveys to assess the perceived image of surgeons and the impact of clerkship on surgical career interest. RESULTS: Among 132 medical students (77 men and 55 women) who rotated through preclinical clerkship in our department, 125 participated in the survey and 66% expressed interest in a surgical career. In the post-clerkship survey, an increased interest in a surgical career was expressed by 79% of the students; notably, including those who initially expressed interest. Approximately 77% of students were satisfied with the practical skill training they received. CONCLUSION: Engaging medical students early in surgical experience through a preclinical clerkship for general surgery appears to promote their interest in a surgical career.