Phosphate additives promote humic acid carbon and nitrogen skeleton formation by regulating precursors and composting bacterial communities.

This study aimed to compare the effect of different phosphate additives including superphosphate (CP) and MP [Mg(OH)2 + H3PO4] on nitrogen conversion, humus fractions formation and bacterial community in food waste compost. The results showed the ratio of humic acid nitrogen in total nitrogen (HA-N/TN) in CP increased by 49 %. Ammonium nitrogen accumulation was increased by 75 % (CP) and 44 % (MP). Spectroscopic techniques proved that phosphate addition facilitated the formation of complex structures in HA. CP enhanced the dominance of Saccharomonospora, while Thermobifida and Bacillus were improved in MP. Structural equation modeling and network analysis demonstrated that ammonium nitrogen can be converted to HA-N and has positive effects on bacterial composition, reducing sugars and amino acids, especially in CP with more clustered network and synergic bacterial interactions. Therefore, the addition of phosphate provides a new idea to regulate the retained nitrogen toward humification in composting.

AnnoSM: An Automated Annotation Tool for Determining the Substituent Modes on the Parent Skeleton Based on a Characteristic MS/MS Fragment Ion Library.

Mass spectrometry (MS) is a powerful technology for the structural elucidation of known or unknown small molecules. However, the accuracy of MS-based structure annotation is still limited due to the presence of numerous isomers in complex matrices. There are still challenges in automatically interpreting the fine structure of molecules, such as the types and positions of substituents (substituent modes, SMs) in the structure. In this study, we employed flavones, flavonols, and isoflavones as examples to develop an automated annotation method for identifying the SMs on the parent molecular skeleton based on a characteristic MS/MS fragment ion library. Importantly, user-friendly software AnnoSM was built for the convenience of researchers with limited computational backgrounds. It achieved 76.87% top-1 accuracy on the 148 authentic standards. Among them, 22 sets of flavonoid isomers were successfully differentiated. Moreover, the developed method was successfully applied to complex matrices. One such example is the extract of Ginkgo biloba L. (EGB), in which 331 possible flavonoids with SM candidates were annotated. Among them, 23 flavonoids were verified by authentic standards. The correct SMs of 13 flavonoids were ranked first on the candidate list. In the future, this software can also be extrapolated to other classes of compounds.

Aralianudaside A, an unusual skeleton triterpenoid saponin with anti-airway inflammatory activity from Aralia elata.

Aralianudaside A, a triterpene saponin with an unusual skeleton of pentacyclic triterpenoid, along with a new triterpene glycoside and six known compounds were obtained from the buds of Aralia elata. Their structures were determined through extensive spectral analysis, including HRESIMS, IR, 1D and 2D NMR, glycolysis and GC. All compounds were evaluated for anti-airway inflammatory activity in lipopolysaccharides (LPS)-induced airway epithelial cells (16HBE), compounds 1, 3, 5, 7 and 8 significantly decreased the expression of pro-inflammatory cytokines IL-1ß and IL-4.

Sensitive lipid biomarker detection for tuberculosis in late Neanderthal skeletons from Subalyuk Cave, Hungary.

Skeletal remains of two Neanderthal individuals, a 25-35 year-old woman and a 3-4 year-old child, were discovered in a Subalyuk Cave in North-Eastern Hungary. Radiocarbon dating of the female and child remains revealed an age of 39,732-39,076 and 36,117-35,387 cal BP, respectively. Paleopathological studies of these Neanderthal remains revealed probable evidence of skeletal mycobacterial infection, including in the sacrum of the adult specimen and the endocranial surface of the child's skull. Application of PCR amplification to the juvenile cranium and a vertebra gave a positive result (IS6110) for tuberculosis, backed up by spoligotyping. Lipid biomarker analyses of the same two specimens revealed definitive signals for C32 mycoserosates, a very characteristic component of the Mycobacterium tuberculosis complex (MTBC). A vertebra from the adult provided weak evidence for mycocerosate biomarkers. The correlation of probable skeletal lesions with characteristic amplified DNA fragments and a proven lipid biomarker points to the presence of tuberculosis in these Neanderthals. In particular, the closely similar biomarker profiles, for two distinct juvenile cranial and vertebral bones, strengthen this diagnosis.

Improvement of the specific surface area of biochar by calcium-precipitated nanoparticles synthesized by microbial induction as a template skeleton: Removal mechanism of tetracycline in water.

The template method is an effective means to improve the specific surface area and porosity of biochar, but the synthesis of template agents and the way they are integrated with biomass materials still need further development. Therefore, the free Pseudomonas sp. Y1 was used to synthesize calcium-precipitated nanoparticles (CPN) on sludge as a fused template skeleton to enlarge the surface area of sludge biochar facilitating the adsorption of tetracycline (TC) in this work. The modified biochar (FBC) showed excellent specific surface area (448.55 m2 g-1) and porosity (0.0053 cm³ g-1), stable morphological structure, abundant active functional groups, and appreciable adsorption capacity (65.43 mg g-1) based on several characterization and adsorption experiments. Moreover, the adsorption model postulated that the removal of TC is mainly a chemisorption-based heat-trapping, disordered multilayer interaction. In detail, this process involved the joint contribution from electrostatic interactions, ligand exchange, hydrogen bonding, π-π bonding, complexation, and pore filling. Meanwhile, the adaptability and stability of FBC were examined by pH and coexisting substances. This template skeleton induced by microorganisms can provide new insight into the modification of biochar with the template method.

Novel 2-Benzo[d]thiazolyl-4-quinolinylphenol Skeleton-Based Turn-on Fluorescent Probe for H2S Detection and its Multiple Applications in Water Environment, Foodstuffs, and Living Organisms.

Hydrogen sulfide (H2S) has comprehensive contributions to maintaining the normal operation and stability of organisms, and it also occurs in the wastewater environment and is related to the deterioration of foodstuffs. Therefore, developing high-sensitive detection techniques for tracing H2S is promising and meaningful. Inspired by this, a novel nopinone-based fluorescent probe NPS for the recognition of H2S was designed and synthesized with excellent sensitivity, low limit of detection (79 nM), good selectivity, and wide pH range (5-9). NPS could emit strong yellow fluorescence and its emission intensity showed a remarkable augmentation at 520 nm upon the supplement of H2S. Furthermore, the recognition mechanism of NPS for H2S was verified by the HRMS analysis, 1H NMR spectra titration, and DFT computation. What is more, NPS also had broad applications in the monitoring of real water samples, red wine, beer, and eggs samples, which showed its development prospect and value in environmental pollution, foodstuffs quality analysis fields. NPS also was applied to monitor trace exogenous H2S and bioimaging in living cells and zebrafish.

Sunlit photocatalytic degradation of organic pollutant by NiCr2O4/Bi2S3/Cr2S3 tracheid skeleton nanocomposite: Mechanism, pathway, reactive sites, genotoxicity and byproduct toxicity evaluation.

In this study, 3D C2S3 (CS) and 2D Bi2S3 (BS) modified NiCr2O4 nanocomposite (NCO-BS-CS NCs) was prepared by sonochemical assisted co-precipitation method for the enhanced photocatalytic activity. Here, NCO-BS-CS NCs showed band gap energy of 2.23 eV and the PL intensity of NCO-BS-CS NCs was lower than NCO, BS, and CS NPs. Thus, the results indicate the fabricated NCO-BS-CS NCs enhance the charge segregation and lower in recombination rate. NCO-BS-CS NCs showed enhanced photodegradation of methyl orange (MO) (95%) and congo red (CR) (99.7%) respectively. The total organic compound (TOC) analysis shows the complete mineralization of about 91 and 98% for MO and CR respectively. Furthermore, the Fukui function was used for the prediction of reactive sites in the photodegradation pathway of MO and CR by NCs. ECOSAR program was done to determine the toxicity of the intermediate and the results conclude that the degraded product shows nontoxic to the environmental organism (fish, daphnia, and algae). Thus, the fabricated NCO-BS-CS NCs can be used for the remediation of toxic organic pollutants from the waste water by photocatalytic degradation.

Status of the Coastal Marine Environment in the Southern Red Sea, Yemen, as Reflected by Elements Accumulated in the Skeletons of Scleractinian (Stony) Corals.

The purpose of this study was to assess the level of anthropogenic contamination as well as to collect more data concerning the mineral composition of scleractinian corals from Southern Red Sea, Hodeidah Governorate, Yemen. The mass fractions of 31 elements were determined in the skeleton of nine coral colonies collected in the vicinity of the south-western coast of Yemen. All measurements were conducted by Instrumental Neutron Activation Analysis (INAA). The final results concerning the distribution of considered elements were comparable and, in some cases, inferior to those reported worldwide. The determined mass fractions of V, Cr, Mn, Fe, Ni, and Zn as Presumably Contaminating Elements (PCE) together with the mass fractions of the same elements previously reported in the literature regarding Red Sea Pleistocene corals permitted calculating the individual Contamination Factor (CF) and collective Pollution Load Index (PLI). In spite of high mass fraction values of Mn and Fe in only a few coral colonies, for all investigated places, the PLI values corresponding to studied areas were less than unit, suggesting the investigated areas could be considered as uncontaminated.

High-Mg calcite nanoparticles within a low-Mg calcite matrix: A widespread phenomenon in biomineralization.

During the process of biomineralization, organisms utilize various biostrategies to enhance the mechanical durability of their skeletons. In this work, we establish that the presence of high-Mg nanoparticles embedded within lower-Mg calcite matrices is a widespread strategy utilized by various organisms from different kingdoms and phyla to improve the mechanical properties of their high-Mg calcite skeletons. We show that such phase separation and the formation of high-Mg nanoparticles are most probably achieved through spinodal decomposition of an amorphous Mg-calcite precursor. Such decomposition is independent of the biological characteristics of the studied organisms belonging to different phyla and even kingdoms but rather, originates from their similar chemical composition and a specific Mg content within their skeletons, which generally ranges from 14 to 48 mol % of Mg. We show evidence of high-Mg calcite nanoparticles in the cases of six biologically different organisms all demonstrating more than 14 mol % Mg-calcite and consider it likely that this phenomenon is immeasurably more prevalent in nature. We also establish the absence of these high-Mg nanoparticles in organisms whose Mg content is lower than 14 mol %, providing further evidence that whether or not spinodal decomposition of an amorphous Mg-calcite precursor takes place is determined by the amount of Mg it contains. The valuable knowledge gained from this biostrategy significantly impacts the understanding of how biominerals, although composed of intrinsically brittle materials, can effectively resist fracture. Moreover, our theoretical calculations clearly suggest that formation of Mg-rich nanoprecipitates greatly enhances the hardness of the biomineralized tissue as well.

Barium alginate as a skeleton coating graphene oxide and bentonite-derived composites: Excellent adsorbent based on predictive design for the enhanced adsorption of methylene blue.

The phenomenon that calcium alginate does not exhibit high adsorption capacity as a carrier material has not been reasonably explained or solved. In this paper, a new viewpoint that the orbital energy level of metal ions and the binding degree of the α-l-guluronate and ß-d-mannuronate units affect the adsorption performance of the composite was innovatively proposed. Taking barium alginate (BA) as an example, the possibility of replacing calcium alginate is discussed. Barium alginate/graphene oxide (BA/GO) membranes and three-dimensional (3D) barium alginate-bentonite-graphene oxide derived (3D-BA) hydrogels were prepared by vacuum freeze-drying to remove methylene blue. The structure and morphology of the prepared adsorbents were characterized by scanning electron microscopy, X-ray photoelectron spectroscopy, thermogravimetry and Fourier transform infrared spectroscopy. The effects of adsorbent dosage, doping ratio, temperature, contact time, pH value and initial dye concentration on the adsorption performance of BA composites were investigated. The adsorption capacities of the BA/GO and 3D-BA materials were 1011.3 and 710.3 mg/g, respectively. The BA/GO membrane exhibited stable filtration performance against high concentrations of dyes. Benefiting from the strong interaction between bentonite, sodium alginate and Ba2+, the 3D-BA hydrogel showed higher thermal stability and better adsorption efficiency than other materials. The Elovich kinetic model and Sips equation can appropriately describe the adsorption process. The results show that barium alginate is a better carrier material than calcium alginate.

Linking isotopic signatures of nitrogen in nearshore coral skeletons with sources in catchment runoff.

We use a multi-tracer approach to identify catchment sources of nitrogen (N) in the skeletons of nearshore Porites corals within the Great Barrier Reef. We measured δ15N, δ13C and C:N ratios of particulate organic matter (POM) sampled from the Pioneer River catchment and identified five distinct end-members: (1) marine planktonic and algal-dominated matter with higher δ15N values from the river mouth and coastal waters; (2) estuarine planktonic and algal matter with lower δ15N values associated with estuarine mixing; (3) lower river freshwater phytoplankton and algal-dominated matter in stratified reservoirs adjacent to catchment weirs, with the 15N-enriched source likely caused by microbial remineralization and denitrification; (4) upper river low δ15N terrigenous soil matter eroded from cane fields bordering waterways; and (5) terrestrial plant detrital matter in forest streams, representing a low δ15N fixed atmospheric nitrogen source. The δ15N values of adjacent, nearshore Porites coral skeletons is reflective of POM composition in coastal waters, with 15N-enriched values reflective of transformed N during flood pulses from the Pioneer River.

Calcite Nanotuned Chitinous Skeletons of Giant Ianthella basta Marine Demosponge.

Marine sponges were among the first multicellular organisms on our planet and have survived to this day thanks to their unique mechanisms of chemical defense and the specific design of their skeletons, which have been optimized over millions of years of evolution to effectively inhabit the aquatic environment. In this work, we carried out studies to elucidate the nature and nanostructural organization of three-dimensional skeletal microfibers of the giant marine demosponge Ianthella basta, the body of which is a micro-reticular, durable structure that determines the ideal filtration function of this organism. For the first time, using the battery of analytical tools including three-dimensional micro-X-ray Fluorescence (3D-µXRF), X-ray diffraction (XRD), infra-red (FTIR), Raman and Near Edge X-ray Fine Structure (NEXAFS) spectroscopy, we have shown that biomineral calcite is responsible for nano-tuning the skeletal fibers of this sponge species. This is the first report on the presence of a calcitic mineral phase in representatives of verongiid sponges which belong to the class Demospongiae. Our experimental data suggest a possible role for structural amino polysaccharide chitin as a template for calcification. Our study suggests further experiments to elucidate both the origin of calcium carbonate inside the skeleton of this sponge and the mechanisms of biomineralization in the surface layers of chitin microfibers saturated with bromotyrosines, which have effective antimicrobial properties and are responsible for the chemical defense of this organism. The discovery of the calcified phase in the chitinous template of I. basta skeleton is expected to broaden the knowledge in biomineralization science where the calcium carbonate is regarded as a valuable material for applications in biomedicine, environmental science, and even in civil engineering.

Review on synthesis of three-dimensional graphene skeletons and their absorption performance for oily wastewater.

Water pollution is a global environmental problem that affects the ecosystem severely. Treatment of oily wastewater and organic pollutants is a major challenge that waits to be solved as soon as possible. Adsorbing is one of the most effective strategies to deal with this problem. Three-dimensional (3D) porous adsorbents made of graphene or graphene-based nanomaterials skeletons had attracted more attention in wastewater treatment because of their large surface area, high porosity, low density, high chemical/thermal stability, and steady mechanical properties, which allow different pollutants to easily access and diffuse into 3D networks of adsorbents. This work presents an extensive summarization of recent progress in the synthesis methodologies and microstructures of 3D graphene foams and 3D graphene-based foams and highlights their adsorption performance for oils and organic solvents. Advantages and disadvantages of various preparation strategies are compared and the corresponded structures of these skeletons are studied in detail. Furthermore, the effects of the structures on oil-adsorption properties are analyzed and some data and parameters of the oil-adsorption properties are listed and studied for easier comparison. At last, the future research directions and technical challenges are prospected, which is hoped that the researchers will be inspired to develop the new graphene-based adsorbents.

The prehistoric roots of Chinese cuisines: Mapping staple food systems of China, 6000 BC-220 AD.

We conducted a meta-analysis of published carbon and nitrogen isotope data from archaeological human skeletal remains (n = 2448) from 128 sites cross China in order to investigate broad spatial and temporal patterns in the formation of staple cuisines. Between 6000-5000 cal BC we found evidence for an already distinct north versus south divide in the use of main crop staples (namely millet vs. a broad spectrum of C3 plant based diet including rice) that became more pronounced between 5000-2000 cal BC. We infer that this pattern can be understood as a difference in the spectrum of subsistence activities employed in the Loess Plateau and the Yangtze-Huai regions, which can be partly explained by differences in environmental conditions. We argue that regional differentiation in dietary tradition are not driven by differences in the conventional "stages" of shifting modes of subsistence (hunting-foraging-pastoralism-farming), but rather by myriad subsistence choices that combined and discarded modes in a number of innovative ways over thousands of years. The introduction of wheat and barley from southwestern Asia after 2000 cal BC resulted in the development of an additional east to west gradient in the degree of incorporation of the different staple products into human diets. Wheat and barley were rapidly adopted as staple foods in the Continental Interior contra the very gradual pace of adoption of these western crops in the Loess Plateau. While environmental and social factors likely contributed to their slow adoption, we explored local cooking practice as a third explanation; wheat and barley may have been more readily folded into grinding-and-baking cooking traditions than into steaming-and-boiling traditions. Changes in these culinary practices may have begun in the female sector of society.

Biochemical characterization of the skeletal matrix of the massive coral, Porites australiensis - The saccharide moieties and their localization.

To construct calcium carbonate skeletons of sophisticated architecture, scleractinian corals secrete an extracellular skeletal organic matrix (SOM) from aboral ectodermal cells. The SOM, which is composed of proteins, saccharides, and lipids, performs functions critical for skeleton formation. Even though polysaccharides constitute the major component of the SOM, its contribution to coral skeleton formation is poorly understood. To this end, we analyzed the SOM of the massive colonial coral, Porites australiensis, the skeleton of which has drawn great research interest because it records environmental conditions throughout the life of the colony. The coral skeleton was extensively cleaned, decalcified with acetic acid, and organic fractions were separated based on solubility. These fractions were analyzed using various techniques, including SDS-PAGE, FT-IR, in vitro crystallization, CHNS analysis, chromatography analysis of monosaccharide and enzyme-linked lectin assay (ELLA). We confirmed the acidic nature of SOM and the presence of sulphate, which is thought to initiate CaCO3 crystallization. In order to analyze glycan structures, we performed ELLA on the soluble SOM for the first time and found that it exhibits strong specificity to Datura stramonium lectin (DSL). Furthermore, using biotinylated DSL with anti-biotin antibody conjugated to nanogold, in situ localization of DSL-binding polysaccharides in the P. australiensis skeleton was performed. Signals were distributed on the surfaces of fiber-like crystals of the skeleton, suggesting that polysaccharides may modulate crystal shape. Our study emphasizes the importance of sugar moieties in biomineralization of scleractinian corals.

Neolithic and medieval virus genomes reveal complex evolution of hepatitis B.

The hepatitis B virus (HBV) is one of the most widespread human pathogens known today, yet its origin and evolutionary history are still unclear and controversial. Here, we report the analysis of three ancient HBV genomes recovered from human skeletons found at three different archaeological sites in Germany. We reconstructed two Neolithic and one medieval HBV genome by de novo assembly from shotgun DNA sequencing data. Additionally, we observed HBV-specific peptides using paleo-proteomics. Our results demonstrated that HBV has circulated in the European population for at least 7000 years. The Neolithic HBV genomes show a high genomic similarity to each other. In a phylogenetic network, they do not group with any human-associated HBV genome and are most closely related to those infecting African non-human primates. The ancient viruses appear to represent distinct lineages that have no close relatives today and possibly went extinct. Our results reveal the great potential of ancient DNA from human skeletons in order to study the long-time evolution of blood borne viruses.

First Report on Chitin in a Non-Verongiid Marine Demosponge: The Mycale euplectellioides Case.

Sponges (Porifera) are recognized as aquatic multicellular organisms which developed an effective biochemical pathway over millions of years of evolution to produce both biologically active secondary metabolites and biopolymer-based skeletal structures. Among marine demosponges, only representatives of the Verongiida order are known to synthetize biologically active substances as well as skeletons made of structural polysaccharide chitin. The unique three-dimensional (3D) architecture of such chitinous skeletons opens the widow for their recent applications as adsorbents, as well as scaffolds for tissue engineering and biomimetics. This study has the ambitious goal of monitoring other orders beyond Verongiida demosponges and finding alternative sources of naturally prestructured chitinous scaffolds; especially in those demosponge species which can be cultivated at large scales using marine farming conditions. Special attention has been paid to the demosponge Mycale euplectellioides(Heteroscleromorpha: Poecilosclerida: Mycalidae) collected in the Red Sea. For the first time, we present here a detailed study of the isolation of chitin from the skeleton of this sponge, as well as its identification using diverse bioanalytical tools. Calcofluor white staining, Fourier-transform Infrared Spcetcroscopy (FTIR), electrospray ionization mass spectrometry (ESI-MS), scanning electron microscopy (SEM), and fluorescence microscopy, as well as a chitinase digestion assay were applied in order to confirm with strong evidence the finding of a-chitin in the skeleton of M. euplectellioides. We suggest that the discovery of chitin within representatives of the Mycale genus is a promising step in their evaluation of these globally distributed sponges as new renewable sources for both biologically active metabolites and chitin, which are of prospective use for pharmacology and biomaterials oriented biomedicine, respectively.

Analysis of Dextromethorphan and Dextrorphan in Skeletal Remains Following Differential Microclimate Exposure: Comparison of Acute vs. Repeated Drug Exposure.

Analysis of dextromethorphan (DXM) and its metabolite dextrorphan (DXT) in skeletal remains of rats following acute (ACU, 75 mg/kg, IP, n = 10) or three repeated (REP, 25 mg/kg, IP, n = 10, 40-min interval) doses of DXM is described. Following dosing and euthanasia, rats decomposed outdoors to skeleton in two different microclimate environments (n = 5 ACU and n = 5 REP at each site): Site A (shaded forest microenvironment) and Site B (rocky substrate exposed to direct sunlight, 600 m from Site A). Two drug-free rats at each site served as negative controls. Skeletal elements (vertebrae, ribs, pelvic girdles, femora, tibiae, skulls and scapulae) were recovered, pulverized and underwent methanolic microwave assisted extraction (MAE). Extracts were analyzed by GC-MS following clean-up by solid-phase extraction (SPE). Drug levels, expressed as mass-normalized response ratios and the ratios of DXT and DXM levels (RRDXT/RRDXM) were compared between drug exposures, microclimate sites, and across skeletal elements. DXM levels differed significantly (P < 0.05) between corresponding bone elements across exposure groups (5/7-site A; 4/7-site B), but no significant differences in DXT levels were observed between corresponding elements. RRDXT/RRDXM differed significantly (P < 0.05) between corresponding bone elements across exposure groups (6/7-site A; 5/7-site B). No significant differences were observed in levels of DXM, DXT or RRDXT/RRDXM between corresponding elements from either group between sites. When data from all bone elements was pooled, levels of DXM and RRDXT/RRDXM differed significantly between exposure groups at each site, while those of DXT did not. For both exposure groups, comparison of pooled data between sites showed no significant differences in levels of DXM, DXT or RRDXT/RRDXM. Different decomposition microclimates did not impede the discrimination of DXM exposure patterns from the analyses of DXM, DXT and RRDXT/RRDXM in bone samples.

Variability in magnesium content in Arctic echinoderm skeletons.

In this study, 235 measurements of magnesium concentration in echinoderm's skeletons were compiled, including 30 species and 216 specimens collected from northern and western Barents Sea. We aimed to reveal the scale of Mg variation in the skeletons of Arctic echinoderms. Furthermore, we attempted to examine whether the Mg concentration in echinoderm skeletons is determined primarily by biological factors or is a passive result of environmental influences. We found that the Mg concentration in echinoderm skeletons was characteristic for particular echinoderm classes or was even species-specific. The highest Mg contents were observed in asteroids, followed by ophiuroids, crinoids, and holothuroids, with the lowest values in echinoids. These results strongly imply that biological factors play an important role in controlling the incorporation of Mg into the skeletons of the studied individuals.

Intra- and Interskeletal Proteome Variations in Fresh and Buried Bones.

Proteomic methods are acquiring greater importance in archaeology and palaeontology due to the longevity of proteins in skeletal remains. There are also developing interests in forensic applications, offering the potential to shed light on post-mortem intervals and age at death estimation. However, our understanding of intra- and interskeletal proteome variations is currently severely limited. Here, we evaluated the proteomes obtained from five distinct subsamples of different skeletal elements from buried pig carcasses to ascertain the extent of variation within and between individuals. We found that reproducibility of data depends on the skeletal element used for sampling and that intrabone differences exceed those observed between the same skeletal element sampled from different individuals. Interestingly, the abundance of several serum proteins appeared to correlate with biological age with relative concentrations of alpha-1 antitrypsin and chromogranin-A increasing and those of fetuin-A decreasing. We also observed a surprising level of divergence in data from different LC-MS/MS runs on aliquots of similar samples analyzed months apart, adding constraints to the comparison of results of such methods across different studies.