Utilizing kinematic analysis of postural instability as an objective measure to aid in distinguishing between normal pressure hydrocephalus and Parkinson's disease.

Objective: Patients with normal pressure hydrocephalus (NPH) and Parkinson's Disease (PD) can clinically appear quite similar at baseline evaluation. We sought to investigate the use of kinematic assessment of postural instability (PI) using inertial measurement units (IMUs) as a mechanism of differentiation between the two disease processes. Methods: 20 patients with NPH, 55 patients with PD, and 56 age-matched, healthy controls underwent quantitative pull test examinations while wearing IMUs at baseline. Center of mass and foot position data were used to compare velocity and acceleration profiles, pull test step length, and reaction times between groups and as a function of Unified Parkinson's disease Rating Scale Pull Test (UPDRSPT) score. Results: Overall, the reactive postural response of NPH patients was characterized by slower reaction times and smaller steps compared to both PD patients and healthy controls. However, when patients were grouped by UPDRSPT scores, no reliable objective difference between groups was detected. Conclusion: At their initial evaluation, very few NPH patients demonstrate "normal" or "mild" PI as they appear to be older upon presentation compared to PD patients. As a result, kinematic assessment utilizing IMUs may not be helpful for differentiating between NPH and PD as a function of UPDRSPT score, but rather as a more fine-tuned method to define disease progression. We emphasize the need for further evaluation of incorporating objective kinematic data collection as a way to evaluate PI and improve patient outcomes.

Divergent role of Mitochondrial Amidoxime Reducing Component 1 (MARC1) in human and mouse.

Recent human genome-wide association studies have identified common missense variants in MARC1, p.Ala165Thr and p.Met187Lys, associated with lower hepatic fat, reduction in liver enzymes and protection from most causes of cirrhosis. Using an exome-wide association study we recapitulated earlier MARC1 p.Ala165Thr and p.Met187Lys findings in 540,000 individuals from five ancestry groups. We also discovered novel rare putative loss of function variants in MARC1 with a phenotype similar to MARC1 p.Ala165Thr/p.Met187Lys variants. In vitro studies of recombinant human MARC1 protein revealed Ala165Thr substitution causes protein instability and aberrant localization in hepatic cells, suggesting MARC1 inhibition or deletion may lead to hepatoprotection. Following this hypothesis, we generated Marc1 knockout mice and evaluated the effect of Marc1 deletion on liver phenotype. Unexpectedly, our study found that whole-body Marc1 deficiency in mouse is not protective against hepatic triglyceride accumulation, liver inflammation or fibrosis. In attempts to explain the lack of the observed phenotype, we discovered that Marc1 plays only a minor role in mouse liver while its paralogue Marc2 is the main Marc family enzyme in mice. Our findings highlight the major difference in MARC1 physiological function between human and mouse.

Changes in Cerebrovascular Procedures and Outcomes During COVID-19 Using the National Surgery Quality Improvement Project.

OBJECTIVE: Explore the consequences of the coronavirus pandemic (COVID-19) on patients suffering from cerebrovascular disorders necessitating interventions. METHODS: Using the National Surgical Quality Improvement Program database, patients with cerebrovascular disease who underwent procedures before (2018-2019) and during (2020-2021) COVID-19 were identified. ICD-10 and Current Procedure Terminology codes were employed to classify diseases and elective cases, respectively. Our study analyzed variations in diagnoses, procedures, demographics, mortality and morbidity likelihood scores, and outcomes. Analysis was conducted using R 4.2.1 with tidyverse, haven, and Ime4 packages. Statistical significance was defined as P < 0.05. RESULTS: There was a significant rise in cerebrovascular accidents (CVAs) (9.96% vs. 12.28%) and a decrease in elective carotid endarterectomies (92.30% vs. 87.22%). Carotid stenting increased significantly (7.63% vs. 12.62%), and mortality probability scores rose for CVAs and carotid interventions. Ethnic (Hispanic) and racial minorities (Asians and Black/African American) were disproportionately affected (P < 0.001). Conditions from delayed care increased, and total operative times rose (117.46 vs. 124.33 minutes). Various patient outcomes worsened (P < 0.05), and multivariate analyses showed Hispanic patients had higher mortality and morbidity probability scores (P < 0.05). CONCLUSIONS: The pandemic led to more severe disease progression and reduced diagnoses due to screening delays, indicating deferred care. Prolonged operative times, extended hospital stays, and worsening outcomes, including infections and thrombotic events, hint at the repercussions of persistent staff shortages in health care facilities. Ethnic and racial minorities faced disproportionate impacts. To minimize harm to patients with cerebrovascular disease in future public health crises, it is crucial to develop policies that address these findings.

Recovery of volitional movement with epidural stimulation after "complete" spinal cord injury due to gunshot: A case report and literature review.

Background: Epidural spinal cord stimulation (eSCS) restores volitional movement and improves autonomic function after nonpenetrating and traumatic spinal cord injury (SCI). There is limited evidence of its utility for penetrating SCI (pSCI). Case Description: A 25-year-old male sustained a gunshot wound (GSW) resulting in T6 motor/sensory paraplegia and complete loss of bowel and bladder function. Following eSCS placement, he regained partial volitional movement and has independent bowel movements 40% of the time. Conclusion: A 25-year-old pSCI patient who, following a GSW resulting in T6-level paraplegia, sustained marked recovery of volitional movement and autonomic function following eSCS placement.

Changes in patterns of traumatic brain injury in the Michigan Trauma Quality Improvement Program database early in the COVID-19 pandemic.

OBJECTIVE: The authors' objective was to investigate the impact of the global COVID-19 pandemic on hospital presentation and process of care for the treatment of traumatic brain injuries (TBIs). Improved understanding of these effects will inform sociopolitical and hospital policies in response to future pandemics. METHODS: The Michigan Trauma Quality Improvement Program (MTQIP) database, which contains data from 36 level I and II trauma centers in Michigan and Minnesota, was queried to identify patients who sustained TBI on the basis of head/neck Abbreviated Injury Scale (AIS) codes during the periods of March 13 through July 2 of 2017-2019 (pre-COVID-19 period) and March 13, 2020, through July 2, 2020 (COVID-19 period). Analyses were performed to detect differences in incidence, patient characteristics, injury severity, and outcomes. RESULTS: There was an 18% decrease in the rate of encounters with TBI in the first 8 weeks (March 13 through May 7), followed by a 16% increase during the last 8 weeks (May 8 through July 2), of our COVID-19 period compared with the pre-COVID-19 period. Cumulatively, there was no difference in the rates of encounters with TBI between the COVID-19 and pre-COVID-19 periods. Severity of TBI, as measured with maximum AIS score for the head/neck region and Glasgow Coma Scale score, was also similar between periods. During the COVID-19 period, a greater proportion of patients with TBI presented more than a day after sustaining their injuries (p = 0.046). COVID-19 was also associated with a doubling in the decubitus ulcer rate from 1.0% to 2.1% (p = 0.002) and change in the distribution of discharge status (p = 0.01). Multivariable analysis showed no differences in odds of death/hospice discharge, intensive care unit stay of at least a day, or need for a ventilator for at least a day between the COVID-19 and pre-COVID-19 periods. CONCLUSIONS: During the early months of the COVID-19 pandemic, the number of patients who presented with TBI was initially lower than in the years 2017-2019 prior to the pandemic. However, there was a subsequent increase in the rate of encounters with TBI, resulting in overall similar rates of TBI between March 13 through July 2 during the COVID-19 period and during the pre-COVID-19 period. The COVID-19 cohort was also associated with negative impacts on time to presentation, rate of decubitus ulcers, and discharge with supervision. Policies in response to future pandemics must consider the resources necessary to care for patients with TBI.

Rates of operative intervention for infection after synthetic or autologous cranioplasty: a National Readmissions Database analysis.

OBJECTIVE: The aim of this study was to characterize the clinical utilization and associated charges of autologous bone flap (ABF) versus synthetic flap (SF) cranioplasty and to characterize the postoperative infection risk of SF versus ABF using the National Readmissions Database (NRD). METHODS: The authors used the publicly available NRD to identify index hospitalizations from October 2015 to December 2018 involving elective ABF or SF cranioplasty after traumatic brain injury (TBI) or stroke. Subsequent readmissions were further characterized if patients underwent neurosurgical intervention for treatment of infection or suspected infection. Survey Cox proportional hazards models were used to assess risk of readmission. RESULTS: An estimated 2295 SF and 2072 ABF cranioplasties were performed from October 2015 to December 2018 in the United States. While the total number of cranioplasty operations decreased during the study period, the proportion of cranioplasties utilizing SF increased (p < 0.001), particularly in male patients (p = 0.011) and those with TBI (vs stroke, p = 0.012). The median total hospital charge for SF cranioplasty was $31,200 more costly than ABF cranioplasty (p < 0.001). Of all first-time readmissions, 20% involved surgical treatment for infectious reasons. Overall, 122 SF patients (5.3%) underwent surgical treatment of infection compared with 70 ABF patients (3.4%) on readmission. After accounting for confounders using a multivariable Cox model, female patients (vs male, p = 0.003), those discharged nonroutinely (vs discharge to home or self-care, p < 0.001), and patients who underwent SF cranioplasty (vs ABF, p = 0.011) were more likely to be readmitted for reoperation. Patients undergoing cranioplasty during more recent years (e.g., 2018 vs 2015) were less likely to be readmitted for reoperation because of infection (p = 0.024). CONCLUSIONS: SFs are increasingly replacing ABFs as the material of choice for cranioplasty, despite their association with increased hospital charges. Female sex, nonroutine discharge, and SF cranioplasty are associated with increased risk for reoperation after cranioplasty.

Racial disparities in recommendations for surgical resection of primary brain tumours: a registry-based cohort analysis.

BACKGROUND: Disparities in treatment and outcomes disproportionately affect minority ethnic and racial populations in many surgical fields. Although substantial research in racial disparities has focused on outcomes, little is known about how surgeon recommendations can be influenced by patient race. The aim of this study was to investigate racial and socioeconomic disparities in the surgical management of primary brain tumors. METHODS: In this registry-based cohort study, we used data from the Surveillance, Epidemiology, and End Results (SEER) database (1975-2016) and the American College of Surgeons National Cancer Database (NCDB) in the USA for independent analysis. Adults (aged ≥20 years) with a new diagnosis of meningioma, glioblastoma, pituitary adenoma, vestibular schwannoma, astrocytoma, and oligodendroglioma, with information on tumour size and surgical recommendation were included in the analysis. The primary outcome of this study was the odds of a surgeon recommending against surgical resection at diagnosis of primary brain neoplasms. This outcome was determined using multivariable logistic regression with clinical, demographic, and socioeconomic factors. FINDINGS: This study included US national data from the SEER (1975-2016) and NCDB (2004-17) databases of adults with a new diagnosis of meningioma (SEER n=63 674; NCDB n=222 673), glioblastoma (n=35 258; n=104 047), pituitary adenoma (n=27 506; n=87 772), vestibular schwannoma (n=11 525; n=30 745), astrocytoma (n=5402; n=10 631), and oligodendroglioma (n=3977; n=9187). Independent of clinical and demographic factors, including insurance status and rural-urban continuum code, Black patients had significantly higher odds of recommendation against surgical resection of meningioma (adjusted odds ratio 1·13, 95% CI 1·06-1·21, p<0·0001), glioblastoma (1·14, 1·01-1·28, p=0·038), pituitary adenoma (1·13, 1·05-1·22, p<0·0001), and vestibular schwannoma (1·48, 1·19-1·84, p<0·0001) when compared with White patients in the SEER dataset. Additionally, patients of unknown race had significantly higher odds of recommendation against surgical resection for pituitary adenoma (1·80, 1·41-2·30, p<0·0001) and vestibular schwannoma (1·49, 1·10-2·04, p=0·011). Performing a validation analysis using the NCDB dataset confirmed these significant results for Black patients with meningioma (1·18, 1·14-1·22, p<0·0001), glioblastoma (1·19, 1·12-1·28, p<0·0001), pituitary adenoma (1·21, 1·16-1·25, p<0·0001), and vestibular schwannoma (1·19, 1·04-1·35, p=0·0085), and indicated and indicated that the findings are independent of patient comorbidities. When further restricted to the most recent decade in SEER, these inequities held true for Black patients, except those with glioblastoma (meningioma [1·18, 1·08-1·28, p<0·0001], pituitary adenoma [1·20, 1·09-1·31, p<0·0001], and vestibular schwannoma [1·54, 1·16-2·04, p=0·0031]). INTERPRETATION: Racial disparities in surgery recommendations in the USA exist for patients with primary brain tumours, independent of potential confounders including clinical, demographic, and select socioeconomic factors. Further studies are needed to understand drivers of this bias and enhance equality in surgical care. FUNDING: None.

Resection of disseminated recurrent myxopapillary ependymoma with more than 4-year follow-up: operative nuance for prolonged prone position. Illustrative case.

BACKGROUND: Symptomatic disseminated myxopapillary ependymoma (MPE) in a young person presents a daunting challenge because the risks of prolonged prone positioning and spinal cord injury may outweigh the likelihood of attaining the benefit of gross total resection. OBSERVATIONS: The authors reported the case of a 15-year-old girl with five discrete recurrent spinal cord ependymomas. The patient received a 25-hour surgical procedure for gross total resection of the tumors and fusion over an approximately 33-hour period. She experienced complete resolution of all preoperative neurological symptoms and subsequently received adjuvant radiation therapy. At 52 months after surgery, she was still experiencing neurologically intact, progression-free survival. This case illustrated one of the most extensive recurrent tumor resections for MPE with prolonged disease-free survival reported to date. It may also represent the longest prone position spinal case reported and was notable for a lack of any of the complications commonly associated with the prolonged prone position. LESSONS: The authors discussed the complexity of surgical decision-making in a symptomatic patient with multiple disseminated metastases, technical considerations for resection of intradural and intramedullary spinal cord tumors, and considerations for avoiding complications during prolonged positioning necessary for spinal surgery.

Maximizing hydrophobic peptide recovery in proteomics and antibody development using a mass spectrometry compatible surfactant.

Peptide loss due to surface absorption can happen at any step in a protein analysis workflow and is sometimes especially deleterious for hydrophobic peptides. In this study, we found the LC-MS compatible surfactant, n-Dodecyl-ß-D-maltoside (DDM), can maximize hydrophobic peptide recovery in various samples including single cell digests, mAb clinical PK samples, and mAb peptide mapping samples. In HeLa single cell proteomics analysis, more than half of all unique peptides identified were found only in DDM prepared samples, most of which had significantly higher hydrophobicities compared to peptides in control samples. In clinical PK studies, DDM enhanced hydrophobic complementarity-determining region (CDR) peptide signals significantly. The fold change of CDR peptides' intensity enhancement in DDM added samples compared to controls correlate with peptide retention time and hydrophobicity, providing guidance for surrogate peptide selection and peptide standard handling in PK studies. For peptide mapping analysis of mAbs, DDM can improve hydrophobic peptide signal and solution stability over 48 h in an autosampler at 4 °C, which can aid method qualification and transfer during drug development. Lastly, maximizing hydrophobic peptide recovery from samples dried in vacuo was achieved by DDM reconstitution, which provided higher signal for later eluting peaks and higher proteome coverage overall.

Readmission risk of malignant brain tumor patients undergoing laser interstitial thermal therapy (LITT) and stereotactic needle biopsy (SNB): a covariate balancing weights analysis of the National Readmissions Database (NRD).

PURPOSE: Despite procedural similarities between laser interstitial thermal therapy (LITT) and stereotactic needle biopsy (SNB), LITT induces delayed, pro-inflammatory responses not associated with SNB that may increase the risk of readmission within 30- or 90- days. Here, we explore this hypothesis. METHODS: We queried the National Readmissions Database (NRD, 2010-18) for malignant brain tumor patients who underwent elective LITT or SNB using International Classification of Diseases codes. Readmissions were defined as non-elective inpatient hospitalizations. Survey regression methods and a weighted analysis were utilized to adjust for demographic and clinical differences between LITT and SNB cohorts. RESULTS: During the study period, an estimated 685 malignant brain patients underwent elective LITT and 15,177 underwent elective SNB. Patients undergoing LITT and SNB exhibited comparable median lengths of hospital stay [IQR; LITT = 2 (1, 3); SNB = 1 (1, 2); p = 0.820]. Likelihood of routine discharge was not significantly different between the two procedures (p = 0.263). No significant differences were observed in the odds of 30- or 90-day unplanned readmission between the LITT and SNB cohorts after multivariable adjustment (all p ≥ 0.177). The covariate balancing weighted analysis confirmed comparable 30 or 90-day readmission risk between LITT and SNB treated patients (all p ≥ 0.201). CONCLUSION: The likelihood of 30- and 90-day readmission for malignant brain tumor patients who underwent LITT or SNB are comparable, supporting the safety profile of LITT as therapy for malignant brain cancers.

High throughput and high confidence sequence variant analysis in therapeutic antibodies using Evosep One liquid chromatography tandem mass spectrometry with synthetic heavy peptides.

Sequence variants are anomalous misincorporations of amino acids into the primary structure of therapeutic antibodies during DNA replication and protein biosynthesis. As these low abundance variants contribute to molecular heterogeneity and could negatively impact the safety and efficacy of a protein therapeutic, analytical methods like liquid chromatography tandem mass spectrometry (LC-MS2) are used to monitor them with the goal of establishing control strategies that limit their occurrence. Current LC-MS2 strategies depend on relatively long gradients that minimize coelution between abundant non-variant peptide peaks and trace-level variants to limit ion suppression that can potentially conceal the latter. However, lengthy LC gradients reduce the number of samples that can be analyzed per day, limiting the practicality of LC-MS2 when analyzing large sample sets. Furthermore, confident variant identification partly depends on capturing rich MS2 spectra that localize any amino acid misincorporations, which can be challenging due to the low abundance of this class of analyte. This work drastically reduces the cycle time to run each therapeutic antibody sample with roughly the same or even more variant identifications, compared to traditional LC-MS2 analysis, by integrating an Evosep One LC platform with an Orbitrap Fusion Lumos mass spectrometer. It also introduces a novel strategy using synthetic peptides that contain heavy isotopes placed near both termini to validate lower confidence variants in one targeted LC-MS2 run according to retention time, precursor mass signal, and MS2 fragment patterns shared with the heavy peptide variant. Taken together, this approach enables high-throughput sequence variant analysis at 30 samples per day as well as validation for lower confidence variants that can be integrated into therapeutic antibody process development and characterization.

The Effects of the COVID-19 Pandemic on Penetrating Neurotrauma at a Level 1 Trauma Center.

BACKGROUND/OBJECTIVE: The COVID-19 pandemic has had a profound impact on the global delivery of health care. Recent data suggest a possible impact of the pandemic on patterns of neurotrauma. The aim was to assess the impact of the pandemic on the incidence of neurotrauma, with a focus on cranial gunshot wounds (cGSWs) at a large Midwestern level 1 trauma center. METHODS: We conducted a retrospective review of our trauma registry from March through September 2020 and compared it to the same months in 2019. Odds ratios were utilized to assess for differences in patient demographics, injury characteristics, rates of neurotrauma, and rates of cGSWs. RESULTS: A total of 1188 patients presented with neurotrauma, 558 in 2019 and 630 in 2020. The majority of patients were male (71.33% in 2019; 68.57% in 2020) and Caucasian (78.67% in 2019; 75.4% in 2020). Patients presented with cGSWs more frequently in 2020 (n = 49, 7.78%) than in 2019 (n = 25, 4.48%). The odds of suffering a cGSW in 2020 was 73.6% higher than those in 2019 (95% confidence interval = [1.0871, 2.7722]; P = 0.0209). The etiology of such injury was most commonly assault (n = 16, 21.62% in 2019; n = 34, 45.95% in 2020), followed by self-inflicted injury (n = 4, 5.41% in 2019; 12, 16.22% in 2020). CONCLUSIONS: Despite the government-mandated shutdown, we observed an increase in the number of neurotrauma cases in 2020. There was a significant increase in the incidence cGSWs in 2020, with an increase in assaults and self-inflicted injuries. Further investigation into socioeconomic factors for the observed increase in cGSWs is warranted.

Testing mechanisms of DNA sliding by architectural DNA-binding proteins: dynamics of single wild-type and mutant protein molecules in vitro and in vivo.

Architectural DNA-binding proteins (ADBPs) are abundant constituents of eukaryotic or bacterial chromosomes that bind DNA promiscuously and function in diverse DNA reactions. They generate large conformational changes in DNA upon binding yet can slide along DNA when searching for functional binding sites. Here we investigate the mechanism by which ADBPs diffuse on DNA by single-molecule analyses of mutant proteins rationally chosen to distinguish between rotation-coupled diffusion and DNA surface sliding after transient unbinding from the groove(s). The properties of yeast Nhp6A mutant proteins, combined with molecular dynamics simulations, suggest Nhp6A switches between two binding modes: a static state, in which the HMGB domain is bound within the minor groove with the DNA highly bent, and a mobile state, where the protein is traveling along the DNA surface by means of its flexible N-terminal basic arm. The behaviors of Fis mutants, a bacterial nucleoid-associated helix-turn-helix dimer, are best explained by mobile proteins unbinding from the major groove and diffusing along the DNA surface. Nhp6A, Fis, and bacterial HU are all near exclusively associated with the chromosome, as packaged within the bacterial nucleoid, and can be modeled by three diffusion modes where HU exhibits the fastest and Fis the slowest diffusion.

Pattern of technology diffusion in the adoption of stereotactic laser interstitial thermal therapy (LITT) in neuro-oncology.

PURPOSE: Understanding factors that influence technology diffusion is central to clinical translation of novel therapies. We characterized the pattern of adoption for laser interstitial thermal therapy (LITT), also known as stereotactic laser ablation (SLA), in neuro-oncology using the National Inpatient Sample (NIS) database. METHODS: We identified patients age ≥ 18 in the NIS (2012-2018) with a diagnosis of primary or metastatic brain tumor that underwent LITT or craniotomy. We compared characteristics and outcomes for patients that underwent these procedures. RESULTS: LITT utilization increased ~ 400% relative to craniotomy during the study period. Despite this increase, the total number of LITT procedures performed for brain tumor was < 1% of craniotomy. After adjusting for this time trend, LITT patients were less likely to have > 2 comorbidities (OR 0.64, CI95 0.51-0.79) or to be older (OR 0.92, CI95 0.86-0.99) and more likely to be female (OR 1.35, CI95 1.08-1.69), Caucasian compared to Black (OR 1.94, CI95 1.12-3.36), and covered by private insurance compared to Medicare or Medicaid (OR 1.38, CI95 1.09-1.74). LITT hospital stays were 50% shorter than craniotomy (IRR 0.52, CI95 0.45-0.61). However, charges related to the procedures were comparable between LITT and craniotomy ($1397 greater for LITT, CI95 $-5790 to $8584). CONCLUSION: For neuro-oncology indications, LITT utilization increased ~ 400% relative to craniotomy. Relative to craniotomy-treated patients, LITT-treated patients were likelier to be young, female, non-Black race, covered by private insurance, or with < 2 comorbidities. While the total hospital charges were comparable, LITT was associated with a shorter hospitalization relative to craniotomy.

Control of the Serine Integrase Reaction: Roles of the Coiled-Coil and Helix E Regions in DNA Site Synapsis and Recombination.

Bacteriophage serine integrases catalyze highly specific recombination reactions between defined DNA segments called att sites. These reactions are reversible depending upon the presence of a second phage-encoded directionality factor. The bipartite C-terminal DNA-binding region of integrases includes a recombinase domain (RD) connected to a zinc-binding domain (ZD), which contains a long flexible coiled-coil (CC) motif that extends away from the bound DNA. We directly show that the identities of the phage A118 integrase att sites are specified by the DNA spacing between the RD and ZD DNA recognition determinants, which in turn directs the relative trajectories of the CC motifs on each subunit of the att-bound integrase dimer. Recombination between compatible dimer-bound att sites requires minimal-length CC motifs and 14 residues surrounding the tip where the pairing of CC motifs between synapsing dimers occurs. Our alanine-scanning data suggest that molecular interactions between CC motif tips may differ in integrative (attP × attB) and excisive (attL × attR) recombination reactions. We identify mutations in 5 residues within the integrase oligomerization helix that control the remodeling of dimers into tetramers during synaptic complex formation. Whereas most of these gain-of-function mutants still require the CC motifs for synapsis, one mutant efficiently, but indiscriminately, forms synaptic complexes without the CC motifs. However, the CC motifs are still required for recombination, suggesting a function for the CC motifs after the initial assembly of the integrase synaptic tetramer. IMPORTANCE The robust and exquisitely regulated site-specific recombination reactions promoted by serine integrases are integral to the life cycle of temperate bacteriophage and, in the case of the A118 prophage, are an important virulence factor of Listeria monocytogenes. The properties of these recombinases have led to their repurposing into tools for genetic engineering and synthetic biology. In this report, we identify determinants regulating synaptic complex formation between correct DNA sites, including the DNA architecture responsible for specifying the identity of recombination sites, features of the unique coiled-coil structure on the integrase that are required to initiate synapsis, and amino acid residues on the integrase oligomerization helix that control the remodeling of synapsing dimers into a tetramer active for DNA strand exchange.

Simple and Sensitive Method for Deep Profiling of Host Cell Proteins in Therapeutic Antibodies by Combining Ultra-Low Trypsin Concentration Digestion, Long Chromatographic Gradients, and BoxCar Mass Spectrometry Acquisition.

Liquid chromatography coupled to mass spectrometry (LC-MS) is a powerful tool for the analysis of host cell proteins (HCP) during antibody drug process development due to its sensitivity, selectivity, and adaptability. However, the enormous dynamic range between the therapeutic antibody and accompanying HCPs poses a significant challenge for LC-MS based detection of these low abundance impurities. To address this challenge, enrichment of HCPs via immunoaffinity, protein A, 2D-LC, or other strategies is typically performed. However, these enrichments are time-consuming and sometimes require a large quantity of sample. Here, we report a simple and sensitive strategy to analyze HCPs in therapeutic antibody samples without cumbersome enrichment by combining an ultra-low trypsin concentration during digestion under nondenaturing conditions, a long chromatographic gradient, and BoxCar acquisition (ULTLB) on a quadrupole-Orbitrap mass spectrometer. Application of this strategy to the NIST monoclonal antibody standard (NISTmAb) resulted in the identification of 453 mouse HCPs, which is a significant increase in the number of identified HCPs without enrichment compared to previous reports. Known amounts of HCPs were spiked into the purified antibody drug substance, demonstrating that the method sensitivity is as low as 0.5 ppm. Thus, the ULTLB method represents a sensitive and simple platform for deep profiling of HCPs in antibodies.

Electron Transfer Dissociation Parameter Optimization Using Design of Experiments Increases Sequence Coverage of Monoclonal Antibodies.

Middle-down analysis of monoclonal antibodies (mAbs) by tandem mass spectrometry (MS2) can provide detailed insight into their primary structure with minimal sample preparation. The middle-down approach uses an enzyme to cleave mAbs into Fc/2, LC, and Fd subunits that are then analyzed by reversed phase liquid chromatography tandem mass spectrometry (RPLC-MS2). As maximum sequence coverage is desired to obtain meaningful structural information at the subunit level, a host of dissociation methods have been developed, and sometimes combined, to bolster fragmentation and increase the number of identified fragments. Here, we present a design of experiments (DOE) approach to optimize MS2 parameters, in particular those that may influence electron transfer dissociation (ETD) efficiency to increase the sequence coverage of antibody subunits. Applying this approach to the NIST monoclonal antibody standard (NISTmAb) using three RPLC-MS2 runs resulted in high sequence coverages of 67%, 67%, and 52% for Fc/2, LC, and Fd subunits, respectively. In addition, we apply this DOE strategy to model the parameters required to maximize the number of fragments produced in "low", "medium", and "high" mass ranges, which ultimately resulted in even higher sequence coverages of NISTmAb subunits (75%, 78%, and 64% for Fc/2, LC, and Fd subunits, respectively). The DOE approach provides high sequence coverage percentages utilizing only one fragmentation method, ETD, and could be extended to other state-of-the-art techniques that combine multiple fragmentation mechanisms to increase coverage.

Integration of liquid chromatography mass spectrometry with a heavy peptide response curve accurately measures unprocessed C-terminal lysine during peptide mapping analysis of therapeutic antibodies in a single run.

Therapeutic monoclonal and bispecific antibodies are susceptible to modification after protein biosynthesis. These post-translational modifications (PTMs) not only contribute to mass and charge heterogeneity, but they can also negatively impact the molecule's activity, half-life, and immunogenicity. Therefore, identification and quantification of PTMs are critical to ensure the safety and efficacy of an antibody therapeutic as well as demonstrate product consistency and process control. Unprocessed C-terminal lysine on the heavy chain (HC) is a prevalent modification that contributes to this charge heterogeneity in antibodies. Peptide mapping through liquid chromatography tandem mass spectrometry (LC-MS2) enjoys higher selectivity and sensitivity for measuring this PTM relative to global PTM methods, but differences in the ionization efficiencies of the unprocessed C-terminal K peptide and the truncated C-terminal K peptide result in its overestimation. Consequently, large discrepancies in this PTM's measured abundance may exist between different characterization assays used in regulatory filings, which can be further compounded by large variability when multiple mass spectrometers are used to quantify C-terminal K during a therapeutic's lifespan. In this study, we propose a simple new method to quantify unprocessed C-terminal K in antibodies in a single LC-MS2 run that incorporates heavy isotopic standards for both the unprocessed and truncated C-terminal K peptide to build a response curve and correct for the disparity in ionization efficiency between these two different peptide sequences. The approach was evaluated across two different Orbitrap-based mass spectrometers using multiple monoclonal and bispecific therapeutic antibodies, resulting in accurate (<10% error, as determined with peptide standards) and precise C-terminal K quantification during peptide mapping analysis.

The HMGB chromatin protein Nhp6A can bypass obstacles when traveling on DNA.

DNA binding proteins rapidly locate their specific DNA targets through a combination of 3D and 1D diffusion mechanisms, with the 1D search involving bidirectional sliding along DNA. However, even in nucleosome-free regions, chromosomes are highly decorated with associated proteins that may block sliding. Here we investigate the ability of the abundant chromatin-associated HMGB protein Nhp6A from Saccharomyces cerevisiae to travel along DNA in the presence of other architectural DNA binding proteins using single-molecule fluorescence microscopy. We observed that 1D diffusion by Nhp6A molecules is retarded by increasing densities of the bacterial proteins Fis and HU and by Nhp6A, indicating these structurally diverse proteins impede Nhp6A mobility on DNA. However, the average travel distances were larger than the average distances between neighboring proteins, implying Nhp6A is able to bypass each of these obstacles. Together with molecular dynamics simulations, our analyses suggest two binding modes: mobile molecules that can bypass barriers as they seek out DNA targets, and near stationary molecules that are associated with neighboring proteins or preferred DNA structures. The ability of mobile Nhp6A molecules to bypass different obstacles on DNA suggests they do not block 1D searches by other DNA binding proteins.

Combination of FAIMS, Protein A Depletion, and Native Digest Conditions Enables Deep Proteomic Profiling of Host Cell Proteins in Monoclonal Antibodies.

Host cell proteins (HCPs) are residual impurities generated by the expression cell line during the production of biopharmaceuticals. Although the majority of these contaminants are removed during purification, HCPs can represent a considerable risk to the efficacy and safety of a therapeutic protein if not actively monitored. The enzyme-linked immunosorbent assay (ELISA) is commonly used throughout production to monitor HCP levels but has limited ability to identify novel HCPs or provide detailed quantification. Liquid chromatography tandem mass spectrometry (LC-MS2) methods are increasingly being used in conjunction with established ELISA techniques to provide rapid adaptability to increasingly complex samples as well as highly quantitative and informative results. However, MS-based methods are still hindered by the large dynamic range between high abundance biopharmaceutical proteins and low abundance HCPs. Here, we propose a multifactorial approach designed to optimize HCP detection in purified monoclonal antibody samples with LC-MS2. By first depleting the sample of antibody on a protein A column, then specifically digesting HCPs while precipitating remaining antibody, and finally reducing spectral complexity through compensation voltage (CV) switching using high-field asymmetric waveform ion mobility spectrometry (FAIMS), we identified multiple-fold more HCPs in the NIST monoclonal antibody standard than any single established mass spectrometry technique reported in the literature. Our analyses consistently identified over 600 high confidence mouse HCPs, a multifold increase over established methods, while maintaining high reproducibility.