Generation of antibodies to an extracellular region of the transporters Glut1/Glut4 by immunization with a designed antigen.

Monoclonal antibodies are one of the fastest growing class of drugs. Nevertheless, relatively few biologics target multispanning membrane proteins because of technical challenges. To target relatively small extracellular regions of multiple membrane-spanning proteins, synthetic peptides, which are composed of amino acids corresponding to an extracellular region of a membrane protein, are often utilized in antibody discovery. However, antibodies to these peptides often do not recognize parental membrane proteins. In this study, we designed fusion proteins in which an extracellular helix of the membrane protein glucose transporter 1 (Glut1) was grafted onto the scaffold protein Adhiron. In the initial design, the grafted fragment did not form a helical conformation. Molecular dynamics simulations of full-length Glut1 suggested the importance of intramolecular interactions formed by surrounding residues in the formation of the helical conformation. A fusion protein designed to maintain such intramolecular interactions did form the desired helical conformation in the grafted region. We then immunized an alpaca with the designed fusion protein and obtained VHH (variable region of heavy-chain antibodies) using the phage display method. The binding of these VHH antibodies to the recombinant Glut1 protein was evaluated by surface plasmon resonance, and their binding to Glut1 on the cell membrane was further validated by flow cytometry. Furthermore, we also succeeded in the generation of a VHH against another integral membrane protein, glucose transporter 4 (Glut4) with the same strategy. These illustrates that our combined biochemical and computational approach can be applied to designing other novel fusion proteins for generating site-specific antibodies.

Structural basis for cross-group recognition of an influenza virus hemagglutinin antibody that targets postfusion stabilized epitope.

Plasticity of influenza virus hemagglutinin (HA) conformation increases an opportunity to generate conserved non-native epitopes with unknown functionality. Here, we have performed an in-depth analysis of human monoclonal antibodies against a stem-helix region that is occluded in native prefusion yet exposed in postfusion HA. A stem-helix antibody, LAH31, provided IgG Fc-dependent cross-group protection by targeting a stem-helix kinked loop epitope, with a unique structure emerging in the postfusion state. The structural analysis and molecular modeling revealed key contact sites responsible for the epitope specificity and cross-group breadth that relies on somatically mutated light chain. LAH31 was inaccessible to the native prefusion HA expressed on cell surface; however, it bound to the HA structure present on infected cells with functional linkage to the Fc-mediated clearance. Our study uncovers a novel non-native epitope that emerges in the postfusion HA state, highlighting the utility of this epitope for a broadly protective antigen design.

Targeting hemoglobin receptors IsdH and IsdB of Staphylococcus aureus with a single VHH antibody inhibits bacterial growth.

Methicillin-resistant Staphylococcus aureus, or MRSA, is one of the major causative agents of hospital-acquired infections worldwide. Novel antimicrobial strategies efficient against antibiotic-resistant strains are necessary and not only against S. aureus. Among those, strategies that aim at blocking or dismantling proteins involved in the acquisition of essential nutrients, helping the bacteria to colonize the host, are intensively studied. A major route for S. aureus to acquire iron from the host organism is the Isd (iron surface determinant) system. In particular, the hemoglobin receptors IsdH and IsdB located on the surface of the bacterium are necessary to acquire the heme moiety containing iron, making them a plausible antibacterial target. Herein, we obtained an antibody of camelid origin that blocked heme acquisition. We determined that the antibody recognized the heme-binding pocket of both IsdH and IsdB with nanomolar order affinity through its second and third complementary-determining regions. The mechanism explaining the inhibition of acquisition of heme in vitro could be described as a competitive process in which the complementary-determining region 3 from the antibody blocked the acquisition of heme by the bacterial receptor. Moreover, this antibody markedly reduced the growth of three different pathogenic strains of MRSA. Collectively, our results highlight a mechanism for inhibiting nutrient uptake as an antibacterial strategy against MRSA.

Functional insights of Tyr37 in framework region 2 directly contributing to the binding affinities and dissociation kinetics in single-domain VHH antibodies.

Single-domain VHH antibody is regarded as one of the promising antibody classes for therapeutic and diagnostic applications. VHH antibodies have amino acids in framework region 2 that are distinct from those in conventional antibodies, such as the Val37Phe/Tyr (V37F/Y) substitution. Correlations between the residue type at position 37 and the conformation of the CDR3 in VHH antigen recognition have been previously reported. However, few studies focused on the meaning of harboring two residue types in position 37 of VHH antibodies, and the concrete roles of Y37 have been little to be elucidated. Here, we investigated the functional states of position 37 in co-crystal structures and performed analyses of three model antibodies with either F or Y at position 37. Our analysis indicates that Y at position 37 enhances the dissociation rate, which is highly correlated with drug efficacy. Our findings help to explain the molecular mechanisms that distinguish VHH antibodies from conventional antibodies.

Biophysical insight into protein-protein interactions in the Interleukin-11/Interleukin-11Rα/glycoprotein 130 signaling complex.

Interleukin-11 (IL-11) is a member of the interleukin-6 (IL-6) family of cytokines. IL-11 is a regulator of multiple events in hematopoiesis, and IL-11-mediated signaling is implicated in inflammatory disease, cancer, and fibrosis. All IL-6 family cytokines signal through the signal-transducing receptor, glycoprotein 130 (gp130), but these cytokines have distinct as well as overlapping biological functions. To understand IL-11 signaling at the molecular level, we performed a comprehensive interaction analysis of the IL-11 signaling complex, comparing it with the IL-6 complex, one of the best-characterized cytokine complexes. Our thermodynamic analysis revealed a clear difference between IL-11 and IL-6. Surface plasmon resonance analysis showed that the interaction between IL-11 and IL-11 receptor α (IL-11Rα) is entropy driven, whereas that between IL-6 and IL-6 receptor α (IL-6Rα) is enthalpy driven. Our analysis using isothermal titration calorimetry revealed that the binding of gp130 to the IL-11/IL-11Rα complex results in entropy loss, but that the interaction of gp130 with the IL-6/IL-6Rα complex results in entropy gain. Our hydrogen-deuterium exchange mass spectrometry experiments suggested that the D2 domain of gp130 was not involved in IL-6-like interactions in the IL-11/IL-11Rα complex. It has been reported that IL-6 interaction with gp130 in the signaling complex was characterized through the hydrophobic interface located in its D2 domain of gp130. Our findings suggest that unique interactions of the IL-11 signaling complex with gp130 are responsible for the distinct biological activities of IL-11 compared to IL-6.

Mechanism of dimerization and structural features of human LI-cadherin.

Liver intestine (LI)-cadherin is a member of the cadherin superfamily, which encompasses a group of Ca2+-dependent cell-adhesion proteins. The expression of LI-cadherin is observed on various types of cells in the human body, such as normal small intestine and colon cells, and gastric cancer cells. Because its expression is not observed on normal gastric cells, LI-cadherin is a promising target for gastric cancer imaging. However, because the cell adhesion mechanism of LI-cadherin has remained unknown, rational design of therapeutic molecules targeting this cadherin has been hampered. Here, we have studied the homodimerization mechanism of LI-cadherin. We report the crystal structure of the LI-cadherin homodimer containing its first four extracellular cadherin repeats (EC1-4). The EC1-4 homodimer exhibited a unique architecture different from that of other cadherins reported so far, driven by the interactions between EC2 of one protein chain and EC4 of the second protein chain. The crystal structure also revealed that LI-cadherin possesses a noncanonical calcium ion-free linker between the EC2 and EC3 domains. Various biochemical techniques and molecular dynamics simulations were employed to elucidate the mechanism of homodimerization. We also showed that the formation of the homodimer observed in the crystal structure is necessary for LI-cadherin-dependent cell adhesion by performing cell aggregation assays. Taken together, our data provide structural insights necessary to advance the use of LI-cadherin as a target for imaging gastric cancer.

Structural basis for antigen recognition by methylated lysine-specific antibodies.

Proteins are modulated by a variety of posttranslational modifications including methylation. Despite its importance, the majority of protein methylation modifications discovered by mass spectrometric analyses are functionally uncharacterized, partly owing to the difficulty in obtaining reliable methylsite-specific antibodies. To elucidate how functional methylsite-specific antibodies recognize the antigens and lead to the development of a novel method to create such antibodies, we use an immunized library paired with phage display to create rabbit monoclonal antibodies recognizing trimethylated Lys260 of MAP3K2 as a representative substrate. We isolated several methylsite-specific antibodies that contained unique complementarity determining region sequence. We characterized the mode of antigen recognition by each of these antibodies using structural and biophysical analyses, revealing the molecular details, such as binding affinity toward methylated/nonmethylated antigens and structural motif that is responsible for recognition of the methylated lysine residue, by which each antibody recognized the target antigen. In addition, the comparison with the results of Western blotting analysis suggests a critical antigen recognition mode to generate cross-reactivity to protein and peptide antigen of the antibodies. Computational simulations effectively recapitulated our biophysical data, capturing the antibodies of differing affinity and specificity. Our exhaustive characterization provides molecular architectures of functional methylsite-specific antibodies and thus should contribute to the development of a general method to generate functional methylsite-specific antibodies by de novo design.

Residue-based program of a ß-peptoid twisted strand shape via a cyclopentane constraint.

N-Substituted peptides, such as peptoids and ß-peptoids, have been reported to have unique structures with diverse functions, like catalysis and manipulation of biomolecular functions. Recently, the preorganization of monomer shape by restricting bond rotations about all backbone dihedral angles has been demonstrated to be useful for de novo design of peptoid structures. Such design strategies are hitherto unexplored for ß-peptoids; to date, no preorganized ß-peptoid monomers have been reported. Here, we report the first design strategy for ß-peptoids, in which all four backbone dihedral angles (ω, ϕ, θ, ψ) are rotationally restricted on a per-residue basis. The introduction of a cyclopentane constraint realized the preorganized monomer structure and led to a ß-peptoid with a stable twisted strand shape.

Taking antithrombic therapy during emergency laparoscopic cholecystectomy for acute cholecystitis does not affect the postoperative outcomes: a propensity score matched study.

BACKGROUND: Continuing antithrombic therapy (ATT) during surgery increases the risk of bleeding. However, it is difficult to discontinue the ATT in emergency surgery. Therefore, safety of emergency laparoscopic cholecystectomy (LC) for acute cholecystitis (AC) is still unclear. We aimed to clarify the affect of ATT during emergency LC for AC. METHODS: Patients with AC were classified into ATT group (n = 30) and non-ATT group (n = 120). Postoperative outcomes were compared after propensity score matching (n = 22). RESULTS: Higher level of c-reactive protein level and shorter activated partial thromboplastin time (APTT) was observed in ATT group than in non-ATT group after matching. No significant difference was found between other patient characteristics and perioperative results. Blood loss over 100 mL was observed in 8 patients. Multivariate analyze showed that APTT was an independent risk factor for bleeding over 100 mL (P = 0.039), while ACT and APT was not. CONCLUSIONS: Taking ATT does not affect the blood loss or complications during emergency LC for AC. Controlling intraoperative bleeding is essential for a safe postoperative outcome.

Preoperative serum CA19-9 predicts postoperative pancreatic fistula in PDAC patients: retrospective analysis at a single institution.

BACKGROUND: Postoperative pancreatic fistula (POPF) is a critical complication of pancreatectomy in patients with pancreatic ductal adenocarcinoma (PDAC). Recent papers reported that serum carbohydrate antigen (CA)19-9 levels predicted long-term prognosis. We investigated whether preoperative serum CA19-9 levels were associated with POPF in PDAC patients. METHODS: This cohort study was conducted at a single institution retrospectively. Clinicopathologic features were determined using medical records. RESULTS: Among of 196 consecutive patients who underwent pancreatectomy against PDAC, 180 patients whose CA19-9 levels were above the measurement sensitivity, were registered in this study. The patients consisted of 122 patients who underwent pancreaticoduodenectomy and 58 patients who underwent distal pancreatectomy. Several clinicopathological factors, including CA 19-9 level, as well as surgical factors were determined retrospectively based on the medical records. Patients with high CA19-9 levels had a significantly higher incidence of POPF than those with low levels (43.9 vs. 13.0%, P < 0.0001). The receiver operating characteristic curves calculated that the cutoff CA19-9 value to predict POPF was 428 U/mL. CA19-9, BMI, curability, and histology were statistically significant risk factors for POPF by univariate analysis. Multivariate analysis showed that CA19-9 and BMI levels were statistically significant independent risk factors for POPF. CA19-9 levels were correlated with both histology and curability. Disease free survival and overall survival of patients with higher levels of CA19-9 were significantly shorter than that of patients with lower levels of preoperative serum CA19-9. CONCLUSIONS: In patients undergoing pancreatectomy for PDAC, higher preoperative CA19-9 levels are a significant predictor for POPF.

Oligo(N-methylalanine) as a Peptide-Based Molecular Scaffold with a Minimal Structure and High Density of Functionalizable Sites.

Functionalizable synthetic molecules with nanometer sizes and defined shapes in water are useful as molecular scaffolds to mimic the functions of biomacromolecules and develop chemical tools for manipulating biomacromolecules. Herein, we propose oligo(N-methylalanine) (oligo-NMA) as a peptide-based molecular scaffold with a minimal structure and a high density of functionalizable sites. Oligo-NMA forms a defined shape in water without hydrogen-bonding networks or ring constraints, which enables the molecule to act as a scaffold with minimal atomic composition. Furthermore, functional groups can be readily introduced on the nitrogens and α-carbons of oligo-NMA. Computational and NMR spectroscopic analysis suggested that the backbone structure of oligo-NMA is not largely affected by functionalization. Moreover, the usefulness of oligo-NMA was demonstrated by the design of protein ligands. The ease of synthesis, minimal structure, and high functionalization flexibility makes oligo-NMA a useful scaffold for chemical and biological applications.

Anion solvation enhanced by positive supercharging mutations preserves thermal stability of an antibody in a wide pH range.

Proteins function through interactions with other molecules. In protein engineering, scientists often engineer proteins by mutating their amino acid sequences on the protein surface to improve various physicochemical properties. "Supercharging" is a method to design proteins by mutating surface residues with charged amino acids. Previous studies demonstrated that supercharging mutations conferred better thermal resistance, solubility, and cell penetration to proteins. Likewise, antibodies recognize antigens through the antigen-binding site on the surface. The genetic and structural diversity of antibodies leads to high specificity and affinity toward antigens, enabling antibodies to be versatile tools in various applications. When assessing therapeutic antibodies, surface charge is an important factor to consider because the isoelectric point plays a role in protein clearance inside the body. In this study, we explored how supercharging mutations affect physicochemical properties of antibodies. Starting from a crystal structure of an antibody with the net charge of -4, we computationally designed a supercharged variant possessing the net charge of +10. The positive-supercharged antibody exhibited marginal improvement in thermal stability, but the secondary structure and the binding affinity to the antigen (net charge of +8) were preserved. We also used physicochemical measurements and molecular dynamics simulations to analyze the effects of supercharging mutations in sodium phosphate buffer with different pH and ion concentrations, which revealed preferential solvation of phosphate ions to the supercharged surface relative to the wild-type surface. These results suggest that supercharging would be a useful approach to preserving thermal stability of antibodies in a wide range of pH, which may enable further diversification of antibody repertoires beyond natural evolution.

Structures of the prefusion form of measles virus fusion protein in complex with inhibitors.

Measles virus (MeV), a major cause of childhood morbidity and mortality, is highly immunotropic and one of the most contagious pathogens. MeV may establish, albeit rarely, persistent infection in the central nervous system, causing fatal and intractable neurodegenerative diseases such as subacute sclerosing panencephalitis and measles inclusion body encephalitis. Recent studies have suggested that particular substitutions in the MeV fusion (F) protein are involved in the pathogenesis by destabilizing the F protein and endowing it with hyperfusogenicity. Here we show the crystal structures of the prefusion MeV-F alone and in complex with the small compound AS-48 or a fusion inhibitor peptide. Notably, these independently developed inhibitors bind the same hydrophobic pocket located at the region connecting the head and stalk of MeV-F, where a number of substitutions in MeV isolates from neurodegenerative diseases are also localized. Since these inhibitors could suppress membrane fusion mediated by most of the hyperfusogenic MeV-F mutants, the development of more effective inhibitors based on the structures may be warranted to treat MeV-induced neurodegenerative diseases.

Per-Residue Program of Multiple Backbone Dihedral Angles of ß-Peptoids via Backbone Substitutions.

Unique folded structures of natural and synthetic oligomers are the most fundamental basis for their unique functions. N-Substituted ß-peptides, or ß-peptoids, are synthetic oligomers with great potential to fold into diverse three-dimensional structures because of the existence of four rotatable bonds in a monomer with highly modular synthetic accessibility. However, the existence of the four rotatable bonds poses a challenge for conformational control of ß-peptoids. Here, we report a strategy for per-residue programming of two dihedral angles of ß-peptoids, which is useful for restricting the conformational space of the oligomers. The oligomer was found to form a unique loop conformation that is stabilized by the backbone rotational restrictions. Circular dichroism and NMR spectroscopic analyses and X-ray crystallographic analysis of the oligomer are presented. The strategy would significantly facilitate the discovery of many more unique folded structures of ß-peptoids.

Phospholipid Membrane Fluidity Alters Ligand Binding Activity of a G Protein-Coupled Receptor by Shifting the Conformational Equilibrium.

The affinity of a ligand for a receptor on the cell surface will be influenced by the membrane composition. Herein, we evaluated the effects of differences in membrane fluidity, controlled by phospholipid composition, on the ligand binding activity of the G protein-coupled receptor human serotonin 2B. Using Nanodisc technology to control membrane properties, we performed biophysical analysis and employed molecular dynamics simulations to demonstrate that increased membrane fluidity shifted the equilibrium toward an active form of the receptor. Our quantitative study will enable development of more realistic in vitro drug discovery assays involving membrane-bound proteins such as G protein-coupled receptors.

A Peptoid with Extended Shape in Water.

The term "peptoids" was introduced decades ago to describe peptide analogues that exhibit better physicochemical and pharmacokinetic properties than peptides. Oligo(N-substituted glycine) (oligo-NSG) was previously proposed as a peptoid due to its high proteolytic resistance and membrane permeability. However, oligo-NSG is conformationally flexible, and ensuring a defined shape in water is difficult. This conformational flexibility severely limits the biological application of oligo-NSG. Here, we propose oligo(N-substituted alanine) (oligo-NSA) as a peptoid that forms a defined shape in water. The synthetic method established in this study enabled the first isolation and conformational study of optically pure oligo-NSA. Computational simulations, crystallographic studies, and spectroscopic analysis demonstrated the well-defined extended shape of oligo-NSA realized by backbone steric effects. This new class of peptoid achieves the constrained conformation without any assistance of N-substituents and serves as a scaffold for displaying functional groups in well-defined three-dimensional space in water, which leads to effective biomolecular recognition.

An epitope-directed antibody affinity maturation system utilizing mammalian cell survival as readout.

Upon developing therapeutically potent antibodies, there are significant requirements, such as increasing their affinity, regulating their epitope, and using native target antigens. Many antibody selection systems, such as a phage display method, have been developed, but it is still difficult to fulfill these requirements at the same time. Here, we propose a novel epitope-directed antibody affinity maturation system utilizing mammalian cell survival as readout. This system is based on the competition of antibody binding, and can target membrane proteins expressed in a native form on a mammalian cell surface. Using this system, we successfully selected an affinity-matured anti-ErbB2 single-chain variable fragment variant, which had the same epitope as the original one. In addition, the affinity was increased mainly due to the decrease in the dissociation rate. This novel cell-based antibody affinity maturation system could contribute to directly obtaining therapeutically potent antibodies that are functional on the cell surface.

Naples Prognostic Score Is a Useful Prognostic Marker in Patients With Metastatic Colorectal Cancer.

BACKGROUND: Systemic inflammation may influence the response to systemic chemotherapy or the prognosis in patients with various cancers. The Naples prognostic score, based on inflammatory and nutritional statuses, is a useful prognostic marker in patients undergoing surgery for colorectal cancer; however, its significance in patients with metastatic colorectal cancer remains unclear. OBJECTIVE: We aimed to evaluate the prognostic significance of the Naples prognostic factor in patients with metastatic colorectal cancer receiving first-line chemotherapy and to compare its prognostic accuracy with the neutrophil:lymphocyte ratio, platelet:lymphocyte ratio, and the systemic immune-inflammatory index. DESIGN: This was a retrospective study of prospectively collected data. SETTINGS: This study was conducted at a university hospital. PATIENTS: A total of 259 patients received first-line systemic chemotherapy for metastatic colorectal cancer. MAIN OUTCOME MEASURES: The Naples prognostic score was calculated by a composite score of albumin and cholesterol concentrations, lymphocyte:monocyte ratio, and neutrophil:lymphocyte ratio. The patients were divided into 3 groups based on increasing Naples scores (groups 0-2), and the associations of the Naples prognostic score with clinicopathologic features and overall survival were evaluated. RESULTS: Higher Naples prognostic score was positively associated with right-sided primary tumors and synchronous metastases and negatively with primary tumor resection. Patients in group 2 (high Naples prognostic score) had significantly shorter overall survival than those in groups 0 and 1 (p = 0.012 and 0.022). Multivariate Cox regression analysis identified the Naples prognostic score as an independent prognostic factor for overall survival (HR = 1.574; p = 0.004). Time-dependent receiver operating characteristic curve analysis showed that Naples prognostic score was more sensitive than other prognostic factors for predicting overall survival. LIMITATIONS: The main limitations are the sample size, single institutional feature, and treatment heterogeneity. CONCLUSIONS: The Naples prognostic score may be a useful prognostic marker in patients with metastatic colorectal cancer receiving systemic chemotherapy. See Video Abstract at http://links.lww.com/DCR/B72. LA PUNTUACIÓN PRONÓSTICA DE NÁPOLES ES UN MARCADOR PRONÓSTICO ÚTIL EN PACIENTES CON CÁNCER COLORRECTAL METASTÁSICO: La inflamación sistémica puede influir en la respuesta a la quimioterapia sistémica o el pronóstico en pacientes con varios tipos de cáncer. La puntuación pronóstica de Nápoles, basada en estados inflamatorios y nutricionales, es un marcador pronóstico útil en pacientes sometidos a cirugía por cáncer colorrectal; sin embargo, su importancia en pacientes con cáncer colorrectal metastásico sigue siendo incierta.El objetivo fue evaluar la importancia pronóstica del factor pronóstico de Nápoles en pacientes con cáncer colorrectal metastásico que reciben quimioterapia de primera línea y comparar su precisión pronóstica con la relación neutrófilos: linfocitos, plaquetas: linfocitos y el índice sistémico inmune-inflamatorio.Este estudio se realizó en un hospital universitario.Este fue un estudio retrospectivo de datos recolectados prospectivamente.Un total de 259 pacientes recibieron quimioterapia sistémica de primera línea para el cáncer colorrectal metastásico.La puntuación pronóstica de Nápoles se calculó mediante una puntuación compuesta de concentraciones de albúmina y colesterol, proporción de linfocitos: monocitos y proporción de neutrófilos: linfocitos. Los pacientes se dividieron en tres grupos basados en el aumento de las puntuaciones de Nápoles (grupos 0-2, respectivamente) y se evaluaron las asociaciones de la puntuación pronóstica de Nápoles con las características clínico-patológicas y la supervivencia general.La puntuación pronóstica de Nápoles es más alta se asoció positivamente con los tumores primarios del lado derecho y metástasis sincrónicas, y negativamente con la resección del tumor primario. Los pacientes del grupo 2 (alto puntaje pronóstico de Nápoles) tuvieron una supervivencia general significativamente menor que los de los grupos 0 y 1 (p = 0.012 y 0.022, respectivamente). El análisis de regresión de Cox multivariado identificó la puntuación pronóstica de Nápoles como un factor pronóstico independiente para la supervivencia global (índice de riesgo = 1.574; p = 0.004). El análisis de la curva característica de funcionamiento del receptor dependiente del tiempo mostró que la puntuación pronóstica de Nápoles era más sensible que otros factores pronósticos para predecir la supervivencia global.Las principales limitaciones son el tamaño de la muestra, la característica institucional única y la heterogeneidad del tratamiento.La puntuación pronóstica de Nápoles puede ser un marcador pronóstico útil en pacientes con cáncer colorrectal metastásico que reciben quimioterapia sistémica. Vea el Abstract del video en http://links.lww.com/DCR/B72.

Large-scale sequence and structural comparisons of human naive and antigen-experienced antibody repertoires.

Elucidating how antigen exposure and selection shape the human antibody repertoire is fundamental to our understanding of B-cell immunity. We sequenced the paired heavy- and light-chain variable regions (VH and VL, respectively) from large populations of single B cells combined with computational modeling of antibody structures to evaluate sequence and structural features of human antibody repertoires at unprecedented depth. Analysis of a dataset comprising 55,000 antibody clusters from CD19(+)CD20(+)CD27(-) IgM-naive B cells, >120,000 antibody clusters from CD19(+)CD20(+)CD27(+) antigen-experienced B cells, and >2,000 RosettaAntibody-predicted structural models across three healthy donors led to a number of key findings: (i) VH and VL gene sequences pair in a combinatorial fashion without detectable pairing restrictions at the population level; (ii) certain VH:VL gene pairs were significantly enriched or depleted in the antigen-experienced repertoire relative to the naive repertoire; (iii) antigen selection increased antibody paratope net charge and solvent-accessible surface area; and (iv) public heavy-chain third complementarity-determining region (CDR-H3) antibodies in the antigen-experienced repertoire showed signs of convergent paired light-chain genetic signatures, including shared light-chain third complementarity-determining region (CDR-L3) amino acid sequences and/or Vκ,λ-Jκ,λ genes. The data reported here address several longstanding questions regarding antibody repertoire selection and development and provide a benchmark for future repertoire-scale analyses of antibody responses to vaccination and disease.

Preservation of physiological passage through the remnant stomach prevents postoperative malnutrition after proximal gastrectomy with double tract reconstruction.

PURPOSE: Double tract reconstruction (DT) after proximal gastrectomy (PG) is considered beneficial for postoperative nutrition status by preserving the physiological passage of food. We conducted this study to assess postoperative nutrition status based on food passage after this operation. METHODS: The subjects of this retrospective study were 63 patients who underwent PG with DT. The patients were divided into two groups according to whether they had postoperative malnutrition (PM) 1 year postoperatively (PM group) or not (non-PM group). PM was defined by both weight loss > 10% and a low body mass index of < 20 or < 22 kg/m2 for patients younger and older than 70 years, respectively. We then evaluated the predictors of PM. RESULTS: There were 33 patients in the PM group. These patients were predominantly female (p < 0.01) and lacked physiological passage through the remnant stomach (PRS) on postoperative fluoroscopy (defined as non-PRS, p = 0.03). Multivariate logistic regression analysis revealed that female gender and non-PRS status were independent predictors of PM (odds ratio [95% CI]; 7.42 [1.33-41.4]; p = 0.02, 6.77 [1.01-45.4]; p = 0.04, respectively). CONCLUSION: Preservation of the physiological passage of food through the remnant stomach prevents PM after PG with DT.