Bronze Age population dynamics and the rise of dairy pastoralism on the eastern Eurasian steppe.

Recent paleogenomic studies have shown that migrations of Western steppe herders (WSH) beginning in the Eneolithic (ca. 3300-2700 BCE) profoundly transformed the genes and cultures of Europe and central Asia. Compared with Europe, however, the eastern extent of this WSH expansion is not well defined. Here we present genomic and proteomic data from 22 directly dated Late Bronze Age burials putatively associated with early pastoralism in northern Mongolia (ca. 1380-975 BCE). Genome-wide analysis reveals that they are largely descended from a population represented by Early Bronze Age hunter-gatherers in the Baikal region, with only a limited contribution (∼7%) of WSH ancestry. At the same time, however, mass spectrometry analysis of dental calculus provides direct protein evidence of bovine, sheep, and goat milk consumption in seven of nine individuals. No individuals showed molecular evidence of lactase persistence, and only one individual exhibited evidence of >10% WSH ancestry, despite the presence of WSH populations in the nearby Altai-Sayan region for more than a millennium. Unlike the spread of Neolithic farming in Europe and the expansion of Bronze Age pastoralism on the Western steppe, our results indicate that ruminant dairy pastoralism was adopted on the Eastern steppe by local hunter-gatherers through a process of cultural transmission and minimal genetic exchange with outside groups.

Targeted Proteomics Guided by Label-free Quantitative Proteome Analysis in Saliva Reveal Transition Signatures from Health to Periodontal Disease.

Periodontal diseases are among the most prevalent worldwide, but largely silent, chronic diseases. They affect the tooth-supporting tissues with multiple ramifications on life quality. Their early diagnosis is still challenging, due to lack of appropriate molecular diagnostic methods. Saliva offers a non-invasively collectable reservoir of clinically relevant biomarkers, which, if utilized efficiently, could facilitate early diagnosis and monitoring of ongoing disease. Despite several novel protein markers being recently enlisted by discovery proteomics, their routine diagnostic application is hampered by the lack of validation platforms that allow for rapid, accurate and simultaneous quantification of multiple proteins in large cohorts. Here we carried out a pipeline of two proteomic platforms; firstly, we applied open ended label-free quantitative (LFQ) proteomics for discovery in saliva (n = 67, including individuals with health, gingivitis, and periodontitis), followed by selected-reaction monitoring (SRM)-targeted proteomics for validation in an independent cohort (n = 82). The LFQ platform led to the discovery of 119 proteins with at least 2-fold significant difference between health and disease. The 65 proteins chosen for the subsequent SRM platform included 50 functionally related proteins derived from the significantly enriched processes of the LFQ data, 11 from literature-mining, and four house-keeping ones. Among those, 60 were reproducibly quantifiable proteins (92% success rate), represented by a total of 143 peptides. Machine-learning modeling led to a narrowed-down panel of five proteins of high predictive value for periodontal diseases with maximum area under the receiver operating curve >0.97 (higher in disease: Matrix metalloproteinase-9, Ras-related protein-1, Actin-related protein 2/3 complex subunit 5; lower in disease: Clusterin, Deleted in Malignant Brain Tumors 1). This panel enriches the pool of credible clinical biomarker candidates for diagnostic assay development. Yet, the quantum leap brought into the field of periodontal diagnostics by this study is the application of the biomarker discovery-through-verification pipeline, which can be used for validation in further cohorts.

A chronic hypoxic response in photoreceptors alters the vitreous proteome in mice.

Reduced oxygenation of the outer retina in the aging eye may activate a chronic hypoxic response in RPE and photoreceptor cells and is considered as a risk factor for the development of age-related macular degeneration (AMD). In mice, a chronically active hypoxic response in the retinal pigment epithelium (RPE) or photoreceptors leads to age-dependent retinal degeneration. To identify proteins that may serve as accessible markers for a chronic hypoxic insult to photoreceptors, we used proteomics to determine the protein composition of the vitreous humor in genetically engineered mice that lack the von Hippel-Lindau tumor suppressor (Vhl) specifically in rods (rodΔVhl) or cones (all-coneΔVhl). Absence of VHL leads to constitutively active hypoxia-inducible transcription factors (HIFs) and thus to a molecular response to hypoxia even in normal room air. To discriminate between the consequences of a local response in photoreceptors and systemic hypoxic effects, we also evaluated the vitreous proteome of wild type mice after exposure to acute hypoxia. 1'043 of the identified proteins were common to all three hypoxia models. 257, 258 and 356 proteins were significantly regulated after systemic hypoxia, in rodΔVhl and in all-coneΔVhl mice, respectively, at least at one of the analyzed time points. Only few of the regulated proteins were shared by the models indicating that the vitreous proteome is differentially affected by systemic hypoxia and the rod or cone-specific hypoxic response. Similarly, the distinct protein compositions in the individual genetic models at early and late time points suggest regulated, cell-specific and time-dependent processes. Among the proteins commonly regulated in the genetic models, guanylate binding protein 2 (GBP2) showed elevated levels in the vitreous that were accompanied by increased mRNA expression in the retina of both rodΔVhl and all-coneΔVhl mice. We hypothesize that some of the differentially regulated proteins at early time points may potentially be used as markers for the detection of a chronic hypoxic response of photoreceptors.

rawDiag: An R Package Supporting Rational LC-MS Method Optimization for Bottom-up Proteomics.

Optimizing methods for liquid chromatography coupled to mass spectrometry (LC-MS) is a nontrivial task. Here we present rawDiag, a software tool supporting rational method optimization by providing MS operator-tailored diagnostic plots of scan-level metadata. rawDiag is implemented as an R package and can be executed on the R command line or through a graphical user interface (GUI) for less experienced users. The code runs platform-independent and can process 100 raw files in <3 min on current consumer hardware, as we show in our benchmark. As a demonstration of the functionality of our package we include a real-world example taken from our daily core facility business.

Comparative Proteomic Analysis of Plant Acclimation to Six Different Long-Term Environmental Changes.

Plants are constantly challenged in their natural environment by a range of changing conditions. We investigated the acclimation processes and adaptive plant responses to various long-term mild changes and compared them directly within one experimental set-up. Arabidopsis thaliana plants were grown in hydroponic culture for 10 d under controlled abiotic stress (15°C, 25°C, salt and osmotic) and in nutrient deficiency (nitrate and phosphate). Plant growth was monitored and proteomic experiments were performed. Resource allocation between tissues altered during the plants' response. The growth patterns and induced changes of the proteomes indicated that the underlying mechanisms of the adaptation processes are highly specific to the respective environmental condition. Our results indicated differential regulation of response to salt and osmotic treatment, while the proteins in the changed temperature regime showed an inverse, temperature-sensitive control. There was a high correlation of protein level between the nutrient-deficient treatments, but the enriched pathways varied greatly. The proteomic analysis also revealed new insights into the regulation of proteins specific to the shoot and the root. Our investigation revealed unique strategies of plant acclimation to the different applied treatments on a physiological and proteome level, and these strategies are quite distinct in tissues below and above ground.

Proteomic evidence of dietary sources in ancient dental calculus.

Archaeological dental calculus has emerged as a rich source of ancient biomolecules, including proteins. Previous analyses of proteins extracted from ancient dental calculus revealed the presence of the dietary milk protein ß-lactoglobulin, providing direct evidence of dairy consumption in the archaeological record. However, the potential for calculus to preserve other food-related proteins has not yet been systematically explored. Here we analyse shotgun metaproteomic data from 100 archaeological dental calculus samples ranging from the Iron Age to the post-medieval period (eighth century BC to nineteenth century AD) in England, as well as 14 dental calculus samples from contemporary dental patients and recently deceased individuals, to characterize the range and extent of dietary proteins preserved in dental calculus. In addition to milk proteins, we detect proteomic evidence of foodstuffs such as cereals and plant products, as well as the digestive enzyme salivary amylase. We discuss the importance of optimized protein extraction methods, data analysis approaches and authentication strategies in the identification of dietary proteins from archaeological dental calculus. This study demonstrates that proteomic approaches can robustly identify foodstuffs in the archaeological record that are typically under-represented due to their poor macroscopic preservation.

Targeted proteomics coming of age - SRM, PRM and DIA performance evaluated from a core facility perspective.

Quantitative mass spectrometry is a rapidly evolving methodology applied in a large number of omics-type research projects. During the past years, new designs of mass spectrometers have been developed and launched as commercial systems while in parallel new data acquisition schemes and data analysis paradigms have been introduced. Core facilities provide access to such technologies, but also actively support the researchers in finding and applying the best-suited analytical approach. In order to implement a solid fundament for this decision making process, core facilities need to constantly compare and benchmark the various approaches. In this article we compare the quantitative accuracy and precision of current state of the art targeted proteomics approaches single reaction monitoring (SRM), parallel reaction monitoring (PRM) and data independent acquisition (DIA) across multiple liquid chromatography mass spectrometry (LC-MS) platforms, using a readily available commercial standard sample. All workflows are able to reproducibly generate accurate quantitative data. However, SRM and PRM workflows show higher accuracy and precision compared to DIA approaches, especially when analyzing low concentrated analytes.

specL--an R/Bioconductor package to prepare peptide spectrum matches for use in targeted proteomics.

MOTIVATION: Targeted data extraction methods are attractive ways to obtain quantitative peptide information from a proteomics experiment. Sequential Window Acquisition of all Theoretical Spectra (SWATH) and Data Independent Acquisition (DIA) methods increase reproducibility of acquired data because the classical precursor selection is omitted and all present precursors are fragmented. However, especially for targeted data extraction, MS coordinates (retention time information precursor and fragment masses) are required for the particular entities (peptide ions). These coordinates are usually generated in a so-called discovery experiment earlier on in the project if not available in public spectral library repositories. The quality of the assay panel is crucial to ensure appropriate downstream analysis. For that, a method is needed to create spectral libraries and to export customizable assay panels. RESULTS: Here, we present a versatile set of functions to generate assay panels from spectral libraries for use in targeted data extraction methods (SWATH/DIA) in the area of proteomics. AVAILABILITY AND IMPLEMENTATION: specL is implemented in the R language and available under an open-source license (GPL-3) in Bioconductor since BioC 3.0 (R-3.1) http://www.bioconductor.org (Trachsel et al., 2015). A vignette with a complete tutorial describing data import/export and analysis is included in the package and can also be found as supplement material of this article. CONTACT: cp@fgcz.ethz.ch or jg@fgcz.ethz.ch SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Proteomic analysis of the thermophilic methylotroph Bacillus methanolicus MGA3.

Bacillus methanolicus MGA3 is a facultative methylotroph of industrial relevance that is able to grow on methanol as its sole source of carbon and energy. The Gram-positive bacterium possesses a soluble NAD(+) -dependent methanol dehydrogenase and assimilates formaldehyde via the ribulose monophosphate (RuMP) cycle. We used label-free quantitative proteomics to generate reference proteome data for this bacterium and compared the proteome of B. methanolicus MGA3 on two different carbon sources (methanol and mannitol) as well as two different growth temperatures (50°C and 37°C). From a total of approximately 1200 different detected proteins, approximately 1000 of these were used for quantification. While the levels of 213 proteins were significantly different at the two growth temperatures tested, the levels of 109 proteins changed significantly when cells were grown on different carbon sources. The carbon source strongly affected the synthesis of enzymes related to carbon metabolism, and in particular, both dissimilatory and assimilatory RuMP cycle enzyme levels were elevated during growth on methanol compared to mannitol. Our data also indicate that B. methanolicus has a functional tricarboxylic acid cycle, the proteins of which are differentially regulated on mannitol and methanol. Other proteins presumed to be involved in growth on methanol were constitutively expressed under the different growth conditions. All MS data have been deposited in the ProteomeXchange with the identifiers PXD000637 and PXD000638 (http://proteomecentral.proteomexchange.org/dataset/PXD000637, http://proteomecentral.proteomexchange.org/dataset/PXD000638).

Too Much of a Good Thing: Severe Hypercalcemia Presenting with Lethargy and Kidney Failure.

INTRODUCTION: We present a case of a 58-year-old man with a history of laryngo-pharyngectomy including bilateral thyroidectomy due to hypopharyngeal cancer presenting with lethargy, acute kidney failure, and hypercalcemia. Milk alkali syndrome was diagnosed given the history of high-dose calcium / vitamin D supplementation after ruling out other causes of hypercalcemia. After initial treatment with normal saline, furosemide and denosumab, the patient developed severe symptomatic hypocalcemia as a rare adverse effect of denosumab.

A venom-derived neurotoxin, CsTx-1, from the spider Cupiennius salei exhibits cytolytic activities.

CsTx-1, the main neurotoxic acting peptide in the venom of the spider Cupiennius salei, is composed of 74 amino acid residues, exhibits an inhibitory cysteine knot motif, and is further characterized by its highly cationic charged C terminus. Venom gland cDNA library analysis predicted a prepropeptide structure for CsTx-1 precursor. In the presence of trifluoroethanol, CsTx-1 and the long C-terminal part alone (CT1-long; Gly-45-Lys-74) exhibit an α-helical structure, as determined by CD measurements. CsTx-1 and CT1-long are insecticidal toward Drosophila flies and destroys Escherichia coli SBS 363 cells. CsTx-1 causes a stable and irreversible depolarization of insect larvae muscle cells and frog neuromuscular preparations, which seem to be receptor-independent. Furthermore, this membranolytic activity could be measured for Xenopus oocytes, in which CsTx-1 and CT1-long increase ion permeability non-specifically. These results support our assumption that the membranolytic activities of CsTx-1 are caused by its C-terminal tail, CT1-long. Together, CsTx-1 exhibits two different functions; as a neurotoxin it inhibits L-type Ca(2+) channels, and as a membranolytic peptide it destroys a variety of prokaryotic and eukaryotic cell membranes. Such a dualism is discussed as an important new mechanism for the evolution of spider venomous peptides.

Structural and biochemical characterization of native and recombinant single insulin-like growth factor-binding domain protein (SIBD-1) from the Central American hunting spider Cupiennius salei (Ctenidae).

Cupiennius salei single insulin-like growth factor binding domain protein (SIBD-1) is an 8.6 kDa Cys-, Pro-, and Gly-rich protein, discovered in the hemocytes of the Central American hunting spider Cupiennius salei. SIBD-1 exhibits high sequence similarity to the N-terminal domain of the insulin-like growth factor-binding protein superfamily and has been reported to play an important role in the spider's immune system. Here, the recombinant expression and the elucidation of the three-dimensional structure of recombinant SIBD-1 and the characterization of the sugar moiety at Thr2 of native SIBD-1 is described in detail.

Bridging data management platforms and visualization tools to enable ad-hoc and smart analytics in life sciences.

Core facilities have to offer technologies that best serve the needs of their users and provide them a competitive advantage in research. They have to set up and maintain instruments in the range of ten to a hundred, which produce large amounts of data and serve thousands of active projects and customers. Particular emphasis has to be given to the reproducibility of the results. More and more, the entire process from building the research hypothesis, conducting the experiments, doing the measurements, through the data explorations and analysis is solely driven by very few experts in various scientific fields. Still, the ability to perform the entire data exploration in real-time on a personal computer is often hampered by the heterogeneity of software, the data structure formats of the output, and the enormous data sizes. These impact the design and architecture of the implemented software stack. At the Functional Genomics Center Zurich (FGCZ), a joint state-of-the-art research and training facility of ETH Zurich and the University of Zurich, we have developed the B-Fabric system, which has served for more than a decade, an entire life sciences community with fundamental data science support. In this paper, we sketch how such a system can be used to glue together data (including metadata), computing infrastructures (clusters and clouds), and visualization software to support instant data exploration and visual analysis. We illustrate our in-daily life implemented approach using visualization applications of mass spectrometry data.

De novo expression of gastrokines in pancreatic precursor lesions impede the development of pancreatic cancer.

Molecular events occurring in stepwise progression from pre-malignant lesions (pancreatic intraepithelial neoplasia; PanIN) to the development of pancreatic ductal adenocarcinoma (PDAC) are poorly understood. Thus, characterization of early PanIN lesions may reveal markers that can help in diagnosing PDAC at an early stage and allow understanding the pathology of the disease. We performed the molecular and histological assessment of patient-derived PanINs, tumor tissues and pancreas from mouse models with PDAC (KC mice that harbor K-RAS mutation in pancreatic tissue), where we noted marked upregulation of gastrokine (GKN) proteins. To further understand the role of gastrokine proteins in PDAC development, GKN-deficient KC mice were developed by intercrossing gastrokine-deficient mice with KC mice. Panc-02 (pancreatic cancer cells of mouse origin) were genetically modified to express GKN1 for further in vitro and in vivo analysis. Our results show that gastrokine proteins were absent in healthy pancreas and invasive cancer, while its expression was prominent in low-grade PanINs. We could detect these proteins in pancreatic juice and serum of KC mice. Furthermore, accelerated PanIN and tumor development were noted in gastrokine deficient KC mice. Loss of gastrokine 1 protein delayed apoptosis during carcinogenesis leading to the development of desmoplastic stroma while loss of gastrokine 2 increased the proliferation rate in precursor lesions. In summary, we identified gastrokine proteins in early pancreatic precursor lesions, where gastrokine proteins delay pancreatic carcinogenesis.

Moderate toxic effects following acute zonisamide overdose.

Zonisamide is an antiepileptic drug that acts on voltage-sensitive sodium and calcium channels, with a modulatory effect on GABA-mediated neuronal inhibition and an inhibitory effect on carbonic anhydrase. It is used mainly for the treatment of partial seizures, and is generally well tolerated at therapeutic doses. The most common reported adverse effects are somnolence, anorexia, dizziness, and headache. There are limited data on zonisamide overdose in the literature, and no case of zonisamide mono-intoxication has been published to date. We describe the first case of zonisamide mono-intoxication in a 25-year-old woman who ingested 12.6 g of this substance with suicidal intent. Despite a plasma zonisamide concentration of 182 mg/L on admission, the patient exhibited a benign clinical course with vomiting and central nervous system depression, requiring brief intubation. Somnolence persisted for 50 hours, and normal-anion-gap metabolic acidosis and polyuria for several days. Complete recovery may be expected with supportive care, even after ingestion of large zonisamide overdoses.

Severe refeeding syndrome after human chorionic gonadotropin diet: a potentially lethal complication.

We present the case of a young male patient who presented with paralysing muscle weakness due to severe hypokalaemia and hypophosphataemia. The initial patient history evaluations could not establish the aetiology. Only after we reviewed the patient's history did he reveal that he had been following a severe calorie-restricted regime, the human chorionic gonadotropin diet, which had ended 2 days prior to developing symptoms. This information then allowed us to diagnose severe refeeding syndrome. As a further complication, the patient developed rhabdomyolysis. After correction of serum electrolytes, symptoms resolved completely. This case emphasises the potential harm of severely calorie-restricted diets, often recommended by online 'experts'. Furthermore, we underline the importance of thorough history taking.

Ancient proteins provide evidence of dairy consumption in eastern Africa.

Consuming the milk of other species is a unique adaptation of Homo sapiens, with implications for health, birth spacing and evolution. Key questions nonetheless remain regarding the origins of dairying and its relationship to the genetically-determined ability to drink milk into adulthood through lactase persistence (LP). As a major centre of LP diversity, Africa is of significant interest to the evolution of dairying. Here we report proteomic evidence for milk consumption in ancient Africa. Using liquid chromatography tandem mass spectrometry (LC-MS/MS) we identify dairy proteins in human dental calculus from northeastern Africa, directly demonstrating milk consumption at least six millennia ago. Our findings indicate that pastoralist groups were drinking milk as soon as herding spread into eastern Africa, at a time when the genetic adaptation for milk digestion was absent or rare. Our study links LP status in specific ancient individuals with direct evidence for their consumption of dairy products.

Dairy pastoralism sustained eastern Eurasian steppe populations for 5,000 years.

Dairy pastoralism is integral to contemporary and past lifeways on the eastern Eurasian steppe, facilitating survival in agriculturally challenging environments. While previous research has indicated that ruminant dairy pastoralism was practiced in the region by circa 1300 BC, the origin, extent and diversity of this custom remain poorly understood. Here, we analyse ancient proteins from human dental calculus recovered from geographically diverse locations across Mongolia and spanning 5,000 years. We present the earliest evidence for dairy consumption on the eastern Eurasian steppe by circa 3000 BC and the later emergence of horse milking at circa 1200 BC, concurrent with the first evidence for horse riding. We argue that ruminant dairying contributed to the demographic success of Bronze Age Mongolian populations and that the origins of traditional horse dairy products in eastern Eurasia are closely tied to the regional emergence of mounted herding societies during the late second millennium BC.

Elucidation of the disulfide bridge pattern of the recombinant human growth and differentiation factor 5 dimer and the interchain Cys/Ala mutant monomer.

Growth and differentiation factor 5 (GDF5) is involved in many developmental processes such as chondrogenesis and joint and bone formation. A recombinant monomeric human GDF5 mutant rGDF5(C84A) is in vitro as potent as the dimeric native form, and clinical investigations of rGDF5(C84A) are in progress. Native homodimeric GDF5 belongs to the transforming growth factor beta (TGF-beta) superfamily; each monomer contains a cystine knot formed by three intrachain disulfide bridges, and the monomers are connected via an interchain disulfide bridge. The disulfide bridge pattern of recombinant homodimeric rGDF5 was recently elucidated by X-ray diffraction. A combination of proteolytic degradation with thermolysin, separation of the generated fragments by reverse-phase high-performance liquid chromatography (RP-HPLC), and subsequent analyses of the disulfide-linked peptides by electrospray-mass spectrometry and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry, amino acid analysis, and Edman degradation led to the unambiguous identification of the disulfide bridge pattern of the monomeric mutant rGDF5(C84A) and of the homodimeric rGDF5 in solution. The cystine knot of homodimeric rGDF5 exhibits the pattern Cys1-Cys5, Cys2-Cys6, and Cys3-Cys7 (three intrachain disulfide bonds), and the monomers are connected by a single interchain disulfide bridge (Cys4-Cys4) in accordance with other members of the TGF-beta superfamily. The monomeric mutant rGDF5(C84A) exhibits the same cystine knot pattern as homodimeric rGDF5.