Structure of the sporulation histidine kinase inhibitor Sda from Bacillus subtilis and insights into its solution state

The crystal structure of the DNA-damage checkpoint inhibitor of sporulation, Sda, from Bacillus subtilis, has been solved by the MAD technique using selenomethionine-substituted protein. The structure closely resembles that previously solved by NMR, as well as the structure of a homologue from Geobacillus stearothermophilus solved in complex with the histidine kinase KinB. The structure contains three molecules in the asymmetric unit. The unusual trimeric arrangement, which lacks simple internal symmetry, appears to be preserved in solution based on an essentially ideal fit to previously acquired scattering data for Sda in solution. This interpretation contradicts previous findings that Sda was monomeric or dimeric in solution. This study demonstrates the difficulties that can be associated with the characterization of small proteins and the value of combining multiple biophysical techniques. It also emphasizes the importance of understanding the physical principles behind these techniques and therefore their limitations

Outer-Sphere Contributions to the Electronic Structure of Type Zero Copper Proteins

Bioinorganic canon states that active-site thiolate coordination promotes rapid electron transfer (ET) to and from type 1 copper proteins. In recent work, we have found that copper ET sites in proteins also can be constructed without thiolate ligation (called “type zero” sites). Here we report multifrequency electron paramagnetic resonance (EPR), magnetic circular dichroism (MCD), and nuclear magnetic resonance (NMR) spectroscopic data together with density functional theory (DFT) and spectroscopy-oriented configuration interaction (SORCI) calculations for type zero Pseudomonas aeruginosa azurin variants. Wild-type (type 1) and type zero copper centers experience virtually identical ligand fields. Moreover, O-donor covalency is enhanced in type zero centers relative that in the C112D (type 2) protein. At the same time, N-donor covalency is reduced in a similar fashion to type 1 centers. QM/MM and SORCI calculations show that the electronic structures of type zero and type 2 are intimately linked to the orientation and coordination mode of the carboxylate ligand, which in turn is influenced by outer-sphere hydrogen bonding

Appetitive Traits associated with Higher and Lower Body Mass Index: Evaluating the Validity of the Adult Eating Behaviour Questionnaire in an Australian Sample

Background: The aims of this study were to evaluate the factor structure of the newly developed Adult Eating Behaviour Questionnaire (AEBQ) (Hunot et al., Appetite 105:356-63, 2016) in an Australian sample, and examine associations between the four food approach and four food avoidance appetitive traits with body mass index (BMI). Methods: Participants (N = 998) recruited between May and October 2016 via a university research participation scheme and online social network sites completed an online version of the AEBQ and self-reported demographic and anthropometric data. Of the sample, 84.8% were females, 29.6% had completed a university degree and the overall mean age was 24.32 years (SD = 8.32). Confirmatory factor analysis (CFA) was used to test three alternative factor structures (derived from issues raised in the original development study): the original 8 factor model, a 7 factor model with Food Responsiveness and Hunger scales combined, and a 7 factor model with the Hunger scale removed. Results: The CFA revealed that the original 8 factor model was a better fit to the data than the 7 factor model in which Food Responsiveness and Hunger scales were combined. However, while reliability estimates for 7 of the 8 scales were good (Cronbach’s α between 0.70-0.86), the reliability of the Hunger scale was modest (0.67) and dropping this factor resulted in a good fitting model. All food avoidance scales (except Food Fussiness) were negatively associated with body mass index (BMI) whereas Emotional Overeating was the only food approach scale positively associated with BMI. Conclusions: The study supports the use of the AEBQ as a reliable and valid measure of food approach and avoidance appetitive traits in adults. Longitudinal studies that examine continuity and stability of appetitive traits across the lifespan will be facilitated by the addition of this measurement tool to the literature

Cognitive mediation in people with dementia:Development, structural, and construct validity of the first dementia‐specific measure

OBJECTIVES: Anxiety and depression are common and deleterious comorbidities in people living with dementia (PLWD). Cognitive behavioural therapy (CBT) is one of the few promising treatments; however, it is unclear whether PLWD have the necessary prerequisites to engage in this. Having an understanding of cognitive mediation, that a thought mediates the relationship between an antecedent event and its emotional consequence, is key for engaging with CBT and is also a critical component of emotion regulation. There are no measures of this construct validated for PLWD. This study aims to adapt and validate an existing measure for this population. A secondary aim is to assess its applicability in older adults (OA) without a recognised neurocognitive impairment. // METHODS: A measure of cognitive mediation was adapted via expert and service user consultation for use in PLWD. A total of 102 PLWD and 77 OA without neurocognitive impairments completed the adapted measure along with two measures of emotion recognition and reasoning. Factor structure was examined separately in both samples, and the measure reduced, with convergent validity assessed. // RESULTS: A final measure of 10 items (named the CM-Dem) was subject to factor analysis yielding a single factor solution. The measure showed good psychometric properties in PLWD, including good model fit, high internal consistency, inter-rater reliability, and moderate convergent validity with related constructs. In contrast, poor validity was found in OA, especially a lack of convergent validity. // CONCLUSIONS: The CM-Dem has clinical and research utility as a measure of cognitive mediation in PLWD, but less so in OA

Genomic mining of prokaryotic repressors for orthogonal logic gates

Genetic circuits perform computational operations based on interactions between freely diffusing molecules within a cell. When transcription factors are combined to build a circuit, unintended interactions can disrupt its function. Here, we apply 'part mining' to build a library of 73 TetR-family repressors gleaned from prokaryotic genomes. The operators of a subset were determined using an in vitro method, and this information was used to build synthetic promoters. The promoters and repressors were screened for cross-reactions. Of these, 16 were identified that both strongly repress their cognate promoter (5- to 207-fold) and exhibit minimal interactions with other promoters. Each repressor-promoter pair was converted to a NOT gate and characterized. Used as a set of 16 NOT/NOR gates, there are >10[superscript 54] circuits that could be built by changing the pattern of input and output promoters. This represents a large set of compatible gates that can be used to construct user-defined circuits.United States. Air Force Office of Scientific Research (Award FA9550-11-C-0028)American Society for Engineering Education. National Defense Science and Engineering Graduate Fellowship (32 CFR 168a)United States. Defense Advanced Research Projects Agency. Chronical of Lineage Indicative of Origins (N66001-12-C-4016)United States. Office of Naval Research (N00014-13-1-0074)National Institutes of Health (U.S.) (GM095765)National Science Foundation (U.S.). Synthetic Biology Engineering Research Center (SA5284-11210

Novel IgG-degrading enzymes of the IgdE protease family link substrate specificity to host tropism of <i>Streptococcus</i> species

Recently we have discovered an IgG degrading enzyme of the endemic pig pathogen S. suis designated IgdE that is highly specific for porcine IgG. This protease is the founding member of a novel cysteine protease family assigned C113 in the MEROPS peptidase database. Bioinformatical analyses revealed putative members of the IgdE protease family in eight other Streptococcus species. The genes of the putative IgdE family proteases of S. agalactiae, S. porcinus, S. pseudoporcinus and S. equi subsp. zooepidemicus were cloned for production of recombinant protein into expression vectors. Recombinant proteins of all four IgdE family proteases were proteolytically active against IgG of the respective Streptococcus species hosts, but not against IgG from other tested species or other classes of immunoglobulins, thereby linking the substrate specificity to the known host tropism. The novel IgdE family proteases of S. agalactiae, S. pseudoporcinus and S. equi showed IgG subtype specificity, i.e. IgdE from S. agalactiae and S. pseudoporcinus cleaved human IgG1, while IgdE from S. equi was subtype specific for equine IgG7. Porcine IgG subtype specificities of the IgdE family proteases of S. porcinus and S. pseudoporcinus remain to be determined. Cleavage of porcine IgG by IgdE of S. pseudoporcinus is suggested to be an evolutionary remaining activity reflecting ancestry of the human pathogen to the porcine pathogen S. porcinus. The IgG subtype specificity of bacterial proteases indicates the special importance of these IgG subtypes in counteracting infection or colonization and opportunistic streptococci neutralize such antibodies through expression of IgdE family proteases as putative immune evasion factors. We suggest that IgdE family proteases might be valid vaccine targets against streptococci of both human and veterinary medical concerns and could also be of therapeutic as well as biotechnological use

Characterization of Xylan Utilization and Discovery of a New Endoxylanase in Thermoanaerobacterium Saccharolyticum through Targeted Gene Deletions

The economical production of fuels and commodity chemicals from lignocellulose requires the utilization of both the cellulose and hemicellulose fractions. Xylanase enzymes allow greater utilization of hemicellulose while also increasing cellulose hydrolysis. Recent metabolic engineering efforts have resulted in a strain of Thermoanaerobacterium saccharolyticum that can convert C5 and C6 sugars, as well as insoluble xylan, into ethanol at high yield. To better understand the process of xylan solubilization in this organism, a series of targeted deletions were constructed in the homoethanologenic T. saccharolyticum strain M0355 to characterize xylan hydrolysis and xylose utilization in this organism. While the deletion of -xylosidase xylD slowed the growth of T. saccharolyticum on birchwood xylan and led to an accumulation of short-chain xylo-oligomers, no other single deletion, including the deletion of the previously characterized endoxylanase XynA, had a phenotype distinct from that of the wild type.This result indicates a multiplicity of xylanase enzymes which facilitate xylan degradation in T. saccharolyticum. Growth on xylan was prevented only when a previously uncharacterized endoxylanase encoded by xynC was also deleted in conjunction with xynA. Sequence analysis of xynC indicates that this enzyme, a low-molecular-weight endoxylanase with homology to glycoside hydrolase family 11 enzymes, is secreted yet untethered to the cell wall. Together, these observations expand our understanding of the enzymatic basis of xylan hydrolysis by T. saccharolyticum