Homotypic and Heterotypic Adhesion Induced by Odorant Receptors and the β2-Adrenergic Receptor

In the mouse olfactory system regulated expression of a large family of G Protein-Coupled Receptors (GPCRs), the Odorant Receptors (ORs), provides each sensory neuron with a single OR identity. In the wiring of the olfactory sensory neuron projections, a complex axon sorting process ensures the segregation of >1,000 subpopulations of axons of the same OR identity into homogeneously innervated glomeruli. ORs are critical determinants in axon sorting, and their presence on olfactory axons raises the intriguing possibility that they may participate in axonal wiring through direct or indirect trans-interactions mediating adhesion or repulsion between axons. In the present work, we used a biophysical assay to test the capacity of ORs to induce adhesion of cell doublets overexpressing these receptors. We also tested the β2 Adrenergic Receptor, a non-OR GPCR known to recapitulate the functions of ORs in olfactory axon sorting. We report here the first evidence for homo- and heterotypic adhesion between cells overexpressing the ORs MOR256-17 or M71, supporting the hypothesis that ORs may contribute to olfactory axon sorting by mediating differential adhesion between axons.     

Optimized assay and storage conditions for enzyme activity profiling of ectomycorrhizae

The aim of a joint effort by different research teams was to provide an improved procedure for enzyme activity profiling of field-sampled ectomycorrhizae, including recommendations on the best conditions and maximum duration for storage of ectomycorrhizal samples. A more simplified and efficient protocol compared to formerly published procedures was achieved by using manufactured 96-filter plates in combination with a vacuum manifold and by optimizing incubation times. Major improvements were achieved by performing the series of eight enzyme assays with a single series of root samples instead of two series, reducing the time needed for sample preparation, minimizing error-prone steps such as pipetting and morphotyping, and facilitating subsequent DNA analyses due to the reduced sequencing effort. The best preservation of samples proved to be storage in soil at 4?C6°C in the form of undisturbed soil cores containing roots. Enzyme activities were maintained for up to 4?weeks under these conditions. Short-term storage of washed roots and ectomycorrhizal tips overnight in water did not cause substantial changes in enzyme activity profiles. No optimal means for longer-term storage by freezing at ?20°C or storage in 100% ethanol were recommended.     

Impact of the use of cryobank samples in a selected cattle breed: a simulation study

Background

High selection pressure on domestic cattle has led to an undesirable increase in inbreeding, as well as to the deterioration of some functional traits which are indirectly selected. Semen stored in a cryobank may be a useful way to redirect selection or limit the loss of genetic diversity in a selected breed. The purpose of this study was to analyse the efficiency of current cryobank sampling methods, by investigating the benefits of using cryopreserved semen in a selection scheme several generations after the semen was collected.

Methods

The theoretical impact of using cryopreserved semen in a selection scheme of a dairy cattle breed was investigated by simulating various scenarios involving two negatively correlated traits and a change in genetic variability of the breed.

Results

Our results indicate that using cryopreserved semen to redirect selection will have an impact on negatively selected traits only if it is combined with major changes in selection objectives or practices. If the purpose is to increase genetic diversity in the breed, it can be a viable option.

Conclusions

Using cryopreserved semen to redirect selection or to improve genetic diversity should be carried out with caution, by considering the pros and cons of prospective changes in genetic diversity and the value of the selected traits. However, the use of genomic information should lead to more interesting perspectives to choose which animals to store in a cryobank and to increase the value of cryobank collections for selected breeds.     

Hedgehog and adipogenesis: fat and fiction

Morphogenes, abundantly described during embryogenesis have recently emerged as crucial modulators of cell differentiation processes. Hedgehog signaling, the dysregulation of which causing several pathologies such as congenital defects and cancer, is involved in several cell differentiation processes including adipogenesis. This review presents an overview of the relations between Hedgehog signaling, adipocyte differentiation and fat mass. While the anti-adipogenic role of Hedgehog signaling seems to be established, the effect of Hedgehog inhibition on adipocyte differentiation in vitro remains debated. Finally, Hedgehog potential as a pharmacological target to treat fat mass disorders is discussed.     

Comparative Analysis of the Secretome from a Model Filarial Nematode (Litomosoides sigmodontis) Reveals Maximal Diversity in Gravid Female Parasites

Filarial nematodes (superfamily Filarioidea) are responsible for an annual global health burden of ∼6.3 million disability-adjusted life-years, which represents the greatest single component of morbidity attributable to helminths affecting humans. No vaccine exists for the major filarial diseases, lymphatic filariasis and onchocerciasis; in part because research on protective immunity against filariae has been constrained by the inability of the human-parasitic species to complete their lifecycles in laboratory mice. However, the rodent filaria Litomosoides sigmodontis has become a popular experimental model, as BALB/c mice are fully permissive for its development and reproduction. Here, we provide a comprehensive analysis of excretory-secretory products from L. sigmodontis across five lifecycle stages and identifications of host proteins associated with first-stage larvae (microfilariae) in the blood. Applying intensity-based quantification, we determined the abundance of 302 unique excretory-secretory proteins, of which 64.6% were present in quantifiable amounts only from gravid adult female nematodes. This lifecycle stage, together with immature microfilariae, released four proteins that have not previously been evaluated as vaccine candidates: a predicted 28.5 kDa filaria-specific protein, a zonadhesin and SCO-spondin-like protein, a vitellogenin, and a protein containing six metridin-like ShK toxin domains. Female nematodes also released two proteins derived from the obligate Wolbachia symbiont. Notably, excretory-secretory products from all parasite stages contained several uncharacterized members of the transthyretin-like protein family. Furthermore, biotin labeling revealed that redox proteins and enzymes involved in purinergic signaling were enriched on the adult nematode cuticle. Comparison of the L. sigmodontis adult secretome with that of the human-infective filarial nematode Brugia malayi (reported previously in three independent published studies) identified differences that suggest a considerable underlying diversity of potential immunomodulators. The molecules identified in L. sigmodontis excretory-secretory products show promise not only for vaccination against filarial infections, but for the amelioration of allergy and autoimmune diseases.Filarial nematodes are the most important helminth parasites of humans in terms of overall impact on public health, with an annual global burden of ∼6.3 million disability-adjusted life-years (1). Lymphatic filariasis (LF)¹ or “elephantiasis,” which affects populations across Africa, South Asia, the Pacific, Latin America, and the Caribbean, accounts for 92% of this toll. The remainder is caused by onchocerciasis or “river blindness,” primarily in sub-Saharan Africa. The major human filarial pathogens are Wuchereria bancrofti (responsible for 90% of LF cases), Brugia malayi and Brugia timori (geographically restricted causes of LF), and Onchocerca volvulus (the sole agent of human onchocerciasis). In addition, Loa loa affects ∼13 million people in West and Central Africa. This parasite usually induces a relatively mild disease, but has been associated with severe and sometimes fatal adverse events following anthelmintic chemotherapy (2). Filarial parasites are primarily drivers of chronic morbidity, which manifests as disabling swelling of the legs, genitals and breasts in LF; or visual impairment and severe dermatitis in onchocerciasis. The filariae are also a major problem in small animal veterinary medicine, with ∼0.5 million dogs in the USA alone infected with Dirofilaria immitis (3), the cause of potentially fatal heartworm disease. However, in domesticated ungulates, filarial infections are generally benign (4).Currently, control of human filarial diseases is almost entirely dependent on three drugs (ivermectin, diethylcarbamazine, and albendazole). Prevention of heartworm also relies on prophylactic treatment of dogs and cats with ivermectin or other macrocyclic lactones. Reports of possible ivermectin resistance in O. volvulus (5) and D. immitis (6) have highlighted the importance of maintaining research efforts in vaccine development against filarial nematodes. However, rational vaccine design has been constrained for several decades (7) by the intrinsic complexity of these metazoan parasites and their multistage lifecycle. Moreover, many filarial species carry obligate bacterial endosymbionts (Wolbachia), which may also stimulate the immune response during infection (8). As part of global efforts to improve prevention and treatment of these diseases, large-scale projects have been undertaken, including sequencing of the nematodes (9–11) and their Wolbachia (10, 12, 13), and proteomic analyses of both whole organisms and excretory-secretory products (ESP) (14, 15). Additionally, two studies (both on B. malayi) have examined lifecycle stage-specific secretomes (16, 17). In the context of vaccine design, the identification of ESP proteins and determination of their expression in each major lifecycle stage can facilitate the prioritization of candidates for efficacy screening in animal models.One barrier to the progression of research in the filarial field is our inability to maintain the full lifecycle of the human parasites in genetically tractable hosts. This lifecycle involves uptake of the first-stage larvae (microfilariae, Mf) by a hematophagous arthropod and two moults in the vector, followed by transmission of third-stage larvae (L3) to a new vertebrate host and two further moults before the nematodes mature as dioecious adults. However, the complete lifecycle of the New World filaria Litomosoides sigmodontis can be maintained in laboratory rodents, including inbred mice (18). This species [incorrectly referred to as L. carinii in the older literature (19)] was first studied in its natural host, the cotton rat (Sigmodon hispidus) (20). Mongolian jirds (Meriones unguiculatus) are also fully permissive for L. sigmodontis infection and are routinely used for maintaining its lifecycle in the laboratory, as they tolerate higher parasite burdens than do laboratory mice. To exploit the full power of murine immunology, including defined knockout strains, L. sigmodontis in mice has been used to address questions regarding the fundamental immunomodulatory mechanisms employed by filarial parasites (21, 22), their ability to mitigate proinflammatory pathology and autoimmune disease (23), and the impact of various vaccine strategies on adult nematode burden and fecundity (24, 25).Using the resource of a newly-determined genome sequence, coupled with a derivative of the intensity-based absolute quantification (iBAQ) proteomic approach, we have examined the stage-specific secretome of L. sigmodontis in vector-derived L3 (vL3), adult males (AM), pre-gravid adult females (pgAF), gravid adult females (gAF), and immature Mf (iMf). In addition to identifying dynamic changes in the ESP profile through the lifecycle, we show important differences in the adult secretomes of L. sigmodontis and B. malayi, especially in the abundance of two novel proteins released by female L. sigmodontis that lack orthologs in B. malayi. As has been observed in other parasitic nematodes, we find transthyretin-like family (TTL) proteins to be particularly dominant in the ESP. Active expulsion of uterine fluid may account for the remarkable diversity of proteins that we detect in gAF ESP, and we highlight several novel proteins that warrant evaluation in vaccine trials and as anti-inflammatory mediators.     

Immunoepidemiological Profiling of Onchocerciasis Patients Reveals Associations with Microfilaria Loads and Ivermectin Intake on Both Individual and Community Levels

Mass drug administration (MDA) programmes against Onchocerca volvulus use ivermectin (IVM) which targets microfilariae (MF), the worm''s offspring. Most infected individuals are hyporesponsive and present regulated immune responses despite high parasite burden. Recently, with MDA programmes, the existence of amicrofilaridermic (a-MF) individuals has become apparent but little is known about their immune responses. Within this immunoepidemiological study, we compared parasitology, pathology and immune profiles in infection-free volunteers and infected individuals that were MF⁺ or a-MF. The latter stemmed from villages in either Central or Ashanti regions of Ghana which, at the time of the study, had received up to eight or only one round of MDA respectively. Interestingly, a-MF patients had fewer nodules and decreased IL-10 responses to all tested stimuli. On the other hand, this patient group displayed contrary IL-5 profiles following in vitro stimulation or in plasma and the dampened response in the latter correlated to reduced eosinophils and associated factors but elevated neutrophils. Furthermore, multivariable regression analysis with covariates MF, IVM or the region (Central vs. Ashanti) revealed that immune responses were associated with different covariates: whereas O. volvulus-specific IL-5 responses were primarily associated with MF, IL-10 secretion had a negative correlation with times of individual IVM therapy (IIT). All plasma parameters (eosinophil cationic protein, IL-5, eosinophils and neutrophils) were highly associated with MF. With regards to IL-17 secretion, although no differences were observed between the groups to filarial-specific or bystander stimuli, these responses were highly associated with the region. These data indicate that immune responses are affected by both, IIT and the rounds of IVM MDA within the community. Consequently, it appears that a lowered infection pressure due to IVM MDA may affect the immune profile of community members even if they have not regularly participated in the programmes.     

Synthesis of non-immunosuppressive cyclophilin-Binding cyclosporin A derivatives as potential anti-HIV-1 drugs

Original cyclosporin A (CsA) derivatives bearing various alkylthio side chains at the sarcosine residue 3 (R configuration) and for the most potent and selective compounds a 4′-hydroxyl group at the Me-Leucine residue 4 were prepared in one or two steps from commercially available CsA. The [2-(dimethyl or diethylamino)-ethylthio-Sar]³-[(4′-OH)MeLeu]⁴-CsA derivatives 3k and 3l displayed potent in vitro anti-HIV-1 (IC₅₀ 46 nM) and low immunosuppressive activities (IC₅₀≥1500 nM).