Program for Integration and Rapid Analysis of Mass Isotopomer Distributions (PIRAMID).

SUMMARY: The analysis of stable isotope labeling experiments requires accurate, efficient, and reproducible quantification of mass isotopomer distributions (MIDs), which is not a core feature of general-purpose metabolomics software tools that are optimized to quantify metabolite abundance. Here, we present PIRAMID (Program for Integration and Rapid Analysis of Mass Isotopomer Distributions), a MATLAB-based tool that addresses this need by offering a user-friendly, graphical user interface-driven program to automate the extraction of isotopic information from mass spectrometry (MS) datasets. This tool can simultaneously extract ion chromatograms for various metabolites from multiple data files in common vendor-agnostic file formats, locate chromatographic peaks based on a targeted list of characteristic ions and retention times, and integrate MIDs for each target ion. These MIDs can be corrected for natural isotopic background based on the user-defined molecular formula of each ion. PIRAMID offers support for datasets acquired from low- or high-resolution MS, and single (MS) or tandem (MS/MS) instruments. It also enables the analysis of single or dual labeling experiments using a variety of isotopes (i.e. 2H, 13C, 15N, 18O, 34S). DATA AVAILABILITY AND IMPLEMENTATION: MATLAB p-code files are freely available for non-commercial use and can be downloaded from https://mfa.vueinnovations.com/. Commercial licenses are also available. All the data presented in this publication are available under the "Help_menu" folder of the PIRAMID software.

COLD REGULATED GENE 27 and 28 antagonize the transcriptional activity of the RVE8/LNK1/LNK2 circadian complex.

Many molecular and physiological processes in plants occur at a specific time of day. These daily rhythms are coordinated in part by the circadian clock, a timekeeper that uses daylength and temperature to maintain rhythms of ∼24 h in various clock-regulated phenotypes. The circadian MYB-like transcription factor REVEILLE 8 (RVE8) interacts with its transcriptional coactivators NIGHT LIGHT-INDUCIBLE AND CLOCK-REGULATED 1 (LNK1) and LNK2 to promote the expression of evening-phased clock genes and cold tolerance factors. While genetic approaches have commonly been used to discover connections within the clock and between clock elements and other pathways, here, we used affinity purification coupled with mass spectrometry (APMS) to identify time-of-day-specific protein interactors of the RVE8-LNK1/LNK2 complex in Arabidopsis (Arabidopsis thaliana). Among the interactors of RVE8/LNK1/LNK2 were COLD-REGULATED GENE 27 (COR27) and COR28, which coprecipitated in an evening-specific manner. In addition to COR27 and COR28, we found an enrichment of temperature-related interactors that led us to establish a previously uncharacterized role for LNK1 and LNK2 in temperature entrainment of the clock. We established that RVE8, LNK1, and either COR27 or COR28 form a tripartite complex in yeast (Saccharomyces cerevisiae) and that the effect of this interaction in planta serves to antagonize transcriptional activation of RVE8 target genes, potentially through mediating RVE8 protein degradation in the evening. Together, these results illustrate how a proteomic approach can be used to identify time-of-day-specific protein interactions. Discovery of the RVE8-LNK-COR protein complex indicates a previously unknown regulatory mechanism for circadian and temperature signaling pathways.

A General Method for Quantification and Discovery of Acyl Groups Attached to Acyl Carrier Proteins in Fatty Acid Metabolism Using LC-MS/MS.

Acyl carrier proteins (ACPs) are the scaffolds for fatty acid biosynthesis in living systems, rendering them essential to a comprehensive understanding of lipid metabolism. However, accurate quantitative methods to assess individual acyl-ACPs do not exist. We developed a robust method to quantify acyl-ACPs to the picogram level. We successfully identified acyl-ACP elongation intermediates (3-hydroxyacyl-ACPs and 2,3-trans-enoyl-ACPs) and unexpected medium-chain (C10:1, C14:1) and polyunsaturated long-chain (C16:3) acyl-ACPs, indicating both the sensitivity of the method and how current descriptions of lipid metabolism and ACP function are incomplete. Such ACPs are likely important to medium-chain lipid production for fuels and highlight poorly understood lipid remodeling events in the chloroplast. The approach is broadly applicable to type II fatty acid synthase systems found in plants and bacteria as well as mitochondria from mammals and fungi because it capitalizes on a highly conserved Asp-Ser-Leu-Asp amino acid sequence in ACPs to which acyl groups attach. Our method allows for sensitive quantification using liquid chromatography-tandem mass spectrometry with de novo-generated standards and an isotopic dilution strategy and will fill a gap in our understanding, providing insights through quantitative exploration of fatty acid biosynthesis processes for optimal biofuels, renewable feedstocks, and medical studies in health and disease.

Ursodeoxycholic acid for prevention of gallstone disease after laparoscopic sleeve gastrectomy: an Atlantic Canada perspective.

BACKGROUND: Prophylactic ursodeoxycholic acid (UDCA) may be beneficial in reducing gallstone disease after bariatric surgery. The American Society for Metabolic and Bariatric Surgery (ASMBS) 2019 guidelines recommend a 6-month course of UDCA for patients undergoing laparoscopic sleeve gastrectomy (LSG). This has not been adopted broadly. This study intends to assess the effect of routine UDCA administration following LSG on symptomatic gallstone disease. METHODS: We performed a retrospective chart review of patients who underwent LSG, between 2009 and 2019, at two tertiary care centers in Atlantic Canada. At one center, UDCA 250 mg oral twice daily was routinely prescribed following LSG for 6 months to patients with an intact gallbladder. At the other center, UDCA was not prescribed. Primary and secondary outcomes were cholecystectomy and endoscopic retrograde cholangiopancreatography (ERCP) rates. Compliance with and side effects of UDCA therapy were analyzed. RESULTS: A total of 751 patients were included in the study. Patients who had prior cholecystectomy or were lost to follow up were excluded. After exclusion criteria were applied, 461 patients were included for analysis: 303 in the UDCA group and 158 in the group who did not receive UDCA. Cholecystectomy rate was not significantly associated with UDCA administration, however there was a trend towards less cholecystectomy in patients who received UDCA (8.3% vs. 13.9%, p = 0.056). ERCP rate was significantly lower in patients who received UDCA (0.3% vs 2.5%, p = 0.031). Rate of gallstone disease requiring intervention, either cholecystectomy or ERCP, was significantly decreased in patients who received UDCA (8.9% vs 15.8%, p = 0.022). The most common barriers to compliance with UDCA were cost (45.4%) and nausea (18.1%). CONCLUSION: This is the first study to demonstrate lower rates of ERCP in patients receiving routine UDCA following LSG. Our findings support the ASMBS 2019 guidelines for administering UDCA after LSG for preventing gallstone disease.

Quantitative Proteomics and Phosphoproteomics Support a Role for Mut9-Like Kinases in Multiple Metabolic and Signaling Pathways in Arabidopsis.

Protein phosphorylation is one of the most prevalent posttranslational modifications found in eukaryotic systems. It serves as a key molecular mechanism that regulates protein function in response to environmental stimuli. The Mut9-like kinases (MLKs) are a plant-specific family of Ser/Thr kinases linked to light, circadian, and abiotic stress signaling. Here we use quantitative phosphoproteomics in conjunction with global proteomic analysis to explore the role of the MLKs in daily protein dynamics. Proteins involved in light, circadian, and hormone signaling, as well as several chromatin-modifying enzymes and DNA damage response factors, were found to have altered phosphorylation profiles in the absence of MLK family kinases. In addition to altered phosphorylation levels, mlk mutant seedlings have an increase in glucosinolate metabolism enzymes. Subsequently, we show that a functional consequence of the changes to the proteome and phosphoproteome in mlk mutant plants is elevated glucosinolate accumulation and increased sensitivity to DNA damaging agents. Combined with previous reports, this work supports the involvement of MLKs in a diverse set of stress responses and developmental processes, suggesting that the MLKs serve as key regulators linking environmental inputs to developmental outputs.

A Customisable Data Acquisition System for Open-Source Hyperspectral Imaging.

Hyperspectral imagers, or imaging spectrometers, are used in many remote sensing environmental studies in fields such as agriculture, forestry, geology, and hydrology. In recent years, compact hyperspectral imagers were developed using commercial-off-the-shelf components, but there are not yet any off-the-shelf data acquisition systems on the market to deploy them. The lack of a self-contained data acquisition system with navigation sensors is a challenge that needs to be overcome to successfully deploy these sensors on remote platforms such as drones and aircraft. Our work is the first successful attempt to deploy an entirely open-source system that is able to collect hyperspectral and navigation data concurrently for direct georeferencing. In this paper, we describe a low-cost, lightweight, and deployable data acquisition device for the open-source hyperspectral imager (OpenHSI). We utilised commercial-off-the-shelf hardware and open-source software to create a compact data acquisition device that can be easily transported and deployed. The device includes a microcontroller and a custom-designed PCB board to interface with ancillary sensors and a Raspberry Pi 4B/NVIDIA Jetson. We demonstrated our data acquisition system on a Matrice M600 drone at a beach in Sydney, Australia, collecting timestamped hyperspectral, navigation, and orientation data in parallel. Using the navigation and orientation data, the hyperspectral data were georeferenced. While the entire system including the pushbroom hyperspectral imager and housing weighed 735 g, it was designed to be easy to assemble and modify. This low-cost, customisable, deployable data acquisition system provides a cost-effective solution for the remote sensing of hyperspectral data for everyone.

Leveraging transcriptome and epigenome landscapes to infer regulatory networks during the onset of sexual maturation.

BACKGROUND: Despite sexual development being ubiquitous to vertebrates, the molecular mechanisms underpinning this fundamental transition remain largely undocumented in many organisms. We designed a time course experiment that successfully sampled the period when Atlantic salmon commence their trajectory towards sexual maturation. RESULTS: Through deep RNA sequencing, we discovered key genes and pathways associated with maturation in the pituitary-ovarian axis. Analyzing DNA methylomes revealed a bias towards hypermethylation in ovary that implicated maturation-related genes. Co-analysis of DNA methylome and gene expression changes revealed chromatin remodeling genes and key transcription factors were both significantly hypermethylated and upregulated in the ovary during the onset of maturation. We also observed changes in chromatin state landscapes that were strongly correlated with fundamental remodeling of gene expression in liver. Finally, a multiomic integrated analysis revealed regulatory networks and identified hub genes including TRIM25 gene (encoding the estrogen-responsive finger protein) as a putative key regulator in the pituitary that underwent a 60-fold change in connectivity during the transition to maturation. CONCLUSION: The study successfully documented transcriptome and epigenome changes that involved key genes and pathways acting in the pituitary - ovarian axis. Using a Systems Biology approach, we identified hub genes and their associated networks deemed crucial for onset of maturation. The results provide a comprehensive view of the spatiotemporal changes involved in a complex trait and opens the door to future efforts aiming to manipulate puberty in an economically important aquaculture species.

Management of the colonic polyps referred for surgery: an opportunity for improvement.

PURPOSE: To examine local practice for non-malignant polyps and to calculate morbidity and mortality associated with bowel resection for this indication. METHODS: This retrospective cohort study was conducted by reviewing our local gastrointestinal pathology database over a five-year period to identify colonic resections performed for benign polyps. Using search terms "polyp" and "adenoma," 272 cases were identified. Exclusion criteria included: cancer diagnosis, emergency surgeries, multiple resections, and subtotal colectomies for polyposis. 106 patients were included in the study. Primary outcome was perioperative mortality. Secondary outcomes included patient morbidity, characteristics of polyps requiring surgery, and the number of patients referred for a second endoscopic opinion prior to proceeding with surgery. RESULTS: 64 male and 42 female patients with a mean age of 65.3 years (± 8.6 years) underwent colon resection for benign polyps. The mean polyp size was 32.7 mm (± 19.5 mm). 30 patients (28.6%) had polyps equal to or less than 2 cm. Most of the polyps described were sessile (n = 55, 51.9%) and located in the right colon (n = 84, 79.3%). Endoscopic resection was attempted in 31 patients (29.2%), and five cases (4.7%) were referred for a second endoscopic opinion prior to proceeding with surgery. Endoscopists incorrectly felt that polyps were malignant in 62 cases (58.5%). Using Clavien-Dindo classification, most patients had no complications n = 36 (34.0%) or minor complications n = 41 (38.7%). Twelve patients (11.3%) had complications that required antibiotics, blood transfusions, or total parental nutrition. Nine patients (8.5%) required surgical or endoscopic management. Six patients (5.7%) required ICU admission. Mortality rate was 1.9% (n = 2). CONCLUSION: Surgery for benign colonic polyps is associated with significant morbidity and mortality. These findings reveal a gap in endoscopic management of benign colonic polyps.

A bifunctional salvage pathway for two distinct S-adenosylmethionine by-products that is widespread in bacteria, including pathogenic Escherichia coli.

S-adenosyl-l-methionine (SAM) is a necessary cosubstrate for numerous essential enzymatic reactions including protein and nucleotide methylations, secondary metabolite synthesis and radical-mediated processes. Radical SAM enzymes produce 5'-deoxyadenosine, and SAM-dependent enzymes for polyamine, neurotransmitter and quorum sensing compound synthesis produce 5'-methylthioadenosine as by-products. Both are inhibitory and must be addressed by all cells. This work establishes a bifunctional oxygen-independent salvage pathway for 5'-deoxyadenosine and 5'-methylthioadenosine in both Rhodospirillum rubrum and Extraintestinal Pathogenic Escherichia coli. Homologous genes for this pathway are widespread in bacteria, notably pathogenic strains within several families. A phosphorylase (Rhodospirillum rubrum) or separate nucleoside and kinase (Escherichia coli) followed by an isomerase and aldolase sequentially function to salvage these two wasteful and inhibitory compounds into adenine, dihydroxyacetone phosphate and acetaldehyde or (2-methylthio)acetaldehyde during both aerobic and anaerobic growth. Both SAM by-products are metabolized with equal affinity during aerobic and anaerobic growth conditions, suggesting that the dual-purpose salvage pathway plays a central role in numerous environments, notably the human body during infection. Our newly discovered bifunctional oxygen-independent pathway, widespread in bacteria, salvages at least two by-products of SAM-dependent enzymes for carbon and sulfur salvage, contributing to cell growth.

Components of leaf-trait variation along environmental gradients.

Leaf area (LA), mass per area (LMA), nitrogen per unit area (Narea ) and the leaf-internal to ambient CO2 ratio (χ) are fundamental traits for plant functional ecology and vegetation modelling. Here we aimed to assess how their variation, within and between species, tracks environmental gradients. Measurements were made on 705 species from 116 sites within a broad north-south transect from tropical to temperate Australia. Trait responses to environment were quantified using multiple regression; within- and between-species responses were compared using analysis of covariance and trait-gradient analysis. Leaf area, the leaf economics spectrum (indexed by LMA and Narea ) and χ (from stable carbon isotope ratios) varied almost independently among species. Across sites, however, χ and LA increased with mean growing-season temperature (mGDD0 ) and decreased with vapour pressure deficit (mVPD0 ) and soil pH. LMA and Narea showed the reverse pattern. Climate responses agreed with expectations based on optimality principles. Within-species variability contributed < 10% to geographical variation in LA but > 90% for χ, with LMA and Narea intermediate. These findings support the hypothesis that acclimation within individuals, adaptation within species and selection among species combine to create predictable relationships between traits and environment. However, the contribution of acclimation/adaptation vs species selection differs among traits.

Effect of an educational intervention on colonoscopy quality outcomes.

BACKGROUND: We aimed to assess the effect of a colonoscopy skills improvement (CSI) course on quality indicators at our institution. METHODS: This retrospective cohort study included ten surgeons and nine gastroenterologists practicing in a tertiary referral center who had undergone CSI training between 2014 and 2015. Procedural data for 50 colonoscopies by each physician was collected immediately before and after CSI training, and again 8 months after training. The primary outcome was adenoma detection rate (ADR) and secondary outcomes included colonoscopy completion rate (CCR), and withdrawal time (WT). Univariate analysis followed by stepwise multivariable logistic regression was performed to assess for predictors of these outcomes. These variables included patient age, gender, indication for colonoscopy, quality of bowel preparation, and CSI training. RESULTS: 2533 colonoscopies were included. There was no improvement in ADR for the entire group immediately after training and at 8 months (31.8% vs. 33.6% vs. 35.3%, p = 0.319). In subgroup analysis, the ADR of surgeons improved non-significantly immediately after completing the course and increased further at 8 months (30.9% vs. 31.6% vs. 37.6%, p = 0.065). The same changes were not observed for the gastroenterology subgroup (32.9% vs. 36.0% vs. 32.8%, p = 0.550). No change was noted in CCR or WT. In multivariate analysis of the surgical subgroup, increased patient age, male gender, and the 8-month time point following CSI training were associated with higher ADR. CONCLUSION: CSI training is associated with an improvement in ADR for surgeons at our institution.

Polygenic and sex specific architecture for two maturation traits in farmed Atlantic salmon.

BACKGROUND: A key developmental transformation in the life of all vertebrates is the transition to sexual maturity, whereby individuals are capable of reproducing for the first time. In the farming of Atlantic salmon, early maturation prior to harvest size has serious negative production impacts. RESULTS: We report genome wide association studies (GWAS) using fish measured for sexual maturation in freshwater or the marine environment. Genotypic data from a custom 50 K single nucleotide polymorphism (SNP) array was used to identify 13 significantly associated SNP for freshwater maturation with the most strongly associated on chromosomes 10 and 11. A higher number of associations (48) were detected for marine maturation, and the two peak loci were found to be the same for both traits. The number and broad distribution of GWAS hits confirmed a highly polygenetic nature, and GWAS performed separately within males and females revealed sex specific genetic behaviour for loci co-located with positional candidate genes phosphatidylinositol-binding clathrin assembly protein-like (picalm) and membrane-associated guanylate kinase, WW and PDZ domain-containing protein 2 (magi2). CONCLUSIONS: The results extend earlier work and have implications for future applied breeding strategies to delay maturation in this important aquaculture species.

Bacteriophage φEf11 ORF28 Endolysin, a Multifunctional Lytic Enzyme with Properties Distinct from All Other Identified Enterococcus faecalis Phage Endolysins.

ϕEf11 is a temperate Siphoviridae bacteriophage that infects strains of Enterococcus faecalis The ϕEf11 genome, encompassing 65 open reading frames (ORFs), is contained within 42,822 bp of DNA. Within this genome, a module of six lysis-related genes was identified. Based upon sequence homology, one of these six genes, ORF28, was predicted to code for an N-acetylmuramoyl-l-alanine amidase endolysin of 46.133 kDa, composed of 421 amino acids. The PCR-amplified ORF28 was cloned and expressed, and the resulting gene product was affinity purified to homogeneity. The purified protein was obtained from a fusion protein that exhibited a molecular mass of 72.5 kDa, consistent with a 46.1-kDa protein combined with a fused 26.5-kDa glutathione S-transferase tag. It produced rapid, profound lysis in E. faecalis populations and was active against 73 of 103 (71%) E. faecalis strains tested. In addition, it caused substantial destruction of E. faecalis biofilms. The lysin was quite stable, retaining its activity for three years in refrigerated storage, was stable over a wide range of pHs, and was unaffected by the presence of a reducing agent; however, it was inhibited by increasing concentrations of Ca2+ Liquid chromatography-mass spectrometry analysis of E. faecalis cell wall digestion products produced by the ORF28 endolysin indicated that the lysin acted as an N-acetylmuramidase, an endo-ß-N-acetylglucosaminidase, and an endopeptidase, rather than an N-acetylmuramoyl-l-alanine amidase. The ϕEf11 ORF28 lysin shared 10% to 37% amino acid identity with the lytic enzymes of all other characterized E. faecalis bacteriophages.IMPORTANCE The emergence of multidrug-resistant pathogenic microorganisms has brought increasing attention to the urgent need for the development of alternative antimicrobial strategies. One such alternative to conventional antibiotics employs lytic enzymes (endolysins) that are produced by bacteriophages in the course of lytic infection. During lytic infection by a bacteriophage, these enzymes hydrolyze the cell wall peptidoglycan, resulting in the lysis of the host cell. However, external endolysin application can result in lysis from without. In this study, we have cloned, expressed, purified, and characterized an endolysin produced by a bacteriophage infecting strains of Enterococcus faecalis The lysin is broadly active against most of the tested E. faecalis strains and exhibits multifunctional enzymatic specificities that differ from all other characterized endolysins produced by E. faecalis bacteriophages.

The validity of optimal leaf traits modelled on environmental conditions.

The ratio of leaf intercellular to ambient CO2 (χ) is modulated by stomatal conductance (gs ). These quantities link carbon (C) assimilation with transpiration, and along with photosynthetic capacities (Vcmax and Jmax ) are required to model terrestrial C uptake. We use optimization criteria based on the growth environment to generate predicted values of photosynthetic and water-use efficiency traits and test these against a unique dataset. Leaf gas-exchange parameters and carbon isotope discrimination were analysed in relation to local climate across a continental network of study sites. Sun-exposed leaves of 50 species at seven sites were measured in contrasting seasons. Values of χ predicted from growth temperature and vapour pressure deficit were closely correlated to ratios derived from C isotope (δ13 C) measurements. Correlations were stronger in the growing season. Predicted values of photosynthetic traits, including carboxylation capacity (Vcmax ), derived from δ13 C, growth temperature and solar radiation, showed meaningful agreement with inferred values derived from gas-exchange measurements. Between-site differences in water-use efficiency were, however, only weakly linked to the plant's growth environment and did not show seasonal variation. These results support the general hypothesis that many key parameters required by Earth system models are adaptive and predictable from plants' growth environments.

Synergism between Inositol Polyphosphates and TOR Kinase Signaling in Nutrient Sensing, Growth Control, and Lipid Metabolism in Chlamydomonas.

The networks that govern carbon metabolism and control intracellular carbon partitioning in photosynthetic cells are poorly understood. Target of Rapamycin (TOR) kinase is a conserved growth regulator that integrates nutrient signals and modulates cell growth in eukaryotes, though the TOR signaling pathway in plants and algae has yet to be completely elucidated. We screened the unicellular green alga Chlamydomonas reinhardtii using insertional mutagenesis to find mutants that conferred hypersensitivity to the TOR inhibitor rapamycin. We characterized one mutant, vip1-1, that is predicted to encode a conserved inositol hexakisphosphate kinase from the VIP family that pyrophosphorylates phytic acid (InsP6) to produce the low abundance signaling molecules InsP7 and InsP8 Unexpectedly, the rapamycin hypersensitive growth arrest of vip1-1 cells was dependent on the presence of external acetate, which normally has a growth-stimulatory effect on Chlamydomonas. vip1-1 mutants also constitutively overaccumulated triacylglycerols (TAGs) in a manner that was synergistic with other TAG inducing stimuli such as starvation. vip1-1 cells had reduced InsP7 and InsP8, both of which are dynamically modulated in wild-type cells by TOR kinase activity and the presence of acetate. Our data uncover an interaction between the TOR kinase and inositol polyphosphate signaling systems that we propose governs carbon metabolism and intracellular pathways that lead to storage lipid accumulation.

Seasonal, interannual and decadal drivers of tree and grass productivity in an Australian tropical savanna.

Tree-grass savannas are a widespread biome and are highly valued for their ecosystem services. There is a need to understand the long-term dynamics and meteorological drivers of both tree and grass productivity separately in order to successfully manage savannas in the future. This study investigated the interannual variability (IAV) of tree and grass gross primary productivity (GPP) by combining a long-term (15 year) eddy covariance flux record and model estimates of tree and grass GPP inferred from satellite remote sensing. On a seasonal basis, the primary drivers of tree and grass GPP were solar radiation in the wet season and soil moisture in the dry season. On an interannual basis, soil water availability had a positive effect on tree GPP and a negative effect on grass GPP. No linear trend in the tree-grass GPP ratio was observed over the 15-year study period. However, the tree-grass GPP ratio was correlated with the modes of climate variability, namely the Southern Oscillation Index. This study has provided insight into the long-term contributions of trees and grasses to savanna productivity, along with their respective meteorological determinants of IAV.

Identification of Evening Complex Associated Proteins in Arabidopsis by Affinity Purification and Mass Spectrometry.

Many species possess an endogenous circadian clock to synchronize internal physiology with an oscillating external environment. In plants, the circadian clock coordinates growth, metabolism and development over daily and seasonal time scales. Many proteins in the circadian network form oscillating complexes that temporally regulate myriad processes, including signal transduction, transcription, protein degradation and post-translational modification. In Arabidopsis thaliana, a tripartite complex composed of EARLY FLOWERING 4 (ELF4), EARLY FLOWERING 3 (ELF3), and LUX ARRHYTHMO (LUX), named the evening complex, modulates daily rhythms in gene expression and growth through transcriptional regulation. However, little is known about the physical interactions that connect the circadian system to other pathways. We used affinity purification and mass spectrometry (AP-MS) methods to identify proteins that associate with the evening complex in A. thaliana. New connections within the circadian network as well as to light signaling pathways were identified, including linkages between the evening complex, TIMING OF CAB EXPRESSION1 (TOC1), TIME FOR COFFEE (TIC), all phytochromes and TANDEM ZINC KNUCKLE/PLUS3 (TZP). Coupling genetic mutation with affinity purifications tested the roles of phytochrome B (phyB), EARLY FLOWERING 4, and EARLY FLOWERING 3 as nodes connecting the evening complex to clock and light signaling pathways. These experiments establish a hierarchical association between pathways and indicate direct and indirect interactions. Specifically, the results suggested that EARLY FLOWERING 3 and phytochrome B act as hubs connecting the clock and red light signaling pathways. Finally, we characterized a clade of associated nuclear kinases that regulate circadian rhythms, growth, and flowering in A. thaliana. Coupling mass spectrometry and genetics is a powerful method to rapidly and directly identify novel components and connections within and between complex signaling pathways.

Discovery of phosphonic acid natural products by mining the genomes of 10,000 actinomycetes.

Although natural products have been a particularly rich source of human medicines, activity-based screening results in a very high rate of rediscovery of known molecules. Based on the large number of natural product biosynthetic genes in microbial genomes, many have proposed "genome mining" as an alternative approach for discovery efforts; however, this idea has yet to be performed experimentally on a large scale. Here, we demonstrate the feasibility of large-scale, high-throughput genome mining by screening a collection of over 10,000 actinomycetes for the genetic potential to make phosphonic acids, a class of natural products with diverse and useful bioactivities. Genome sequencing identified a diverse collection of phosphonate biosynthetic gene clusters within 278 strains. These clusters were classified into 64 distinct groups, of which 55 are likely to direct the synthesis of unknown compounds. Characterization of strains within five of these groups resulted in the discovery of a new archetypical pathway for phosphonate biosynthesis, the first (to our knowledge) dedicated pathway for H-phosphinates, and 11 previously undescribed phosphonic acid natural products. Among these compounds are argolaphos, a broad-spectrum antibacterial phosphonopeptide composed of aminomethylphosphonate in peptide linkage to a rare amino acid N(5)-hydroxyarginine; valinophos, an N-acetyl l-Val ester of 2,3-dihydroxypropylphosphonate; and phosphonocystoximate, an unusual thiohydroximate-containing molecule representing a new chemotype of sulfur-containing phosphonate natural products. Analysis of the genome sequences from the remaining strains suggests that the majority of the phosphonate biosynthetic repertoire of Actinobacteria has been captured at the gene level. This dereplicated strain collection now provides a reservoir of numerous, as yet undiscovered, phosphonate natural products.

Mesencephalic Astrocyte-Derived Neurotrophic Factor as a Urine Biomarker for Endoplasmic Reticulum Stress-Related Kidney Diseases.

Endoplasmic reticulum (ER) stress and disrupted proteostasis contribute to the pathogenesis of a variety of glomerular and tubular diseases. Thus, it is imperative to develop noninvasive biomarkers for detecting ER stress in podocytes or tubular cells in the incipient stage of disease, when a kidney biopsy is not yet clinically indicated. Mesencephalic astrocyte-derived neurotrophic factor (MANF) localizes to the ER lumen and is secreted in response to ER stress in several cell types. Here, using mouse models of human nephrotic syndrome caused by mutant laminin ß2 protein-induced podocyte ER stress and AKI triggered by tunicamycin- or ischemia-reperfusion-induced tubular ER stress, we examined MANF as a potential urine biomarker for detecting ER stress in podocytes or renal tubular cells. ER stress upregulated MANF expression in podocytes and tubular cells. Notably, urinary MANF excretion concurrent with podocyte or tubular cell ER stress preceded clinical or histologic manifestations of the corresponding disease. Thus, MANF can potentially serve as a urine diagnostic or prognostic biomarker in ER stress-related kidney diseases to help stratify disease risk, predict disease progression, monitor treatment response, and identify subgroups of patients who can be treated with ER stress modulators in a highly targeted manner.

In Vivo Studies in Rhodospirillum rubrum Indicate That Ribulose-1,5-bisphosphate Carboxylase/Oxygenase (Rubisco) Catalyzes Two Obligatorily Required and Physiologically Significant Reactions for Distinct Carbon and Sulfur Metabolic Pathways.

All organisms possess fundamental metabolic pathways to ensure that needed carbon and sulfur compounds are provided to the cell in the proper chemical form and oxidation state. For most organisms capable of using CO2 as sole source of carbon, ribulose-1,5-bisphosphate (RuBP) carboxylase/oxygenase (Rubisco) catalyzes primary carbon dioxide assimilation. In addition, sulfur salvage pathways are necessary to ensure that key sulfur-containing compounds are both available and, where necessary, detoxified in the cell. Using knock-out mutations and metabolomics in the bacterium Rhodospirillum rubrum, we show here that Rubisco concurrently catalyzes key and essential reactions for seemingly unrelated but physiologically essential central carbon and sulfur salvage metabolic pathways of the cell. In this study, complementation and mutagenesis studies indicated that representatives of all known extant functional Rubisco forms found in nature are capable of simultaneously catalyzing reactions required for both CO2-dependent growth as well as growth using 5-methylthioadenosine as sole sulfur source under anaerobic photosynthetic conditions. Moreover, specific inactivation of the CO2 fixation reaction did not affect the ability of Rubisco to support anaerobic 5-methylthioadenosine metabolism, suggesting that the active site of Rubisco has evolved to ensure that this enzyme maintains both key functions. Thus, despite the coevolution of both functions, the active site of this protein may be differentially modified to affect only one of its key functions.