Comparative Analysis of Protein Quantification by the SomaScan Assay versus Orthogonal Methods in Urine from People with Diabetic Kidney Disease.

To our knowledge, calibration curves or other validations for thousands of SomaScan aptamers are not publicly available. Moreover, the abundance of urine proteins obtained from these assays is not routinely validated with orthogonal methods (OMs). We report an in-depth comparison of SomaScan readout for 23 proteins in urine samples from patients with diabetic kidney disease (n = 118) vs OMs, including liquid chromatography-targeted mass spectrometry (LC-MS), ELISA, and nephelometry. Pearson correlation between urine abundance of the 23 proteins from SomaScan 3.2 vs OMs ranged from -0.58 to 0.86, with a median (interquartile ratio, [IQR]) of 0.49 (0.18, 0.53). In multivariable linear regression, the SomaScan readout for 6 of the 23 examined proteins (26%) was most strongly associated with the OM-derived abundance of the same (target) protein. For 3 of 23 (13%), the SomaScan and OM-derived abundance of each protein were significantly associated, but the SomaScan readout was more strongly associated with OM-derived abundance of one or more "off-target" proteins. For the remaining 14 proteins (61%), the SomaScan readouts were not significantly associated with the OM-derived abundance of the targeted proteins. In 6 of the latest group, the SomaScan readout was not associated with urine abundance of any of the 23 quantified proteins. To sum, over half of the SomaScan results could not be confirmed by independent orthogonal methods.

Specific circulating miRNAs are associated with plasma lipids in a healthy American cohort.

Low-density lipoprotein cholesterol (LDL-c) is both a therapeutic target and a risk factor for cardiovascular disease (CVD). MicroRNA (miRNA) has been shown to regulate cholesterol homeostasis, and miRNA in blood circulation has been linked to hypercholesterolemia. However, few studies to date have associated miRNA with phenotypes like LDL-c in a healthy population. To this end, we analyzed circulating miRNA in relation to LDL-c in a healthy cohort of 353 participants using two separate bioinformatic approaches. The first approach found that miR-15b-5p and miR-16-5p were upregulated in individuals with at-risk levels of LDL-c. The second approach identified two miRNA clusters, one that positively and a second that negatively correlated with LDL-c. Included in the cluster that positively correlated with LDL-c were miR-15b-5p and miR-16-5p, as well as other miRNA from the miR-15/107, miR-30, and let-7 families. Cross-species analyses suggested that several miRNAs that associated with LDL-c are conserved between mice and humans. Finally, we examined the influence of diet on circulating miRNA. Our results robustly linked circulating miRNA with LDL-c, suggesting that miRNA could be used as biomarkers for hypercholesterolemia or targets for developing cholesterol-lowering drugs.NEW & NOTEWORTHY This study explored the association between circulating microRNA (miRNA) and low-density lipoprotein cholesterol (LDL-c) in a healthy population of 353 participants. Two miRNAs, miR-15b-5p and miR-16-5p, were upregulated in individuals with at-risk LDL-c levels. Several miRNA clusters were positively and negatively correlated with LDL-c and are known to target mRNA involved in lipid metabolism. The study also investigated the influence of diet on circulating miRNA, suggesting potential biomarkers for hypercholesterolemia.

Androgen receptor transcriptional activity is required for heregulin-1ß-mediated nuclear localization of the HER3/ErbB3 receptor tyrosine kinase.

Prostate cancer is initially regulated by the androgen receptor (AR), a ligand-activated, transcription factor, and is in a hormone-dependent state (hormone-sensitive prostate cancer (HSPC)), but eventually becomes androgen-refractory (castration-resistant prostate cancer (CRPC)) because of mechanisms that bypass the AR, including by activation of ErbB3, a member of the epidermal growth factor receptor family. ErbB3 is synthesized in the cytoplasm and transported to the plasma membrane for ligand binding and dimerization, where it regulates downstream signaling, but nuclear forms are reported. Here, we demonstrate in prostatectomy samples that ErbB3 nuclear localization is observed in malignant, but not benign prostate, and that cytoplasmic (but not nuclear) ErbB3 correlated positively with AR expression but negatively with AR transcriptional activity. In support of the latter, androgen depletion upregulated cytoplasmic, but not nuclear ErbB3, while in vivo studies showed that castration suppressed ErbB3 nuclear localization in HSPC, but not CRPC tumors. In vitro treatment with the ErbB3 ligand heregulin-1ß (HRG) induced ErbB3 nuclear localization, which was androgen-regulated in HSPC but not in CRPC. In turn, HRG upregulated AR transcriptional activity in CRPC but not in HSPC cells. Positive correlation between ErbB3 and AR expression was demonstrated in AR-null PC-3 cells where stable transfection of AR restored HRG-induced ErbB3 nuclear transport, while AR knockdown in LNCaP reduced cytoplasmic ErbB3. Mutations of ErbB3's kinase domain did not affect its localization but was responsible for cell viability in CRPC cells. Taken together, we conclude that AR expression regulated ErbB3 expression, its transcriptional activity suppressed ErbB3 nuclear translocation, and HRG binding to ErbB3 promoted it.

Untargeted metabolomic, and proteomic analysis identifies metabolic biomarkers and pathway alterations in individuals with 22q11.2 deletion syndrome.

INTRODUCTION: The chromosome 22q11.2 deletion syndrome (22q11.2DS) is characterized by a well-defined microdeletion and is associated with a wide range of brain-related phenotypes including schizophrenia spectrum disorders (SCZ), autism spectrum disorders (ASD), anxiety disorders and attention deficit disorders (ADHD). The typically deleted region in 22q11.2DS contains multiple genes which haploinsufficiency has the potential of altering the protein and the metabolic profiles. OBJECTIVES: Alteration in metabolic processes and downstream protein pathways during the early brain development may help to explain the increased prevalence of the observed neurodevelopmental phenotypes in 22q11.2DS. However, relatively little is known about the correlation of dysregulated protein/metabolite expression and neurobehavioral impairments in individuals who developed them over time. METHODS: In this study, we performed untargeted metabolic and proteomic analysis in plasma samples derived from 30 subjects including 16 participants with 22q11.2DS and 14 healthy controls (TD) enrolled in a longitudinal study, aiming to identify a metabolic and protein signature informing about the underlying mechanisms involved in disease development and progression. The metabolic and proteomic profiles were also compared between the participants with 22q11.2DS with and without various comorbidities, such as medical involvement, psychiatric conditions, and autism spectrum disorder (ASD) to detect potential changes among multiple specimens, collected overtime, with the aim to understand the basic underlying mechanisms involved in disease development and progression. RESULTS: We observed a large number of statistically significant differences in metabolites between the two groups. Among them, the levels of taurine and arachidonic acid were significantly lower in 22q11.2DS compared to the TD group. In addition, we identified 16 proteins that showed significant changes in expression levels (adjusted P < 0.05) in 22q11.2DS as compared to TD, including those involved in 70 pathways such as gene expression, the PI3K-Akt signaling pathway and the complement system. Within participants with 22q11.2DS, no significant changes in those with and without medical or psychiatric conditions were observed. CONCLUSION: To our knowledge, this is the first report on plasma metabolic and proteomic profiling and on the identification of unique biomarkers in 22q11.2DS. These findings may suggest the potential role of the identified metabolites and proteins as biomarkers for the onset of comorbid conditions in 22q11.2DS. Ultimately, the altered protein pathways in 22q11.2DS may provide insights of the biological mechanisms underlying the neurodevelopmental phenotype and may provide missing molecular outcome measures in future clinical trials to assess early-diagnosis treatment and the efficacy of response to targeted treatment.

Human Keratinocyte Responses to Woodsmoke Chemicals.

Air pollution consists of complex mixtures of chemicals with serious deleterious health effects from acute and chronic exposure. To help understand the mechanisms by which adverse effects occur, the present work examines the responses of cultured human epidermal keratinocytes to specific chemicals commonly found in woodsmoke. Our earlier findings with liquid smoke flavoring (aqueous extract of charred wood) revealed that such extracts stimulated the expression of genes associated with oxidative stress and proinflammatory response, activated the aryl hydrocarbon receptor, thereby inducing cytochrome P4501A1 activity, and induced cross-linked envelope formation, a lethal event ordinarily occurring during terminal differentiation. The present results showed that furfural produced transcriptional responses resembling those of liquid smoke, cyclohexanedione activated the aryl hydrocarbon receptor, and several chemicals induced envelope formation. Of these, syringol permeabilized the cells to the egress of lactate dehydrogenase at a concentration close to that yielding envelope formation, while furfural induced envelope formation without permeabilization detectable in this way. Furfural (but not syringol) stimulated the incorporation of amines into cell proteins in extracts in the absence of transglutaminase activity. Nevertheless, both chemicals substantially increased the amount of cellular protein incorporated into envelopes and greatly altered the envelope protein profile. Moreover, the proportion of keratin in the envelopes was dramatically increased. These findings are consistent with the chemically induced protein cross-linking in the cells. Elucidating mechanisms by which this phenomenon occurs may help understand how smoke chemicals interact with proteins to elicit cellular responses, interpret bioassays of complex pollutant mixtures, and suggest additional sensitive ways to monitor exposures.

Metabolic dysfunctions predict the development of Alzheimer's disease: Statistical and machine learning analysis of EMR data.

INTRODUCTION: The incidence of Alzheimer's disease (AD) and obesity rise concomitantly. This study examined whether factors affecting metabolism, race/ethnicity, and sex are associated with AD development. METHODS: The analyses included patients ≥ 65 years with AD diagnosis in six University of California hospitals between January 2012 and October 2023. The controls were race/ethnicity, sex, and age matched without dementia. Data analyses used the Cox proportional hazards model and machine learning (ML). RESULTS: Hispanic/Latino and Native Hawaiian/Pacific Islander, but not Black subjects, had increased AD risk compared to White subjects. Non-infectious hepatitis and alcohol abuse were significant hazards, and alcohol abuse had a greater impact on women than men. While underweight increased AD risk, overweight or obesity reduced risk. ML confirmed the importance of metabolic laboratory tests in predicting AD development. DISCUSSION: The data stress the significance of metabolism in AD development and the need for racial/ethnic- and sex-specific preventive strategies. HIGHLIGHTS: Hispanics/Latinos and Native Hawaiians/Pacific Islanders show increased hazards of Alzheimer's disease (AD) compared to White subjects. Underweight individuals demonstrate a significantly higher hazard ratio for AD compared to those with normal body mass index. The association between obesity and AD hazard differs among racial groups, with elderly Asian subjects showing increased risk compared to White subjects. Alcohol consumption and non-infectious hepatitis are significant hazards for AD. Machine learning approaches highlight the potential of metabolic panels for AD prediction.

Apolipoproteine and KLOTHO Gene Variants Do Not Affect the Penetrance of Fragile X-Associated Tremor/Ataxia Syndrome.

In this study, the potential role and interaction of the APOε and KLOTHO genes on the penetrance of fragile X-associated tremor/ataxia syndrome (FXTAS) and on the IQ trajectory were investigated. FXTAS was diagnosed based on molecular, clinical and radiological criteria. Males with the premutation (PM) over 50 years, 165 with and 34 without an FXTAS diagnosis, were included in this study and were compared based on their APO (ε2-ε3-ε4) and KLOTHO variant (KL-VS) genotypes. The effect of APOε4 on FXTAS stage and on diagnosis did not differ significantly by KL-VS genotype with interaction effect p = 0.662 and p = 0.91, respectively. In the FXTAS individuals with an APOε2 allele, a marginal significance was observed towards a larger decline in verbal IQ (VIQ) in individuals with an APOε4 allele compared to those without an APOε4 allele (p = 0.071). In conclusion, our findings suggest that the APOε4 and KL-VS genotypes alone or through their interaction effect do not appear to predispose to either FXTAS diagnosis or stage in male carriers of the PM allele. A further study is needed to establish the trend of IQ decline in the FXTAS individuals who carry APOε4 with APOε2 compared to those without APOε4.

Blood Proteome Profiling Reveals Biomarkers and Pathway Alterations in Fragile X PM at Risk for Developing FXTAS.

Fragile X-associated Tremor/Ataxia Syndrome (FXTAS) is a neurodegenerative disorder associated with the FMR1 premutation. Currently, it is not possible to determine when and if individual premutation carriers will develop FXTAS. Thus, with the aim to identify biomarkers for early diagnosis, development, and progression of FXTAS, along with associated dysregulated pathways, we performed blood proteomic profiling of premutation carriers (PM) who, as part of an ongoing longitudinal study, emerged into two distinct groups: those who developed symptoms of FXTAS (converters, CON) over time (at subsequent visits) and those who did not (non-converters, NCON). We compared these groups to age-matched healthy controls (HC). We assessed CGG repeat allele size by Southern blot and PCR analysis. The proteomic profile was obtained by liquid chromatography mass spectrometry (LC-MS/MS). We identified several significantly differentiated proteins between HC and the PM groups at Visit 1 (V1), Visit 2 (V2), and between the visits. We further reported the dysregulated protein pathways, including sphingolipid and amino acid metabolism. Our findings are in agreement with previous studies showing that pathways involved in mitochondrial bioenergetics, as observed in other neurodegenerative disorders, are significantly altered and appear to contribute to the development of FXTAS. Lastly, we compared the blood proteome of the PM who developed FXTAS over time with the CSF proteome of the FXTAS patients recently reported and found eight significantly differentially expressed proteins in common. To our knowledge, this is the first report of longitudinal proteomic profiling and the identification of unique biomarkers and dysregulated protein pathways in FXTAS.

Neuroinflammatory transcriptional programs induced in rhesus pre-frontal cortex white matter during acute SHIV infection.

BACKGROUND: Immunosurveillance of the central nervous system (CNS) is vital to resolve infection and injury. However, immune activation within the CNS in the setting of chronic viral infections, such as HIV-1, is strongly linked to progressive neurodegeneration and cognitive decline. Establishment of HIV-1 in the CNS early following infection underscores the need to delineate features of acute CNS immune activation, as these early inflammatory events may mediate neurodegenerative processes. Here, we focused on elucidating molecular programs of neuroinflammation in brain regions based on vulnerability to neuroAIDS and/or neurocognitive decline. To this end, we assessed transcriptional profiles within the subcortical white matter of the pre-frontal cortex (PFCw), as well as synapse dense regions from hippocampus, superior temporal cortex, and caudate nucleus, in rhesus macaques following infection with Simian/Human Immunodeficiency Virus (SHIV.C.CH505). METHODS: We performed RNA extraction and sequenced RNA isolated from 3 mm brain punches. Viral RNA was quantified in the brain and cerebrospinal fluid by RT-qPCR assays targeting SIV Gag. Neuroinflammation was assessed by flow cytometry and multiplex ELISA assays. RESULTS: RNA sequencing and flow cytometry data demonstrated immune surveillance of the rhesus CNS by innate and adaptive immune cells during homeostasis. Following SHIV infection, viral entry and integration within multiple brain regions demonstrated vulnerabilities of key cognitive and motor function brain regions to HIV-1 during the acute phase of infection. SHIV-induced transcriptional alterations were concentrated to the PFCw and STS with upregulation of gene expression pathways controlling innate and T-cell inflammatory responses. Within the PFCw, gene modules regulating microglial activation and T cell differentiation were induced at 28 days post-SHIV infection, with evidence for stimulation of immune effector programs characteristic of neuroinflammation. Furthermore, enrichment of pathways regulating mitochondrial respiratory capacity, synapse assembly, and oxidative and endoplasmic reticulum stress were observed. These acute neuroinflammatory features were substantiated by increased influx of activated T cells into the CNS. CONCLUSIONS: Our data show pervasive immune surveillance of the rhesus CNS at homeostasis and reveal perturbations of important immune, neuronal, and synaptic pathways within key anatomic regions controlling cognition and motor function during acute HIV infection. These findings provide a valuable framework to understand early molecular features of HIV associated neurodegeneration.

Investigating Grapevine Red Blotch Virus Infection in Vitis vinifera L. cv. Cabernet Sauvignon Grapes: A Multi-Omics Approach.

Grapevine red blotch virus (GRBV) is a recently identified virus. Previous research indicates primarily a substantial impact on berry ripening in all varieties studied. The current study analyzed grapes' primary and secondary metabolism across grapevine genotypes and seasons to reveal both conserved and variable impacts to GRBV infection. Vitis vinifera cv. Cabernet Sauvignon (CS) grapevines grafted on two different rootstocks (110R and 420A) were analyzed in 2016 and 2017. Metabolite profiling revealed a considerable impact on amino acid and malate acid levels, volatile aroma compounds derived from the lipoxygenase pathway, and anthocyanins synthesized in the phenylpropanoid pathway. Conserved transcriptional responses to GRBV showed induction of auxin-mediated pathways and photosynthesis with inhibition of transcription and translation processes mainly at harvest. There was an induction of plant-pathogen interactions at pre-veraison, for all genotypes and seasons, except for CS 110R in 2017. Lastly, differential co-expression analysis revealed a transcriptional shift from metabolic synthesis and energy metabolism to transcription and translation processes associated with a virus-induced gene silencing transcript. This plant-derived defense response transcript was only significantly upregulated at veraison for all genotypes and seasons, suggesting a phenological association with disease expression and plant immune responses.

Reoperation rates for stress urinary incontinence and pelvic organ prolapse in women after undergoing Mid-Urethral sling with or without concomitant colporrhaphy in academic centers within the United States.

In this study, the rates of reoperation for stress urinary incontinence (SUI) and pelvic organ prolapse (POP) in women who underwent a mid-urethral sling (MUS) with or without concurrent colporrhaphy were evaluated. An academic faculty practice consortium database was used to identify a cohort of patients treated surgically for SUI with or without concurrent POP repair (apical, anterior, posterior, or a combination of the three) with or without hysterectomy between 2009 and 2011. A total of 20,484 patients matched the criteria. Of patients who underwent a MUS, 7.2% underwent secondary surgery, with a higher rate of 8.6% associated with those who underwent concurrent prolapse repair (Apical repair HR 1.84, p < .01; Anterior compartment repair HR 1.47, p < .01). Concurrent hysterectomy was associated with a lower hazard of secondary prolapse surgery (HR 0.48; p < .01) if the initial surgery involved a complete POP repair. Prolapse mesh repair resulted in a higher hazard of additional surgery (HR 1.43, p < .01). Medicaid insurance was also associated with an increased hazard ratio compared to commercial insurance for secondary surgery (HR 1.32, p < .01). For women undergoing MUS with complete prolapse repair, concurrent hysterectomy is associated with lower secondary surgery rates. Concurrent prolapse repair with mesh is associated with higher secondary surgery rates.Synopsis: For women undergoing MUS (mid-urethral sling) with complete prolapse repair, concurrent hysterectomy is associated with lower secondary surgery rates. Concurrent prolapse repair with mesh is associated with higher secondary surgery rates.Impact StatementWhat is already known on this subject? Stress urinary incontinence (SUI) and pelvic organ prolapse (POP) can present at the same time and negatively impact patients' quality of life. There is little data regarding reoperation rates for patients who undergo both MUS and colporrhaphy in one setting.What do the results of this study add? This study found that patients who undergo concurrent MUS (mid-urethral sling) and complete POP repair with the addition of hysterectomy had a lower risk of secondary surgery.What are the implications of these findings for clinical practice and/or further research? Our data can be used by surgeons to counsel patients on the risks of re-operation for SUI for those who would like to undergo concurrent POP repair with or without hysterectomy.

Hepatic transcriptional profile reveals the role of diet and genetic backgrounds on metabolic traits in female progenitor strains of the Collaborative Cross.

Mice have provided critical mechanistic understandings of clinical traits underlying metabolic syndrome (MetSyn) and susceptibility to MetSyn in mice is known to vary among inbred strains. We investigated the diet- and strain-dependent effects on metabolic traits in the eight Collaborative Cross (CC) founder strains (A/J, C57BL/6J, 129S1/SvImJ, NOD/ShiLtJ, NZO/HILtJ, CAST/EiJ, PWK/PhJ, and WSB/EiJ). Liver transcriptomics analysis showed that both atherogenic diet and host genetics have profound effects on the liver transcriptome, which may be related to differences in metabolic traits observed between strains. We found strain differences in circulating trimethylamine N-oxide (TMAO) concentration and liver triglyceride content, both of which are traits associated with metabolic diseases. Using a network approach, we identified a module of transcripts associated with TMAO and liver triglyceride content, which was enriched in functional pathways. Interrogation of the module related to metabolic traits identified NADPH oxidase 4 (Nox4), a gene for a key enzyme in the production of reactive oxygen species, which showed a strong association with plasma TMAO and liver triglyceride. Interestingly, Nox4 was identified as the highest expressed in the C57BL/6J and NZO/HILtJ strains and the lowest expressed in the CAST/EiJ strain. Based on these results, we suggest that there may be genetic variation in the contribution of Nox4 to the regulation of plasma TMAO and liver triglyceride content. In summary, we show that liver transcriptomic analysis identified diet- or strain-specific pathways for metabolic traits in the Collaborative Cross (CC) founder strains.

Reoperation after Hypospadias Repair: Long-Term Analysis.

PURPOSE: While the true incidence of secondary hypospadias repair is unknown, the current literature cites a 3.3% to 6.7% reoperation rate after distal hypospadias repair and an association with age. We hypothesized that secondary surgery rates are associated with patient factors and have been underreported due to limited followup. MATERIALS AND METHODS: We used an academic practice plan consortium database to identify a population that underwent primary hypospadias repair in 2009 and 2010. Secondary surgeries between 2009 and 2019 were captured. The association of variables such as age, insurance type, region, surgeon volume and surgeon years in practice with all-inclusive and specific secondary surgery procedures were analyzed using mixed effects multiple logistic regression models. RESULTS: We identified 5,178 boys who had primary hypospadias repair performed by 84 pediatric urologists at 46 hospitals in 2009 and 2010. During the ensuing 9 to 10 years, distal, proximal and perineal hypospadias repair had a 12.6%, 37.9% and 46.6% rate of secondary surgery, respectively. After adjusting for all other variables in the model, patients with noncommercial insurance had a 26% (OR 1.26, p=0.04) increased odds of secondary surgery. Patient age and surgeon years in practice were not associated with all-inclusive secondary surgery except for endoscopic treatment. Surgeon volume was not associated with secondary surgery. CONCLUSIONS: This study demonstrated that secondary surgery rates are underreported if followup is limited to less than 6 years. Patient age, surgeon volume and experience did not associate with all-inclusive reoperation rates whereas insurance status was a major predictor of reoperation.

Cerebral Microbleeds in Fragile X-Associated Tremor/Ataxia Syndrome.

BACKGROUND: Fragile X-associated tremor/ataxia syndrome is a neurodegenerative disease of late onset developed by carriers of the premutation in the fragile x mental retardation 1 (FMR1) gene. Pathological features of neurodegeneration in fragile X-associated tremor/ataxia syndrome include toxic levels of FMR1 mRNA, ubiquitin-positive intranuclear inclusions, white matter disease, iron accumulation, and a proinflammatory state. OBJECTIVE: The objective of this study was to analyze the presence of cerebral microbleeds in the brains of patients with fragile X-associated tremor/ataxia syndrome and investigate plausible causes for cerebral microbleeds in fragile X-associated tremor/ataxia syndrome. METHODS: We collected cerebral and cerebellar tissue from 15 fragile X-associated tremor/ataxia syndrome cases and 15 control cases carrying FMR1 normal alleles. We performed hematoxylin and eosin, Perls and Congo red stains, ubiquitin, and amyloid ß protein immunostaining. We quantified the number of cerebral microbleeds, amount of iron, presence of amyloid ß within the capillaries, and number of endothelial cells containing intranuclear inclusions. We evaluated the relationships between pathological findings using correlation analysis. RESULTS: We found intranuclear inclusions in the endothelial cells of capillaries and an increased number of cerebral microbleeds in the brains of those with fragile X-associated tremor/ataxia syndrome, both of which are indicators of cerebrovascular dysfunction. We also found a suggestive association between the amount of capillaries that contain amyloid ß in the cerebral cortex and the rate of disease progression. CONCLUSION: We propose microangiopathy as a pathologic feature of fragile X-associated tremor/ataxia syndrome. © 2021 International Parkinson and Movement Disorder Society.

Identifying Toxicologically Significant Compounds in Urban Wildfire Ash Using In Vitro Bioassays and High-Resolution Mass Spectrometry.

Urban wildfires may generate numerous unidentified chemicals of toxicity concern. Ash samples were collected from burned residences and from an undeveloped upwind reference site, following the Tubbs fire in Sonoma County, California. The solvent extracts of ash samples were analyzed using GC- and LC-high-resolution mass spectrometry (HRMS) and using a suite of in vitro bioassays for their bioactivity toward nuclear receptors [aryl hydrocarbon receptor (AhR), estrogen receptor (ER), and androgen receptor (AR)], their influence on the expression of genetic markers of stress and inflammation [interleukin-8 (IL-8) and cyclooxygenase-2 (COX-2)], and xenobiotic metabolism [cytochrome P4501A1 (CYP1A1)]. Genetic markers (CYP1A1, IL-8, and COX-2) and AhR activity were significantly higher with wildfire samples than in solvent controls, whereas AR and ER activities generally were unaffected or reduced. The bioassay responses of samples from residential areas were not significantly different from the samples from the reference site despite differing chemical compositions. Suspect and nontarget screening was conducted to identify the chemicals responsible for elevated bioactivity using the multiple streams of HRMS data and open-source data analysis workflows. For the bioassay endpoint with the largest available database of pure compound results (AhR), nontarget features statistically related to whole sample bioassay response using Spearman's rank-order correlation coefficients or elastic net regression were significantly more likely (by 10 and 15 times, respectively) to be known AhR agonists than the overall population of compounds tentatively identified by nontarget analysis. The findings suggest that a combination of nontarget analysis, in vitro bioassays, and statistical analysis can identify bioactive compounds in complex mixtures.

MeCP2 isoform e1 mutant mice recapitulate motor and metabolic phenotypes of Rett syndrome.

Mutations in the X-linked gene MECP2 cause the majority of Rett syndrome (RTT) cases. Two differentially spliced isoforms of exons 1 and 2 (MeCP2-e1 and MeCP2-e2) contribute to the diverse functions of MeCP2, but only mutations in exon 1, not exon 2, are observed in RTT. We previously described an isoform-specific MeCP2-e1-deficient male mouse model of a human RTT mutation that lacks MeCP2-e1 while preserving expression of MeCP2-e2. However, RTT patients are heterozygous females that exhibit delayed and progressive symptom onset beginning in late infancy, including neurologic as well as metabolic, immune, respiratory and gastrointestinal phenotypes. Consequently, we conducted a longitudinal assessment of symptom development in MeCP2-e1 mutant females and males. A delayed and progressive onset of motor impairments was observed in both female and male MeCP2-e1 mutant mice, including hind limb clasping and motor deficits in gait and balance. Because these motor impairments were significantly impacted by age-dependent increases in body weight, we also investigated metabolic phenotypes at an early stage of disease progression. Both male and female MeCP2-e1 mutants exhibited significantly increased body fat compared to sex-matched wild-type littermates prior to weight differences. Mecp2e1-/y males exhibited significant metabolic phenotypes of hypoactivity, decreased energy expenditure, increased respiratory exchange ratio, but decreased food intake compared to wild-type. Untargeted analysis of lipid metabolites demonstrated a distinguishable profile in MeCP2-e1 female mutant liver characterized by increased triglycerides. Together, these results demonstrate that MeCP2-e1 mutation in mice of both sexes recapitulates early and progressive metabolic and motor phenotypes of human RTT.

Early motor phenotype detection in a female mouse model of Rett syndrome is improved by cross-fostering.

Rett syndrome (RTT) is an X-linked neurodevelopmental disorder caused by mutations in the gene encoding methyl CpG binding protein 2 (MeCP2) that occur sporadically in 1:10,000 female births. RTT is characterized by a period of largely normal development followed by regression in language and motor skills at 6-18 months of age. Mecp2 mutant mice recapitulate many of the clinical features of RTT, but the majority of behavioral assessments have been conducted in male Mecp2 hemizygous null mice as offspring of heterozygous dams. Given that RTT patients are predominantly female, we conducted a systematic analysis of developmental milestones, sensory abilities, and motor deficits, following the longitudinal decline of function from early postnatal to adult ages in female Mecp2 heterozygotes of the conventional Bird line (Mecp2tm1.1bird-/+), as compared to their female wildtype littermate controls. Further, we assessed the impact of postnatal maternal environment on developmental milestones and behavioral phenotypes. Cross-fostering to CD1 dams accelerated several developmental milestones independent of genotype, and induced earlier onset of weight gain in adult female Mecp2tm1.1bird-/+ mice. Cross-fostering improved the sensitivity of a number of motor behaviors that resulted in observable deficits in Mecp2tm1.1bird-/+ mice at much earlier (6-7 weeks) ages than were previously reported (6-9 months). Our findings indicate that female Mecp2tm1.1bird-/+ mice recapitulate many of the motor aspects of RTT syndrome earlier than previously appreciated. In addition, rearing conditions may impact the phenotypic severity and improve the ability to detect genotype differences in female Mecp2 mutant mice.

Pictorial Urgency Scale: A New Tool for Evaluating Bladder Urgency in Children.

PURPOSE: Bladder fullness and urgency are difficult for some patients to express. We hypothesized that images on a pictorial urgency scale would correlate with International Continence Society standard verbal descriptors and bladder volume. MATERIALS AND METHODS: The study population consisted of 267 toilet trained children with a mean age of 7.2 years and their parents (91 adults). Patients were excluded if they had a history of urinary infection, voiding dysfunction, genitourinary surgery or reflux. Participants were read each of the 4 descriptors and asked to point to an image. Correlation between descriptors and figures was analyzed using a mixed effects proportional odds logistic regression model (aim 1 of study). In addition, 73 children undergoing voiding cystourethrography were asked to point to the images during bladder filling. Correlation between percent of expected capacity and image was analyzed using a linear mixed effects model (aim 2 of study). RESULTS: Correlation between descriptors and images (aim 1) was 0.87 (95% CI 0.84 to 0.89) for all participants, 0.84 (95% CI 0.81 to 0.88) for patients younger than age 6 years and 0.88 (95% CI 0.85 to 0.90) for patients 6 to 17 years old. Sequencing of the images was appropriate for increasing degree of urgency. In 73 children undergoing voiding cystourethrography correlation between image and percent of expected capacity (aim 2) was 0.75 (95% CI 0.67 to 0.81, p <0.001). CONCLUSIONS: Figures on the pictorial urgency scale correlate with standard verbal descriptors and bladder volume. The pictorial scale could be a supplemental tool to improve communication of urgency sensation in younger children.

Impact of pre-analytical variables on deep learning accuracy in histopathology.

AIMS: Machine learning (ML) binary classification in diagnostic histopathology is an area of intense investigation. Several assumptions, including training image quality/format and the number of training images required, appear to be similar in many studies irrespective of the paucity of supporting evidence. We empirically compared training image file type, training set size, and two common convolutional neural networks (CNNs) using transfer learning (ResNet50 and SqueezeNet). METHODS AND RESULTS: Thirty haematoxylin and eosin (H&E)-stained slides with carcinoma or normal tissue from three tissue types (breast, colon, and prostate) were photographed, generating 3000 partially overlapping images (1000 per tissue type). These lossless Portable Networks Graphics (PNGs) images were converted to lossy Joint Photographic Experts Group (JPG) images. Tissue type-specific binary classification ML models were developed by the use of all PNG or JPG images, and repeated with a subset of 500, 200, 100, 50, 30 and 10 images. Eleven models were generated for each tissue type, at each quantity of training images, for each file type, and for each CNN, resulting in 924 models. Internal accuracies and generalisation accuracies were compared. There was no meaningful significant difference in accuracies between PNG and JPG models. Models trained with more images did not invariably perform better. ResNet50 typically outperformed SqueezeNet. Models were generalisable within a tissue type but not across tissue types. CONCLUSIONS: Lossy JPG images were not inferior to lossless PNG images in our models. Large numbers of unique H&E-stained slides were not required for training optimal ML models. This reinforces the need for an evidence-based approach to best practices for histopathological ML.

Corneocyte proteomics: Applications to skin biology and dermatology.

Advances in mass spectrometry-based proteomics now permit analysis of complex cellular structures. Application to epidermis and its appendages (nail plate, hair shaft) has revealed a wealth of information about their protein profiles. The results confirm known site-specific differences in levels of certain keratins and add great depth to our knowledge of site specificity of scores of other proteins, thereby connecting anatomy and pathology. An example is the evident overlap in protein profiles of hair shaft and nail plate, helping rationalize their sharing of certain dystrophic syndromes distinct from epidermis. In addition, interindividual differences in protein level are manifest as would be expected. This approach permits characterization of altered profiles as a result of disease, where the magnitude of perturbation can be quantified and monitored during treatment. Proteomic analysis has also clarified the nature of the isopeptide cross-linked residual insoluble material after vigorous extraction with protein denaturants, nearly intractable to analysis without fragmentation. These structures, including the cross-linked envelope of epidermal corneocytes, are comprised of hundreds of protein constituents, evidence for strengthening the terminal structure complementary to disulphide bonding. Along with other developing technologies, proteomic analysis is anticipated to find use in disease risk stratification, detection, diagnosis and prognosis after the discovery phase and clinical validation.