Divergent evolution of NLR genes in the genus Glycine: impacts of annuals and perennials' life history strategies.

Within the family Fabaceae, the genus Glycine is composed of two subgenera annuals (2n=40) and perennials. This life strategy transition may have differentially affected the evolution of various gene families. Its cultivated species G. max has high level of susceptibility to major pathogens including viruses, bacteria and fungi. Understanding nucleotide-binding domain leucine-rich repeat (NLR) genes evolution in soybean is of paramount importance due to their central role in plant immunity and their potential in improving disease resistance in soybean cultivars. In this study, we investigated the significance of this annual-perennial transition on the macroevolution of NLR genes in the genus Glycine. Our results reveal a remarkable distinction between annual species such as Glycine max and Glycine soja, which exhibit an expanded NLRome compared to perennial species (G. cyrtoloba, G. stenophita, G. dolichocarpa, G. falcata, G. syndetika, G. latifolia and G. tomentella). Our evolutionary timescale analysis pinpoints recent accelerated gene duplication events for this expansion, which occurred between 0.1 and 0.5 million years ago, driven predominantly by lineage-specific and terminal duplications. In contrast, perennials initially experienced significant contraction during the diploidisation phase following the Glycine-specific whole-genome duplication event (~10 million years ago). Despite the reduction in the NLRome, perennial lineages exhibit a unique and highly diversified repertoire of NLR genes with limited interspecies synteny. The investigation of gene gain and loss ratios revealed that this diversification resulted from the birth of novel genes following individual speciation events. Among perennials, G. latifolia, a well-known resistance resource, has the highest ratio of these novel genes in the tertiary gene pool. Our study suggests evolutionary mechanisms, including recombination and transposition, as potential drivers for the emergence of these novel genes. This study also provides evidence for the unbalanced expansion of the NLRome in the Dt subgenome compared with the At subgenome in the young allopolyploid G. dolichocarpa. To the best of our knowledge, this is the first study to investigate the effect of annuality and perenniality life transition on the evolution of NLR genes in the genus Glycine to identify its genomics resources for improving the resistance of soybean crop with global importance on the economy and food security.

Investigation of Resistance Genes in Genus Vigna Reveals Highly Variable NLRome in Parallel Domesticated Member Species.

Vigna is a unique genus that consist of multiple crop species that are domesticated in parallel fashion between 7-10 thousand years ago. Here we studied the evolution of nucleotide-binding site leucine-rich repeat receptor (NLR) genes across five crop species of genus Vigna. In total identified 286, 350, 234, 250, 108 and 161 NLR genes were from Phaseolous vulgaris, Vigna. unguiculata, Vigna mungo, Vigna radiata, Vigna angularis and Vigna umbellata respectively. Comprehensive phylogenetic and clusterization analysis reveals the presence of seven subgroups of Coiled coil like NLRs (CC-NLR) genes and four distinct lineages of Toll interleukin receptor like NLRs (TIR-NLR). Subgroup CCG10-NLR shows large scale diversification among Vigna species suggesting genus specific distinct duplication pattern in Vigna species. Mainly birth of new NLR gene families and higher rate of terminal duplication is the major determinants for expansion of NLRome in genus Vigna. Recent expansion of NLRome in V. anguiculata and V. radiata was also observed which might suggest that domestication have supported their duplication of lineage specific NLR genes. In short, large scale difference in the architecture of NLRome were observed in diploid plant species. Our findings allowed us to hypothesized that independent parallel domestication is the major drivers of highly divergent evolution of NLRome in genus Vigna.

Evolution of NLR genes in genus Arachis reveals asymmetric expansion of NLRome in wild and domesticated tetraploid species.

Arachis hypogaea is an allotetraploid crop widely grown in the world. Wild relatives of genus Arachis are the rich source of genetic diversity and high levels of resistance to combat pathogens and climate change. The accurate identification and characterization of plant resistance gene, nucleotide binding site leucine rich repeat receptor (NLRs) substantially contribute to the repertoire of resistances and improve production. In the current study, we have studied the evolution of NLR genes in genus Arachis and performed their comparative genomics among four diploids (A. duranensis, A. ipaensis, A. cardenasii, A. stenosperma) and two tetraploid (wild: A. monticola and domesticated: A. hypogaea) species. In total 521, 354, 284, 794, 654, 290 NLR genes were identified from A. cardenasii, A. stenosperma and A. duranensis, A. hypogaea, A. monticola and A. ipaensis respectively. Phylogenetic analysis and classification of NLRs revealed that they belong to 7 subgroups and specific subgroups have expanded in each genome leading towards divergent evolution. Gene gain and loss, duplication assay reveals that wild and domesticated tetraploids species have shown asymmetric expansion of NLRome in both sub-genome (AA and BB). A-subgenome of A. monticola exhibited significant contraction of NLRome while B-subgenome shows expansion and vice versa in case of A. hypogaea probably due to distinct natural and artificial selection pressure. In addition, diploid species A. cardenasii revealed the largest repertoire of NLR genes due to higher frequency of gene duplication and selection pressure. A. cardenasii and A. monticola can be regarded as putative resistance resources for peanut breeding program for introgression of novel resistance genes. Findings of this study also emphasize the application neo-diploids and polyploids due to higher quantitative expression of NLR genes. To the best of our knowledge, this is the first study that studied the effect of domestication and polyploidy on the evolution of NLR genes in genus Arachis to identify genomic resources for improving resistance of polyploid crop with global importance on economy and food security.

Investigation of NLR Genes Reveals Divergent Evolution on NLRome in Diploid and Polyploid Species in Genus Trifolium.

Crop wild relatives contain a greater variety of phenotypic and genotypic diversity compared to their domesticated counterparts. Trifolium crop species have limited genetic diversity to cope with biotic and abiotic stresses due to artificial selection for consumer preferences. Here, we investigated the distribution and evolution of nucleotide-binding site leucine-rich repeat receptor (NLR) genes in the genus of Trifolium with the objective to identify reference NLR genes. We identified 412, 350, 306, 389 and 241 NLR genes were identified from Trifolium. subterraneum, T. pratense, T. occidentale, subgenome-A of T. repens and subgenome-B of T. repens, respectively. Phylogenetic and clustering analysis reveals seven sub-groups in genus Trifolium. Specific subgroups such as G4-CNL, CCG10-CNL and TIR-CNL show distinct duplication patterns in specific species, which suggests subgroup duplications that are the hallmarks of their divergent evolution. Furthermore, our results strongly suggest the overall expansion of NLR repertoire in T. subterraneum is due to gene duplication events and birth of gene families after speciation. Moreover, the NLRome of the allopolyploid species T. repens has evolved asymmetrically, with the subgenome -A showing expansion, while the subgenome-B underwent contraction. These findings provide crucial background data for comprehending NLR evolution in the Fabaceae family and offer a more comprehensive analysis of NLR genes as disease resistance genes.

Dynamic Evolution of NLR Genes in Dalbergioids.

Dalbergioid is a large group within the family Fabaceae that consists of diverse plant species distributed in distinct biogeographic realms. Here, we have performed a comprehensive study to understand the evolution of the nucleotide-binding leucine-rich repeats (NLRs) gene family in Dalbergioids. The evolution of gene families in this group is affected by a common whole genome duplication that occurred approximately 58 million years ago, followed by diploidization that often leads to contraction. Our study suggests that since diploidization, the NLRome of all groups of Dalbergioids is expanding in a clade-specific manner with fewer exceptions. Phylogenetic analysis and classification of NLRs revealed that they belong to seven subgroups. Specific subgroups have expanded in a species-specific manner, leading to divergent evolution. Among the Dalbergia clade, the expansion of NLRome in six species of the genus Dalbergia was observed, with the exception of Dalbergia odorifera, where a recent contraction of NLRome occurred. Similarly, members of the Pterocarpus clade genus Arachis revealed a large-scale expansion in the diploid species. In addition, the asymmetric expansion of NLRome was observed in wild and domesticated tetraploids after recent duplications in the genus Arachis. Our analysis strongly suggests that whole genome duplication followed by tandem duplication after divergence from a common ancestor of Dalbergioids is the major cause of NLRome expansion. To the best of our knowledge, this is the first ever study to provide insight toward the evolution of NLR genes in this important tribe. In addition, accurate identification and characterization of NLR genes is a substantial contribution to the repertoire of resistances among members of the Dalbergioids species.

Using Exogenous Melatonin, Glutathione, Proline, and Glycine Betaine Treatments to Combat Abiotic Stresses in Crops.

Abiotic stresses, such as drought, salinity, heat, cold, and heavy metals, are associated with global climate change and hamper plant growth and development, affecting crop yields and quality. However, the negative effects of abiotic stresses can be mitigated through exogenous treatments using small biomolecules. For example, the foliar application of melatonin provides the following: it protects the photosynthetic apparatus; it increases the antioxidant defenses, osmoprotectant, and soluble sugar levels; it prevents tissue damage and reduces electrolyte leakage; it improves reactive oxygen species (ROS) scavenging; and it increases biomass, maintains the redox and ion homeostasis, and improves gaseous exchange. Glutathione spray upregulates the glyoxalase system, reduces methylglyoxal (MG) toxicity and oxidative stress, decreases hydrogen peroxide and malondialdehyde accumulation, improves the defense mechanisms, tissue repairs, and nitrogen fixation, and upregulates the phytochelatins. The exogenous application of proline enhances growth and other physiological characteristics, upregulates osmoprotection, protects the integrity of the plasma lemma, reduces lipid peroxidation, increases photosynthetic pigments, phenolic acids, flavonoids, and amino acids, and enhances stress tolerance, carbon fixation, and leaf nitrogen content. The foliar application of glycine betaine improves growth, upregulates osmoprotection and osmoregulation, increases relative water content, net photosynthetic rate, and catalase activity, decreases photorespiration, ion leakage, and lipid peroxidation, protects the oxygen-evolving complex, and prevents chlorosis. Chemical priming has various important advantages over transgenic technology as it is typically more affordable for farmers and safe for plants, people, and animals, while being considered environmentally acceptable. Chemical priming helps to improve the quality and quantity of the yield. This review summarizes and discusses how exogenous melatonin, glutathione, proline, and glycine betaine can help crops combat abiotic stresses.

Genome Wide Analysis of Family-1 UDP Glycosyltransferases in Populus trichocarpa Specifies Abiotic Stress Responsive Glycosylation Mechanisms.

Populus trichocarpa (Black cottonwood) is a dominant timber-yielding tree that has become a notable model plant for genome-level insights in forest trees. The efficient transport and solubility of various glycoside-associated compounds is linked to Family-1 UDP-glycosyltransferase (EC 2.4.1.x; UGTs) enzymes. These glycosyltransferase enzymes play a vital role in diverse plant functions, such as regulation of hormonal homeostasis, growth and development (seed, flower, fiber, root, etc.), xenobiotic detoxification, stress response (salt, drought, and oxidative), and biosynthesis of secondary metabolites. Here, we report a genome-wide analysis of the P. trichocarpa genome that identified 191 putative UGTs distributed across all chromosomes (with the exception of chromosome 20) based on 44 conserved plant secondary product glycosyltransferase (PSPG) motif amino acid sequences. Phylogenetic analysis of the 191 Populus UGTs together with 22 referenced UGTs from Arabidopsis and maize clustered the putative UGTs into 16 major groups (A-P). Whole-genome duplication events were the dominant pattern of duplication among UGTs in Populus. A well-conserved intron insertion was detected in most intron-containing UGTs across eight examined eudicots, including Populus. Most of the UGT genes were found preferentially expressed in leaf and root tissues in general. The regulation of putative UGT expression in response to drought, salt and heat stress was observed based on microarray and available RNA sequencing datasets. Up- and down-regulated UGT expression models were designed, based on transcripts per kilobase million values, confirmed their maximally varied expression under drought, salt and heat stresses. Co-expression networking of putative UGTs indicated their maximum co-expression with cytochrome P450 genes involved in triterpenoid biosynthesis. Our results provide an important resource for the identification of functional UGT genes to manipulate abiotic stress responsive glycosylation in Populus.

Advances in Metabolomics-Driven Diagnostic Breeding and Crop Improvement.

Climate change continues to threaten global crop output by reducing annual productivity. As a result, global food security is now considered as one of the most important challenges facing humanity. To address this challenge, modern crop breeding approaches are required to create plants that can cope with increased abiotic/biotic stress. Metabolomics is rapidly gaining traction in plant breeding by predicting the metabolic marker for plant performance under a stressful environment and has emerged as a powerful tool for guiding crop improvement. The advent of more sensitive, automated, and high-throughput analytical tools combined with advanced bioinformatics and other omics techniques has laid the foundation to broadly characterize the genetic traits for crop improvement. Progress in metabolomics allows scientists to rapidly map specific metabolites to the genes that encode their metabolic pathways and offer plant scientists an excellent opportunity to fully explore and rationally harness the wealth of metabolites that plants biosynthesize. Here, we outline the current application of advanced metabolomics tools integrated with other OMICS techniques that can be used to: dissect the details of plant genotype-metabolite-phenotype interactions facilitating metabolomics-assisted plant breeding for probing the stress-responsive metabolic markers, explore the hidden metabolic networks associated with abiotic/biotic stress resistance, facilitate screening and selection of climate-smart crops at the metabolite level, and enable accurate risk-assessment and characterization of gene edited/transgenic plants to assist the regulatory process. The basic concept behind metabolic editing is to identify specific genes that govern the crucial metabolic pathways followed by the editing of one or more genes associated with those pathways. Thus, metabolomics provides a superb platform for not only rapid assessment and commercialization of future genome-edited crops, but also for accelerated metabolomics-assisted plant breeding. Furthermore, metabolomics can be a useful tool to expedite the crop research if integrated with speed breeding in future.

De-novo Domestication for Improving Salt Tolerance in Crops.

Global agriculture production is under serious threat from rapidly increasing population and adverse climate changes. Food security is currently a huge challenge to feed 10 billion people by 2050. Crop domestication through conventional approaches is not good enough to meet the food demands and unable to fast-track the crop yields. Also, intensive breeding and rigorous selection of superior traits causes genetic erosion and eliminates stress-responsive genes, which makes crops more prone to abiotic stresses. Salt stress is one of the most prevailing abiotic stresses that poses severe damages to crop yield around the globe. Recent innovations in state-of-the-art genomics and transcriptomics technologies have paved the way to develop salinity tolerant crops. De novo domestication is one of the promising strategies to produce superior new crop genotypes through exploiting the genetic diversity of crop wild relatives (CWRs). Next-generation sequencing (NGS) technologies open new avenues to identifying the unique salt-tolerant genes from the CWRs. It has also led to the assembly of highly annotated crop pan-genomes to snapshot the full landscape of genetic diversity and recapture the huge gene repertoire of a species. The identification of novel genes alongside the emergence of cutting-edge genome editing tools for targeted manipulation renders de novo domestication a way forward for developing salt-tolerance crops. However, some risk associated with gene-edited crops causes hurdles for its adoption worldwide. Halophytes-led breeding for salinity tolerance provides an alternative strategy to identify extremely salt tolerant varieties that can be used to develop new crops to mitigate salinity stress.

Next-Generation Breeding Strategies for Climate-Ready Crops.

Climate change is a threat to global food security due to the reduction of crop productivity around the globe. Food security is a matter of concern for stakeholders and policymakers as the global population is predicted to bypass 10 billion in the coming years. Crop improvement via modern breeding techniques along with efficient agronomic practices innovations in microbiome applications, and exploiting the natural variations in underutilized crops is an excellent way forward to fulfill future food requirements. In this review, we describe the next-generation breeding tools that can be used to increase crop production by developing climate-resilient superior genotypes to cope with the future challenges of global food security. Recent innovations in genomic-assisted breeding (GAB) strategies allow the construction of highly annotated crop pan-genomes to give a snapshot of the full landscape of genetic diversity (GD) and recapture the lost gene repertoire of a species. Pan-genomes provide new platforms to exploit these unique genes or genetic variation for optimizing breeding programs. The advent of next-generation clustered regularly interspaced short palindromic repeat/CRISPR-associated (CRISPR/Cas) systems, such as prime editing, base editing, and de nova domestication, has institutionalized the idea that genome editing is revamped for crop improvement. Also, the availability of versatile Cas orthologs, including Cas9, Cas12, Cas13, and Cas14, improved the editing efficiency. Now, the CRISPR/Cas systems have numerous applications in crop research and successfully edit the major crop to develop resistance against abiotic and biotic stress. By adopting high-throughput phenotyping approaches and big data analytics tools like artificial intelligence (AI) and machine learning (ML), agriculture is heading toward automation or digitalization. The integration of speed breeding with genomic and phenomic tools can allow rapid gene identifications and ultimately accelerate crop improvement programs. In addition, the integration of next-generation multidisciplinary breeding platforms can open exciting avenues to develop climate-ready crops toward global food security.

Rewilding crops for climate resilience: economic analysis and de novo domestication strategies.

To match predicted population growth, annual food production should be doubled by 2050. This is not achievable by current agronomical and breeding practices, due to the impact of climate changes and associated abiotic stresses on agricultural production systems. Here, we analyze the impact of global climate trends on crop productivity and show that the overall loss in crop production from climate-driven abiotic stresses may exceed US$170 billion year-1 and represents a major threat to global food security. We also show that abiotic stress tolerance had been present in wild progenitors of modern crops but was lost during their domestication. We argue for a major shift in our paradigm of crop breeding, focusing on climate resilience, and call for a broader use of wild relatives as a major tool in this process. We argue that, while molecular tools are currently in place to harness the potential of climate-resilient genes present in wild relatives, the complex polygenic nature of tolerance traits remains a major bottleneck in this process. Future research efforts should be focused not only on finding appropriate wild relatives but also on development of efficient cell-based high-throughput phenotyping platforms allowing assessment of the in planta operation of key genes.

Metabolomics: A Powerful Tool to Study the Complexity of Wheat Metabolome.

Wheat is a widely cultivated cereal, consumed by nearly 80% of the total population in the world. Although wheat is growing on 215 million hectares annually, its production is still inadequate to meet the future demand of feeding the 10 billion human population. Global food security is the biggest challenge as climate change is threatening crop production. There is a need to fast-- track the wheat breeding by devising modern biotechnological tools. Climate-smart wheat having greater stress resilience, better adaptability and improved agronomic traits are vital to guarantee food security. Substantial understanding and knowledge of vital biochemical pathways and regulatory networks is required for achieving stress resilience in wheat. Metabolomics has emerged as a fascinating technology to speed up the crop improvement programs by deciphering unique metabolic pathways for abiotic/biotic stress tolerance. State-of-the-art metabolomics tools such as nuclear magnetic resonance (NMR) and advanced mass spectrometry (MS) has opened new horizons for detailed analysis of wheat metabolome. The identification of unique metabolic pathways offers various types of stress tolerance and helps to screen the elite wheat cultivars. In this review, we summarize the applications of metabolomics to probe the stress-responsive metabolites and stress-inducive regulatory pathways that govern abiotic/biotic stress tolerance in wheat and highlight the significance of metabolic profiling to characterize wheat agronomics traits. Furthermore, we also describe the potential of metabolomics-assisted speed breeding for wheat improvement and propose future directions.

Metabolomics: A Way Forward for Crop Improvement.

Metabolomics is an emerging branch of "omics" and it involves identification and quantification of metabolites and chemical footprints of cellular regulatory processes in different biological species. The metabolome is the total metabolite pool in an organism, which can be measured to characterize genetic or environmental variations. Metabolomics plays a significant role in exploring environment-gene interactions, mutant characterization, phenotyping, identification of biomarkers, and drug discovery. Metabolomics is a promising approach to decipher various metabolic networks that are linked with biotic and abiotic stress tolerance in plants. In this context, metabolomics-assisted breeding enables efficient screening for yield and stress tolerance of crops at the metabolic level. Advanced metabolomics analytical tools, like non-destructive nuclear magnetic resonance spectroscopy (NMR), liquid chromatography mass-spectroscopy (LC-MS), gas chromatography-mass spectrometry (GC-MS), high performance liquid chromatography (HPLC), and direct flow injection (DFI) mass spectrometry, have sped up metabolic profiling. Presently, integrating metabolomics with post-genomics tools has enabled efficient dissection of genetic and phenotypic association in crop plants. This review provides insight into the state-of-the-art plant metabolomics tools for crop improvement. Here, we describe the workflow of plant metabolomics research focusing on the elucidation of biotic and abiotic stress tolerance mechanisms in plants. Furthermore, the potential of metabolomics-assisted breeding for crop improvement and its future applications in speed breeding are also discussed. Mention has also been made of possible bottlenecks and future prospects of plant metabolomics.

Modern Trends in Plant Genome Editing: An Inclusive Review of the CRISPR/Cas9 Toolbox.

Increasing agricultural productivity via modern breeding strategies is of prime interest to attain global food security. An array of biotic and abiotic stressors affect productivity as well as the quality of crop plants, and it is a primary need to develop crops with improved adaptability, high productivity, and resilience against these biotic/abiotic stressors. Conventional approaches to genetic engineering involve tedious procedures. State-of-the-art OMICS approaches reinforced with next-generation sequencing and the latest developments in genome editing tools have paved the way for targeted mutagenesis, opening new horizons for precise genome engineering. Various genome editing tools such as transcription activator-like effector nucleases (TALENs), zinc-finger nucleases (ZFNs), and meganucleases (MNs) have enabled plant scientists to manipulate desired genes in crop plants. However, these approaches are expensive and laborious involving complex procedures for successful editing. Conversely, CRISPR/Cas9 is an entrancing, easy-to-design, cost-effective, and versatile tool for precise and efficient plant genome editing. In recent years, the CRISPR/Cas9 system has emerged as a powerful tool for targeted mutagenesis, including single base substitution, multiplex gene editing, gene knockouts, and regulation of gene transcription in plants. Thus, CRISPR/Cas9-based genome editing has demonstrated great potential for crop improvement but regulation of genome-edited crops is still in its infancy. Here, we extensively reviewed the availability of CRISPR/Cas9 genome editing tools for plant biotechnologists to target desired genes and its vast applications in crop breeding research.

Recombinant mouse prion protein alone or in combination with lipopolysaccharide alters expression of innate immunity genes in the colon of mice.

The objectives of this study were to test whether recombinant mouse (mo)PrP alone or in combination with LPS or under simulated endotoxemia would affect expression of genes related to host inflammatory and antimicrobial responses. To test our hypotheses colon tissues were collected from 16 male mice (FVB/N strain) and mounted in an Ussing chamber. Application of moPrP to the mucosal side of the colon affected genes related to TLR- and NLR- signaling and antimicrobial responses. When LPS was added on the mucosal side of the colon, genes related to TLR, Nlrp3 inflammasome, and iron transport proteins were over-expressed. Addition of LPS to the serosal side of the colon up-regulated genes related to TLR- and NLR-signaling, Nlrp3 inflammasome, and a chemokine. Treatment with both moPrP and LPS to the mucosal side of the colon upregulated genes associated with TLR, downstream signal transduction (DST), inflammatory response, attraction of dendritic cells to the site of inflammation, and the JNK-apoptosis pathway. Administration of moPrP to the mucosal side and LPS to the serosal side of the colon affected genes related to TLR- and NLR-signaling, DST, apoptosis, inflammatory response, cytokines, chemokines, and antimicrobial peptides. Overall this study suggests a potential role for moPrP as an endogenous 'danger signal' associated with activation of colon genes related to innate immunity and antibacterial responses.

Lipopolysaccharide induced conversion of recombinant prion protein.

The conformational conversion of the cellular prion protein (PrP(C)) to the ß-rich infectious isoform PrP(Sc) is considered a critical and central feature in prion pathology. Although PrP(Sc) is the critical component of the infectious agent, as proposed in the "protein-only" prion hypothesis, cellular components have been identified as important cofactors in triggering and enhancing the conversion of PrP(C) to proteinase K resistant PrP(Sc). A number of in vitro systems using various chemical and/or physical agents such as guanidine hydrochloride, urea, SDS, high temperature, and low pH, have been developed that cause PrP(C) conversion, their amplification, and amyloid fibril formation often under non-physiological conditions. In our ongoing efforts to look for endogenous and exogenous chemical mediators that might initiate, influence, or result in the natural conversion of PrP(C) to PrP(Sc), we discovered that lipopolysaccharide (LPS), a component of gram-negative bacterial membranes interacts with recombinant prion proteins and induces conversion to an isoform richer in ß sheet at near physiological conditions as long as the LPS concentration remains above the critical micelle concentration (CMC). More significant was the LPS mediated conversion that was observed even at sub-molar ratios of LPS to recombinant ShPrP (90-232).

The human urine metabolome.

Urine has long been a "favored" biofluid among metabolomics researchers. It is sterile, easy-to-obtain in large volumes, largely free from interfering proteins or lipids and chemically complex. However, this chemical complexity has also made urine a particularly difficult substrate to fully understand. As a biological waste material, urine typically contains metabolic breakdown products from a wide range of foods, drinks, drugs, environmental contaminants, endogenous waste metabolites and bacterial by-products. Many of these compounds are poorly characterized and poorly understood. In an effort to improve our understanding of this biofluid we have undertaken a comprehensive, quantitative, metabolome-wide characterization of human urine. This involved both computer-aided literature mining and comprehensive, quantitative experimental assessment/validation. The experimental portion employed NMR spectroscopy, gas chromatography mass spectrometry (GC-MS), direct flow injection mass spectrometry (DFI/LC-MS/MS), inductively coupled plasma mass spectrometry (ICP-MS) and high performance liquid chromatography (HPLC) experiments performed on multiple human urine samples. This multi-platform metabolomic analysis allowed us to identify 445 and quantify 378 unique urine metabolites or metabolite species. The different analytical platforms were able to identify (quantify) a total of: 209 (209) by NMR, 179 (85) by GC-MS, 127 (127) by DFI/LC-MS/MS, 40 (40) by ICP-MS and 10 (10) by HPLC. Our use of multiple metabolomics platforms and technologies allowed us to identify several previously unknown urine metabolites and to substantially enhance the level of metabolome coverage. It also allowed us to critically assess the relative strengths and weaknesses of different platforms or technologies. The literature review led to the identification and annotation of another 2206 urinary compounds and was used to help guide the subsequent experimental studies. An online database containing the complete set of 2651 confirmed human urine metabolite species, their structures (3079 in total), concentrations, related literature references and links to their known disease associations are freely available at http://www.urinemetabolome.ca.

Detailed biophysical characterization of the acid-induced PrP(c) to PrP(ß) conversion process.

Prions are believed to spontaneously convert from a native, monomeric highly helical form (called PrP(c)) to a largely ß-sheet-rich, multimeric and insoluble aggregate (called PrP(sc)). Because of its large size and insolubility, biophysical characterization of PrP(sc) has been difficult, and there are several contradictory or incomplete models of the PrP(sc) structure. A ß-sheet-rich, soluble intermediate, called PrP(ß), exhibits many of the same features as PrP(sc) and can be generated using a combination of low pH and/or mild denaturing conditions. Studies of the PrP(c) to PrP(ß) conversion process and of PrP(ß) folding intermediates may provide insights into the structure of PrP(sc). Using a truncated, recombinant version of Syrian hamster PrP(ß) (shPrP(90-232)), we used NMR spectroscopy, in combination with other biophysical techniques (circular dichroism, dynamic light scattering, electron microscopy, fluorescence spectroscopy, mass spectrometry, and proteinase K digestion), to characterize the pH-driven PrP(c) to PrP(ß) conversion process in detail. Our results show that below pH 2.8 the protein oligomerizes and conversion to the ß-rich structure is initiated. At pH 1.7 and above, the oligomeric protein can recover its native monomeric state through dialysis to pH 5.2. However, when conversion is completed at pH 1.0, the large oligomer "locks down" irreversibly into a stable, ß-rich form. At pH values above 3.0, the protein is amenable to NMR investigation. Chemical shift perturbations, NOE, amide line width, and T(2) measurements implicate the putative "amylome motif" region, "NNQNNF" as the region most involved in the initial helix-to-ß conversion phase. We also found that acid-induced PrP(ß) oligomers could be converted to fibrils without the use of chaotropic denaturants. The latter finding represents one of the first examples wherein physiologically accessible conditions (i.e., only low pH) were used to achieve PrP conversion and fibril formation.

Palmitoylation gates phosphorylation-dependent regulation of BK potassium channels.

Large conductance calcium- and voltage-gated potassium (BK) channels are important regulators of physiological homeostasis and their function is potently modulated by protein kinase A (PKA) phosphorylation. PKA regulates the channel through phosphorylation of residues within the intracellular C terminus of the pore-forming alpha-subunits. However, the molecular mechanism(s) by which phosphorylation of the alpha-subunit effects changes in channel activity are unknown. Inhibition of BK channels by PKA depends on phosphorylation of only a single alpha-subunit in the channel tetramer containing an alternatively spliced insert (STREX) suggesting that phosphorylation results in major conformational rearrangements of the C terminus. Here, we define the mechanism of PKA inhibition of BK channels and demonstrate that this regulation is conditional on the palmitoylation status of the channel. We show that the cytosolic C terminus of the STREX BK channel uniquely interacts with the plasma membrane via palmitoylation of evolutionarily conserved cysteine residues in the STREX insert. PKA phosphorylation of the serine residue immediately upstream of the conserved palmitoylated cysteine residues within STREX dissociates the C terminus from the plasma membrane, inhibiting STREX channel activity. Abolition of STREX palmitoylation by site-directed mutagenesis or pharmacological inhibition of palmitoyl transferases prevents PKA-mediated inhibition of BK channels. Thus, palmitoylation gates BK channel regulation by PKA phosphorylation. Palmitoylation and phosphorylation are both dynamically regulated; thus, cross-talk between these 2 major posttranslational signaling cascades provides a mechanism for conditional regulation of BK channels. Interplay of these distinct signaling cascades has important implications for the dynamic regulation of BK channels and physiological homeostasis.