An Age-Wise Comparison of Human Airway Smooth Muscle Proliferative Capacity

We compared the proliferation of neonatal and adult airway smooth muscle cells (ASMC) with no/moderate lung disease, in glucose- (energy production by glycolysis) or glucose-free medium (ATP production from mitochondrial oxidative phosphorylations only), in response to 10% fetal calf serum (FCS) and PDGF-AA. In the presence of glucose, cell counts were significantly greater in neonatal vs. adult ASMC. Similarly, neonatal ASMC DNA synthesis in 10% FCS and PDGF-AA, and [Ca²⁺]i responses in the presence of histamine were significantly enhanced vs. adults. In glucose-free medium, cell proliferation was preserved in neonatal cells, unlike in adult cells, with concomitant increased porin (an indicator of mitochondrial activity) protein expression. Compared to adults, stimulated neonatal human ASMC are in a rapid and robust proliferative phase and have the capacity to respond disproportionately under abnormal environmental conditions, through increased mitochondrial biogenesis and altered calcium homeostasis.     

Comparison of Photoacclimation in Twelve Freshwater Photoautotrophs (Chlorophyte,Bacillaryophyte, Cryptophyte and Cyanophyte) Isolated from a Natural Community

Different representative of algae and cyanobacteria were isolated from a freshwater habitat and cultivated in laboratory to compare their photoacclimation capacity when exposed to a wide range of light intensity and to understand if this factor may modify natural community dominance. All species successfully acclimated to all light intensities and the response of phytoplankton to increased light intensity was similar and included a decrease of most photosynthetic pigments accompanied by an increase in photoprotective pigment content relative to Chl a. Most species also decreased their light absorption efficiency on a biovolume basis. This decrease not only resulted in a lower fraction of energy absorbed by the cell, but also to a lower transfer of energy to PSII and PSI. Furthermore, energy funnelled to PSII or PSI was also rearranged in favour of PSII. High light acclimated organisms also corresponded to high non-photochemical quenching and photosynthetic electron transport reduction state and to a low Φ''_M. Thus photoacclimation processes work toward reducing the excitation pressure in high light environment through a reduction of light absorption efficiency, but also by lowering conversion efficiency. Interestingly, all species of our study followed that tendency despite being of different functional groups (colonial, flagellated, different sizes) and of different phylogeny demonstrating the great plasticity and adaptation ability of freshwater phytoplankton to their light environment. These adjustments may explain the decoupling between growth rate and photosynthesis observed above photosynthesis light saturation point for all species. Even if some species did reach higher growth rate in our conditions and thus, should dominate in natural environment with respect to light intensity, we cannot exclude that other environmental factors also influence the population dynamic and make the outcome harder to predict.     

Whole-bacterium ribosome display selection for isolation of highly specific anti-Staphyloccocus aureus Affitins for detection- and capture-based biomedical applications

Detection and capture methods using antibodies have been developed to ensure identification of pathogens in biological samples. Though antibodies have many attractive properties, they also have limitations and there are needs to expand the panel of available affinity proteins with different properties. Affitins, that we developed from the Sul7d proteins, are a solid class of affinity proteins, which can be used as substitutes to antibodies or to complement them. We report the generation and characterization of antibacterial Affitins with high specificity for Staphylococcus aureus. For the first time, ribosome display selections were carried out using whole-living-cell and naïve combinatorial libraries, which avoid production of protein targets and immunization of animals. We showed that Affitin C5 exclusively recognizes S. aureus among dozens of strains, including clinical ones. C5 binds staphylococcal Protein A (SpA) with a K _D of 108 ± 2 nM and has a high thermostability (T _m = 77.0°C). Anti-S. aureus C5 binds SpA or bacteria in various detection and capture applications, including ELISA, western blot analysis, bead-fishing, and fluorescence imaging. Thus, novel anti-bacteria Affitins which are cost-effective, stable, and small can be rapidly and fully designed in vitro with high affinity and specificity for a surface-exposed marker. This class of reagents can be useful in diagnostic and biomedical applications.     

Motor-dependent microtubule disassembly driven by tubulin tyrosination

In cells, stable microtubules (MTs) are covalently modified by a carboxypeptidase, which removes the C-terminal Tyr residue of α-tubulin. The significance of this selective detyrosination of MTs is not understood. In this study, we report that tubulin detyrosination in fibroblasts inhibits MT disassembly. This inhibition is relieved by overexpression of the depolymerizing motor mitotic centromere-associated kinesin (MCAK). Conversely, suppression of MCAK expression prevents disassembly of normal tyrosinated MTs in fibroblasts. Detyrosination of MTs suppresses the activity of MCAK in vitro, apparently as the result of a decreased affinity of the adenosine diphosphate (ADP)–inorganic phosphate- and ADP-bound forms of MCAK for the MT lattice. Detyrosination also impairs MT disassembly in neurons and inhibits the activity of the neuronal depolymerizing motor KIF2A in vitro. These results indicate that MT depolymerizing motors are directly inhibited by the detyrosination of tubulin, resulting in the stabilization of cellular MTs. Detyrosination of transiently stabilized MTs may give rise to persistent subpopulations of disassembly-resistant polymers to sustain subcellular cytoskeletal differentiation.     

Swi5-Sfr1 protein stimulates Rad51-mediated DNA strand exchange reaction through organization of DNA bases in the presynaptic filament

The Swi5-Sfr1 heterodimer protein stimulates the Rad51-promoted DNA strand exchange reaction, a crucial step in homologous recombination. To clarify how this accessory protein acts on the strand exchange reaction, we have analyzed how the structure of the primary reaction intermediate, the Rad51/single-stranded DNA (ssDNA) complex filament formed in the presence of ATP, is affected by Swi5-Sfr1. Using flow linear dichroism spectroscopy, we observe that the nucleobases of the ssDNA are more perpendicularly aligned to the filament axis in the presence of Swi5-Sfr1, whereas the bases are more randomly oriented in the absence of Swi5-Sfr1. When using a modified version of the natural protein where the N-terminal part of Sfr1 is deleted, which has no affinity for DNA but maintained ability to stimulate the strand exchange reaction, we still observe the improved perpendicular DNA base orientation. This indicates that Swi5-Sfr1 exerts its activating effect through interaction with the Rad51 filament mainly and not with the DNA. We propose that the role of a coplanar alignment of nucleobases induced by Swi5-Sfr1 in the presynaptic Rad51/ssDNA complex is to facilitate the critical matching with an invading double-stranded DNA, hence stimulating the strand exchange reaction.     

Multivalent nanobodies targeting death receptor 5 elicit superior tumor cell killing through efficient caspase induction

Multiple therapeutic agonists of death receptor 5 (DR5) have been developed and are under clinical evaluation. Although these agonists demonstrate significant anti-tumor activity in preclinical models, the clinical efficacy in human cancer patients has been notably disappointing. One possible explanation might be that the current classes of therapeutic molecules are not sufficiently potent to elicit significant response in patients, particularly for dimeric antibody agonists that require secondary cross-linking via Fcγ receptors expressed on immune cells to achieve optimal clustering of DR5. To overcome this limitation, a novel multivalent Nanobody approach was taken with the goal of generating a significantly more potent DR5 agonist. In the present study, we show that trivalent DR5 targeting Nanobodies mimic the activity of natural ligand, and furthermore, increasing the valency of domains to tetramer and pentamer markedly increased potency of cell killing on tumor cells, with pentamers being more potent than tetramers in vitro. Increased potency was attributed to faster kinetics of death-inducing signaling complex assembly and caspase-8 and caspase-3 activation. In vivo, multivalent Nanobody molecules elicited superior anti-tumor activity compared to a conventional DR5 agonist antibody, including the ability to induce tumor regression in an insensitive patient-derived primary pancreatic tumor model. Furthermore, complete responses to Nanobody treatment were obtained in up to 50% of patient-derived primary pancreatic and colon tumor models, suggesting that multivalent DR5 Nanobodies may represent a significant new therapeutic modality for targeting death receptor signaling.     

New horizons in schizophrenia treatment: autophagy protection is coupled with behavioral improvements in a mouse model of schizophrenia

Autophagy plays a key role in the pathophysiology of schizophrenia as manifested by a 40% decrease in BECN1/Beclin 1 mRNA in postmortem hippocampal tissues relative to controls. This decrease was coupled with the deregulation of the essential ADNP (activity-dependent neuroprotector homeobox), a binding partner of MAP1LC3B/LC3B (microtubule-associated protein 1 light chain 3 β) another major constituent of autophagy. The drug candidate NAP (davunetide), a peptide fragment from ADNP, enhanced the ADNP-LC3B interaction. Parallel genetic studies have linked allelic variation in the gene encoding MAP6/STOP (microtubule-associated protein 6) to schizophrenia, along with altered MAP6/STOP protein expression in the schizophrenic brain and schizophrenic-like behaviors in Map6-deficient mice. In this study, for the first time, we reveal significant decreases in hippocampal Becn1 mRNA and reversal by NAP but not by the antipsychotic clozapine (CLZ) in Map6-deficient (Map6^+/−) mice. Normalization of Becn1 expression by NAP was coupled with behavioral protection against hyperlocomotion and cognitive deficits measured in the object recognition test. CLZ reduced hyperlocomotion below control levels and did not significantly affect object recognition. The combination of CLZ and NAP resulted in normalized outcome behaviors. Phase II clinical studies have shown NAP-dependent augmentation of functional activities of daily living coupled with brain protection. The current studies provide a new mechanistic pathway and a novel avenue for drug development.     

Enantiopure ethyl 2,3-dibromopropionate: Enantioselective synthesis vs preparative HPLC enantioseparation of racemate on multigram scale

Racemic ethyl 2,3-dibromopropionate, commercially available at low price, is a key intermediate used in the synthesis of several heterocycle fragments, which are present in many biologically active compounds. Surprisingly, the enantiomers are not commercially available and have never been described in the literature. In this work, we undertook two different strategies to obtain these enantiomers, which are enantioselective synthesis and preparative HPLC enantioseparation of commercially available racemate on multigram scale. The first strategy has proved inadequate because racemization occurred during the synthesis (ee ≈ 9-50%). Conversely, the second strategy produced a very good enantioseparation of commercially available racemate (ee > 99.5% for both enantiomers) on multigram scale.