Evolutionary biology through the lens of budding yeast comparative genomics.

The budding yeast Saccharomyces cerevisiae is a highly advanced model system for studying genetics, cell biology and systems biology. Over the past decade, the application of high-throughput sequencing technologies to this species has contributed to this yeast also becoming an important model for evolutionary genomics. Indeed, comparative genomic analyses of laboratory, wild and domesticated yeast populations are providing unprecedented detail about many of the processes that govern evolution, including long-term processes, such as reproductive isolation and speciation, and short-term processes, such as adaptation to natural and domestication-related environments.

Systematic identification of signal integration by protein kinase A.

Cellular processes and homeostasis control in eukaryotic cells is achieved by the action of regulatory proteins such as protein kinase A (PKA). Although the outbound signals from PKA directed to processes such as metabolism, growth, and aging have been well charted, what regulates this conserved regulator remains to be systematically identified to understand how it coordinates biological processes. Using a yeast PKA reporter assay, we identified genes that influence PKA activity by measuring protein-protein interactions between the regulatory and the two catalytic subunits of the PKA complex in 3,726 yeast genetic-deletion backgrounds grown on two carbon sources. Overall, nearly 500 genes were found to be connected directly or indirectly to PKA regulation, including 80 core regulators, denoting a wide diversity of signals regulating PKA, within and beyond the described upstream linear pathways. PKA regulators span multiple processes, including the antagonistic autophagy and methionine biosynthesis pathways. Our results converge toward mechanisms of PKA posttranslational regulation by lysine acetylation, which is conserved between yeast and humans and that, we show, regulates protein complex formation in mammals and carbohydrate storage and aging in yeast. Taken together, these results show that the extent of PKA input matches with its output, because this kinase receives information from upstream and downstream processes, and highlight how biological processes are interconnected and coordinated by PKA.

Evolutionary rescue by compensatory mutations is constrained by genomic and environmental backgrounds.

Since deleterious mutations may be rescued by secondary mutations during evolution, compensatory evolution could identify genetic solutions leading to therapeutic targets. Here, we tested this hypothesis and examined whether these solutions would be universal or would need to be adapted to one's genetic and environmental make-ups. We performed experimental evolutionary rescue in a yeast disease model for the Wiskott-Aldrich syndrome in two genetic backgrounds and carbon sources. We found that multiple aspects of the evolutionary rescue outcome depend on the genotype, the environment, or a combination thereof. Specifically, the compensatory mutation rate and type, the molecular rescue mechanism, the genetic target, and the associated fitness cost varied across contexts. The course of compensatory evolution is therefore highly contingent on the initial conditions in which the deleterious mutation occurs. In addition, these results reveal biologically favored therapeutic targets for the Wiskott-Aldrich syndrome, including the target of an unrelated clinically approved drug. Our results experimentally illustrate the importance of epistasis and environmental evolutionary constraints that shape the adaptive landscape and evolutionary rate of molecular networks.

Gene coexpression networks reveal key drivers of phenotypic divergence in lake whitefish.

A functional understanding of processes involved in adaptive divergence is one of the awaiting opportunities afforded by high-throughput transcriptomic technologies. Functional analysis of coexpressed genes has succeeded in the biomedical field in identifying key drivers of disease pathways. However, in ecology and evolutionary biology, functional interpretation of transcriptomic data is still limited. Here, we used Weighted Gene Co-Expression Network Analysis (WGCNA) to identify modules of coexpressed genes in muscle and brain tissue of a lake whitefish backcross progeny. Modules were connected to gradients of known adaptive traits involved in the ecological speciation process between benthic and limnetic ecotypes. Key drivers, that is, hub genes of functional modules related to reproduction, growth, and behavior were identified, and module preservation was assessed in natural populations. Using this approach, we identified modules of coexpressed genes involved in phenotypic divergence and their key drivers, and further identified a module part specifically rewired in the backcross progeny. Functional analysis of transcriptomic data can significantly contribute to the understanding of the mechanisms underlying ecological speciation. Our findings point to bone morphogenetic protein and calcium signaling as common pathways involved in coordinated evolution of trophic behavior, trophic morphology (gill rakers), and reproduction. Results also point to pathways implicating hemoglobins and constitutive stress response (HSP70) governing growth in lake whitefish.

High-throughput characterization of the effect of sodium chloride and potassium chloride on 31 lactic acid bacteria and their co-cultures.

Salt (NaCl) is associated with a risk of hypertension and the development of coronary heart disease, so its consumption should be limited. However, salt plays a key role in the quality and safety of food by controlling undesirable microorganisms. Since studies have focused primarily on the effect of salts on the overall counts of the lactic acid bacteria (LAB) group, we have not yet understood how salt stress individually affects the strains and the interactions between them. In this study, we characterized the effect of sodium chloride (NaCl) and potassium chloride (KCl) on the growth and acidification of 31 LAB strains. In addition, we evaluated the effect of salts on a total of 93 random pairwise strain combinations. Strains and co-cultures were tested at 3% NaCl, 5% NaCl, and 3% KCl on solid medium using an automated approach and image analysis. The results showed that the growth of LAB was significantly reduced by up to 68% at 5% NaCl (p < 0.0001). For the co-cultures, a reduction of up to 57% was observed at 5% NaCl (p < 0.0001). However, acidification was less affected by salt stress, whether for monocultures or co-cultures. Furthermore, KCl had a lesser impact on both growth and acidification compared to NaCl. Indeed, some strains showed a significant increase in growth at 3% KCl, such as Lactococcus lactis subsp. lactis 74310 (23%, p = 0.01). More importantly, co-cultures appeared to be more resilient and had more varied responses to salt stress than the monocultures, as several cases of suppression of the significant effect of salts on acidification and growth were detected. Our results highlight that while salts can modulate microbial interactions, these latter can also attenuate the effect of salts on LAB.

Nonparallelism in MHCIIß diversity accompanies nonparallelism in pathogen infection of lake whitefish (Coregonus clupeaformis) species pairs as revealed by next-generation sequencing.

Major histocompatibility (MHC) immune system genes may evolve in response to pathogens in the environment. Because they also may affect mate choice, they are candidates for having great importance in ecological speciation. Here, we use next-generation sequencing to test the general hypothesis of parallelism in patterns of MHCIIß diversity and bacterial infections among five dwarf and normal whitefish sympatric pairs. A second objective was to assess the functional relationships between specific MHCIIß alleles and pathogens in natural conditions. Each individual had between one and four alleles, indicating two paralogous loci. In Cliff Lake, the dwarf ecotype was monomorphic for the most common allele. In Webster Lake, the skew in the allelic distribution was towards the same allele but in the normal ecotype, underscoring the nonparallel divergence among lakes. Our signal of balancing selection matched putative peptide binding region residues in some cases, but not in others, supporting other recent findings of substantial functional differences in fish MHCIIß compared with mammals. Individuals with fewer alleles were less likely to be infected; thus, we found no evidence for the heterozygote advantage hypothesis. MHCIIß alleles and pathogenic bacteria formed distinct clusters in multivariate analyses, and clusters of certain alleles were associated with clusters of pathogens, or sometimes the absence of pathogens, indicating functional relationships at the individual level. Given that patterns of MHCIIß and bacteria were nonparallel among dwarf and normal whitefish pairs, we conclude that pathogens driving MHCIIß evolution did not play a direct role in their parallel phenotypic evolution.

The Physicochemical Characterization and In Vitro Digestibility of Maple Sugar Sand and Downgraded Maple Syrups.

The maple syrup industry generates substandard syrups and sugar sand as by-products, which are underused. In this study, we conducted a comprehensive analysis of the physicochemical composition of these products to assess their potential for valorization. Using HPLC analysis, we measured sugar and organic acid content as well as total polyphenol content using the Folin-Ciocalteu method. Additionally, we evaluated the in vitro digestibility using the TIM-1 model. We showed that the composition of ropy and buddy downgraded syrups is comparable to that of standard maple syrup, whereas sugar sand's composition is highly variable, with carbohydrate content ranging from 5.01 mg/g to 652.89 mg/g and polyphenol content ranging from 11.30 µg/g to 120.95 µg/g. In vitro bioaccessibility reached 70% of total sugars for all by-products. Organic acid bioaccessibility from sugar sand and syrup reached 76% and 109% relative to standard maple syrup, respectively. Polyphenol bioaccessibility exceeded 100% during digestion. This can be attributed to favorable extraction conditions, the breakdown of complex polyphenol forms and the food matrix. In conclusion, our study demonstrates that sugar sand and downgraded maple syrups exhibit digestibility comparable to that of standard maple syrup. Consequently, they hold potential as a source of polyphenols, sugar or organic acids for applications such as industrial fermentation or livestock feeds.

Correlation of maple sap composition with bacterial and fungal communities determined by multiplex automated ribosomal intergenic spacer analysis (MARISA).

During collection, maple sap is contaminated by bacteria and fungi that subsequently colonize the tubing system. The bacterial microbiota has been more characterized than the fungal microbiota, but the impact of both components on maple sap quality remains unclear. This study focused on identifying bacterial and fungal members of maple sap and correlating microbiota composition with maple sap properties. A multiplex automated ribosomal intergenic spacer analysis (MARISA) method was developed to presumptively identify bacterial and fungal members of maple sap samples collected from 19 production sites during the tapping period. Results indicate that the fungal community of maple sap is mainly composed of yeast related to Mrakia sp., Mrakiella sp., Guehomyces pullulans, Cryptococcus victoriae and Williopsis saturnus. Mrakia, Mrakiella and Guehomyces peaks were identified in samples of all production sites and can be considered dominant and stable members of the fungal microbiota of maple sap. A multivariate analysis based on MARISA profiles and maple sap chemical composition data showed correlations between Candida sake, Janthinobacterium lividum, Williopsis sp., Leuconostoc mesenteroides, Mrakia sp., Rhodococcus sp., Pseudomonas tolaasii, G. pullulans and maple sap composition at different flow periods. This study provides new insights on the relationship between microbial community and maple sap quality.

Leveraging Experimental Strategies to Capture Different Dimensions of Microbial Interactions.

Microorganisms are a fundamental part of virtually every ecosystem on earth. Understanding how collectively they interact, assemble, and function as communities has become a prevalent topic both in fundamental and applied research. Owing to multiple advances in technology, answering questions at the microbial system or network level is now within our grasp. To map and characterize microbial interaction networks, numerous computational approaches have been developed; however, experimentally validating microbial interactions is no trivial task. Microbial interactions are context-dependent, and their complex nature can result in an array of outcomes, not only in terms of fitness or growth, but also in other relevant functions and phenotypes. Thus, approaches to experimentally capture microbial interactions involve a combination of culture methods and phenotypic or functional characterization methods. Here, through our perspective of food microbiologists, we highlight the breadth of innovative and promising experimental strategies for their potential to capture the different dimensions of microbial interactions and their high-throughput application to answer the question; are microbial interaction patterns or network architecture similar along different contextual scales? We further discuss the experimental approaches used to build various types of networks and study their architecture in the context of cell biology and how they translate at the level of microbial ecosystem.

A continuously changing selective context on microbial communities associated with fish, from egg to fork.

Fast increase of fish aquaculture production to meet consumer demands is accompanied by important ecological concerns such as disease outbreaks. Meanwhile, food waste is an important concern with fish products since they are highly perishable. Recent aquaculture and fish product microbiology, and more recently, microbiota research, paved the way to a highly integrated approach to understand complex relationships between host fish, product and their associated microbial communities at health/disease and preservation/spoilage frontiers. Microbial manipulation strategies are increasingly validated as promising tools either to replace or to complement traditional veterinary and preservation methods. In this review, we consider evolutionary forces driving fish microbiota assembly, in particular the changes in the selective context along the production chain. We summarize the current knowledge concerning factors governing assembly and dynamics of fish hosts and food microbial communities. Then, we discuss the current microbial community manipulation strategies from an evolutionary standpoint to provide a perspective on the potential for risks, conflict and opportunities. Finally, we conclude that to harness evolutionary forces in the development of sustainable microbiota manipulation applications in the fish industry, an integrated knowledge of the controlling abiotic and especially biotic factors is required.

Competition experiments in a soil microcosm reveal the impact of genetic and biotic factors on natural yeast populations.

The ability to measure microbial fitness directly in natural conditions and in interaction with other microbes is a challenge that needs to be overcome if we want to gain a better understanding of microbial fitness determinants in nature. Here we investigate the influence of the natural microbial community on the relative fitness of the North American populations SpB, SpC and SpC* of the wild yeast Saccharomyces paradoxus using DNA barcodes and a soil microcosm derived from soil associated with oak trees. We find that variation in fitness among these genetically distinct groups is influenced by the microbial community. Altering the microbial community load and diversity with an irradiation treatment significantly diminishes the magnitude of fitness differences among populations. Our findings suggest that microbial interactions could affect the evolution of yeast lineages in nature by modulating variation in fitness.

A systems biology approach to explore the impact of maple tree dormancy release on sap variation and maple syrup quality.

Maple sap is a complex nutrient matrix collected during spring to produce maple syrup. The characteristics of sap change over the production period and its composition directly impacts syrup quality. This variability could in part be attributed to changes in tree metabolism following dormancy release, but little is known about these changes in deciduous trees. Therefore, understanding the variation in sap composition associated with dormancy release could help pinpoint the causes of some defects in maple syrup. In particular, a defect known as "buddy", is an increasing concern for the industry. This off-flavor appears around the time of bud break, hence its name. To investigate sap variation related to bud break and the buddy defect, we monitored sap variation with respect to a dormancy release index (Sbb) and syrup quality. First, we looked at variation in amino acid content during this period. We observed a shift in amino acid relative proportions associated with dormancy release and found that most of them increase rapidly near the point of bud break, correlating with changes in syrup quality. Second, we identified biological processes that respond to variation in maple sap by performing a competition assay using the barcoded Saccharomyces cerevisiae prototroph deletion collection. This untargeted approach revealed that the organic sulfur content may be responsible for the development of the buddy off-flavor, and that dormancy release is necessary for the appearance of the defect, but other factors such as microbial activity may also be contributing.

Identification of the fitness determinants of budding yeast on a natural substrate.

The budding yeasts are prime models in genomics and cell biology, but the ecological factors that determine their success in non-human-associated habitats is poorly understood. In North America Saccharomyces yeasts are present on the bark of deciduous trees, where they feed on bark and sap exudates. In the North East, Saccharomyces paradoxus is found on maples, which makes maple sap a natural substrate for this species. We measured growth rates of S. paradoxus natural isolates on maple sap and found variation along a geographical gradient not explained by the inherent variation observed under optimal laboratory conditions. We used a functional genomic screen to reveal the ecologically relevant genes and conditions required for optimal growth in this substrate. We found that the allantoin degradation pathway is required for optimal growth in maple sap, in particular genes necessary for allantoate utilization, which we demonstrate is the major nitrogen source available to yeast in this environment. Growth with allantoin or allantoate as the sole nitrogen source recapitulated the variation in growth rates in maple sap among strains. We also show that two lineages of S. paradoxus display different life-history traits on allantoin and allantoate media, highlighting the ecological relevance of this pathway.

Multi-scale perturbations of protein interactomes reveal their mechanisms of regulation, robustness and insights into genotype-phenotype maps.

Cellular architectures and signaling machineries are organized through protein-protein interactions (PPIs). High-throughput methods to study PPIs in yeast have opened a new perspective on the organization of the cell by allowing the study of whole protein interactomes. Recent investigations have moved from the description of this organization to the analysis of its dynamics by experimenting how protein interaction networks (PINs) are rewired in response to perturbations. Here we review studies that have used the budding yeast as an experimental system to explore these altered networks. Given the large space of possible PPIs and the diversity of potential genetic and environmental perturbations, high-throughput methods are an essential requirement to survey PIN perturbations on a large scale. Network perturbations are typically conceptualized as the removal of entire proteins (nodes), the modification of single PPIs (edges) or changes in growth conditions. These studies have revealed mechanisms of PPI regulation, PIN architectural organization, robustness and sensitivity to perturbations. Despite these major advances, there are still inherent limits to current technologies that lead to a trade-off between the number of perturbations and the number of PPIs that can be considered simultaneously. Nevertheless, as we exemplify here, targeted approaches combined with the existing resources remain extremely powerful to explore the inner organization of cells and their responses to perturbations.

Measuring the severity of depression and remission in primary care: validation of the HAMD-7 scale.

BACKGROUND: Symptomatic remission is the optimal outcome in depression. A brief, validated tool for symptom measurement that can indicate when remission has occurred in mental health and primary care settings is unavailable. We evaluated a 7-item abbreviated version (HAMD-7) of the 17-item Hamilton Depression Rating Scale (HAMD-17) in a randomized controlled clinical trial of patients with major depressive disorder being cared for in primary care settings. METHODS: We enrolled 454 patients across 47 primary care settings who met DSM-IV-TR criteria for a major depressive disorder. Of these, 410 patients requiring antidepressant medication were randomized to have their symptoms rated with either HAMD-7 (n = 205) or HAMD-17 (n = 205) as the primary measurement tool. The primary outcome was the proportion of patients who achieved a-priori defined responses to 8 weeks of therapy using each instrument. RESULTS: Of the 205 participants per group, 67% of those evaluated with HAMD-7 were classified as having responded to therapy (defined as a > or = 50% reduction from the pretreatment score), compared with 74% of those evaluated with HAMD-17 (p = 0.43). The difference between the groups' changes in scores from baseline (pretreatment) to endpoint was significant (p < 0.001), without a main effect of group (p = 0.84) or group-by-time (p = 0.83) interaction. The HAMD-7 test was brief to administer (e.g., 3-4 min for 85% of the primary care physicians evaluated), which facilitated the efficient and structured evaluation of salient depressive symptoms. INTERPRETATION: The abbreviated HAMD-7 depression scale is equivalent to the HAMD-17 in assessing remission in patients with a major depressive disorder undergoing drug therapy.

Feedback regulation between autophagy and PKA.

Protein kinase A (PKA) controls diverse cellular processes and homeostasis in eukaryotic cells. Many processes and substrates of PKA have been described and among them are direct regulators of autophagy. The mechanisms of PKA regulation and how they relate to autophagy remain to be fully understood. We constructed a reporter of PKA activity in yeast to identify genes affecting PKA regulation. The assay systematically measures relative protein-protein interactions between the regulatory and catalytic subunits of the PKA complex in a systematic set of genetic backgrounds. The candidate PKA regulators we identified span multiple processes and molecular functions (autophagy, methionine biosynthesis, TORC signaling, protein acetylation, and DNA repair), which themselves include processes regulated by PKA. These observations suggest the presence of many feedback loops acting through this key regulator. Many of the candidate regulators include genes involved in autophagy, suggesting that not only does PKA regulate autophagy but that autophagy also sends signals back to PKA.

Genome-wide protein-protein interaction screening by protein-fragment complementation assay (PCA) in living cells.

Proteins are the building blocks, effectors and signal mediators of cellular processes. A protein's function, regulation and localization often depend on its interactions with other proteins. Here, we describe a protocol for the yeast protein-fragment complementation assay (PCA), a powerful method to detect direct and proximal associations between proteins in living cells. The interaction between two proteins, each fused to a dihydrofolate reductase (DHFR) protein fragment, translates into growth of yeast strains in presence of the drug methotrexate (MTX). Differential fitness, resulting from different amounts of reconstituted DHFR enzyme, can be quantified on high-density colony arrays, allowing to differentiate interacting from non-interacting bait-prey pairs. The high-throughput protocol presented here is performed using a robotic platform that parallelizes mating of bait and prey strains carrying complementary DHFR-fragment fusion proteins and the survival assay on MTX. This protocol allows to systematically test for thousands of protein-protein interactions (PPIs) involving bait proteins of interest and offers several advantages over other PPI detection assays, including the study of proteins expressed from their endogenous promoters without the need for modifying protein localization and for the assembly of complex reporter constructs.

Treatment-resistant depression: definitions, review of the evidence, and algorithmic approach.

BACKGROUND: Most adults with major depressive disorder (MDD) fail to achieve remission with index pharmacological treatment. Moreover, at least half will not achieve and sustain remission following multiple pharmacological approaches. Herein, we succinctly review treatment modalities proven effective in treatment-resistant depression (TRD). METHODS: We conducted a review of computerized databases (PubMed, Google Scholar) from 1980 to April 2013. Articles selected for review were based on author consensus, adequacy of sample size, the use of a standardized experimental procedure, validated assessment measures and overall manuscript quality. RESULTS: The evidence base supporting augmentation of conventional antidepressants with atypical antipsychotics (i.e., aripiprazole, quetiapine, and olanzapine) is the most extensive and rigorous of all pharmacological approaches in TRD. Emerging evidence supports the use of some psychostimulants (i.e., lisdexamfetamine) as well as aerobic exercise. In addition, treatments informed by pathogenetic disease models provide preliminary evidence for the efficacy of immune-inflammatory based therapies and metabolic interventions. Manual based psychotherapies remain a treatment option, with the most compelling evidence for cognitive behavioral therapy. Disparate neurostimulation strategies are also available for individuals insufficiently responsive to pharmacotherapy and/or psychosocial interventions. LIMITATIONS: Compared to non-treatment-resistant depression, TRD has been less studied. Most clinical studies on TRD have focused on pharmacotherapy-resistant depression, with relatively fewer studies evaluating "next choice" treatments in individuals who do not initially respond to psychosocial and/or neurostimulatory treatments. CONCLUSION: The pathoetiological heterogeneity of MDD/TRD invites the need for mechanistically dissimilar, and empirically validated, treatment approaches for TRD.

Integrative avenues for exploring the dynamics and evolution of protein interaction networks.

Over the past decade, the study of protein interaction networks (PINs) has shed light on the organizing principles of living cells. However, PINs have been mostly mapped in one single condition. We outline three of the most promising avenues of investigation in this field, namely the study of first, how PINs are rewired by mutations and environmental perturbations; secondly, how inter-species interactions affect PIN achitectures; thirdly, what mechanisms and forces drive PIN evolution. These investigations will unravel the dynamics and condition dependence of PINs and will thus lead to a better functional annotation of network architecture. One major challenge to reach these goals is the integration of PINs with other cellular regulatory networks in the context of complex cellular phenotypes.

Maple sap predominant microbial contaminants are correlated with the physicochemical and sensorial properties of maple syrup.

Maple sap processing and microbial contamination are significant aspects that affect maple syrup quality. In this study, two sample sets from 2005 and 2008 were used to assess the maple syrup quality variation and its relationship to microbial populations, with respect to processing, production site and harvesting period. The abundance of maple sap predominant bacteria (Pseudomonas fluorescens group and two subgroups, Rahnella spp., Janthinobacterium spp., Leuconostoc mesenteroides) and yeast (Mrakia spp., Mrakiella spp.,Guehomyces pullulans) was assessed by quantitative PCR. Maple syrup properties were analyzed by physicochemical and sensorial methods. Results indicate that P. fluorescens, Mrakia spp., Mrakiella spp. G. pullulans and Rahnella spp. are stable contaminants of maple sap, as they were found for every production site throughout the flow period. Multiple factor analysis reports a link between the relative abundance of P. fluorescens group and Mrakia spp. in maple sap with maple and vanilla odor as well as flavor of maple syrup. This evidence supports the contribution of these microorganisms or a consortium of predominant microbial contaminants to the characteristic properties of maple syrup.