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.

Tactile estimation of hedonic and sensory properties during active touch: An electroencephalography study.

Perceptual judgements about our physical environment are informed by somatosensory information. In real-world exploration, this often involves dynamic hand movements to contact surfaces, termed active touch. The current study investigated cortical oscillatory changes during active exploration to inform the estimation of surface properties and hedonic preferences of two textured stimuli: smooth silk and rough hessian. A purpose-built touch sensor quantified active touch, and oscillatory brain activity was recorded from 129-channel electroencephalography. By fusing these data streams at a single trial level, oscillatory changes within the brain were examined while controlling for objective touch parameters (i.e., friction). Time-frequency analysis was used to quantify changes in cortical oscillatory activity in alpha (8-12 Hz) and beta (16-24 Hz) frequency bands. Results reproduce findings from our lab, whereby active exploration of rough textures increased alpha-band event-related desynchronisation in contralateral sensorimotor areas. Hedonic processing of less preferred textures resulted in an increase in temporoparietal beta-band and frontal alpha-band event-related desynchronisation relative to most preferred textures, suggesting that higher order brain regions are involved in the hedonic processing of texture. Overall, the current study provides novel insight into the neural mechanisms underlying texture perception during active touch and how this process is influenced by cognitive tasks.

Impact of data source choice on multimorbidity measurement: a comparison study of 2.3 million individuals in the Welsh National Health Service.

BACKGROUND: Measurement of multimorbidity in research is variable, including the choice of the data source used to ascertain conditions. We compared the estimated prevalence of multimorbidity and associations with mortality using different data sources. METHODS: A cross-sectional study of SAIL Databank data including 2,340,027 individuals of all ages living in Wales on 01 January 2019. Comparison of prevalence of multimorbidity and constituent 47 conditions using data from primary care (PC), hospital inpatient (HI), and linked PC-HI data sources and examination of associations between condition count and 12-month mortality. RESULTS: Using linked PC-HI compared with only HI data, multimorbidity was more prevalent (32.2% versus 16.5%), and the population of people identified as having multimorbidity was younger (mean age 62.5 versus 66.8 years) and included more women (54.2% versus 52.6%). Individuals with multimorbidity in both PC and HI data had stronger associations with mortality than those with multimorbidity only in HI data (adjusted odds ratio 8.34 [95% CI 8.02-8.68] versus 6.95 (95%CI 6.79-7.12] in people with ≥ 4 conditions). The prevalence of conditions identified using only PC versus only HI data was significantly higher for 37/47 and significantly lower for 10/47: the highest PC/HI ratio was for depression (14.2 [95% CI 14.1-14.4]) and the lowest for aneurysm (0.51 [95% CI 0.5-0.5]). Agreement in ascertainment of conditions between the two data sources varied considerably, being slight for five (kappa < 0.20), fair for 12 (kappa 0.21-0.40), moderate for 16 (kappa 0.41-0.60), and substantial for 12 (kappa 0.61-0.80) conditions, and by body system was lowest for mental and behavioural disorders. The percentage agreement, individuals with a condition identified in both PC and HI data, was lowest in anxiety (4.6%) and highest in coronary artery disease (62.9%). CONCLUSIONS: The use of single data sources may underestimate prevalence when measuring multimorbidity and many important conditions (especially mental and behavioural disorders). Caution should be used when interpreting findings of research examining individual and multiple long-term conditions using single data sources. Where available, researchers using electronic health data should link primary care and hospital inpatient data to generate more robust evidence to support evidence-based healthcare planning decisions for people with multimorbidity.

Examining the social networks of older adults receiving informal or formal care: a systematic review.

PURPOSE: To address the care needs of older adults, it is important to identify and understand the forms of care support older adults received. This systematic review aims to examine the social networks of older adults receiving informal or formal care and the factors that influenced their networks. METHODS: A systematic review was conducted by searching six databases from inception to January 31, 2023. The review included primary studies focusing on older adults receiving long-term care, encompassing both informal and formal care. To assess the risk of bias in the included studies, validated appraisal tools specifically designed for different study types were utilized. Network analysis was employed to identify the grouping of study concepts, which subsequently formed the foundation for describing themes through narrative synthesis. RESULTS: We identified 121 studies relating to the formal and informal care of older adults' networks. A variety of social ties were examined by included studies. The most commonly examined sources of care support were family members (such as children and spouses) and friends. Several factors were consistently reported to influence the provision of informal care, including the intensity of networks, reciprocity, and geographical proximity. In terms of formal care utilization, older age and poor health status were found to be associated with increased use of healthcare services. Additionally, physical limitations and cognitive impairment were identified as factors contributing to decreased social engagement. CONCLUSION: This review found that older people were embedded within a diverse network. The findings of this review emphasize the importance of recognizing and incorporating the diversity of social networks in care plans and policies to enhance the effectiveness of interventions and improve the overall well-being of older adults.

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.

An x-ray microanalytical method for measuring in vivo element and water concentrations, relating to osmoregulation, in cells and tissues of the posterior eye.

Osmoregulation is critical for cell and tissue survival yet there are relatively few methods available to determine osmotic gradients from water and elemental concentration either in single cells or across multiple cellular layers of tissue. X-ray microanalysis of frozen-hydrated preparations in a scanning electron microscope is one such powerful, sensitive, nondestructive technique. Here we use x-ray microanalysis to quantitatively analyse intracellular element concentrations and oxygen concentrations, as a proxy for water concentrations, in selected individual cells of the posterior eye. Using frozen-hydrated preparations of the retinal complex of chicken eyes, it is shown that structural preservation is sufficient to identify cell layers and individual cells. The quantitative analysis of selected areas in the photoreceptor layer, inner nuclear layer and ganglion cell layer, where specific cell types were known to be present, provided measurements of intracellular element concentrations comparable with the analysis of individual cells. It is also shown that in the cells of the retinal pigment epithelium and outer photoreceptor segments elemental analyses were reasonably consistent at the cellular level in different depth levels of the same sample. Comparison of oxygen concentrations, as a proxy for water concentration, at two accelerating voltages (15 and 5 kV) indicated that at 15 kV oxygen concentration was largely derived from intracellular water. Water concentrations could be calculated and concentrations of diffusible elements (Na, K) could be defined in mmol/L. From the latter it is possible to calculate osmotic concentrations of individual cells and osmotic gradients across the tissue. LAY DESCRIPTION: Understanding many cellular processes, in both healthy and diseased states, depends on knowing how the water content of cells and their surrounding fluids is controlled. The transport of water is generally down its concentration gradient or against the osmotic concentration gradient defined by solutes such as sodium, potassium and chloride dissolved in the water. We have refined a microanalytical method, that detects the x-rays emitted from specific elements when they are bombarded by electrons in a scanning electron microscope, to apply it to the analysis of the retina of the eye. The method facilitates the measurement of the elemental composition, water and osmotic concentration gradients of cells and tissues in the eye, that may be involved in the development of myopia, or short sightedness, a condition that afflicts many people including some 80 - 90% of children in Asia.

Structural switch of lysyl-tRNA synthetase between translation and transcription.

Lysyl-tRNA synthetase (LysRS), a component of the translation apparatus, is released from the cytoplasmic multi-tRNA synthetase complex (MSC) to activate the transcription factor MITF in stimulated mast cells through undefined mechanisms. Here we show that Ser207 phosphorylation provokes a new conformer of LysRS that inactivates its translational function but activates its transcriptional function. The crystal structure of an MSC subcomplex established that LysRS is held in the MSC by binding to the N terminus of the scaffold protein p38/AIMP2. Phosphorylation-created steric clashes at the LysRS domain interface disrupt its binding grooves for p38/AIMP2, releasing LysRS and provoking its nuclear translocation. This alteration also exposes the C-terminal domain of LysRS to bind to MITF and triggers LysRS-directed production of the second messenger Ap(4)A that activates MITF. Thus our results establish that a single conformational change triggered by phosphorylation leads to multiple effects driving an exclusive switch of LysRS function from translation to transcription.

FluNet: An AI-Enabled Influenza-Like Warning System.

Influenza is an acute viral respiratory disease that is currently causing severe financial and resource strains worldwide. With the COVID-19 pandemic exceeding 153 million cases worldwide, there is a need for a low-cost and contactless surveillance system to detect symptomatic individuals. The objective of this study was to develop FluNet, a novel, proof-of-concept, low-cost and contactless device for the detection of high-risk individuals. The system conducts face detection in the LWIR with a precision rating of 0.98, a recall of 0.91, an F-score of 0.96, and a mean intersection over union of 0.74 while sequentially taking the temperature trend of faces with a thermal accuracy of ± 1 K. In parallel, determining if someone is coughing by using a custom lightweight deep convolutional neural network with a precision rating of 0.95, a recall of 0.92, an F-score of 0.94 and an AUC of 0.98. We concluded this study by testing the accuracy of the direction of arrival estimation for the cough detection revealing an error of ± 4.78°. If a subject is symptomatic, a photo is taken with a specified region of interest using a visible light camera. Two datasets have been constructed, one for face detection in the LWIR consisting of 250 images of 20 participants' faces at various rotations and coverings, including face masks. The other for the real-time detection of coughs comprised of 40,482 cough / not cough sounds. These findings could be helpful for future low-cost edge computing applications for influenza-like monitoring.

Probing the Impact of the Knob-into-Hole Mutations on the Structure and Function of a Therapeutic Antibody.

Bispecific antibodies (BsAbs) have drawn increasing interest in the biopharmaceutical industry due to their advantage to bind two distinct antigens simultaneously. The knob-into-hole approach is an effective way to produce bispecific antibodies by driving heterodimerization with mutations in the CH3 domain of each half antibody. To better understand the conformational impact by the knob and hole mutations, we combined size-exclusion chromatography (SEC), differential scanning calorimetry (DSC), and hydrogen-deuterium exchange mass spectrometry (H/D exchange MS), to characterize the global and peptide-level conformational changes. We found no significant alteration in structure or conformational dynamics induced by the knob-into-hole framework, and the conformational stability is similar to the wild-type (WT) IgG4 molecules (except for some small difference in the CH3 domain) expressed in E. coli. Functional studies including antigen-binding and neonatal fragment crystallizable (Fc) receptor (FcRn) binding demonstrated no difference between the knob-into-hole and WT IgG4 molecules in E. coli.

Role of Molecular Structure in the Production of Water-Soluble Species by Photo-oxidation of Petroleum.

Asphaltenes are high-boiling and recalcitrant compounds that are generally minor components of crude oil (∼0.1-15.0 wt %) but dominate the composition of heavily weathered spilled petroleum. These solid residues exhibit a high structural complexity, comprised of polycyclic aromatic hydrocarbons (PAHs) that are a mixture of single-core (island) and multicore (archipelago) structural motifs. The mass fraction of each motif is sample-dependent. Thus, knowledge of a potential structural dependence (single- versus multicore) on the production of water-soluble species from asphaltene samples is key to understanding the contribution of photochemically generated dissolved organic matter from oil spills. In this work, asphaltene samples with enriched mass fractions of either island (single-core) or archipelago (multicore) structural motifs are photo-oxidized on artificial seawater by the use of a solar simulator. Molecular characterization of oil- and water-soluble photoproducts, conducted by Fourier transform ion cyclotron resonance mass spectrometry, reveals that island motifs exhibit very limited production of water-soluble species, and their oil-soluble products reflect the molecular composition of the starting material. Conversely, archipelago motifs yield a water-soluble compositional continuum of Ox, SxOy, and NxOy containing hydrocarbons species that exhibit the typical molecular fingerprint of dissolved organic matter (DOM). The lower carbon number and aromaticity of the archipelago-derived asphaltene photoproducts suggest the occurrence of photofragmentation (or photolysis) reactions. To investigate the possibility of the opposite reaction (photopolymerization), the photo-oxidation of small PAHs isolated from a low-boiling petroleum distillation cut was also performed. It yielded water-soluble compounds with carbon number and aromaticity up to 2-fold higher than the starting material, strongly suggesting that polymerization (addition reactions) occurs. Collectively, the results indicate that the presence of archipelago motifs and the occurrence of cracking/polymerization reactions are central in the production of dissolved organic matter from fossil fuels.

Characterization of an Asphalt Binder and Photoproducts by Fourier Transform Ion Cyclotron Resonance Mass Spectrometry Reveals Abundant Water-Soluble Hydrocarbons.

Road asphalt is comprised of aggregate (rocks) mixed with a binder composed of high-boiling petroleum-derived compounds, which have been thought to be relatively inert (unreactive) and thus leach small amounts of polyaromatic hydrocarbons (PAHs) into water from the built environment. However, recent studies have demonstrated that petroleum readily undergoes photooxidation and generates water-soluble oxygen-containing hydrocarbons. Therefore, here, we investigate the effects of solar irradiation on an asphalt binder. Upon irradiation in a photooxidation microcosm, thin films of the asphalt binder produce abundant oil- and water-soluble oxygenated hydrocarbons, which we hypothesize are also leached from roads and highways through photooxidation reactions. Ultrahigh-resolution Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) enables extensive compositional characterization of the virgin asphalt binder, irradiated asphalt binder, and the water-soluble photoproducts. The results reveal the production of water-soluble species that resemble the molecular composition of petroleum-derived dissolved organic matter, including abundant hydrocarbons and S-containing species with up to 18 oxygen atoms. The results also confirm photo-induced oxidation, fragmentation, and potentially polymerization as active processes involved in the production of water-soluble organic pollutants from asphalt.

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.

Diagnosis of Hemoglobinopathy and ß-Thalassemia by 21 Tesla Fourier Transform Ion Cyclotron Resonance Mass Spectrometry and Tandem Mass Spectrometry of Hemoglobin from Blood.

BACKGROUND: Hemoglobinopathies and thalassemias are the most common genetically determined disorders. Current screening methods include cation-exchange HPLC and electrophoresis, the results of which can be ambiguous because of limited resolving power. Subsequently, laborious genetic testing is required for confirmation. METHODS: We performed a top-down tandem mass spectrometry (MS/MS) approach with a fast data acquisition (3 min), ultrahigh mass accuracy, and extensive residue cleavage by use of positive electrospray ionization 21 Tesla Fourier transform ion cyclotron resonance-tandem mass spectrometry (21 T FT-ICR MS/MS) for hemoglobin (Hb) variant de novo sequencing and ß-thalassemia diagnosis. RESULTS: We correctly identified all Hb variants in blind analysis of 18 samples, including the first characterization of homozygous Hb Himeji variant. In addition, an Hb heterozygous variant with isotopologue mass spacing as small as 0.0194 Da (Hb AD) was resolved in both precursor ion mass spectrum (MS1) and product ion mass spectrum (MS2). In blind analysis, we also observed that the abundance ratio between intact δ and ß subunits (δ/ß) or the abundance ratio between intact δ and α subunits (δ/α) could serve to diagnose ß-thalassemia trait caused by a mutation in 1 HBB gene. CONCLUSIONS: We found that 21 T FT-ICR MS/MS provides a benchmark for top-down MS/MS analysis of blood Hb. The present method has the potential to be translated to lower resolving power mass spectrometers (lower field FT-ICR mass spectrometry and Orbitrap) for Hb variant analysis (by MS1 and MS2) and ß-thalassemia diagnosis (MS1).

Combating selective ionization in the high resolution mass spectral characterization of complex mixtures.

Direct "dilute and shoot" mass spectral analysis of complex naturally-occurring mixtures has become the "standard" analysis in environmental and petrochemical science, as well as in many other areas of research. Despite recent advances in ionization methods, that approach still suffers several limitations for the comprehensive characterization of compositionally complex matrices. Foremost, the selective ionization of highly acidic (negative electrospray ionization ((-) ESI)) and/or basic (positive electrospray ionization ((+) ESI)) species limits the detection of weakly acidic/basic species, and similar issues (matrix effects) complicate atmospheric pressure photo-ionization (APPI)/atmospheric pressure chemical ionization (APCI) analyses. Furthermore, given the wide range of chemical functionalities and structural motifs in these compositionally complex mixtures, aggregation can similarly limit the observed species to a small (10-20%) mass fraction of the whole sample. Finally, irrespective of the ionization method, the mass analyzer must be capable of resolving tens-of-thousands of mass spectral peaks and provide the mass accuracy (typically 50-300 ppb mass measurement error) required for elemental composition assignment, and thus is generally limited to high-field Fourier transform ion cyclotron mass spectrometry (FT-ICR MS). Here, we describe three approaches to combat the above issues for (+) ESI, (-) ESI, and (+) APPI FT-ICR MS analysis of petroleum samples. Each approach relies on chromatographic fractionation to help reduce selective ionization discrimination and target either specific chemical functionalities (pyridinic and pyrrolic species (nitrogen) or carboxylic acids (oxygen)) or specific structural motifs (single aromatic core (island) or multi-core aromatics (archipelago)) known to be related to ionization efficiency. Each fractionation method yields a 2-10-fold increase in the compositional coverage, exposes species that are undetectable using direct "dilute and shoot" analysis, and provides coarse selectivity in chemical functionalities that can both increase the assignment confidence and optimize ionization conditions to maximize compositional coverage.

Nanostructure of Gasification Charcoal (Biochar).

In this work, we investigate the molecular composition and nanostructure of gasification charcoal (biochar) by comparing it with heat-treated fullerene arc-soot. Using ultrahigh resolution Fourier transform ion-cyclotron resonance and laser desorption ionization time-of-flight mass spectrometry, Raman spectroscopy, and high resolution transmission electron microscopy we analyzed charcoal of low tar content obtained from gasification. Mass spectrometry revealed no magic number fullerenes such as C60 or C70 in the charcoal. The positive molecular ion m/ z 701, previously considered a graphitic part of the nanostructure, was found to be a breakdown product of pyrolysis and not part of the nanostructure. A higher mass distribution of ions similar to that found in thermally treated fullerene soot indicates that they share a nanostructure. Recent insights into the formation of all carbon fullerenes reveal that conditions in charcoal formation are not optimal for the formation of fullerenes, but instead, curved carbon structures coalesce into fulleroid-like structures. Microscopy and spectroscopy support such a stacked, fulleroid-like nanostructure, which was explored using reactive molecular dynamics simulations.

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.

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.

Spontaneous Calcium-Independent Dimerization of the Isolated First Domain of Neural Cadherin.

Cadherins are calcium-dependent, transmembrane adhesion molecules that assemble through direct noncovalent association of their N-terminal extracellular modular domains. As the transmembrane component of adherens junctions, they indirectly link adherent cells' actin cytoskeletons. Here, we investigate the most distal extracellular domain of neural cadherin (N-cadherin), a protein required at excitatory synapses, the site of long-term potentiation. This domain is the site of the adhesive interface, and it forms a dimer spontaneously without binding calcium, a surprising finding given that calcium binding is required for proper physiological function. A critical tryptophan at position 2, W2, provides a spectroscopic probe for the "closed" monomer and strand-swapped dimer. Spectroscopic studies show that W2 remains docked in the two forms but has a different apparent interaction with the hydrophobic pocket. Size-exclusion chromatography was used to measure the levels of the monomer and dimer over time to study the kinetics and equilibria of the unexpected spontaneous dimer formation ( Kd = 130 µM; τ = 2 days at 4 °C). Our results support the idea that NCAD1 is missing critical contacts that facilitate the rapid exchange of the ßA-strand. Furthermore, the monomer and dimer have equivalent and exceptionally high intrinsic stability for a 99-residue Ig-like domain with no internal disulfides ( Tm = 77 °C; Δ H = 85 kcal/mol). Ultimately, a complete analysis of synapse dynamics requires characterization of the kinetics and equilibria of N-cadherin. The studies reported here take a reductionist approach to understanding the essential biophysics of an atypical Ig-like domain that is the site of the adhesive interface of N-cadherin.

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.