Carbon dioxide/bicarbonate is required for sensitive inactivation of mammalian glyceraldehyde-3-phosphate dehydrogenase by hydrogen peroxide.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) contains an active site Cys and is one of the most sensitive cellular enzymes to oxidative inactivation and redox regulation. Here, we show that inactivation by hydrogen peroxide is strongly enhanced in the presence of carbon dioxide/bicarbonate. Inactivation of isolated mammalian GAPDH by H2O2 increased with increasing bicarbonate concentration and was sevenfold faster in 25 mM (physiological) bicarbonate compared with bicarbonate-free buffer of the same pH. H2O2 reacts reversibly with CO2 to form a more reactive oxidant, peroxymonocarbonate (HCO4-), which is most likely responsible for the enhanced inactivation. However, to account for the extent of enhancement, we propose that GAPDH must facilitate formation and/or targeting of HCO4- to promote its own inactivation. Inactivation of intracellular GAPDH was also strongly enhanced by bicarbonate: treatment of Jurkat cells with 20 µM H2O2 in 25 mM bicarbonate buffer for 5 min caused almost complete GAPDH inactivation, but no loss of activity when bicarbonate was not present. H2O2-dependent GAPDH inhibition in bicarbonate buffer was observed even in the presence of reduced peroxiredoxin 2 and there was a significant increase in cellular glyceraldehyde-3-phosphate/dihydroxyacetone phosphate. Our results identify an unrecognized role for bicarbonate in enabling H2O2 to influence inactivation of GAPDH and potentially reroute glucose metabolism from glycolysis to the pentose phosphate pathway and NAPDH production. They also demonstrate what could be wider interplay between CO2 and H2O2 in redox biology and the potential for variations in CO2 metabolism to influence oxidative responses and redox signaling.

Secreted Amyloid Precursor Protein Alpha (sAPPα) Regulates the Cellular Proteome and Secretome of Mouse Primary Astrocytes.

Secreted amyloid precursor protein alpha (sAPPα), processed from a parent mammalian brain protein, amyloid precursor protein, can modulate learning and memory. Recently it has been shown to modulate the transcriptome and proteome of human neurons, including proteins with neurological functions. Here, we analysed whether the acute administration of sAPPα facilitated changes in the proteome and secretome of mouse primary astrocytes in culture. Astrocytes contribute to the neuronal processes of neurogenesis, synaptogenesis and synaptic plasticity. Cortical mouse astrocytes in culture were exposed to 1 nM sAPPα, and changes in both the whole-cell proteome (2 h) and the secretome (6 h) were identified with Sequential Window Acquisition of All Theoretical Fragment Ion Spectra-Mass Spectrometry (SWATH-MS). Differentially regulated proteins were identified in both the cellular proteome and secretome that are involved with neurologically related functions of the normal physiology of the brain and central nervous system. Groups of proteins have a relationship to APP and have roles in the modulation of cell morphology, vesicle dynamics and the myelin sheath. Some are related to pathways containing proteins whose genes have been previously implicated in Alzheimer's disease (AD). The secretome is also enriched in proteins related to Insulin Growth Factor 2 (IGF2) signaling and the extracellular matrix (ECM). There is the promise that a more specific investigation of these proteins will help to understand the mechanisms of how sAPPα signaling affects memory formation.

Comparison of SPEED, S-Trap, and In-Solution-Based Sample Preparation Methods for Mass Spectrometry in Kidney Tissue and Plasma.

Mass spectrometry is a powerful technique for investigating renal pathologies and identifying biomarkers, and efficient protein extraction from kidney tissue is essential for bottom-up proteomic analyses. Detergent-based strategies aid cell lysis and protein solubilization but are poorly compatible with downstream protein digestion and liquid chromatography-coupled mass spectrometry, requiring additional purification and buffer-exchange steps. This study compares two well-established detergent-based methods for protein extraction (in-solution sodium deoxycholate (SDC); suspension trapping (S-Trap)) with the recently developed sample preparation by easy extraction and digestion (SPEED) method, which uses strong acid for denaturation. We compared the quantitative performance of each method using label-free mass spectrometry in both sheep kidney cortical tissue and plasma. In kidney tissue, SPEED quantified the most unique proteins (SPEED 1250; S-Trap 1202; SDC 1197). In plasma, S-Trap produced the most unique protein quantifications (S-Trap 150; SDC 148; SPEED 137). Protein quantifications were reproducible across biological replicates in both tissue (R2 = 0.85-0.90) and plasma (SPEED R2 = 0.84; SDC R2 = 0.76, S-Trap R2 = 0.65). Our data suggest SPEED as the optimal method for proteomic preparation in kidney tissue and S-Trap or SPEED as the optimal method for plasma, depending on whether a higher number of protein quantifications or greater reproducibility is desired.

A lipopolysaccharide-dependent phage infects a pseudomonad phytopathogen and can evolve to evade phage resistance.

Bacterial pathogens are major causes of crop diseases, leading to significant production losses. For instance, kiwifruit canker, caused by the phytopathogen Pseudomonas syringae pv. actinidiae (Psa), has posed a global challenge to kiwifruit production. Treatment with copper and antibiotics, whilst initially effective, is leading to the rise of bacterial resistance, requiring new biocontrol approaches. Previously, we isolated a group of closely related Psa phages with biocontrol potential, which represent environmentally sustainable antimicrobials. However, their deployment as antimicrobials requires further insight into their properties and infection strategy. Here, we provide an in-depth examination of the genome of ΦPsa374-like phages and show that they use lipopolysaccharides (LPS) as their main receptor. Through proteomics and cryo-electron microscopy of ΦPsa374, we revealed the structural proteome and that this phage possess a T = 9 capsid triangulation, unusual for myoviruses. Furthermore, we show that ΦPsa374 phage resistance arises in planta through mutations in a glycosyltransferase involved in LPS synthesis. Lastly, through in vitro evolution experiments we showed that phage resistance is overcome by mutations in a tail fibre and structural protein of unknown function in ΦPsa374. This study provides new insight into the properties of ΦPsa374-like phages that informs their use as antimicrobials against Psa.

Identifying Candidate Protein Markers of Acute Kidney Injury in Acute Decompensated Heart Failure.

One-quarter of patients with acute decompensated heart failure (ADHF) experience acute kidney injury (AKI)-an abrupt reduction or loss of kidney function associated with increased long-term mortality. There is a critical need to identify early and real-time markers of AKI in ADHF; however, to date, no protein biomarkers have exhibited sufficient diagnostic or prognostic performance for widespread clinical uptake. We aimed to identify novel protein biomarkers of AKI associated with ADHF by quantifying changes in protein abundance in the kidneys that occur during ADHF development and recovery in an ovine model. Relative quantitative protein profiling was performed using sequential window acquisition of all theoretical fragment ion spectra-mass spectrometry (SWATH-MS) in kidney cortices from control sheep (n = 5), sheep with established rapid-pacing-induced ADHF (n = 8), and sheep after ~4 weeks recovery from ADHF (n = 7). Of the 790 proteins quantified, we identified 17 candidate kidney injury markers in ADHF, 1 potential kidney marker of ADHF recovery, and 2 potential markers of long-term renal impairment (differential abundance between groups of 1.2-2.6-fold, adjusted p < 0.05). Among these 20 candidate protein markers of kidney injury were 6 candidates supported by existing evidence and 14 novel candidates not previously implicated in AKI. Proteins of differential abundance were enriched in pro-inflammatory signalling pathways: glycoprotein VI (activated during ADHF development; adjusted p < 0.01) and acute phase response (repressed during recovery from ADHF; adjusted p < 0.01). New biomarkers for the early detection of AKI in ADHF may help us to evaluate effective treatment strategies to prevent mortality and improve outcomes for patients.

Identification of Active Site Residues of the Siderophore Synthesis Enzyme PvdF and Evidence for Interaction of PvdF with a Substrate-Providing Enzyme.

The problematic opportunistic pathogen Pseudomonas aeruginosa secretes a siderophore, pyoverdine. Pyoverdine scavenges iron needed by the bacteria for growth and for pathogenicity in a range of different infection models. PvdF, a hydroxyornithine transformylase enzyme, is essential for pyoverdine synthesis, catalysing synthesis of formylhydroxyornithine (fOHOrn) that forms part of the pyoverdine molecule and provides iron-chelating hydroxamate ligands. Using a mass spectrometry assay, we confirm that purified PvdF catalyses synthesis of fOHOrn from hydroxyornithine and formyltetrahydrofolate substrates. Site directed mutagenesis was carried out to investigate amino acid residues predicted to be required for enzymatic activity. Enzyme variants were assayed for activity in vitro and also in vivo, through measuring their ability to restore pyoverdine production to a pvdF mutant strain. Variants at two putative catalytic residues N168 and H170 greatly reduced enzymatic activity in vivo though did not abolish activity in vitro. Change of a third residue D229 abolished activity both in vivo and in vitro. A change predicted to block entry of N10-formyltetrahydrofolate (fTHF) to the active site also abolished activity both in vitro and in vivo. A co-purification assay showed that PvdF binds to an enzyme PvdA that catalyses synthesis of hydroxyornithine, with this interaction likely to increase the efficiency of fOHOrn synthesis. Our findings advance understanding of how P. aeruginosa synthesises pyoverdine, a key factor in host-pathogen interactions.

A SWATH-MS analysis of Myalgic Encephalomyelitis/Chronic Fatigue Syndrome peripheral blood mononuclear cell proteomes reveals mitochondrial dysfunction.

BACKGROUND: Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is a serious and complex physical illness that affects all body systems with a multiplicity of symptoms, but key hallmarks of the disease are pervasive fatigue and 'post-exertional malaise', exacerbation after physical and/or mental activity of the intrinsic fatigue and other symptoms that can be highly debilitating and last from days to months. Although the disease can vary widely between individuals, common symptoms also include pain, cognitive deficits, sleep dysfunction, as well as immune, neurological and autonomic symptoms. Typically, it is a very isolating illness socially, carrying a stigma because of the lack of understanding of the cause and pathophysiology. METHODS: To gain insight into the pathophysiology of ME/CFS, we examined the proteomes of peripheral blood mononuclear cells (PBMCs) by SWATH-MS analysis in a small well-characterised group of patients and matched controls. A principal component analysis (PCA) was used to stratify groups based on protein abundance patterns, which clearly segregated the majority of the ME/CFS patients (9/11) from the controls. This majority subgroup of ME/CFS patients was then further compared to the control group. RESULTS: A total of 60 proteins in the ME/CFS patients were differentially expressed (P < 0.01, Log10 (Fold Change) > 0.2 and < -0.2). Comparison of the PCA selected subgroup of ME/CFS patients (9/11) with controls increased the number of proteins differentially expressed to 99. Of particular relevance to the core symptoms of fatigue and post-exertional malaise experienced in ME/CFS, a proportion of the identified proteins in the ME/CFS groups were involved in mitochondrial function, oxidative phosphorylation, electron transport chain complexes, and redox regulation. A significant number were also involved in previously implicated disturbances in ME/CFS, such as the immune inflammatory response, DNA methylation, apoptosis and proteasome activation. CONCLUSIONS: The results from this study support a model of deficient ATP production in ME/CFS, compensated for by upregulation of immediate pathways upstream of Complex V that would suggest an elevation of oxidative stress. This study and others have found evidence of a distinct pathology in ME/CFS that holds promise for developing diagnostic biomarkers.

Ovarian fluid proteome variation associates with sperm swimming speed in an externally fertilizing fish.

Sperm velocity is a key trait that predicts the outcome of sperm competition. By promoting or impeding sperm velocity, females can control fertilization via postcopulatory cryptic female choice. In Chinook salmon, ovarian fluid (OF), which surrounds the ova, mediates sperm velocity according to male and female identity, biasing the outcome of sperm competition towards males with faster sperm. Past investigations have revealed proteome variation in OF, but the specific components of OF that differentially mediate sperm velocity have yet to be characterized. Here we use quantitative proteomics to investigate whether OF protein composition explains variation in sperm velocity and fertilization success. We found that OF proteomes from six females robustly clustered into two groups and that these groups are distinguished by the abundance of a restricted set of proteins significantly associated with sperm velocity. Exposure of sperm to OF from females in group I had faster sperm compared to sperm exposed to the OF of group II females. Overall, OF proteins that distinguished between these groups were enriched for vitellogenin and calcium ion interactions. Our findings suggest that these proteins may form the functional basis for cryptic female choice via the biochemical and physiological mediation of sperm velocity.

The exon-intron gene structure upstream of the initiation codon predicts translation efficiency.

Introns in mRNA leaders are common in complex eukaryotes, but often overlooked. These introns are spliced out before translation, leaving exon-exon junctions in the mRNA leaders (leader EEJs). Our multi-omic approach shows that the number of leader EEJs inversely correlates with the main protein translation, as does the number of upstream open reading frames (uORFs). Across the five species studied, the lowest levels of translation were observed for mRNAs with both leader EEJs and uORFs (29%). This class of mRNAs also have ribosome footprints on uORFs, with strong triplet periodicity indicating uORF translation. Furthermore, the positions of both leader EEJ and uORF are conserved between human and mouse. Thus, the uORF, in combination with leader EEJ predicts lower expression for nearly one-third of eukaryotic proteins.

Overexpression of a newly identified d-amino acid transaminase in Mycobacterium smegmatis complements glutamate racemase deletion.

Glutamate racemase (MurI) has been proposed as a target for anti-tuberculosis drug development based on the inability of ΔmurI mutants of Mycobacterium smegmatis to grow in the absence of d-glutamate. In this communication, we identify ΔmurI suppressor mutants that are detected during prolonged incubation. Whole genome sequencing of these ΔmurI suppressor mutants identified the presence of a SNP, located in the promoter region of MSMEG_5795. RT-qPCR and transcriptional fusion analyses revealed that the ΔmurI suppressor mutant overexpressed MSMEG_5795 14-fold compared to the isogenic wild-type. MSMEG_5795, which is annotated as 4-amino-4-deoxychorismate lyase (ADCL) but which also has homology to d-amino acid transaminase (d-AAT), was expressed, purified and found to have d-AAT activity and to be capable of producing d-glutamate from d-alanine. Consistent with its d-amino acid transaminase function, overexpressed MSMEG_5795 is able to complement both ΔmurI deletion mutants and alanine racemase (Δalr) deletion mutants, thus confirming a multifunctional role for this enzyme in M. smegmatis.

A molecular war: convergent and ontogenetic evidence for adaptive host manipulation in related parasites infecting divergent hosts.

Mermithids (phylum Nematoda) and hairworms (phylum Nematomorpha) somehow drive their arthropod hosts into water, which is essential for the worms' survival after egression. The mechanisms behind this behavioural change have been investigated in hairworms, but not in mermithids. Establishing a similar mechanistic basis for host behavioural change between these two distantly related parasitic groups would provide strong convergent evidence for adaptive manipulation and insight into how these parasites modify and/or create behaviour. Here, we search for this convergence, and also contrast changes in physiology between hosts infected with immature and mature mermithids to provide the first ontogenetic evidence for adaptive manipulation by disentangling host response and pathology from the parasite's apparent manipulative effects. We used SWATH-mass spectrometry on brains of Forficula auricularia (earwig) and Bellorchestia quoyana (sandhopper), infected with the mermithids Mermis nigrescens and Thaumamermis zealandica, respectively, at both immature and mature stages of infection, to quantify proteomic changes resulting from mermithid infection. Across both hosts (and hairworm-infected hosts, from earlier studies), the general function of dysregulated proteins was conserved. Proteins involved in energy generation/mobilization were dysregulated, corroborating reports of erratic/hyperactive behaviour in infected hosts. Dysregulated proteins involved in axon/dendrite and synapse modulation were also common to all hosts, suggesting neuronal manipulation is involved in inducing positive hydrotaxis. Furthermore, downregulation of CamKII and associated proteins suggest manipulation of memory also contributes to the behavioural shift.

Ribosomal frameshifting and dual-target antiactivation restrict quorum-sensing-activated transfer of a mobile genetic element.

Symbiosis islands are integrative and conjugative mobile genetic elements that convert nonsymbiotic rhizobia into nitrogen-fixing symbionts of leguminous plants. Excision of the Mesorhizobium loti symbiosis island ICEMlSym(R7A) is indirectly activated by quorum sensing through TraR-dependent activation of the excisionase gene rdfS. Here we show that a +1 programmed ribosomal frameshift (PRF) fuses the coding sequences of two TraR-activated genes, msi172 and msi171, producing an activator of rdfS expression named Frameshifted excision activator (FseA). Mass-spectrometry and mutational analyses indicated that the PRF occurred through +1 slippage of the tRNA(phe) from UUU to UUC within a conserved msi172-encoded motif. FseA activated rdfS expression in the absence of ICEMlSym(R7A), suggesting that it directly activated rdfS transcription, despite being unrelated to any characterized DNA-binding proteins. Bacterial two-hybrid and gene-reporter assays demonstrated that FseA was also bound and inhibited by the ICEMlSym(R7A)-encoded quorum-sensing antiactivator QseM. Thus, activation of ICEMlSym(R7A) excision is counteracted by TraR antiactivation, ribosomal frameshifting, and FseA antiactivation. This robust suppression likely dampens the inherent biological noise present in the quorum-sensing autoinduction circuit and ensures that ICEMlSym(R7A) transfer only occurs in a subpopulation of cells in which both qseM expression is repressed and FseA is translated. The architecture of the ICEMlSym(R7A) transfer regulatory system provides an example of how a set of modular components have assembled through evolution to form a robust genetic toggle that regulates gene transcription and translation at both single-cell and cell-population levels.

The cysteine dioxygenase homologue from Pseudomonas aeruginosa is a 3-mercaptopropionate dioxygenase.

Thiol dioxygenation is the initial oxidation step that commits a thiol to important catabolic or biosynthetic pathways. The reaction is catalyzed by a family of specific non-heme mononuclear iron proteins each of which is reported to react efficiently with only one substrate. This family of enzymes includes cysteine dioxygenase, cysteamine dioxygenase, mercaptosuccinate dioxygenase, and 3-mercaptopropionate dioxygenase. Using sequence alignment to infer cysteine dioxygenase activity, a cysteine dioxygenase homologue from Pseudomonas aeruginosa (p3MDO) has been identified. Mass spectrometry of P. aeruginosa under standard growth conditions showed that p3MDO is expressed in low levels, suggesting that this metabolic pathway is available to the organism. Purified recombinant p3MDO is able to oxidize both cysteine and 3-mercaptopropionic acid in vitro, with a marked preference for 3-mercaptopropionic acid. We therefore describe this enzyme as a 3-mercaptopropionate dioxygenase. Mössbauer spectroscopy suggests that substrate binding to the ferrous iron is through the thiol but indicates that each substrate could adopt different coordination geometries. Crystallographic comparison with mammalian cysteine dioxygenase shows that the overall active site geometry is conserved but suggests that the different substrate specificity can be related to replacement of an arginine by a glutamine in the active site.

Proteomic characterization of macro-, micro- and nano-extracellular vesicles derived from the same first trimester placenta: relevance for feto-maternal communication.

STUDY QUESTION: What proteins are carried by extracellular vesicles (EVs) released from normal first trimester placentae? SUMMARY ANSWER: One thousand five hundred and eighty-five, 1656 and 1476 proteins were characterized in macro-, micro- and nano-vesicles, respectively, from first trimester placentae, with all EV fractions being enriched for proteins involved in vesicle transport and inflammation. WHAT IS KNOWN ALREADY: Placental EVs are being increasingly recognized as important mediators of both healthy and pathological pregnancies. However, current research has focused on detecting changes in specific proteins in particular fractions of vesicles during disease. This is the first study to investigate the full proteome of different-sized fractions of EVs from the same first trimester placenta and highlights the differences/similarities between the vesicle fractions. STUDY DESIGN, SIZE, DURATION: A well-established ex vivo placental explant culture model was used to generate macro-, micro- and nano-vesicles from 56 first trimester placentae. Vesicle fractions were collected by differential ultracentrifugation, quantified and characterized. PARTICIPANTS/MATERIALS, SETTING, METHODS: Placental macro-, micro- and nano-vesicles were characterized by microscopy, dynamic light scattering and nanoparticle tracking analysis. The proteome of each EV fraction was interrogated using liquid chromatography-coupled tandem mass spectrometry. Results were validated by semi-quantitative western blotting. MAIN RESULTS AND THE ROLE OF CHANCE: A total of 1585, 1656 and 1476 proteins were identified in macro-, micro- and nano-vesicles, respectively. One thousand one hundred and twenty-five proteins were shared between all three fractions while up to 223 proteins were unique to each fraction. Gene Ontology pathway analysis showed an enrichment of proteins involved in vesicle transport and inflammation in all three fractions of EVs. The expression levels of proteins involved in internalization of vesicles (annexin V, calreticulin, CD31, CD47), the complement pathway [C3, decay-accelerating factor (DAF), membrane cofactor protein (MCP), protectin] and minor histocompatibility antigens [ATP-dependent RNA helicase (DDX3), ribosomal protein S4 (RPS4)] were different between different-sized EVs. LIMITATIONS, REASONS FOR CAUTION: This study is largely hypothesis-generating in nature. It is important to validate these findings using EVs isolated from maternal plasma and the function of the different EV fractions would need further investigation. WIDER IMPLICATIONS OF THE FINDINGS: Our results support the concept that various EV factions can interact with different maternal cells and have unique effects to mediate feto-maternal communication during early pregnancy. This study also provides a list of candidate proteins, which may inform the identification of robust markers that can be used to isolate placental vesicles from the maternal blood in the future. STUDY FUNDING/COMPETING INTERESTS: M.T. is a recipient of the University of Auckland Health Research Doctoral Scholarship and the Freemasons Postgraduate Scholarship. This project was supported by a School of Medicine Performance-based research fund (PBRF) grant awarded to L.W.C. No authors have any conflicts of interest to disclose.

Proteomic analysis of early-stage embryos: implications for egg quality in hapuku (Polyprion oxygeneios).

In order to develop biomarkers that may help predict the egg quality of captive hapuku (Polyprion oxygeneios) and provide potential avenues for its manipulation, the present study (1) sequenced the proteome of early-stage embryos using isobaric tag for relative and absolute quantification analysis, and (2) aimed to establish the predictive value of the abundance of identified proteins with regard to egg quality through regression analysis. Egg quality was determined for eight different egg batches by blastomere symmetry scores. In total, 121 proteins were identified and assigned to one of nine major groups according to their function/pathway. A mixed-effects model analysis revealed a decrease in relative protein abundance that correlated with (decreasing) egg quality in one major group (heat-shock proteins). No differences were found in the other protein groups. Linear regression analysis, performed for each identified protein separately, revealed seven proteins that showed a significant decrease in relative abundance with reduced blastomere symmetry: two correlates that have been named in other studies (vitellogenin, heat-shock protein-70) and a further five new candidate proteins (78 kDa glucose-regulated protein, elongation factor-2, GTP-binding nuclear protein Ran, iduronate 2-sulfatase and 6-phosphogluconate dehydrogenase). Notwithstanding issues associated with multiple statistical testing, we conclude that these proteins, and especially iduronate 2-sulfatase and the generic heat-shock protein group, could serve as biomarkers of egg quality in hapuku.

C-type natriuretic peptide (CNP) signal peptide fragments are present in the human circulation.

BACKGROUND: Signal peptides may be novel biomarkers in cardiovascular diseases. METHODS: We developed a novel immunoassay to the signal peptide of preproCNP (CNPsp) and used this to document circulating venous concentrations of CNPsp in normal healthy volunteers (n=109), regional plasma CNPsp concentrations in patients undergoing clinically indicated catheterisation (n=24) and temporal CNPsp concentrations in patients with ST-elevation myocardial infarction (STEMI) <4h after symptom onset (n=8). The structure/sequence of circulating CNPsp was confirmed by tandem mass spectrometry (MS/MS). RESULTS: In normal human plasma, CNPsp was detectable at levels higher than NT-proCNP (74±17 vs. 20 ± 5.5 pmol/L). There was no correlation between NTproCNP and CNPsp, but plasma concentrations of sibling signal peptides - CNPsp and BNPsp - were strongly correlated (r=0.532, P<0.001). In patients undergoing catheterisation, there were significant arterio-venous step-ups in CNPsp concentrations across the heart (P<0.01) and kidney (P<0.01). Arterial concentrations of CNPsp significantly correlated with heart rate (r=0.446, P<0.05). In STEMI patients, plasma concentrations of CNPsp showed a biphasic elevation pattern between 6 and 12h after symptom onset, with 12h values significantly elevated (∼ 3-fold) compared with levels at presentation (P<0.05). MS/MS verified circulating CNPsp to be preproCNP(14-23) and preproCNP(16-23) peptides. CONCLUSIONS: This is the first report of a circulating preproCNP derived signal peptide. Given the clear cardiac and renal secretion profiles of CNPsp and its response in STEMI patients, further studies on potential biological functions and biomarker applications of CNPsp in cardiovascular disease are warranted.

Regulation of ubiquitin transfer by XIAP, a dimeric RING E3 ligase.

RING domains of E3 ligases promote transfer of Ub (ubiquitin) from the E2~Ub conjugate to target proteins. In many cases interaction of the E2~Ub conjugate with the RING domain requires its prior dimerization. Using cross-linking experiments we show that E2 conjugated ubiquitin contacts the RING homodimer interface of the IAP (inhibitor of apoptosis) proteins, XIAP (X-linked IAP) and cIAP (cellular IAP) 2. Structural and biochemical analysis of the XIAP RING dimer shows that an aromatic residue at the dimer interface is required for E2~Ub binding and Ub transfer. Mutation of the aromatic residue abolishes Ub transfer, but not interaction with Ub. This indicates that nuleophilic attack on the thioester bond depends on precise contacts between Ub and the RING domain. RING dimerization is a critical activating step for the cIAP proteins; however, our analysis shows that the RING domain of XIAP forms a stable dimer and its E3 ligase activity does not require an activation step.

Comparing Walker's (2008) skull trait sex estimation standard to proteomic sex estimation for a group of South Asian individuals.

This research assesses the potential for misidentification of sex in individuals of South Asian ancestry using the Walker (2008) morphological skull sex estimation standard [1]. Chromosomal sex was assessed using proteomic analysis targeting sex chromosome-specific amylogenic peptides. Results showed that the Walker method produced incorrect classification for 36.7 % of individuals. Overwhelmingly, those incorrectly assigned were chromosomally male. Misidentification was due to males within the group having lower trait scores (i.e., more gracile traits) than the standard would predict. There was also a high level of overlap in trait scores between male and females indicating reduced expression of sexual dimorphism. The use of established multivariate statistical techniques improved accuracy of sex estimation in some cases, but larger osteological data sets from South Asian individuals are required to develop population-specific standards. We suggest that peptide analysis may provide a useful tool for the forensic anthropologist when assessing sex in populations without population specific osteological standards.

Comprehensive Matrisome Profiling of Human Adipose Tissue for Soft Tissue Reconstruction.

For effective translation of research from tissue engineering and regenerative medicine domains, the cell-instructive extracellular matrix (ECM) of specific tissues must be accurately realized. As adipose tissue is gaining traction as a biomaterial for soft tissue reconstruction, with highly variable clinical outcomes obtained, a quantitative investigation of the adipose tissue matrisome is overdue. In this study, the human adipose tissue matrisome is profiled using quantitative sequential windowed acquisition of all theoretical fragment ion spectra - mass spectrometry (SWATH-MS) proteomics across a cohort of 13 fat-grafting patients, to provide characterization of ECM proteins within the tissue, and to understand human population variation. There are considerable differences in the expression of matrisome proteins across the patient cohort, with age and lipoaspirate collection technique contributing to the greatest variation across the core matrisome. A high abundance of basement membrane proteins (collagen IV and heparan sulfate proteoglycan) is detected, as well as fibrillar collagens I and II, reflecting the hierarchical structure of the tissue. This study provides a comprehensive proteomic evaluation of the adipose tissue matrisome and contributes to an enhanced understanding of the influence of the matrisome in adipose-related pathologies by providing a healthy reference cohort and details an experimental pipeline that can be further exploited for future biomaterial development.

Extracting the truth through chemical analyses: Early life histories of Victorian-era dental patients in Aotearoa New Zealand.

OBJECTIVES: There are few bioarcheological analyses of life experiences in colonial period Aotearoa New Zealand, despite this being a time of major adaptation and social change. In our study, early life histories are constructed from multi-isotope and enamel peptide analysis of permanent first molars associated with Victorian era dental practices operating between AD 1881 and 1905 in Invercargill. Chemical analyses of the teeth provide insight into the childhood feeding practices, diet, and mobility of the people who had their teeth extracted. MATERIALS AND METHODS: Four permanent left mandibular first molars were analyzed from a cache of teeth discovered at the Leviathan Gift Depot site during excavations in 2019. The methods used were: (1) enamel peptide analysis to assess chromosomal sex; (2) bulk (δ13 Ccarbonate ) and incremental (δ13 Ccollagen and δ15 N) isotope analysis of dentin to assess childhood diet; and (3) strontium (87 Sr/86 Sr) and oxygen (δ18 O) isotope analysis of enamel to assess childhood residency. Two modern permanent first molars from known individuals were analyzed as controls. RESULTS: The archaeological teeth were from three chromosomal males and one female. The protein and whole diets were predominately based on C3 -plants and domestic animal products (meat and milk). A breastfeeding signal was only identified in one historic male. All individuals likely had childhood residences in Aotearoa. DISCUSSION: Unlike most bioarcheological studies that rely on the remains of the dead, the teeth analysed in this study were extracted from living people. We suggest that the dental patients were likely second or third generation colonists to Aotearoa, with fairly similar childhood diets. They were potentially lower-class individuals either living in, or passing through, the growing colonial center of Invercargill.