The role of carotid stenosis ultrasound scale in the prediction of ischemic stroke.

INTRODUCTION: To improve the accuracy of ultrasound techniques for the assessment of carotid stenosis, we designed a novel carotid artery stenosis ultrasound scale (CASUS), and evaluated its accuracy, reliability, and its value in predicting the occurrence of cardiovascular and cerebrovascular diseases in a prospective study. METHODS: A total of 750 patients with first-time ischemic stroke and hospitalized within 24 h were enrolled in the study. Using color Doppler ultrasound (CDUS), the degree of stenosis and blood flow (BF) in bilateral internal carotid arteries (ICA) and the V1-V3 segment of vertebral arteries (VA) was assessed. Cubic simulation curves for BF and global blood flow (GBF) over the stenosis score (SS), total stenosis score (TSS), and radiological imaging- total stenosis score (RI-TSS) were fitted and compared. The receiver operating characteristic (ROC) curves using TSS, RI-TSS, or GBF to predict various ischemic stroke endpoints were also analyzed and compared. RESULTS: There was a linear relationship between SS and BF both ICA and VA (R2 were 0.734 and 0.783, respectively, both P < 0.05). Both TSS and RI-TSS with GBF showed an inverse "S" curve relationship (R2 was 0.839 and 0.843, all P < 0.05). The AUC values of TSS-based and RI-TSS-based predictions of each endpoint were all greater than 0.7 (all P < 0.05), but the differences of the AUC values between TSS, RI-TSS, and GBF were not statistically significant (all P > 0.05). CONCLUSIONS: The novel CASUS can better reflect the level of cerebral reperfusion in patients with ischemic stroke and can better predict the occurrence of cardiovascular and cerebrovascular diseases.

Biophysical properties of the clinical-stage antibody landscape.

Antibodies are a highly successful class of biological drugs, with over 50 such molecules approved for therapeutic use and hundreds more currently in clinical development. Improvements in technology for the discovery and optimization of high-potency antibodies have greatly increased the chances for finding binding molecules with desired biological properties; however, achieving drug-like properties at the same time is an additional requirement that is receiving increased attention. In this work, we attempt to quantify the historical limits of acceptability for multiple biophysical metrics of "developability." Amino acid sequences from 137 antibodies in advanced clinical stages, including 48 approved for therapeutic use, were collected and used to construct isotype-matched IgG1 antibodies, which were then expressed in mammalian cells. The resulting material for each source antibody was evaluated in a dozen biophysical property assays. The distributions of the observed metrics are used to empirically define boundaries of drug-like behavior that can represent practical guidelines for future antibody drug candidates.

Cytosolic delivery of siRNA by ultra-high affinity dsRNA binding proteins.

Protein-based methods of siRNA delivery are capable of uniquely specific targeting, but are limited by technical challenges such as low potency or poor biophysical properties. Here, we engineered a series of ultra-high affinity siRNA binders based on the viral protein p19 and developed them into siRNA carriers targeted to the epidermal growth factor receptor (EGFR). Combined in trans with a previously described endosome-disrupting agent composed of the pore-forming protein Perfringolysin O (PFO), potent silencing was achieved in vitro with no detectable cytotoxicity. Despite concerns that excessively strong siRNA binding could prevent the discharge of siRNA from its carrier, higher affinity continually led to stronger silencing. We found that this improvement was due to both increased uptake of siRNA into the cell and improved pharmacodynamics inside the cell. Mathematical modeling predicted the existence of an affinity optimum that maximizes silencing, after which siRNA sequestration decreases potency. Our study characterizing the affinity dependence of silencing suggests that siRNA-carrier affinity can significantly affect the intracellular fate of siRNA and may serve as a handle for improving the efficiency of delivery. The two-agent delivery system presented here possesses notable biophysical properties and potency, and provide a platform for the cytosolic delivery of nucleic acids.

Effects of Blood Pressure in the Early Phase of Ischemic Stroke and Stroke Subtype on Poststroke Cognitive Impairment.

BACKGROUND AND PURPOSE: Blood pressure (BP) control in the early phase of stroke is controversial to reduce the risk of poststroke cognitive impairment (PSCI). This study was to investigate the impact of BP levels in the early phase of ischemic stroke and stroke subtype on PSCI. METHODS: Seven hundred and ninety-six patients with acute ischemic stroke were included. Cognitive function was assessed after stroke onset using the Montreal Cognitive Assessment. Patients were divided into quintiles according to systolic BP and diastolic BP levels in the early phase. Subtype analyses were according to Trial of ORG 10172 in Acute Stroke Treatment classification (infarct cause) and Oxfordshire Community Stroke Project classification (infarct location). RESULTS: After adjusting for multiple variables, the quintiles with the lowest systolic BP (Q1, 102-127 mm Hg) and with the highest systolic BP (Q5, 171-215 mm Hg) were associated with increased PSCI risk (odds ratio, 1.83; 95% confidence interval, 1.64-2.28; P=0.007 in Q1; odds ratio, 2.32; 95% confidence interval, 1.74-2.90; P<0.001 in Q5) at 3 months as compared with the middle quintile (Q3, 143-158 mm Hg). Similar association was found in diastolic BP quintiles. The analysis of cerebral infarction subtype demonstrated that both large artery atherosclerosis and total anterior circulation infarct were associated with increased risk of PSCI at 3 months after adjusting for multiple variables (large artery atherosclerosis: odds ratio, 1.42; 95% confidence interval, 1.06-1.90; P=0.031; total anterior circulation infarct: odds ratio, 1.68; 95% confidence interval, 1.32-2.15; P=0.001). CONCLUSIONS: Lower or higher BP in the early phase of ischemic stroke was correlated with increased PSCI risk at 3 months. Maintaining systolic/diastolic BP in the levels of 143 to 158/93 to 102 mm Hg might be beneficial to reduce the occurrence of PSCI. Moreover, large artery atherosclerosis subtype and total anterior circulation infarct subtype were correlated with increased PSCI risk at 3 months. CLINICAL TRIAL REGISTRATION: URL: https://www.chictr.org. Unique identifier: ChiCTR-TRC-14004804.

Prediction of delayed retention of antibodies in hydrophobic interaction chromatography from sequence using machine learning.

MOTIVATION: The hydrophobicity of a monoclonal antibody is an important biophysical property relevant for its developability into a therapeutic. In addition to characterizing heterogeneity, Hydrophobic Interaction Chromatography (HIC) is an assay that is often used to quantify the hydrophobicity of an antibody to assess downstream risks. Earlier studies have shown that retention times in this assay can be correlated to amino-acid or atomic propensities weighted by the surface areas obtained from protein 3-dimensional structures. The goal of this study is to develop models to enable prediction of delayed HIC retention times directly from sequence. RESULTS: We utilize the randomforest machine learning approach to estimate the surface exposure of amino-acid side-chains in the variable region directly from the antibody sequence. We obtain mean-absolute errors of 4.6% for the prediction of surface exposure. Using experimental HIC data along with the estimated surface areas, we derive an amino-acid propensity scale that enables prediction of antibodies likely to have delayed retention times in the assay. We achieve a cross-validation Area Under Curve of 0.85 for the Receiver Operating Characteristic curve of our model. The low computational expense and high accuracy of this approach enables real-time assessment of hydrophobic character to enable prioritization of antibodies during the discovery process and rational engineering to reduce hydrophobic liabilities. AVAILABILITY AND IMPLEMENTATION: Structure data, aligned sequences, experimental data and prediction scores for test-cases, and R scripts used in this work are provided as part of the Supplementary Material. CONTACT: tushar.jain@adimab.com. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Risk factors of acute ischemic stroke and the role of angiotensin I in predicting prognosis of patients undergoing endovascular thrombectomy.

Purpose: The interaction between the renin-angiotensin system (RAS) and the acute ischemic stroke (AIS) is definite but not fully understood. This study aimed to analyze the risk factors of AIS and explore the role of serum indicators such as angiotensin I (Ang I) in the prognosis of patients undergoing endovascular thrombectomy (EVT). Patients and methods: Patients with AIS who underwent EVT and healthy controls were retrospectively enrolled in this study, and the patients were divided into a good or a poor prognosis group. We compared Ang I, blood routine indexes, biochemical indexes, electrolyte indexes, and coagulation indexes between patients and controls. We used univariate and multivariate logistic regression analyses to evaluate possible risk factors for AIS and the prognosis of patients undergoing EVT. Independent risk factors for the prognosis of patients undergoing EVT were identified through multifactorial logistic regression analyses to construct diagnostic nomograms, further assessed by receiver operating characteristic curves (ROC). Results: Consistent with previous studies, advanced age, high blood glucose, high D-dimer, and high prothrombin activity are risk factors for AIS. In addition, Ang I levels are lower in AIS compared to the controls. The level of Ang I was higher in the good prognosis group. Furthermore, we developed a nomogram to evaluate its ability to predict the prognosis of AIS after EVT. The AUC value of the combined ROC model (Ang I and albumin-globulin ratio (AGR)) was 0.859. Conclusions: In conclusion, advanced age, high blood glucose, high D-dimer, and high prothrombin activity are risk factors for AIS. The combined Ang I and AGR model has a good predictive ability for the prognosis of AIS patients undergoing arterial thrombectomy.

Serum and glucocorticoid-induced kinase (SGK) 1 and the epithelial sodium channel are regulated by multiple with no lysine (WNK) family members.

The four WNK (with no lysine (K)) protein kinases affect ion balance and contain an unusual protein kinase domain due to the unique placement of the active site lysine. Mutations in two WNKs cause a heritable form of ion imbalance culminating in hypertension. WNK1 activates the serum- and glucocorticoid-induced protein kinase SGK1; the mechanism is noncatalytic. SGK1 increases membrane expression of the epithelial sodium channel (ENaC) and sodium reabsorption via phosphorylation and sequestering of the E3 ubiquitin ligase neural precursor cell expressed, developmentally down-regulated 4-2 (Nedd4-2), which otherwise promotes ENaC endocytosis. Questions remain about the intrinsic abilities of WNK family members to regulate this pathway. We find that expression of the N termini of all four WNKs results in modest to strong activation of SGK1. In reconstitution experiments in the same cell line all four WNKs also increase sodium current blocked by the ENaC inhibitor amiloride. The N termini of the WNKs also have the capacity to interact with SGK1. More detailed analysis of activation by WNK4 suggests mechanisms in common with WNK1. Further evidence for the importance of WNK1 in this process comes from the ability of Nedd4-2 to bind to WNK1 and the finding that endogenous SGK1 has reduced activity if WNK1 is knocked down by small interfering RNA.

Generation of a safe and efficacious llama single-domain antibody fragment (vHH) targeting the membrane-proximal region of 4-1BB for engineering therapeutic bispecific antibodies for cancer.

BACKGROUND: The discovery of checkpoint inhibitors towards cytotoxic T-lymphocyte protein 4 (CTLA-4) and programmed cell death protein 1 (PD-1) has been revolutionary for the treatment of cancers. These therapies have only offered an average of 20%-30% response rates across the tumor spectrum and the combination of agonists towards the tumor-necrosis superfamily members, such as 4-1BB and CD40, has shown potent efficacy in preclinical studies; however, these agonists have exhibited high degrees of toxicity with limited efficacy in human trials. In this study, we have generated a single-domain antibody towards a unique epitope of 4-1BB that limits its potential on-target toxicity while maintaining sufficient potency. This 4-1BB binder is ideal for use in the engineering of multispecific antibodies to localize 4-1BB activation within the tumor microenvironment, as shown here by a anti-PD-L1/4-1BB bispecific candidate (PM1003). METHODS: To determine the functional activity of the 4-1BB- and PD-L1-binding elements of PM1003, in vitro luciferase reporter and primary cell assays were used to test the potency of programmed cell death 1 ligand 1 (PD-L1) blockade and PD-L1-mediated 4-1BB activation via cross-bridging. X-ray crystallography was conducted to resolve the binding epitopes of the respective binding arms, and accurate binding kinetics were determined using standard affinity measurement techniques. Human 4-1BB and/or PD-L1 knock-in mice were used in cancer models for testing the in vivo antitumor efficacy of PM1003, and safety was evaluated further. RESULTS: PM1003 shows potent activation of 4-1BB and blockade of PD-L1 in cell-based assays. 4-1BB activation was exerted through the bridging of PD-L1 on target cells and 4-1BB on effector cells. No PD-L1-independent activation of 4-1BB was observed. Through X-ray crystallography, a unique binding epitope in the cysteine-rich domain 4 (CRD4) region was resolved that provides high potency and potentially low on-target toxicity as determined by primary immune cell assays and toxicity evaluation in vivo. CONCLUSIONS: A unique single-domain antibody was discovered that binds to the CRD4 domain of 4-1BB. When incorporated into a 4-1BB/PD-L1 bispecific (PM1003), we have shown the potent inhibition of PD-L1 activity with 4-1BB agonism upon cross-bridging with PD-L1 in vitro. Antitumor activity with minimal toxicity was found in vivo. Thus, PM1003 is a uniquely differentiating and next generation therapeutic agent for cancer therapy.

The uniqueness of flow in probing the aggregation behavior of clinically relevant antibodies.

The development of therapeutic monoclonal antibodies (mAbs) can be hindered by their tendency to aggregate throughout their lifetime, which can illicit immunogenic responses and render mAb manufacturing unfeasible. Consequently, there is a need to identify mAbs with desirable thermodynamic stability, solubility, and lack of self-association. These behaviors are assessed using an array of in silico and in vitro assays, as no single assay can predict aggregation and developability. We have developed an extensional and shear flow device (EFD), which subjects proteins to defined hydrodynamic forces which mimic those experienced in bioprocessing. Here, we utilize the EFD to explore the aggregation propensity of 33 IgG1 mAbs, whose variable domains are derived from clinical antibodies. Using submilligram quantities of material per replicate, wide-ranging EFD-induced aggregation (9-81% protein in pellet) was observed for these mAbs, highlighting the EFD as a sensitive method to assess aggregation propensity. By comparing the EFD-induced aggregation data to those obtained previously from 12 other biophysical assays, we show that the EFD provides distinct information compared with current measures of adverse biophysical behavior. Assessing a candidate's liability to hydrodynamic force thus adds novel insight into the rational selection of developable mAbs that complements other assays.

Assessing the binding properties of the anti-PD-1 antibody landscape using label-free biosensors.

Here we describe an industry-wide collaboration aimed at assessing the binding properties of a comprehensive panel of monoclonal antibodies (mAbs) against programmed cell death protein 1 (PD-1), an important checkpoint protein in cancer immunotherapy and validated therapeutic target, with well over thirty unique mAbs either in clinical development or market-approved in the United States, the European Union or China. The binding kinetics of the PD-1/mAb interactions were measured by surface plasmon resonance (SPR) using a Carterra LSA instrument and the results were compared to data collected on a Biacore 8K. The effect of chip type on the SPR-derived binding rate constants and affinities were explored and the results compared with solution affinities from Meso Scale Discovery (MSD) and Kinetic Exclusion Assay (KinExA) experiments. When using flat chip types, the LSA and 8K platforms yielded near-identical kinetic rate and affinity constants that matched solution phase values more closely than those produced on 3D-hydrogels. Of the anti-PD-1 mAbs tested, which included a portion of those known to be in clinical development or approved, the affinities spanned from single digit picomolar to nearly 425 nM, challenging the dynamic range of our methods. The LSA instrument was also used to perform epitope binning and ligand competition studies which revealed over ten unique competitive binding profiles within this group of mAbs.

A novel biparatopic hybrid antibody-ACE2 fusion that blocks SARS-CoV-2 infection: implications for therapy.

In the absence of a proven effective vaccine preventing infection by SARS-CoV-2, or a proven drug to treat COVID-19, the positive results of passive immune therapy using convalescent serum provide a strong lead. We have developed a new class of tetravalent, biparatopic therapy, 89C8-ACE2. It combines the specificity of a monoclonal antibody (89C8) that recognizes the relatively conserved N-terminal domain of the viral Spike (S) glycoprotein, and the ectodomain of ACE2, which binds to the receptor-binding domain of S. This molecule shows exceptional performance in vitro, inhibiting the interaction of recombinant S1 to ACE2 and transduction of ACE2-overexpressing cells by S-pseudotyped lentivirus with IC50s substantially below 100 pM, and with potency approximately 100-fold greater than ACE2-Fc itself. Moreover, 89C8-ACE2 was able to neutralize authentic viral infection in a standard 96-h co-incubation assay at low nanomolar concentrations, making this class of molecule a promising lead for therapeutic applications.

Deamidation and isomerization liability analysis of 131 clinical-stage antibodies.

Contemporary in vivo and in vitro discovery platform technologies greatly increase the odds of identifying high-affinity monoclonal antibodies (mAbs) towards essentially any desired biologically relevant epitope. Lagging discovery throughput is the ability to select for highly developable mAbs with drug-like properties early in the process. Upstream consideration of developability metrics should reduce the frequency of failures in later development stages. As the field moves towards incorporating biophysical screening assays in parallel to discovery processes, similar approaches should also be used to ensure robust chemical stability. Optimization of chemical stability in the early stages of discovery has the potential to reduce complications in formulation development and improve the potential for successful liquid formulations. However, at present, our knowledge of the chemical stability characteristics of clinical-stage therapeutic mAbs is fragmented and lacks comprehensive comparative assessment. To address this knowledge gap, we produced 131 mAbs with amino acid sequences corresponding to the variable regions of clinical-stage mAbs, subjected these to low and high pH stresses and identified the resulting modifications at amino acid-level resolution via tryptic peptide mapping. Among this large set of mAbs, relatively high frequencies of asparagine deamidation events were observed in CDRs H2 and L1, while CDRs H3, H2 and L1 contained relatively high frequencies of instances of aspartate isomerization.

Affinity Maturation Enhances Antibody Specificity but Compromises Conformational Stability.

Monoclonal antibodies (mAbs) have recently emerged as one of the most promising classes of biotherapeutics. A potential advantage of B cell-derived mAbs as therapeutic agents is that they have been subjected to natural filtering mechanisms, which may enrich for B cell receptors (BCRs) with favorable biophysical properties. Here, we evaluated 400 human mAbs for polyreactivity, hydrophobicity, and thermal stability using high-throughput screening assays. Overall, mAbs derived from memory B cells and long-lived plasma cells (LLPCs) display reduced levels of polyreactivity, hydrophobicity, and thermal stability compared with naive B cell-derived mAbs. Somatic hypermutation (SHM) is inversely associated with all three biophysical properties, as well as BCR expression levels. Finally, the developability profiles of the human B cell-derived mAbs are comparable with those observed for clinical mAbs, suggesting their high therapeutic potential. The results provide insight into the biophysical consequences of affinity maturation and have implications for therapeutic antibody engineering and development.

Structure, heterogeneity and developability assessment of therapeutic antibodies.

Increasing attention has been paid to developability assessment with the understanding that thorough evaluation of monoclonal antibody lead candidates at an early stage can avoid delays during late-stage development. The concept of developability is based on the knowledge gained from the successful development of approximately 80 marketed antibody and Fc-fusion protein drug products and from the lessons learned from many failed development programs over the last three decades. Here, we reviewed antibody quality attributes that are critical to development and traditional and state-of-the-art analytical methods to monitor those attributes. Based on our collective experiences, a practical workflow is proposed as a best practice for developability assessment including in silico evaluation, extended characterization and forced degradation using appropriate analytical methods that allow characterization with limited material consumption and fast turnaround time.

Corpus callosum infarction with cognitive dysfunction: two case reports and literature review.

INTRODUCTION: The incidence of corpus callosum infarction is low, and sudden cognitive dysfunction caused by corpus callosum infarction is very rare. We report two cases of acute corpus callosum infarction with sudden cognitive impairment, and the related basis, clinical manifestations, diagnosis, differential diagnosis, treatment, and prognosis of this disease are reviewed. CASES PRESENTATION: The two patients had sudden and severe memory impairment and spatial orientation disorder. Their cognitive function scores were significantly lower, and their MRI demonstrated clear corpus callosum infarction. Through treatment, the symptoms improved significantly. DISCUSSION: This paper reports two cases with corpus callosum infarction with sudden cognitive impairment, and its relevant background is also reviewed, which will help doctors with the classification diagnosis of cerebral infarction and understanding of corpus callosum infarction.

Mid-Term Blood Pressure Variability Is Associated With Clinical Outcome After Ischemic Stroke.

BACKGROUND: The aim of this study was to investigate the relation between mid-term blood pressure (BP) variability (BPV) within 7 days of onset and the prognosis in acute stroke patients. METHODS: Total 873 acute ischemic stroke patients were included in this study. Mid-term BPV was evaluated through the calculations of coefficient of variation (CV) of the systolic BP (SBP) and diastolic BP (DBP) within 7 days of onset. Clinical outcomes were assessed using the recovery situations of neurological function at 3 months, the primary outcome (symptomatic recurrent stroke) and the secondary outcomes (recurrent stroke, all-cause mortality, and the composite of cardiovascular events) within 12 months. RESULTS: Among 873 patients with ischemic stroke, 83 died, 125 developed recurrent stroke, and 212 developed cardiovascular events during 12 months' follow-up. At 3 months, systolic or diastolic BPV (within 7 days of onset) was associated with the recovery situations of neurological function in three models (all P < 0.05). Both higher CV of SBP and CV of DBP were significantly correlated with the increased risk of recurrent stroke (hazard ratio [HR] = 2.32, 95% confidence interval [CI]: 1.29-4.18, P = 0.005 for CV of SBP; HR = 2.33, 95% CI: 1.29-4.19, P = 0.005 for CV of DBP) and composite cardiovascular events (HR = 2.22, 95% CI: 1.41-3.48, P = 0.001 for CV of SBP; HR = 2.21, 95% CI: 1.41-3.47, P = 0.001 for CV of DBP) during 12 months' follow-up. CONCLUSIONS: After acute ischemic stroke, high systolic or diastolic BPV within 7 days of onset was associated with the recovery situations of neurological function at 3 months, and recurrent stroke and composite cardiovascular events within 12 months. CLINICAL TRIAL REGISTRATION: URL: http://www.chictr.org.cn. Unique identifier: ChiCTR-TRC-14004804.

Rapid assessment of oxidation via middle-down LCMS correlates with methionine side-chain solvent-accessible surface area for 121 clinical stage monoclonal antibodies.

Susceptibility of methionine to oxidation is an important concern for chemical stability during the development of a monoclonal antibody (mAb) therapeutic. To minimize downstream risks, leading candidates are usually screened under forced oxidation conditions to identify oxidation-labile molecules. Here we report results of forced oxidation on a large set of in-house expressed and purified mAbs with variable region sequences corresponding to 121 clinical stage mAbs. These mAb samples were treated with 0.1% H2O2 for 24 hours before enzymatic cleavage below the hinge, followed by reduction of inter-chain disulfide bonds for the detection of the light chain, Fab portion of heavy chain (Fd) and Fc by liquid chromatography-mass spectrometry. This high-throughput, middle-down approach allows detection of oxidation site(s) at the resolution of 3 distinct segments. The experimental oxidation data correlates well with theoretical predictions based on the solvent-accessible surface area of the methionine side-chains within these segments. These results validate the use of upstream computational modeling to predict mAb oxidation susceptibility at the sequence level.

Database-Centric Method for Automated High-Throughput Deconvolution and Analysis of Kinetic Antibody Screening Data.

The state-of-the-art industrial drug discovery approach is the empirical interrogation of a library of drug candidates against a target molecule. The advantage of high-throughput kinetic measurements over equilibrium assessments is the ability to measure each of the kinetic components of binding affinity. Although high-throughput capabilities have improved with advances in instrument hardware, three bottlenecks in data processing remain: (1) intrinsic molecular properties that lead to poor biophysical quality in vitro are not accounted for in commercially available analysis models, (2) processing data through a user interface is time-consuming and not amenable to parallelized data collection, and (3) a commercial solution that includes historical kinetic data in the analysis of kinetic competition data does not exist. Herein, we describe a generally applicable method for the automated analysis, storage, and retrieval of kinetic binding data. This analysis can deconvolve poor quality data on-the-fly and store and organize historical data in a queryable format for use in future analyses. Such database-centric strategies afford greater insight into the molecular mechanisms of kinetic competition, allowing for the rapid identification of allosteric effectors and the presentation of kinetic competition data in absolute terms of percent bound to antigen on the biosensor.

Midterm Blood Pressure Variability Is Associated with Poststroke Cognitive Impairment: A Prospective Cohort Study.

OBJECTIVE: The aim of this study was to investigate the relationship between blood pressure variability (BPV) and poststroke cognitive impairment (PSCI). METHODS: Seven-hundred ninety-six patients with acute ischemic stroke were included in this study. Midterm BPV was evaluated by calculating the SD and coefficient of variation (CV, 100 × SD/mean) of systolic blood pressure (SBP) and diastolic blood pressure during the 7 days after stroke onset. Cognitive function was assessed using the Montreal Cognitive Assessment (MoCA) at admission and at all follow-up visits. Patients with MoCA scores <26 were considered to have PSCI. RESULTS: The incidence of PSCI reached its peak (72%) 3 months after stroke onset and decreased to 30.3% at 12 months poststroke. After adjusting for covariables, the increase in the prevalence of PSCI at 3 months was independently associated with increases in the CV of blood pressure during the 7 days after stroke [odds ratios and 95% CI for patients in the second to fifth quintiles of SBP CV were 2.28 (1.18, 4.39), 2.33 (1.18, 4.62), 2.69 (1.31, 5.53), and 4.76 (1.95, 11.67), respectively]. Sub-analysis of the MoCA scores revealed that the patients had impairments in visuoperceptual abilities and executive functions, as well as in naming and delayed recall (p < 0.05). CONCLUSION: Midterm BPV during the early phase of acute ischemic stroke is independently associated with PSCI, especially in the visuoperceptual, executive, and delayed recall domains. CLINICAL TRIAL REGISTRATION: http://www.chictr.org.cn, identifier ChiCTR-TRC-14004804.

Broad epitope coverage of a human in vitro antibody library.

Successful discovery of therapeutic antibodies hinges on the identification of appropriate affinity binders targeting a diversity of molecular epitopes presented by the antigen. Antibody campaigns that yield such broad "epitope coverage" increase the likelihood of identifying candidates with the desired biological functions. Accordingly, epitope binning assays are employed in the early discovery stages to partition antibodies into epitope families or "bins" and prioritize leads for further characterization and optimization. The collaborative program described here, which used hen egg white lysozyme (HEL) as a model antigen, combined 3 key capabilities: 1) access to a diverse panel of antibodies selected from a human in vitro antibody library; 2) application of state-of-the-art high-throughput epitope binning; and 3) analysis and interpretation of the epitope binning data with reference to an exhaustive set of published antibody:HEL co-crystal structures. Binning experiments on a large merged panel of antibodies containing clones from the library and the literature revealed that the inferred epitopes for the library clones overlapped with, and extended beyond, the known structural epitopes. Our analysis revealed that nearly the entire solvent-exposed surface of HEL is antigenic, as has been proposed for protein antigens in general. The data further demonstrated that synthetic antibody repertoires provide as wide epitope coverage as those obtained from animal immunizations. The work highlights molecular insights contributed by increasingly higher-throughput binning methods and their broad utility to guide the discovery of therapeutic antibodies representing a diverse set of functional epitopes.