Preference For Acids Research Articles

Metabolic Heterogeneity of Cerebral Cortical and Cerebellar Astrocytes.

Astrocytes play critical roles in regulating neuronal synaptogenesis, maintaining blood-brain barrier integrity, and recycling neurotransmitters. Increasing numbers of studies have suggested astrocyte heterogeneity in morphology, gene profile, and function. However, metabolic phenotype of astrocytes in different brain regions have not been explored. In this paper, we investigated the metabolic signature of cortical and cerebellar astrocytes using primary astrocyte cultures. We observed that cortical astrocytes were larger than cerebellar astrocytes, whereas cerebellar astrocytes had more and longer processes than cortical astrocytes. Using a Seahorse extracellular flux analyzer, we demonstrated that cortical astrocytes had higher mitochondrial respiration and glycolysis than cerebellar astrocytes. Cerebellar astrocytes have lower spare capacity of mitochondrial respiration and glycolysis as compared with cortical astrocytes. Consistently, cortical astrocytes have higher mitochondrial oxidation and glycolysis-derived ATP content than cerebellar astrocytes. In addition, cerebellar astrocytes have a fuel preference for glutamine and fatty acid, whereas cortical astrocytes were more dependent on glucose to meet energy demands. Our study indicated that cortical and cerebellar astrocytes display distinct metabolic phenotypes. Future studies on astrocyte metabolic heterogeneity and brain function in aging and neurodegeneration may lead to better understanding of the role of astrocyte in brain aging and neurodegenerative disorders.

Characterization of the enzymatic properties of human RNPEPL1/aminopeptidase Z.

It is now evident that the M1 family of aminopeptidases play important roles in many pathophysiological processes. Among them, the enzymatic properties of arginyl aminopeptidase-like 1 (RNPEPL1) are characterized only by its truncated form. No peptide substrate has been identified. To characterize the enzymatic properties of RNPEPL1 in more detail, the full-length protein was expressed in Escherichia coli and purified to homogeneity. The full-length RNPEPL1 showed rather restricted substrate specificity and basic amino acid preference towards synthetic substrates, which was different from the previously reported specificity characterized by the truncated form. Searching for peptide substrates, we found that several peptides, such as Met-enkephalin and kallidin, were cleaved. RNPEPL1 cleaved bradykinin to de-[Arg]-bradykinin despite the presence of proline at the P2'-position. The enzyme cleaved Met-enkephalin but not dynorphin A1-17. Similar to aminopeptidase B, the full-length RNPEPL1 showed basic amino acid preference towards both synthetic and peptide substrates. In addition to the unusual cleavage of bradykinin, this enzyme shows chain length-dependent cleavage of peptide substrates sharing N-terminal amino acid sequence. This is the first study to report the enzymatic properties of the full-length human RNPEPL1 as an aminopeptidase enzyme.

Exploring Multifunctional Hydrogen-Bonded Organic Framework Materials.

ConspectusHydrogen-bonded organic framework (HOF) materials have provided a new dimension and bright promise as a new platform for developing multifunctional materials. They can be readily self-assembled from their corresponding organic molecules with diverse functional sites such as carboxylic acid and amine groups for their hydrogen bonding and aromatic ones for their weak π···π interactions to stabilize the frameworks. Compared with those established porous materials such as zeolites, metal-organic frameworks (MOFs), and covalent-organic frameworks (COFs), it is much more difficult to stabilize HOFs and thus establish their permanent porosities given the fact that hydrogen bonds are typically weaker than ionic, coordination, and covalent bonds. But it provides the uniqueness of HOF materials in which they can be easily recovered and regenerated through simple recrystallization. HOF materials can also be easily and straightforwardly processed and very compatible with the biomolecules, making them potentially very useful materials for industrial and biomedical applications. The reversible and weak bonding nature of the hydrogen bonds can be readily utilized to construct flexible porous HOF materials in which we can tune the temperature and pressure to control their porosities and, thus, their diverse applications, for example, on gas separations, gas storage, drug delivery, and sensing. Some specific organic functional groups are quite directional for the hydrogen bond formations; for example, carboxylic acid prefers to form a directional dimer, which has enabled us to readily construct reticular porous HOF materials whose pores can be systematically tuned. In this Account, we outline our journey of exploring this new type of porous material by establishing one of the first porous HOFs in 2011 and thus developing its diverse applications. We have been able to use organic molecules with different functional sites, including 2,4-diaminotriazine (DAT), carboxylic acid (COOH), aldehyde (CHO), and cyano (CN), to construct porous HOFs. Through tuning the pore sizes, introducing specific binding sites, and making use of the framework flexibility, we have realized a series of HOF materials for the gas separations of C2H2/C2H4, C2H4/C2H6, C3H6/C3H8, C2H2/CO2, CO2/N2, and Xe/Kr and enantioselective separation of alcohols. To make use of optically active organic molecules, we have developed HOF materials for their luminescent sensing and optical lasing. Our research endeavors on multifunctional HOF materials have initiated extensive research in this emerging research topic among chemistry and materials sciences communities. We foresee that not only many more HOF materials will be developed but novel functions will be fulfilled beyond our imaginations soon.

Structural Analysis and Diversity of Calmodulin-Binding Domains in Membrane and Intracellular Ca2+-ATPases.

The plasma membrane and autoinhibited Ca2+-ATPases contribute to the Ca2+ homeostasis in a wide variety of organisms. The enzymatic activity of these pumps is stimulated by calmodulin, which interacts with the target protein through the calmodulin-binding domain (CaMBD). Most information about this region is related to all calmodulin modulated proteins, which indicates general chemical properties and there is no established relation between Ca2+ pump sequences and taxonomic classification. Thus, the aim of this study was to perform an in silico analysis of the CaMBD from several Ca2+-ATPases, in order to determine their diversity and to detect specific patterns and amino acid selection in different species. Patterns related to potential and confirmed CaMBD were detected using sequences retrieved from the literature. The occurrence of these patterns was determined across 120 sequences from 17 taxonomical classes, which were analyzed by a phylogenetic tree to establish phylogenetic groups. Predicted physicochemical characteristics including hydropathy and net charge were calculated for each group of sequences. 22 Ca2+-ATPases sequences from animals, unicellular eukaryotes, and plants were retrieved from bioinformatic databases. These sequences allow us to establish the Patterns 1(GQILWVRGLTRLQTQ), 3(KNPSLEALQRW), and 4(SRWRRLQAEHVKK), which are present at the beginning of putative CaMBD of metazoan, parasites, and land plants. A pattern 2 (IRVVNAFR) was consistently found at the end of most analyzed sequences. The amino acid preference in the CaMBDs changed depending on the phylogenetic groups, with predominance of several aliphatic and charged residues, to confer amphiphilic properties. The results here displayed show a conserved mechanism to contribute to the Ca2+ homeostasis across evolution and may help to detect putative CaMBDs.

Box-Behnken Optimization and Theoretical Study for Copper Leaching from Scrap Laptop Computer Printed Circuit Board by Malonic Acid and Hydrogen Peroxide

The hydrometallurgical method of copper leaching from scrap electronics is commonly undertaken using pulverized printed circuit boards. Though studies on the use of organic acids like citric and oxalic acids for copper removal are well documented, those on the use of malonic acid are rare. Herein, we report on the investigation of the efficiencies of mixtures of malonic acid and hydrogen peroxide for copper leaching from unpulverized scrap PCB by the Box-Behnken design and the theoretical determination of the most stable copper-malonic acid complex. The average copper content in each unpulverized printed circuit board was found to be (0.84 ± 0.13) mg/g following aqua-regia digestion and atomic absorption spectrophotometry measurement. The conditions at which the highest leaching efficiency of 51.3% was obtained were a malonic acid concentration of 0.5 M, a hydrogen peroxide concentration of 1.0 M, and a contact time of 75 min. The contact time was the most significant individual parameter that influenced the efficiency of copper leached with a p-value of 0.001. Theoretical calculations show that malonic acid prefers to coordinate with copper in a 2:1 malonic acid: copper ratio. This study shows that appreciable amounts of copper can be leached from the minimally processed unpulverized printed circuit board.

Enhancing lactose recognition of a key enzyme in 2'-fucosyllactose synthesis: α-1,2-fucosyltransferase.

2'-Fucosyllactose, a representative oligosaccharide in human milk, is an emerging and promising food and pharmaceutical ingredient due to its powerful health benefits, such as participating in immune regulation, regulation of intestinal flora, etc. To enable economically viable production of 2'-fucosyllactose, different biosynthesis strategies using precursors and pathway enzymes have been developed. The α-1,2-fucosyltransferases are an essential part involved in these strategies, but their strict substrate selectivity and unsatisfactory substrate tolerance are one of the key roadblocks limiting biosynthesis. To tackle this issue, a semi-rational manipulation combining computer-aided designing and screening with biochemical experiments were adopted. The mutant had a 100-fold increase in catalytic efficiency compared to the wild-type. The highest 2'-fucosyllactose yield was up to 0.65 mol mol-1 lactose with a productivity of 2.56 g mL-1 h-1 performed by enzymatic catalysis in vitro. Further analysis revealed that the interactions between the mutant and substrates were reduced. The crucial contributions of wild-type and mutant to substrate recognition ability were closely related to their distinct phylotypes in terms of amino acid preference. It is envisioned that the engineered α-1,2-fucosyltransferase could be harnessed to relieve constraints imposed on the bioproduction of 2'-fucosyllactose and lay a theoretical foundation for elucidating the substrate recognition mechanisms of fucosyltransferases. © 2022 Society of Chemical Industry.

Manganese chloride (MnCl2) induced novel model of Parkinson’s disease in adult Zebrafish; Involvement of oxidative stress, neuroinflammation and apoptosis pathway

Parkinson’s disease (PD) is a progressive neurodegenerative disorder imposing a severe health and socioeconomic burden worldwide. Existing pharmacological approaches for developing PD are poorly developed and do not represent all the characteristics of disease pathology. Developing cost-effective, reliable Zebrafish (ZF) model will meet this gap. The present study was conceived to develop a reliable PD model in the ZF using manganese chloride (MnCl2). Here, we report that chronic exposure to 2 mM MnCl2 for 21 days produced non-motor and motor PD-like symptoms in adult ZF. Compared with control fish, MnCl2-treated fish showed reduced locomotory activity, indicating a deficit in motor function. In the light-dark box test, MnCl2-treated fish exhibited anxiety and depression-like behavior. MnCl2-treated fish exhibited a less olfactory preference for amino acids, indicating olfactory dysfunction. These behavioral symptoms were associated with decreased dopamine and increased DOPAC levels. Furthermore, oxidative stress-mediated apoptotic pathway, decreased brain derived neurotropic factor (BDNF) and increased pro-inflammatory cytokines levels were observed upon chronic exposure to MnCl2 in the brain of ZF. Thus, MnCl2-induced PD in ZF can be a cost-effective PD model in the drug discovery process. Moreover, this model could be potentially utilized to investigate the molecular pathways underlying the multifaceted pathophysiology which leads to PD using relatively inexpensive species. MnCl2 being heavy metal may have other side effects in addition to neurotoxicity. Our model recapitulates most of the hallmarks of PD, but not all pathological processes are involved. Future studies are required to recapitulate the complete pathophysiology of PD.

Karakteristik dan Tingkat Kesukaan Es Krim Tape Ketan (Oryza sativa) dan Singkong (Manihot esculenta) Probiotik

Tape is a traditional fermented food whose benefits can be increased by adding probiotic cells such as Saccharomyces boulardii and Lactobacillus plantarum Dad-13. Tape tends to be easily damaged, so to maintain the viability of probiotics and as a food diversification, tape can be substituted for ice cream dough. The purpose of this research was to determine the effect of substitution and the proportion of substitution of glutinous rice and cassava tape in the manufacture of ice cream on the characteristics and preference level of probiotic ice cream. This research method was carried out using a completely randomized design with a factorial pattern with two factors, the first factor was the type of tape, and the second factor was the proportion of substitution. The type of tape used are white cassava tape, yellow cassava tape, white glutinous rice tape, and black glutinous rice tape with substitution proportions of 0%, 10%, 20%, 30%, and 40%. Analysis of probiotic sticky rice tape and cassava ice cream included water content, total solids, melting time, antioxidant activity, total lactic acid bacteria, total yeast, and preference level. The data obtained will be calculated using the Anova statistikal method. If there is a significant difference between treatments, it is carried out with Duncan's Multiple Range Test significant difference test at a confidence level of α= 5%. The results showed that glutinous rice and cassava tape types and their substitution proportions affected the water content, total solids, melting time, antioxidant activity, total lactic acid bacteria, total yeast, and preference level. Based on the preference level and antioxidant activity, the best ice cream was produced with the substitution of 20% white cassava tape or 20% yellow cassava tape, which had an antioxidant activity of 32.41% and 33.17% RSA and total lactic acid bacteria of 1.79 x 108 and 1.67 x 108 CFU/g, respectively.

Cathepsin B Dipeptidyl Carboxypeptidase and Endopeptidase Activities Demonstrated across a Broad pH Range.

Cathepsin B is a lysosomal protease that participates in protein degradation. However, cathepsin B is also active under neutral pH conditions of the cytosol, nuclei, and extracellular locations. The dipeptidyl carboxypeptidase (DPCP) activity of cathepsin B, assayed with the Abz-GIVR↓AK(Dnp)-OH substrate, has been reported to display an acidic pH optimum. In contrast, the endopeptidase activity, monitored with Z-RR-↓AMC, has a neutral pH optimum. These observations raise the question of whether other substrates can demonstrate cathepsin B DPCP activity at neutral pH and endopeptidase activity at acidic pH. To address this question, global cleavage profiling of cathepsin B with a diverse peptide library was conducted under acidic and neutral pH conditions. Results revealed that cathepsin B has (1) major DPCP activity and modest endopeptidase activity under both acidic and neutral pH conditions and (2) distinct pH-dependent amino acid preferences adjacent to cleavage sites for both DPCP and endopeptidase activities. The pH-dependent cleavage preferences were utilized to design a new Abz-GnVR↓AK(Dnp)-OH DPCP substrate, with norleucine (n) at the P3 position, having improved DPCP activity of cathepsin B at neutral pH compared to the original Abz-GIVR↓AK(Dnp)-OH substrate. The new Z-VR-AMC and Z-ER-AMC substrates displayed improved endopeptidase activity at acidic pH compared to the original Z-RR-AMC. These findings illustrate the new concept that cathepsin B possesses DPCP and endopeptidase activities at both acidic and neutral pH values. These results advance understanding of the pH-dependent cleavage properties of the dual DPCP and endopeptidase activities of cathepsin B that function under different cellular pH conditions.

Possible Charged Residue Switch for Acylglycerol Selectivity of Lipase MAS1.

The amino acid residues lining the substrate binding pocket play quite an important role during the lipase catalytic process. The conversion of those residues might cause a dramatic change in the lipase properties, such as the substrate selectivity of lipase. In our study, T237 residue sitting on the entrance of the catalytic pocket in lipase MAS1 was important for the catalytic performance. When replacing polar Thr with the positively charged Arg, the synthesis ratio of partial glycerides/triglycerides increases to 6.32 rather than 1.21 of MAS1 wild type (WT), as the substrate ratio of glycerol and fatty acids is 1:3. And the fatty acid preference shifted to long-chain substrates for mutant T237R rather than middle-chain substrates for MAS1 WT. Molecular docking analysis revealed that hydrophobic and side chain properties of Arg might contribute to the change of the MAS1 lipase catalytic performance. This work would pave a way for the accurate rational transformation of the lipases to produce value lipid for industrial application.

Severe Asymptomatic Essential Fatty Acid Deficiency (EFAD) in a Patient Enrolled in a Quality Improvement Project for HPN Patients (QIP-PN)

ObjectivesIntroduction. A 97-year-old, 47 kg female with a history of dysphasia, and failed enteral feeding was on HPN for 35 months. PN provided 150 grams dextrose, 75 grams amino acids, electrolytes, MVI and trace elements. Intravenous lipid emulsion (ILE) was held because liver dysfunction and line infections.MethodsThe patient enrolled in Amerita QIP-PN program. Essential fatty acid and trace element levels were obtained.ResultsSevere deficiencies of linoleic (c18:2w6; 524.3; (nl) 2653.4–6130.3 umol/L) and ἀ-linolenic acids (c18:3w3; 4.12; nl 26.1–150.1) were detected. Eicosatrienoic (Mead) acid (c20:3w9) was markedly elevated at 135.33 (nl 10.3–41.3). The eicosatrienoic to arachidonic acid (triene to tetraene) ratio was markedly elevated (0.27; nl 0.02–0.05). Stearic (267.2; nl 590.2 – 1377.2) and arachidic (8.86; nl 16.8 – 38.5) acids were low. Total polyunsaturated (1.6; nl 3.57 – 8.11) and total n-6 fatty acids (1.3; nl 3.3 – 7.1) were low. Seven fatty acid levels were elevated, apparently from de novo lipogenesis in the presence of insulin: myristic = 299.15 (nl 39.4 – 258.2), hexadecanoic = 300.41(nl 19.82 – 59.93), palmetoleic = 1197.60 (nl 68.5 – 570.2), vaccenic = 458.11 (nl 84.82 – 260.8), docosapentanoic = 32.8 (nl 9.2–32.1), docosenoic = 20.88 (nl 5.73 – 11.92). Interventions. Four-oil ILE was added as 20 grams once weekly, providing ∼4 grams of linoleic and ∼0.5 grams of ἀ-linolenic acids. Follow up free fatty acids showed normalization of ἀ-linolenic (27.07) and improvement in linoleic (889.74) and mead (60.34) acids. The triene to tetraene ratio improved to 0.17.ConclusionsAn elevated Mead acid level and elevated triene to tetraene ratio confirmed severe EFAD. Mead acid is produced from the elongation of oleic acid (n-9). The n-3, n-6, and n-9 fatty acids all compete for the same desaturases. But the desaturase enzymes have a preference for the fatty acids based on their n-terminal structure. The sequence is n-3 > n-6 > n-9. Therefore, increased Mead acid (20:3n-9) synthesis occurs only with very low availability of n-3 and n-6 fatty acids. In severe EFAD, Mead acid serves as a precursor to specific prostaglandins and leukotrienes via the cycloxygenase and lipoxygenase pathways. This case highlights the value of the QIP-PN in identifying EFAD in long-term HPN patients.Funding SourcesNone.

Proteomic analysis reveals USP7 as a novel regulator of palmitic acid-induced hepatocellular carcinoma cell death

Nutrient surplus and consequent free fatty acid accumulation in the liver cause hepatosteatosis. The exposure of free fatty acids to cultured hepatocyte and hepatocellular carcinoma cell lines induces cellular stress, organelle adaptation, and subsequent cell death. Despite many studies, the mechanism associated with lipotoxicity and subsequent cell death still remains poorly understood. Here, we have used the proteomics approach to circumvent the mechanism for lipotoxicity using hepatocellular carcinoma cells as a model. Our quantitative proteomics data revealed that ectopic lipids accumulation in cells severely affects the ubiquitin-proteasomal system. The palmitic acid (PA) partially lowered the expression of deubiquitinating enzyme USP7 which subsequently destabilizes p53 and promotes mitotic entry of cells. Our global phosphoproteomics analysis also provides strong evidence of an altered cell cycle checkpoint proteins’ expression that abrogates early G2/M checkpoints recovery with damaged DNA and induced mitotic catastrophe leading to hepatocyte death. We observe that palmitic acid prefers apoptosis-inducing factor (AIF) mediated cell death by depolarizing mitochondria and translocating AIF to the nucleus. In summary, the present study provides evidence of PA-induced hepatocellular death mediated by deubiquitinase USP7 downregulation and subsequent mitotic catastrophe.

T cells discriminate between groups C1 and C2 HLA-C.

Dimorphic amino acids at positions 77 and 80 delineate HLA-C allotypes into two groups, C1 and C2, which associate with disease through interactions with C1 and C2-specific natural killer cell receptors. How the C1/C2 dimorphism affects T cell recognition is unknown. Using HLA-C allotypes that differ only by the C1/C2-defining residues, we found that KRAS-G12D neoantigen-specific T cell receptors (TCRs) discriminated between C1 and C2 presenting the same KRAS-G12D peptides. Structural and functional experiments, and immunopeptidomics analysis revealed that Ser77 in C1 and Asn77 in C2 influence amino acid preference near the peptide C-terminus (pΩ), including the pΩ-1 position, in which C1 favors small and C2 prefers large residues. This resulted in weaker TCR affinity for KRAS-G12D-bound C2-HLA-C despite conserved TCR contacts. Thus, the C1/C2 dimorphism on its own impacts peptide presentation and HLA-C-restricted T cell responses, with implications in disease, including adoptive T cell therapy targeting KRAS-G12D-induced cancers.

The repertoire of the elongation of very long-chain fatty acids (Elovl) protein family is conserved in tambaqui (Colossoma macropomum): Gene expression profiles offer insights into the sexual differentiation process

Elongation of very long-chain fatty acids (Elovl) proteins are critical players in the regulation of the length of a fatty acid. At present, eight members of the Elovl family (Elovl1–8), displaying a characteristic fatty acid substrate specificity, have been identified in vertebrates, including teleost fish. In general, Elovl1, Elovl3, Elovl6 and Elovl7 exhibit a substrate preference for saturated and monounsaturated fatty acids, while Elovl2, Elovl4, Elovl5 and Elovl8 use polyunsaturated fatty acids (PUFA) as substrates. PUFA elongases have received considerable attention in aquatic animals due to their involvement in the conversion of C18 PUFAs to long-chain polyunsaturated fatty acids (LC-PUFA). Here, we identified the full repertoire of elovl genes in the tambaqui Colossoma macropomum genome. A detailed phylogenetic and synteny analysis suggests a conservation of these genes among teleosts. Furthermore, based on RNAseq gene expression data, we discovered a gender bias expression of elovl genes during sex differentiation of tambaqui, toward future males. Our findings suggest a role of Elovl enzymes and fatty acid metabolism in tambaqui sexual differentiation.

Quantitative structure–activity relationship modeling reveals the minimal sequence requirement and amino acid preference of sirtuin-1’s deacetylation substrates in diabetes mellitus

Sirtuin 1 (SIRT1) is a nicotinamide adenine dinucleotide ([Formula: see text]-dependent deacetylase involved in multiple glucose metabolism pathways and plays an important role in the pathogenesis of diabetes mellitus (DM). The enzyme specifically recognizes its deacetylation substrates' peptide segments containing a central acetyl-lysine residue as well as a number of amino acids flanking the central residue. In this study, we attempted to ascertain the minimal sequence requirement (MSR) around the central acetyl-lysine residue of SIRT1 substrate-recognition sites as well as the amino acid preference (AAP) at different residues of the MSR window through quantitative structure-activity relationship (QSAR) strategy, which would benefit our understanding of SIRT1 substrate specificity at the molecular level and is also helpful to rationally design substrate-mimicking peptidic agents against DM by competitively targeting SIRT1 active site. In this procedure, a large-scale dataset containing 6801 13-mer acetyl-lysine peptides (and their SIRT1-catalyized deacetylation activities) were compiled to train 10 QSAR regression models developed by systematic combination of machine learning methods (PLS and SVM) and five amino acids descriptors (DPPS, T-scale, MolSurf, [Formula: see text]-score, and FASGAI). The two best QSAR models (PLS+FASGAI and SVM+DPPS) were then employed to statistically examine the contribution of residue positions to the deacetylation activity of acetyl-lysine peptide substrates, revealing that the MSR can be represented by 5-mer acetyl-lysine peptides that meet a consensus motif [Formula: see text][Formula: see text][Formula: see text](AcK)0[Formula: see text]. Structural analysis found that the [Formula: see text] and (AcK)0 residues are tightly packed against the enzyme active site and confer both stability and specificity for the enzyme-substrate complex, whereas the [Formula: see text], [Formula: see text] and [Formula: see text] residues are partially exposed to solvent but can also effectively stabilize the complex system. Subsequently, a systematic deacetylation activity change profile (SDACP) was created based on QSAR modeling, from which the AAP for each residue position of MSR was depicted. With the profile, we were able to rationally design an SDACP combinatorial library with promising deacetylation activity, from which nine MSR acetyl-lysine peptides as well as two known SIRT1 acetyl-lysine peptide substrates were tested by using SIRT1 deacetylation assay. It is revealed that the designed peptides exhibit a comparable or even higher activity than the controls, although the former is considerably shorter than the latter.

Sialoglycan-binding patterns of bacterial AB5 toxin B subunits correlate with host range and toxicity, indicating evolution independent of A subunits

Many pathogenic bacteria secrete AB5 toxins that can be virulence factors. Cytotoxic A subunits are delivered to the cytosol following B subunit binding to specific host cell surface glycans. Some B subunits are not associated with A subunits, for example, YpeB of Yersinia pestis, the etiologic agent of plague. Plague cannot be eradicated because of Y. pestis' adaptability to numerous hosts. We previously showed selective binding of other B5 pentamers to a sialoglycan microarray, with sialic acid (Sia) preferences corresponding to those prominently expressed by various hosts, for example, N-acetylneuraminic acid (Neu5Ac; prominent in humans) or N-glycolylneuraminic acid (Neu5Gc; prominent in ruminant mammals and rodents). Here, we report that A subunit phylogeny evolved independently of B subunits and suggest a future B subunit nomenclature based on bacterial species names. We also found via phylogenetic analysis of B subunits, which bind Sias, that homologous molecules show poor correlation with species phylogeny. These data indicate ongoing lateral gene transfers between species, including mixing of A and B subunits. Consistent with much broader host range of Y. pestis, we show that YpeB recognizes all mammalian Sia types, except for 4-O-acetylated ones. Notably, YpeB alone causes dose-dependent cytotoxicity, which is abolished by a mutation (Y77F) eliminating Sia recognition, suggesting that cell proliferation and death are promoted via lectin-like crosslinking of cell surface sialoglycoconjugates. These findings help explain the host range of Y. pestis and could be important for pathogenesis. Overall, our data indicate ongoing rapid evolution of both host Sias and pathogen toxin-binding properties.

Characterization of the class IIa histone deacetylases substrate specificity.

Class IIa histone deacetylases (HDACs) play critical roles in vertebrate development and physiology, yet direct evidence of their intrinsic deacetylase activity and on substrate specificity regarding the peptide sequence is still missing. In this study, we designed and synthesized a combinatorial peptide library allowing us to profile class IIa HDACs sequence specificity at positions +3 through -3 from the central lysine modified by the well-accepted trifluoroacetyl function. Our data revealed a strong preference for bulky aromatic acids directly flanking the central trifluoroacetyllysine, while all class IIa HDACs disfavor positively charged residues and proline at the +1/-1 positions. The chemical nature of amino acid residues N-terminally to the central trifluoroacetyllysine has a more profound effect on substrate recognition as compared to residues located C-terminally. These findings were validated by designing selected favored and disfavored peptide sequences, with the favored ones are accepted with catalytic efficacy of 75000 and 525000M-1 s-1 for HDAC7 and HDAC5, respectively. Results reported here could help in developing class IIa HDACs inhibitors and also in the search for new natural class IIa HDACs substrates.

Cytochrome P450 168A1 from Pseudomonas aeruginosa is involved in the hydroxylation of biologically relevant fatty acids.

The cytochrome P450 CYP168A1 from Pseudomonas aeruginosa was cloned and expressed in Escherichia coli followed by purification and characterization of function. CYP168A1 is a fatty acid hydroxylase that hydroxylates saturated fatty acids, including myristic (0.30 min-1), palmitic (1.61 min-1) and stearic acids (1.24 min-1), at both the ω-1- and ω-2-positions. However, CYP168A1 only hydroxylates unsaturated fatty acids, including palmitoleic (0.38 min-1), oleic (1.28 min-1) and linoleic acids (0.35 min-1), at the ω-1-position. CYP168A1 exhibited a catalytic preference for palmitic, oleic and stearic acids as substrates in keeping with the phosphatidylcholine-rich environment deep in the lung that is colonized by P. aeruginosa.

Soil phytoremediation reveals alteration in soil microbial metabolic activities along time gradient of cover crop mulching

The vitality and diversity of soil microbial metabolism are the core of soil function expression, cover crop is an environmentally friendly agricultural production practice; however, shifts in soil microbial metabolic activities along time gradient of cover crop remain unclear. Here, we used metagenomic and biological techniques to investigate soil microbial potential function and carbon (C) source utilization capacity in the time series of white clover (WC, Trifolium repens L.) for 6, 10, and 15 years in a typical semiarid apple orchard. Conventional tillage (CT) was taken as the control. This study demonstrated that living mulch 6 years of WC had little effect on soil microbial functions. However, after 10 and 15 years of crop cover, an enrichment of genes related to amino acid metabolism, carbon cycle, and nitrogen metabolism was observed in soil microorganisms. Furthermore, average well color development (AWCD) was increased in 10 and 15 years of cover crop, soil microbiome exhibited a stronger preference for carbohydrates, amino acids, and polymers as C sources. The results mainly provided insight into the variation character of microbial metabolic function under increasing duration of cover crop.

Evolution of Amino Acid Propensities under Stability-Mediated Epistasis.

Site-specific amino acid preferences are influenced by the genetic background of the protein. The preferences for resident amino acids are expected to, on average, increase over time because of replacements at other sites—a nonadaptive phenomenon referred to as the “evolutionary Stokes shift.” Alternatively, decreases in resident amino acid propensity have recently been viewed as evidence of adaptations to external environmental changes. Using population genetics theory and thermodynamic stability constraints, we show that nonadaptive evolution can lead to both positive and negative shifts in propensities following the fixation of an amino acid, emphasizing that the detection of negative shifts is not conclusive evidence of adaptation. By examining propensity shifts from when an amino acid is first accepted at a site until it is subsequently replaced, we find that 50% of sites show a decrease in the propensity for the newly resident amino acid while the remaining sites show an increase. Furthermore, the distributions of the magnitudes of positive and negative shifts were comparable. Preferences were often conserved via a significant negative autocorrelation in propensity changes—increases in propensities often followed by decreases, and vice versa. Lastly, we explore the underlying mechanisms that lead propensities to fluctuate. We observe that stabilizing replacements increase the mutational tolerance at a site and in doing so decrease the propensity for the resident amino acid. In contrast, destabilizing substitutions result in more rugged fitness landscapes that tend to favor the resident amino acid. In summary, our results characterize propensity trajectories under nonadaptive stability-constrained evolution against which evidence of adaptations should be calibrated.