Age‐dependent alterations of the NMDA receptor developmental profile and adult behavior in postnatally ketamine‐treated mice

Ketamine is a NMDA receptor (NMDAR) antagonist used in pediatric anesthesia. Given the role of glutamatergic signaling during brain maturation, we studied the effects of a single ketamine injection (40 mg/kg s.c) in mouse neonates depending on postnatal age at injection (P2, P5, or P10) on cortical NMDAR subunits expression and association with Membrane‐Associated Guanylate Kinases PSD95 and SAP102. The effects of ketamine injection at P2, P5, or P10 on motor activity were compared in adulthood. Ketamine increased GluN2A and GluN2B mRNA levels in P2‐treated mice without change in proteins, while it decreased GluN2B protein in P10‐treated mice without change in mRNA. Ketamine reduced GluN2A mRNA and protein levels in P5‐treated mice without change in GluN2B and GluN1. Ketamine affected the GluN2A/PSD95 association regardless of the age at injection, while GluN2B/PSD95 association was enhanced only in P5‐treated mice. Microdissection of ketamine‐treated mouse cortex showed a decrease in GluN2A mRNA level in superficial layers (I–IV) and an increase in all subunit expressions in deep layers (V–VI) in P5‐ and P10‐treated mice, respectively. Our data suggest that ketamine impairs cortical NMDAR subunit developmental profile and delays the synaptic targeting of GluN2A‐enriched NMDAR. Ketamine injection at P2 or P10 resulted in hyperlocomotion in adult male mice in an open field, without change in females. Voluntary running‐wheel exercise showed age‐ and sex‐dependent alterations of the mouse activity, especially during the dark phase. Overall, a single neonatal ketamine exposure led to short‐term NMDAR cortical developmental profile impairments and long‐term motor activity alterations persisting in adulthood. © 2014 Wiley Periodicals, Inc. Develop Neurobiol 75: 315–333, 2015     

Human dopamine transporter: the first implementation of a combined in silico/in vitro approach revealing the substrate and inhibitor specificities

Parkinson’s disease (PD) is characterized by the loss of dopamine-generating neurons in the substantia nigra and corpus striatum. Current treatments alleviate PD symptoms rather than exerting neuroprotective effect on dopaminergic neurons. New drugs targeting the dopaminergic neurons by specific uptake through the human dopamine transporter (hDAT) could represent a viable strategy for establishing selective neuroprotection. Molecules able to increase the bioactive amount of extracellular dopamine, thereby enhancing and compensating a loss of dopaminergic neurotransmission, and to exert neuroprotective response because of their accumulation in the cytoplasm, are required. By means of homology modeling, molecular docking, and molecular dynamics simulations, we have generated 3D structure models of hDAT in complex with substrate and inhibitors. Our results clearly reveal differences in binding affinity of these compounds to the hDAT in the open and closed conformations, critical for future drug design. The established in silico approach allowed the identification of promising substrate compounds that were subsequently analyzed for their efficiency in inhibiting hDAT-dependent fluorescent substrate uptake, through in vitro live cell imaging experiments. Taken together, our work presents the first implementation of a combined in silico/in vitro approach enabling the selection of promising dopaminergic neuron-specific substrates.     

Cytotaxonomy of autumnal flowering species of Hyacinthaceae from Algeria

Cytotaxonomic investigations of the autumn-flowering squills, Prospero autumnale (L.) Speta ≡ Scilla autumnalis L., Prospero obtusifolium (Poir.) Speta ≡ Scilla obtusifolia Poir., Barnardia numidica (Poir.) Speta ≡ Scilla numidica Poir., and Hyacinthoides lingulata (Poir.) Rothm. ≡ Scilla lingulata Poir. were performed in 20 populations from northern Algeria located between Tipasa and La Vieille Calle. Various chromosome numbers were found, including a new cytotype, 2n = 8, for the flora of Algeria, concerning plants identified as Prospero obtusifolium (Poir.) Speta [including P. fallax (Steinh.) Speta = S. autumnalis L. ssp. fallax (Steinh.) Batt.]. The numbers 2n = 14, 28, and 42 correspond, respectively, to diploid, tetraploid, and hexaploid levels of P. autumnale s.l. [including P. pulchellum (Munby) Speta ≡ Scilla pulchella Munby = S. autumnalis var. pulchella (Munby) Batt.], with x = 7. The cytotypes of Barnardia numidica (Poir.) Speta with 2n = 18 and Hyacinthoides lingulata (Poir.) Rothm. with 2n = 16 chromosomes were confirmed.     

Synthetic antibodies designed on natural sequence landscapes

We present a method for synthetic antibody library generation that combines the use of high-throughput immune repertoire analysis and a novel synthetic technology. The library design recapitulates positional amino acid frequencies observed in natural antibody repertoires. V-segment diversity in four heavy (V_H) and two kappa (V_κ) germlines was introduced based on the analysis of somatically hypermutated donor-derived repertoires. Complementarity-determining region 3 length and amino acid designs were based on aggregate frequencies of all V_H and V_κ sequences in the data set. The designed libraries were constructed through an adaptation of a novel gene synthesis technology that enables precise positional control of amino acid composition and incorporation frequencies. High-throughput pyrosequencing was used to monitor the fidelity of construction and characterize genetic diversity in the final 3.6 × 10¹⁰ transformants. The library exhibited Fab expression superior to currently reported synthetic approaches of equivalent diversity, with greater than 93% of clones observed to successfully display both a correctly folded heavy chain and a correctly folded light chain. Genetic diversity in the library was high, with 95% of 7.0 × 10⁵ clones sequenced observed only once. The obtained library diversity explores a comparable sequence space as the donor-derived natural repertoire and, at the same time, is able to access novel recombined diversity due to lack of segmental linkage. The successful isolation of low- and subnanomolar-affinity antibodies against a diverse panel of receptors, growth factors, enzymes, antigens from infectious reagents, and peptides confirms the functional viability of the design strategy.     

A cost-benefit analysis of multidimensional fractionation of affinity purification-mass spectrometry samples

Affinity purification coupled to mass spectrometry (AP-MS) is gaining widespread use for the identification of protein-protein interactions. It is unclear, however, whether typical AP sample complexity is limiting for the identification of all protein components using standard one-dimensional LC-MS/MS. Multidimensional sample separation is useful for reducing sample complexity prior to MS analysis and increases peptide and protein coverage of complex samples. Here, we monitored the effects of upstream protein or peptide separation techniques on typical mammalian AP-MS samples, generated by FLAG affinity purification of four baits with different biological functions and/or subcellular distribution. As a first separation step, we employed SDS-PAGE, strong cation exchange LC, or reversed-phase LC at basic pH. We also analyzed the benefits of using an instrument with a faster scan rate, the new TripleTOF 5600 mass spectrometer. While all multidimensional approaches yielded a clear increase in spectral counts, the increase in unique peptides and additional protein identification was modest and came at the cost of increased instrument and handling time. The use of a high duty-cycle instrument achieved similar benefits without these drawbacks. An increase in spectral counts is beneficial when data analysis methods relying on spectral counts, including Significance Analysis of INTeractome (SAINT), are used.     

Effects of a Skin Neuropeptide (Substance P) on Cutaneous Microflora

Background

Skin is the largest human neuroendocrine organ and hosts the second most numerous microbial population but the interaction of skin neuropeptides with the microflora has never been investigated. We studied the effect of Substance P (SP), a peptide released by nerve endings in the skin on bacterial virulence.

Methodology/Principal Findings

Bacillus cereus, a member of the skin transient microflora, was used as a model. Exposure to SP strongly stimulated the cytotoxicity of B. cereus (+553±3% with SP 10⁻⁶ M) and this effect was rapid (<5 min). Infection of keratinocytes with SP treated B. cereus led to a rise in caspase1 and morphological alterations of the actin cytoskeleton. Secretome analysis revealed that SP stimulated the release of collagenase and superoxide dismutase. Moreover, we also noted a shift in the surface polarity of the bacteria linked to a peel-off of the S-layer and the release of S-layer proteins. Meanwhile, the biofilm formation activity of B. cereus was increased. The Thermo unstable ribosomal Elongation factor (Ef-Tu) was identified as the SP binding site in B. cereus. Other Gram positive skin bacteria, namely Staphylococcus aureus and Staphylococcus epidermidis also reacted to SP by an increase of virulence. Thermal water from Uriage-les-Bains and an artificial polysaccharide (Teflose®) were capable to antagonize the effect of SP on bacterial virulence.

Conclusions/Significance

SP is released in sweat during stress and is known to be involved in the pathogenesis of numerous skin diseases through neurogenic inflammation. Our study suggests that a direct effect of SP on the skin microbiote should be another mechanism.     

Plasma Lysosphingomyelin Demonstrates Great Potential as a Diagnostic Biomarker for Niemann-Pick Disease Type C in a Retrospective Study

Niemann-Pick disease type C (NP-C) is a devastating, neurovisceral lysosomal storage disorder which is characterised by variable manifestation of visceral signs, progressive neuropsychiatric deterioration and premature death, caused by mutations in the NPC1 and NPC2 genes. Due to the complexity of diagnosis and the availability of an approved therapy in the EU, improved detection of NP-C may have a huge impact on future disease management. At the cellular level dysfunction or deficiency of either the NPC1 or NPC2 protein leads to a complex intracellular endosomal/lysosomal trafficking defect, and organ specific patterns of sphingolipid accumulation. Lysosphingolipids have been shown to be excellent biomarkers of sphingolipidosis in several enzyme deficient lysosomal storage disorders. Additionally, in a recent study the lysosphingolipids, lysosphingomyelin (SPC) and glucosylsphingosine (GlcSph), appeared to be elevated in the plasma of three adult NP-C patients. In order to investigate the clinical utility of SPC and GlcSph as diagnostic markers, an in-depth fit for purpose biomarker assay validation for measurement of these biomarkers in plasma by liquid chromatography-tandem mass spectrometry was performed. Plasma SPC and GlcSph are stable and can be measured accurately, precisely and reproducibly. In a retrospective analysis of 57 NP-C patients and 70 control subjects, median plasma SPC and GlcSph were significantly elevated in NP-C by 2.8-fold and 1.4-fold respectively. For miglustat-naïve NP-C patients, aged 2–50 years, the area under the ROC curve was 0.999 for SPC and 0.776 for GlcSph. Plasma GlcSph did not correlate with SPC levels in NP-C patients. The data indicate excellent potential for the use of lysosphingomyelin in NP-C diagnosis, where it could be used to identify NP-C patients for confirmatory genetic testing.     

Exploring blocking assays using Octet,ProteOn, and Biacore biosensors

We demonstrate the use of label-free real-time optical biosensors in competitive binding assays by epitope binning a panel of antibodies. We describe three assay orientations that we term in tandem, premix, and classical sandwich blocking, and we perform each of them on three platforms: ForteBio’s Octet QK, Bio-Rad’s ProteOn XPR36, and GE Healthcare’s Biacore 3000. By testing whether antibodies block one another’s binding to their antigen in a pairwise fashion, we establish a blocking profile for each antibody relative to the others in the panel. The blocking information is then used to create “bins” of antibodies with similar epitopes. The advantages and disadvantages of each biosensor, factors to consider when deciding on the most appropriate blocking assay orientation for a particular interaction system, and tips for dealing with ambiguous data are discussed. The data from our different assay orientations and biosensors agree very well, establishing these machines as valuable tools for characterizing antibody epitopes and multiprotein complexes of biological significance.