Extractability and chromatographic separation of proteins from potato (Solanum tuberosum L.) trimmings.

By-products from the potato processing industry, like potato trimmings, are sustainable sources of proteins. Here, a size-exclusion high performance liquid chromatography (SE-HPLC) method was applied to simultaneously determine the extractability and aggregation state of proteins from three batches of potato trimmings of different cultivars. Obtained SE-HPLC profiles allowed distinguishing between the patatin and protease inhibitor fractions of potato proteins. Moreover, only 75% of the crude proteins could be extracted in phosphate buffer containing sodium dodecyl sulfate and a reducing agent, indicating the presence of physical extraction barriers. Ball milling for 5 min significantly increased protein extractability, but prolonged treatment resulted in aggregation of native patatin and a reduced protein extractability. Microwave-dried trimmings had a lower protein extractability than freeze-dried trimmings. In future research, the SE-HPLC method can be used to examine changes in potato protein (fractions) as a result of processing.

Phenotyping, genetics, and "-omics" approaches to unravel and introgress enhanced resistance against apple scab (Venturia inaequalis) in apple cultivars (Malus × domestica).

Apple scab disease, caused by the fungus Venturia inaequalis, endangers commercial apple production globally. It is predominantly managed by frequent fungicide sprays that can harm the environment and promote the development of fungicide-resistant strains. Cultivation of scab-resistant cultivars harboring diverse qualitative Rvi resistance loci and quantitative trait loci associated with scab resistance could reduce the chemical footprint. A comprehensive understanding of the host-pathogen interaction is, however, needed to efficiently breed cultivars with enhanced resistance against a variety of pathogenic strains. Breeding efforts should not only encompass pyramiding of Rvi loci and their corresponding resistance alleles that directly or indirectly recognize pathogen effectors, but should also integrate genes that contribute to effective downstream defense mechanisms. This review provides an overview of the phenotypic and genetic aspects of apple scab resistance, and currently known corresponding defense mechanisms. Implementation of recent "-omics" approaches has provided insights into the complex network of physiological, molecular, and signaling processes that occur before and upon scab infection, thereby revealing the importance of both constitutive and induced defense mechanisms. Based on the current knowledge, we outline advances toward more efficient introgression of enhanced scab resistance into novel apple cultivars by conventional breeding or genetic modification techniques. However, additional studies integrating different "-omics" approaches combined with functional studies will be necessary to unravel effective defense mechanisms as well as key regulatory genes underpinning scab resistance in apple. This crucial information will set the stage for successful knowledge-based breeding for enhanced scab resistance.

Enhanced protein secretion in reduced genome strains of Streptomyces lividans.

BACKGROUND: S. lividans TK24 is a popular host for the production of small molecules and the secretion of heterologous protein. Within its large genome, twenty-nine non-essential clusters direct the biosynthesis of secondary metabolites. We had previously constructed ten chassis strains, carrying deletions in various combinations of specialized metabolites biosynthetic clusters, such as those of the blue actinorhodin (act), the calcium-dependent antibiotic (cda), the undecylprodigiosin (red), the coelimycin A (cpk) and the melanin (mel) clusters, as well as the genes hrdD, encoding a non-essential sigma factor, and matAB, a locus affecting mycelial aggregation. Genome reduction was aimed at reducing carbon flow toward specialized metabolite biosynthesis to optimize the production of secreted heterologous protein. RESULTS: Two of these S. lividans TK24 derived chassis strains showed ~ 15% reduction in biomass yield, 2-fold increase of their total native secretome mass yield and enhanced abundance of several secreted proteins compared to the parental strain. RNAseq and proteomic analysis of the secretome suggested that genome reduction led to cell wall and oxidative stresses and was accompanied by the up-regulation of secretory chaperones and of secDF, a Sec-pathway component. Interestingly, the amount of the secreted heterologous proteins mRFP and mTNFα, by one of these strains, was 12 and 70% higher, respectively, than that secreted by the parental strain. CONCLUSION: The current study described a strategy to construct chassis strains with enhanced secretory abilities and proposed a model linking the deletion of specialized metabolite biosynthetic clusters to improved production of secreted heterologous proteins.

Integrative multi-omics analyses of date palm (Phoenix dactylifera) roots and leaves reveal how the halophyte land plant copes with sea water.

Date palm (Phoenix dactylifera L.) is able to grow and complete its life cycle while being rooted in highly saline soils. Which of the many well-known salt-tolerance strategies are combined to fine-tune this remarkable resilience is unknown. The precise location, whether in the shoot or the root, where these strategies are employed remains uncertain, leaving us unaware of how the various known salt-tolerance mechanisms are integrated to fine-tune this remarkable resilience. To address this shortcoming, we exposed date palm to a salt stress dose equivalent to seawater for up to 4 weeks and applied integrative multi-omics analyses followed by targeted metabolomics, hormone, and ion analyses. Integration of proteomic into transcriptomic data allowed a view beyond simple correlation, revealing a remarkably high degree of convergence between gene expression and protein abundance. This sheds a clear light on the acclimatization mechanisms employed, which depend on reprogramming of protein biosynthesis. For growth in highly saline habitats, date palm effectively combines various salt-tolerance mechanisms found in both halophytes and glycophytes: "avoidance" by efficient sodium and chloride exclusion at the roots, and "acclimation" by osmotic adjustment, reactive oxygen species scavenging in leaves, and remodeling of the ribosome-associated proteome in salt-exposed root cells. Combined efficiently as in P. dactylifera L., these sets of mechanisms seem to explain the palm's excellent salt stress tolerance.

Proteomic and Low-Polar Metabolite Profiling Reveal Unique Dynamics in Fatty Acid Metabolism during Flower and Berry Development of Table Grapes.

Grapevine development and ripening are complex processes that involve several biochemical pathways, including fatty acid and lipid metabolism. Fatty acids are essential components of lipids, which play crucial roles in fruit maturation and flavor development. However, the dynamics of fatty acid metabolism in grape flowers and berries are poorly understood. In this study, we present those dynamics and investigate the mechanisms of fatty acid homeostasis on 'Thompson Seedless' berries using metabolomic and proteomic analyses. Low-polar metabolite profiling indicated a higher abundance of fatty acids at the pre-flowering and pre-veraison stages. Proteomic analyses revealed that grape flowers and berries display unique profiles of proteins involved in fatty acid biosynthesis, triacylglycerol assembly, fatty acid ß-oxidation, and lipid signaling. These findings show, for the first time, that fatty acid metabolism also plays an important role in the development of non-oil-rich tissues, opening new perspectives about lipid function and its relation to berry quality.

Unravelling the diversity in water usage among wild banana species in response to vapour pressure deficit.

The rise in global temperature is not only affecting plant functioning directly, but is also increasing air vapour pressure deficit (VPD). The yield of banana is heavily affected by water deficit but so far breeding programs have never addressed the issue of water deficit caused by high VPD. A reduction in transpiration at high VPD has been suggested as a key drought tolerance breeding trait to avoid excessive water loss, hydraulic failure and to increase water use efficiency. In this study, stomatal and transpiration responses under increasing VPD at the leaf and whole-plant level of 8 wild banana (sub)species were evaluated, displaying significant differences in stomatal reactivity. Three different phenotypic groups were identified under increasing VPD. While (sub)species of group III maintained high transpiration rates under increasing VPD, M. acuminata ssp. errans (group I), M. acuminata ssp. zebrina (group II) and M. balbisiana (group II) showed the highest transpiration rate limitations to increasing VPD. In contrast to group I, group II only showed strong reductions at high VPD levels, limiting the cost of reduced photosynthesis and strongly increasing their water use efficiency. M. acuminata ssp. zebrina and M. balbisiana thus show the most favourable responses. This study provides a basis for the identification of potential parent material in gene banks for breeding future-proof bananas that cope better with lack of water.

Peptides From the Variable Domain of Immunoglobulin G as Biomarkers in Chronic Inflammatory Demyelinating Polyradiculoneuropathy.

BACKGROUND AND OBJECTIVES: Chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) is a clinically heterogeneous immune-mediated disease. Diagnostic biomarkers for CIDP are currently lacking. Peptides derived from the variable domain of circulating immunoglobulin G (IgG) have earlier been shown to be shared among patients with the same immunologic disease. Because humoral immune factors are hypothesized to be involved in the pathogenesis of CIDP, we evaluated IgG variable domain-derived peptides as diagnostic biomarkers in CIDP (primary objective) and whether IgG-derived peptides could cluster objective clinical entities in CIDP (secondary objective). METHODS: IgG-derived peptides were determined in prospectively collected sera of patients with CIDP and neurologic controls by means of mass spectrometry. Peptides of interest were selected through statistical analysis in a discovery cohort followed by sequence determination and confirmation. Diagnostic performance was evaluated for individual selected peptides and for a multipeptide model incorporating selected peptides, followed by performance reassessment in a validation cohort. Clustering of patients with CIDP based on IgG-derived peptides was evaluated through unsupervised sparse principal component analysis followed by k-means clustering. RESULTS: Sixteen peptides originating from the IgG variable domain were selected as candidate biomarkers in a discovery cohort of 44 patients with CIDP and 29 neurologic controls. For all 16 peptides, univariate logistic regressions and ROC curve analysis demonstrated increasing peptide abundances to associate with increased odds for CIDP (area under the curves [AUCs] ranging from 64.6% to 79.6%). When including age and sex in the logistic regression models, this remained the case for 13/16 peptides. A model composed of 5/16 selected peptides showed strong discriminating performance between patients with CIDP and controls (AUC 91.5%; 95% CI 84.6%-98.4%; p < 0.001). In the validation cohort containing 45 patients and 43 controls, 2/16 peptides demonstrated increasing abundances to associate with increased odds for CIDP, while the five-peptide model demonstrated an AUC of 61.2% (95% CI 49.3%-73.2%; p = 0.064). Peptide-based patient clusters did not associate with clinical features. DISCUSSION: IgG variable domain-derived peptides showed a valid source for diagnostic biomarkers in CIDP, albeit with challenges toward replication. Our proof-of-concept findings warrant further study of IgG-derived peptides as biomarkers in more homogeneous cohorts of patients with CIDP and controls. CLASSIFICATION OF EVIDENCE: This study provides Class III evidence that the pattern of serum IgG-derived peptide clusters may help differentiate between patients with CIDP and those with other peripheral neuropathies.

Pharmacokinetics during therapeutic hypothermia in neonates: from pathophysiology to translational knowledge and physiologically-based pharmacokinetic (PBPK) modeling.

INTRODUCTION: Perinatal asphyxia (PA) still causes significant morbidity and mortality. Therapeutic hypothermia (TH) is the only effective therapy for neonates with moderate to severe hypoxic-ischemic encephalopathy after PA. These neonates need additional pharmacotherapy, and both PA and TH may impact physiology and, consequently, pharmacokinetics (PK) and pharmacodynamics (PD). AREAS COVERED: This review provides an overview of the available knowledge in PubMed (until November 2022) on the pathophysiology of neonates with PA/TH. In vivo pig models for this setting enable distinguishing the effect of PA versus TH on PK and translating this effect to human neonates. Available asphyxia pig models and methodological considerations are described. A summary of human neonatal PK of supportive pharmacotherapy to improve neurodevelopmental outcomes is provided. EXPERT OPINION: To support drug development for this population, knowledge from clinical observations (PK data, real-world data on physiology), preclinical (in vitro and in vivo (minipig)) data, and molecular and cellular biology insights can be integrated into a predictive physiologically-based PK (PBPK) framework, as illustrated by the I-PREDICT project (Innovative physiology-based pharmacokinetic model to predict drug exposure in neonates undergoing cooling therapy). Current knowledge, challenges, and expert opinion on the future directions of this research topic are provided.

Development of a neonatal Göttingen Minipig model for dose precision in perinatal asphyxia: technical opportunities, challenges, and potential further steps.

Animal models provide useful information on mechanisms in human disease conditions, but also on exploring (patho)physiological factors affecting pharmacokinetics, safety, and efficacy of drugs in development. Also, in pediatric patients, nonclinical data can be critical for better understanding the disease conditions and developing new drug therapies in this age category. For perinatal asphyxia (PA), a condition defined by oxygen deprivation in the perinatal period and possibly resulting in hypoxic ischemic encephalopathy (HIE) or even death, therapeutic hypothermia (TH) together with symptomatic drug therapy, is the standard approach to reduce death and permanent brain damage in these patients. The impact of the systemic hypoxia during PA and/or TH on drug disposition is largely unknown and an animal model can provide useful information on these covariates that cannot be assessed separately in patients. The conventional pig is proven to be a good translational model for PA, but pharmaceutical companies do not use it to develop new drug therapies. As the Göttingen Minipig is the commonly used pig strain in nonclinical drug development, the aim of this project was to develop this animal model for dose precision in PA. This experiment consisted of the instrumentation of 24 healthy male Göttingen Minipigs, within 24 h of partus, weighing approximately 600 g, to allow the mechanical ventilation and the multiple vascular catheters inserted for maintenance infusion, drug administration and blood sampling. After premedication and induction of anesthesia, an experimental protocol of hypoxia was performed, by decreasing the inspiratory oxygen fraction (FiO2) at 15%, using nitrogen gas. Blood gas analysis was used as an essential tool to evaluate oxygenation and to determine the duration of the systemic hypoxic insult to approximately 1 h. The human clinical situation was mimicked for the first 24 h after birth in case of PA, by administering four compounds (midazolam, phenobarbital, topiramate and fentanyl), frequently used in a neonatal intensive care unit (NICU). This project aimed to develop the first neonatal Göttingen Minipig model for dose precision in PA, allowing to separately study the effect of systemic hypoxia versus TH on drug disposition. Furthermore, this study showed that several techniques that were thought to be challenging or even impossible in these very small animals, such as endotracheal intubation and catheterization of several veins, are feasible by trained personnel. This is relevant information for laboratories using the neonatal Göttingen Minipig for other disease conditions or drug safety testing.

Complementarity determining regions in SARS-CoV-2 hybrid immunity.

Pre-vaccination SARS-CoV-2 infection can boost protection elicited by COVID-19 vaccination and post-vaccination breakthrough SARS-CoV-2 infection can boost existing immunity conferred by COVID-19 vaccination. Such 'hybrid immunity' is effective against SARS-CoV-2 variants. In order to understand 'hybrid immunity' at the molecular level we studied the complementarity determining regions (CDR) of anti-RBD (receptor binding domain) antibodies isolated from individuals with 'hybrid immunity' as well as from 'naive' (not SARS-CoV-2 infected) vaccinated individuals. CDR analysis was done by liquid chromatography/mass spectrometry-mass spectrometry. Principal component analysis and partial least square differential analysis showed that COVID-19 vaccinated people share CDR profiles and that pre-vaccination SARS-CoV-2 infection or breakthrough infection further shape the CDR profile, with a CDR profile in hybrid immunity that clustered away from the CDR profile in vaccinated people without infection. Thus, our results show a CDR profile in hybrid immunity that is distinct from the vaccination-induced CDR profile.

Antinuclear antibodies in individuals with COVID-19 reflect underlying disease: Identification of new autoantibodies in systemic sclerosis (CDK9) and malignancy (RNF20, RCC1, TRIP13).

A high prevalence of antinuclear antibodies (ANA) in COVID-19 has been insinuated, but the nature of the target antigens is poorly understood. We studied ANA by indirect immunofluorescence in 229 individuals with COVID-19. The target antigens of high titer ANA (≥1:320) were determined by immunoprecipitation (IP) combined with liquid-chromatography-mass spectrometry (MS). High titer ANA (≥1:320) were found in 14 (6%) of the individuals with COVID-19. Of the 14 COVID-19 cases with high titer ANA, 6 had an underlying autoimmune disease and 5 a malignancy. IP-MS revealed known target antigens associated with autoimmune disease as well as novel autoantigens, including CDK9 (in systemic sclerosis) and RNF20, RCC1 and TRIP13 (in malignancy). The novel autoantigens were confirmed by IP-Western blotting. In conclusion, in depth analysis of the targets of high titer ANA revealed novel autoantigens in systemic sclerosis and in malignant disease.

Mitochondria metabolism sets the species-specific tempo of neuronal development.

Neuronal development in the human cerebral cortex is considerably prolonged compared with that of other mammals. We explored whether mitochondria influence the species-specific timing of cortical neuron maturation. By comparing human and mouse cortical neuronal maturation at high temporal and cell resolution, we found a slower mitochondria development in human cortical neurons compared with that in the mouse, together with lower mitochondria metabolic activity, particularly that of oxidative phosphorylation. Stimulation of mitochondria metabolism in human neurons resulted in accelerated development in vitro and in vivo, leading to maturation of cells weeks ahead of time, whereas its inhibition in mouse neurons led to decreased rates of maturation. Mitochondria are thus important regulators of the pace of neuronal development underlying human-specific brain neoteny.

Proteomic and metabolomic integration reveals the effects of pre-flowering cytokinin applications on central carbon metabolism in table grape berries.

Consumers around the world prefer high quality table grapes. To achieve higher quality traits at ripening, grapevine producers apply different plant growth regulators. The synthetic cytokinin forchlorfenuron N-(2-chloro-4-pyridinyl)-N'-phenylurea (CPPU) is widely used, its effect on grape quality is poorly understood. We hypothesized that the use of CPPU in pre-flowering can lead to changes in the metabolism that affects grape quality at harvest. Therefore, we investigated the role of CPPU applications on the quality of grapes by integrating proteomics and metabolomics. CPPU-treated grapevines showed a significant increase in berry size and firmness. Proteomic analyses indicated that CPPU-treated berries accumulated enzymes associated with carbohydrate metabolism, glycolysis, and tricarboxylic acid (TCA) cycle at harvest. Metabolomic analyses showed shifts in the abundance of compounds associated with carbohydrate metabolism and TCA cycle in CPPU-treated grapes. These findings suggest that CPPU applications modulate central carbon metabolism, improving grape berry quality.

A multiomics integrative analysis of color de-synchronization with softening of 'Hass' avocado fruit: A first insight into a complex physiological disorder.

Exocarp color de-synchronization with softening of 'Hass' avocado is a relevant recurrent problem for the avocado supply chain. This study aimed to unravel the mechanisms driving this de-synchronization integrating omics datasets from avocado exocarp of different storage conditions and color phenotypes. In addition, we propose potential biomarkers to predict color synchronized/de-synchronized fruit. Integration of transcriptomics, proteomics and metabolomics and network analysis revealed eight transcription factors associated with differentially regulated genes between regular air (RA) and controlled atmosphere (CA) and twelve transcription factors related to avocado fruit color de-synchronization control in ready-to-eat stage. CA was positively correlated to auxins, ethylene, cytokinins and brassinosteroids-related genes, while RA was characterized by enrichment of cell wall remodeling and abscisic acid content associated genes. At ready-to-eat higher contents of flavonoids, abscisic acid and brassinosteroids were associated with color-softening synchronized avocados. In contrast, de-synchronized fruit revealed increases of jasmonic acid, salicylic acid and auxin levels.

Hybridization, missing wild ancestors and the domestication of cultivated diploid bananas.

Hybridization and introgressions are important evolutionary forces in plants. They contribute to the domestication of many species, including understudied clonal crops. Here, we examine their role in the domestication of a clonal crop of outmost importance, banana (Musa ssp.). We used genome-wide SNPs generated for 154 diploid banana cultivars and 68 samples of the wild M. acuminata to estimate and geo-localize the contribution of the different subspecies of M. acuminata to cultivated banana. We further investigated the wild to domesticate transition in New Guinea, an important domestication center. We found high levels of admixture in many cultivars and confirmed the existence of unknown wild ancestors with unequal contributions to cultivated diploid. In New Guinea, cultivated accessions exhibited higher diversity than their direct wild ancestor, the latter recovering from a bottleneck. Introgressions, balancing selection and positive selection were identified as important mechanisms for banana domestication. Our results shed new lights on the radiation of M. acuminata subspecies and on how they shaped banana domestication. They point candidate regions of origin for two unknown ancestors and suggest another contributor in New Guinea. This work feed research on the evolution of clonal crops and has direct implications for conservation, collection, and breeding.

Investigating the Sequence Determinants of the Curling of Amyloid Fibrils Using Ovalbumin as a Case Study.

Highly ordered, straight amyloid fibrils readily lend themselves to structure determination techniques and have therefore been extensively characterized. However, the less ordered curly fibrils remain relatively understudied, and the structural organization underlying their specific characteristics remains poorly understood. We found that the exemplary curly fibril-forming protein ovalbumin contains multiple aggregation prone regions (APRs) that form straight fibrils when isolated as peptides or when excised from the full-length protein through hydrolysis. In the context of the intact full-length protein, however, the regions separating the APRs facilitate curly fibril formation. In fact, a meta-analysis of previously reported curly fibril-forming proteins shows that their inter-APRs are significantly longer and more hydrophobic when compared to straight fibril-forming proteins, suggesting that they may cause strain in the amyloid state. Hence, inter-APRs driving curly fibril formation may not only apply to our model protein but rather constitute a more general mechanism.

From fruit growth to ripening in plantain: a careful balance between carbohydrate synthesis and breakdown.

In this study, we aimed to investigate for the first time different fruit development stages in plantain banana in order gain insights into the order of appearance and dominance of specific enzymes and fluxes. We examined fruit development in two plantain banana cultivars during the period between 2-12 weeks after bunch emergence using high-throughput proteomics, quantification of major metabolites, and analyses of metabolic fluxes. Starch synthesis and breakdown are processes that take place simultaneously. During the first 10 weeks fruits accumulated up to 48% of their dry weight as starch, and glucose 6-phosphate and fructose were important precursors. We found a unique amyloplast transporter and hypothesize that it facilitates the import of fructose. We identified an invertase originating from the Musa balbisiana genome that would enable carbon flow back to growth and starch synthesis and maintain a high starch content even during ripening. Enzymes associated with the initiation of ripening were involved in ethylene and auxin metabolism, starch breakdown, pulp softening, and ascorbate biosynthesis. The initiation of ripening was cultivar specific, with faster initiation being particularly linked to the 1-aminocyclopropane-1-carboxylate oxidase and 4-alpha glucanotransferase disproportionating enzymes. Information of this kind is fundamental to determining the optimal time for picking the fruit in order to reduce post-harvest losses, and has potential applications for breeding to improve fruit quality.

High-throughput phenotyping reveals differential transpiration behaviour within the banana wild relatives highlighting diversity in drought tolerance.

Crop wild relatives, the closely related species of crops, may harbour potentially important sources of new allelic diversity for (a)biotic tolerance or resistance. However, to date, wild diversity is only poorly characterized and evaluated. Banana has a large wild diversity but only a narrow proportion is currently used in breeding programmes. The main objective of this study was to evaluate genotype-dependent transpiration responses in relation to the environment. By applying continuous high-throughput phenotyping, we were able to construct genotype-specific transpiration response models in relation to light, VPD and soil water potential. We characterized and evaluated six (sub)species and discerned four phenotypic clusters. Significant differences were observed in leaf area, cumulative transpiration and transpiration efficiency. We confirmed a general stomatal-driven 'isohydric' drought avoidance behaviour, but discovered genotypic differences in the onset and intensity of stomatal closure. We pinpointed crucial genotype-specific soil water potentials when drought avoidance mechanisms were initiated and when stress kicked in. Differences between (sub)species were dependent on environmental conditions, illustrating the need for high-throughput dynamic phenotyping, modelling and validation. We conclude that the banana wild relatives contain useful drought tolerance traits, emphasising the importance of their conservation and potential for use in breeding programmes.

A digital sensor to measure real-time leaf movements and detect abiotic stress in plants.

Plant and plant organ movements are the result of a complex integration of endogenous growth and developmental responses, partially controlled by the circadian clock, and external environmental cues. Monitoring of plant motion is typically done by image-based phenotyping techniques with the aid of computer vision algorithms. Here we present a method to measure leaf movements using a digital inertial measurement unit (IMU) sensor. The lightweight sensor is easily attachable to a leaf or plant organ and records angular traits in real-time for two dimensions (pitch and roll) with high resolution (measured sensor oscillations of 0.36 ± 0.53° for pitch and 0.50 ± 0.65° for roll). We were able to record simple movements such as petiole bending, as well as complex lamina motions, in several crops, ranging from tomato to banana. We also assessed growth responses in terms of lettuce rosette expansion and maize seedling stem movements. The IMU sensors are capable of detecting small changes of nutations (i.e. bending movements) in leaves of different ages and in different plant species. In addition, the sensor system can also monitor stress-induced leaf movements. We observed that unfavorable environmental conditions evoke certain leaf movements, such as drastic epinastic responses, as well as subtle fading of the amplitude of nutations. In summary, the presented digital sensor system enables continuous detection of a variety of leaf motions with high precision, and is a low-cost tool in the field of plant phenotyping, with potential applications in early stress detection.

Odorant-binding proteins in canine anal sac glands indicate an evolutionarily conserved role in mammalian chemical communication.

BACKGROUND: Chemical communication is an important aspect of the behavioural ecology of a wide range of mammals. In dogs and other carnivores, anal sac glands are thought to convey information to conspecifics by secreting a pallet of small volatile molecules produced by symbiotic bacteria. Because these glands are unique to carnivores, it is unclear how their secretions relate to those of other placental mammals that make use of different tissues and secretions for chemical communication. Here we analyse the anal sac glands of domestic dogs to verify the secretion of proteins and infer their evolutionary relationship to those involved in the chemical communication of non-carnivoran mammals. RESULTS: Proteomic analysis of anal sac gland secretions of 17 dogs revealed the consistently abundant presence of three related proteins. Homology searches against online databases indicate that these proteins are evolutionary related to 'odorant binding proteins' (OBPs) found in a wide range of mammalian secretions and known to contribute to chemical communication. Screening of the dog's genome sequence show that the newly discovered OBPs are encoded by a single cluster of three genes in the pseudoautosomal region of the X-chromosome. Comparative genomic screening indicates that the same locus is shared by a wide range of placental mammals and that it originated at least before the radiation of extant placental orders. Phylogenetic analyses suggest a dynamic evolution of gene duplication and loss, resulting in large gene clusters in some placental taxa and recurrent loss of this locus in others. The homology of OBPs in canid anal sac glands and those found in other mammalian secretions implies that these proteins maintained a function in chemical communication throughout mammalian evolutionary history by multiple shifts in expression between secretory tissues involved in signal release and nasal mucosa involved in signal reception. CONCLUSIONS: Our study elucidates a poorly understood part of the biology of a species that lives in close association with humans. In addition, it shows that the protein repertoire underlying chemical communication in mammals is more evolutionarily stable than the variation of involved glands and tissues would suggest.