Molecular characterization of the Salmonella typhimurium parE gene.

The DNA sequence of the wild type S. typhimurium parE gene was determined. The predicted protein has 96.7% amino acid identity with the ParE protein of E.coli, but is 29 amino acids longer, due to an additional basepair in the 3' end of the S. typhimurium gene. Subclones of the S. typhimurium parE gene localized the sites of four heat sensitive mutations within parE. The parE206 and parE374 mutations are identical (Val67-Met) and lie in a highly conserved region corresponding to the ATP binding pocket of GyrB. Two additional heat sensitive mutations were sequenced and predict the following amino acid substitutions: parE377 (Gly399-Ser) and parE493 (Thr583-Pro). All of the heat sensitive mutations lie in regions with strong amino acid homology to GyrB.     

Developing Regenerate: A circular economy engagement tool for the assessment of new and existing buildings

The transition toward a circular economy (CE) is key in decarbonizing the built environment. Despite this, knowledge of—and engagement with—CE philosophies remains limited within the construction industry. Discussion with practitioners reveals this to be contributed to by a lack of clarity regarding CE principles, with numerous organizations recommending implementation of differing and sometimes conflicting principles. In addition, a systematic assessment of how building designs consider CE is made difficult by the multiple design areas required to be considered and the large amount of design data required to do so. The absence of a systematic CE assessment causes a lack of comparability across designs, preventing benchmarking of CE practices in building design at present. This paper details the development of Regenerate, a CE engagement tool for the assessment of new and existing buildings, established in an effort to overcome the aforementioned barriers to the adoption of CE within the construction sector. A CE design workflow for the built environment is proposed, comprising four overarching circularity principles (Design for Adaptability; Design for Deconstructability; Circular Material Selection; Resource Efficiency) and contributing design actions. In addition to engaging stakeholders by enabling the assessment of building designs, the tool retrieves key data for further research. Information on completed design actions as well as recycling and waste metrics is collected to facilitate future CE benchmarking. “Bill of materials” data (i.e., material quantities) is also compiled, with this being key in material stock modeling research and embodied carbon benchmarking.     

A molecular perspective on the phylogeny of placental mammals based on mitochondrial 12S rDNA sequences,with special reference to the problem of the Paenungulata

Part of the 12S rDNA gene was amplified and sequenced for 11 placental mammals, 3 marsupials, and 2 monotremes. Multiple alignments for these sequences and nine additional placental sequences taken from GenBank were obtained using CLUSTAL. Phylogenetic analyses were performed using standard parsimony, transversion parsimony, and Lake's method of invariants. All of our analyses uniteLoxodontia withDugong. Procavia, in turn, is a sister group to these taxa, thus supporting the monophyly of the Paenungulata. Perissodactyls are a sister group to paenungulates when transitions and transversions are both included but not when transitions are omitted. Likewise, cetaceans are a sister group to artiodactyls on minimum length trees under standard parsimony but not under transversion parsimony. Rodent monophyly and bat monophyly also receive mixed support, as does a putative alliance between primates and lagomorphs. Interestingly, the percentage divergence between the echidna and the platypus is less than for the rat and mouse.     

Antitumor effects of an antibody-carboxypeptidase g2 conjugate in combination with a benzoic acid mustard prodrug

The F(ab’)₂ fragment of the antitumor monoclonal antibody, A5B7, was covalently linked to the bacterial enzyme carboxypeptidase G2 (CPG2). The resulting conjugate was used in combination with a prodrug of a benzoic acid mustard alkylating agent to treat human colon tumor xenografts in a two-step targeting strategy, antibody-directed enzyme produrug therapy (ADEPT). The prodrug, 4-[(2-chloroethyl) (2-mesyloxyethyl) amino]-benzoyl-l-glutamic acid is rapidly converted by CPG2 to a drug that is at least 15x more toxic in vitro against LS174T colorectal tumor cells than the prodrug. Optimal tumor/ blood ratios of the A5B7-CPG2 were achieved 72 h after administration of the conjugate to athymic mice bearing established LS174T tumor xenografts. Significant antitumor activity was seen in LS174T tumor-bearing mice treated with the conjugate followed 3 d later by the prodrug. In contrast, prodrug, conjugate, or active drug alone did not result in any antitumor activity in this tumor model. These studies demonstrate the advantage of a two-step ADEPT system for the treatment of colorectal cancer.     

Loss of DNA repair mechanisms in cardiac myocytes induce dilated cardiomyopathy

Cardiomyopathy is a progressive disease of the myocardium leading to impaired contractility. Genotoxic cancer therapies are known to be potent drivers of cardiomyopathy, whereas causes of spontaneous disease remain unclear. To test the hypothesis that endogenous genotoxic stress contributes to cardiomyopathy, we deleted the DNA repair gene Ercc1 specifically in striated muscle using a floxed allele of Ercc1 and mice expressing Cre under control of the muscle-specific creatinine kinase (Ckmm) promoter or depleted systemically (Ercc1^−/D mice). Ckmm-Cre^+/−;Ercc1^−/fl mice expired suddenly of heart disease by 7 months of age. As young adults, the hearts of Ckmm-Cre^+/−;Ercc1^−/fl mice were structurally and functionally normal, but by 6-months-of-age, there was significant ventricular dilation, wall thinning, interstitial fibrosis, and systolic dysfunction indicative of dilated cardiomyopathy. Cardiac tissue from the tissue-specific or systemic model showed increased apoptosis and cardiac myocytes from Ckmm-Cre^+/-;Ercc1^−/fl mice were hypersensitive to genotoxins, resulting in apoptosis. p53 levels and target gene expression, including several antioxidants, were increased in cardiac tissue from Ckmm-Cre^+/−;Ercc1^−/fl and Ercc1^−/D mice. Despite this, cardiac tissue from older mutant mice showed evidence of increased oxidative stress. Genetic or pharmacologic inhibition of p53 attenuated apoptosis and improved disease markers. Similarly, overexpression of mitochondrial-targeted catalase improved disease markers. Together, these data support the conclusion that DNA damage produced endogenously can drive cardiac disease and does so mechanistically via chronic activation of p53 and increased oxidative stress, driving cardiac myocyte apoptosis, dilated cardiomyopathy, and sudden death.     

Phenotypic effects of overexpression of Agrobacterium rhizogenes T-DNA ORF13 in transgenic tobacco plants are mediated by diffusible factor(s)

Nicotiana tabacum cv. Xanthi transgenic plants expressing ORF13 of Agrobacterium rhizogenes 8196 T-DNA under the 35S RNA promoter from the cauliflower mosaic virus displayed developmental abnormalities. They were small, with short and variable internodal lengths, their root systems were poorly developed; leaves were small, asymmetric, rounded, wrinkled and dark green; flowers were short, and irregularly shaped. They exhibited reduced apical dominance and regularly produced offshoots at the base of the plant. This phenotype was also exhibited by offshoots of normal N. tabacum cv. Xanthi stock grafted with a transgenic scion indicating that expression of ORF13 influences plant development via diffusible factor(s).     

Pattern-based clustering of daily weigh-in trajectories using dynamic time warping

“Smart”-scales are a new tool for frequent monitoring of weight change as well as weigh-in behavior. These scales give researchers the opportunity to discover patterns in the frequency that individuals weigh themselves over time, and how these patterns are associated with overall weight loss. Our motivating data come from an 18-month behavioral weight loss study of 55 adults classified as overweight or obese who were instructed to weigh themselves daily. Adherence to daily weigh-in routines produces a binary times series for each subject, indicating whether a participant weighed in on a given day. To characterize weigh-in by time-invariant patterns rather than overall adherence, we propose using hierarchical clustering with dynamic time warping (DTW). We perform an extensive simulation study to evaluate the performance of DTW compared to Euclidean and Jaccard distances to recover underlying patterns in adherence time series. In addition, we compare cluster performance using cluster validation indices (CVIs) under the single, average, complete, and Ward linkages and evaluate how internal and external CVIs compare for clustering binary time series. We apply conclusions from the simulation to cluster our real data and summarize observed weigh-in patterns. Our analysis finds that the adherence trajectory pattern is significantly associated with weight loss.     

Specificity and regulation of the mutator transposable element system in maize

The Mutator transposable element system is exceptional in many of its basic attributes. The high frequency and low specificity of mutant induction are both unusual and useful characteristics of the Mutator system. Other basic features are at least equally fascinating: the existence of multiple Mu element subfamilies with apparently unrelated internal sequences; the lack of correlation between Mu element transposition and excision; the complex inheritance of Mutator activity; the tight developmental regulation of Afufaror‐conditioned events; and the coordinated processes of element modification/inactivation, to name a few.

Molecular and genetic studies over the last 10 years have begun to explain many of these interesting properties and have uncovered new mysteries of Mutator biology. Both positive and negative regulators of the system have been identified and characterized to varying degrees. Insertion specificity has been observed at several levels. Recent accomplishments include the isolation of an autonomous Mu element and the discovery of maize lines with altered developmental regulation of Mutator‐derived mutability. This review defines the Mutator system, describes the status of current experimentation in the Mutator field, proposes models that may explain some aspects of Mutator behavior, and details future studies that will help elucidate the nature of the Mutator phenomenon.     

Physiological effects of translation initiation factor IF3 and ribosomal protein L20 limitation inEscherichia coli

To investigate the physiological roles of translation initiation factor IF3 and ribosomal protein L20 inEscherichia coli, theinfC, rpmI andrpIT genes encoding IF3, L35 and L20, respectively, were placed under the control oflac promoter/operator sequences. Thus, their expression is dependent upon the amount of inducer isopropyl thiogalactoside (IPTG) in the medium. Lysogenic strains were constructed with recombinant lambda phages that express eitherrpmI andrplT orinfC andrpmI in trans, thereby allowing depletion of only IF3 or L20 at low IPTG concentrations. At low IPTG concentrations in the IF3-limited strain, the cellular concentration of IF3, but not L20, decreases and the growth rate slows. Furthermore, ribosomes run off polysomes, indicating that IF3 functions during the initiation phase of protein synthesis in vivo. During slow growth, the ratio of RNA to protein increases rather than decreases as occurs with control strains, indicating that IF3 limitation disrupts feedback inhibition of rRNA synthesis. As IF3 levels drop, expression from an AUU-infC-lacZ fusion increases, whereas expression decreases from an AUG-infC-lacZ fusion, thereby confirming the model of autogenous regulation ofinfC. The effects of L20 limitation are similar; cells grown in low concentrations of IPTG exhibited a decrease in the rate of growth, a decrease in cellular L20 concentration, no change in IF3 concentration, and a small increase in the ratio of RNA to protein. In addition, a decrease in 50S subunits and the appearance of an aberrant ribosome peak at approximately 41–43S is seen. Previous studies have shown that the L20 protein negatively controls its own gene expression. Reduction of the cellular concentration of L20 derepresses the expression of anrplT-lacZ gene fusion, thus confirming autogenous regulation by L20.