A Dual Read-Out Assay to Evaluate the Potency of Compounds Active against Mycobacterium tuberculosis

Tuberculosis is a serious global health problem caused by the bacterium Mycobacterium tuberculosis. There is an urgent need for discovery and development of new treatments, but this can only be accomplished through rapid and reproducible M. tuberculosis assays designed to identify potent inhibitors. We developed an automated 96-well assay utilizing a recombinant strain of M. tuberculosis expressing a far-red fluorescent reporter to determine the activity of novel compounds; this allowed us to measure growth by monitoring both optical density and fluorescence. We determined that optical density and fluorescence were correlated with cell number during logarithmic phase growth. Fluorescence was stably maintained without antibiotic selection over 5 days, during which time cells remained actively growing. We optimized parameters for the assay, with the final format being 5 days’ growth in 96-well plates in the presence of 2% w/v DMSO. We confirmed reproducibility using rifampicin and other antibiotics. The dual detection method allows for a reproducible calculation of the minimum inhibitory concentration (MIC), at the same time detecting artefacts such as fluorescence quenching or compound precipitation. We used our assay to confirm anti-tubercular activity and establish the structure activity relationship (SAR) around the imidazo[1,2-a]pyridine-3-carboxamides, a promising series of M. tuberculosis inhibitors.     

Toward Modern Classification of Eustigmatophytes,Including the Description of Neomonodaceae Fam. Nov. and Three New Genera1

The class Eustigmatophyceae includes mostly coccoid, freshwater algae, although some genera are common in terrestrial habitats and two are primarily marine. The formal classification of the class, developed decades ago, does not fit the diversity and phylogeny of the group as presently known and is in urgent need of revision. This study concerns a clade informally known as the Pseudellipsoidion group of the order Eustigmatales, which was initially known to comprise seven strains with oval to ellipsoidal cells, some bearing a stipe. We examined those strains as well as 10 new ones and obtained 18S rDNA and rbcL gene sequences. The results from phylogenetic analyses of the sequence data were integrated with morphological data of vegetative and motile cells. Monophyly of the Pseudellipsoidion group is supported in both 18S rDNA and rbcL trees. The group is formalized as the new family Neomonodaceae comprising, in addition to Pseudellipsoidion, three newly erected genera. By establishing Neomonodus gen. nov. (with type species Neomonodus ovalis comb. nov.), we finally resolve the intricate taxonomic history of a species originally described as Monodus ovalis and later moved to the genera Characiopsis and Pseudocharaciopsis. Characiopsiella gen. nov. (with the type species Characiopsiella minima comb. nov.) and Munda gen. nov. (with the type species Munda aquilonaris) are established to accommodate additional representatives of the polyphyletic genus Characiopsis. A morphological feature common to all examined Neomonodaceae is the absence of a pyrenoid in the chloroplasts, which discriminates them from other morphologically similar yet unrelated eustigmatophytes (including other Characiopsis-like species).     

Protease-activated Receptor 1 (PAR1) and PAR4 Heterodimers Are Required for PAR1-enhanced Cleavage of PAR4 by α-Thrombin

Thrombin is a potent platelet agonist that activates platelets and other cells of the cardiovascular system by cleaving its G-protein-coupled receptors, protease-activated receptor 1 (PAR1), PAR4, or both. We now show that cleaving PAR1 and PAR4 with α-thrombin induces heterodimer formation. PAR1-PAR4 heterodimers were not detected when unstimulated; however, when the cells were stimulated with 10 nm α-thrombin, we were able to detect a strong interaction between PAR1 and PAR4 by bioluminescence resonance energy transfer. In contrast, activating the receptors without cleavage using PAR1 and PAR4 agonist peptides (TFLLRN and AYPGKF, respectively) did not enhance heterodimer formation. Preventing PAR1 or PAR4 cleavage with point mutations or hirugen also prevented the induction of heterodimers. To further characterize the PAR1-PAR4 interactions, we mapped the heterodimer interface by introducing point mutations in transmembrane helix 4 of PAR1 or PAR4 that prevented heterodimer formation. Finally, we show that mutations in PAR1 or PAR4 at the heterodimer interface prevented PAR1-assisted cleavage of PAR4. These data demonstrate that PAR1 and PAR4 require allosteric changes induced via receptor cleavage by α-thrombin to mediate heterodimer formation, and we have determined the PAR1-PAR4 heterodimer interface. Our findings show that PAR1 and PAR4 have dynamic interactions on the cell surface that should be taken into account when developing and characterizing PAR antagonists.     

Regional registration of [6‐14C]glucose metabolism during brain activation of α‐syntrophin knockout mice

α‐Syntrophin is a component of the dystrophin scaffold‐protein complex that serves as an adaptor for recruitment of key proteins to the cytoplasmic side of plasma membranes. α‐Syntrophin knockout (KO) causes loss of the polarized localization of aquaporin4 (AQP4) at astrocytic endfeet and interferes with water and K⁺ homeostasis. During brain activation, release of ions and metabolites from endfeet is anticipated to increase perivascular fluid osmolarity, AQP4‐mediated osmotic water flow from endfeet, and metabolite washout from brain. This study tests the hypothesis that reduced levels of endfoot AQP4 increase retention of [¹⁴C]metabolites during sensory stimulation. Conscious KO and wild‐type mice were pulse‐labeled with [6‐¹⁴C] glucose during unilateral acoustic stimulation or bilateral acoustic plus whisker stimulation, and label retention was assayed by computer‐assisted brain imaging or analysis of [¹⁴C]metabolites in extracts, respectively. High‐resolution autoradiographic assays detected a 17% side‐to‐side difference (p < 0.05) in inferior colliculus of KO mice, not wild‐type mice. However, there were no labeling differences between KO and wild‐type mice for five major HPLC fractions from four dissected regions, presumably because of insufficient anatomical resolution. The results suggest a role for AQP4‐mediated water flow in support of washout of metabolites, and underscore the need for greater understanding of astrocytic water and metabolite fluxes.     

Alpha-Synuclein Induces Lysosomal Rupture and Cathepsin Dependent Reactive Oxygen Species Following Endocytosis

α-synuclein dysregulation is a critical aspect of Parkinson''s disease pathology. Recent studies have observed that α-synuclein aggregates are cytotoxic to cells in culture and that this toxicity can be spread between cells. However, the molecular mechanisms governing this cytotoxicity and spread are poorly characterized. Recent studies of viruses and bacteria, which achieve their cytoplasmic entry by rupturing intracellular vesicles, have utilized the redistribution of galectin proteins as a tool to measure vesicle rupture by these organisms. Using this approach, we demonstrate that α-synuclein aggregates can induce the rupture of lysosomes following their endocytosis in neuronal cell lines. This rupture can be induced by the addition of α-synuclein aggregates directly into cells as well as by cell-to-cell transfer of α-synuclein. We also observe that lysosomal rupture by α-synuclein induces a cathepsin B dependent increase in reactive oxygen species (ROS) in target cells. Finally, we observe that α-synuclein aggregates can induce inflammasome activation in THP-1 cells. Lysosomal rupture is known to induce mitochondrial dysfunction and inflammation, both of which are well established aspects of Parkinson''s disease, thus connecting these aspects of Parkinson''s disease to the propagation of α-synuclein pathology in cells.     

Real‐Time Investigation of Intercalation and Structure Evolution in Printed Polymer:Fullerene Bulk Heterojunction Thin Films

The complex intermixing morphology is critical for the performance of the nanostructured polymer:fullerene bulk heterojunction (BHJ) solar cells. Here, time resolved in situ grazing incidence X‐ray diffraction and grazing incidence small angle X‐ray scattering are used to track the structure formation of BHJ thin films formed from the donor polymer poly(2,5‐bis(3‐hexadecylthiophen‐2‐yl)thieno[3,2‐b]thiophene) with different fullerene derivative acceptors. The formation of stable bimolecular crystals through the intercalation of fullerene molecules between the side chains of polymer crystallites is investigated. Such systems exhibit more efficient exciton dissociation but lower photo‐conductance and faster decay of charges. On the basis of the experimental observations, intercalation obviously takes place before or with the formation of the crystalline polymer domains. It results in more stable structures whose volume remains constant upon further drying. Three distinct periods of drying are observed and the formation of unidimensional fullerene channels along the π‐stacking direction of polymer crystallites is confirmed.     

Effects of Central Corneal Stromal Thickness and Epithelial Thickness on Intraocular Pressure Using Goldmann Applanation and Non-Contact Tonometers

Purpose

To investigate whether corneal thickness parameters measured by optical coherence tomography (OCT), such as central corneal thickness (CCT), central corneal stromal thickness (CCST), and central corneal epithelial thickness (CCET), influence the intraocular pressure (IOP) difference measured by Goldmann applanation tonometry (GAT) and non-contact tonometry (NCT).

Methods

In total, 50 eyes from 50 subjects without glaucomatous defects were included in this retrospective, cross-sectional study. We measured IOP using GAT and NCT and calculated the difference between the two methods. CCT was measured by a Cirrus HD-OCT device using anterior segment imaging. The basement membrane of the epithelium, which was seen as a high-reflection line in the OCT image, was taken as a reference line to measure CCST and CCET.

Results

The mean IOP measured by GAT and NCT was 16.7 ± 3.0 and 18.1 ± 3.8 mmHg, respectively. The mean IOP difference was 1.5 ± 1.7 mmHg, and the IOP measured by NCT was 8.4% ± 11.3% higher than that measured by GAT. The CCET and CCST were 57.9 ± 5.6 and 501.7 ± 33.8 μm, respectively. CCT showed a positive correlation with both GAT IOP (r = 0.648, P < 0.001) and NCT IOP (r = 0.676, P < 0.001). Although CCST showed a significant correlation with GAT IOP and NCT IOP, CCET did not. The difference between GAT IOP and NCT IOP increased with CCT (r = 0.333, P = 0.018), and CCET was positively correlated with the IOP difference between GAT and NCT (r = 0.435, P = 0.002).

Conclusions

IOP increased with greater CCT, and CCST seemed to have a more important role than CCET. CCET also increased with greater CCT, and this may be a possible explanation for the increasing difference in IOP between GAT and NCT with increasing CCT.     

Conserved regulation of MAP kinase expression by PUF RNA-binding proteins

Mitogen-activated protein kinase (MAPK) and PUF (for Pumilio and FBF [fem-3 binding factor]) RNA-binding proteins control many cellular processes critical for animal development and tissue homeostasis. In the present work, we report that PUF proteins act directly on MAPK/ERK-encoding mRNAs to downregulate their expression in both the Caenorhabditis elegans germline and human embryonic stem cells. In C. elegans, FBF/PUF binds regulatory elements in the mpk-1 3′ untranslated region (3′ UTR) and coprecipitates with mpk-1 mRNA; moreover, mpk-1 expression increases dramatically in FBF mutants. In human embryonic stem cells, PUM2/PUF binds 3′UTR elements in both Erk2 and p38α mRNAs, and PUM2 represses reporter constructs carrying either Erk2 or p38α 3′ UTRs. Therefore, the PUF control of MAPK expression is conserved. Its biological function was explored in nematodes, where FBF promotes the self-renewal of germline stem cells, and MPK-1 promotes oocyte maturation and germ cell apoptosis. We found that FBF acts redundantly with LIP-1, the C. elegans homolog of MAPK phosphatase (MKP), to restrict MAPK activity and prevent apoptosis. In mammals, activated MAPK can promote apoptosis of cancer cells and restrict stem cell self-renewal, and MKP is upregulated in cancer cells. We propose that the dual negative regulation of MAPK by both PUF repression and MKP inhibition may be a conserved mechanism that influences both stem cell maintenance and tumor progression.