Mechanistic Investigation of the Time-Dependent Aldehyde Oxidase Inhibitor Hydralazine.

The anti-hypertensive agent hydralazine is a time-dependent inhibitor of the cytosolic drug-metabolizing enzyme aldehyde oxidase (AO). Glutathione (GSH) was found to suppress the inhibition of AO by hydralazine in multiple enzyme sources (human liver and kidney cytosol, human liver S9, rat liver S9, and recombinant human AO) and with different AO substrates (zoniporide, O6 -benzylguanine, and dantrolene). Hydralazine-induced AO inactivation was unaffected when GSH was added to the incubation mixture after pre-incubation of hydralazine with AO (rather than during the pre-incubation), suggesting that GSH traps a hydralazine reactive intermediate prior to enzyme inactivation. Consistent with previous reports of 1-phthalazylmercapturic acid formation when hydralazine was incubated with N-acetylcysteine, we detected a metabolite producing an MS/MS spectrum consistent with a 1-phthalazyl-GSH conjugate. O6 -Benzylguanine, an AO substrate, did not protect against hydralazine-induced AO inactivation, implying that hydralazine does not compete with O6 -benzylguanine for binding to the AO active site. Catalase also failed to protect AO from hydralazine-induced inactivation, suggesting that hydrogen peroxide is not involved. However, an allosteric AO inhibitor (thioridazine) offered some protection, indicating a catalytic role for AO in the bioactivation of hydralazine. AO inhibition by phthalazine (a substrate and inhibitor of AO and a metabolite of hydralazine) was unaffected by the presence of GSH. GSH also prevented hydralazine from inhibiting the nitro-reduction of dantrolene by AO. Furthermore, the GSH-hydralazine combination stimulated dantrolene reduction. Phthalazine inhibited only oxidation reactions, not reduction of dantrolene. Together, these results support the hypothesis that hydralazine is converted to a reactive intermediate that inactivates AO. SIGNIFICANCE STATEMENT: These studies suggest that a reactive intermediate of hydralazine plays a primary role in the mechanism of aldehyde oxidase (AO) inactivation. Inactivation was attenuated by glutathione and unaffected by catalase. Phthalazine (hydralazine metabolite) inhibited AO regardless of the presence of glutathione; however, phthalazine inhibited only oxidation reactions, while hydralazine inhibited both oxidation and reduction reactions. This report advances our mechanistic understanding of hydralazine as an AO inhibitor and provides information to facilitate appropriate use of hydralazine when probing AO metabolism.

Discrimination, the Model Minority Stereotype, and Peer Relationships Across the High School Years.

Ethnic stereotyping can profoundly influence youth adjustment; however, little work has addressed how the model minority stereotype may affect adolescent social adjustment. This study examined Asian American adolescents' peer relationships over time and how perceived discrimination and model minority stereotyping are associated with positive (support) and negative (criticism) qualities in these relationships. Multi-wave survey data were collected from 175 Asian adolescents in the Southeast over three time points. Participants were 60% female (freshmen Mage = 14.42 years, SD = 0.64 and sophomores Mage = 15.56 years, SD = 0.74). They were 75% US-born and represented various heritage groups (e.g., Hmong, East/Southeast Asian, South Asian). Within-person, year-to-year associations between variables were explored. Criticism from White and other-ethnic peers decreased over time. Discrimination was associated with higher criticism over time, and links between model minority stereotyping and support were found. With White peers, when stereotyping experiences increased, both positive and negative relationship qualities increased. Experiences of stereotyping and discrimination interacted, exacerbating each other with regard to criticism. The discussion compares model minority stereotyping and discrimination, both likely to create strained relationships.

Long-Term Quality of Life Among Survivors of Severe Sepsis: Analyses of Two International Trials.

OBJECTIVES: To describe the quality of life among sepsis survivors. DESIGN: Secondary analyses of two international, randomized clinical trials (A Controlled Comparison of Eritoran and placebo in patients with Severe Sepsis [derivation cohort] and PROWESS-SHOCK [validation cohort]). SETTING: ICUs in North and South America, Europe, Africa, Asia, and Australia. PATIENTS: Adults with severe sepsis. We analyzed only patients who were functional and living at home without help before sepsis hospitalization (n = 1,143 and 987 from A Controlled Comparison of Eritoran and placebo in patients with Severe Sepsis and PROWESS-SHOCK, respectively). INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: In A Controlled Comparison of Eritoran and placebo in patients with Severe Sepsis and PROWESS-SHOCK, the average age of patients living at home independently was 63 and 61 years; 400 (34.9%) and 298 (30.2%) died by 6 months. In A Controlled Comparison of Eritoran and placebo in patients with Severe Sepsis, 580 patients had a quality of life measured using EQ-5D at 6 months. Of these, 41.6% could not live independently (22.7% were home but required help, 5.1% were in nursing home or rehabilitation facilities, and 5.3% were in acute care hospitals). Poor quality of life at 6 months, as evidenced by problems in mobility, usual activities, and self-care domains were reported in 37.4%, 43.7%, and 20.5%, respectively, and the high incidence of poor quality of life was also seen in patients in PROWESS-SHOCK. Over 45% of patients with mobility and self-care problems at 6 months in A Controlled Comparison of Eritoran and placebo in patients with Severe Sepsis died or reported persistent problems at 1 year. CONCLUSIONS: Among individuals enrolled in a clinical trial who lived independently prior to severe sepsis, one third had died and of those who survived, a further one third had not returned to independent living by 6 months. Both mortality and quality of life should be considered when designing new interventions and considering endpoints for sepsis trials.

Model Minority Stereotyping, Perceived Discrimination, and Adjustment Among Adolescents from Asian American Backgrounds.

The model minority image is a common and pervasive stereotype that Asian American adolescents must navigate. Using multiwave data from 159 adolescents from Asian American backgrounds (mean age at initial recruitment = 15.03, SD = .92; 60 % female; 74 % US-born), the current study targeted unexplored aspects of the model minority experience in conjunction with more traditionally measured experiences of negative discrimination. When examining normative changes, perceptions of model minority stereotyping increased over the high school years while perceptions of discrimination decreased. Both experiences were not associated with each other, suggesting independent forms of social interactions. Model minority stereotyping generally promoted academic and socioemotional adjustment, whereas discrimination hindered outcomes. Moreover, in terms of academic adjustment, the model minority stereotype appears to protect against the detrimental effect of discrimination. Implications of the complex duality of adolescents' social interactions are discussed.

Effectiveness of a care bundle for primary prevention of intraventricular hemorrhage in high-risk neonates: a Bayesian analysis.

OBJECTIVE: To determine whether an intraventricular hemorrhage (IVH) prevention bundle featuring midline-elevated positioning reduced IVH among high-risk infants. STUDY DESIGN: In a retrospective study design, we compared outcomes of infants <1250 grams birth weight or <30 weeks gestation before (N = 205) and after (N = 360) implementation of an IVH prevention bundle, using Bayesian and frequentist logistic regression to determine whether the intervention decreased any grade IVH. RESULTS: In both the Bayesian and frequentist analyses, there was no difference in odds of any grade IVH before and after the implementation of the prevention bundle (OR 0.993; 95% Credible Interval 0.751-1.323 and OR 1.23; 95% Confidence Interval 0.818-1.864 respectively). Bias analyses suggested that these results were robust to bias from potential deaths attributable to IVH. CONCLUSION: In this retrospective analysis, we found no evidence for a protective effect of an IVH prevention bundle on IVH incidence among high-risk neonates at a level IV NICU.

An optimized approach for multiplexing single-nuclear ATAC-seq using oligonucleotide-conjugated antibodies.

BACKGROUND: Single-cell technologies to analyze transcription and chromatin structure have been widely used in many research areas to reveal the functions and molecular properties of cells at single-cell resolution. Sample multiplexing techniques are valuable when performing single-cell analysis, reducing technical variation and permitting cost efficiencies. Several commercially available methods have been used in many scRNA-seq studies. On the other hand, while several methods have been published, multiplexing techniques for single nuclear assay for transposase-accessible chromatin (snATAC)-seq assays remain under development. We developed a simple nucleus hashing method using oligonucleotide-conjugated antibodies recognizing nuclear pore complex proteins, NuHash, to perform snATAC-seq library preparations by multiplexing. RESULTS: We performed multiplexing snATAC-seq analyses on a mixture of human and mouse cell samples (two samples, 2-plex, and four samples, 4-plex) using NuHash. The analyses on nuclei with at least 10,000 read counts showed that the demultiplexing accuracy of NuHash was high, and only ten out of 9144 nuclei (2-plex) and 150 of 12,208 nuclei (4-plex) had discordant classifications between NuHash demultiplexing and discrimination using reference genome alignments. The differential open chromatin region (OCR) analysis between female and male samples revealed that male-specific OCRs were enriched in chromosome Y (four out of nine). We also found that five female-specific OCRs (20 OCRs) were on chromosome X. A comparative analysis between snATAC-seq and deeply sequenced bulk ATAC-seq on the same samples revealed that the bulk ATAC-seq signal intensity was positively correlated with the number of cell clusters detected in snATAC-seq. Moreover, when we categorized snATAC-seq peaks based on the number of cell clusters in which the peak was present, we observed different distributions over different genomic features between the groups. This result suggests that the peak intensities of bulk ATAC-seq can be used to identify different types of functional loci. CONCLUSIONS: Our multiplexing method using oligo-conjugated anti-nuclear pore complex proteins, NuHash, permits high-accuracy demultiplexing of samples. The NuHash protocol is straightforward, works on frozen samples, and requires no modifications for snATAC-seq library preparation.

Study the forest, not only the trees: Environmental exposures, not genomes, generate most health disparities.

As sequencing and analysis techniques provide increasingly detailed data at a plummeting cost, it is increasingly popular to seek the answers to medical and public health challenges in the DNA sequences of affected populations. This is methodologically attractive in its simplicity, but a genomics-only approach ignores environmentally mediated health disparities, which are well-documented at multiple national and global scales. While genetic differences exist among populations, it is unlikely that these differences overcome social and environmental factors in driving the gap in health outcomes between privileged and oppressed communities. We advocate for following the lead of communities in addressing their self-identified interests, rather than treating widespread suffering as a convenient natural experiment.

Master Transcription Regulators and Transcription Factors Regulate Immune-Associated Differences Between Patients of African and European Ancestry With Colorectal Cancer.

Background and Aims: Individuals of African (AFR) ancestry have a higher incidence of colorectal cancer (CRC) than those of European (EUR) ancestry and exhibit significant health disparities. Previous studies have noted differences in the tumor microenvironment between AFR and EUR patients with CRC. However, the molecular regulatory processes that underpin these immune differences remain largely unknown. Methods: Multiomics analysis was carried out for 55 AFR and 456 EUR patients with microsatellite-stable CRC using The Cancer Genome Atlas. We evaluated the tumor microenvironment by using gene expression and methylation data, transcription factor, and master transcriptional regulator analysis to identify the cell signaling pathways mediating the observed phenotypic differences. Results: We demonstrate that downregulated genes in AFR patients with CRC showed enrichment for canonical pathways, including chemokine signaling. Moreover, evaluation of the tumor microenvironment showed that cytotoxic lymphocytes and neutrophil cell populations are significantly decreased in AFR compared with EUR patients, suggesting AFR patients have an attenuated immune response. We further demonstrate that molecules called "master transcriptional regulators" (MTRs) play a critical role in regulating the expression of genes impacting key immune processes through an intricate signal transduction network mediated by disease-associated transcription factors (TFs). Furthermore, a core set of these MTRs and TFs showed a positive correlation with levels of cytotoxic lymphocytes and neutrophils across both AFR and EUR patients with CRC, thus suggesting their role in driving the immune infiltrate differences between the two ancestral groups. Conclusion: Our study provides an insight into the intricate regulatory landscape of MTRs and TFs that orchestrate the differences in the tumor microenvironment between patients with CRC of AFR and EUR ancestry.

Vitamin D receptor activation and left ventricular hypertrophy in advanced kidney disease.

BACKGROUND: In chronic kidney disease (CKD), left ventricular hypertrophy (LVH) is prevalent and is associated with increased cardiovascular morbidity and mortality. Vitamin D receptor (VDR) activation attenuates LVH progression in animal models. METHODS: PRIMO is a multinational, randomized, double-blinded trial with oral paricalcitol in subjects with stages 3-4 CKD, mild-to-moderate LVH and an LV ejection fraction >50%. The primary endpoint is change in the left ventricular mass index (LVMI) compared with placebo after 48 weeks of treatment. The main secondary endpoints are changes in diastolic function parameters. In this paper, we report baseline characteristics from this study. RESULTS: LVMI was 33.0 ± 7.5 g/m(2.7) for males and 30.8 ± 7.2 g/m(2.7) for females (p = 0.04). LVMI correlated with systolic blood pressure (r = 0.24), urine albumin creatinine ratio (r = 0.39), troponin T (r = 0.29), high-sensitivity C-reactive protein (r = 0.25) and plasma levels of B-type brain natriuretic peptide (r = 0.22); all p < 0.01. In multiple linear regression, each remained independently associated with LVMI. The early diastolic velocity of the lateral mitral annulus (E') was 8.1 ± 2.4 cm/s. E' was inversely correlated with age in univariate (r = -0.14, p = 0.04) and multivariable (p = 0.02) analysis. CONCLUSION: Among 227 multinational subjects with stages 3-4 CKD, baseline LVMI correlates with baseline blood pressure, urine albumin creatinine ratio and cardiac biomarkers, and baseline diastolic function correlates with age. This research was funded by Abbott Laboratories; ClinicalTrials.gov No. NCT00497146.

The use of group sequential, information-based sample size re-estimation in the design of the PRIMO study of chronic kidney disease.

BACKGROUND: Chronic kidney disease is associated with a marked increase in risk for left ventricular hypertrophy and cardiovascular mortality compared with the general population. Therapy with vitamin D receptor activators has been linked with reduced mortality in chronic kidney disease and an improvement in left ventricular hypertrophy in animal studies. PURPOSE: PRIMO (Paricalcitol capsules benefits in Renal failure Induced cardia MOrbidity) is a multinational, multicenter randomized controlled trial to assess the effects of paricalcitol (a selective vitamin D receptor activator) on mild to moderate left ventricular hypertrophy in patients with chronic kidney disease. METHODS: Subjects with mild-moderate chronic kidney disease are randomized to paricalcitol or placebo after confirming left ventricular hypertrophy using a cardiac echocardiogram. Cardiac magnetic resonance imaging is then used to assess left ventricular mass index at baseline, 24 and 48 weeks, which is the primary efficacy endpoint of the study. Because of limited prior data to estimate sample size, a maximum information group sequential design with sample size re-estimation is implemented to allow sample size adjustment based on the nuisance parameter estimated using the interim data. An interim efficacy analysis is planned at a pre-specified time point conditioned on the status of enrollment. The decision to increase sample size depends on the observed treatment effect. A repeated measures analysis model, using available data at Week 24 and 48 with a backup model of an ANCOVA analyzing change from baseline to the final nonmissing observation, are pre-specified to evaluate the treatment effect. Gamma-family of spending function is employed to control family-wise Type I error rate as stopping for success is planned in the interim efficacy analysis. LIMITATIONS: If enrollment is slower than anticipated, the smaller sample size used in the interim efficacy analysis and the greater percent of missing week 48 data might decrease the parameter estimation accuracy, either for the nuisance parameter or for the treatment effect, which might in turn affect the interim decision-making. CONCLUSIONS: The application of combining a group sequential design with a sample-size re-estimation in clinical trial design has the potential to improve efficiency and to increase the probability of trial success while ensuring integrity of the study.

Bedside quantification of dead-space fraction using routine clinical data in patients with acute lung injury: secondary analysis of two prospective trials.

INTRODUCTION: Dead-space fraction (Vd/Vt) has been shown to be a powerful predictor of mortality in acute lung injury (ALI) patients. The measurement of Vd/Vt is based on the analysis of expired CO2 which is not a part of standard practice thus limiting widespread clinical application of this method. The objective of this study was to determine prognostic value of Vd/Vt estimated from routinely collected pulmonary variables. METHODS: Secondary analysis of the original data from two prospective studies of ALI patients. Estimated Vd/Vt was calculated using the rearranged alveolar gas equation: Vd/Vt=1-[(0.86×VCO2est)/(VE×PaCO2)] where VCO2est is the estimated CO2 production calculated from the Harris Benedict equation, minute ventilation (VE) is obtained from the ventilator rate and expired tidal volume and PaCO2 from arterial gas analysis. Logistic regression models were created to determine the prognostic value of estimated Vd/Vt. RESULTS: One hundred and nine patients in Mayo Clinic validation cohort and 1896 patients in ARDS-net cohort demonstrated an increase in percent mortality for every 10% increase in Vd/Vt in a dose response fashion. After adjustment for non-pulmonary and pulmonary prognostic variables, both day 1 (adjusted odds ratio-OR = 1.07, 95%CI 1.03 to 1.13) and day 3 (OR = 1.12, 95% CI 1.06 to 1.18) estimated dead-space fraction predicted hospital mortality. CONCLUSIONS: Elevated estimated Vd/Vt predicts mortality in ALI patients in a dose response manner. A modified alveolar gas equation may be of clinical value for a rapid bedside estimation of Vd/Vt, utilizing routinely collected clinical data.

Development of Freeze-Drying Cycles for Pharmaceutical Products Using a Micro Freeze-Dryer.

This article aims to investigate how a small-scale freeze-dryer can be used for process design. The system encompasses a temperature controlled metallic ring that surrounds a small batch of vials, in contact with the external vials through removable thermal conductors. The temperature of the ring can be modified to keep a constant difference with the temperature of one or more vials of the batch. In this article, an extensive validation of the system is given, considering 10% w/w sucrose and 5% w/w mannitol solutions, processed in different types of vials (6 R and 20 R) and in different operating conditions. The micro freeze-dryer was also shown to be able to provide accurate estimates of the overall heat transfer coefficient from the shelf to the product in the vials (Kv) and of the resistance of the dried cake to vapor flux (Rp): both values appeared to be very close to those obtained for the same case studies in a pilot-scale unit. Finally, the use of the micro freeze-dryer to control product temperature and drying time values to simulate a pilot-scale unit was addressed, thus demonstrating the adequacy of this system for process scaleup.

Evaluation of a Treatment Package to Increase Mean Length of Utterances for Children with Autism.

Skinner's (1957) classification of mand responses has spawned decades of research related to teaching individuals with developmental disabilities. However, few studies have evaluated how to teach individuals with autism to progress from simple to more complex mands for desired items and activities. The present study used a treatment package consisting of errorless teaching, differential reinforcement, and systematic decision rules to increase the number of words per mand utterance used by 6 children with autism. Daily probes were conducted in the absence of prompting and differential reinforcement throughout every stage of the treatment. Results showed that all children showed significant developmental gains in the mean length of utterances. Increased rates of manding, increased emission of mand frames, and decreased instances of indicating responses (i.e., pointing, reaching) in the absence of mands were also observed. Implications regarding the feasibility of intensive mand training in practice are discussed.

On the use of a micro freeze-dryer for the investigation of the primary drying stage of a freeze-drying process.

This paper deals with the use of a small-scale freeze-dryer, where very few vials are loaded (e.g. 19, each 10 mL, or 7, each 20 mL), for freeze-drying cycle investigation. The system has a metallic ring surrounding the batch of vials, in contact with the external ones, and its temperature is manipulated independently from that of the shelf on the basis of the temperature of the product measured by thermocouples in some vials of the batch. The experimental study was carried out using two sucrose solutions (5% and 10% w/w), aiming to verify the homogeneity of the batch. Both product temperature and the weight loss after 6 h from the onset of the primary drying stage were selected as key parameters. Experiments were carried out according to a 2 N design of experiments, with two values of chamber pressure (60 and 90 mTorr) and two values of shelf temperature (-20 and 0 °C). Satisfactory results may be obtained by selecting a ring temperature 5 °C lower than that of the monitored samples in case of both products investigated. Besides, the system appears to be useful for the estimation of the coefficient of heat transfer to the product (Kv) and of the resistance of the dried cake to vapour flux (Rp), thus enabling the use of mathematical modelling for process design and optimization.

The Use of Matrix Training to Teach Color-Shape Tacts to Children with Autism.

Matrix training consists of preplanning instruction by arranging components of desired skills across a minimum of two axes. In the current study, three matrices were developed for each participant (e.g., Matrix 1, Generalization Matrix 1, and Generalization Matrix 2) with known color and shape components. Following baseline, nonoverlapping (i.e., diagonal) training was conducted with Matrix 1. Results of posttests were used to determine the extent of emergence of untrained color-shape combinations across all matrices. Results from all six participants indicated that mastery criteria were eventually met for Matrix 1. For five participants, mastery criteria were also eventually met for generalization matrices. Results replicate findings from prior studies and offer a simple approach for both testing emergence of untrained skills and remediating responding.

Teaching mands for information using speech generating devices: A replication and extension.

Approximately 30% of individuals diagnosed with autism spectrum disorder (ASD) fail to develop vocal communication and, therefore, use some form of augmentative or alternative communication system. The current study replicates and extends previous research on teaching "Who?" and "Which?" mands for information to 3 young children diagnosed with ASD using a speech generating device. Procedures were evaluated using a multiple baseline across participants design. All participants learned to mand for information and, subsequently, used the information to access preferred items.

Functional genetic variants can mediate their regulatory effects through alteration of transcription factor binding.

Functional variants in the genome are usually identified by their association with local gene expression, DNA methylation or chromatin states. DNA sequence motif analysis and chromatin immunoprecipitation studies have provided indirect support for the hypothesis that functional variants alter transcription factor binding to exert their effects. In this study, we provide direct evidence that functional variants can alter transcription factor binding. We identify a multifunctional variant within the TBC1D4 gene encoding a canonical NFκB binding site, and edited it using CRISPR-Cas9 to remove this site. We show that this editing reduces TBC1D4 expression, local chromatin accessibility and binding of the p65 component of NFκB. We then used CRISPR without genomic editing to guide p65 back to the edited locus, demonstrating that this re-targeting, occurring ~182 kb from the gene promoter, is enough to restore the function of the locus, supporting the central role of transcription factors mediating the effects of functional variants.