Insight into the Carboxyl Transferase Domain Mechanism of Pyruvate Carboxylase from \u3cem\u3eRhizobium etli\u3c/em\u3e

The effects of mutations in the active site of the carboxyl transferase domain of Rhizobium etli pyruvate carboxylase have been determined for the forward reaction to form oxaloacetate, the reverse reaction to form MgATP, the oxamate-induced decarboxylation of oxaloacetate, the phosphorylation of MgADP by carbamoyl phosphate, and the bicarbonate-dependent ATPase reaction. Additional studies with these mutants examined the effect of pyruvate and oxamate on the reactions of the biotin carboxylase domain. From these mutagenic studies, putative roles for catalytically relevant active site residues were assigned and a more accurate description of the mechanism of the carboxyl transferase domain is presented. The T882A mutant showed no catalytic activity for reactions involving the carboxyl transferase domain but surprisingly showed 7- and 3.5-fold increases in activity, as compared to that of the wild-type enzyme, for the ADP phosphorylation and bicarbonate-dependent ATPase reactions, respectively. Furthermore, the partial inhibition of the T882A-catalyzed BC domain reactions by oxamate and pyruvate further supports the critical role of Thr882 in the proton transfer between biotin and pyruvate in the carboxyl transferase domain. The catalytic mechanism appears to involve the decarboxylation of carboxybiotin and removal of a proton from Thr882 by the resulting biotin enolate with either a concerted or subsequent transfer of a proton from pyruvate to Thr882. The resulting enolpyruvate then reacts with CO2 to form oxaloacetate and complete the reaction

Low Connexin Channel-Dependent Intercellular Communication in Human Adult Hematopoietic Progenitor/Stem Cells: Probing Mechanisms of Autologous Stem Cell Therapy

Human bone marrow is a clinical source of autologous progenitor stem cells showing promise for cardiac repair following ischemic insult. Functional improvements following delivery of adult bone marrow CD34+ cells into heart tissue may require metabolic/electrical communication between participating cells. Since connexin43 (Cx43) channels are implicated in cardiogenesis and provide intercellular connectivity in the heart, the authors analyzed the expression of 20 connexins (Cx) in CD34+ cells and in monocytes and granulocytes in bone marrow and spinal cord. Reverse transcriptase-polymerase chain reaction (RT-PCR) detected only low expression of Cx43 and Cx37. Very low level dye coupling was detected by flow cytometry between CD34+ cells and other Cx43 expressing cells, including HL-1 cardiac cells, and was not inhibited by specific gap junction inhibitors. The results indicate that CD34+ cells are unlikely to communicate via gap junctions and the authors conclude that use of CD34+ cells to repair damaged hearts is unlikely to involve gap junctions. The results concur with the hypothesis that bone marrow cells elicit improved cardiac function through release of undefined paracrine mediators

Probing the Catalytic Roles of Arg548 and Gln552 in the Carboxyl Transferase Domain of the \u3cem\u3eRhizobium etli\u3c/em\u3e Pyruvate Carboxylase by Site-directed Mutagenesis

The roles of Arg548 and Gln552 residues in the active site of the carboxyl transferase domain of Rhizobium etli pyruvate carboxylase were investigated using site-directed mutagenesis. Mutation of Arg548 to alanine or glutamine resulted in the destabilization of the quaternary structure of the enzyme, suggesting that this residue has a structural role. Mutations R548K, Q552N, and Q552A resulted in a loss of the ability to catalyze pyruvate carboxylation, biotin-dependent decarboxylation of oxaloacetate, and the exchange of protons between pyruvate and water. These mutants retained the ability to catalyze reactions that occur at the active site of the biotin carboxylase domain, i.e., bicarbonate-dependent ATP cleavage and ADP phosphorylation by carbamoyl phosphate. The effects of oxamate on the catalysis in the biotin carboxylase domain by the R548K and Q552N mutants were similar to those on the catalysis of reactions by the wild-type enzyme. However, the presence of oxamate had no effect on the reactions catalyzed by the Q552A mutant. We propose that Arg548 and Gln552 facilitate the binding of pyruvate and the subsequent transfer of protons between pyruvate and biotin in the partial reaction catalyzed in the active site of the carboxyl transferase domain of Rhizobium etli pyruvate carboxylase

Comparison of the salivary and dentinal microbiome of children with severe-early childhood caries to the salivary microbiome of caries-free children

peer-reviewedBackground The main objectives of this study were to describe and compare the microbiota of 1) deep dentinal lesions of deciduous teeth of children affected with severe early childhood caries (S-ECC) and 2) the unstimulated saliva of these children and 3) the unstimulated saliva of caries-free children, and to compare microbiota compositional differences and diversity of taxa in these sampled sites. Methods Children with S-ECC and without S-ECC were recruited. The saliva of all children with and without S-ECC was sampled along with the deep dentinal microbiota from children affected by S-ECC. The salivary microbiota of children affected by S-ECC (n = 68) was compared to that of caries-free children (n = 70), by Illumina MiSeq sequencing of 16S rRNA amplicons. Finally, the caries microbiota of deep dentinal lesions of those children with S-ECC was investigated. Results Using two beta diversity metrics (Bray Curtis dissimilarity and UniFrac distance), the caries microbiota was found to be distinct from that of either of the saliva groups (caries-free & caries-active) when bacterial abundance was taken into account. However, when the comparison was made by measuring only presence and absence of bacterial taxa, all three microbiota types separated. While the alpha diversity of the caries microbiota was lowest, the diversity difference between the caries samples and saliva samples was statistically significant (p < 0.001). The major phyla of the caries active dentinal microbiota were Firmicutes (median abundance value 33.5%) and Bacteroidetes (23.2%), with Neisseria (10.3%) being the most abundant genus, followed by Prevotella (10%). The caries-active salivary microbiota was dominated by Proteobacteria (median abundance value 38.2%) and Bacteroidetes (27.8%) with the most abundant genus being Neisseria (16.3%), followed by Porphyromonas (9.5%). Caries microbiota samples were characterized by high relative abundance of Streptococcus mutans, Prevotella spp., Bifidobacterium and Scardovia spp. Conclusions Distinct differences between the caries microbiota and saliva microbiota were identified, with separation of both salivary groups (caries-active and caries-free) whereby rare taxa were highlighted. While the caries microbiota was less diverse than the salivary microbiota, the presence of these rare taxa could be the difference between health and disease in these children

PSR J0045-7319: A DUAL-LINE BINARY RADIO PULSAR

Binary radio pulsars are superb tools for mapping binary orbits, because of the precision of the pulse timing method (Taylor and Weisberg 1989). To date, all orbital parameters for binary pulsars have been derived from observations of the pulsar alone. We present the first observations of the radial velocity variations due to the binary motion of a companion to a radio pulsar. Our results demonstrate that the companion to the Small Magellanic Cloud pulsar PSR J0045-7319 is the B1V star identified by Kaspi et al. (1994). The mass ratio of the system is 6.3 +/- 1.2, which, for a neutron star mass of 1.4 Mo, implies a mass of 8.8 +/- 1.8 Mo for the companion, consistent with the mass expected for a B1V star. The inclination angle for the binary system is therefore 44 +/- 5 degrees, and the projected rotational velocity of the companion is 113 +/- 10 km/s. The heliocentric radial velocity of the binary system is consistent with that of other stars and gas in the same region of the Small Magellanic Cloud.Comment: 4 pages, Z-compressed postscript, figure included, accepted for publication in ApJ L in July 199

TotalSegmentator: robust segmentation of 104 anatomical structures in CT images

We present a deep learning segmentation model that can automatically and robustly segment all major anatomical structures in body CT images. In this retrospective study, 1204 CT examinations (from the years 2012, 2016, and 2020) were used to segment 104 anatomical structures (27 organs, 59 bones, 10 muscles, 8 vessels) relevant for use cases such as organ volumetry, disease characterization, and surgical or radiotherapy planning. The CT images were randomly sampled from routine clinical studies and thus represent a real-world dataset (different ages, pathologies, scanners, body parts, sequences, and sites). The authors trained an nnU-Net segmentation algorithm on this dataset and calculated Dice similarity coefficients (Dice) to evaluate the model's performance. The trained algorithm was applied to a second dataset of 4004 whole-body CT examinations to investigate age dependent volume and attenuation changes. The proposed model showed a high Dice score (0.943) on the test set, which included a wide range of clinical data with major pathologies. The model significantly outperformed another publicly available segmentation model on a separate dataset (Dice score, 0.932 versus 0.871, respectively). The aging study demonstrated significant correlations between age and volume and mean attenuation for a variety of organ groups (e.g., age and aortic volume; age and mean attenuation of the autochthonous dorsal musculature). The developed model enables robust and accurate segmentation of 104 anatomical structures. The annotated dataset (https://doi.org/10.5281/zenodo.6802613) and toolkit (https://www.github.com/wasserth/TotalSegmentator) are publicly available.Comment: Accepted at Radiology: Artificial Intelligenc

Floodplain management in temperate regions : is multifunctionality enhancing biodiversity?

Background: Floodplains are among the most diverse, dynamic, productive and populated but also the most threatened ecosystems on Earth. Threats are mainly related to human activities that alter the landscape and disrupt fluvial processes to obtain benefits related to multiple ecosystem services (ESS). Floodplain management therefore requires close coordination among interest groups with competing claims and poses multi-dimensional challenges to policy-makers and project managers. The European Commission proposed in its recent Biodiversity Strategy to maintain and enhance European ecosystems and their services by establishing green infrastructure (GI). GI is assumed to provide multiple ecosystem functions and services including the conservation of biodiversity in the same spatial area. However, evidence for biodiversity benefits of multifunctional floodplain management is scattered and has not been synthesised. Methods/design: This protocol specifies the methods for conducting a systematic review to answer the following policy-relevant questions: a) what is the impact of floodplain management measures on biodiversity; b) how does the impact vary according to the level of multifunctionality of the measures; c) is there a difference in the biodiversity impact of floodplain management across taxa; d) what is the effect of the time since implementation on the impact of the most important measures; and e) are there any other factors that significantly modify the biodiversity impact of floodplain management measures? Within this systematic review we will assess multifunctionality in terms of ESS that are affected by an implemented intervention. Biodiversity indicators included in this systematic review will be related to the diversity, richness and abundance of species, other taxa or functional groups. We will consider if organisms are typical for and native to natural floodplain ecosystems. Specific inclusion criteria have been developed and the wide range of quality of primary literature will be evaluated with a tailor-made system for assessing susceptibility to bias and the reliability of the studies. The review is intended to bridge the science-policy interface and will provide a useful synthesis of knowledge for decision-makers at all governance levels

A baseline for the genetic stock identification of Atlantic herring, Clupea harengus, in ICES Divisions 6.a, 7.b-c

Atlantic herring in International Council for Exploration of the Sea (ICES) Divisions 6.a, 7.b-c comprises at least three populations, distinguished by temporal and spatial differences in spawning, which have until recently been managed as two stocks defined by geographical delineators. Outside of spawning the populations form mixed aggregations, which are the subject of acoustic surveys. The inability to distinguish the populations has prevented the development of separate survey indices and separate stock assessments. A panel of 45 single-nucleotide polymorphisms, derived from whole-genome sequencing, were used to genotype 3480 baseline spawning samples (2014-2021). A temporally stable baseline comprising 2316 herring from populations known to inhabit Division 6.a was used to develop a genetic assignment method, with a self-assignment accuracy greater than 90%. The long-term temporal stability of the assignment model was validated by assigning archive (2003-2004) baseline samples (270 individuals) with a high level of accuracy. Assignment of non-baseline samples (1514 individuals) from Divisions 6.a, 7.b-c indicated previously unrecognized levels of mixing of populations outside of the spawning season. The genetic markers and assignment models presented constitute a 'toolbox' that can be used for the assignment of herring caught in mixed survey and commercial catches in Division 6.a into their population of origin with a high level of accuracy