Distinct bacterial dissemination and disease outcome in mice subcutaneously infected with Borrelia burgdorferi in the midline of the back and the footpad

Subcutaneous inoculation of mice with Borrelia burgdorferi, the causative agent of Lyme disease, results in established infection and the development of acute arthritis and carditis, hallmarks of human disease. Because conflicting results may originate from the site of subcutaneous inoculation, we addressed the dissemination capacity of spirochetes injected in the shoulder region versus the footpad. Spirochetes inoculated in the footpad disseminated to a lesser extent to distant organs, such as the ear and the heart. This resulted in distinct degrees of joint and cardiac inflammation at the peak of the disease. The differences eventually leveled out. These results suggest that caution must be exercised in the interpretation of results obtained with routes of inoculation that do not closely represent the natural site of infection.     

Mutations that cause osteoglophonic dysplasia define novel roles for FGFR1 in bone elongation

Activating mutations in the genes for fibroblast growth factor receptors 1-3 (FGFR1-3) are responsible for a diverse group of skeletal disorders. In general, mutations in FGFR1 and FGFR2 cause the majority of syndromes involving craniosynostosis, whereas the dwarfing syndromes are largely associated with FGFR3 mutations. Osteoglophonic dysplasia (OD) is a "crossover" disorder that has skeletal phenotypes associated with FGFR1, FGFR2, and FGFR3 mutations. Indeed, patients with OD present with craniosynostosis, prominent supraorbital ridge, and depressed nasal bridge, as well as the rhizomelic dwarfism and nonossifying bone lesions that are characteristic of the disorder. We demonstrate here that OD is caused by missense mutations in highly conserved residues comprising the ligand-binding and transmembrane domains of FGFR1, thus defining novel roles for this receptor as a negative regulator of long-bone growth.     

Strategy and tactics of disarming GHG at the source: N2O reductase crops

Nitrous oxide (N(2)O), the third most abundant greenhouse gas (GHG), is highly stable and plays a significant role in stratospheric ozone destruction. The primary anthropogenic source of N(2)O stems from use of nitrogen fertilizers in soil. The bacterial enzyme nitrous oxide reductase (N(2)OR), naturally found in some soils, is the only known enzyme capable of catalyzing the final step of the denitrification pathway, conversion of N(2)O to N(2). In this opinion, we discuss potential biology-based strategies to reduce N(2)O by amplifying the amount of available enzyme catalyst in agri-system environments during crop growth and in post-harvest detritus. N(2)OR from Pseudomonas stutzeri has been tested in transgenic plants with promising results. Such seed-borne phytoremediation systems targeted towards GHGs merit field testing.     

Effect of canagliflozin on renal threshold for glucose, glycemia, and body weight in normal and diabetic animal models

Background

Canagliflozin is a sodium glucose co-transporter (SGLT) 2 inhibitor in clinical development for the treatment of type 2 diabetes mellitus (T2DM).

Methods

¹⁴C-alpha-methylglucoside uptake in Chinese hamster ovary-K cells expressing human, rat, or mouse SGLT2 or SGLT1; ³H-2-deoxy-d-glucose uptake in L6 myoblasts; and 2-electrode voltage clamp recording of oocytes expressing human SGLT3 were analyzed. Graded glucose infusions were performed to determine rate of urinary glucose excretion (UGE) at different blood glucose (BG) concentrations and the renal threshold for glucose excretion (RT_G) in vehicle or canagliflozin-treated Zucker diabetic fatty (ZDF) rats. This study aimed to characterize the pharmacodynamic effects of canagliflozin in vitro and in preclinical models of T2DM and obesity.

Results

Treatment with canagliflozin 1 mg/kg lowered RT_G from 415±12 mg/dl to 94±10 mg/dl in ZDF rats while maintaining a threshold relationship between BG and UGE with virtually no UGE observed when BG was below RT_G. Canagliflozin dose-dependently decreased BG concentrations in db/db mice treated acutely. In ZDF rats treated for 4 weeks, canagliflozin decreased glycated hemoglobin (HbA1c) and improved measures of insulin secretion. In obese animal models, canagliflozin increased UGE and decreased BG, body weight gain, epididymal fat, liver weight, and the respiratory exchange ratio.

Conclusions

Canagliflozin lowered RT_G and increased UGE, improved glycemic control and beta-cell function in rodent models of T2DM, and reduced body weight gain in rodent models of obesity.     

Trans-4-hydroxy-2-hexenal is a neurotoxic product of docosahexaenoic (22:6; n-3) acid oxidation

Lipid peroxidation of docosahexaenoic (22:6; n-3) acid (DHA) is elevated in the CNS in patients with Alzheimer's disease and in animal models of seizure and ethanol withdrawal. One product of DHA oxidation is trans -4-hydroxy-2-hexenal (HHE), a six carbon analog of the n-6 fatty acid derived trans -4-hydroxy-2-nonenal (HNE). In this work, we studied the neurotoxic potential of HHE. HHE and HNE were toxic to primary cultures of cerebral cortical neurons with LD₅₀'s of 23 and 18 μmol/L, respectively. Toxicity was prevented by the addition of thiol scavengers. HHE and HNE depleted neuronal GSH content identically with depletion observed with 10 μmol/L of either compound. Using an antibody raised against HHE–protein adducts, we show that HHE modified specific proteins of 75, 50, and 45 kDa in concentration- and time-dependent manners. The time-dependent formation of HHE differed from that of F₄-neuroprostanes following in vitro DHA oxidation likely as a result of the different oxidation pathways involved. Using purified mitochondrial aldehyde dehydrogenase ALDH5A, we found that HHE was oxidized 6.5-fold less efficiently than HNE. Our data demonstrate that HHE and HNE have similarities but also differences in their neurotoxic mechanisms and metabolism.     

Microbial oxidation of naphthalene to cis-1,2-naphthalene dihydrodiol using naphthalene dioxygenase in biphasic media

The selective oxidation of aryl substrates to chiral cis-1,2-dihydrodiols is an industrially important reaction for the production of intermediates that can be used to produce fine chemicals, pharmaceuticals, and many other bioactive natural products. More specifically, the oxidation of naphthalene to produce optically pure (+)-cis-(1R,2S)-1,2-napthalene dihydrodiol (NDHD) to be used as a chiral synthon for specialty chemicals has gained much interest. Escherichia coli JM109(DE3) pDTG141 expresses naphthalene dioxygenase which catalyzes this reaction. Poor substrate solubility and substrate toxicity are barriers to using the power of these enzymes in large-scale aqueous whole cell systems. A biphasic reaction system was chosen to overcome these barriers. The optimal biphasic conditions for E. coli JM109(DE3) pDTG141 were determined to be 20% dodecane as the organic solvent containing 40 g/L naphthalene. The productivity of the biotransformation using resting cells was 1.75 g-diol/g-cdw/h for the first 6 h with 20% organic phase, which was increased from 0.59 g-diol/g-cdw/h for growing cells with 40% organic phase. The biocatalytic activity was retained for at least 12 h. The biocatalyst could be recycled for at least four runs in both suspended and immobilized form. The stability of the 12 h recycle was improved by immobilization in calcium alginate beads. The process has been improved both environmentally and economically by reducing the amount of solvent used and by recycling the biocatalyst.     

Amylase partitioning and extractive bioconversion of starch using thermoseparating aqueous two-phase systems.

The effectiveness of thermoseparating polymer-based aqueous two-phase systems (ATPS) in the enzymatic hydrolysis of starch was investigated. In this work, the phase diagrams of PEO-PPO-2500/ammonium sulfate and PEO-PPO-2500/magnesium sulfate systems were determined at 25 degrees C. The partition behavior of pure alpha-amylase and amyloglucosidase in four ATPS, namely, PEO-PPO/(NH(4))(2)SO(4), PEO-PPO/MgSO(4), polyethylene glycol (PEG)/(NH(4))(2)SO(4), and PEG/MgSO(4), was evaluated. The effects of phase-forming component concentrations on the enzyme activity and partitioning were assessed. Partitioning of a recombinant, thermostable alpha-amylase (MJA1) from the hyperthermophile, Methanococcus jannaschii was also investigated. All of the studied enzymes partitioned unevenly in these polymer/salt systems. The PEO-PPO-2500/MgSO(4) system was extremely attractive for starch hydrolysis. Polymer-based starch hydrolysis experiments containing PEO-PPO-2500/MgSO(4) indicated that the use of ATPS had a significant effect on soluble starch hydrolysis. Batch starch hydrolysis experiments with PEO-PPO/salt two-phase systems resulted in higher production of maltose or glucose and exhibited remarkably faster hydrolysis. A 22% gain in maltose yield was obtained as a result of the increased productivity. This work is the first reported application of thermoseparating polymer ATPS in the processing of starches. These results reveal the potential for thermoseparating polymer-enhanced extractive bioconversion of starch as a practical technology.     

Reconstruction of a beech population bottleneck using archival demographic information and Bayesian analysis of genetic data

Range expansion and contraction has occurred in the history of most species and can seriously impact patterns of genetic diversity. Historical data about range change are rare and generally appropriate for studies at large scales, whereas the individual pollen and seed dispersal events that form the basis of geneflow and colonization generally occur at a local scale. In this study, we investigated range change in Fagus sylvatica on Mont Ventoux, France, using historical data from 1838 to the present and approximate Bayesian computation (ABC) analyses of genetic data. From the historical data, we identified a population minimum in 1845 and located remnant populations at least 200 years old. The ABC analysis selected a demographic scenario with three populations, corresponding to two remnant populations and one area of recent expansion. It also identified expansion from a smaller ancestral population but did not find that this expansion followed a population bottleneck, as suggested by the historical data. Despite a strong support to the selected scenario for our data set, the ABC approach showed a low power to discriminate among scenarios on average and a low ability to accurately estimate effective population sizes and divergence dates, probably due to the temporal scale of the study. This study provides an unusual opportunity to test ABC analysis in a system with a well-documented demographic history and identify discrepancies between the results of historical, classical population genetic and ABC analyses. The results also provide valuable insights into genetic processes at work at a fine spatial and temporal scale in range change and colonization.     

The link between the PDL1 costimulatory pathway and Th17 in fetomaternal tolerance

Fetomaternal tolerance has been shown to depend both on regulatory T cells (Tregs) and negative signals from the PD1-PDL1 costimulatory pathway. More recently, IL-17-producing T cells (Th17) have been recognized as a barrier in inducing tolerance in transplantation. In this study, we investigate the mechanisms of PDL1-mediated regulation of fetomaternal tolerance using an alloantigen-specific CD4(+) TCR transgenic mouse model system (ABM-tg mouse). PDL1 blockade led to an increase in embryo resorption and a reduction in litter size. This was associated with a decrease in Tregs, leading to a lower Treg/effector T cell ratio. Moreover, PDL1 blockade inhibited Ag-specific alloreactive T cell apoptosis and induced apoptosis of Tregs and a shift toward higher frequency of Th17 cells, breaking fetomaternal tolerance. These Th17 cells arose predominantly from CD4(+)Foxp3(-) cells, rather than from conversion of Tregs. Locally in the placenta, similar decrease in regulatory and apoptotic markers was observed by real-time PCR. Neutralization of IL-17 abrogated the anti-PDL1 effect on fetal survival rate and restored Treg numbers. Finally, the adoptive transfer of Tregs was also able to improve fetal survival in the setting of PDL1 blockade. This is to our knowledge the first report using an alloantigen-specific model that establishes a link between PDL1, Th17 cells, and fetomaternal tolerance.     

17β-Estradiol Directly Lowers Mitochondrial Membrane Microviscosity and Improves Bioenergetic Function in Skeletal Muscle

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