A gap analysis of UK biobank publications reveals SNPs associated with intrinsic subtypes of breast cancer.

Breast cancer is a multifaceted disease and a leading cause of cancer morbidity and mortality in females across the globe. In 2020 alone, 2.3 million women were diagnosed and 685,000 died of breast cancer worldwide. With the number of diagnoses projected to increase to 3 million per year by 2040 it is essential that new methods of detection and disease stratification are sought to decrease this global cancer burden. Although significant improvements have been made in breast cancer diagnosis and treatment, the prognosis of breast cancer remains poor in some patient groups (i.e. triple negative breast cancer), necessitating research into better patient stratification, diagnosis and drug discovery. The UK Biobank, a comprehensive biomedical and epidemiological database with a wide variety of multiomics data (genomics, proteomics, metabolomics) offers huge potential to uncover groundbreaking discoveries in breast cancer research leading to improved patient stratification. Combining genomic, proteomic, and metabolic profiles of breast cancer in combination with histological classification, can aid treatment decisions through accurate diagnosis and prognosis prediction of tumor behaviour. Here, we systematically reviewed PubMed publications reporting the analysis of UK Biobank data in breast cancer research. Our analysis of UK Biobank studies in the past five years identified 125 publications, of which 76 focussed on genomic data analysis. Interestingly, only two studies reported the analysis of metabolomics and proteomics data, with none performing multiomics analysis of breast cancer. A meta-analysis of the 76 publications identified 2870 genetic variants associated with breast cancer across 445 genes. Subtype analysis revealed differential genetic alteration in 13 of the 445 genes and the identification of 59 well-established breast cancer genes. in differential pathways. Pathway interaction analyses illuminated their involvement in general cancer biomolecular pathways (e.g. DNA damage repair, Gene expression). While our meta-analysis only measured genetic differences in breast cancer due to current usage of UK Biobank data, minimal multi-omics analyses have been performed and the potential for harnessing multi-omics strategies within the UK Biobank cohort holds promise for unravelling the biological signatures of distinct breast cancer subtypes further in the future.

Younger but sicker? Cohort trends in disease accumulation among middle-aged and older adults in Scotland using health-linked data from the Scottish Longitudinal Study.

BACKGROUND: In the United Kingdom, rising prevalence of multimorbidity-the co-occurrence of two or more chronic conditions- is coinciding with stagnation in life expectancy. We investigate patterns of disease accumulation and how they vary by birth cohort, social and environmental inequalities in Scotland, a country which has long suffered from excess mortality and poorer health outcomes relative to its neighbours. METHODS: Using a dataset which links census data from 1991, 2001 and 2011 to disease registers and hospitalization data, we follow cohorts of adults aged 30-69 years for 18 years. We model physical and mental disease accumulation using linear mixed-effects models. RESULTS: Recent cohorts experience higher levels of chronic disease accumulation compared to their predecessors at the same ages. Moreover, in more recently born cohorts we observe socioeconomic status disparities emerging earlier in the life course, which widen over time and with every successive cohort. Patterns of chronic conditions are also changing, and the most common diseases suffered by later born cohorts are cancer, hypertension, asthma, drug and alcohol problems and depression. CONCLUSION: We recommend policies which target prevention of chronic disease in working age adults, considering how and why certain conditions are becoming more prevalent across time and space.

Low-Temperature X-ray Microanalysis Sheds New Light on Mineral Nutrition Aspects of Insect Leaf Galling.

Manipulation of host plant physiology by leaf-galling insects is a multifaceted process. Among fundamental knowledge gaps surrounding this scientifically intriguing phenomenon is the appropriation of plant mineral nutrients and moisture for galling advantage. Small, soluble mineral ions and watery cell contents in dense gall tissues risk disruption during routine sample preparations. In this study, an X-ray microanalysis was applied to investigate gall mineral nutrition. Morphologically diverse leaf galls were sampled from three Australian rainforest tree species. Using cryo-analytical scanning electron microscopy, real-time X-ray analytical maps of cellular mineral nutrients and water were integrated with anatomical images of gall and leaf cross-sectional surfaces. A comparison of host-leaf and gall anatomies bore direct evidence of drastic changes to leaf cells through the galling process. Distinct "wet" and "dry" regions within galls were anatomically and/or chemically differentiated, suggesting specific functionality. "Wet" regions comprising hydrated cells including soft gall-cavity linings where larvae are known to feed contained soluble plant mineral nutrients, while C-rich "dry" tissues largely devoid of mineral nutrients likely contribute structural support. Mapping immobile nutrients such as Mn may provide a means of "matching" specific gall cell types to those in ungalled host-leaf tissues. The findings here provided otherwise inaccessible insights into leaf-gall mineral nutrition.

Complex Mixture Analysis of Emerging Contaminants Generated from Coal Tar- and Petroleum-Derived Pavement Sealants: Molecular Compositions and Correlations with Toxicity Revealed by Fourier Transform Ion Cyclotron Resonance Mass Spectrometry.

Pavement sealants are of environmental concern because of their complex petroleum-based chemistry and potential toxicity. Specifically, coal tar-derived sealants contain high concentrations of toxic/carcinogenic polycyclic aromatic hydrocarbons (PAHs) that, when weathered, can be transferred into the surrounding environment. Previous studies have demonstrated the effects of coal tar sealants on PAH concentration in nearby waterways and their harmful effects in aquatic ecosystems. Here, we investigate and compare the molecular composition of two different pavement sealants, petroleum asphalt- and coal tar-derived, and their photoproducts, by positive-ion (+) atmospheric pressure photoionization (APPI) and negative-ion (-) electrospray ionization (ESI) coupled with ultrahigh-resolution Fourier transform ion cyclotron resonance mass spectrometry to address species (high-boiling and/or high oxygen content) that lie outside the analytical window of other techniques due to ultra-high molecular complexity. In addition, we evaluate the toxicity of the water-soluble photoproducts by use of Microtox bioassay. The results demonstrate that the coal tar sealant contains higher amounts of PAHs and produces abundant water-soluble compounds, relative to unweathered materials, with a high abundance of PAH-like molecules of high toxicity. By comparison, the asphalt sealant produces fewer toxic water-soluble species, with molecular compositions that are consistent with natural dissolved organic matter. These results capture the mass, chemical diversity, toxicity, and source/photoproduct relationship of these compositionally complex emerging contaminants from the built environment.

Molecular-Level Characterization of Oil-Soluble Ketone/Aldehyde Photo-Oxidation Products by Fourier Transform Ion Cyclotron Resonance Mass Spectrometry Reveals Similarity Between Microcosm and Field Samples.

We present a solid-phase extraction method followed by derivatization with a charged tag to characterize ketone/aldehyde-containing functionalities (proposed photo-oxidation transformation products) in weathered petroleum by Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS). A photo-oxidation-only microcosm mimics solar irradiation of crude oil in the environment after an oil spill. A biodegradation-only microcosm enables independent determination as to which of the two weathering processes contributes to the formation of oil-soluble ketone/aldehyde species. Results confirm that photo-oxidation produces ketones/aldehydes in crude oil when exposed to solar radiation in laboratory experiments, whereas biodegraded oil samples do not produce ketone/aldehyde compounds. Field samples collected after different time periods and locations after the Deepwater Horizon oil spill are also shown to contain ketones/aldehydes, and comparison of field and photo-oxidation-only microcosm transformation products reveal remarkable similarity. These results indicate that the photo-oxidation microcosm comprehensively represents ketone/aldehyde-formation products in the field, whereas the biodegradation microcosm does not. Solid-phase extraction coupled with derivatization leads to selective identification of ketone/aldehyde species by MS. Although improved dynamic range and slightly reduced mass spectral complexity is achieved by separation/derivatization, comprehensive molecular characterization still requires mass resolving power and mass accuracy provided by FT-ICR MS.

Classification of Plasma Cell Disorders by 21 Tesla Fourier Transform Ion Cyclotron Resonance Top-Down and Middle-Down MS/MS Analysis of Monoclonal Immunoglobulin Light Chains in Human Serum.

The current five-year survival rate for systemic AL amyloidosis or multiple myeloma is ∼51%, indicating the urgent need for better diagnosis methods and treatment plans. Here, we describe highly specific and sensitive top-down and middle-down MS/MS methods owning the advantages of fast sample preparation, ultrahigh mass accuracy, and extensive residue cleavages with 21 telsa FT-ICR MS/MS. Unlike genomic testing, which requires bone marrow aspiration and may fail to identify all monoclonal immunoglobulins produced by the body, the present method requires only a blood draw. In addition, circulating monoclonal immunoglobulins spanning the entire population are analyzed and reflect the selection of germline sequence by B cells. The monoclonal immunoglobulin light chain FR2-CDR2-FR3 was sequenced by database-aided de novo MS/MS and 100% matched the gene sequencing result, except for two amino acids with isomeric counterparts, enabling accurate germline sequence classification. The monoclonal immunoglobulin heavy chains were also classified into specific germline sequences based on the present method. This work represents the first application of top/middle-down MS/MS sequencing of endogenous human monoclonal immunoglobulins with polyclonal immunoglobulins background.

Migration Status and Smoking Behaviors in Later-Life in China-Evidence From the China Health and Retirement Longitudinal Study (CHARLS).

Background: China is the biggest consumer of tobacco in the world, with a high prevalence of smoking especially among men. Along with the rapid demographic change in China, the burden of diseases attributable to health behaviors, particularly smoking is steadily increasing. So, smoking has become a major risk factor for mortality in China. Smoking behaviors may be related to migration processes, as a result of both who migrates and post-migration experiences related to socioeconomic position, stress and acculturation. Existing studies that have examined smoking and migration in China have, however, only focused on temporary rural-to-urban migrants and focused on relatively younger migrants. This paper examines the association between smoking behaviors and a comprehensive assessment of migration status in later-life in China. Methods: Using the China Health and Retirement Longitudinal Study (CHARLS), a nationally representative dataset, this paper studies smoking behaviors of rural-to-urban migrants, urban-to-urban migrants, rural return migrants, and urban return migrants. We compare them with corresponding non-migrant groups in both rural and urban locations in China. Using a model that controls for demographic factors, early-life circumstances, socioeconomic factors, and factors related to migration, we examine both the decision to start smoking and the decision to quit smoking. In addition, we also address pre-migration selection in our analyses. Results: The results show rural-to-urban migrants are no more likely to start smoking compared with rural non-migrants, but they are more likely to quit smoking. While urban-to-urban migrants are more likely to start smoking compared with urban non-migrants, this effect is explained by the factors we include in the full model. Urban-to-urban migrants are, however, less likely to quit smoking. Moreover, both rural return migrants and urban return migrants seem to be more likely to start smoking and less likely to quit smoking compared with non-migrant groups. Conclusion: There are strong associations between migration status and later-life smoking behaviors in China; these associations vary greatly according to different migration status and point to populations and factors that public health activities should focus on.

Middle-Down Characterization of the Cell Cycle Dependence of Histone H4 Posttranslational Modifications and Proteoforms.

Post-translational modifications (PTMs) of histones are important epigenetic regulatory mechanisms that are often dysregulated in cancer. We employ middle-down proteomics to investigate the PTMs and proteoforms of histone H4 during cell cycle progression. We use pH gradient weak cation exchange-hydrophilic interaction liquid chromatography (WCX-HILIC) for on-line liquid chromatography-mass spectrometry analysis to separate and analyze the proteoforms of histone H4. This procedure provides enhanced separation of proteoforms, including positional isomers, and simplifies downstream data analysis. We use ultrahigh mass accuracy and resolution Fourier transform-ion cyclotron resonance (FT-ICR) mass spectrometer to unambiguously distinguish between acetylation and tri-methylation (∆m = 0.036 Da). In total, we identify and quantify 233 proteoforms of histone H4 in two breast cancer cell lines. We observe significant increases in S1 phosphorylation during mitosis, implicating an important role in mitotic chromatin condensation. A decrease of K20 unmodified proteoforms is observed as the cell cycle progresses, corresponding to an increase of K20 mono- and di-methylation. Acetylation at K5, K8, K12, and K16 declines as cells traverse from S phase to mitosis, suggesting cell cycle-dependence and an important role during chromatin replication and condensation. These new insights into the epigenetics of the cell cycle may provide new diagnostic and prognostic biomarkers.

Mechanistic Origins of Enzyme Activation in Human Glucokinase Variants Associated with Congenital Hyperinsulinism.

Human glucokinase (GCK) acts as the body's primary glucose sensor and plays a critical role in glucose homeostatic maintenance. Gain-of-function mutations in gck produce hyperactive enzyme variants that cause congenital hyperinsulinism. Prior biochemical and biophysical studies suggest that activated disease variants can be segregated into two mechanistically distinct classes, termed α-type and ß-type. Steady-state viscosity variation studies indicate that the kcat values of wild-type GCK and an α-type variant are partially diffusion-limited, whereas the kcat value of a ß-type variant is viscosity-independent. Transient-state chemical quench-flow analyses demonstrate that wild-type GCK and the α-type variant display burst kinetics, whereas the ß-type variant lacks a burst phase. Comparative hydrogen-deuterium exchange mass spectrometry of unliganded enzymes demonstrates that a disordered active site loop, which folds upon binding of glucose, is protected from exchange in the α-type variant. The α-type variant also displays an increased level of exchange within a ß-strand located near the enzyme's hinge region, which becomes more solvent-exposed upon glucose binding. In contrast, ß-type activation causes no substantial difference in global or local exchange relative to that of unliganded, wild-type GCK. Together, these results demonstrate that α-type activation results from a shift in the conformational ensemble of unliganded GCK toward a state resembling the glucose-bound conformation, whereas ß-type activation is attributable to an accelerated rate of product release. This work elucidates the molecular basis of naturally occurring, activated GCK disease variants and provides insight into the structural and dynamic origins of GCK's unique kinetic cooperativity.

Linking Natural Oil Seeps from the Gulf of Mexico to Their Origin by Use of Fourier Transform Ion Cyclotron Resonance Mass Spectrometry.

We report chemical characterization of natural oil seeps from the Gulf of Mexico by Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) and Gas Chromatography/Atmospheric Pressure Chemical Ionization Mass Spectrometry (GC/APCI-MS), to highlight how FT-ICR MS can also be employed as a means to determine petroleum connectivity, in addition to traditional GC/MS techniques. The source of petroleum is the Green Canyon (GC) 600 lease block in the Gulf of Mexico. Within GC600, two natural oil seepage zones, Mega Plume and Birthday Candles, continuously release hydrocarbons and develop persistent oil slicks at the sea surface above them. We chemically trace the petroleum from the surface oil slicks to the Mega Plume seep itself, and further to a petroleum reservoir 5 km away in lease block GC645 (Holstein Reservoir). We establish the connectivity between oil samples and confirm a common geological origin for the oil slicks, oil seep, and reservoir oil. The ratios of seven common petroleum biomarkers detected by GC/APCI-MS display clear similarity between the GC600 and GC645 samples, as well as a distinct difference from another reservoir oil collected ∼300 km away (Macondo crude oil from MC252 lease block). FT-ICR MS and principal component analysis (PCA) demonstrate further similarities between the GC600 and GC645 samples that distinctly differ from MC252. A common geographical origin is postulated for the GC600/GC645 samples, with petroleum migrating from the GC645 reservoir to the oil seeps found in GC600 and up through the water column to the sea surface as an oil slick.

Pih1p-Tah1p Puts a Lid on Hexameric AAA+ ATPases Rvb1/2p.

The Saccharomyces cerevisiae (Sc) R2TP complex affords an Hsp90-mediated and nucleotide-driven chaperone activity to proteins of small ribonucleoprotein particles (snoRNPs). The current lack of structural information on the ScR2TP complex, however, prevents a mechanistic understanding of this biological process. We characterized the structure of the ScR2TP complex made up of two AAA+ ATPases, Rvb1/2p, and two Hsp90 binding proteins, Tah1p and Pih1p, and its interaction with the snoRNP protein Nop58p by a combination of analytical ultracentrifugation, isothermal titration calorimetry, chemical crosslinking, hydrogen-deuterium exchange, and cryoelectron microscopy methods. We find that Pih1p-Tah1p interacts with Rvb1/2p cooperatively through the nucleotide-sensitive domain of Rvb1/2p. Nop58p further binds Pih1p-Tahp1 on top of the dome-shaped R2TP. Consequently, nucleotide binding releases Pih1p-Tah1p from Rvb1/2p, which offers a mechanism for nucleotide-driven binding and release of snoRNP intermediates.

Mapping the contact surfaces in the Lamin A:AIMP3 complex by hydrogen/deuterium exchange FT-ICR mass spectrometry.

Aminoacyl-tRNA synthetases-interacting multifunctional protein3 (AIMP3/p18) is involved in the macromolecular tRNA synthetase complex via its interaction with several aminoacyl-tRNA synthetases. Recent reports reveal a novel function of AIMP3 as a tumor suppressor by accelerating cellular senescence and causing defects in nuclear morphology. AIMP3 specifically mediates degradation of mature Lamin A (LmnA), a major component of the nuclear envelope matrix; however, the mechanism of how AIMP3 interacts with LmnA is unclear. Here we report solution-phase hydrogen/deuterium exchange (HDX) for AIMP3, LmnA, and AIMP3 in association with the LmnA C-terminus. Reversed-phase LC coupled with LTQ 14.5 T Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) results in high mass accuracy and resolving power for comparing the D-uptake profiles for AIMP3, LmnA, and their complex. The results show that the AIMP3-LmnA interaction involves one of the two putative binding sites and an adjacent novel interface on AIMP3. LmnA binds AIMP3 via its extreme C-terminus. Together these findings provide a structural insight for understanding the interaction between AIMP3 and LmnA in AIMP3 degradation.

Molecular Communication over Gas Stream Channels using Portable Mass Spectrometry.

The synthetic generation/coding and transmission of olfactory information over a gas stream or an odor network is a new and unexplored field. Application areas vary from the entertainment or advertisement industry to security and telemedicine. However, current technological limitations frustrate the accurate reproduction of decoded and transmitted olfactory data. This study describes the development, testing, and characterization of a novel odor emitter (OE) that is used to investigate the generation-encoding of gaseous standards with odorous characteristics with a regulatable way, for scent transmission purposes. The calibration and the responses of a developed OE were examined using a portable quadrupole mass spectrometer (MS). Experiments were undertaken for a range of volatile organic compounds (VOCs) at different temperatures and flow rates. Individual compounds and mixtures were tested to investigate periodic and dynamic transmission characteristics within two different size tubular containers for distances up to 3 m. Olfactory information transmission is demonstrated using MS as the main molecular sensor for odor detection and monitoring and for the first time spatial encryption of olfactory information is shown. Graphical Abstract ᅟ.

Analysis of Monoclonal Antibodies in Human Serum as a Model for Clinical Monoclonal Gammopathy by Use of 21 Tesla FT-ICR Top-Down and Middle-Down MS/MS.

With the rapid growth of therapeutic monoclonal antibodies (mAbs), stringent quality control is needed to ensure clinical safety and efficacy. Monoclonal antibody primary sequence and post-translational modifications (PTM) are conventionally analyzed with labor-intensive, bottom-up tandem mass spectrometry (MS/MS), which is limited by incomplete peptide sequence coverage and introduction of artifacts during the lengthy analysis procedure. Here, we describe top-down and middle-down approaches with the advantages of fast sample preparation with minimal artifacts, ultrahigh mass accuracy, and extensive residue cleavages by use of 21 tesla FT-ICR MS/MS. The ultrahigh mass accuracy yields an RMS error of 0.2-0.4 ppm for antibody light chain, heavy chain, heavy chain Fc/2, and Fd subunits. The corresponding sequence coverages are 81%, 38%, 72%, and 65% with MS/MS RMS error ~4 ppm. Extension to a monoclonal antibody in human serum as a monoclonal gammopathy model yielded 53% sequence coverage from two nano-LC MS/MS runs. A blind analysis of five therapeutic monoclonal antibodies at clinically relevant concentrations in human serum resulted in correct identification of all five antibodies. Nano-LC 21 T FT-ICR MS/MS provides nonpareil mass resolution, mass accuracy, and sequence coverage for mAbs, and sets a benchmark for MS/MS analysis of multiple mAbs in serum. This is the first time that extensive cleavages for both variable and constant regions have been achieved for mAbs in a human serum background. Graphical Abstract ᅟ.

Physical activity and trajectories of frailty among older adults: Evidence from the English Longitudinal Study of Ageing.

BACKGROUND: Frail older adults are heavy users of health and social care. In order to reduce the costs associated with frailty in older age groups, safe and cost-effective strategies are required that will reduce the incidence and severity of frailty. OBJECTIVE: We investigated whether self-reported intensity of physical activity (sedentary, mild, moderate or vigorous) performed at least once a week can significantly reduce trajectories of frailty in older adults who are classified as non-frail at baseline (Rockwood's Frailty Index [FI] ≤ 0.25). METHODS: Multi-level growth curve modelling was used to assess trajectories of frailty in 8649 non-frail adults aged 50 and over and according to baseline self-reported intensity of physical activity. Frailty was measured in five-year age cohorts based on age at baseline (50-54; 55-59; 60-64; 65-69; 70-74; 75-79; 80+) on up to 6 occasions, providing an average of 10 years of follow-up. All models were adjusted for baseline sex, education, wealth, cohabitation, smoking, and alcohol consumption. RESULTS: Compared with the sedentary reference group, mild physical activity was insufficient to significantly slow the progression of frailty, moderate physical activity reduced the progression of frailty in some age groups (particularly ages 65 and above) and vigorous activity significantly reduced the trajectory of frailty progression in all older adults. CONCLUSION: Healthy non-frail older adults require higher intensities of physical activity for continued improvement in frailty trajectories.

Label-Free Relative Quantitation of Isobaric and Isomeric Human Histone H2A and H2B Variants by Fourier Transform Ion Cyclotron Resonance Top-Down MS/MS.

Histone variants are known to play a central role in genome regulation and maintenance. However, many variants are inaccessible by antibody-based methods or bottom-up tandem mass spectrometry due to their highly similar sequences. For many, the only tractable approach is with intact protein top-down tandem mass spectrometry. Here, ultra-high-resolution FT-ICR MS and MS/MS yield quantitative relative abundances of all detected HeLa H2A and H2B isobaric and isomeric variants with a label-free approach. We extend the analysis to identify and relatively quantitate 16 proteoforms from 12 sequence variants of histone H2A and 10 proteoforms of histone H2B from three other cell lines: human embryonic stem cells (WA09), U937, and a prostate cancer cell line LaZ. The top-down MS/MS approach provides a path forward for more extensive elucidation of the biological role of many previously unstudied histone variants and post-translational modifications.

Quantitative Mass Spectrometry Reveals Changes in Histone H2B Variants as Cells Undergo Inorganic Arsenic-Mediated Cellular Transformation.

Exposure to inorganic arsenic, a ubiquitous environmental toxic metalloid, leads to carcinogenesis. However, the mechanism is unknown. Several studies have shown that inorganic arsenic exposure alters specific gene expression patterns, possibly through alterations in chromatin structure. While most studies on understanding the mechanism of chromatin-mediated gene regulation have focused on histone post-translational modifications, the role of histone variants remains largely unknown. Incorporation of histone variants alters the functional properties of chromatin. To understand the global dynamics of chromatin structure and function in arsenic-mediated carcinogenesis, analysis of the histone variants incorporated into the nucleosome and their covalent modifications is required. Here we report the first global mass spectrometric analysis of histone H2B variants as cells undergo arsenic-mediated epithelial to mesenchymal transition. We used electron capture dissociation-based top-down tandem mass spectrometry analysis validated with quantitative reverse transcription real-time polymerase chain reaction to identify changes in the expression levels of H2B variants in inorganic arsenic-mediated epithelial-mesenchymal transition. We identified changes in the expression levels of specific histone H2B variants in two cell types, which are dependent on dose and length of exposure of inorganic arsenic. In particular, we found increases in H2B variants H2B1H/1K/1C/1J/1O and H2B2E/2F, and significant decreases in H2B1N/1D/1B as cells undergo inorganic arsenic-mediated epithelial-mesenchymal transition. The analysis of these histone variants provides a first step toward an understanding of the functional significance of the diversity of histone structures, especially in inorganic arsenic-mediated gene expression and carcinogenesis.

Proinflammatory genotype is associated with the frailty phenotype in the English Longitudinal Study of Ageing.

BACKGROUND: Frailty is a state of increased vulnerability to poor resolution of homeostasis after a stressor event, which increases the risk of adverse outcomes including falls, disability and death. The underlying pathophysiological pathways of frailty are not known but the hypothalamic-pituitary-adrenal axis and heightened chronic systemic inflammation appear to be major contributors. METHODS: We used the English Longitudinal Study of Ageing dataset of 3160 individuals over the age of 50 and assessed their frailty status according to the Fried-criteria. We selected single nucleotide polymorphisms in genes involved in the steroid hormone or inflammatory pathways and performed linear association analysis using age and sex as covariates. To support the biological plausibility of any genetic associations, we selected biomarker levels for further analyses to act as potential endophenotypes of our chosen genetic loci. RESULTS: The strongest association with frailty was observed in the Tumor Necrosis Factor (TNF) (rs1800629, P = 0.001198, ß = 0.0894) and the Protein Tyrosine Phosphatase, Receptor type, J (PTPRJ) (rs1566729, P = 0.001372, ß = 0.09397) genes. Rs1800629 was significantly associated with decreased levels of high-density lipoprotein (HDL) (P = 0.00949) and cholesterol levels (P = 0.00315), whereas rs1566729 was associated with increased levels of HDL (P = 0.01943). After correcting for multiple testing none of the associations remained significant. CONCLUSIONS: We provide potential evidence for the involvement of a multifunctional proinflammatory cytokine gene (TNF) in the frailty phenotype. The implication of this gene is further supported by association with the endophenotype biomarker results.

Quantitative Mass Spectrometry Reveals that Intact Histone H1 Phosphorylations are Variant Specific and Exhibit Single Molecule Hierarchical Dependence.

Breast cancer was the second leading cause of cancer related mortality for females in 2014. Recent studies suggest histone H1 phosphorylation may be useful as a clinical biomarker of breast and other cancers because of its ability to recognize proliferative cell populations. Although monitoring a single phosphorylated H1 residue is adequate to stratify high-grade breast tumors, expanding our knowledge of how H1 is phosphorylated through the cell cycle is paramount to understanding its role in carcinogenesis. H1 analysis by bottom-up MS is challenging because of the presence of highly homologous sequence variants expressed by most cells. These highly basic proteins are difficult to analyze by LC-MS/MS because of the small, hydrophilic nature of peptides produced by tryptic digestion. Although bottom-up methods permit identification of several H1 phosphorylation events, these peptides are not useful for observing the combinatorial post-translational modification (PTM) patterns on the protein of interest. To complement the information provided by bottom-up MS, we utilized a top-down MS/MS workflow to permit identification and quantitation of H1 proteoforms related to the progression of breast cells through the cell cycle. Histones H1.2 and H1.4 were observed in MDA-MB-231 metastatic breast cells, whereas an additional histone variant, histone H1.3, was identified only in nonneoplastic MCF-10A cells. Progressive phosphorylation of histone H1.4 was identified in both cell lines at mitosis (M phase). Phosphorylation occurred first at S172 followed successively by S187, T18, T146, and T154. Notably, phosphorylation at S173 of histone H1.2 and S172, S187, T18, T146, and T154 of H1.4 significantly increases during M phase relative to S phase, suggesting that these events are cell cycle-dependent and may serve as markers for proliferation. Finally, we report the observation of the H1.2 SNP variant A18V in MCF-10A cells.

The N-terminal Domain of Escherichia coli Assimilatory NADPH-Sulfite Reductase Hemoprotein Is an Oligomerization Domain That Mediates Holoenzyme Assembly.

Assimilatory NADPH-sulfite reductase (SiR) from Escherichia coli is a structurally complex oxidoreductase that catalyzes the six-electron reduction of sulfite to sulfide. Two subunits, one a flavin-binding flavoprotein (SiRFP, the α subunit) and the other an iron-containing hemoprotein (SiRHP, the ß subunit), assemble to make a holoenzyme of about 800 kDa. How the two subunits assemble is not known. The iron-rich cofactors in SiRHP are unique because they are a covalent arrangement of a Fe4S4 cluster attached through a cysteine ligand to an iron-containing porphyrinoid called siroheme. The link between cofactor biogenesis and SiR stability is also ill-defined. By use of hydrogen/deuterium exchange and biochemical analysis, we show that the α8ß4 SiR holoenzyme assembles through the N terminus of SiRHP and the NADPH binding domain of SiRFP. By use of small angle x-ray scattering, we explore the structure of the SiRHP N-terminal oligomerization domain. We also report a novel form of the hemoprotein that occurs in the absence of its cofactors. Apo-SiRHP forms a homotetramer, also dependent on its N terminus, that is unable to assemble with SiRFP. From these results, we propose that homotetramerization of apo-SiRHP serves as a quality control mechanism to prevent formation of inactive holoenzyme in the case of limiting cellular siroheme.