Search

Abstract Background Correct classification of estrogen receptor (ER) status is essential for prognosis and treatment planning in patients with breast cancer (BC). Therefore, it is recommended to sample tumor tissue from an accessible metastasis. However, ER expression can show intra- and intertumoral heterogeneity. 16α-[18F]fluoroestradiol ([18F]FES) Positron Emission Tomography/Computed Tomography (PET/CT) allows noninvasive whole-body (WB) identification of ER distribution and is usually performed as a single static image 60 min after radiotracer injection. Using dynamic whole-body (D-WB) PET imaging, we examine [18F]FES kinetics and explore whether Patlak parametric images ( $${K}_{i}$$ K i ) are quantitative and improve lesion visibility. Results This prospective study included eight patients with metastatic ER-positive BC scanned using a D-WB PET acquisition protocol. The kinetics of [18F]FES were best characterized by the irreversible two-tissue compartment model in tumor lesions and in the majority of organ tissues. $${K}_{i}$$ K i values from Patlak parametric images correlated with $${K}_{i}$$ K i values from the full kinetic analysis, r2 = 0.77, and with the semiquantitative mean standardized uptake value (SUVmean), r2 = 0.91. Furthermore, parametric $${K}_{i}$$ K i images had the highest target-to-background ratio (TBR) in 162/164 metastatic lesions and the highest contrast-to-noise ratio (CNR) in 99/164 lesions compared to conventional SUV images. TBR was 2.45 (95% confidence interval (CI): 2.25–2.68) and CNR 1.17 (95% CI: 1.08–1.26) times higher in $${K}_{i}$$ K i images compared to SUV images. These quantitative differences were seen as reduced background activity in the $${K}_{i}$$ K i images. Conclusion [18F]FES uptake is best described by an irreversible two-tissue compartment model. D-WB [18F]FES PET/CT scans can be used for direct reconstruction of parametric $${K}_{i}$$ K i images, with superior lesion visibility and $${K}_{i}$$ K i values comparable to $${K}_{i}$$ K i values found from full kinetic analyses. This may aid correct ER classification and treatment decisions. Trial registration ClinicalTrials.gov: NCT04150731, https://clinicaltrials.gov/study/NCT04150731

Introduction: Increased theta and delta power and decreased alpha and beta power, measured with quantitative electroencephalography (EEG), have been demonstrated to have utility for predicting the development of dementia in patients with Parkinson’s disease (PD). Noradrenaline modulates cortical activity and optimizes cognitive processes. We claim that the loss of noradrenaline may explain cognitive impairment and the pathological slowing of EEG waves. Here, we test the relationship between the number of noradrenergic α2 adrenoceptors and changes in the spectral EEG ratio in patients with PD. Methods: We included nineteen patients with PD and thirteen healthy control (HC) subjects in the study. We used positron emission tomography (PET) with [11C]yohimbine to quantify α2 adrenoceptor density. We used EEG power in the delta (δ, 1.5–3.9 Hz), theta (θ, 4–7.9 Hz), alpha (α, 8–12.9 Hz) and beta (β, 13–30 Hz) bands in regression analyses to test the relationships between α2 adrenoceptor density and EEG band power. Results: PD patients had higher power in the theta and delta bands compared to the HC volunteers. Patients’ theta band power was inversely correlated with α2 adrenoceptor density in the frontal cortex. In the HC subjects, age was correlated with, and occipital background rhythm frequency (BRF) was inversely correlated with, α2 adrenoceptor density in the frontal cortex, while occipital BRF was inversely correlated with α2 adrenoceptor density in the thalamus. Conclusions: The findings support the claim that the loss or dysfunction of noradrenergic neurotransmission may relate to the parallel processes of cognitive decline and EEG slowing.

Estrogen receptors (ERs) play a multitude of roles in brain function and are implicated in various brain disorders. The use of positron emission tomography (PET) tracers for the visualization of ERs’ intricate landscape has shown promise in oncology but remains limited in the context of brain disorders. Despite recent progress in the identification and development of more selective ligands for various ERs subtypes, further optimization is necessary to enable the reliable and efficient imaging of these receptors. In this perspective, we briefly touch upon the significance of estrogen signaling in the brain and raise the setbacks associated with the development of PET tracers for identification of specific ERs subtypes in the brain. We then propose avenues for developing efficient PET tracers to non-invasively study the dynamics of ERs in the brain, as well as neuropsychiatric diseases associated with their malfunction in a longitudinal manner. This perspective puts several potential candidates on the table and highlights the unmet needs and areas requiring further research to unlock the full potential of PET tracers for ERs imaging, ultimately aiding in deepening our understanding of ERs and forging new avenues for potential therapeutic strategies.

The noradrenaline system attracts attention for its role in mood disorders and neurodegenerative diseases but the lack of well-validated methods impairs our understanding when assessing its function and release in vivo. This study combines simultaneous positron emission tomography (PET) and microdialysis to explore if [11C]yohimbine, a selective antagonist radioligand of the α2 adrenoceptors, may be used to assess in vivo changes in synaptic noradrenaline during acute pharmacological challenges. Anesthetised Göttingen minipigs were positioned in a head holder in a PET/CT device. Microdialysis probes were placed in the thalamus, striatum and cortex and dialysis samples were collected every 10 min. Three 90 min [11C]yohimbine scans were acquired: at baseline and at two timepoints after the administration of amphetamine (1–10 mg/kg), a non-specific releaser of dopamine and noradrenaline, or nisoxetine (1 mg/kg), a specific noradrenaline transporter inhibitor. [11C]yohimbine volumes of distribution (VT) were obtained using the Logan kinetic model. Both challenges induced a significant decrease in yohimbine VT, with time courses reflecting their different mechanisms of action. Dialysis samples revealed a significant increase in noradrenaline extracellular concentrations after challenge and an inverse correlation with changes in yohimbine VT. These data suggest that [11C]yohimbine can be used to evaluate acute variations in synaptic noradrenaline concentrations after pharmacological challenges.

Abstract Background In vitro experiments using radiolabeled molecules is fundamental for Positron emission tomography (PET) or single photon emission computed tomography (SPECT) tracer development and various metabolic assays, but no consensus on appropriate incubation conditions exists. Specifically, the use of shaking versus non-shaking conditions, cell number to medium volume and the choice of cell plating material may unintentionally influence cellular oxygenation and medium composition. This is problematic when testing the oxygen-dependence of tracers including 18F-fluoro-2-deoxyglucose ([18F]FDG) and hypoxia-selective 2-nitroimidazoles (e.g., 18F-fluoroazomycin-arabinoside, [18F]FAZA) or when doing prolonged experiments. The purpose of this study was to assess the influence of various experimental conditions on tracer retention. Methods Tumor cells were seeded in a) Glass or standard Polystyrene Petri dishes or as b) discrete droplets in polystyrene Petri dishes or on 9 mm glass coverslips positioned in glass Petri dishes. When confluent, cells were pre-equilibrated for 2 h to 21%, 0.5% or 0% O2 and [18F] FDG or [18F] FAZA was added, followed by cell harvest and analysis of radioactivity 1 h ([18F]FDG) or 3 h ([18F]FAZA) after. Experiments were conducted with/without orbital shaking. Results The influence of hypoxia on tracer retention varied widely among cell lines, but shaking-induced convection did not influence uptake. In contrast, hypoxia-driven [18F] FAZA, and to some extent [18F] FDG, retention was much lower in cells grown on polyethylene than glass. Scaling-down the number of cells did not compromise accuracy. Conclusions Tracer retention was similar under stagnant and forced convection conditions suggesting that the former approach may be appropriate even when accurate control of oxygen and tracer availability is required. In contrast, conventional plasticware should be used with caution when studying tracers and drugs that are metabolized and retained or activated at low O2 levels. Downscaling of cell number, by reducing the effective growth area, was feasible, without compromising accuracy.

Accumulation of uremic toxins may lead to the life-threatening condition “uremic syndrome” in patients with advanced chronic kidney disease (CKD) requiring renal replacement therapy. Clinical evaluation of proximal tubular secretion of organic cations (OC), of which some are uremic toxins, is desired, but difficult. The biomedical knowledge on OC secretion and cellular transport partly relies on studies using the fluorescent tracer 4-dimethylaminostyryl)-N-methylpyridinium (ASP+), which has been used in many studies of renal excretion mechanisms of organic ions and which could be a candidate as a PET tracer. This study is aimed at expanding the knowledge of the tracer characteristics of ASP+ by recording the distribution and intensity of ASP+ signals in vivo both by fluorescence and by positron emission tomography (PET) imaging and at investigating if the fluorescence signal of ASP+ is influenced by the presence of albumin. Two-photon in vivo microscopy of male Münich Wistar Frömter rats showed that a bolus injection of ASP+ conferred a fluorescence signal to the blood plasma lasting for about 30 minutes. In the renal proximal tubule, the bolus resulted in a complex pattern of fluorescence including a rapid and strong transient signal at the brush border, a very low signal in the luminal fluid, and a slow transient intracellular signal. PET imaging using 11C-labelled ASP+ showed accumulation in the liver, heart, and kidney. Fluorescence emission spectra recorded in vitro of ASP+ alone and in the presence of albumin using both 1-photon excitation and two-photon excitation showed that albumin strongly enhance the emission from ASP+ and induce a shift of the emission maximum from 600 to 570 nm. Conclusion. The renal pattern of fluorescence observed from ASP+ in vivo is likely affected by the local concentration of albumin, and quantification of ASP+ fluorescent signals in vivo cannot be directly translated to ASP+ concentrations.

In order to examine attitudes in the field of domestic violence toward mandated reporter laws, a survey was administered to domestic violence shelter workers. The respondents overwhelmingly supported the mandate to report abuse of a child. However, a mandate to report child witnessing of domestic violence was opposed by a slight majority. When compared with the group opposing the mandate, the group in support perceived more frequent occurrence of a report's positive impact and less frequent occurrence of a report's negative impacts. These results have implications for advocacy efforts that are focused on mandated reporter laws. [ABSTRACT FROM AUTHOR]

The purpose of this study was to assess the preparation of human service organizations for the minimization of risk and the maximization of opportunity. Three primary areas were examined: political connectivity (which aids in the maximization of opportunity), documentation of agency policies (which aids in the minimization of risk), and comprehensive strategic planning (which aids in both the maximization of opportunity and the minimization of risk). A survey of organizational characteristics was conducted with agencies known for provision of sexual education services to adolescents within Florida. This study revealed some of the strengths and deficits of the organizations in the sample. Strengths included fairly high rates of political connectivity and written documentation of personnel policies. There were deficits in the documentation of programmatic policies and the comprehensiveness of strategic plans. [ABSTRACT FROM AUTHOR]

Abstract Evidence suggests that synapses are affected first in Parkinson’s disease (PD). Here, we tested the claim that pathological accumulation of α-synuclein, and subsequent synaptic disruption, occur in absence of dopaminergic neuron loss in PD. We determined early synaptic changes in rats that overexpress human α-synuclein by local injection of viral-vectors in midbrain. We aimed to achieve α-synuclein levels sufficient to induce terminal pathology without significant loss of nigral neurons. We tested synaptic disruption in vivo by analyzing motor defects and binding of a positron emission tomography (PET) radioligand to the vesicular monoamine transporter 2, (VMAT2), [11C]dihydrotetrabenazine (DTBZ). Animals overexpressing α-synuclein had progressive motor impairment and, 12 weeks post-surgery, showed asymmetric in vivo striatal DTBZ binding. The PET images matched ligand binding in post-mortem tissue, and histological markers of dopaminergic integrity. Histology confirmed the absence of nigral cell death with concomitant significant loss of striatal terminals. Progressive aggregation of proteinase-K resistant and Ser129-phosphorylated α-synuclein was observed in dopaminergic terminals, in dystrophic swellings that resembled axonal spheroids and contained mitochondria and vesicular proteins. In conclusion, pathological α-synuclein in nigro-striatal axonal terminals leads to early axonal pathology, synaptic disruption, dysfunction of dopaminergic neurotransmission, motor impairment, and measurable change of VMAT2 in the absence of cell loss.

The purpose of this article is to identify, assess, and offer solutions to common measurement errors found in sexual abstinence education evaluation. A critical review of the methodology of adolescent sexuality research was performed. "Gold standards" of their measurement strategy were derived and applied against 14 selected studies. Many of the articles reviewed had substantial limitations in their measurement strategies. However, several articles demonstrated excellence and serve as models for future efforts. Sexual abstinence education evaluation is plagued by the inherent weaknesses of self-report and health outcome measures. However, with careful adherence to the gold standards proposed, it is possible to limit the threat from these weaknesses, maximizing the benefit of self-report surveys and county-level health indicators. [ABSTRACT FROM AUTHOR]

Introduction: Despite the decades long use of [ 11 C]palmitate positron emission tomography (PET)/computed tomography in basic metabolism studies, only personal communications regarding dosimetry and biodistribution data have been published. Methods: Dosimetry and biodistribution studies were performed in 2 pigs and 2 healthy volunteers by whole-body [ 11 C]palmitate PET scans. Metabolite studies were performed in 40 participants (healthy and with type 2 diabetes) under basal and hyperinsulinemic conditions. Metabolites were estimated using 2 approaches and subsequently compared: Indirect [ 11 C]CO 2 release and parent [ 11 C]palmitate measured by a solid-phase extraction (SPE) method. Finally, myocardial fatty acid uptake was calculated in a patient cohort using input functions derived from individual metabolite correction compared with population-based metabolite correction. Results: In humans, mean effective dose was 3.23 (0.02) µSv/MBq, with the liver and myocardium receiving the highest absorbed doses. Metabolite correction using only [ 11 C]CO 2 estimates underestimated the fraction of metabolites in studies lasting more than 20 minutes. Population-based metabolite correction showed excellent correlation with individual metabolite correction in the cardiac PET validation cohort. Conclusion: First, mean effective dose of [ 11 C]palmitate is 3.23 (0.02) µSv/MBq in humans allowing multiple scans using ∼300 MBq [ 11 C]palmitate, and secondly, population-based metabolite correction compares well with individual correction.

Abstract Background The developing mouse kidney is currently the best-characterized model of organogenesis at a transcriptional level. Detailed spatial maps have been generated for gene expression profiling combined with systematic in situ screening. These studies, however, fall short of capturing the transcriptional complexity arising from each locus due to the limited scope of microarray-based technology, which is largely based on "gene-centric" models. Results To address this, the polyadenylated RNA and microRNA transcriptomes of the 15.5 dpc mouse kidney were profiled using strand-specific RNA-sequencing (RNA-Seq) to a depth sufficient to complement spatial maps from pre-existing microarray datasets. The transcriptional complexity of RNAs arising from mouse RefSeq loci was catalogued; including 3568 alternatively spliced transcripts and 532 uncharacterized alternate 3' UTRs. Antisense expressions for 60% of RefSeq genes was also detected including uncharacterized non-coding transcripts overlapping kidney progenitor markers, Six2 and Sall1, and were validated by section in situ hybridization. Analysis of genes known to be involved in kidney development, particularly during mesenchymal-to-epithelial transition, showed an enrichment of non-coding antisense transcripts extended along protein-coding RNAs. Conclusion The resulting resource further refines the transcriptomic cartography of kidney organogenesis by integrating deep RNA sequencing data with locus-based information from previously published expression atlases. The added resolution of RNA-Seq has provided the basis for a transition from classical gene-centric models of kidney development towards more accurate and detailed "transcript-centric" representations, which highlights the extent of transcriptional complexity of genes that direct complex development events.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is characterized by highly heterogeneous manifestations ranging from asymptomatic cases to death for still incompletely understood reasons. As part of the IMmunoPhenotyping Assessment in a COVID-19 Cohort study, we mapped the plasma proteomes of 1117 hospitalized patients with COVID-19 from 15 hospitals across the United States. Up to six samples were collected within ~28 days of hospitalization resulting in one of the largest COVID-19 plasma proteomics cohorts with 2934 samples. Using perchloric acid to deplete the most abundant plasma proteins allowed for detecting 2910 proteins. Our findings show that increased levels of neutrophil extracellular trap and heart damage markers are associated with fatal outcomes. Our analysis also identified prognostic biomarkers for worsening severity and death. Our comprehensive longitudinal plasma proteomics study, involving 1117 participants and 2934 samples, allowed for testing the generalizability of the findings of many previous COVID-19 plasma proteomics studies using much smaller cohorts.

Tau protein aggregation is associated with posttranslational modifications (PTMs) in 75% of all dementia cases. The distribution of tau pathology and the presence of specific tau phosphorylation sites of interest are typically visualized and measured using antibodies. However, previous knowledge of the target epitopes is required. Additionally, antibodies can be used in a semi-quantitative manner but cannot be used to determine the absolute amount of tau or the extent of the modifications at specific sites or domains. Here we present a discovery assay that characterizes the global qualitative and quantitative tau modification landscape of a sample without a priori knowledge. Our workflow uses sarkosyl fractionation to extract the pathological tau species from sample-limited brain specimens, followed by mass spectrometry (MS) to characterize and quantify tau PTMs. The two-step MS-based proteomics approach includes an exploratory tau PTM analysis and a targeted full-length expressed stable isotope-labeled tau assay, which monitors specific unmodified tau peptides using a heavy isotope-labeled internal standard as a reference. This enables the absolute quantification of the respective tau peptides and the total tau amount in the sample, thus providing the modification extent of tau PTMs. This approach provides precise, comprehensive, qualitative and quantitative tau PTM profiling of the sample. It also enables the detailed molecular comparison of tau across multiple experiments, including a comparison between neurodegenerative diseases, stages of the disease, human patient heterogeneity and characterization of animal models. The approach is useful for studying the molecular features of pathological tau in neurodegeneration. The procedure requires 7-8 d and is suitable for users with expertise in targeted and untargeted MS-based protein analysis., (© 2024. Springer Nature Limited.)

Aggregation prone molecules, such as tau, form both historically well characterized fibrillar deposits (neurofibrillary tangles) and recently identified phosphate-buffered saline (PBS) extract species called proteopathic seeds. Both can cause normal endogenous tau to undergo templated misfolding. The relationship of these seeds to the fibrils that define tau-related diseases is unknown. We characterized the aqueous extractable and sarkosyl insoluble fibrillar tau species derived from human Alzheimer brain using mass spectrometry and in vitro bioassays. Post-translational modifications (PTMs) including phosphorylation, acetylation and ubiquitination are identified in both preparations. PBS extract seed competent tau can be distinguished from sarkosyl insoluble tau by the presence of overlapping, but less abundant, PTMs and an absence of some PTMs unique to the latter. The presence of ubiquitin and other PTMs on the PBS-extracted tau species correlates with the amount of tau in the seed competent size exclusion fractions, with the bioactivity and with the aggressiveness of clinical disease. These results demonstrate that the PTMs present on bioactive, seed competent PBS extract tau species are closely related to, but distinct from, the PTMs of mature paired helical filaments, consistent with the idea that they are a forme fruste of tau species that ultimately form fibrils., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Characterizing perturbation of molecular pathways in congenital Zika virus (ZIKV) infection is critical for improved therapeutic approaches. Leveraging integrative systems biology, proteomics, and RNA-seq, we analyzed embryonic brain tissues from an immunocompetent, wild-type congenital ZIKV infection mouse model. ZIKV induced a robust immune response accompanied by the downregulation of critical neurodevelopmental gene programs. We identified a negative correlation between ZIKV polyprotein abundance and host cell cycle-inducing proteins. We further captured the downregulation of genes/proteins, many of which are known to be causative for human microcephaly, including Eomesodermin/T-box Brain Protein 2 (EOMES/TBR2) and Neuronal Differentiation 2 (NEUROD2). Disturbances of distinct molecular pathways in neural progenitors and post-mitotic neurons may contribute to complex brain phenotype of congenital ZIKV infection. Overall, this report on protein- and transcript-level dynamics enhances understanding of the ZIKV immunopathological landscape through characterization of fetal immune response in the developing brain., Competing Interests: The authors declare no conflicts of interest., (© 2023.)

To develop therapies for Alzheimer's disease, we need accurate in vivo diagnostics. Multiple proteomic studies mapping biomarker candidates in cerebrospinal fluid (CSF) resulted in little overlap. To overcome this shortcoming, we apply the rarely used concept of proteomics meta-analysis to identify an effective biomarker panel. We combine ten independent datasets for biomarker identification: seven datasets from 150 patients/controls for discovery, one dataset with 20 patients/controls for down-selection, and two datasets with 494 patients/controls for validation. The discovery results in 21 biomarker candidates and down-selection in three, to be validated in the two additional large-scale proteomics datasets with 228 diseased and 266 control samples. This resulting 3-protein biomarker panel differentiates Alzheimer's disease (AD) from controls in the two validation cohorts with areas under the receiver operating characteristic curve (AUROCs) of 0.83 and 0.87, respectively. This study highlights the value of systematically re-analyzing previously published proteomics data and the need for more stringent data deposition., Competing Interests: Declaration of interests R.S. is member of the European Behavioral Pharmacology Society. H.Z. is chair of the Alzheimer’s Association Global Biomarker Standardization Consortium and the Alzheimer’s Association Biofluid-Based Biomarker PIA. J.A.S. reports patents for tau therapeutics and biomarkers. H.S., J.A.S., and P.W.v.Z. have submitted a patent application for markers described in this manuscript. H.S. and J.A.S. report additional patent applications (unrelated to the work described in the manuscript) around tau-PTM-based biomarkers for AD and other tauopathies., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: Mouse models that overexpress human mutant Tau (P301S and P301L) are commonly used in preclinical studies of Alzheimer's Disease (AD) and while several drugs showed therapeutic effects in these mice, they were ineffective in humans. This leads to the question to which extent the murine models reflect human Tau pathology on the molecular level., Methods: We isolated insoluble, aggregated Tau species from two common AD mouse models during different stages of disease and characterized the modification landscape of the aggregated Tau using targeted and untargeted mass spectrometry-based proteomics. The results were compared to human AD and to human patients that suffered from early onset dementia and that carry the P301L Tau mutation., Results: Both mouse models accumulate insoluble Tau species during disease. The Tau aggregation is driven by progressive phosphorylation within the proline rich domain and the C-terminus of the protein. This is reflective of early disease stages of human AD and of the pathology of dementia patients carrying the P301L Tau mutation. However, Tau ubiquitination and acetylation, which are important to late-stage human AD are not represented in the mouse models., Conclusion: AD mouse models that overexpress human Tau using risk mutations are a suitable tool for testing drug candidates that aim to intervene in the early formation of insoluble Tau species promoted by increased phosphorylation of Tau., (© 2023. The Author(s).)

In liquid-chromatography-tandem-mass-spectrometry-based proteomics, information about the presence and stoichiometry of protein modifications is not readily available. To overcome this problem, we developed multiFLEX-LF, a computational tool that builds upon FLEXIQuant, which detects modified peptide precursors and quantifies their modification extent by monitoring the differences between observed and expected intensities of the unmodified precursors. multiFLEX-LF relies on robust linear regression to calculate the modification extent of a given precursor relative to a within-study reference. multiFLEX-LF can analyze entire label-free discovery proteomics data sets in a precursor-centric manner without preselecting a protein of interest. To analyze modification dynamics and coregulated modifications, we hierarchically clustered the precursors of all proteins based on their computed relative modification scores. We applied multiFLEX-LF to a data-independent-acquisition-based data set acquired using the anaphase-promoting complex/cyclosome (APC/C) isolated at various time points during mitosis. The clustering of the precursors allows for identifying varying modification dynamics and ordering the modification events. Overall, multiFLEX-LF enables the fast identification of potentially differentially modified peptide precursors and the quantification of their differential modification extent in large data sets using a personal computer. Additionally, multiFLEX-LF can drive the large-scale investigation of the modification dynamics of peptide precursors in time-series and case-control studies. multiFLEX-LF is available at https://gitlab.com/SteenOmicsLab/multiflex-lf.

Background: Multigene panel tests for breast cancer predisposition routinely include ATM as it is now a well-established breast cancer predisposition gene., Methods: We included ATM in a multigene panel test applied to the Australian Breast Cancer Family Registry (ABCFR), a population-based case-control-family study of breast cancer, with the purpose of estimating the prevalence and penetrance of heterozygous ATM pathogenic variants from the family data, using segregation analysis., Results: The estimated breast cancer hazard ratio for carriers of pathogenic ATM variants in the ABCFR was 1.32 (95% confidence interval 0.45-3.87; P = 0.6). The estimated cumulative risk of breast cancer to age 80 years for heterozygous ATM pathogenic variant carriers was estimated to be 13% (95% CI 4.6-30)., Conclusions: Although ATM has been definitively identified as a breast cancer predisposition gene, further evidence, such as variant-specific penetrance estimates, are needed to inform risk management strategies for carriers of pathogenic variants to increase the clinical utility of population testing of this gene., (© 2022. The Author(s).)

Introduction: Current techniques to diagnose and/or monitor critically ill neonates with bronchopulmonary dysplasia (BPD) require invasive sampling of body fluids, which is suboptimal in these frail neonates. We tested our hypothesis that it is feasible to use noninvasively collected urine samples for proteomics from extremely low gestational age newborns (ELGANs) at risk for BPD to confirm previously identified proteins and biomarkers associated with BPD., Methods: We developed a robust high-throughput urine proteomics methodology that requires only 50 μL of urine. We utilized the methodology with a proof-of-concept study validating proteins previously identified in invasively collected sample types such as blood and/or tracheal aspirates on urine collected within 72 h of birth from ELGANs (gestational age [26 ± 1.2] weeks) who were admitted to a single Neonatal Intensive Care Unit (NICU), half of whom eventually developed BPD (n = 21), while the other half served as controls (n = 21)., Results: Our high-throughput urine proteomics approach clearly identified several BPD-associated changes in the urine proteome recapitulating expected blood proteome changes, and several urinary proteins predicted BPD risk. Interestingly, 16 of the identified urinary proteins are known targets of drugs approved by the Food and Drug Administration., Conclusion: In addition to validating numerous proteins, previously found in invasively collected blood, tracheal aspirate, and bronchoalveolar lavage, that have been implicated in BPD pathophysiology, urine proteomics also suggested novel potential therapeutic targets. Ease of access to urine could allow for sequential proteomic evaluations for longitudinal monitoring of disease progression and impact of therapeutic intervention in future studies., (© 2022 S. Karger AG, Basel.)

Population-based estimates of breast cancer risk for carriers of pathogenic variants identified by gene-panel testing are urgently required. Most prior research has been based on women selected for high-risk features and more data is needed to make inference about breast cancer risk for women unselected for family history, an important consideration of population screening. We tested 1464 women diagnosed with breast cancer and 862 age-matched controls participating in the Australian Breast Cancer Family Study (ABCFS), and 6549 healthy, older Australian women enroled in the ASPirin in Reducing Events in the Elderly (ASPREE) study for rare germline variants using a 24-gene-panel. Odds ratios (ORs) were estimated using unconditional logistic regression adjusted for age and other potential confounders. We identified pathogenic variants in 11.1% of the ABCFS cases, 3.7% of the ABCFS controls and 2.2% of the ASPREE (control) participants. The estimated breast cancer OR [95% confidence interval] was 5.3 [2.1-16.2] for BRCA1, 4.0 [1.9-9.1] for BRCA2, 3.4 [1.4-8.4] for ATM and 4.3 [1.0-17.0] for PALB2. Our findings provide a population-based perspective to gene-panel testing for breast cancer predisposition and opportunities to improve predictors for identifying women who carry pathogenic variants in breast cancer predisposition genes., (© 2021. The Author(s).)

Background: Diphtheria is a potentially fatal respiratory disease caused by toxigenic Corynebacterium diphtheriae. Although resistance to erythromycin has been recognized, β-lactam resistance in toxigenic diphtheria has not been described. Here, we report a case of fatal respiratory diphtheria caused by toxigenic C. diphtheriae resistant to penicillin and all other β-lactam antibiotics, and describe a novel mechanism of inducible carbapenem resistance associated with the acquisition of a mobile resistance element., Methods: Long-read whole-genome sequencing was performed using Pacific Biosciences Single Molecule Real-Time sequencing to determine the genome sequence of C. diphtheriae BQ11 and the mechanism of β-lactam resistance. To investigate the phenotypic inducibility of meropenem resistance, short-read sequencing was performed using an Illumina NextSeq500 sequencer on the strain both with and without exposure to meropenem., Results: BQ11 demonstrated high-level resistance to penicillin (benzylpenicillin minimum inhibitory concentration [MIC] ≥ 256 μg/ml), β-lactam/β-lactamase inhibitors and cephalosporins (amoxicillin/clavulanic acid MIC ≥ 256 μg/mL; ceftriaxone MIC ≥ 8 μg/L). Genomic analysis of BQ11 identified acquisition of a novel transposon carrying the penicillin-binding protein (PBP) Pbp2c, responsible for resistance to penicillin and cephalosporins. When strain BQ11 was exposed to meropenem, selective pressure drove amplification of the transposon in a tandem array and led to a corresponding change from a low-level to a high-level meropenem-resistant phenotype., Conclusions: We have identified a novel mechanism of inducible antibiotic resistance whereby isolates that appear to be carbapenem susceptible on initial testing can develop in vivo resistance to carbapenems with repeated exposure. This phenomenon could have significant implications for the treatment of C. diphtheriae infection, and may lead to clinical failure., (© The Author(s) 2020. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

Background: The androgen receptor (AR) pathway-associated gene nuclear receptor coactivator 2 (NCOA2) has an established oncogenic role in early prostate cancer and likewise is a driver of metastatic disease and castration-resistant prostate cancer. However, its significance as a biomarker in metastatic castration-resistant prostate cancer (mCRPC), both alone and in conjunction with co-occurring AR alterations using a liquid biopsy approach has not been investigated., Methods: Ninety-one patients were included in this study, (n = 68 receiving an androgen receptor pathway inhibitor and n = 23 receiving taxane chemotherapy). Up to 30 ml of peripheral blood was collected before commencing treatment from each patient. Plasma cell-free DNA, along with a matched germline sample, underwent targeted next-generation sequencing using a validated, highly sensitive in-house prostate cancer panel. Variants in AR and NCOA2 were identified and correlated with clinical outcomes., Results: Plasma AR and NCOA2 aberrations were identified in 35% and 13% of the cohort, respectively, whilst 8% had concurrent AR and NCOA2 alterations. NCOA2 copy number gain and any NCOA2 aberration predicted for lower prostate-specific antigen (PSA) response rates. Likewise, median overall survival was shorter for NCOA2 gain (10.1 vs. 18.3 months; p = .004), remaining significant after adjusting for covariates including circulating tumor DNA fraction and tumor suppressor gene alterations. Importantly, dual AR and NCOA2 aberrations were also associated with inferior outcomes, including no PSA responses in patients treated with AR pathway inhibitors (0% vs. 64%; p = .02)., Conclusions: These data highlight the importance of identifying multiple markers of AR pathway modulation in mCRPC and represent the first instance of the assessment of plasma NCOA2 status as a prognostic biomarker for standard-of-care therapies. Further assessment is warranted to determine if NCOA2 aberrations are a marker of primary resistance to AR pathway inhibitors., (© 2021 Wiley Periodicals LLC.)

While gene panel sequencing is becoming widely used for cancer risk prediction, its clinical utility with respect to predicting aggressive prostate cancer (PrCa) is limited by our current understanding of the genetic risk factors associated with predisposition to this potentially lethal disease phenotype. This study included 837 men diagnosed with aggressive PrCa and 7261 controls (unaffected men and men who did not meet criteria for aggressive PrCa). Rare germline pathogenic variants (including likely pathogenic variants) were identified by targeted sequencing of 26 known or putative cancer predisposition genes. We found that 85 (10%) men with aggressive PrCa and 265 (4%) controls carried a pathogenic variant ( p < 0.0001). Aggressive PrCa odds ratios (ORs) were estimated using unconditional logistic regression. Increased risk of aggressive PrCa (OR (95% confidence interval)) was identified for pathogenic variants in BRCA2 (5.8 (2.7-12.4)), BRCA1 (5.5 (1.8-16.6)), and ATM (3.8 (1.6-9.1)). Our study provides further evidence that rare germline pathogenic variants in these genes are associated with increased risk of this aggressive, clinically relevant subset of PrCa. These rare genetic variants could be incorporated into risk prediction models to improve their precision to identify men at highest risk of aggressive prostate cancer and be used to identify men with newly diagnosed prostate cancer who require urgent treatment.

Case-control studies of breast cancer have consistently shown that pathogenic variants in CHEK2 are associated with about a 3-fold increased risk of breast cancer. Information about the recurrent protein-truncating variant CHEK2 c.1100delC dominates this estimate. There have been no formal estimates of age-specific cumulative risk of breast cancer for all CHEK2 pathogenic (including likely pathogenic) variants combined. We conducted a population-based case-control-family study of pathogenic CHEK2 variants (26 families, 1071 relatives) and estimated the age-specific cumulative risk of breast cancer using segregation analysis. The estimated hazard ratio for carriers of pathogenic CHEK2 variants (combined) was 4.9 (95% CI 2.5-9.5) relative to non-carriers. The HR for carriers of the CHEK2 c.1100delC variant was estimated to be 3.5 (95% CI 1.02-11.6) and the HR for carriers of all other CHEK2 variants combined was estimated to be 5.7 (95% CI 2.5-12.9). The age-specific cumulative risk of breast cancer was estimated to be 18% (95% CI 11-30%) and 33% (95% CI 21-48%) to age 60 and 80 years, respectively. These findings provide important information for the clinical management of breast cancer risk for women carrying pathogenic variants in CHEK2 .

VTRNA2-1 is a metastable epiallele with accumulating evidence that methylation at this region is heritable, modifiable and associated with disease including risk and progression of cancer. This study investigated the influence of genetic variation and other factors such as age and adult lifestyle on blood DNA methylation in this region. We first sequenced the VTRNA2-1 gene region in multiple-case breast cancer families in which VTRNA2-1 methylation was identified as heritable and associated with breast cancer risk. Methylation quantitative trait loci (mQTL) were investigated using a prospective cohort study (4500 participants with genotyping and methylation data). The cis -mQTL analysis (334 variants ± 50 kb of the most heritable CpG site) identified 43 variants associated with VTRNA2-1 methylation ( p < 1.5 × 10 -4 ); however, these explained little of the methylation variation (R 2 < 0.5% for each of these variants). No genetic variants elsewhere in the genome were found to strongly influence VTRNA2-1 methylation. SNP-based heritability estimates were consistent with the mQTL findings (h 2 = 0, 95%CI: -0.14 to 0.14). We found no evidence that age, sex, country of birth, smoking, body mass index, alcohol consumption or diet influenced blood DNA methylation at VTRNA2-1 . Genetic factors and adult lifestyle play a minimal role in explaining methylation variability at the heritable VTRNA2-1 cluster.

To elucidate the role of Tau isoforms and post-translational modification (PTM) stoichiometry in Alzheimer's disease (AD), we generated a high-resolution quantitative proteomics map of 95 PTMs on multiple isoforms of Tau isolated from postmortem human tissue from 49 AD and 42 control subjects. Although Tau PTM maps reveal heterogeneity across subjects, a subset of PTMs display high occupancy and frequency for AD, suggesting importance in disease. Unsupervised analyses indicate that PTMs occur in an ordered manner, leading to Tau aggregation. The processive addition and minimal set of PTMs associated with seeding activity was further defined by analysis of size-fractionated Tau. To summarize, features in the Tau protein critical for disease intervention at different stages of disease are identified, including enrichment of 0N and 4R isoforms, underrepresentation of the C terminus, an increase in negative charge in the proline-rich region (PRR), and a decrease in positive charge in the microtubule binding domain (MBD)., Competing Interests: Declaration of Interests B.T.H. is a member of the SAB and owns shares in Dewpoint. He also serves on an advisory panel for Biogen, and his laboratory has current research funding from AbbVie. His wife is an employee and shareholder of Novartis., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Improvements in LC-MS/MS methods and technology have enabled the identification of thousands of modified peptides in a single experiment. However, protein regulation by post-translational modifications (PTMs) is not binary, making methods to quantify the modification extent crucial to understanding the role of PTMs. Here, we introduce FLEXIQuant-LF, a software tool for large-scale identification of differentially modified peptides and quantification of their modification extent without knowledge of the types of modifications involved. We developed FLEXIQuant-LF using label-free quantification of unmodified peptides and robust linear regression to quantify the modification extent of peptides. As proof of concept, we applied FLEXIQuant-LF to data-independent-acquisition (DIA) data of the anaphase promoting complex/cyclosome (APC/C) during mitosis. The unbiased FLEXIQuant-LF approach to assess the modification extent in quantitative proteomics data provides a better understanding of the function and regulation of PTMs. The software is available at https://github.com/SteenOmicsLab/FLEXIQuantLF., Competing Interests: CS, KK, JM, RC, BR, JS, HS No competing interests declared, (© 2020, Schlaffner et al.)

Few genetic risk factors have been demonstrated to be specifically associated with aggressive prostate cancer (PrCa). Here, we report a case-case study of PrCa comparing the prevalence of germline pathogenic/likely pathogenic (P/LP) genetic variants in 787 men with aggressive disease and 769 with nonaggressive disease. Overall, we observed P/LP variants in 11.4% of men with aggressive PrCa and 9.8% of men with nonaggressive PrCa (two-tailed Fisher's exact tests, P = .28). The proportion of BRCA2 and ATM P/LP variant carriers in men with aggressive PrCa exceeded that observed in men with nonaggressive PrCa; 18/787 carriers (2.3%) and 4/769 carriers (0.5%), P = .004, and 14/787 carriers (0.02%) and 5/769 carriers (0.01%), P = .06, respectively. Our findings contribute to the extensive international effort to interpret the genetic variation identified in genes included on gene-panel tests, for which there is currently an insufficient evidence-base for clinical translation in the context of PrCa risk., (© 2020 UICC.)

Purpose: To characterize the spectrum of BRCA1 and BRCA2 pathogenic germline variants in women from south-west Poland and west Ukraine affected with breast or ovarian cancer. Testing in women at high risk of breast and ovarian cancer in these regions is currently mainly limited to founder mutations., Methods: Unrelated women affected with breast and/or ovarian cancer from Poland (n = 337) and Ukraine (n = 123) were screened by targeted sequencing. Excluded from targeted sequencing were 34 Polish women who had previously been identified as carrying a founder mutation in BRCA1. No prior testing had been conducted among the Ukrainian women. Thus, this study screened BRCA1 and BRCA2 in the germline DNA of 426 women in total., Results: We identified 31 and 18 women as carriers of pathogenic/likely pathogenic (P/LP) genetic variants in BRCA1 and BRCA2, respectively. We observed five BRCA1 and eight BRCA2 P/LP variants (13/337, 3.9%) in the Polish women. Combined with the 34/337 (10.1%) founder variants identified prior to this study, the overall P/LP variant frequency in the Polish women was thus 14% (47/337). Among the Ukrainian women, 16/123 (13%) women were identified as carrying a founder mutation and 20/123 (16.3%) were found to carry non-founder P/LP variants (10 in BRCA1 and 10 in BRCA2)., Conclusions: These results indicate that genetic testing in women at high risk of breast and ovarian cancer in Poland and Ukraine should not be limited to founder mutations. Extended testing will enhance risk stratification and management for these women and their families.

Plasma circulating tumor DNA (ctDNA) analysis has emerged as a minimally invasive means to perform molecular tumor typing. Here we developed a custom ultra-sensitive ctDNA next-generation sequencing assay using molecular barcoding technology and off-the-shelf reagents combined with bioinformatics tools for enhanced ctDNA analysis. Assay performance was assessed via a spike-in experiment and the technique was applied to analyze 41 plasma samples from men with advanced prostate cancer. Orthogonal validation was performed using a commercial assay. Sensitivity and specificity of 93 and 99.5% were recorded for ultra-rare somatic variants (<1%), with high concordance observed between the in-house and commercial assays. The optimized protocol dramatically improved the efficiency of the assay and enabled the detection of low-frequency somatic variants from plasma cell-free DNA (cfDNA).

Alzheimer's disease (AD) causes unrelenting, progressive cognitive impairments, but its course is heterogeneous, with a broad range of rates of cognitive decline 1 . The spread of tau aggregates (neurofibrillary tangles) across the cerebral cortex parallels symptom severity 2,3 . We hypothesized that the kinetics of tau spread may vary if the properties of the propagating tau proteins vary across individuals. We carried out biochemical, biophysical, MS and both cell- and animal-based-bioactivity assays to characterize tau in 32 patients with AD. We found striking patient-to-patient heterogeneity in the hyperphosphorylated species of soluble, oligomeric, seed-competent tau. Tau seeding activity correlates with the aggressiveness of the clinical disease, and some post-translational modification (PTM) sites appear to be associated with both enhanced seeding activity and worse clinical outcomes, whereas others are not. These data suggest that different individuals with 'typical' AD may have distinct biochemical features of tau. These data are consistent with the possibility that individuals with AD, much like people with cancer, may have multiple molecular drivers of an otherwise common phenotype, and emphasize the potential for personalized therapeutic approaches for slowing clinical progression of AD.

The advent of gene panel testing is challenging the previous practice of using clinically defined cancer family syndromes to inform single-gene genetic screening. Individual and family cancer histories that would have previously indicated testing of a single gene or a small number of related genes are now, increasingly, leading to screening across gene panels that contain larger numbers of genes. We have applied a gene panel test that included four DNA mismatch repair (MMR) genes (MLH1, MSH2, MSH6 and PMS2) to an Australian population-based case-control-family study of breast cancer. Altogether, eight pathogenic variants in MMR genes were identified: six in 1421 case-families (0.4%, 4 MSH6 and 2 PMS2) and two in 833 control-families (0.2%, one each of MLH1 and MSH2). This testing highlights the current and future challenges for clinical genetics in the context of anticipated gene panel-based population-based screening that includes the MMR genes. This testing is likely to provide additional opportunities for cancer prevention via cascade testing for Lynch syndrome and precision medicine for breast cancer treatment.

Limisphaera ngatamarikiensis NGM72.4 T is a thermophilic representative of the class Verrucomicrobiae Isolated from geothermally heated subaqueous clay sediments from a Ngatamariki hotspring in Aotearoa New Zealand, the 3,908,748-bp genome was sequenced using the Illumina HiSeq 2500 platform. Annotation revealed 3,083 coding sequences, including 3,031 proteins, 3 rRNA genes, and 46 tRNA genes., (Copyright © 2020 Carere et al.)