Connections Network: Harnessing the Collective Influence of Grassroots Leaders to Address Health-Related Problems in Hawkins and Hancock County, TN

In March 2021, grassroots leaders in two counties in northeast Tennessee formed a new network called Connections. Leaders are working to strengthen the capacity of the network and member organizations by promoting partnerships as vital to address effectively rural social determinants of health. Connections provides network members with capacity-building tools and resources, including two funding opportunities, to achieve their missions and sustain impact. Network members are also aligning around common goals to address the socioeconomic conditions affecting health outcomes. Connections will utilize findings from network activities and collaborations to identify synergies that can accelerate improvements in community health and well-being

How accurate and statistically robust are catalytic site predictions based on closeness centrality?

<p>Abstract</p> <p>Background</p> <p>We examine the accuracy of enzyme catalytic residue predictions from a network representation of protein structure. In this model, amino acid α-carbons specify vertices within a graph and edges connect vertices that are proximal in structure. Closeness centrality, which has shown promise in previous investigations, is used to identify important positions within the network. Closeness centrality, a global measure of network centrality, is calculated as the reciprocal of the average distance between vertex <it>i </it>and all other vertices.</p> <p>Results</p> <p>We benchmark the approach against 283 structurally unique proteins within the Catalytic Site Atlas. Our results, which are inline with previous investigations of smaller datasets, indicate closeness centrality predictions are statistically significant. However, unlike previous approaches, we specifically focus on residues with the very best scores. Over the top five closeness centrality scores, we observe an average true to false positive rate ratio of 6.8 to 1. As demonstrated previously, adding a solvent accessibility filter significantly improves predictive power; the average ratio is increased to 15.3 to 1. We also demonstrate (for the first time) that filtering the predictions by residue identity improves the results even more than accessibility filtering. Here, we simply eliminate residues with physiochemical properties unlikely to be compatible with catalytic requirements from consideration. Residue identity filtering improves the average true to false positive rate ratio to 26.3 to 1. Combining the two filters together has little affect on the results. Calculated p-values for the three prediction schemes range from 2.7E-9 to less than 8.8E-134. Finally, the sensitivity of the predictions to structure choice and slight perturbations is examined.</p> <p>Conclusion</p> <p>Our results resolutely confirm that closeness centrality is a viable prediction scheme whose predictions are statistically significant. Simple filtering schemes substantially improve the method's predicted power. Moreover, no clear effect on performance is observed when comparing ligated and unligated structures. Similarly, the CC prediction results are robust to slight structural perturbations from molecular dynamics simulation.</p

Changes in Lysozyme Flexibility upon Mutation Are Frequent, Large and Long-Ranged

We investigate changes in human c-type lysozyme flexibility upon mutation via a Distance Constraint Model, which gives a statistical mechanical treatment of network rigidity. Specifically, two dynamical metrics are tracked. Changes in flexibility index quantify differences within backbone flexibility, whereas changes in the cooperativity correlation quantify differences within pairwise mechanical couplings. Regardless of metric, the same general conclusions are drawn. That is, small structural perturbations introduced by single point mutations have a frequent and pronounced affect on lysozyme flexibility that can extend over long distances. Specifically, an appreciable change occurs in backbone flexibility for 48% of the residues, and a change in cooperativity occurs in 42% of residue pairs. The average distance from mutation to a site with a change in flexibility is 17–20 Å. Interestingly, the frequency and scale of the changes within single point mutant structures are generally larger than those observed in the hen egg white lysozyme (HEWL) ortholog, which shares 61% sequence identity with human lysozyme. For example, point mutations often lead to substantial flexibility increases within the β-subdomain, which is consistent with experimental results indicating that it is the nucleation site for amyloid formation. However, β-subdomain flexibility within the human and HEWL orthologs is more similar despite the lowered sequence identity. These results suggest compensating mutations in HEWL reestablish desired properties

Hydrogen bond networks determine emergent mechanical and thermodynamic properties across a protein family

<p>Abstract</p> <p>Background</p> <p>Gram-negative bacteria use periplasmic-binding proteins (bPBP) to transport nutrients through the periplasm. Despite immense diversity within the recognized substrates, all members of the family share a common fold that includes two domains that are separated by a conserved hinge. The hinge allows the protein to cycle between open (apo) and closed (ligated) conformations. Conformational changes within the proteins depend on a complex interplay of mechanical and thermodynamic response, which is manifested as an increase in thermal stability and decrease of flexibility upon ligand binding.</p> <p>Results</p> <p>We use a distance constraint model (DCM) to quantify the give and take between thermodynamic stability and mechanical flexibility across the bPBP family. Quantitative stability/flexibility relationships (QSFR) are readily evaluated because the DCM links mechanical and thermodynamic properties. We have previously demonstrated that QSFR is moderately conserved across a mesophilic/thermophilic RNase H pair, whereas the observed variance indicated that different enthalpy-entropy mechanisms allow similar mechanical response at their respective melting temperatures. Our predictions of heat capacity and free energy show marked diversity across the bPBP family. While backbone flexibility metrics are mostly conserved, cooperativity correlation (long-range couplings) also demonstrate considerable amount of variation. Upon ligand removal, heat capacity, melting point, and mechanical rigidity are, as expected, lowered. Nevertheless, significant differences are found in molecular cooperativity correlations that can be explained by the detailed nature of the hydrogen bond network.</p> <p>Conclusion</p> <p>Non-trivial mechanical and thermodynamic variation across the family is explained by differences within the underlying H-bond networks. The mechanism is simple; variation within the H-bond networks result in altered mechanical linkage properties that directly affect intrinsic flexibility. Moreover, varying numbers of H-bonds and their strengths control the likelihood for energetic fluctuations as H-bonds break and reform, thus directly affecting thermodynamic properties. Consequently, these results demonstrate how unexpected large differences, especially within cooperativity correlation, emerge from subtle differences within the underlying H-bond network. This inference is consistent with well-known results that show allosteric response within a family generally varies significantly. Identifying the hydrogen bond network as a critical determining factor for these large variances may lead to new methods that can predict such effects.</p

Calculating Ensemble Averaged Descriptions of Protein Rigidity without Sampling

Previous works have demonstrated that protein rigidity is related to thermodynamic stability, especially under conditions that favor formation of native structure. Mechanical network rigidity properties of a single conformation are efficiently calculated using the integer body-bar Pebble Game (PG) algorithm. However, thermodynamic properties require averaging over many samples from the ensemble of accessible conformations to accurately account for fluctuations in network topology. We have developed a mean field Virtual Pebble Game (VPG) that represents the ensemble of networks by a single effective network. That is, all possible number of distance constraints (or bars) that can form between a pair of rigid bodies is replaced by the average number. The resulting effective network is viewed as having weighted edges, where the weight of an edge quantifies its capacity to absorb degrees of freedom. The VPG is interpreted as a flow problem on this effective network, which eliminates the need to sample. Across a nonredundant dataset of 272 protein structures, we apply the VPG to proteins for the first time. Our results show numerically and visually that the rigidity characterizations of the VPG accurately reflect the ensemble averaged properties. This result positions the VPG as an efficient alternative to understand the mechanical role that chemical interactions play in maintaining protein stability

Coevolved mutations reveal distinct architectures for two core proteins in the bacterial flagellar motor

Switching of bacterial flagellar rotation is caused by large domain movements of the FliG protein triggered by binding of the signal protein CheY to FliM. FliG and FliM form adjacent multi-subunit arrays within the basal body C-ring. The movements alter the interaction of the FliG C-terminal (FliGC) "torque" helix with the stator complexes. Atomic models based on the Salmonella entrovar C-ring electron microscopy reconstruction have implications for switching, but lack consensus on the relative locations of the FliG armadillo (ARM) domains (amino-terminal (FliGN), middle (FliGM) and FliGC) as well as changes during chemotaxis. The generality of the Salmonella model is challenged by the variation in motor morphology and response between species. We studied coevolved residue mutations to determine the unifying elements of switch architecture. Residue interactions, measured by their coevolution, were formalized as a network, guided by structural data. Our measurements reveal a common design with dedicated switch and motor modules. The FliM middle domain (FliMM) has extensive connectivity most simply explained by conserved intra and inter-subunit contacts. In contrast, FliG has patchy, complex architecture. Conserved structural motifs form interacting nodes in the coevolution network that wire FliMM to the FliGC C-terminal, four-helix motor module (C3-6). FliG C3-6 coevolution is organized around the torque helix, differently from other ARM domains. The nodes form separated, surface-proximal patches that are targeted by deleterious mutations as in other allosteric systems. The dominant node is formed by the EHPQ motif at the FliMMFliGM contact interface and adjacent helix residues at a central location within FliGM. The node interacts with nodes in the N-terminal FliGc α-helix triad (ARM-C) and FliGN. ARM-C, separated from C3-6 by the MFVF motif, has poor intra-network connectivity consistent with its variable orientation revealed by structural data. ARM-C could be the convertor element that provides mechanistic and species diversity.JK was supported by Medical Research Council grant U117581331. SK was supported by seed funds from Lahore University of Managment Sciences (LUMS) and the Molecular Biology Consortium

Detection of Botulinum Neurotoxin Serotype B at Sub Mouse LD50 Levels by a Sandwich Immunoassay and Its Application to Toxin Detection in Milk

Botulinum neurotoxin (BoNT), the causative agent of botulism, a serious neuroparylatic disease, is produced by the anaerobic bacterium Clostridium botulinum and consists of a family of seven serotypes (A-H). We previously reported production of high-affinity monoclonal antibodies to BoNT serotype A.Recombinant peptide fragments of the light chain, the transmembrane and receptor-binding domains of the heavy chain of botulinum neurotoxin type B (BoNT/B) were expressed in Escherichia coli as GST-fusion proteins and purified. These proteins were used to immunize BALB/cJ mice for the generation of monoclonal antibodies (mAbs). Antibody-producing hybridomas were detected using either a direct binding ELISA binding to plate-immobilized BoNT/B, or with a capture-capture ELISA whereby the capacity of the antibody to capture BoNT/B from solution was tested. A total of five mAbs were selected, two of which bound the toxin light chain and three bound the receptor-binding domain of BoNT/B heavy chain. MAb MCS6-27 was identified via capture-capture ELISA and was the only mAb able to bind BoNT/B in solution under physiological conditions. MAbs F24-1, F26-16, F27-33 and F29-40 were identified via direct binding ELISA, and were able to capture BoNT/B in solution only in the presence of 0.5-0.9 mM sodium dodecyl sulphate (SDS). MAb MCS6-27 and an anti-BoNT/B polyclonal antibody were incorporated into a sandwich ELISA that did not require SDS.We report here the generation of monoclonal antibodies to serotype B and the subsequent development of a sensitive sandwich immunoassay. This immunoassay has a detection limit of 100 fg BoNT/B, fifty times more sensitive than the mouse bioassay detection limit of 5 pg BoNT/B. Additionally, this assay detected as little as 39 pg/mL of toxin in skim, 2% and whole milk

Mechanisms of Allergen-Antibody Interaction of Cockroach Allergen Bla g 2 with Monoclonal Antibodies That Inhibit IgE Antibody Binding

BACKGROUND: Cockroach allergy is strongly associated with asthma, and involves the production of IgE antibodies against inhaled allergens. Reports of conformational epitopes on inhaled allergens are limited. The conformational epitopes for two specific monoclonal antibodies (mAb) that interfere with IgE antibody binding were identified by X-ray crystallography on opposite sites of the quasi-symmetrical cockroach allergen Bla g 2. METHODOLOGY/PRINCIPAL FINDINGS: Mutational analysis of selected residues in both epitopes was performed based on the X-ray crystal structures of the allergen with mAb Fab/Fab' fragments, to investigate the structural basis of allergen-antibody interactions. The epitopes of Bla g 2 for the mAb 7C11 or 4C3 were mutated, and the mutants were analyzed by SDS-PAGE, circular dichroism, and/or mass spectrometry. Mutants were tested for mAb and IgE antibody binding by ELISA and fluorescent multiplex array. Single or multiple mutations of five residues from both epitopes resulted in almost complete loss of mAb binding, without affecting the overall folding of the allergen. Preventing glycosylation by mutation N268Q reduced IgE binding, indicating a role of carbohydrates in the interaction. Cation-π interactions, as well as electrostatic and hydrophobic interactions, were important for mAb and IgE antibody binding. Quantitative differences in the effects of mutations on IgE antibody binding were observed, suggesting heterogeneity in epitope recognition among cockroach allergic patients. CONCLUSIONS/SIGNIFICANCE: Analysis by site-directed mutagenesis of epitopes identified by X-ray crystallography revealed an overlap between monoclonal and IgE antibody binding sites and provided insight into the B cell repertoire to Bla g 2 and the mechanisms of allergen-antibody recognition, including involvement of carbohydrates

Modeling-Dependent Protein Characterization of the Rice Aldehyde Dehydrogenase (ALDH) Superfamily Reveals Distinct Functional and Structural Features

The completion of the rice genome sequence has made it possible to identify and characterize new genes and to perform comparative genomics studies across taxa. The aldehyde dehydrogenase (ALDH) gene superfamily encoding for NAD(P)+-dependent enzymes is found in all major plant and animal taxa. However, the characterization of plant ALDHs has lagged behind their animal- and prokaryotic-ALDH homologs. In plants, ALDHs are involved in abiotic stress tolerance, male sterility restoration, embryo development and seed viability and maturation. However, there is still no structural property-dependent functional characterization of ALDH protein superfamily in plants. In this paper, we identify members of the rice ALDH gene superfamily and use the evolutionary nesting events of retrotransposons and protein-modeling–based structural reconstitution to report the genetic and molecular and structural features of each member of the rice ALDH superfamily in abiotic/biotic stress responses and developmental processes. Our results indicate that rice-ALDHs are the most expanded plant ALDHs ever characterized. This work represents the first report of specific structural features mediating functionality of the whole families of ALDHs in an organism ever characterized