Chemoproteomic discovery of a covalent allosteric inhibitor of WRN helicase.

WRN helicase is a promising target for treatment of cancers with microsatellite instability (MSI) due to its essential role in resolving deleterious non-canonical DNA structures that accumulate in cells with faulty mismatch repair mechanisms1-5. Currently there are no approved drugs directly targeting human DNA or RNA helicases, in part owing to the challenging nature of developing potent and selective compounds to this class of proteins. Here we describe the chemoproteomics-enabled discovery of a clinical-stage, covalent allosteric inhibitor of WRN, VVD-133214. This compound selectively engages a cysteine (C727) located in a region of the helicase domain subject to interdomain movement during DNA unwinding. VVD-133214 binds WRN protein cooperatively with nucleotide and stabilizes compact conformations lacking the dynamic flexibility necessary for proper helicase function, resulting in widespread double-stranded DNA breaks, nuclear swelling and cell death in MSI-high (MSI-H), but not in microsatellite-stable, cells. The compound was well tolerated in mice and led to robust tumour regression in multiple MSI-H colorectal cancer cell lines and patient-derived xenograft models. Our work shows an allosteric approach for inhibition of WRN function that circumvents competition from an endogenous ATP cofactor in cancer cells, and designates VVD-133214 as a promising drug candidate for patients with MSI-H cancers.

Driving Under the Influence of Cannabis: Impact of Combining Toxicology Testing with Field Sobriety Tests.

BACKGROUND: Cannabis is increasingly used both medically and recreationally. With widespread use, there is growing concern about how to identify cannabis-impaired drivers. METHODS: A placebo-controlled randomized double-blinded protocol was conducted to study the effects of cannabis on driving performance. One hundred ninety-one participants were randomized to smoke ad libitum a cannabis cigarette containing placebo or delta-9-tetrahydrocannabinol (THC) (5.9% or 13.4%). Blood, oral fluid (OF), and breath samples were collected along with longitudinal driving performance on a simulator (standard deviation of lateral position [SDLP] and car following [coherence]) over a 5-hour period. Law enforcement officers performed field sobriety tests (FSTs) to determine if participants were impaired. RESULTS: There was no relationship between THC concentrations measured in blood, OF, or breath and SDLP or coherence at any of the timepoints studied (P > 0.05). FSTs were significant (P < 0.05) for classifying participants into the THC group vs the placebo group up to 188 minutes after smoking. Seventy-one minutes after smoking, FSTs classified 81% of the participants who received active drug as being impaired. However, 49% of participants who smoked placebo (controls) were also deemed impaired at this same timepoint. Combining a 2 ng/mL THC cutoff in OF with positive findings on FSTs reduced the number of controls classified as impaired to zero, 86 minutes after smoking the placebo. CONCLUSIONS: Requiring a positive toxicology result in addition to the FST observations substantially improved the classification accuracy regarding possible driving under the influence of THC by decreasing the percentage of controls classified as impaired.

A Rapid Assay for SARS-CoV-2 Neutralizing Antibodies That Is Insensitive to Antiretroviral Drugs.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike pseudotyped virus (PSV) assays are widely used to measure neutralization titers of sera and of isolated neutralizing Abs (nAbs). PSV neutralization assays are safer than live virus neutralization assays and do not require access to biosafety level 3 laboratories. However, many PSV assays are nevertheless somewhat challenging and require at least 2 d to carry out. In this study, we report a rapid (<30 min), sensitive, cell-free, off-the-shelf, and accurate assay for receptor binding domain nAb detection. Our proximity-based luciferase assay takes advantage of the fact that the most potent SARS-CoV-2 nAbs function by blocking the binding between SARS-CoV-2 and angiotensin-converting enzyme 2. The method was validated using isolated nAbs and sera from spike-immunized animals and patients with coronavirus disease 2019. The method was particularly useful in patients with HIV taking antiretroviral therapies that interfere with the conventional PSV assay. The method provides a cost-effective and point-of-care alternative to evaluate the potency and breadth of the predominant SARS-CoV-2 nAbs elicited by infection or vaccines.

Implementing Guidelines for NICU Parent Presence: Effects on Parent and Infant Stress.

BACKGROUND: Relationships between parents and infants are essential for mitigating stressors encountered in neonatal intensive care units (NICUs) and are supported by parent presence and engagement. PURPOSE: The purpose of this study was to compare NICU parent and infant outcomes pre- and postimplementation of an intervention aimed at increasing parent presence and engagement in the NICU. This family-centered care intervention consisted of communicating specific guidelines for parent presence. METHODS: Data related to parent presence, skin-to-skin care, and breastfeeding; parental stress; infant outcomes including weight gain, length of stay, feeding status at discharge, and stress; and unit-level outcomes were collected from a convenience sample of 40 NICU families recruited preimplementation and compared with data for 38 NICU families recruited postimplementation of specific guidelines for parent presence. To establish comparability of groups, infants were assigned scores using the Neonatal Medical Index. RESULTS: Parent presence, engagement in skin-to-skin care, and breastfeeding rates were not significantly different between groups. Stress-related outcomes were significantly decreased in NICU mothers, fathers, and infants, and infant feeding outcomes were improved in the postintervention group. CONCLUSIONS: Specific guidelines for parent presence may represent an invitation for parents to engage with their NICU infants and may positively impact parent and infant stress.

Commercial Serology Assays Predict Neutralization Activity against SARS-CoV-2.

BACKGROUND: It is unknown whether a positive serology result correlates with protective immunity against SARS-CoV-2. There are also concerns regarding the low positive predictive value of SARS-CoV-2 serology tests, especially when testing populations with low disease prevalence. METHODS: A neutralization assay was validated in a set of PCR-confirmed positive specimens and in a negative cohort. In addition, 9530 specimens were screened using the Diazyme SARS-CoV-2 IgG serology assay and all positive results (N = 164 individuals) were reanalyzed using the neutralization assay, the Roche total immunoglobin assay, and the Abbott IgG assay. The relationship between the magnitude of a positive SARS-CoV-2 serology result and neutralizing activity was determined. Neutralizing antibody titers (50% inhibitory dilution, ID50) were also longitudinally monitored in patients confirmed to have SARS-CoV-2 by PCR. RESULTS: The SARS-CoV-2 neutralization assay had a positive percentage agreement (PPA) of 96.6% with a SARS-CoV-2 PCR test and a negative percentage agreement (NPA) of 98.0% across 100 negative control individuals. ID50 neutralization titers positively correlated with all 3 clinical serology platforms. Longitudinal monitoring of hospitalized PCR-confirmed patients with COVID-19 demonstrated they made high neutralization titers against SARS-CoV-2. PPA between the Diazyme IgG assay alone and the neutralization assay was 50.6%, while combining the Diazyme IgG assay with either the Roche or Abbott platforms increased the PPA to 79.2 and 78.4%, respectively. CONCLUSIONS: These 3 clinical serology assays positively correlate with SARS-CoV-2 neutralization activity observed in patients with COVID-19. All patients confirmed SARS-CoV-2 positive by PCR develop neutralizing antibodies.

Dermatologic support for oncology: Quantifying the consultative services received by hospitalized oncology patients.

Hospitalized oncology patients often require multidisciplinary care. Inpatient consultative dermatologists can provide expertise in the management of cutaneous complications that patients with cancer may experience. The goal of this study was to quantify the types of consults received by hospitalized oncology patients to better understand the utilization of dermatology consults in this population. Hospital billing codes were used to identify inpatient oncology patients and the types of consults they received at a single quaternary care hospital center. Between July 1, 2015, and January 31, 2020, 14,175 patients were admitted to an oncology service for more than 24 hours, and 5,243 (37%) of these patients received at least 1 consultation during their hospital admission. These patients received a total of 10,492 consults from 101 different services. Dermatology had the fifth-highest number of consults (n = 623; 5.9%). Among patients receiving consults, 608 (11.6%) received inpatient dermatology consults. Infectious disease was the service with the most consults (n = 1,485; 14.2%) and was also the service most commonly co-consulted with dermatology (n = 262; 42.1%). The inpatient consultative dermatology service is highly utilized among hospitalized oncology patients, suggesting that expertise in dermatologic care is valued by oncology teams.

Calibrating from Within: Multipoint Internal Calibration of a Quantitative Mass Spectrometric Assay of Serum Methotrexate.

BACKGROUND: Clinical LC-MS/MS assays traditionally require that samples be run in batches with calibration curves in each batch. This approach is inefficient and presents a barrier to random access analysis. We developed an alternative approach called multipoint internal calibration (MPIC) that eliminated the need for batch-mode analysis. METHODS: The new approach used 4 variants of 13C-labeled methotrexate (0.026-10.3 µM) as an internal calibration curve within each sample. One site carried out a comprehensive validation, which included an evaluation of interferences and matrix effects, lower limit of quantification (LLOQ), and 20-day precision. Three sites evaluated assay precision and linearity. MPIC was also compared with traditional LC-MS/MS and an immunoassay. RESULTS: Recovery of spiked analyte was 93%-102%. The LLOQ was validated to be 0.017 µM. Total variability, determined in a 20-day experiment, was 11.5%CV. In a 5-day variability study performed at each site, total imprecision was 3.4 to 16.8%CV. Linearity was validated throughout the calibrator range (r2 > 0.995, slopes = 0.996-1.01). In comparing 40 samples run in each laboratory, the median interlaboratory imprecision was 6.55%CV. MPIC quantification was comparable to both traditional LC-MS/MS and immunoassay (r2 = 0.96-0.98, slopes = 1.04-1.06). Bland-Altman analysis of all comparisons showed biases rarely exceeding 20% when MTX concentrations were >0.4 µM. CONCLUSION: The MPIC method for serum methotrexate quantification was validated in a multisite proof-of-concept study and represents a big step toward random-access LC-MS/MS analysis, which could change the paradigm of mass spectrometry in the clinical laboratory.

Validation of a liquid chromatography tandem mass spectrometry (LC-MS/MS) method to detect cannabinoids in whole blood and breath.

Background The widespread availability of cannabis raises concerns regarding its effect on driving performance and operation of complex equipment. Currently, there are no established safe driving limits regarding ∆9-tetrahydrocannabinol (THC) concentrations in blood or breath. Daily cannabis users build up a large body burden of THC with residual excretion for days or weeks after the start of abstinence. Therefore, it is critical to have a sensitive and specific analytical assay that quantifies THC, the main psychoactive component of cannabis, and multiple metabolites to improve interpretation of cannabinoids in blood; some analytes may indicate recent use. Methods A liquid chromatography tandem mass spectrometry (LC-MS/MS) method was developed to quantify THC, cannabinol (CBN), cannabidiol (CBD), 11-hydroxy-THC (11-OH-THC), (±)-11-nor-9-carboxy-Δ9-THC (THCCOOH), (+)-11-nor-Δ9-THC-9-carboxylic acid glucuronide (THCCOOH-gluc), cannabigerol (CBG), and tetrahydrocannabivarin (THCV) in whole blood (WB). WB samples were prepared by solid-phase extraction (SPE) and quantified by LC-MS/MS. A rapid and simple method involving methanol elution of THC in breath collected in SensAbues® devices was optimized. Results Lower limits of quantification ranged from 0.5 to 2 µg/L in WB. An LLOQ of 80 pg/pad was achieved for THC concentrations in breath. Calibration curves were linear (R2>0.995) with calibrator concentrations within ±15% of their target and quality control (QC) bias and imprecision ≤15%. No major matrix effects or drug interferences were observed. Conclusions The methods were robust and adequately quantified cannabinoids in biological blood and breath samples. These methods will be used to identify cannabinoid concentrations in an upcoming study of the effects of cannabis on driving.

Risk factors for keratinocyte carcinoma skin cancer in nonwhite individuals: A retrospective analysis.

BACKGROUND: Because most of the US population will consist of nonwhite individuals by the year 2043, it is essential that both physicians and patients are educated about skin cancer in nonwhite persons. OBJECTIVE: To update the epidemiology, investigate specific risk factors, and facilitate earlier diagnosis and intervention of keratinocyte carcinoma in nonwhite individuals. METHODS: Institutional review board-approved retrospective chart review of all nonwhite patients who had received a biopsy-proven diagnosis of skin cancer at Drexel Dermatology during June 2008-June 2015. RESULTS: Squamous cell carcinoma (SCC) was the most commonly diagnosed skin cancer in black and Asian populations, and basal cell carcinoma was the most common skin cancer in Hispanics. Black persons exhibited the majority of their SCC lesions in sun-protected areas, particularly the anogenital area. On average, current smokers received skin cancer diagnoses 12.27 years earlier than former smokers and 9.36 years earlier than nonsmokers. LIMITATIONS: Single-center design and interpractitioner variability of skin examination. CONCLUSION: The importance of lesions in photoprotected areas in nonwhite individuals should not go overlooked. However, emphasis should also be placed on active examination of sun-protected areas in nonwhite persons and recognition of the relationship between human papillomavirus and genital SCC lesions. Smoking cessation should be integrated in dermatologic counseling of all patients. Interventions tailored to each of these ethnic groups are needed.

Hypopigmented Macules as Manifestation of Lichen Planus and Lichen Planopilaris.

Lichen planus (LP) is an idiopathic inflammatory disease of the skin, hair, nails, and mucous membranes. Classic cutaneous LP is characterized by violaceous flat-topped papules that typically favor the extremities. LP on the scalp, otherwise known as lichen planopilaris, classically presents with scarring alopecia, perifollicular erythema and follicular prominence. Although LP pigmentosus presents primarily as hyperpigmentation, there is only one previous report of hypopigmented LP. In this report, the authors report 2 cases of LP that presented primarily as hypopigmented macules in 2 African American men. The first patient presented with hypopigmented macules on face and scalp as well as trunk and extremities. The second patient presented with hypopigmented macules on scalp with associated alopecia. Histopathological examination from both patients showed features of LP. The authors propose a new variant of LP that presents acutely as hypopigmented lesions.

Contact dermatitis: one for the books.

Allergic contact dermatitis (ACD) is a frequent problem, often caused from repeated exposure to an object or substance related to the patient's routine activities. We present a case of a well-demarcated, erythematous, scaly plaque on a finger caused from reading with an e-book device. Although metal from mobile devices can cause ACD, mobile device cases may cause irritation or contain additives that can also cause contact dermatitis. Similar presentations of contact dermatitis may become more common as technology use increases.

Comparison of Quantitative Mass Spectrometry Platforms for Monitoring Kinase ATP Probe Uptake in Lung Cancer.

Recent developments in instrumentation and bioinformatics have led to new quantitative mass spectrometry platforms including LC-MS/MS with data-independent acquisition (DIA) and targeted analysis using parallel reaction monitoring mass spectrometry (LC-PRM), which provide alternatives to well-established methods, such as LC-MS/MS with data-dependent acquisition (DDA) and targeted analysis using multiple reaction monitoring mass spectrometry (LC-MRM). These tools have been used to identify signaling perturbations in lung cancers and other malignancies, supporting the development of effective kinase inhibitors and, more recently, providing insights into therapeutic resistance mechanisms and drug repurposing opportunities. However, detection of kinases in biological matrices can be challenging; therefore, activity-based protein profiling enrichment of ATP-utilizing proteins was selected as a test case for exploring the limits of detection of low-abundance analytes in complex biological samples. To examine the impact of different MS acquisition platforms, quantification of kinase ATP uptake following kinase inhibitor treatment was analyzed by four different methods: LC-MS/MS with DDA and DIA, LC-MRM, and LC-PRM. For discovery data sets, DIA increased the number of identified kinases by 21% and reduced missingness when compared with DDA. In this context, MRM and PRM were most effective at identifying global kinome responses to inhibitor treatment, highlighting the value of a priori target identification and manual evaluation of quantitative proteomics data sets. We compare results for a selected set of desthiobiotinylated peptides from PRM, MRM, and DIA and identify considerations for selecting a quantification method and postprocessing steps that should be used for each data acquisition strategy.

Evaluating kinase ATP uptake and tyrosine phosphorylation using multiplexed quantification of chemically labeled and post-translationally modified peptides.

Cancer biologists and other healthcare researchers face an increasing challenge in addressing the molecular complexity of disease. Biomarker measurement tools and techniques now contribute to both basic science and translational research. In particular, liquid chromatography-multiple reaction monitoring mass spectrometry (LC-MRM) for multiplexed measurements of protein biomarkers has emerged as a versatile tool for systems biology. Assays can be developed for specific peptides that report on protein expression, mutation, or post-translational modification; discovery proteomics data rapidly translated into multiplexed quantitative approaches. Complementary advances in affinity purification enrich classes of enzymes or peptides representing post-translationally modified or chemically labeled substrates. Here, we illustrate the process for the relative quantification of hundreds of peptides in a single LC-MRM experiment. Desthiobiotinylated peptides produced by activity-based protein profiling (ABPP) using ATP probes and tyrosine-phosphorylated peptides are used as examples. These targeted quantification panels can be applied to further understand the biology of human disease.

Rituximab Use in Pediatric Dermatology.

Rituximab, an anti CD20 monoclonal antibody leading to transitory B cell depletion, is used to treat a wide variety of immune system tumors and immune mediated diseases. While most of data supporting the efficacy and safety of rituximab in treating autoimmune patients is focused on the adult population, the utilization of rituximab (RTX) for a wide range of pediatric conditions is also increasing. While there are a number of published case reports, a comprehensive review of the various uses for rituximab in pediatric dermatology is lacking. To better assess the therapeutic role of rituximab in the management of skin disease in children, here we comprehensively document reported cases of use including details regarding specific treatment regimens, efficacy and safety profile. Evaluation of the data supports consideration for the initiation of rituximab at early time points in the treatment ladder, before certain diseases become refractory to conventional treatment.

J Drugs Dermatol. 2016;15(7):821-829.

Would you be surprised? Prospective multicenter study of the Surprise Question as a screening tool to predict mortality in trauma patients.

BACKGROUND: The Surprise Question (SQ) ("Would I be surprised if the patient died within the next year?") is a validated tool used to identify patients with limited life expectancy. Because it may have potential to expedite palliative care interventions per American College of Surgeons Trauma Quality Improvement Program Palliative Care Best Practices Guidelines, we sought to determine if trauma team members could use the SQ to accurately predict 1-year mortality in trauma patients. METHODS: A multicenter, prospective, cohort study collected data (August 2020 to February 2021) on trauma team members' responses to the SQ at 24 hours from admission. One-year mortality was obtained via social security death index records. Positive/negative predictive values and accuracy were calculated overall, by provider role and by patient age. RESULTS: Ten Level I/II centers enrolled 1,172 patients (87.9% blunt). The median age was 57 years (interquartile range, 36-74 years), and the median Injury Severity Score was 10 (interquartile range, 5-14 years). Overall 1-year mortality was 13.3%. Positive predictive value was low (30.5%) regardless of role. Mortality prediction minimally improved as age increased (positive predictive value highest between 65 and 74 years old, 34.5%) but consistently trended to overprediction of death, even in younger patients. CONCLUSION: Trauma team members' ability to forecast 1-year mortality using the SQ at 24 hours appears limited perhaps because of overestimation of injury effects, preinjury conditions, and/or team bias. This has implications for the Trauma Quality Improvement Program Guidelines and suggests that more research is needed to determine the optimal time to screen trauma patients with the SQ. LEVEL OF EVIDENCE: Prognostic and Epidemiological; Level III.