GABARAPL1 antibodies: target one protein, get one free!

Atg8 is a yeast protein involved in the autophagic process and in particular in the elongation of autophagosomes. In mammals, several orthologs have been identified and are classed into two subfamilies: the LC3 subfamily and the GABARAP subfamily, referred to simply as the LC3 or GABARAP families. GABARAPL1 (GABARAP-like protein 1), one of the proteins belonging to the GABARAP (GABA(A) receptor-associated protein) family, is highly expressed in the central nervous system and implicated in processes such as receptor and vesicle transport as well as autophagy. The proteins that make up the GABARAP family demonstrate conservation of their amino acid sequences and protein structures. In humans, GABARAPL1 shares 86% identity with GABARAP and 61% with GABARAPL2 (GATE-16). The identification of the individual proteins is thus very limited when working in vivo due to a lack of unique peptide sequences from which specific antibodies can be developed. Actually, and to our knowledge, there are no available antibodies on the market that are entirely specific to GABARAPL1 and the same may be true of the anti-GABARAP antibodies. In this study, we sought to examine the specificity of three antibodies targeted against different peptide sequences within GABARAPL1: CHEM-CENT (an antibody raised against a short peptide sequence within the center of the protein), PTG-NTER (an antibody raised against the N-terminus of the protein) and PTG-FL (an antibody raised against the full-length protein). The results described in this article demonstrate the importance of testing antibody specificity under the conditions for which it will be used experimentally, a caution that should be taken when studying the expression of the GABARAP family proteins.     

Altered expression of Bcl-2 and Bax in follicles within dehydroepiandrosterone-induced polycystic ovaries in rats

PCOS (polycystic ovary syndrome) is a heterogeneous disease characterized by hyperandrogenaemia, hirsutism, oligo- or amenorrhea, insulin resistance and anovulation. The aim of the present study was to evaluate if the balance between the ovarian expression of Bax (proapoptotic protein) and Bcl-2 (antiapoptotic protein) is altered in a PCOS model developed in rats by DHEA (dehydroepiandrosterone) administration. In addition, the ovarian morphology and the circulating progesterone levels were evaluated. Histological studies confirmed the presence of follicular cysts, atretic follicles and the absence of corpora lutea in the ovaries from the PCOS group and a significant decrease in circulating progesterone levels. Immunohistochemical studies showed that the expression of Bcl-2 and Bax were mainly localized in granulosa cells of AFs (antral follicles) in both groups. Bax expression was greater in preantral and AFs from PCOS ovarian sections than in the controls. In contrast, intense Bcl-2 immunostaining was observed in the control AFs, while Bcl-2 protein was either absent in PFs (preantral follicles) or weakly expressed in AFs from PCOS rats. These results were partially confirmed by Western studies. Data revealed that the ovarian level of Bcl-2 protein was lower in PCOS than in the control and that there were no differences in Bax ovarian levels between groups. However, Bax/Bcl-2 ratio was significantly higher in PCOS group than in the control group. In conclusion, an increase in ovarian apoptosis through an imbalance among the Bcl-2 family members may be involved in the transformation of growing follicles in cystic follicles in the ovaries from DHEA-induced PCOS rats.     

The GD1a glycan is a cellular receptor for adenoviruses causing epidemic keratoconjunctivitis

Adenovirus type 37 (Ad37) is a leading cause of epidemic keratoconjunctivitis (EKC), a severe and highly contagious ocular disease. Whereas most other adenoviruses infect cells by engaging CD46 or the coxsackie and adenovirus receptor (CAR), Ad37 binds previously unknown sialic acid-containing cell surface molecules. By glycan array screening, we show here that the receptor-recognizing knob domain of the Ad37 fiber protein specifically binds a branched hexasaccharide that is present in the GD1a ganglioside and that features two terminal sialic acids. Soluble GD1a glycan and GD1a-binding antibodies efficiently prevented Ad37 virions from binding and infecting corneal cells. Unexpectedly, the receptor is constituted by one or more glycoproteins containing the GD1a glycan motif rather than the ganglioside itself, as shown by binding, infection and flow cytometry experiments. Molecular modeling, nuclear magnetic resonance and X-ray crystallography reveal that the two terminal sialic acids dock into two of three previously established sialic acid-binding sites in the trimeric Ad37 knob. Surface plasmon resonance analysis shows that the knob-GD1a glycan interaction has high affinity. Our findings therefore form a basis for the design and development of sialic acid-containing antiviral drugs for topical treatment of EKC.     

miR-141 and miR-200a act on ovarian tumorigenesis by controlling oxidative stress response

Although there is evidence that redox regulation has an essential role in malignancies, its impact on tumor prognosis remains unclear. Here we show crosstalk between oxidative stress and the miR-200 family of microRNAs that affects tumorigenesis and chemosensitivity. miR-141 and miR-200a target p38α and modulate the oxidative stress response. Enhanced expression of these microRNAs mimics p38α deficiency and increases tumor growth in mouse models, but it also improves the response to chemotherapeutic agents. High-grade human ovarian adenocarcinomas that accumulate miR-200a have low concentrations of p38α and an associated oxidative stress signature. The miR200a-dependent stress signature correlates with improved survival of patients in response to treatment. Therefore, the role of miR-200a in stress could be a predictive marker for clinical outcome in ovarian cancer. In addition, although oxidative stress promotes tumor growth, it also sensitizes tumors to treatment, which could account for the limited success of antioxidants in clinical trials.     

HIV-2 Integrase Variation in Integrase Inhibitor-Na?ve Adults in Senegal,West Africa

Background

Antiretroviral therapy for HIV-2 infection is hampered by intrinsic resistance to many of the drugs used to treat HIV-1. Limited studies suggest that the integrase inhibitors (INIs) raltegravir and elvitegravir have potent activity against HIV-2 in culture and in infected patients. There is a paucity of data on genotypic variation in HIV-2 integrase that might confer intrinsic or transmitted INI resistance.

Methods

We PCR amplified and analyzed 122 HIV-2 integrase consensus sequences from 39 HIV-2–infected, INI-naive adults in Senegal, West Africa. We assessed genetic variation and canonical mutations known to confer INI-resistance in HIV-1.

Results

No amino acid-altering mutations were detected at sites known to be pivotal for INI resistance in HIV-1 (integrase positions 143, 148 and 155). Polymorphisms at several other HIV-1 INI resistance-associated sites were detected at positions 72, 95, 125, 154, 165, 201, 203, and 263 of the HIV-2 integrase protein.

Conclusion

Emerging genotypic and phenotypic data suggest that HIV-2 is susceptible to the new class of HIV integrase inhibitors. We hypothesize that intrinsic HIV-2 integrase variation at “secondary” HIV-1 INI-resistance sites may affect the genetic barrier to HIV-2 INI resistance. Further studies will be needed to assess INI efficacy as part of combination antiretroviral therapy in HIV-2–infected patients.     

Is Aboriginal Food Less Allergenic? Comparing IgE-Reactivity of Eggs from Modern and Ancient Chicken Breeds in a Cohort of Allergic Children

Background

Hen''s egg allergy ranks among the most frequent primary food allergies in children. We aimed to investigate sensitization profiles of egg allergic patients and compare in vitro IgE reactivities of eggs from ancient chicken breeds (Araucana and Maran) with those from conventional laying hen hybrids.

Methodology

Egg allergic children (n = 25) were subjected to skin prick test, double blind placebo controlled food challenge, and sensitization profiles to Gal d 1–5 were determined by allergen microarray. IgE binding and biological activity of eggs from different chicken breeds were investigated by immunoblot, ELISA, and mediator release assays.

Principal Findings

We found that Gal d 1 and Gal d 2 are generally major egg allergens, whereas Gal d 3–5 displayed high sensitization prevalence only in patients reacting to both, egg white and yolk. It seems that the onset of egg allergy is mediated by egg white allergens expanding to yolk sensitization in later stages of disease. Of note, egg white/yolk weight ratios were reduced in eggs from Auraucana and Maran chicken. As determined in IgE immunoblots and mass analysis, eggs from ancient chicken breeds did not differ in their protein composition. Similar IgE-binding was observed for all egg white preparations, while an elevated allergenicity was detected in egg yolk from Araucana chicken.

Conclusion/Significance

Our results on allergenicity and biological activity do not confirm the common assumption that aboriginal food might be less allergenic. Comprehensive diagnosis of egg allergy should distinguish between reactivity to hen''s egg white and yolk fractions to avoid unnecessary dietary restrictions to improve life quality of the allergic child and its family.     

Snake population venomics and antivenomics of Bothrops atrox: Paedomorphism along its transamazonian dispersal and implications of geographic venom variability on snakebite management

We describe two geographically differentiated venom phenotypes across the wide distribution range of Bothrops atrox, from the Colombian Magdalena Medio Valley through Puerto Ayacucho and El Paují, in the Venezuelan States of Amazonas and Orinoquia, respectively, and S?o Bento in the Brazilian State of Maranh?o. Colombian and Venezuelan venoms show an ontogenetic toxin profile phenotype whereas Brazilian venoms exhibit paedomorphic phenotypes. Venoms from each of the 16 localities sampled contain both population-specific toxins and proteins shared by neighboring B. atrox populations. Mapping the molecular similarity between conspecific populations onto a physical map of B. atrox range provides clues for tracing dispersal routes that account for the current biogeographic distribution of the species. The proteomic pattern is consistent with a model of southeast and southwest dispersal and allopatric fragmentation northern of the Amazon Basin, and trans-Amazonian expansion through the Andean Corridor and across the Amazon river between Monte Alegre and Santarém. An antivenomic approach applied to assess the efficacy towards B. atrox venoms of two antivenoms raised in Costa Rica and Brazil using Bothrops venoms different than B. atrox in the immunization mixtures showed that both antivenoms immunodepleted very efficiently the major toxins (PIII-SVMPs, serine proteinases, CRISP, LAO) of paedomorphic venoms from Puerto Ayacucho (Venezuelan Amazonia) through S?o Bento, but had impaired reactivity towards PLA(2) and P-I SVMP molecules abundantly present in ontogenetic venoms. The degree of immunodepletion achieved suggests that each of these antivenoms may be effective against envenomations by paedomorphic, and some ontogenetic, B. atrox venoms.     

Biophysical characteristics reveal neural stem cell differentiation potential

Background

Distinguishing human neural stem/progenitor cell (huNSPC) populations that will predominantly generate neurons from those that produce glia is currently hampered by a lack of sufficient cell type-specific surface markers predictive of fate potential. This limits investigation of lineage-biased progenitors and their potential use as therapeutic agents. A live-cell biophysical and label-free measure of fate potential would solve this problem by obviating the need for specific cell surface markers.

Methodology/Principal Findings

We used dielectrophoresis (DEP) to analyze the biophysical, specifically electrophysiological, properties of cortical human and mouse NSPCs that vary in differentiation potential. Our data demonstrate that the electrophysiological property membrane capacitance inversely correlates with the neurogenic potential of NSPCs. Furthermore, as huNSPCs are continually passaged they decrease neuron generation and increase membrane capacitance, confirming that this parameter dynamically predicts and negatively correlates with neurogenic potential. In contrast, differences in membrane conductance between NSPCs do not consistently correlate with the ability of the cells to generate neurons. DEP crossover frequency, which is a quantitative measure of cell behavior in DEP, directly correlates with neuron generation of NSPCs, indicating a potential mechanism to separate stem cells biased to particular differentiated cell fates.

Conclusions/Significance

We show here that whole cell membrane capacitance, but not membrane conductance, reflects and predicts the neurogenic potential of human and mouse NSPCs. Stem cell biophysical characteristics therefore provide a completely novel and quantitative measure of stem cell fate potential and a label-free means to identify neuron- or glial-biased progenitors.     

Synthesis and characterization of mononuclear oxovanadium(IV) complexes and their enzyme inhibition studies with a carbohydrate metabolic enzyme phosphodiesterase I

The increasing interest in vanadium coordination chemistry is based on its well-established chemical and biological functions. A beta-diketonato complex of oxovanadium(IV) is known to be having numerous catalytic applications and also exhibits promising insulin mimetic properties. In continuation of our structure activity relationship studies of metal complexes, we report herein the synthesis and characterization of the vanadium complexes of beta-diketonato ligand system with systematic variations of electronic and steric factors. Two complexes, VO(tmh)(2) (tmh = 2,2,6,6,-tetramethyl-3,5-heptanedione), and VO(hd)(2) (hd = 3,5-heptanedione) were synthesized and characterized by using different spectroscopic techniques. Elemental and mass spectral analysis supports the presence of two beta-diketonato ligands per VO(2+) unit. UV-Vis spectra in different solvents indicate coordination of coordinating solvent molecules at sixth position resulting in red shift of the band I transition. NMR and IR spectra reveal binding of coordinating solvent molecule at vacant sixth position trans to oxo group without releasing beta-diketonato ligands. Enzyme inhibition studies of these and other related oxovanadium(IV) complexes with beta-diketonato ligand system are conducted with snake venom phosphodiesterase I (SPVDE). All of these complexes showed significant inhibitory potential and were found to be non-competitive inhibitors against this enzyme.