Antisense-mediated isoform switching of steroid receptor coactivator-1 in the central nucleus of the amygdala of the mouse brain

Background

Antisense oligonucleotide (AON)-mediated exon skipping is a powerful tool to manipulate gene expression. In the present study we investigated the potential of exon skipping by local injection in the central nucleus of the amygdala (CeA) of the mouse brain. As proof of principle we targeted the splicing of steroid receptor coactivator-1 (SRC-1), a protein involved in nuclear receptor function. This nuclear receptor coregulator exists in two splice variants (SRC-1a and SRC-1e) which display differential distribution and opposing activities in the brain, and whose mRNAs differ in a single SRC-1e specific exon.

Methods

For proof of principle of feasibility, we used immunofluorescent stainings to study uptake by different cell types, translocation to the nucleus and potential immunostimulatory effects at different time points after a local injection in the CeA of the mouse brain of a control AON targeting human dystrophin with no targets in the murine brain. To evaluate efficacy we designed an AON targeting the SRC-1e-specific exon and with qPCR analysis we measured the expression ratio of the two splice variants.

Results

We found that AONs were taken up by corticotropin releasing hormone expressing neurons and other cells in the CeA, and translocated into the cell nucleus. Immune responses after AON injection were comparable to those after sterile saline injection. A successful shift of the naturally occurring SRC-1a:SRC-1e expression ratio in favor of SRC-1a was observed, without changes in total SRC-1 expression.

Conclusions

We provide a proof of concept for local neuropharmacological use of exon skipping by manipulating the expression ratio of the two splice variants of SRC-1, which may be used to study nuclear receptor function in specific brain circuits. We established that exon skipping after local injection in the brain is a versatile and useful tool for the manipulation of splice variants for numerous genes that are relevant for brain function.     

Fluorescent Microcrystals Obtained from Coumarin 6 Using the Reprecipitation Method

The reprecipitation method was applied to Coumarin 6. A concentrated solution of the dye in acetone was mixed at room temperature with a large volume of water, and microcrystals rapidly appeared in the medium. Their size and shape were visualized by fluorescence microscopy, as well as by transmission electron microscopy. The absorption and fluorescence properties were studied on the suspensions and compared with those of the dissolved dye. This study is aimed at clarifying the influence of the reprecipitation process upon the fluorescence properties of the dye in the solid state.     

A magnetic resonance imaging-based articulatory and acoustic study of "retroflex" and "bunched" American English /r/

Speakers of rhotic dialects of North American English show a range of different tongue configurations for /r/. These variants produce acoustic profiles that are indistinguishable for the first three formants [Delattre, P., and Freeman, D. C., (1968). "A dialect study of American English r's by x-ray motion picture," Linguistics 44, 28-69; Westbury, J. R. et al. (1998), "Differences among speakers in lingual articulation for American English /r/," Speech Commun. 26, 203-206]. It is puzzling why this should be so, given the very different vocal tract configurations involved. In this paper, two subjects whose productions of "retroflex" /r/ and "bunched" /r/ show similar patterns of F1-F3 but very different spacing between F4 and F5 are contrasted. Using finite element analysis and area functions based on magnetic resonance images of the vocal tract for sustained productions, the results of computer vocal tract models are compared to actual speech recordings. In particular, formant-cavity affiliations are explored using formant sensitivity functions and vocal tract simple-tube models. The difference in F4/F5 patterns between the subjects is confirmed for several additional subjects with retroflex and bunched vocal tract configurations. The results suggest that the F4/F5 differences between the variants can be largely explained by differences in whether the long cavity behind the palatal constriction acts as a half- or a quarter-wavelength resonator.     

Components of the ubiquitin-proteasome pathway compete for surfaces on Rad23 family proteins

Background  

The delivery of ubiquitinated proteins to the proteasome for degradation is a key step in the regulation of the ubiquitin-proteasome pathway, yet the mechanisms underlying this step are not understood in detail. The Rad23 family of proteins is known to bind ubiquitinated proteins through its two ubiquitin-associated (UBA) domains, and may participate in the delivery of ubiquitinated proteins to the proteasome through docking via the Rad23 ubiquitin-like (UBL) domain.     

Facile synthesis of novel indolo[3,2-b]carbazole derivatives and a chromogenic-sensing 5,12-dihydroindolo[3,2-b]carbazole

Novel indolo[3,2-b]carbazole derivatives and a chromogenic-sensing 5,12-dihydroindolo[3,2-b]carbazole have been synthesized starting from tetra-tert-butylated 6,12-diaryl-5,11-dihydroindolo[3,2-b]carbazoles, which were prepared via an efficient tert-butylation of 6,12-diaryl-5,11-dihydroindolo[3,2-b]carbazoles.     

Mechanical and frictional properties of nanoparticle monolayers grafted on functionalized mica substrates

Normal and lateral forces between two opposing monolayers of grafted polymer nanoparticles (NPs) were measured using the Surface Forces Apparatus in a humid atmosphere. The NPs made of N, N-diethylacrylamide and 2-hydroxyethyl methacrylate have a hydrodynamic diameter of ca. 660 nm at 25 degrees C. The effect of surface roughness was studied by creating surface asperities using different NP grafting densities ranging from 0.41 to 2.63 NPs/mum (2). An increase in the NPs grafting density gave rise to an increase in surface roughness and to a deformation of the nanoparticles caused by the lateral pressure between neighboring particles. An elastoplastic behavior of the nanoparticles was observed for large grafting densities, while a purely elastic behavior was observed for small grafting densities. The lateral forces measured between two opposing NP monolayers sliding past each other followed Amontons' law for all grafting densities. The friction coefficient between the surfaces appeared to increase significantly with an increase in surface roughness, which was inherent to an increase in the elastoplastic behavior of the NP monolayers.     

Stability of silanols and grafted alkylsilane monolayers on plasma-activated mica surfaces

We investigated the effect of physical and chemical modifications of mica surfaces induced by water vapor-based plasma treatments on the stability of silanols and grafted alkylsilane monolayers. The plasma-activated substrates were characterized using XPS, TOF-SIMS, and contact angle measurements. They revealed a large surface coverage of silanol groups (Si-OH) and a loss of aluminum atoms compared to freshly cleaved mica surfaces. The stability of plasma-induced silanol groups was investigated by contact angle measurements using ethylene glycol as a probe liquid. The Si-OH surface coverage decreased rapidly under vacuum or thermal treatment to give rise to hydrophobic dehydrated surfaces. The stability of end-grafted monofunctionalized n-alkylsilanes was investigated in different solvents and at different pH using water contact angle measurements. The degrafting of alkylsilanes from the activated mica was promoted in acidic aqueous solutions. This detachment was associated with the hydrolysis of covalent bonds between the alkylsilanes and the mica surface. The monolayer stability was enhanced by increasing the length of the alkyl chains that probably act as a hydrophobic protective layer against hydrolysis reactions. Stable alkylsilane monolayers in water with pH greater than 5.5 were obtained on mica surfaces activated at low plasma pressure. We attributed this stability to the loss of surface Al atoms induced by the plasma treatment.     

Understanding the two-step spin-transition phenomenon in Iron(II) 1D chain materials

Three analogous one dimensional (1D) polymeric iron(II) spin crossover (SCO) materials containing the new ligand 4,6-bis(2',2'-pyridyl)pyrazine (bdpp) have been comprehensively characterised magnetically (thermal and light-induced) and structurally. Within this series are two polymorphs of the formula [Fe(NCS)(2)(bdpp)], 1 and 2 a, which differ magnetically in that phase 1 undergoes a full two-step SCO (T(1/2(1))=135 K and T(1/2(2))=90 K) whereas phase 2 a remains high spin (HS) over all temperatures. The central distinction between these two materials lies in the presence of intermolecular pi-pi interactions generated by the crystal packing in 1, which are absent in 2 a. The isostructural selenocyanate analogue of 2 a, [Fe(NCSe)(2)(bdpp)], 2 b, undergoes a full two-step SCO (T(1/2(1))=200 K and T(1/2(2))=125 K). Structural analyses of 1 and 2 b at a range of temperatures provide deep insight into their two-step SCO nature. Structural analysis of 1 at 25 K (1(LS-LS)), 123 K (1(LS-HS)) and 250 K (1(HS-HS)) reveals two distinct iron(II) centres at each temperature, with ordered, alternating HS and LS (low spin) sites at the intermediate plateau (IP) temperatures. In contrast, structural analysis of 2 b at 90 K (2 b(LS)), 150 K (2 b(LS/HS)) and 250 K (2 b(HS)) reveals one unique iron(II) centre at each temperature with an "averaged" LS/HS character at the IP temperature. Weak planes of diffuse scattering in the single-crystal X-ray diffraction patterns were observed for this phase at 90 and 150 K, indicating that 1D long range ordering of alternating HS/LS iron(II) centres occurs along the 1D coordination chains, but that there is no correlation between chains. The lack of observable diffuse scattering at 250 K suggests that the onset of the 1D structural ordering in the chain direction corresponds to the first step of the SCO and that this structural transition is electronically driven. The photomagnetic properties of both 1 and 2 b have been investigated and show approximately 62 and 53 % photo-excitation of a HS metastable state at low temperatures and T(LIESST) values of 55 and 49 K, respectively. Relaxation studies on the HS fraction in 2 b fitted well to a stretched exponential model with kinetic parameters indicative of weak cooperativity.     

Redirecting the cyclization steps of fungal polyketide synthase

Regiospecific cyclizations of the nascent poly-beta-ketone backbones dictate the structures of polyketide natural products. The fungal iterative megasynthases use terminal thioesterase/claisen cyclase (TE/CLC) domains to direct the fate of the polyketide chains. In this work, we present two strategies toward redirecting the cyclization steps of fungal PKSs using the Gibberella fujikuroi PKS4. First, inactivation or removal of the TE/CLC domain resulted in the synthesis of the new polyketide SMA93 2. Complementation of the mutant PKS4 with a standalone TE/CLC domain restored the regioselective cyclization steps of PKS4 and led to the synthesis of SMA76 1, demonstrating that cyclization enzymes can interact with the megasynthase in trans. This led to the second approach in which various dissociated, bacterial tailoring enzymes were added to the megasynthase in trans. Addition of the act KR led to the synthesis of mutactin 3, while the addition of first ring and second ring cyclases yielded anthraquinone compounds DMAC 5 and SEK26 6. The cooperative activities of fungal and bacterial PKS components are especially important and enable synthesis of polyketides utilizing enzymes from two distinct families of PKSs.     

Modeling the resting state of oxalate oxidase and oxalate decarboxylase enzymes

In view of the biological and commercial interest in models for Oxalate Decarboxylases (OxDC) and Oxalate Oxidases (OxOx), we have synthesized and characterized three new Mn (II) complexes ( 1- 3) employing N3O-donor amino-carboxylate ligands (TCMA, 1,4,7-triazacyclononane- N-acetic acid; K (i) Pr 2TCMA, potassium 1,4-diisopropyl-1,4,7-triazacyclononane- N-acetate; and KBPZG, potassium N,N-bis(3,5-dimethylpyrazolyl methyl)glycinate). These complexes were characterized by several techniques including X-ray crystallographic analysis, X-band electron paramagnetic resonance (EPR), electrospray ionization mass spectrometry (ESI-MS), and cyclic voltammetry. The crystal structures of 1 and 3 revealed that both form infinite polymeric chains of Mn (II) complexes linked by the pendant carboxylate arms of the TCMA (-) and the BPZG (-) ligands in a syn-antipattern. Complex 2 crystallizes as a mononuclear Mn (II) cation, six-coordinate in a distorted octahedral geometry. Although complexes 1 and 3 crystallize as polymeric chains, all compounds present the same N3O-donor set atoms around the metal center as observed in the crystallographically characterized OxDC and OxOx. Moreover, complex 2 also contains two water molecules coordinated to the Mn center as observed in the active site of OxDC and OxOx. ESI-MS spectrometry, combined with EPR, were useful techniques to establish that complexes 1- 3 are present as mononuclear Mn (II) species in solution. Finally, complexes 1- 3 are able to model the resting state active sites, with special attention focused on complex 2 which provides the first exact first coordination sphere ligand structural model for the resting states of both OxDC and OxOx.