High-Throughput Multi-attribute Analysis of Antibody-Drug Conjugates Enabled by Trapped Ion Mobility Spectrometry and Top-Down Mass Spectrometry.

Antibody-drug conjugates (ADCs) are one of the fastest growing classes of anticancer therapies. Combining the high targeting specificity of monoclonal antibodies (mAbs) with cytotoxic small molecule drugs, ADCs are complex molecular entities that are intrinsically heterogeneous. Primary sequence variants, varied drug-to-antibody ratio (DAR) species, and conformational changes in the starting mAb structure upon drug conjugation must be monitored to ensure the safety and efficacy of ADCs. Herein, we have developed a high-throughput method for the analysis of cysteine-linked ADCs using trapped ion mobility spectrometry (TIMS) combined with top-down mass spectrometry (MS) on a Bruker timsTOF Pro. This method can analyze ADCs (∼150 kDa) by TIMS followed by a three-tiered top-down MS characterization strategy for multi-attribute analysis. First, the charge state distribution and DAR value of the ADC are monitored (MS1). Second, the intact mass of subunits dissociated from the ADC by low-energy collision-induced dissociation (CID) is determined (MS2). Third, the primary sequence for the dissociated subunits is characterized by CID fragmentation using elevated collisional energies (MS3). We further automate this workflow by directly injecting the ADC and using MS segmentation to obtain all three tiers of MS information in a single 3-min run. Overall, this work highlights a multi-attribute top-down MS characterization method that possesses unparalleled speed for high-throughput characterization of ADCs.

Rapid Analysis of Reduced Antibody Drug Conjugate by Online LC-MS/MS with Fourier Transform Ion Cyclotron Resonance Mass Spectrometry.

Antibody drug conjugates (ADCs), which harness the high targeting specificity of monoclonal antibodies (mAb) with the potency of small molecule therapeutics, are one of the fastest growing pharmaceutical classes. Nevertheless, ADC conjugation techniques and processes may introduce intrinsic heterogeneity including primary sequence variants, varied drug-to-antibody ratio (DAR) species, and drug positional isomers, which must be monitored to ensure the safety and efficacy of ADCs. Liquid chromatography coupled to mass spectrometry (LC-MS) is a powerful tool for characterization of ADCs. However, the conventional bottom-up MS analysis workflows require an enzymatic digestion step which can be time consuming and may introduce artifactual modifications. Herein, we develop an online LC-MS/MS method for rapid analysis of reduced ADCs without digestion, enabling determination of DAR, characterization of the primary sequence, and localization of the drug conjugation site of the ADC using high-resolution Fourier transform ion cyclotron resonance (FTICR) MS. Specifically, a model cysteine-linked ADC was reduced to generate six unique subunits: light chain (Lc) without drug (Lc0), Lc with 1 drug (Lc1), heavy chain (Hc) without drug (Hc0), and Hc with 1-3 drugs (Hc1-3, respectively). A concurrent reduction strategy is applied to assess ADC subunits in both the partially reduced (intrachain disulfide bonds remain intact) and fully reduced (all disulfide bonds are cleaved) forms. The entire procedure including the sample preparation and LC-MS/MS takes less than 55 min, enabling rapid multiattribute analysis of ADCs.

Declined circulating Elabela levels in patients with essential hypertension and its association with impaired vascular function: A preliminary study.

Background: Elabela (ELA) is a newly identified endogenous ligand of apelin receptor (APJ) which has been confirmed to be implicated in the pathogenesis of hypertension. Previous experiments have revealed the critical role of ELA in eliciting vasodilation and lowering blood pressure. However, the role of plasma ELA levels in hypertensive patients and its relationship with vascular function have not been investigated.Method: Thirty-one patients with essential hypertension (EH) and 31 age-matched healthy subjects as controls were recruited in the study. Plasma ELA concentration and vascular function parameters including brachial artery flow-mediated dilation (FMD) and brachial-ankle pulse wave velocity (baPWV) were measured.Results: We observed remarkably lower plasma ELA concentration in hypertensive patients as compared with controls (1.29 ± 0.56 ng/ml vs. 1.79 ± 0.55 ng/ml; P = 0.001). Linear correlation analysis showed that ELA was negatively correlated with systolic blood pressure (r = -0.388, P = 0.002) and diastolic blood pressure (r = -0.321, P = 0.011) and positively correlated with FMD (r = 0.319, P = 0.011). There was no statistically significant relationship between ELA and baPWV (r = 0.234, P = 0.067). Stepwise multiple linear analysis also identified a close association of plasma ELA levels with endothelial function.Conclusion: The present study demonstrates for the first time that circulating ELA levels are reduced in patients with EH. The fall in endogenous ELA levels may be involved in the pathogenesis of hypertension-related vascular damage.

Native Reversed-Phase Liquid Chromatography: A Technique for LCMS of Intact Antibody-Drug Conjugates.

The synthesis of antibody-drug conjugates (ADCs) using the interchain cysteines of the antibody inherently gives a mixture of proteins with varying drug-to-antibody ratio. The drug distribution profiles of ADCs are routinely characterized by hydrophobic interaction chromatography (HIC). Because HIC is not in-line compatible with mass spectrometry (MS) due to the high salt levels, it is laborious to identify the constituents of HIC peaks. An MS-compatible alternative to HIC is reported here: native reversed phase liquid chromatography (nRPLC). This novel technique employs a mobile phase 50 mM ammonium acetate for high sensitivity in MS and elution with a gradient of water/isopropanol. The key to the enhancement is a bonded phase giving weaker drug-surface interactions compared to the noncovalent interactions holding the antibody-drug conjugates together. The hydrophobicity of the bonded phase is varied, and the least hydrophobic bonded phase in the series, poly(methyl methacrylate), is found to resolve the intact constituents of a model ADC (Ab095-PZ) and a commercial ADC (brentuximab vedotin) under the MS-compatible conditions. The nRPLC-MS data show that all species, ranging from drug-to-antibody ratios of 1 to 8, remained intact in the column. Another desired advantage of the nRPLC is the ability of resolving multiple positional isomers of ADC that are not well-resolved in other chromatographic modes. This supports the premise that lower hydrophobicity of the bonded phase is the key to enabling online nRPLC-MS analysis of antibody-drug conjugates.

Middle-Down Multi-Attribute Analysis of Antibody-Drug Conjugates with Electron Transfer Dissociation.

Antibody-drug conjugates (ADCs) are designed to combine the target specificity of monoclonal antibodies and potent cytotoxin drugs to achieve better therapeutic outcomes. Comprehensive evaluation of the quality attributes of ADCs is critical for drug development but remains challenging due to heterogeneity of the construct. Currently, peptide mapping with reversed-phase liquid chromatography (RPLC) coupled to mass spectrometry (MS) is the predominant approach to characterize ADCs. However, it is suboptimal for sequence characterization and quantification of ADCs because it lacks a comprehensive view of coexisting variants and suffers from varying ionization effects of drug-conjugated peptides compared to unconjugated counterparts. Here, we present the first middle-down RPLC-MS analysis of both cysteine (Adcetris; BV) and lysine (Kadcyla; T-DM1) conjugated ADCs at the subunit level (∼25 kDa) with electron transfer dissociation (ETD). We successfully achieved high-resolution separation of subunit isomers arising from different drug conjugation and subsequently localized the conjugation sites. Moreover, we obtained a comprehensive overview of the microvariants associated with each subunits and characterized them such as oxidized variants with different sites. Furthermore, we observed relatively high levels of conjugation near complementarity-determining regions (CDRs) from the heavy chain but no drug conjugation near CDRs of light chain (Lc) from lysine conjugated T-DM1. Based on the extracted ion chromatograms, we accurately measured average drug to antibody ratio (DAR) values and relative occupancy of drug-conjugated subunits. Overall, the middle-down MS approach enables the evaluation of multiple quality attributes including DAR, positional isomers, conjugation sites, occupancy, and microvariants, which potentially opens up a new avenue to characterize ADCs.

Online Hydrophobic Interaction Chromatography-Mass Spectrometry for the Analysis of Intact Monoclonal Antibodies.

Therapeutic monoclonal antibodies (mAbs) are an important class of drugs for a wide spectrum of human diseases. Liquid chromatography (LC) coupled to mass spectrometry (MS) is one of the techniques in the forefront for comprehensive characterization of analytical attributes of mAbs. Among various protein chromatography modes, hydrophobic interaction chromatography (HIC) is a popular offline nondenaturing separation technique utilized to purify and analyze mAbs, typically with the use of non-MS-compatible mobile phases. Herein we demonstrate for the first time, the application of direct HIC-MS and HIC-tandem MS (MS/MS) with electron capture dissociation (ECD) for analyzing intact mAbs on quadrupole-time-of-flight (Q-TOF) and Fourier transform ion cyclotron resonance (FTICR) mass spectrometers, respectively. Our method allows for rapid determination of relative hydrophobicity, intact masses, and glycosylation profiles of mAbs as well as sequence and structural characterization of the complementarity-determining regions in an online configuration.

Control Strategy for Small Molecule Impurities in Antibody-Drug Conjugates.

Antibody-drug conjugates (ADCs) are an emerging class of biopharmaceuticals. As such, there are no specific guidelines addressing impurity limits and qualification requirements. The current ICH guidelines on impurities, Q3A (Impurities in New Drug Substances), Q3B (Impurities in New Drug Products), and Q6B (Specifications: Test Procedures and Acceptance Criteria for Biotechnological/Biological Products) do not adequately address how to assess small molecule impurities in ADCs. The International Consortium for Innovation and Quality in Pharmaceutical Development (IQ) formed an impurities working group (IWG) to discuss this issue. This white paper presents a strategy for evaluating the impact of small molecule impurities in ADCs. This strategy suggests a science-based approach that can be applied to the design of control systems for ADC therapeutics. The key principles that form the basis for this strategy include the significant difference in molecular weights between small molecule impurities and the ADC, the conjugation potential of the small molecule impurities, and the typical dosing concentrations and dosing schedule. The result is that exposure to small impurities in ADCs is so low as to often pose little or no significant safety risk.

Low prevalence of lipid metabolism abnormalities in APOE ε2-genotype and male patients 60 years or older with schizophrenia.

BACKGROUND: Schizophrenia is a serious mental disorder largely manageable with atypical antipsychotics; however, these drugs have been associated with glucose/lipid metabolism issues such as diabetes and hyperlipidaemia. Apolipoprotein E (APOE) is the most abundant apolipoprotein, and APOE genotypes have been correlated with lipid metabolism phenotypes in an age-dependent manner. Studies examining the relationship between the APOE genotype and lipid abnormalities in patients with schizophrenia have been inconclusive, but primarily focused on adult patient populations. Therefore, we explored the correlations between the APOE genotype and glucose/lipid metabolism indicators and abnormalities in hospitalized patients 60 years or older with schizophrenia with a history of long-term antipsychotics use. METHODS: We assessed APOE genotype, age, weight, height, blood glucose, triglycerides, cholesterol, high-density lipoprotein, and low-density lipoprotein in a total of 294 patients. APOE genotypes were divided into three groups: APOE ε2 (ε2/ε2 and ε2/ε3), APOE ε3 (ε3/ε3), and APOE ε4 (ε3/ε4 and ε4/ε4), and comparisons were conducted among these groups or according to ε2 carrier status. RESULTS: APOE ε3/ε3 was the most common genotype (68.3%) and at least one ε3 allele was present in 81.8% of patients. There were no differences in antipsychotics type or dose according to the APOE genotype, but serum cholesterol values varied near significantly (P = 0.052) and low-density lipoprotein values varied significantly according to genotype (P < 0.05, lowest in the APOE ε2 genotype). Men had lower cholesterol and low-density lipoprotein levels (P < 0.05) than women. Compared to patients administered typical antipsychotics, those administered atypical antipsychotics had higher triglyceride, cholesterol, and low-density lipoprotein levels (P < 0.05). Stepwise linear regressions showed that cholesterol and low-density lipoprotein levels were influenced by sex, the APOE ε2 genotype, and atypical antipsychotics use. CONCLUSIONS: In the context of atypical antipsychotics use, carriers of the APOE ε2-genotype and male patients with schizophrenia 60 years or older may be less likely to develop a lipid metabolism abnormality.

Mobilization of Circulating Endothelial Progenitor Cells by dl-3-n-Butylphthalide in Acute Ischemic Stroke Patients.

OBJECTIVE: The research aim was to investigate the effects of dl-3-n-butylphthalide (NBP) on the level of circulating endothelial progenitor cells (EPCs) and clinical outcome in patients with acute ischemic stroke (AIS). MATERIALS AND METHODS: A total of 170 patients were included and randomly assigned to NBP group and control group. All patients were administrated a basic antiplatelet and lipid-lowering therapy. Among the patients, 86 received additional NBP administration for 30 days, whereas 84 received only basic therapy (the control). The level of circulating EPCs (marked with CD34(+)/CD133(+)/KDR(+)) was determined by flow cytometry at baseline and days 7, 14, and 30 after therapy. Impairment of neurological function was evaluated by the National Institutes of Health Stroke Scale (NIHSS) on days 7, 14, 30, and 90 after therapy. The association between the increased level of circulating EPCs and improvement of NIHSS score was evaluated by Pearson analysis. The clinical outcome was evaluated by modified Rankin Scale (mRS) on day 90. During the observation period, any adverse events related to drugs were reported. RESULTS: The levels of circulating EPCs on days 14 and 30 were significantly higher in the NBP group than in the control group. In contrast, NIHSS score was notably lower in NBP group on day 14, 30 and day 90. Pearson correlation analysis revealed a significant association between the increased level of EPCs and improvement of NIHSS score. Also, the mRS score in the NBP group was lower on day 90. Importantly, the reported adverse events in the 2 groups were comparable. CONCLUSION: NBP significantly increases the circulating level and improves clinical outcome in patients with AIS.

Three-dimensional sparse image reconstruction for terahertz surface layer holography with random step frequency.

In this Letter, a sparse image reconstruction approach is proposed for three-dimensional (3D) terahertz (THz) surface layer holography by a sharply dwindled amount of frequency samples, without reducing the high quality of the final reconstructed 3D THz images. To avoid the range ambiguity resulting from the reduction of frequency samples, a random step frequency method is adopted to evaluate the rough range profile of the 3D surface layer. With the obtained range profile, a de-ambiguity procedure is proposed to demodulate the sparse echoed data to greatly compress the maximum nonambiguous range and recover all the information for 3D holography image reconstruction. Proof-of-state experiments are performed in 0.2-THz band. The results verify the effectiveness and the efficiency of the sparse imaging scheme for THz surface layer 3D holography.

Chromosome-scale genome, together with transcriptome and metabolome, provides insights into the evolution and anthocyanin biosynthesis of Rubus rosaefolius Sm. (Rosaceae).

Rubus rosaefolius is a kind of red raspberry possessing high nutritional and pharmaceutical value. Here we present a chromosome-level draft genome of R. rosaefolius. Of the total 131 assembled scaffolds, 70 with a total size of 219.02 Mb, accounting for 99.33% of the estimated genome size, were anchored to seven pseudochromosomes. We traced a whole-genome duplication (WGD) event shared among members of the Rosaceae family, from which were derived 5090 currently detectable duplicated gene pairs (dgps). Of the WGD-dgps 75.09% underwent purifying selection, and approximately three-quarters of informative WGD-dgps expressed their two paralogs with significant differences. We detected a wide variety of anthocyanins in the berries of R. rosaefolius, and their total concentration remained relatively stable during berry development but increased rapidly during the ripening stage, mainly because of the contributions of pelargonidin-3-O-glucoside and pelargonidin-3-O-(6″-O-malonyl)glucoside. We identified many structural genes that encode enzymes, such as RrDFR, RrF3H, RrANS, and RrBZ1, and play key roles in anthocyanin biosynthesis. The expression of some of these genes significantly increased or decreased with the accumulation of pelargonidin-3-O-glucoside and pelargonidin-3-O-(6″-O-malonyl)glucoside. We also identified some transcription factors and specific methylase-encoding genes that may play a role in regulating anthocyanin biosynthesis by targeting structural genes. In conclusion, our findings provide deeper insights into the genomic evolution and molecular mechanisms underlying anthocyanin biosynthesis in berries of R. rosaefolius. This knowledge may significantly contribute to the targeted domestication and breeding of Rubus species.

A CLRN3-Based CD8+ T-Related Gene Signature Predicts Prognosis and Immunotherapy Response in Colorectal Cancer.

BACKGROUND: Colorectal cancer (CRC) ranks among the most prevalent malignancies affecting the gastrointestinal tract. The infiltration of CD8+ T cells significantly influences the prognosis and progression of tumor patients. METHODS: This study establishes a CRC immune risk model based on CD8+ T cell-related genes. CD8+ T cell-related genes were identified through Weighted Gene Co-expression Network Analysis (WGCNA), and the enriched gene sets were annotated via Gene Ontology (GO) and Reactome pathway analysis. Employing machine learning methods, including the Least Absolute Shrinkage and Selection Operator (LASSO) algorithm and Random Forest (RF), we identified nine genes associated with CD8+ T-cell infiltration. The infiltration levels of immune cells in CRC tissues were assessed using the ssGSEA algorithm. RESULTS: These genes provide a foundation for constructing a prognostic model. The TCGA-CRC sample model's prediction scores were categorized, and the prediction models were validated through Cox regression analysis and Kaplan-Meier curve analysis. Notably, although CRC tissues with higher risk scores exhibited elevated levels of CD8+ T-cell infiltration, they also demonstrated heightened expression of immune checkpoint genes. Furthermore, comparison of microsatellite instability (MSI) and gene mutations across the immune subgroups revealed notable gene variations, particularly with APC, TP53, and TNNT1 showing higher mutation frequencies. Finally, the predictive model's efficacy was corroborated through the use of Tumor Immune Dysfunction and Exclusion (TIDE), Immune Profiling Score (IPS), and immune escape-related molecular markers. The predictive model was validated through an external cohort of CRC and the Bladder Cancer Immunotherapy Cohort. CLRN3 expression levels in tumor and adjacent normal tissues were assessed using quantitative real-time polymerase chain reaction (qRT-PCR) and western blot. Subsequent in vitro and in vivo experiments demonstrated that CLRN3 knockdown significantly attenuated the malignant biological behavior of CRC cells, while overexpression had the opposite effect. CONCLUSIONS: This study presents a novel prognostic model for CRC, providing a framework for enhancing the survival rates of CRC patients by targeting CD8+ T-cell infiltration.

Vestibular Migraine and Recurrent Vertigo in Children: A Diagnostic Focus From a Tertiary Hospital Study.

BACKGROUND: To improve diagnostic precision in pediatric vertigo, particularly in Vestibular Migraine of Childhood (VMC), probable VMC (pVMC), Recurrent Vertigo of Childhood (RVC), and unspecified categories, by delineating clinical characteristics and prevalence to refine diagnostics and treatments. METHODS: Retrospective analysis of 102 pediatric patients (five to 18 years; 46 females, 56 males) at the Dizziness Center of the Otolaryngology Department in a tertiary-level hospital from January 2019 to December 2023. Patients were classified into VMC, pVMC, RVC, and indeterminate groups. Evaluations included audiometry and vestibular tests (video head impulse test [vHIT] or caloric testing), conducted in the audiology unit and vestibular testing laboratory. Data were analyzed using IBM SPSS 20.0. RESULTS: Diagnoses were 8.8% VMC, 31.4% pVMC, 51.0% RVC, and 8.8% indeterminate. Nausea and vomiting were common in VMC and pVMC; cochlear symptoms like tinnitus and hearing loss predominated in VMC. Although vestibular testing showed no significant group differences, VMC had more vHIT abnormalities and RVC had more caloric test anomalies. CONCLUSIONS: This study highlights the need for comprehensive diagnostics in pediatric vestibular disorders, revealing unique and overlapping traits across VMC, pVMC, and RVC. Insights call for further research to refine diagnostic criteria and improve treatment methods.

Simulated seasonal diets alter yak rumen microbiota structure and metabolic function.

Yak is the only ruminant on the Qinghai-Tibetan Plateau that grazes year-round. Although previous research has shown that yak rumen microbiota fluctuates in robust patterns with seasonal foraging, it remains unclear whether these dynamic shifts are driven by changes in environment or nutrient availability. The study examines the response of yak rumen microbiota (bacteria, fungi, and archaea) to simulated seasonal diets, excluding the contribution of environmental factors. A total of 18 adult male yaks were randomly divided into three groups, including a nutrition stress group (NSG, simulating winter pasture), a grazing simulation group (GSG, simulating warm season pasture), and a supplementation group (SG, simulating winter pasture supplemented with feed concentrates). Volatile fatty acids (VFAs) profiling showed that ruminal acetate, propionate and total VFA contents were significantly higher (p < 0.05) in GSG rumen. Metagenomic analysis showed that Bacteroidetes (53.9%) and Firmicutes (37.1%) were the dominant bacterial phyla in yak rumen across dietary treatments. In GSG samples, Actinobacteriota, Succinivibrionaceae_UCG-002, and Ruminococcus albus were the most abundant, while Bacteroides was significantly more abundant in NSG samples (p < 0.05) than that in GSG. The known fiber-degrading fungus, Neocallimastix, was significantly more abundant in NSG and SG samples, while Cyllamyces were more prevalent in NSG rumen than in the SG rumen. These findings imply that a diverse consortium of microbes may cooperate in response to fluctuating nutrient availability, with depletion of known rumen taxa under nutrient deficiency. Archaeal community composition showed less variation between treatments than bacterial and fungal communities. Additionally, Orpinomyces was significantly positively correlated with acetate levels, both of which are prevalent in GSG compared with other groups. Correlation analysis between microbial taxa and VFA production or between specific rumen microbes further illustrated a collective response to nutrient availability by gut microbiota and rumen VFA metabolism. PICRUSt and FUNGuild functional prediction analysis indicated fluctuation response of the function of microbial communities among groups. These results provide a framework for understanding how microbiota participate in seasonal adaptations to forage availability in high-altitude ruminants, and form a basis for future development of probiotic supplements to enhance nutrient utilization in livestock.

Targeted brain delivery of RVG29-modified rifampicin-loaded nanoparticles for Alzheimer's disease treatment and diagnosis.

Alzheimer's disease (AD) is an aging-related neurodegenerative disease. The main pathological features of AD are ß-amyloid protein (Aß) deposition and tau protein hyperphosphorylation. Currently, there are no effective drugs for the etiological treatment of AD. Rifampicin (RIF) is a semi-synthetic broad-spectrum antibiotic with anti-ß-amyloid deposition, anti-inflammatory, anti-apoptosis, and neuroprotective effects, but its application in AD treatment has been limited for its strong hydrophobicity, high toxicity, short half-life, low bioavailability, and blood-brain barrier hindrance. We designed a novel brain-targeted and MRI-characteristic nanomedicine via loading rabies virus protein 29 (RVG29), rifampicin, and Gd on poly (l-lactide) nanoparticles (RIF@PLA-PEG-Gd/Mal-RVG29). The cytotoxicity assay demonstrated that RIF@PLA-PEG-Gd/Mal-RVG29 had favorable biocompatibility and security. Fluorescence imaging in vivo showed that PLA-PEG-Gd/Mal-RVG29 could deliver rifampicin into the brain by enhancing cellular uptake and brain targeting performance, leading to improvement of the bioavailability of rifampicin. In in vivo study, RIF@PLA-PEG-Gd/Mal-RVG29 improved the spatial learning and memory capability of APP/PS1 mice in the Morris water maze, as compared to rifampicin. Immunofluorescence, TEM, immunoblotting, and H&E staining revealed that RIF@PLA-PEG-Gd/Mal-RVG29 reduced Aß deposition in hippocampal and cortex of APP/PS1 mice, improved the damage of synaptic ultrastructure, increased the expression level of PSD95 and SYP, as well as reduced the necrosis of neurons. These findings suggest that RIF@PLA-PEG-Gd/Mal-RVG29 may be an effective strategy for the treatment of AD.

Association Between Long-Term Visit-to-Visit Hemoglobin A1c and Cardiovascular Risk in Type 2 Diabetes: The ACCORD Trial.

Background: To explore the association between visit-to-visit variability of glycated hemoglobin (HbA1c) and cardiovascular outcomes in the patients with type 2 diabetes mellitus (T2DM) of the Action to Control Cardiovascular Risk in Diabetes (ACCORD) study. Methods: We conducted a post-hoc analysis on the ACCORD population including 9,544 participants with T2DM. Visit-to-visit variability of HbA1c was defined as the individual SD, coefficient of variation (CV), and variability independent of the mean (VIM) across HbA1c measurements. The clinical measurements included primary outcome [the first occurrence of non-fatal myocardial infarction (MI), non-fatal stroke or cardiovascular death], total mortality, cardiovascular death, non-fatal MI event, non-fatal stroke, total stroke, heart failure, macrovascular events, and major coronary events (CHD). Results: Over a median follow-up of 4.85 years, 594 and 268 participants experienced all-cause mortality and cardiovascular mortality, respectively. After adjusting for baseline HbA1c levels and confounding factors, the adjusted hazard ratio (HR) comparing patients in the highest vs. the lowest quartile CV of HbA1c variability was 1.61 (95% CI 1.29-2.00) for the primary outcome. Similar trends for secondary outcome were also observed. There was no association between HbA1c fluctuation and non-fatal stroke. Noticeably, there was 66% greater risk for the all-cause mortality among patients in the highest vs. the lowest quartile (HR 1.66, 95% CI 1.27-2.17). Conclusions: Greater variability of HbA1c is associated with higher risk for cardiovascular complications and all-cause death in T2DM. Our study stresses the significance of well-controlled glycemic levels for improving cardiovascular outcomes. Further randomized clinical trials are required to confirm these findings.

Conjugation Site Analysis by MS/MS Protein Sequencing.

In-depth knowledge about the site of drug-linker conjugation is important for the understanding of the conjugation efficiency and the exact locations of payloads for antibody-drug conjugates (ADCs). Here we describe a peptide mapping-based protocol, covering sample preparation procedure, LC-MS/MS setup, and data processing (auto and manual), to determine the locations of drug-linker attachment on mAbs. In comparison with classical mAb peptide mapping, some improvements will be highlighted for maintaining hydrophobic drug-loaded peptides in solution, enabling efficient chromatographic separation and mass spectrometric detection, and allowing for their unambiguous identification in LC-MS/MS map by using diagnostic fragmentation ions of the payload.

Assessing localized conformational stability of antibody-drug conjugate by protein conformation assay.

Antibody-drug conjugates (ADCs) are a class of attractive therapeutic agents to fight cancer with conjugation of potent chemical agents on target-selective antibodies. The conceptually elegant approach has encountered mounting practical challenges in combining the mAb and potent drug while maintaining the conformational and physiochemical stability of the bioconjugates. The attachment of hydrophobic drug-linker with antibody could potentially alter the antibody conformational scaffold, locally or globally. Here we propose to use a protein conformation assay (PCA) to measure the higher-order structure of antibodies upon drug-linker conjugation. The PCA analysis provides insights into the formation of partially unfolded ADCs, which may correlate with protein stability and aggregation propensity. To further elucidate the cause of the unfolding events, in-depth peptide mapping combined with the PCA conformational footprints were performed on a commercial ADC trastuzumab emtansine in this study. The locally altered conformational hot-spots observed in PCA matched with conjugation sites with high occupancy rate identified in peptide mapping. In summary, by combining PCA and in-depth peptide mapping, a snapshot of ADC structural conformation and stability profile could be obtained and provide a swift and convenient measurement of the 'fitness' of ADC to facilitate payload selection, conjugation process development and early predictive developability assessment.

Electromyographic Comparison of the Efficacy of Ultrasound-guided Suprainguinal and Infrainguinal Fascia Iliaca Compartment Block for Blockade of the Obturator Nerve in Total Knee Arthroplasty: A Prospective Randomized Controlled Trial.

BACKGROUND AND OBJECTIVES: The knee is innervated by the femoral, obturator, and sciatic nerves. An infrainguinal fascia iliaca compartment block (FICB) is often used as a technique for pain management after hip and knee arthroplasty. This approach blocks the femoral nerve, lateral femoral cutaneous nerve, and obturator nerve. Previous studies show suprainguinal FICB achieves improved postoperative analgesia compared with infrainguinal FICB after hip fracture. However, the analgesic effects of suprainguinal or infrainguinal FICB on the obturator nerve after total knee arthroplasty (TKA) remain to be established. This study compared the efficacy of suprainguinal versus infrainguinal FICB for the blockade of the obturator nerve using electromyography and quantification of total opioid consumption during the 24 hours after TKA. METHODS: This prospective, randomized controlled clinical study enrolled 74 patients scheduled to undergo TKA. Patients were randomized 1:1 to receive suprainguinal FICB (group S) or infrainguinal FICB (group I) with 30 mL of 0.375% ropivacaine. The primary endpoint was the mean amplitude of the adductor longus compound muscle action potential (CMAP) at 0 (before the block), 10, 20, and 30 minutes after FICB. The secondary endpoint was total opioid consumption during the 24 hours after TKA. RESULTS: Data from 62 patients were included in the analysis. The mean amplitude of the adductor longus CMAP was significantly lower in group S compared with group I (repeated-measures analysis of variance; F=4.73, P=0.034). At 24 hours after TKA, mean (SD) total opioid consumption was significantly lower in group S, compared with group I (131.5±76.8 vs. 201.5±85.1 µg) (P=0.001). CONCLUSIONS: Suprainguinal FICB significantly increased the incidence of successful obturator nerve block and significantly decreased fentanyl consumption 24 hours after TKA compared with infrainguinal FICB.