Dynamics of an incoherent feedforward loop drive ERK-dependent pattern formation in the early Drosophila embryo.

Positional information in development often manifests as stripes of gene expression, but how stripes form remains incompletely understood. Here, we use optogenetics and live-cell biosensors to investigate the posterior brachyenteron (byn) stripe in early Drosophila embryos. This stripe depends on interpretation of an upstream ERK activity gradient and the expression of two target genes, tailless (tll) and huckebein (hkb), that exert antagonistic control over byn. We find that high or low doses of ERK signaling produce transient or sustained byn expression, respectively. Although tll transcription is always rapidly induced, hkb converts graded ERK inputs into a variable time delay. Nuclei thus interpret ERK amplitude through the relative timing of tll and hkb transcription. Antagonistic regulatory paths acting on different timescales are hallmarks of an incoherent feedforward loop, which is sufficient to explain byn dynamics and adds temporal complexity to the steady-state model of byn stripe formation. We further show that 'blurring' of an all-or-none stimulus through intracellular diffusion non-locally produces a byn stripe. Overall, we provide a blueprint for using optogenetics to dissect developmental signal interpretation in space and time.

B-cell-directed CAR T-cell therapy activates CD8+ cytotoxic CARneg bystander T cells in patients and nonhuman primates.

ABSTRACT: Chimeric antigen receptor (CAR) T cells hold promise as a therapy for B-cell-derived malignancies, and despite their impressive initial response rates, a significant proportion of patients ultimately experience relapse. Although recent studies have explored the mechanisms of in vivo CAR T-cell function, little is understood about the activation of surrounding CARneg bystander T cells and their potential to enhance tumor responses. We performed single-cell RNA sequencing on nonhuman primate (NHP) and patient-derived T cells to identify the phenotypic and transcriptomic hallmarks of bystander activation of CARneg T cells following B-cell-targeted CAR T-cell therapy. Using a highly translatable CD20 CAR NHP model, we observed a distinct population of activated CD8+ CARneg T cells emerging during CAR T-cell expansion. These bystander CD8+ CARneg T cells exhibited a unique transcriptional signature with upregulation of natural killer-cell markers (KIR3DL2, CD160, and KLRD1), chemokines, and chemokine receptors (CCL5, XCL1, and CCR9), and downregulation of naïve T-cell-associated genes (SELL and CD28). A transcriptionally similar population was identified in patients after a tisagenlecleucel infusion. Mechanistic studies revealed that interleukin-2 (IL-2) and IL-15 exposure induced bystander-like CD8+ T cells in a dose-dependent manner. In vitro activated and patient-derived T cells with a bystander phenotype efficiently killed leukemic cells through a T-cell receptor-independent mechanism. Collectively, to our knowledge, these data provide the first comprehensive identification and profiling of CARneg bystander CD8+ T cells following B-cell-targeting CAR T-cell therapy and suggest a novel mechanism through which CAR T-cell infusion might trigger enhanced antileukemic responses. Patient samples were obtained from the trial #NCT03369353, registered at www.ClinicalTrials.gov.

Integrating Data Across Multiple Sites in the Northeastern United States to Examine Associations Between a Prenatal Metal Mixture and Child Cognition.

We applied a novel hierarchical Bayesian weighted quantile sum (HBWQS) regression to combine data across 3 study sites to examine associations between prenatal exposure to metals and cognitive functioning in childhood. Data from 326 mother-child dyads enrolled in an ongoing cohort study, the Programming of Intergenerational Stress Mechanisms (PRISM) Study, based in New York, New York (recruitment in 2013-2020) and Boston, Massachusetts (recruitment 2011-2013), and the First Thousand Days of Life (FTDL) cohort study (recruitment 2012-2019), based in northern Virginia, were used. Arsenic, cadmium, manganese, lead, and antimony were measured in urine collected during pregnancy. Cognitive functioning was assessed in children aged 3-11 years using the National Institutes of Health Toolbox Cognition Battery. The HBWQS regression showed a negative association between the urinary metal mixture and the Cognition Early Childhood Composite Score in the PRISM New York City (ß = -3.67, 95% credible interval (CrI): -7.61, -0.01) and FTDL (ß = -3.76, 95% CrI: -7.66, -0.24) samples, with a similar trend in the PRISM Boston sample (ß = -3.24, 95% CrI: -6.77, 0.144). We did not detect these associations in traditionally pooled models. HBWQS regression allowed us to account for site heterogeneity and detect associations between prenatal metal-mixture exposure and cognitive outcomes in childhood. Given the ubiquity of metals exposure, interventions aimed at reducing prenatal exposure may improve cognitive outcomes in children. This article is part of a Special Collection on Environmental Epidemiology.

Hedgehog signaling and the primary cilium: implications for spatial and temporal constraints on signaling.

The mechanisms of vertebrate Hedgehog signaling are linked to the biology of the primary cilium, an antenna-like organelle that projects from the surface of most vertebrate cell types. Although the advantages of restricting signal transduction to cilia are often noted, the constraints imposed are less frequently considered, and yet they are central to how Hedgehog signaling operates in developing tissues. In this Review, we synthesize current understanding of Hedgehog signal transduction, ligand secretion and transport, and cilia dynamics to explore the temporal and spatial constraints imposed by the primary cilium on Hedgehog signaling in vivo.

Surgery for internal rotation contracture in infancy may obviate the need for brachial plexus nerve reconstruction: early experience.

BACKGROUND: Shoulder internal rotation contracture and subluxation in the first year of life has long been recognized in some patients with brachial plexus birth injury (BPBI). Surgical management of shoulder pathology has traditionally been undertaken following nerve reconstruction as necessary. In some patients; however, shoulder pathology may impair or obscure functional neuromuscular recovery of the upper extremity. As a proof of concept, we report a highly selected subset of patients with BPBI in whom shoulder surgery undertaken before one year of age obviated the need for neuroma resection and nerve grafting. METHODS: A retrospective review was performed of all patients with upper trunk BPBI who underwent shoulder surgery before one year of age from 2015 to 2018. Upper extremity motor function was evaluated with preoperative and postoperative Active Movement Scale scores, Cookie tests, and the requirement for subsequent neuroma resection and nerve grafting. RESULTS: Fifteen patients with BPBI meeting the inclusion criteria underwent shoulder surgery (including a subscapularis slide and tendon transfers of the teres major and latissimus dorsi muscles) before 1 year of age. Preoperatively, no patients of the appropriate age passed the Cookie test for elbow flexion. Thirteen patients either passed the Cookie test or scored Active Movement Scale score 7 for elbow flexion at or before the last available follow-up undertaken at a median age of 3.4 [1.4, 5.2] years. One of those 13 patients underwent single fascicular distal nerve transfer to improve elbow flexion before subsequently passing the Cookie test. Two patients did not have sufficient follow-up to assess elbow flexion. CONCLUSION: Although the exact role of shoulder surgery in infancy for BPBI remains to be defined, the findings from this study provide proof of concept that early, targeted surgical treatment of the shoulder may obviate the need for brachial plexus nerve reconstruction in a highly selected group of infants with BPBI.

Evaluation of the NIH Toolbox Odor Identification Test across normal cognition, amnestic mild cognitive impairment, and dementia due to Alzheimer's disease.

INTRODUCTION: Olfactory decline is associated with cognitive decline in aging, amnestic mild cognitive impairment (aMCI), and amnestic dementia associated with Alzheimer's disease neuropathology (ADd). The National Institutes of Health Toolbox Odor Identification Test (NIHTB-OIT) may distinguish between these clinical categories. METHODS: We compared NIHTB-OIT scores across normal cognition (NC), aMCI, and ADd participants (N = 389, ≥65 years) and between participants positive versus negative for AD biomarkers and the APOE ε4 allele. RESULTS: NIHTB-OIT scores decreased with age (p < 0.001) and were lower for aMCI (p < 0.001) and ADd (p < 0.001) compared to NC participants, correcting for age and sex. The NIHTB-OIT detects aMCI (ADd) versus NC participants with 49.4% (56.5%) sensitivity and 88.8% (89.5%) specificity. NIHTB-OIT scores were lower for participants with positive AD biomarkers (p < 0.005), but did not differ based on the APOE ε4 allele (p > 0.05). DISCUSSION: The NIHTB-OIT distinguishes clinically aMCI and ADd participants from NC participants. HIGHLIGHTS: National Institutes of Health Toolbox Odor Identification Test (NIHTB-OIT) discriminated normal controls from mild cognitive impairment. NIHTB-OIT discriminated normal controls from Alzheimer's disease dementia. Rate of olfactory decline with age was similar across all diagnostic categories. NIHTB-OIT scores were lower in participants with positive Alzheimer's biomarker tests. NIHTB-OIT scores did not differ based on APOE genotype.

Evaluating the feasibility of implementing the Sup-ER protocol for infants with brachial plexus birth injury.

BACKGROUND: The Sup-ER protocol involves a repositioning program for infants with brachial plexus birth injury to position the shoulder in external rotation (ER) to address progressive loss in passive range of motion (PROM). The British Columbia Children's Hospital (BCCH) eligibility criteria for this protocol are infants aged 4-8 weeks with decreased shoulder ER PROM and/or Active Movement Scale (AMS) shoulder ER and/or supination scores ≤2. The resources needed to implement this protocol in large clinics have not been studied. PURPOSE: This study aims to evaluate the BCCH criteria that are used to identify appropriate candidates for the Sup-ER protocol. STUDY DESIGN: A retrospective cohort study was conducted to identify the percentage of infants who would have been recommended the Sup-ER protocol based on their PROM and AMS scores between 4 and 8 weeks of age. METHODS: A sensitivity and specificity evaluation was used to describe the BCCH criteria's ability to identify infants in this historical cohort who went on to have incomplete shoulder function (ie, true positive) vs infants who had functional shoulder outcome at 9 months of age (ie, false positive). RESULTS: At a mean of 5.8 weeks (95% confidence interval [CI] 5.3, 6.3), 46 of the 87 (53%) infants satisfied the BCCH Sup-ER protocol criteria. Forty-four (51%) were female, half (n = 45) were left side affected, and 88% had upper plexus injury. The BCCH Sup-ER protocol criteria had sensitivity of 100% and specificity of 71% to identify infants with incomplete shoulder function. Removing the AMS supination ≤2 score criterion from the criteria improves the specificity to 84%, while sensitivity remains high (97%). CONCLUSIONS: Modifying the BCCH criteria to all infants aged 4-8 weeks with AMS shoulder ER ≤2 and/or decreased shoulder ER PROM improves the precision of identifying infants who would benefit from the Sup-ER protocol.

Prevalence and predictors of elbow flexion contractures during early childhood following brachial plexus birth injury.

BACKGROUND: Elbow flexion contracture development in school-age children with a brachial plexus birth injury (BPBI) is common. Reports indicate onset between 2 and 4 years; however, little is known about early childhood prevalence, development, and trajectory of these contractures. PURPOSE: To determine the prevalence and predictors of BPBI elbow flexion contractures during early childhood. STUDY DESIGN: A retrospective cross-sectional study. METHODS: Demographic, diagnostic, treatment, and elbow contracture data were collected for children with a BPBI <4 years between 2015 and 2019 from a prospectively collected database. Spinal root motor contributions and injury were determined using Active Movement Scale (AMS) scores at 6 weeks of age and used to predict contracture development. RESULTS: Of the 171 children that met inclusion criteria, 87% (n = 149) had upper plexus injuries. The mean age at the time of evaluation for an elbow contracture was 21.4 ± 12.7 months. The prevalence of elbow flexion contractures was 22% (n = 38), with mean onset at 13.4 ± 11.0 months. Mean contracture degree was -10.8 ± -6.9 degrees with 76% (n = 29) <-10 degrees. AMS shoulder abduction, flexion, and external rotation; elbow flexion; forearm supination; and wrist extension scores at a mean 2.3 ± 1.4 months were significantly lower in children who developed elbow flexion contractures (p < 0.001). Logistic regression found that low AMS elbow flexion with high elbow extension scores were a significant (p < 0.003) predictor of elbow contracture development. CONCLUSIONS: The prevalence of elbow flexion contractures in early childhood is greater than previously understood. These findings indicate that C5-C6 injury affecting elbow flexion with relative preservation of elbow extension is a predictor of contracture development. Further research is needed to investigate the nature and sequelae of C5-C6 injury and its effects on elbow flexion contracture development.

Chemoproteomic Profiling Reveals that Anticancer Natural Product Dankastatin†B Covalently Targets Mitochondrial VDAC3.

Chlorinated gymnastatin and dankastatin alkaloids derived from the fungal strain Gymnascella dankaliensis have been reported to possess significant anticancer activity but their mode of action is unknown. These members possess electrophilic functional groups that can might undergo covalent bond formation with specific proteins to exert their biological activity. To better understand the mechanism of action of this class of natural products, we mapped the proteome-wide cysteine reactivity of the most potent of these alkaloids, dankastatin B, by using activity-based protein profiling chemoproteomic approaches. We identified a primary target of dankastatin B in breast cancer cells as cysteine C65 of the voltage-dependent anion-selective channel on the outer mitochondrial membrane VDAC3. We demonstrated direct and covalent interaction of dankastatin B with VDAC3. VDAC3 knockdown conferred hypersensitivity to dankastatin B-mediated antiproliferative effects in breast cancer cells, thus indicating that VDAC3 was at least partially involved in the anticancer effects of this natural product. Our study reveals a potential mode of action of dankastatin B through covalent targeting of VDAC3 and highlights the utility of chemoproteomic approaches in gaining mechanistic understanding of electrophilic natural products.

Zinc supplementation alters tissue distribution of arsenic in Mus musculus.

Arsenic occurs naturally in the environment and humans can be exposed through food, drinking water and inhalation of air-borne particles. Arsenic exposure is associated with cardiovascular, pulmonary, renal, immunologic, and developmental toxicities as well as carcinogenesis. Arsenic displays dose-depen toxicities in target organs or tissues with elevated levels of arsenic. Zinc is an essential micronutrient with proposed protective benefits due to its antioxidant properties, integration into zinc-containing proteins and zinc-related immune signaling. In this study, we tested levels of arsenic and zinc in plasma, kidney, liver, and spleen as model tissues after chronic (42-day) treatment with either arsenite, zinc, or in combination. Arsenite exposure had minimal impact on tissue zinc levels with the exception of the kidney. Conversely, zinc supplementation of arsenite-exposed mice reduced the amount of arsenic detected in all tissues tested. Expression of transporters associated with zinc or arsenic influx and efflux were evaluated under each treatment condition. Significant effects of arsenite exposure on zinc transporter expression displayed tissue selectivity for liver and kidney, and was restricted to Zip10 and Zip14, respectively. Arsenite also interacted with zinc co-exposure for Zip10 expression in liver tissue. Pairwise comparisons show neither arsenite nor zinc supplementation alone significantly altered expression of transporters utilized by arsenic. However, significant interactions between arsenite and zinc were evident for Aqp7 and Mrp1 in a tissue selective manner. These findings illustrate interactions between arsenite and zinc leading to changes in tissue metal level and suggest a potential mechanism by which zinc may offer protection from arsenic toxicities.

Advancing nutrition science to meet evolving global health needs.

Populations in crisis!A global overview of health challenges and policy efforts within the scope of current nutrition issues, from persistent forms of undernutrition, including micronutrient deficiency, to diet-related chronic diseases. Nutrition science has evolved from a therapeutic and prevention emphasis to include a focus on diets and food systems. Working and consensus definitions are needed, as well as guidance related to healthy diets and the emerging issues that require further research and consensus building. Between nutrient deficiency and chronic disease, nutrition has evolved from focusing exclusively on the extremes of overt nutrient deficiency and chronic disease prevention, to equipping bodies with the ability to cope with physiologic, metabolic, and psychological stress. Just what is 'optimal nutrition', is that a valid public health goal, and what terminology is being provided by the nutrition science community? Nutrition research on 'healthspan', resilience, and intrinsic capacity may provide evidence to support optimal nutrition. Finally, experts provide views on ongoing challenges of achieving consensus or acceptance of the various definitions and interventions for health promotion, and how these can inform government health policies.Nutrition topics that receive particular focus in these proceedings include choline, NAD-replenishment in neurodegenerative diseases, and xanthophyll carotenoids. Choline is a crucial nutrient essential for cellular metabolism, requiring consumption from foods or supplements due to inadequate endogenous synthesis. Maternal choline intake is vital for fetal and infant development to prevent neural tube defects. Neurodegenerative diseases pose a growing health challenge, lacking effective therapies. Nutrition, including NAD-replenishing nutrients, might aid prevention. Emerging research indicates xanthophyll carotenoids enhance vision and cognition, potentially impacting age-related diseases.

Measuring individual true change with PROMIS using IRT-based plausible values.

AIMS: A primary advantage of IRT-based patient-reported outcome measures such as PROMIS short forms and computer-adaptive tests is that each estimate of the latent trait comes with a standard error. Such measurement error needs to be acknowledged, in particular when monitoring individual patients over time. In this study, we use plausible values to account for measurement error and analyze the probability of true within-individual change. METHODS: We use a longitudinal, observational study of stable and exacerbated COPD patients (N = 185), providing PROMIS Physical Function and Fatigue T-scores over 3 months. At each measurement, we imputed 1000 plausible values from the scores' posterior distribution. These were then used to calculate probability of true change using a pre-specified threshold such as minimally important difference supported by the literature, or [Formula: see text] > 0. We demonstrate assessment of change in individuals and in groups, across different measures (Short Forms and CATs), and at various levels of confidence. RESULTS: Using plausible value imputation and with 95% certainty, 47.5% of participants in the exacerbated group reported less fatigue, compared with 26.5% of participants in the stable group. Comparison of Short Forms and CATs suggests that CATs have better ability to detect change compared to short forms. We also illustrate this method using an individual's probability of change at different time points. CONCLUSION: Plausible values offer a flexible way to include measurement error in analysis of individuals and on sample level. Assessment of probability of true change can complement existing distribution-based approaches and facilitates interpretation of improvement or decline.

Baseline characterization of the ARMADA (Assessing Reliable Measurement in Alzheimer's Disease) study cohorts.

INTRODUCTION: The National Institutes of Health (NIH) Toolbox (NIHTB) provides computerized measures of cognition, emotion, sensation, and motor abilities across the lifespan. The ARMADA (Assessing Reliable Measurement in Alzheimer's Disease and Cognitive Aging) study validated the NIHTB in individuals across the cognitive aging spectrum. This article reports the characteristics of our sample of participants. METHODS: Participants were recruited across nine sites and classified clinically as cognitively normal (NC), with mild cognitive impairment (MCI), or with dementia of the Alzheimer's type (DAT.) They completed the NIHTB at multiple time points and many had at least one Alzheimer's biomarker previously obtained. RESULTS: Groups differed with respect to dementia severity levels, as anticipated, but were well-matched across many demographic characteristics. DISCUSSION: The ARMADA study demographics and baseline characteristics provide a suitable sample for validating the NIHTB across the cognitive aging spectrum. Other enriched samples (African American participants, Spanish NIHTB, 85+ years of age) will be reported elsewhere. HIGHLIGHTS: There is a need for assessments that can detect the early stages of cognitive decline in older adults. The ARMADA (Assessing Reliable Measurement in Alzheimer's Disease and Cognitive Aging) study will validate the National Institutes of Health (NIH) Toolbox across the aging spectrum, including mild cognitive impairment (MCI) and dementia of the Alzheimer's type (DAT). Here we report the characteristics of participants. Groups were well-matched across most demographic characteristics, and clinical characteristics differed as expected. ARMADA study cohorts reflect their respective clinical syndromes for validating the NIH Toolbox.

Prenatal exposure to PM2.5 and childhood cognition: Accounting for between-site heterogeneity in a pooled analysis of ECHO cohorts in the Northeastern United States.

BACKGROUND: Emerging studies have investigated the adverse health effects of PM2.5 using data from multiple cohorts, and results often are not generalizable across cohorts. We aimed to assess associations between prenatal PM2.5 and childhood cognition in two U.S. cohorts while accounting for between-site heterogeneity. METHODS: Analyses included 348 mother-child dyads enrolled in the dual site (New York City and Boston) PRogramming of Intergenerational Stress Mechanisms (PRISM) cohort and in the First Thousand Days of Life (FTDL) study (Northern Virginia) participating in the Environmental influences on Child Health Outcomes (ECHO) national consortium. Residential prenatal PM2.5 exposure was estimated using a validated satellite-based model and childhood cognition was measured using the NIH Toolbox Cognition Battery at three to eight years of age. We used a log-linear model applied to contingency tables formed by cross-classifying covariates by site to examine between-site heterogeneity using 3rd trimester PM2.5 exposure, age-corrected cognition scores, and covariates potentially causing heterogeneities. Multivariable linear regression models informed by the combinability analysis were used to estimate the coefficients and 95% confidence intervals (CIs) for the association between 3rd trimester PM2.5 exposure and age-corrected cognition scores (mean = 100, SD = 15). RESULTS: The log-linear model indicated that inter-study associations were similar between PRISM-NYC and FTDL, which were different from those in PRISM-Boston. Accordingly, we combined the data of PRISM-NYC and FTDL cohorts. We observed associations between 3rd trimester PM2.5 and cognition scores, findings were varying by site, childsex, and test. For example, a 1 µg/m3 increase of 3rd trimester PM2.5 was associated with -4.35 (95% CI = -8.73, -0.25) mean early childhood cognition scores in females in PRISM-Boston. In the pooled NYC + FTDL site, the association between PM2.5 and childhood cognition may be modified by maternal education and urbanicity. CONCLUSIONS: We found associations between prenatal PM2.5 and impaired childhood cognition. Since multi-site analyses are increasingly conducted, our findings suggest the needed awareness of between-site heterogeneity.

ARMADA: Assessing reliable measurement in Alzheimer's disease and cognitive aging project methods.

INTRODUCTION: Early detection of cognitive decline in older adults is a public health priority. Advancing Reliable Measurement in Alzheimer's Disease and Cognitive Aging (ARMADA), a multisite study, is validating cognition, emotion, motor, and sensory modules of the National Institutes of Health Toolbox for Assessment of Neurological and Behavioral Function (NIHTB) in the aging spectrum from cognitively normal to dementia of the Alzheimer's type (DAT). METHODS: Participants 65 to 85 years old, in demographic groups racially proportional to the general US population, are recruited in one of three groups to validate the NIHTB: cognitively normal, amnestic mild cognitive impairment (aMCI), or mild DAT. Additional special emphasis cohorts include (1) Blacks in the three clinical groups; (2) Spanish-speakers in the three clinical groups; (3) cognitively normal, population-proportional, over age 85. DISCUSSION: Longitudinal study will determine whether NIHTB can predict cognitive decline and is associated with Alzheimer's disease biomarkers. Here, we detail the methods for the ARMADA study.

HIV Viremia and Risk of Stroke Among People Living with HIV Who Are Using Antiretroviral Therapy.

BACKGROUND: Rates of stroke are higher in people living with HIV compared with age-matched uninfected individuals. Causes of elevated stroke risk, including the role of viremia, are poorly defined. METHODS: Between 1 January 2006 and 31 December 2014, we identified incident strokes among people living with HIV on antiretroviral therapy at five sites across the United States. We considered three parameterizations of viral load (VL) including (1) baseline (most recent VL before study entry), (2) time-updated, and (3) cumulative VL (copy-days/mL of virus). We used Cox proportional hazards models to estimate hazard ratios (HRs) for stroke risk comparing the 75th percentile ("high VL") to the 25th percentile ("low VL") of baseline and time-updated VL. We used marginal structural Cox models, with most models adjusted for traditional stroke risk factors, to estimate HRs for stroke associated with cumulative VL. RESULTS: Among 15,974 people living with HIV, 139 experienced a stroke (113 ischemic; 18 hemorrhagic; eight were unknown type) over a median follow-up of 4.2 years. Median baseline VL was 38 copies/mL (interquartile interval: 24, 3,420). High baseline VL was associated with increased risk of both ischemic (HR: 1.3; 95% CI = 0.96-1.7) and hemorrhagic stroke (HR: 3.1; 95% CI = 1.6-5.9). In time-updated models, high VL was also associated with an increased risk of any stroke (HR: 1.8; 95% CI = 1.4-2.3). We observed no association between cumulative VL and stroke risk. CONCLUSIONS: Our findings are consistent with the hypothesis that elevated HIV VL may increase stroke risk, regardless of previous VL levels.

3,3'-Diindolylmethane Exhibits Significant Metabolism after Oral Dosing in Humans.

3,3'-Diindolylmethane (DIM), a major phytochemical derived from ingestion of cruciferous vegetables, is also a dietary supplement. In preclinical models, DIM is an effective cancer chemopreventive agent and has been studied in a number of clinical trials. Previous pharmacokinetic studies in preclinical and clinical models have not reported DIM metabolites in plasma or urine after oral dosing, and the pharmacological actions of DIM on target tissues is assumed to be solely via the parent compound. Seven subjects (6 males and 1 female) ranging from 26-65 years of age, on a cruciferous vegetable-restricted diet prior to and during the study, took 2 BioResponse DIM 150-mg capsules (45.3 mg DIM/capsule) every evening for one week with a final dose the morning of the first blood draw. A complete time course was performed with plasma and urine collected over 48 hours and analyzed by UPLC-MS/MS. In addition to parent DIM, two monohydroxylated metabolites and 1 dihydroxylated metabolite, along with their sulfate and glucuronide conjugates, were present in both plasma and urine. Results reported here are indicative of significant phase 1 and phase 2 metabolism and differ from previous pharmacokinetic studies in rodents and humans, which reported only parent DIM present after oral administration. 3-((1H-indole-3-yl)methyl)indolin-2-one, identified as one of the monohydroxylated products, exhibited greater potency and efficacy as an aryl hydrocarbon receptor agonist when tested in a xenobiotic response element-luciferase reporter assay using Hepa1 cells. In addition to competitive phytochemical-drug adverse reactions, additional metabolites may exhibit pharmacological activity highlighting the importance of further characterization of DIM metabolism in humans. SIGNIFICANCE STATEMENT: 3,3'-Diindolylmethane (DIM), derived from indole-3-carbinol in cruciferous vegetables, is an effective cancer chemopreventive agent in preclinical models and a popular dietary supplement currently in clinical trials. Pharmacokinetic studies to date have found little or no metabolites of DIM in plasma or urine. In marked contrast, we demonstrate rapid appearance of mono- and dihydroxylated metabolites in human plasma and urine as well as their sulfate and glucuronide conjugates. The 3-((1H-indole-3-yl)methyl)indolin-2-one metabolite exhibited significant aryl hydrocarbon receptor agonist activity, emphasizing the need for further characterization of the pharmacological properties of DIM metabolites.

Effects of zinc status on age-related T cell dysfunction and chronic inflammation.

Age-related T cell dysfunction contributes to immunosenescence and chronic inflammation. Aging is also associated with a progressive decline in zinc status. Zinc is an essential micronutrient critical for immune function. A significant portion of the older populations are at risk for marginal zinc deficiency. The combined impact of dietary zinc deficiency and age on immune dysfunction has not been well explored despite the common occurrence together in the elderly population. We hypothesize that age-related zinc loss contributes to T cell dysfunction and chronic inflammation in the elderly and is exacerbated by inadequate dietary intake and improved with zinc supplementation. Using an aging mouse model, the effects of marginal zinc deficiency and zinc supplementation on Th1/Th17/proinflammatory cytokine profiles and CD4+ T cell naïve/memory phenotypes were examined. In the first study, young (2 months) and old (24 months) C57BL/6 mice were fed a zinc adequate (ZA) or marginally zinc deficient (MZD) diets for 6 weeks. In the second study, mice were fed a ZA or zinc supplemented (ZS) diet for 6 weeks. MZD old mice had significant increase in LPS-induced IL6 compared to ZA old mice. In contrast, ZS old mice had significantly reduced plasma MCP1 levels, reduced T cell activation-induced IFNγ, IL17, and TNFα response, as well as increased naïve CD4+ T-cell subset compared to ZA old mice. Our data suggest that zinc deficiency is an important contributing factor in immune aging, and improving zinc status can in part reverse immune dysfunction and reduce chronic inflammation associated with aging.

Long-Term Hand Function Outcomes of the Surgical Management of Complete Brachial Plexus Birth Injury.

PURPOSE: Hand function outcomes of primary nerve reconstruction for total brachial plexus birth injury (BPBI) are confounded by nerve roots left in continuity, inclusion of secondary procedures, and no assessment of the ability to perform activities of daily living. The purpose of this study was to evaluate the long-term hand function outcomes in a cohort of patients with a complete BPBI who had no nerve root in continuity prior to primary nerve reconstruction targeting the lower trunk. METHODS: This single-center retrospective case series of complete BPBI included patients who underwent primary nerve reconstruction. The outcomes were assessed using the active movement scale (AMS) and brachial plexus outcome measure preoperatively and at the age of 4 and 8 years. RESULTS: Fifty patients with a complete BPBI, of whom 82% (41/50) had an avulsion of C8-T1, underwent primary nerve reconstruction at a mean age of 4.1 months. Compared with the preoperative AMS scores, a statistically significant increase of AMS scores was observed at 4 and 8 years of age for all movements except forearm pronation. Between 4 and 8 years of age, there was a statistically significant improvement of external rotation of the shoulder and elbow flexion as well as diminution of thumb flexion. In the brachial plexus outcome measure assessment, there were 83% (24/29) at 4 years and 81% (21/26) at 8 years who had sufficient functional movement to perform wrist, finger, and thumb activities. CONCLUSIONS: Functional hand outcome was restored to sufficiently perform bimanual activity tasks in 81% (21/26) of patients with a complete BPBI at 8 years of age. This affirmed that primary nerve reconstruction reinnervating the lower trunk can result in a functional extremity. TYPE OF STUDY/LEVEL OF EVIDENCE: Therapeutic IV.

Sulforaphane Bioavailability and Chemopreventive Activity in Men Presenting for Biopsy of the Prostate Gland: A Randomized Controlled Trial.

Previous studies suggest compounds such as sulforaphane (SFN) derived from cruciferous vegetables may prevent prostate cancer development and progression. This study evaluated the effect of broccoli sprout extract (BSE) supplementation on blood histone deacetylase (HDAC) activity, prostate RNA gene expression, and tissue biomarkers (histone H3 lysine 18 acetylation (H3K18ac), HDAC3, HDAC6, Ki67, and p21). A total of 98 men scheduled for prostate biopsy were allocated into either BSE (200 µmol daily) or a placebo in our double-blind, randomized controlled trial. We used nonparametric tests to evaluate the differences of blood HDAC activity and prostate tissue immunohistochemistry biomarkers between treatment groups. Further, we performed RNA-Seq analysis on the prostate biopsies and identified 40 differentially expressed genes correlated with BSE treatment, including downregulation of two genes previously implicated in prostate cancer development, AMACR and ARLNC1. Although urine and plasma SFN isothiocyanates and individual SFN metabolites were statistically higher in the treatment group, our results did not show a significant difference in HDAC activity or prostate tissue biomarkers. This study indicates BSE supplementation correlates with changes in gene expression but not with several other prostate cancer biomarkers. More research is required to fully understand the chemopreventive effects of BSE supplementation on prostate cancer.