Recognition of a 10 base pair sequence of DNA and stereochemical control of the binding affinity of chiral hairpin polyamide-Hoechst 33258 conjugates

Chiral hairpin polyamides linked to a Hoechst 33258 analogue at the -position of the hairpin turn amino acid (1, 2) were synthesized on solid phase by adopting Fmoc and ivDde techniques. The DNA-binding properties of enantiomeric conjugates 1 and 2, and N-terminal linked conjugate 3 for 8–14 bp sequences were determined by spectrofluorometric and thermal melting studies. Conjugates 1 and 2 recognize a 10 bp sequence, while conjugate 3 recognizes a 9 bp sequence. Interestingly, R-enantiomer 1 exhibited 10- to 30-fold higher binding affinities than S-enantiomer 2 for the DNA sequences studied. These binding differences were accounted for by molecular modeling studies, which revealed that the amide proton nearest to the chiral center in R-conjugate 1 is better positioned to form hydrogen bonds to the DNA bases, while S-conjugate 2 does not.     

The sub-lip domain--a distinct pathway for myotome precursors that demonstrate rostral-caudal migration

We have previously reported that the myotome is formed by a first wave of pioneer cells generated from all along the dorsomedial portion of the epithelial somite and a second wave of cells issued from all four edges of the dermomyotome. Cells from the extreme rostral and caudal edges directly generate myofibers that elongate towards the opposite pole of each segment and along the pre-existing myotomal scaffold. In contrast, cells from the dorsomedial and ventrolateral lips first reach the extreme edges and then contribute to myofiber formation. The mechanism by which these epithelial cells translocate remained unknown and was the goal of the present study. We have found that epithelial cells along the dorsomedial and ventrolateral lips of the dermomyotome first delaminate into the immediate underlayer of the corresponding lips, the sub-lip domain, then migrate longitudinally along this pathway until reaching the extreme edges from which they differentiate into myofibers. Cells of the sub-lip domain are negative for Pax3 and desmin but express MyoD, Myf5 and FREK, suggesting that they are specific myogenic progenitors.     

The third wave of myotome colonization by mitotically competent progenitors: regulating the balance between differentiation and proliferation during muscle development

The myotome is formed by a first wave of pioneer cells originating from the entire dorsomedial region of epithelial somites and a second wave that derives from all four lips of the dermomyotome but generates myofibers from only the rostral and caudal edges. Because the precedent progenitors exit the cell cycle upon myotome colonization, subsequent waves must account for consecutive growth. In this study, double labeling with CM-DiI and BrdU revealed the appearance of a third wave of progenitors that enter the myotome as mitotically active cells from both rostral and caudal dermomyotome edges. These cells express the fibroblast growth factor (FGF) receptor FREK and treatment with FGF4 promotes their proliferation and redistribution towards the center of the myotome. Yet, they are negative for MyoD, Myf5 and FGF4, which are, however, expressed in myofibers. The proliferating progenitors first appear around the 30-somite stage in cervical-level myotomes and their number continuously increases, making up 85% of total muscle nuclei by embryonic day (E)4. By this stage, generation of second-wave myofibers, which also enter from the extreme lips is still under way. Formation of the latter fibers peaks at 30 somites and progressively decreases with age until E4. Thus, cells in these dermomyotome lips generate simultaneously distinct types of muscle progenitors in changing proportions as a function of age. Consistent with a heterogeneity in the cellular composition of the extreme lips, MyoD is normally expressed in only a subset of these epithelial cells. Treatment with Sonic hedgehog drives most of them to become MyoD positive and then to become myofibers, with a concurrent reduction in the proportion of proliferating muscle precursors.     

LGN-dependent orientation of cell divisions in the dermomyotome controls lineage segregation into muscle and dermis

The plane of cell divisions is pivotal for differential fate acquisition. Dermomyotome development provides an excellent system with which to investigate the link between these processes. In the central sheet of the early dermomyotome, single epithelial cells divide with a planar orientation. Here, we report that in the avian embryo, in addition to self-renewing, a subset of progenitors translocates into the myotome where they generate differentiated myocytes. By contrast, in the late epithelium, individual progenitors divide perpendicularly to produce both mitotic myoblasts and dermis. To examine whether spindle orientations influence fate segregation, early planar divisions were randomized and/or shifted to a perpendicular orientation by interfering with LGN function or by overexpressing inscuteable. Clones derived from single transfected cells exhibited an enhanced proportion of mixed dermomyotome/myotome progeny at the expense of `like' daughter cells in either domain. Loss of LGN or Gαi1 function in the late epithelium randomized otherwise perpendicular mitoses and favored muscle development at the expense of dermis. Hence, LGN-dependent early planar divisions are required for the proper allocation of progenitors into either dermomyotome or myotome, whereas late perpendicular divisions are necessary for the normal balance between muscle and dermis production.     

BrainTV: a novel approach for online mapping of human brain functions

Our understanding of the brain's functional organisation has greatly benefited from occasional exploratory sessions during electrophysiological studies, trying various manipulations of an animal's environment to trigger responses in particular neurons. Famous examples of such exploration have unveiled various unexpected response properties, such as those of mirror neurons. This approach, which relies on the possibility to test online the reactivity of precise neural populations has no equivalent so far in humans. The present study proposes and applies a radically novel framework for mapping human brain functions in ecological situations based on a combination of a) exploratory sessions, using real-time electrophysiology to formulate hypotheses about the functional role of precise cortical regions and b) controlled experimental protocols specifically adapted to test these hypotheses. Using this two-stage approach with an epileptic patient candidate for surgery and implanted with intracerebral electrodes, we were able to precisely map high-level auditory functions in the patients' superior temporal lobe. We propose that this procedure constitutes at the least a useful complement of electrical cortical stimulations to map eloquent brain areas in epileptic patients before their surgery, but also a path of discovery for human functional brain mapping.     

A rapid assay for drug sensitivity of glioblastoma stem cells

Glioblastoma (GBM) is a highly infiltrating, aggressive brain cancer with no available curative treatment. We developed a rapid assay for assessing the effect of various drugs on GBM stem cells. The assay uses a small number of separated CD133+ cells (20,000 in 0.2 ml) in 96-well plate that form neurospheres within 1-2 days. Various drugs disperse the neurospheres within 24-36 h, which can be quantified microscopically. We used the GBM cell line A-172 to develop the conditions for the assay, utilizing Gleevec, the gamma-secretase inhibitor DAPT, and the anti-bacterial peptide amph1D. The results show dispersion of the neurospheres leading to cell death, at relatively low drugs concentrations (<25 microM). Drug combination showed a synergistic effect and disruption of neurospheres under lower concentrations. We applied this assay to the CD133+ cells of surgical specimens from three patients that showed similar results. This assay facilitates a rapid test of drugs on small amounts of fractionated patient's GBM stem cells.     

Depletion of B cells rejuvenates the peripheral B‐cell compartment but is insufficient to restore immune competence in aging

Aging is associated with increasing prevalence and severity of infections caused by a decline in bone marrow (BM) lymphopoiesis and reduced B‐cell repertoire diversity. The current study proposes a strategy to enhance immune responsiveness in aged mice and humans, through rejuvenation of the B lineage upon B‐cell depletion. We used hCD20Tg mice to deplete peripheral B cells in old and young mice, analyzing B‐cell subsets, repertoire and cellular functions in vitro, and immune responsiveness in vivo. Additionally, elderly patients, previously treated with rituximab healthy elderly and young individuals, were vaccinated against hepatitis B (HBV) after undergoing a detailed analysis for B‐cell compartments. B‐cell depletion in old mice resulted in rejuvenated B‐cell population that was derived from de novo synthesis in the bone marrow. The rejuvenated B cells exhibited a "young"‐like repertoire and cellular responsiveness to immune stimuli in vitro. Yet, mice treated with B‐cell depletion did not mount enhanced antibody responses to immunization in vivo, nor did they survive longer than control mice in "dirty" environment. Consistent with these results, peripheral B cells from elderly depleted patients showed a "young"‐like repertoire, population dynamics, and cellular responsiveness to stimulus. Nevertheless, the response rate to HBV vaccination was similar between elderly depleted and nondepleted subjects, although antibody titers were higher in depleted patients. This study proposes a proof of principle to rejuvenate the peripheral B‐cell compartment in aging, through B‐cell depletion. Further studies are warranted in order to apply this approach for enhancing humoral immune responsiveness among the elderly population.     

Photophysical and photodynamic properties of Pyronin Y in micellar media at different temperatures

In this study, photophysics and photodynamical properties of Pyronin Y (PyY) in different liquid media were investigated. Interactions of PyY, which is a positively charged pigment compound pertaining to the xanthene derivatives with surfactants possessing distinct charges, were determined by using the molecular absorption and fluorescence spectroscopy techniques. It was observed that band intensities of absorption and fluorescence spectra belonging to PyY increase in proportion to the water when compared to three micelle systems, cetyltrimethylammonium bromide (CTAB), sodium dodecyl sulfate (SDS) and Triton X‐100 (TX‐100). This suggests that interactions in micelle systems are different from those in deionized water, and solvation and surface interactions modify. It is determined that the strongest interaction occurs between PyY dye and SDS, anionic surfactant, and this interaction arises from the electrostatic forces. Calculated photophysical parameters indicated that the microenvironment of PyY in SDS micelle is different to that of other systems. In temperature studies, it was reported that increasing the temperature of the samples increased non‐radiative transitions. Steady‐state fluorescence anisotropy values were calculated by using fluorescence intensities of PyY compound in pre‐micellar, micellar and post‐micellar systems. Once the PyY fluorescence probe is added to the surfactant containing solutions below the critical micelle concentrations, the measured anisotropy values were found to be low because the probe remains in the deionized water phase. When the surfactant concentration of the medium becomes closer to the critical micelle concentrations, the steady‐state anisotropy value prominently increases. This is because of the restrictions on the rotational diffusion of the probe in micellar solution. It is observed that positively charged PyY shows a higher affinity to the negatively charged SDS compared with the positively charged CTAB and neutral TX‐100 surfactants. This can be explained by Coulombic interactions.     

Antimicrobial lipopolypeptides composed of palmitoyl Di- and tricationic peptides: in vitro and in vivo activities, self-assembly to nanostructures, and a plausible mode of action

Antimicrobial lipopeptides are produced nonribosomally in bacteria and fungi during cultivation. They are composed of a cationic or an anionic peptide covalently bound to a specifically modified aliphatic chain. Most of the peptidic moieties have complex cyclic structures. Here we report that conjugation of a palmitic acid to the N-terminus of very short cationic di- and tripeptides composed of all l- and d, l-amino acids endowed them with potent antimicrobial activities. Interestingly, cell specificity was determined by the sequence of the short peptidic chain. Palmitoyllysine served as a control and was inactive toward all microorganisms tested. Replacing an l-amino acid with its d-enantiomer did not affect the activity of the corresponding lipopeptides. Importantly, selected lipopeptides were also potent in vivo in a mouse model of Candida albicans infection. Bacterial leakage experiments and negative staining electron microscopy suggest that their mode of action involves permeation and disintegration of the microorganism's membrane, similar to many long antimicrobial peptides and lipopeptides. Interestingly, each lipopeptide assembled in solution into a nanostructure with a unique morphology which could partially explain differences in their biological activity. Besides adding important information on the parameters necessary for antimicrobial lipopeptides to kill microorganisms, the simple composition of these minilipopeptides and their diverse cell specificities make them attractive candidates for various applications.