Integrative annotation and knowledge discovery of kinase post-translational modifications and cancer-associated mutations through federated protein ontologies and resources.

Many bioinformatics resources with unique perspectives on the protein landscape are currently available. However, generating new knowledge from these resources requires interoperable workflows that support cross-resource queries. In this study, we employ federated queries linking information from the Protein Kinase Ontology, iPTMnet, Protein Ontology, neXtProt, and the Mouse Genome Informatics to identify key knowledge gaps in the functional coverage of the human kinome and prioritize understudied kinases, cancer variants and post-translational modifications (PTMs) for functional studies. We identify 32 functional domains enriched in cancer variants and PTMs and generate mechanistic hypotheses on overlapping variant and PTM sites by aggregating information at the residue, protein, pathway and species level from these resources. We experimentally test the hypothesis that S768 phosphorylation in the C-helix of EGFR is inhibitory by showing that oncogenic variants altering S768 phosphorylation increase basal EGFR activity. In contrast, oncogenic variants altering conserved phosphorylation sites in the \u27hydrophobic motif\u27 of PKCβII (S660F and S660C) are loss-of-function in that they reduce kinase activity and enhance membrane translocation. Our studies provide a framework for integrative, consistent, and reproducible annotation of the cancer kinomes. Sci Rep 2018 Apr 25; 8(1):6518

Composition and diversity of the subgingival microbiome and its relationship with age in postmenopausal women: an epidemiologic investigation

Abstract Background The extent to which the composition and diversity of the oral microbiome varies with age is not clearly understood. Methods The 16S rRNA gene of subgingival plaque in 1219 women, aged 53–81 years, was sequenced and its taxonomy annotated against the Human Oral Microbiome Database (v.14.5). Composition of the subgingival microbiome was described in terms of centered log(2)-ratio (CLR) transformed OTU values, relative abundance, and prevalence. Correlations between microbiota abundance and age were evelauted using Pearson Product Moment correlations. P-values were corrected for multiple testing using the Bonferroni method. Results Of the 267 species identified overall, Veillonella dispar was the most abundant bacteria when described by CLR OTU (mean 8.3) or relative abundance (mean 8.9%); whereas Streptococcus oralis, Veillonella dispar and Veillonella parvula were most prevalent (100%, all) when described as being present at any amount. Linear correlations between age and several CLR OTUs (Pearson r = − 0.18 to 0.18), of which 82 (31%) achieved statistical significance (P < 0.05). The correlations lost significance following Bonferroni correction. Twelve species that differed across age groups (each corrected P < 0.05); 5 (42%) were higher in women ages 50–59 compared to ≥70 (corrected P < 0.05), and 7 (48%) were higher in women 70 years and older. Conclusions We identified associations between several bacterial species and age across the age range of postmenopausal women studied. Understanding the functions of these bacteria could identify intervention targets to enhance oral health in later life.https://deepblue.lib.umich.edu/bitstream/2027.42/152202/1/12903_2019_Article_906.pd

Safety and immunogenicity of the protein-based PHH-1V compared to BNT162b2 as a heterologous SARS-CoV-2 booster vaccine in adults vaccinated against COVID-19 : a multicentre, randomised, double-blind, non-inferiority phase IIb trial

A SARS-CoV-2 protein-based heterodimer vaccine, PHH-1V, has been shown to be safe and well-tolerated in healthy young adults in a first-in-human, Phase I/IIa study dose-escalation trial. Here, we report the interim results of the Phase IIb HH-2, where the immunogenicity and safety of a heterologous booster with PHH-1V is assessed versus a homologous booster with BNT162b2 at 14, 28 and 98 days after vaccine administration. The HH-2 study is an ongoing multicentre, randomised, active-controlled, double-blind, non-inferiority Phase IIb trial, where participants 18 years or older who had received two doses of BNT162b2 were randomly assigned in a 2:1 ratio to receive a booster dose of vaccine-either heterologous (PHH-1V group) or homologous (BNT162b2 group)-in 10 centres in Spain. Eligible subjects were allocated to treatment stratified by age group (18-64 versus ≥65 years) with approximately 10% of the sample enrolled in the older age group. The primary endpoints were humoral immunogenicity measured by changes in levels of neutralizing antibodies (PBNA) against the ancestral Wuhan-Hu-1 strain after the PHH-1V or the BNT162b2 boost, and the safety and tolerability of PHH-1V as a boost. The secondary endpoints were to compare changes in levels of neutralizing antibodies against different variants of SARS-CoV-2 and the T-cell responses towards the SARS-CoV-2 spike glycoprotein peptides. The exploratory endpoint was to assess the number of subjects with SARS-CoV-2 infections ≥14 days after PHH-1V booster. This study is ongoing and is registered with , . From 15 November 2021, 782 adults were randomly assigned to PHH-1V (n = 522) or BNT162b2 (n = 260) boost vaccine groups. The geometric mean titre (GMT) ratio of neutralizing antibodies on days 14, 28 and 98, shown as BNT162b2 active control versus PHH-1V, was, respectively, 1.68 (p < 0.0001), 1.31 (p = 0.0007) and 0.86 (p = 0.40) for the ancestral Wuhan-Hu-1 strain; 0.62 (p < 0.0001), 0.65 (p < 0.0001) and 0.56 (p = 0.003) for the Beta variant; 1.01 (p = 0.92), 0.88 (p = 0.11) and 0.52 (p = 0.0003) for the Delta variant; and 0.59 (p ≤ 0.0001), 0.66 (p < 0.0001) and 0.57 (p = 0.0028) for the Omicron BA.1 variant. Additionally, PHH-1V as a booster dose induced a significant increase of CD4 + and CD8 + T-cells expressing IFN-γ on day 14. There were 458 participants who experienced at least one adverse event (89.3%) in the PHH-1V and 238 (94.4%) in the BNT162b2 group. The most frequent adverse events were injection site pain (79.7% and 89.3%), fatigue (27.5% and 42.1%) and headache (31.2 and 40.1%) for the PHH-1V and the BNT162b2 groups, respectively. A total of 52 COVID-19 cases occurred from day 14 post-vaccination (10.14%) for the PHH-1V group and 30 (11.90%) for the BNT162b2 group (p = 0.45), and none of the subjects developed severe COVID-19. Our interim results from the Phase IIb HH-2 trial show that PHH-1V as a heterologous booster vaccine, when compared to BNT162b2, although it does not reach a non-inferior neutralizing antibody response against the Wuhan-Hu-1 strain at days 14 and 28 after vaccination, it does so at day 98. PHH-1V as a heterologous booster elicits a superior neutralizing antibody response against the previous circulating Beta and the currently circulating Omicron BA.1 SARS-CoV-2 variants in all time points assessed, and for the Delta variant on day 98 as well. Moreover, the PHH-1V boost also induces a strong and balanced T-cell response. Concerning the safety profile, subjects in the PHH-1V group report significantly fewer adverse events than those in the BNT162b2 group, most of mild intensity, and both vaccine groups present comparable COVID-19 breakthrough cases, none of them severe. HIPRA SCIENTIFIC, S.L.U

Selective Synthesis of Site-Differentiated Fe4S4 and Fe6S6 Clusters

Obtaining rational control over the structure and nuclearity of metalloclusters is an ongoing challenge in synthetic Fe-S cluster chemistry. We report a new family of tridentate imidazolin-2-imine ligands L(NIm R ) 3 that can bind [Fe 4 S 4 ] 2+ or [Fe 6 S 6 ] 3+ clusters, depending on the steric profile of the ligand and the reaction stoichiometry. A high-yielding synthetic route to L(NIm R ) 3 ligands (where R is the imidazolyl N substituents) from trianiline and 2-chloroimidazolium precursors is described. For L(NIm Me ) 3 (tris(1,3,5-(3-(N,N-dimethyl-4,5-diphenylimidazolin-2-imino)phenylmethyl))benzene), metalation with 1 equiv of [Ph 4 P] 2 [Fe 4 S 4 Cl 4 ] and 3 equiv of NaBPh 4 furnishes a mixture of products, but adjusting the stoichiometry to 1.5 equiv of [Ph 4 P] 2 [Fe 4 S 4 Cl 4 ] provides (L(NIm Me ) 3 )Fe 6 S 6 Cl 6 in high yield. Formation of an [Fe 6 S 6 ] 3+ cluster using L(NIm Tol ) 3 (tris(1,3,5-(3-(N,N-bis(4-methylphenyl)-4,5-diphenylimidazolin-2-imino)phenylmethyl))benzene) is not observed; instead, the [Fe 4 S 4 ] 2+ cluster [(L(NIm Tol ) 3 )(Fe 4 S 4 Cl)][BPh 4 ] is cleanly generated when 1 equiv of [Ph 4 P] 2 [Fe 4 S 4 Cl 4 ] is employed. The selectivity for cluster nuclearity is rationalized by the orientation of the imidazolyl rings whereby long N-imidazolyl substituents preclude formation of [Fe 6 S 6 ] 3+ clusters but not [Fe 4 S 4 ] 2+ clusters. Thus, the structure and nuclearity of L(NIm R ) 3 -bound Fe-S clusters may be selectively controlled through rational modification the ligand's substituents

Additional file 2: of Classifying kinase conformations using a machine learning approach

Machine learning dataset. This file contains the measured Φ, Ψ, χ1, and pseudo-dihedral angles used in the analysis. (ZIP 15492 kb

Adipocyte progenitor cells initiate monocyte chemoattractant protein-1-mediated macrophage accumulation in visceral adipose tissue

Objective: Macrophages are important producers of obesity-induced MCP-1; however, initial obesity-induced increases in MCP-1 production precede M1 macrophage accumulation in visceral adipose tissue (VAT). The initial cellular source of obesity-induced MCP-1 in vivo is currently unknown. Preliminary reports based on in vitro studies of preadipocyte cell lines and adherent stroma-vascular fraction cells suggest that resident stromal cells express MCP-1. In the past several years, elegant methods of identifying adipocyte progenitor cells (AdPCs) have become available, making it possible to study these cells in vivo. We have previously published that global deletion of transcription factor Inhibitor of Differentiation 3 (Id3) attenuates high fat diet-induced obesity, but it is unclear if Id3 plays a role in diet-induced MCP-1 production. We sought to determine the initial cellular source of MCP-1 and identify molecular regulators mediating MCP-1 production. Methods: Id3+/+ and Id3−/− mice were fed either a standard chow or HFD for varying lengths of time. Flow cytometry, semi-quantitative real-time PCR, ELISAs and adoptive transfers were used to assess the importance of AdPCs during diet-induced obesity. Flow cytometry was also performed on a cohort of 14 patients undergoing bariatric surgery. Results: Flow cytometry identified committed CD45−CD31−Ter119−CD29+CD34+Sca-1+CD24− adipocyte progenitor cells as producers of high levels of MCP-1 in VAT. High-fat diet increased AdPC numbers, an effect dependent on Id3. Loss of Id3 increased p21Cip1 levels and attenuated AdPC proliferation, resulting in reduced MCP-1 and M1 macrophage accumulation in VAT, compared to Id3+/+ littermate controls. AdPC rescue by adoptive transfer of 50,000 Id3+/+ AdPCs into Id3−/− recipient mice increased MCP-1 levels and M1 macrophage number in VAT. Additionally, flow cytometry identified MCP-1-producing CD45−CD31−CD34+CD44+CD90+ AdPCs in human omental and subcutaneous adipose tissue, with a higher percentage in omental adipose. Furthermore, high surface expression of CD44 marked abundant MCP-1 producers, only in visceral adipose tissue. Conclusions: This study provides the first in vivo evidence, to our knowledge, that committed AdPCs in VAT are the initial source of obesity-induced MCP-1 and identifies the helix-loop-helix transcription factor Id3 as a critical regulator of p21Cip1 expression, AdPC proliferation, MCP-1 expression and M1 macrophage accumulation in VAT. Inhibition of Id3 and AdPC expansion, as well as CD44 expression in human AdPCs, may serve as unique therapeutic targets for the regulation of adipose tissue inflammation

Characterization by ENDOR Spectroscopy of the Iron–Alkyl Bond in a Synthetic Counterpart of Organometallic Intermediates in Radical SAM Enzymes

Members of the radical S-adenosyl-l-methionine (SAM) enzyme superfamily initiate a broad spectrum of radical transformations through reductive cleavage of SAM by a [4Fe–4S]1+ cluster it coordinates to generate the reactive 5′-deoxyadenosyl radical (5′-dAdo•). However, 5′-dAdo• is not directly liberated for reaction and instead binds to the unique Fe of the cluster to create the catalytically competent S = 1/2 organometallic intermediate Ω. An alternative mode of reductive SAM cleavage, especially seen photochemically, instead liberates CH3•, which forms the analogous S = 1/2 organometallic intermediate with an Fe–CH3 bond, ΩM. The presence of a covalent Fe–C bond in both structures was established by the ENDOR observation of 13C and 1H hyperfine couplings to the alkyl groups that show isotropic components indicative of Fe–C bond covalency. The synthetic [Fe4S4]3+–CH3 cluster, M-CH3, is a crystallographically characterized analogue to ΩM that exhibits the same [Fe4S4]3+ cluster state as Ω and ΩM, and thus an analysis of its spectroscopic propertiesand comparison with those of Ω and ΩMcan be grounded in its crystal structure. We report cryogenic (2 K) EPR and 13C/1/2H ENDOR measurements on isotopically labeled M-CH3. At low temperatures, the complex exhibits EPR spectra from two distinct conformers/subpopulations. ENDOR shows that at 2 K, one contains a static methyl, but in the other, the methyl undergoes rapid tunneling/hopping rotation about the Fe–CH3 bond. This generates an averaged hyperfine coupling tensor whose analysis requires an extended treatment of rotational averaging. The methyl group 13C/1/2H hyperfine couplings are compared with the corresponding values for Ω and ΩM

Subgingival microbiome is associated with alveolar bone loss measured 5 years later in postmenopausal women

BackgroundThe aim of this study was to quantify the association between subgingival microbiota and periodontal disease progression in older women, for which limited published data exist.MethodsA total of 1016 postmenopausal women, aged 53 to 81 years, completed baseline (1997 to 2001) and 5- year (2002 to 2006) dental exams that included probing depth, clinical attachment level, gingival bleeding, and radiographic alveolar crestal height (ACH). Baseline microbiota were measured in subgingival plaque using 16S rRNA sequencing. Associations between 52 microbiota we previously found statistically significantly associated with clinical periodontal disease at baseline, were examined with disease progression. The traditional Socransky microbiota complexes also were evaluated. Side- by- side radiograph comparisons were used to define progression as - ¥2 teeth with - ¥1 mm ACH loss or - ¥1 new tooth loss to periodontitis. The association between baseline centered log(2) ratio transformed microbial relative abundances and 5- year periodontal disease progression was measured with generalized linear models.ResultsOf 36 microbiota we previously showed were elevated in moderate/severe disease at baseline, 24 had statistically significantly higher baseline mean relative abundance in progressing compared with non- progressing women (P < .05, all); which included all Socransky red bacteria (P. gingivalis, T. forsythia, T. denticola). Of 16 microbiota elevated in none/mild disease at baseline, five had statistically significantly lower baseline abundance in non- progressing compared with progressing women (P < 0.05, all), including one Socransky yellow bacteria (S. oralis). When adjusted for baseline age, socioeconomic status, and self- rated general health status, odds ratios for 5- year progression ranged from 1.18 to 1.51 (per 1- standard deviation increment in relative abundance) for microbiota statistically significantly (P < 0.05) positively associated with progression, and from 0.77 to 0.82 for those statistically significantly (P < 0.05) inversely associated with progression. These associations were similar when stratified on baseline levels of pocket depth, gingival bleeding, ACH, and smoking status.ConclusionsThese prospective results affirm clearly that subgingival microbiota are measurably elevated several years prior to progression of alveolar bone loss, and include antecedent elevations in previously undocumented taxa additional to known Socransky pathogenic complexes.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/167800/1/jper10670_am.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/167800/2/jper10670.pd

Subgingival microbiome is associated with alveolar bone loss measured 5 years later in postmenopausal women

BackgroundThe aim of this study was to quantify the association between subgingival microbiota and periodontal disease progression in older women, for which limited published data exist.MethodsA total of 1016 postmenopausal women, aged 53 to 81 years, completed baseline (1997 to 2001) and 5- year (2002 to 2006) dental exams that included probing depth, clinical attachment level, gingival bleeding, and radiographic alveolar crestal height (ACH). Baseline microbiota were measured in subgingival plaque using 16S rRNA sequencing. Associations between 52 microbiota we previously found statistically significantly associated with clinical periodontal disease at baseline, were examined with disease progression. The traditional Socransky microbiota complexes also were evaluated. Side- by- side radiograph comparisons were used to define progression as - ¥2 teeth with - ¥1 mm ACH loss or - ¥1 new tooth loss to periodontitis. The association between baseline centered log(2) ratio transformed microbial relative abundances and 5- year periodontal disease progression was measured with generalized linear models.ResultsOf 36 microbiota we previously showed were elevated in moderate/severe disease at baseline, 24 had statistically significantly higher baseline mean relative abundance in progressing compared with non- progressing women (P < .05, all); which included all Socransky red bacteria (P. gingivalis, T. forsythia, T. denticola). Of 16 microbiota elevated in none/mild disease at baseline, five had statistically significantly lower baseline abundance in non- progressing compared with progressing women (P < 0.05, all), including one Socransky yellow bacteria (S. oralis). When adjusted for baseline age, socioeconomic status, and self- rated general health status, odds ratios for 5- year progression ranged from 1.18 to 1.51 (per 1- standard deviation increment in relative abundance) for microbiota statistically significantly (P < 0.05) positively associated with progression, and from 0.77 to 0.82 for those statistically significantly (P < 0.05) inversely associated with progression. These associations were similar when stratified on baseline levels of pocket depth, gingival bleeding, ACH, and smoking status.ConclusionsThese prospective results affirm clearly that subgingival microbiota are measurably elevated several years prior to progression of alveolar bone loss, and include antecedent elevations in previously undocumented taxa additional to known Socransky pathogenic complexes.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/167800/1/jper10670_am.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/167800/2/jper10670.pd