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.     

The spatio-temporal dynamics of PKA activity profile during mitosis and its correlation to chromosome segregation

The cyclic adenosine monophosphate dependent kinase protein (PKA) controls a variety of cellular processes including cell cycle regulation. Here, we took advantages of genetically encoded FRET-based biosensors, using an AKAR-derived biosensor to characterize PKA activity during mitosis in living HeLa cells using a single-cell approach. We measured PKA activity changes during mitosis. HeLa cells exhibit a substantial increase during mitosis, which ends with telophase. An AKAREV T>A inactive form of the biosensor and H89 inhibitor were used to ascertain for the specificity of the PKA activity measured. On a spatial point of view, high levels of activity near to chromosomal plate during metaphase and anaphase were detected. By using the PKA inhibitor H89, we assessed the role of PKA in the maintenance of a proper division phenotype. While this treatment in our hands did not impaired cell cycle progression in a drastic manner, inhibition of PKA leads to a dramatic increase in chromososme misalignement on the spindle during metaphase that could result in aneuploidies. Our study emphasizes the insights that can be gained with genetically encoded FRET-based biosensors, which enable to overcome the shortcomings of classical methologies and unveil in vivo PKA spatiotemporal profiles in HeLa cells.     

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.     

The nucleoporin Nup205/NPP-3 is lost near centrosomes at mitotic onset and can modulate the timing of this process in Caenorhabditis elegans embryos

Regulation of mitosis in time and space is critical for proper cell division. We conducted an RNA interference-based modifier screen to identify novel regulators of mitosis in Caenorhabditis elegans embryos. Of particular interest, this screen revealed that the Nup205 nucleoporin NPP-3 can negatively modulate the timing of mitotic onset. Furthermore, we discovered that NPP-3 and nucleoporins that are associated with it are lost from the nuclear envelope (NE) in the vicinity of centrosomes at the onset of mitosis. We demonstrate that centrosomes are both necessary and sufficient for NPP-3 local loss, which also requires the activity of the Aurora-A kinase AIR-1. Our findings taken together support a model in which centrosomes and AIR-1 promote timely onset of mitosis by locally removing NPP-3 and associated nucleoporins from the NE.     

Evaluation of C-reactive protein, Haptoglobin and cardiac Troponin 1 levels in brachycephalic dogs with upper airway obstructive syndrome

ABSTRACT: BACKGROUND: Brachycephalic dogs have unique upper respiratory anatomy with abnormal breathing patterns similar to those in humans with obstructive sleep apnea syndrome (OSAS). The objective of this study was to evaluate the correlation between anatomical components, clinical signs and several biomarkers, used to determine systemic inflammation and myocardial damage (C-reactive protein, CRP; Haptoglobin, Hp; cardiac troponin I, cTnI), in dogs with brachycephalic upper airway obstructive syndrome (BAOS). RESULTS: Fifty brachycephalic dogs were included in the study and the following information was studied: signalment, clinical signs, thoracic radiographs, blood work, ECG, components of BAOS, and CRP, Hp and cTnI levels. A high proportion of dogs with BAOS (88%) had gastrointestinal signs. The prevalence of anatomic components of BAOS was: elongated soft palate (100%), stenotic nares (96%), everted laryngeal saccules (32%) and tracheal hypoplasia (29.1%). Increased serum levels of biomarkers were found in a variable proportion of dogs: 14% (7/50) had values of CRP > 20 mg/L, 22.9% (11/48) had values of Hp > 3 g/L and 47.8% (22/46) had levels of cTnI > 0.05 ng/dl. Dogs with everted laryngeal saccules had more severe respiratory signs (p<0.02) and higher values of CRP (p<0.044). No other statistical association between biomarkers levels and severity of clinical signs was found. CONCLUSIONS: According to the low percentage of patients with elevated levels of CRP and Hp, BAOS does not seem to cause an evident systemic inflammatory status. Some degree of myocardial damage may occur in dogs with BAOS that can be detected by cTnI concentration.     

Ruthenium bis-terpyridine complexes connected to an oligothiophene unit for dry dye-sensitised solar cells.

The preparation of a series of new heteroleptic ruthenium complexes containing a 4'-phosphonic acid terpyridine and a terpyridine substituted in 4' position by a bithiophene or tert-thiophene are described. The UV-Vis absorption and emission spectra along the redox potentials of these complexes are reported. These new complexes were also tested as sensitizers in dry dye-sensitised solar cell using regioregular polyoctylthiophene as a solid hole conductor. It has been shown that the complexes substituted with thiophene chain exhibit poor photovoltaic efficiency most probably due to low electron injection efficiency. This latter property can be rationalised by the fact that the LUMO orbitals in these complexes are localised in the terpyridine substituted by the thiophene chain and not in the terpyridine phosphonic which is bonded to the TiO2 photoanode.     

Potential for metabolic engineering of resveratrol biosynthesis

Resveratrol, an interesting plant phenolic compound, is found in red wine but is not widely distributed in other common food sources. Health benefits of resveratrol include prevention of cardiovascular diseases and cancers, and--as discovered more recently--promotion of longevity in several animal systems. The pathway and enzymes for resveratrol biosynthesis are well characterized. Furthermore, metabolic engineering of this compound has been achieved in plants, microbes and animals. This review attempts to summarize current understanding of resveratrol pathway-engineering in various systems, to outline the challenges in commercial applications and to identify future opportunities for resveratrol bioengineering.     

Crystal structure of Bordetella pertussis BugD solute receptor unveils the basis of ligand binding in a new family of periplasmic binding proteins

Periplasmic binding proteins of a new family particularly well represented in Bordetella pertussis have been called Bug receptors. One B.pertussis Bug protein is part of a tripartite tricarboxylate transporter while the functions of the other 77 are unknown. We report the first structure of a Bug receptor, BugD. It adopts the characteristic Venus flytrap motif observed in other periplasmic binding proteins, with two globular domains bisected by a deep cleft. BugD displays a closed conformation resulting from the fortuitous capture of a ligand, identified from the electron density as an aspartate. The structure reveals a distinctive alpha carboxylate-binding motif, involving two water molecules that bridge the carboxylate oxygen atoms to the protein. Both water molecules are hydrogen bonded to a common carbonyl group from Ala14, and each forms a hydrogen bond with one carboxylate oxygen atom of the ligand. Additional hydrogen bonds are found between the ligand alpha carboxylate oxygen atoms and protein backbone amide groups and with a threonine hydroxyl group. This specific ligand-binding motif is highly conserved in Bug proteins, indicating that they may all be receptors of amino acids or other carboxylated solutes, with a similar binding mode. The present structure thus unveils the bases of ligand binding in this large family of periplasmic binding proteins, several hundred members of which have been identified in various bacterial species.     

Solid-state dye-sensitized TiO(2) solar cells based on a sensitizer covalently wired to a hole conducting polymer.

We report on the molecular wiring efficiency of a ruthenium polypyridine complex acting as a sensitizer connected to a poly(3-hexyl)thiophene chain acting as hole transporting material. We have developed an efficient synthetic strategy to covalently connect via an ethanyl spacer a regioregular poly(3-hexyl)thiophene chain to a ruthenium complex. Solid-state dye-sensitized solar cells were prepared either with the latter system or with a similar ruthenium sensitizer but lacking the polymer chain (reference system). The comparison of the photocurrent-photovoltage characteristics of the cells recorded under AM1.5 indicates a two fold improvement of the overall photoconversion efficiencies when the sensitizer is grafted to the hole transporting material (eta = 0.27%) relative to the reference system (eta = 0.13%). The higher photovoltaic performance can be attributed to the better diffusion-like propagation of the holes from the sensitizer to the counter electrode through the covalently linked polythiophene chain.