Development and assessment of sensitive immuno‐PCR assays for the quantification of cerebrospinal fluid three‐ and four‐repeat tau isoforms in tauopathies

Characteristic tau isoform composition of the insoluble fibrillar tau inclusions define tauopathies, including Alzheimer's disease (AD), progressive supranuclear palsy (PSP) and frontotemporal dementia with parkinsonism linked to chromosome 17/frontotemporal lobar degeneration‐tau (FTDP‐17/FTLD‐tau). Exon 10 splicing mutations in the tau gene, MAPT, in familial FTDP‐17 cause elevation of tau isoforms with four microtubule‐binding repeat domains (4R‐tau) compared to those with three repeats (3R‐tau). On the basis of two well‐characterised monoclonal antibodies against 3R‐ and 4R‐tau, we developed novel, sensitive immuno‐PCR assays for measuring the trace amounts of these isoforms in CSF. This was with the aim of assessing if CSF tau isoform changes reflect the pathological changes in tau isoform homeostasis in the degenerative brain and if these would be relevant for differential clinical diagnosis. Initial analysis of clinical CSF samples of PSP (n = 46), corticobasal syndrome (CBS; n = 22), AD (n = 11), Parkinson's disease with dementia (PDD; n = 16) and 35 controls revealed selective decreases of immunoreactive 4R‐tau in CSF of PSP and AD patients compared with controls, and lower 4R‐tau levels in AD compared with PDD. These decreases could be related to the disease‐specific conformational masking of the RD4‐binding epitope because of abnormal folding and/or aggregation of the 4R‐tau isoforms in tauopathies or increased sequestration of the 4R‐tau isoforms in brain tau pathology.     

New benzothieno[2,3-c]pyridines as non-steroidal CYP17 inhibitors: design,synthesis, anticancer screening,apoptosis induction,and in silico ADME profile studies

A series of [1]benzothieno[2,3-c]pyridines was synthesised. Most compounds were chosen by NCI-USA to evaluate their anticancer activity. Compounds 5a–c showed prominent growth inhibition against most cell lines. 5c was selected at five dose concentration levels. It exhibited potent broad-spectrum anticancer activity with a GI₅₀ of 4 nM–37 µM. Cytotoxicity of 5a–c was further evaluated against prostate, renal, and breast cancer cell lines. 5c showed double and quadruple the activity of staurosporine and abiraterone, respectively, against the PC-3 cell line with IC₅₀ 2.08 µM. The possible mechanism of anti-prostate cancer was explored via measuring the CYP17 enzyme activity in mice prostate cancer models compared to abiraterone. The results revealed that 5c suppressed the CYP17 enzyme to 15.80 nM. Moreover, it was found to be equipotent to abiraterone in testosterone production. Cell cycle analysis and apoptosis were performed. Additionally, the ADME profile of compound 5c demonstrated both good oral bioavailability and metabolic stability.     

Macrotrachela quadricornifera featured in a space experiment

Macrotrachela quadricornifera, a bdelloid rotifer, is the animal model of an experiment scheduled on the International Space Station (ISS). The focus of the experiment is the role of the cytoskeleton during oogenesis and early development. Bdelloids will fly desiccated, be rehydrated on the ISS and cultivated for five generations. We present the outline of the ISS experiment, the expectations and the state-of-the-art of ground-based research run to date on the major topics of the planned experiment: anhydrobiosis and embryogenesis. Anhydrobiosis focuses on two major aspects, storage conditions that enhance recovery rate and comparison of the resistance between dormant and active rotifers to UV radiation and other environmental injuries. Embryogenesis has been approached at the morphological level under ground conditions: developing embryos have been studied by light and confocal microscopes.     

Novel heme ligand displacement by CO in the soluble hemophore HasA and its proximal ligand mutants: implications for heme uptake and release

HasASM, a hemophore secreted by the Gram-negative bacteria Serratia marcescens, extracts heme from host hemoproteins and shuttles it to HasRSM, a specific hemophore outer membrane receptor. Heme iron in HasASM is in a six-coordinate ferric state. It is linked to the protein by the heretofore uncommon axial ligand set, His32 and Tyr75. A third residue of the heme pocket, His83, plays a crucial role in heme ligation through hydrogen bonding to Tyr75. The vibrational frequencies of coordinated carbon monoxide constitute a sensitive probe of trans ligand field, FeCO structure, and electrostatic landscape of the distal heme pockets of heme proteins. In this study, carbonyl complexes of wild-type (WT) HasASM and its heme pocket mutants His32Ala, Tyr75Ala, and His83Ala were characterized by resonance Raman spectroscopy. The CO complexes of WT HasASM, HasASM(His32Ala), and HasASM(His83Ala) exhibit similar spectral features and fall above the line that correlates nuFe-CO and nuC-O for proteins having a proximal imidazole ligand. This suggests that the proximal ligand field in these CO adducts is weaker than that for heme-CO proteins bearing a histidine axial ligand. In contrast, the CO complex of HasASM(Tyr75Ala) has resonance Raman signatures consistent with ImH-Fe-CO ligation. These results reveal that in WT HasASM, the axial ImH side chain of His32 is displaced by CO. This is in contrast to other heme proteins known to have the His/Tyr axial ligand set, wherein the phenolic side chain of the Tyr ligand dissociates upon CO addition. The displacement of His32 and its stabilization in an unbound state is postulated to be relevant to heme uptake and/or release.     

Modulation by substrates of the interaction between the HasR outer membrane receptor and its specific TonB-like protein, HasB

TonB is a cytoplasmic membrane protein required for active transport of various essential substrates such as heme and iron siderophores through the outer membrane receptors of Gram-negative bacteria. This protein spans the periplasm, contacts outer membrane transporters by its C-terminal domain, and transduces energy from the protonmotive force to the transporters. The TonB box, a relatively conserved sequence localized on the periplasmic side of the transporters, has been shown to directly contact TonB.While Serratia marcescens TonB functions with various transporters, HasB, a TonB-like protein, is dedicated to the HasR transporter. HasR acquires heme either freely or via an extracellular heme carrier, the hemophore HasA, that binds to HasR and delivers heme to the transporter. Here, we study the interaction of HasR with a HasB C-terminal domain and compare it with that obtained with a TonB C-terminal fragment. Analysis of the thermodynamic parameters reveals that the interaction mode of HasR with HasB differs from that with TonB, the difference explaining the functional specificity of HasB for HasR. We also demonstrate that the presence of the substrate on the extracellular face of the transporter modifies, via enthalpy-entropy compensation, the interaction with HasB on the periplasmic face. The transmitted signal depends on the nature of the substrate. While the presence of heme on the transporter modifies only slightly the nature of interactions involved between HasR and HasB, hemophore binding on the transporter dramatically changes the interactions and seems to locally stabilize some structural motifs. In both cases, the HasR TonB box is the target for those modifications.     

A rapid method combining Golgi and Nissl staining to study neuronal morphology and cytoarchitecture.

The Golgi silver impregnation technique gives detailed information on neuronal morphology of the few neurons it labels, whereas the majority remain unstained. In contrast, the Nissl staining technique allows for consistent labeling of the whole neuronal population but gives very limited information on neuronal morphology. Most studies characterizing neuronal cell types in the context of their distribution within the tissue slice tend to use the Golgi silver impregnation technique for neuronal morphology followed by deimpregnation as a prerequisite for showing that neuron's histological location by subsequent Nissl staining. Here, we describe a rapid method combining Golgi silver impregnation with cresyl violet staining that provides a useful and simple approach to combining cellular morphology with cytoarchitecture without the need for deimpregnating the tissue. Our method allowed us to identify neurons of the facial nucleus and the supratrigeminal nucleus, as well as assessing cellular distribution within layers of the dorsal cochlear nucleus. With this method, we also have been able to directly compare morphological characteristics of neuronal somata at the dorsal cochlear nucleus when labeled with cresyl violet with those obtained with the Golgi method, and we found that cresyl violet-labeled cell bodies appear smaller at high cellular densities. Our observation suggests that cresyl violet staining is inadequate to quantify differences in soma sizes.     

Blockade of antimicrobial proteins S100A8 and S100A9 inhibits phagocyte migration to the alveoli in streptococcal pneumonia

We investigated the roles of the potent, chemotactic antimicrobial proteins S100A8, S100A9, and S100A8/A9 in leukocyte migration in a model of streptococcal pneumonia. We first observed differential secretion of S100A8, S100A9, and S100A8/A9 that preceded neutrophil recruitment. This is partially explained by the expression of S100A8 and S100A9 proteins by pneumocytes in the early phase of Streptococcus pneumoniae infection. Pretreatment of mice with anti-S100A8 and anti-S100A9 Abs, alone or in combination had no effect on bacterial load or mice survival, but caused neutrophil and macrophage recruitment to the alveoli to diminish by 70 and 80%, respectively, without modifying leukocyte blood count, transendothelial migration or neutrophil sequestration in the lung vasculature. These decreases were also associated with a 68% increase of phagocyte accumulation in lung tissue and increased expression of the chemokines CXCL1, CXCL2, and CCL2 in lung tissues and bronchoalveolar lavages. These results show that S100A8 and S100A9 play an important role in leukocyte migration and strongly suggest their involvement in the transepithelial migration of macrophages and neutrophils. They also indicate the importance of antimicrobial proteins, as opposed to classical chemotactic factors such as chemokines, in regulating innate immune responses in the lung.     

Reversible blockade of thymic output: an inherent part of TLR ligand-mediated immune response

TLRs constitute a first set of sensors that detect viral nucleic acids including dsRNA which triggers TLR3. We report the early, direct, and detrimental effect of polyinosine-polycytidilic acid treatment on T cell development. Inhibition of thymopoiesis was targeted to several thymocyte subpopulations. First, both a blockade of the double negative (DN)1-DN2 transition and a severe down-regulation of DN3-DN4 thymocyte proliferation were observed. In addition, an important decrease in the absolute numbers of double-positive thymocytes, concomitant with an increase in frequencies of apoptotic cells in this population were shown. This inhibition of thymopoiesis resulted in a reduced thymic output, as evidenced by a drop of the absolute numbers of naive T cells and TCR excision circles levels. The decrease in thymic cellularity and defects in thymic development were severely reduced, but not completely abolished in IFN-alpha/betaR(-/-) mice, showing a direct contribution of type I IFNs, known to be massively up-regulated in viral infections, to the inhibition of T cell development. Strikingly, the TCR repertoire in treated mice was biased toward shorter CDR3 lengths as a result of a decreased expression of TdT and Rag2. However, thymic integrity remained intact since thymopoiesis was restored both quantitatively and qualitatively 14 days after the cessation of polyinosine-polycytidilic acid treatment. These results demonstrate a novel immunomodulatory role for virally encoded TLR ligands and RNA sensors; they further illustrate the diversity of mechanisms that viruses use to interfere with the development of a pathogen-specific immune responses.     

Evolution of protein-coding genes in Drosophila

Several contributing factors have been implicated in evolutionary rate heterogeneity among proteins, but their evolutionary mechanisms remain poorly characterized. The recently sequenced 12 Drosophila genomes provide a unique opportunity to shed light on these unresolved issues. Here, we focus on the role of natural selection in shaping evolutionary rates. We use the Drosophila genomic data to distinguish between factors that increase the strength of purifying selection on proteins and factors that affect the amount of positive selection experienced by proteins. We confirm the importance of translational selection in shaping protein evolution in Drosophila and show that factors such as tissue bias in expression, gene essentiality, intron number, and recombination rate also contribute to evolutionary rate variation among proteins.