Calorie restriction and SIRT3 trigger global reprogramming of the mitochondrial protein acetylome.

Calorie restriction (CR) extends life span in diverse species. Mitochondria play a key role in CR adaptation; however, the molecular details remain elusive. We developed and applied a quantitative mass spectrometry method to probe the liver mitochondrial acetyl-proteome during CR versus control diet in mice that were wild-type or lacked the protein deacetylase SIRT3. Quantification of 3,285 acetylation sites-2,193 from mitochondrial proteins-rendered a comprehensive atlas of the acetyl-proteome and enabled global site-specific, relative acetyl occupancy measurements between all four experimental conditions. Bioinformatic and biochemical analyses provided additional support for the effects of specific acetylation on mitochondrial protein function. Our results (1) reveal widespread reprogramming of mitochondrial protein acetylation in response to CR and SIRT3, (2) identify three biochemically distinct classes of acetylation sites, and (3) provide evidence that SIRT3 is a prominent regulator in CR adaptation by coordinately deacetylating proteins involved in diverse pathways of metabolism and mitochondrial maintenance.

Antimicrobial Residue Accumulation Contributes to Higher Levels of Rhodococcus equi Carrying Resistance Genes in the Environment of Horse-Breeding Farms.

Antimicrobial residues excreted in the environment following antimicrobial treatment enhance resistant microbial communities in the environment and have long-term effects on the selection and maintenance of antimicrobial resistance genes (AMRGs). In this study, we focused on understanding the impact of antimicrobial use on antimicrobial residue pollution and antimicrobial resistance (AMR) in the environment of horse-breeding farms. Rhodococcus equi is an ideal microbe to study these associations because it lives naturally in the soil, exchanges AMRGs with other bacteria in the environment, and can cause disease in animals and humans. The environment is the main source of R. equi infections in foals; therefore, higher levels of multidrug-resistant (MDR) R. equi in the environment contribute to clinical infections with MDR R. equi. We found that macrolide residues in the environment of horse-breeding farms and the use of thoracic ultrasonographic screening (TUS) for early detection of subclinically affected foals with R. equi infections were strongly associated with the presence of R. equi carrying AMRGs in the soil. Our findings indicate that the use of TUS contributed to historically higher antimicrobial use in foals, leading to the accumulation of antimicrobial residues in the environment and enhancing MDR R. equi.

Biochar and surfactant synergistically enhanced PFAS destruction in UV/sulfite system at neutral pH.

The UV/sulfite-based advanced reduction process (ARP) emerges as an effective strategy to combat per- and polyfluoroalkyl substances (PFAS) pollution in water. Yet, the UV/sulfite-ARP typically operates at highly alkaline conditions (e.g., pH > 9 or even higher) since the generated reductive radicals for PFAS degradation can be quickly sequestered by protons (H+). To overcome the associated challenges, we prototyped a biochar-surfactant-system (BSS) to synergistically enhance PFAS sorption and degradation by UV/sulfite-ARP. The degradation and defluorination efficiencies of perfluorooctanoic acid (PFOA) depended on solution pH, and concentrations of surfactant (cetyltrimethylammonium bromide; CTAB), sulfite, and biochar. At high pH (8-10), adding biochar and BSS showed no or even small inhibitory effect on PFOA degradation, since the degradation efficiencies were already high enough that cannot be differentiated. However, at acidic and neutral pH (6-7), an evident enhancement of PFOA degradation and defluorination efficiencies occurred. This is due to the synergies between biochar and CTAB that create favorable microenvironments for enhanced PFOA sorption and deeper destruction by prolonging the longevity of reductive radicals (e.g., SO3•-), which is less affected by ambient pH conditions. The performance of UV/sulfite/BSS was further optimized and used for the degradation of four PFAS. At the optimal experimental condition, the UV/sulfite/BSS system can completely degrade PFOA with >30% defluorination efficiency for up to five continuous cycles (n = 5). Overall, our BSS provides a cost-effective and sustainable technique to effectively degrade PFAS in water under environmentally relevant pH conditions. The BSS-enabled ARP technique can be easily tied into PFAS treatment train technology (e.g., advanced oxidation process) for more efficient and deeper defluorination of various PFAS in water.

Interplay among subunit identity, subunit proportion, chain length, and stereochemistry in the activity profile of sequence-random peptide mixtures.

Fmoc-based solid-phase synthesis methodology was used to prepare peptide mixtures containing one type of hydrophobic residue and one type of cationic residue. Each mixture was random in terms of sequence but highly controlled in terms of length. Analysis of the antibacterial and hemolytic properties of these mixtures revealed that selective antibacterial activity can be achieved with heterochiral binary mixtures but not homochiral binary mixture, if the proper amino acid residues are used.

Technical note: simultaneous determination of amino thiols in pig tissue by ultra-high performance liquid chromatography with fluorescence detection.

Sulfur amino acid nutrition and metabolism are linked to animal disease. While validated methods for the determination of amino thiol levels in plasma or serum are available, there is a dearth of validated methods for their measurement in tissue. A robust and reproducible ultra-high performance liquid chromatography method has been validated for the simultaneous determination of concentrations of cysteine (Cys), cysteinylglycine (CysGly), homocysteine (Hcys), γ-glutamylcysteine (γ-GluCys), and glutathione (GSH) in pig tissue. Tissue was homogenized and deproteinized with trichloroacetic acid. Amino thiols in the acid-soluble fraction of the tissue homogenate were reduced with tris-(2-carboxyethyl)-phosphine hydrochloride and derivatized with 4-(aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole (ABD-F). Amino thiols were resolved under reversed-phase gradient conditions on a Waters Acquity BEH C18 column (1.7 µm, 2.1 mm × 100 mm) within 4.5 min and detected with fluorescence. The peak area ratio of analyte to 2-mercaptopropionylglycine internal standard, added to external calibration standards and samples, was used to develop linear calibration curves. Linear calibrations were performed over the range of 15-1,500 nmol/g for Cys, CysGly, Hcys, and γ-GluCys and 150-15,000 nmol/g for GSH. Linearity, lower limit of detection, lower limit of quantitation, accuracy, precision, sample stability, and carryover were evaluated. We demonstrate excellent linearity for all analytes within their respective concentration range (r2 > 0.99) and excellent recovery of amino thiols from spiked samples (mean ± SD across tissues; Cys, 100.0 ± 2.2%; CysGly, 95.4 ± 5.1%; Hcys, 96.6 ± 2.0%; γ-GluCys, 102.2 ± 2.7%; and GSH, 100.6 ± 3.3%). The intra-day and inter-day precisions did not exceed 5% and 10%, respectively. Repeated freezing and thawing of tissue homogenate did not affect measured amino thiol concentrations, ABD-labeled amino thiols were stable for 1 wk after derivatization, and there was no sample carryover across consecutive injections. We confirm the identity of each ABD-labeled amino thiol with Orbitrap mass spectrometry. Finally, we apply the method to the determination of amino thiol concentrations in liver and jejunum tissues in newly weaned pigs and show that despite elevated Cys and maintained GSH concentrations in liver, both γ-GluCys and GSH decline in jejunum of weaned pigs.

The synthesis of glutathione, a major intracellular antioxidant, in animal tissue accounts for a considerable fraction of the intake of the sulfur amino acids methionine and cysteine. Animal scientists accordingly need methods suitable for measuring the abundance of metabolites related to sulfur amino acid metabolism in solid tissue. However, methods currently available are either validated for measuring these metabolites in plasma, serum, or urine, do not fully describe all procedures needed to prepare tissue samples for analysis, or are validated for measuring only cysteine and glutathione in tissue. The focus of this work was to describe the sample preparation and analysis methods needed to measure these metabolites in solid tissue. Sample preparation time is less than 2 h and sample analysis time is less than 5 min. The method is robust and reproducible and is applied to identify weaning-induced differences in sulfur amino acid metabolism in liver and small intestine in pigs. The method will also help evaluate the impact of diet, stress, or inflammation on cysteine and glutathione metabolism on a tissue-by-tissue basis to help optimize levels of sulfur amino acids in swine diets.

Anti-Toxoplasma gondii activity of Trametes versicolor (Turkey tail) mushroom extract.

Toxoplasma gondii (T. gondii) infection continues to rise globally in humans and animals with high socioeconomic and public health challenges. Current medications used against T. gondii infection are limited in efficacy, safety, and affordability. This research was conducted to assess the higher fungi extract effect on T. gondii tachyzoites growth in vitro and possibly decipher its mechanism of action. Furthermore, we evaluated the extract's effect on human foreskin fibroblast viability. The methanol extracts of Turkey tail (TT) mushroom was tested against T. gondii tachyzoites growth using an RH-RFP type I strain that expresses red fluorescent protein throughout culture in a dose-dependent manner using a fluorescent plate reader. Similarly, we tested the effect of the extract on host cell viability. We observed that TT extract inhibited tachyzoites growth with a 50% minimum inhibitory concentration (IC50s), IC50 = 5.98 ± 1.22 µg/mL, and 50% cytotoxic concentration (CC50s), CC50 ≥ 100 µg/mL. It was discovered that TT extract induced strong mitochondria superoxide and  reactive oxygen species production and disrupted mitochondria membrane potential in T. gondii tachyzoites. Additionally, scanning electron microscopy depicted that TT extract and pyrimethamine (PY) caused a morphological deformation of tachyzoites in vitro. In conclusion, TT methanol extract made up of phytosterols, bioactive sphingolipids, peptides, phenolic acids, and lactones could be a promising source of new compounds for the future development of anti-Toxoplasma gondii drugs. Extracts were non-cytotoxic, even at higher concentrations.

Evaluation of diverse α/ß-backbone patterns for functional α-helix mimicry: analogues of the Bim BH3 domain.

Peptidic oligomers that contain both α- and ß-amino acid residues, in regular patterns throughout the backbone, are emerging as structural mimics of α-helix-forming conventional peptides (composed exclusively of α-amino acid residues). Here we describe a comprehensive evaluation of diverse α/ß-peptide homologues of the Bim BH3 domain in terms of their ability to bind to the BH3-recognition sites on two partner proteins, Bcl-x(L) and Mcl-1. These proteins are members of the anti-apoptotic Bcl-2 family, and both bind tightly to the Bim BH3 domain itself. All α/ß-peptide homologues retain the side-chain sequence of the Bim BH3 domain, but each homologue contains periodic α-residue → ß(3)-residue substitutions. Previous work has shown that the ααßαααß pattern, which aligns the ß(3)-residues in a 'stripe' along one side of the helix, can support functional α-helix mimicry, and the results reported here strengthen this conclusion. The present study provides the first evaluation of functional mimicry by ααß and αααß patterns, which cause the ß(3)-residues to spiral around the helix periphery. We find that the αααß pattern can support effective mimicry of the Bim BH3 domain, as manifested by the crystal structure of an α/ß-peptide bound to Bcl-x(L), affinity for a variety of Bcl-2 family proteins, and induction of apoptotic signaling in mouse embryonic fibroblast extracts. The best αααß homologue shows substantial protection from proteolytic degradation relative to the Bim BH3 α-peptide.

Sorption and recovery of phenolic compounds from aqueous phase of sewage sludge hydrothermal liquefaction using bio-char.

Bio-char, a by-product of thermochemical conversion processes, has a great potential in phenolic compounds sorption from the waste aqueous phase produced from the hydrothermal liquefaction (HTL) process while being a low-cost sorbent. This study investigated the effect of temperature, pH, bio-char concentration, and mixing speed on two types of bio-char sorption of phenolic compounds using Taguchi's design of experiment and response surface method. Isothermal kinetics and thermodynamic properties were also evaluated to explain the sorption mechanism. The experimental results were well described by the pseudo-second-order kinetic model for both types of bio-char. The Langmuir isotherm model was found to be more suitable at high sorption temperatures, while the Freundlich isotherm model was better at low temperatures. Finally, the alkaline desorption and regeneration experiments were examined, and the eluents with phenolic compounds were characterized using a liquid chromatography-mass spectrometer.

Spontaneous In Vitro and In Vivo Interaction of (-)-Oleocanthal with Glycine in Biological Fluids: Novel Pharmacokinetic Markers.

Since the first discovery of its ibuprofen-like anti-inflammatory activity in 2005, the olive phenolic (-)-oleocanthal gained great scientific interest and popularity due to its reported health benefits. (-)-Oleocanthal is a monophenolic secoiridoid exclusively occurring in extra-virgin olive oil (EVOO). While several groups have investigated oleocanthal pharmacokinetics (PK) and disposition, none was able to detect oleocanthal in biological fluids or identify its PK profile that is essential for translational research studies. Besides, oleocanthal could not be detected following its addition to any fluid containing amino acids or proteins such as plasma or culture media, which could be attributed to its unique structure with two highly reactive aldehyde groups. Here, we demonstrate that oleocanthal spontaneously reacts with amino acids, with high preferential reactivity to glycine compared to other amino acids or proteins, affording two products: an unusual glycine derivative with a tetrahydropyridinium skeleton that is named oleoglycine, and our collective data supported the plausible formation of tyrosol acetate as the second product. Extensive studies were performed to validate and confirm oleocanthal reactivity, which were followed by PK disposition studies in mice, as well as cell culture transport studies to determine the ability of the formed derivatives to cross physiological barriers such as the blood-brain barrier. To the best of our knowledge, we are showing for the first time that (-)-oleocanthal is biochemically transformed to novel products in amino acids/glycine-containing fluids, which were successfully monitored in vitro and in vivo, creating a completely new perspective to understand the well-documented bioactivities of oleocanthal in humans.

Prospective evaluation of pretreatment executive cognitive impairment and depression in patients referred for radiotherapy.

PURPOSE: Cancer patients are at risk of cognitive impairment and depression. We sought to ascertain the prevalence of executive, visuospatial, memory, and general cognitive performance deficits before radiotherapy in a radiation oncology clinic referral population and correlate the neurocognitive measures with the depression symptom burden. METHODS AND MATERIALS: A total of 122 sequential patients referred for radiotherapy evaluation were administered a test battery composed of the Executive Interview (EXIT25), Executive Clock Drawing Task (CLOX1 and CLOX2), Mini Mental State Examination (MMSE), Memory Impairment Screen (MIS), and Geriatric Depression Scale (GDS). The mean age +/- standard deviation was 58 +/- 17 years. Of 122 patients, 24 (20%) had been referred for breast cancer, 21 (17%) for gastrointestinal cancer, 17 (14%) for genitourinary disease, and 8 (7%) for brain lesions; the rest were a variety of tumor sites. The cognitive performance among the tumor cohorts was compared using Bonferroni-corrected analysis of variance and Tukey-Kramer tests. Pearson correlation coefficients were determined between each cognitive instrument and the GDS. RESULTS: Of the 122 patients, 52 (43%) exhibited a detectable executive cognition decrement on one or more test measures. Five percent had poor memory performance (MIS), 18% had poor visuospatial performance (CLOX2), and 13% had poor global cognition (MMSE). Patients with brain tumors performed substantially worse on the EXIT25. No between-group differences were found for CLOX1, CLOX2, MIS, or GDS performance. The EXIT25 scores correlated significantly with the GDS scores (r = 0.26, p = 0.005). CONCLUSIONS: The results of this study have shown that patients referred for radiotherapy exhibit cognitive impairment profiles comparable to those observed in acutely ill medical inpatients. Executive control impairment appears more prevalent than global cognitive deficits, visuospatial impairment, or depression.

Dual recognition of H3K4me3 and H3K27me3 by a plant histone reader SHL.

The ability of a cell to dynamically switch its chromatin between different functional states constitutes a key mechanism regulating gene expression. Histone mark "readers" display distinct binding specificity to different histone modifications and play critical roles in regulating chromatin states. Here, we show a plant-specific histone reader SHORT LIFE (SHL) capable of recognizing both H3K27me3 and H3K4me3 via its bromo-adjacent homology (BAH) and plant homeodomain (PHD) domains, respectively. Detailed biochemical and structural studies suggest a binding mechanism that is mutually exclusive for either H3K4me3 or H3K27me3. Furthermore, we show a genome-wide co-localization of SHL with H3K27me3 and H3K4me3, and that BAH-H3K27me3 and PHD-H3K4me3 interactions are important for SHL-mediated floral repression. Together, our study establishes BAH-PHD cassette as a dual histone methyl-lysine binding module that is distinct from others in recognizing both active and repressive histone marks.

EBS is a bivalent histone reader that regulates floral phase transition in Arabidopsis.

The ability of cells to perceive and translate versatile cues into differential chromatin and transcriptional states is critical for many biological processes1-5. In plants, timely transition to a flowering state is crucial for successful reproduction6-9. EARLY BOLTING IN SHORT DAY (EBS) is a negative transcriptional regulator that prevents premature flowering in Arabidopsis thaliana10,11. We found that EBS contains bivalent bromo-adjacent homology (BAH)-plant homeodomain (PHD) reader modules that bind H3K27me3 and H3K4me3, respectively. We observed co-enrichment of a subset of EBS-associated genes with H3K4me3, H3K27me3, and Polycomb repressor complex 2 (PRC2). Notably, EBS adopted an autoinhibition mode to mediate its switch in binding preference between H3K27me3 and H3K4me3. This binding balance was critical because disruption of either EBS-H3K27me3 or EBS-H3K4me3 interaction induced early floral transition. Our results identify a bivalent chromatin reader capable of recognizing two antagonistic histone marks, and we propose a distinct mechanism of interaction between active and repressive chromatin states.

Identifying Dysregulated Epigenetic Enzyme Activity in Castrate-Resistant Prostate Cancer Development.

There is a tremendous need for novel strategies aimed at directly assessing activities of histone modifiers to probe epigenetic determinants associated with disease progression. Here, we developed a high-throughput peptide microarray assay to identify altered histone lysine (de)acetylation activity in prostate cancer (PCa). This microarray-based activity assay revealed up-regulated histone acetyltransferase (HAT) activity against specific histone H3 sites in a castrate-resistant (CR) PCa cell line compared to its hormone-sensitive (HS) isogenic counterpart. NAD+-dependent deacetylation assays revealed down-regulated sirtuin activity in validated CR lines. Levels of acetyltransferases GCN5, PCAF, CBP, and p300 were unchanged between matched HS and CR cell lines. However, autoacetylation of p300 at K1499, a modification known to enhance HAT activity and a target of deacetylation by SIRT2, was highly elevated in CR cells, while SIRT2 protein level was reduced in CR cells. Interrogation of HS and matched CR xenograft lines reveals that H3K18 hyperacetylation, increased p300 activity, and decreased SIRT2 expression are associated with progression to CR in 8/12 (66%). Tissue microarray analysis revealed that hyperacetylation of H3K18 is a feature of CRPC. Inhibition of p300 results in lower H3K18ac levels and increased expression of androgen receptors. Thus, a novel histone array identifies altered enzyme activities during the progression to CRPC and may be utilized in a personalized medicine approach. Reduced SIRT2 expression and increased p300 activity lead to a concerted mechanism of hyperacetylation at specific histone lysine sites (H3K9, H3K14, and H3K18) in CRPC.

Reader domain specificity and lysine demethylase-4 family function.

The KDM4 histone demethylases are conserved epigenetic regulators linked to development, spermatogenesis and tumorigenesis. However, how the KDM4 family targets specific chromatin regions is largely unknown. Here, an extensive histone peptide microarray analysis uncovers trimethyl-lysine histone-binding preferences among the closely related KDM4 double tudor domains (DTDs). KDM4A/B DTDs bind strongly to H3K23me3, a poorly understood histone modification recently shown to be enriched in meiotic chromatin of ciliates and nematodes. The 2.28 Å co-crystal structure of KDM4A-DTD in complex with H3K23me3 peptide reveals key intermolecular interactions for H3K23me3 recognition. Furthermore, analysis of the 2.56 Å KDM4B-DTD crystal structure pinpoints the underlying residues required for exclusive H3K23me3 specificity, an interaction supported by in vivo co-localization of KDM4B and H3K23me3 at heterochromatin in mammalian meiotic and newly postmeiotic spermatocytes. In vitro demethylation assays suggest H3K23me3 binding by KDM4B stimulates H3K36 demethylation. Together, these results provide a possible mechanism whereby H3K23me3-binding by KDM4B directs localized H3K36 demethylation during meiosis and spermatogenesis.

A Cyclic Peptide Mimic of the ß-Amyloid Binding Domain on Transthyretin.

Self-association of ß-amyloid (Aß) into oligomers and fibrils is associated with Alzheimer's disease (AD), motivating the search for compounds that bind to and inhibit Aß oligomerization and/or neurotoxicity. Peptides are an attractive class of such compounds, with potential advantages over small molecules in affinity and specificity. Self-complementation and peptide library screening are two strategies that have been employed in the search for peptides that bind to Aß. Alternatively, one could design Aß-binding peptides based on knowledge of complementary binding proteins. One candidate protein, transthyretin (TTR), binds Aß, inhibits aggregation, and reduces its toxicity. Previously, strand G of TTR was identified as part of a specific Aß binding domain, and G16, a 16-mer peptide with a sequence that spans strands G and H of TTR, was synthesized and tested. Although both TTR and G16 bound to Aß, they differed significantly in their effect on Aß aggregation, and G16 was less effective than TTR at protecting neurons from Aß toxicity. G16 lacks the ß-strand/loop/ß-strand structure of TTR's Aß binding domain. To enforce proper residue alignment, we transplanted the G16 sequence onto a ß-hairpin template. Two peptides with 18 and 22 amino acids were synthesized using an orthogonally protected glutamic acid derivative, and an N-to-C cyclization reaction was carried out to further restrict conformational flexibility. The cyclized 22-mer (but not the noncyclized 22-mer nor the 18-mer) strongly suppressed Aß aggregation into fibrils, and protected neurons against Aß toxicity. The imposition of structural constraints generated a much-improved peptidomimetic of the Aß binding epitope on TTR.

Residue-Based Preorganization of BH3-Derived α/ß-Peptides: Modulating Affinity, Selectivity and Proteolytic Susceptibility in α-Helix Mimics.

We report progress toward a general strategy for mimicking the recognition properties of specific α-helices within natural proteins through the use of oligomers that are less susceptible than conventional peptides to proteolysis. The oligomers contain both α- and ß-amino acid residues, with the density of the ß subunits low enough that an α-helix-like conformation can be adopted but high enough to interfere with protease activity. Previous studies with a different protein-recognition system that suggested ring-constrained ß residues can be superior to flexible ß residues in terms of maximizing α/ß-peptide affinity for a targeted protein surface. Here, we use mimicry of the 18-residue Bim BH3 domain to expand the scope of this strategy. Two significant advances have been achieved. First, we have developed and validated a new ring-constrained ß residue that bears an acidic side chain, which complements previously known analogues that are either hydrophobic or basic. Second, we have discovered that placing cyclic ß residues at sites that make direct contact with partner proteins can lead to substantial discrimination between structurally homologous binding partners, the proteins Bcl-xL and Mcl-1. Overall, this study helps to establish that α/ß-peptides containing ring-preorganized ß residues can reliably provide proteolytically resistant ligands for proteins that naturally evolved to recognize α-helical partners.

ChIP-less analysis of chromatin states.

BACKGROUND: Histone post-translational modifications (PTMs) are key epigenetic regulators in chromatin-based processes. Increasing evidence suggests that vast combinations of PTMs exist within chromatin histones. These complex patterns, rather than individual PTMs, are thought to define functional chromatin states. However, the ability to interrogate combinatorial histone PTM patterns at the nucleosome level has been limited by the lack of direct molecular tools. RESULTS: Here we demonstrate an efficient, quantitative, antibody-free, chromatin immunoprecipitation-less (ChIP-less) method for interrogating diverse epigenetic states. At the heart of the workflow are recombinant chromatin reader domains, which target distinct chromatin states with combinatorial PTM patterns. Utilizing a newly designed combinatorial histone peptide microarray, we showed that three reader domains (ATRX-ADD, ING2-PHD and AIRE-PHD) displayed greater specificity towards combinatorial PTM patterns than corresponding commercial histone antibodies. Such specific recognitions were employed to develop a chromatin reader-based affinity enrichment platform (matrix-assisted reader chromatin capture, or MARCC). We successfully applied the reader-based platform to capture unique chromatin states, which were quantitatively profiled by mass spectrometry to reveal interconnections between nucleosomal histone PTMs. Specifically, a highly enriched signature that harbored H3K4me0, H3K9me2/3, H3K79me0 and H4K20me2/3 within the same nucleosome was identified from chromatin enriched by ATRX-ADD. This newly reported PTM combination was enriched in heterochromatin, as revealed by the associated DNA. CONCLUSIONS: Our results suggest the broad utility of recombinant reader domains as an enrichment tool specific to combinatorial PTM patterns, which are difficult to probe directly by antibody-based approaches. The reader affinity platform is compatible with several downstream analyses to investigate the physical coexistence of nucleosomal PTM states associated with specific genomic loci. Collectively, the reader-based workflow will greatly facilitate our understanding of how distinct chromatin states and reader domains function in gene regulatory mechanisms.

Metabolomic profiling reveals suppression of oxylipin biosynthesis during the early stages of legume-rhizobia symbiosis.

The establishment of symbiosis between leguminous plants and rhizobial bacteria requires rapid metabolic changes in both partners. We utilized untargeted quantitative mass spectrometry to perform metabolomic profiling of small molecules in extracts of the model legume Medicago truncatula treated with rhizobial Nod factors. One metabolite closely resembling the 9(R)-HODE class of oxylipins reproducibly showed a decrease in concentration within the first hour of in planta nod factor treatment. Oxylipins are precursors of the jasmonic acid biosynthetic pathway and we showed that both this metabolite and jasmonic acid inhibit Nod factor signaling. Since, oxylipins have been implicated as antimicrobial compounds produced by plants, these observations suggest that the oxylipin pathway may play multiple roles in facilitating Nod factor signaling during the early stages of symbiosis.

Hydrophile scanning as a complement to alanine scanning for exploring and manipulating protein-protein recognition: application to the Bim BH3 domain.

Alanine scanning has been widely employed as a method of identifying side chains that play important roles in protein-protein and protein-peptide interactions. Here we show how an analogous and complementary technique, hydrophile scanning, can provide additional insight on such interactions. Mutation of a wild-type residue to alanine removes most of the side-chain atoms, and the effect of this removal is typically interpreted to indicate contribution of the deleted side chain to the stability of the complex. Hydrophile scanning involves systematic mutation of wild-type residues to a cationic or anionic residue (lysine or glutamic acid, in this case). We find that the results of these mutations provide insights on interactions between polypeptide surfaces that are complementary to the information obtained via alanine scanning. We have applied this technique to a peptide that corresponds to the BH3 domain of the pro-apoptotic protein Bim. The wild-type Bim BH3 domain binds strongly to the anti-apoptotic proteins Bcl-x(L) and Mcl-1. Combining information from the alanine, lysine, and glutamic acid scans has enabled us to identify Bim BH3 domain mutants containing only two or three sequence changes that bind very selectively either to Bcl-x(L) or Mcl-1. Our findings suggest that hydrophile scanning may prove to be a broadly useful tool for revealing sources of protein-protein recognition and for engineering selectivity into natural sequences.