Validation of a universal set of primers to study animal-associated microeukaryotic communities.

The application of metabarcoding to study animal-associated microeukaryotes has been restricted because the universal barcode used to study microeukaryotic ecology and distribution in the environment, the Small Subunit of the Ribosomal RNA gene (18S rRNA), is also present in the host. As a result, when host-associated microbial eukaryotes are analysed by metabarcoding, the reads tend to be dominated by host sequences. We have done an in silico validation against the SILVA 18S rRNA database of a non-metazoan primer set (primers that are biased against the metazoan 18S rRNA) that recovers only 2.6% of all the metazoan sequences, while recovering most of the other eukaryotes (80.4%). Among metazoans, the non-metazoan primers are predicted to amplify 74% of Porifera sequences, 4% of Ctenophora, and 15% of Cnidaria, while amplifying almost no sequences within Bilateria. In vivo, these non-metazoan primers reduce significantly the animal signal from coral and human samples, and when compared against universal primers provide at worst a 2-fold decrease in the number of metazoan reads and at best a 2800-fold decrease. This easy, inexpensive, and near-universal method for the study of animal-associated microeukaryotes diversity will contribute to a better understanding of the microbiome.

Trypsin and N-aminopeptidase (APN) activities in the hepatopancreas of an intertidal euryhaline crab: Biochemical characteristics and differential modulation by histamine and salinity.

No studies are available about biochemical characteristics and modulation (i.e. by endogenous and/or environmental cues) of trypsin (a key digestive endoprotease) in hepatopancreas of intertidal euryhaline crabs neither on the possible concomitant modulation of key ectoproteases such as aminopeptidase-N (APN) involved in final steps of protein digestion. Furthermore, nothing is still known in decapods crustaceans about the role of histamine as primary chemical messenger for modulation of main components of digestive process (i.e. proteases). We determined biochemical characteristics and investigated the effect of histamine injections; of histamine in vitro and of acclimation of individuals to low and high salinity on trypsin and aminopeptidase-N (APN) activities in the hepatopancreas of the euryhaline crab Cyrtograpsus angulatus (Dana 1851). Trypsin activity was maximal at pH7.4 and at 45°C. APN activity increased from pH6.6 to 7.6-9.0 and was maintained high at 37-45°C. Both activities exhibited Michaelis-Menten kinetics (apparent Km: trypsin=0.36mM; APN=0.07mM). The injection of 10-4M histamine decreased trypsin activity (about 40%) in hepatopancreas while did not affect APN activity. Similarly, in vitro 10-4M histamine decreased trypsin activity (about 52%) in hepatopancreas but not APN activity. Trypsin activity in the hepatopancreas was not affected by acclimation of crabs to low (10psu) or high (40psu) environmental salinity while APN activity was increased (about 200%) in 10psu. The results show the differential modulation of trypsin and APN by distinct cues and point to histamine as modulator of intracellular trypsin by direct action on the hepatopancreas.

Digestive strategies in the South American subterranean rodent Ctenomys talarum.

Ctenomys talarum is a subterranean herbivorous rodent which due to its particular life style is frequently exposed to variations in surface environmental conditions (i.e. food quality and availability, temperature). Thus, unlike other subterranean rodents, C. talarum has to buffer both the surface and burrow challenging environmental conditions. We studied the occurrence of digestive strategies at different levels of C. talarum living in their natural habitat. We determined the dimensions of different parts of the gastrointestinal tract and organs along as the activity of key digestive enzymes (disaccharidase, N-aminopeptidase) in different parts of the gut in individuals seasonally caught. The results show that C. talarum exhibits characteristics in the gut at the biochemical level (high disaccharidase activities in small intestine, high N-aminopeptidase activity in the caecum and large intestine, and a seasonal differential modulation of N-aminopeptidase activity in small and large intestines), which could represent adaptive strategies to face seasonal variations in key environmental factors.

Glucose homeostasis in the euryhaline crab Cytograpsus angulatus: Effects of the salinity in the amylase, maltase and sucrase activities in the hepatopancreas and in the carbohydrate reserves in different tissues.

We studied the existence, biochemical characteristics and response to different environmental salinities of amylase, maltase and sucrase activity in the intertidal euryhaline crab Cyrtograpsus angulatus (Dana, 1852) along with the response to distinct salinities of glycogen and free glucose content in storage organs. Amylase, maltase and sucrase activities were kept over a broad range of pH and temperature and exhibited Michaelis-Menten kinetics. Zymography showed the existence of two amylase forms in crabs exposed to 35 (osmoconformation) and low (6-10psu; hyper-regulation) or high (40psu) (hypo-regulation) salinities. Carbohydrases activity in the hepatopancreas and glycemia were not affected in crab exposed to different environmental salinities. In 6 and 40psu, the glycogen content in anterior gills was lower than in 35psu. In 6, 10 and 40psu, glycogen concentration in hepatopancreas, muscle and posterior gills were similar to that in 35psu. Free glucose concentration in chela muscle was higher in 6 and 40psu than in 35psu. The existence and biochemical characteristics of carbohydrases activity and the adjustments in concentration of glycogen in anterior gills and free glucose in chela muscle suggests the ability to perform complete hydrolysis of glycogenic substrates and to keep glucose homeostasis in relation to acclimation to different salinity conditions.

Evaluation of PCR Approaches for Detection of Bartonella bacilliformis in Blood Samples.

BACKGROUND: The lack of an effective diagnostic tool for Carrion's disease leads to misdiagnosis, wrong treatments and perpetuation of asymptomatic carriers living in endemic areas. Conventional PCR approaches have been reported as a diagnostic technique. However, the detection limit of these techniques is not clear as well as if its usefulness in low bacteriemia cases. The aim of this study was to evaluate the detection limit of 3 PCR approaches. METHODOLOGY/PRINCIPAL FINDINGS: We determined the detection limit of 3 different PCR approaches: Bartonella-specific 16S rRNA, fla and its genes. We also evaluated the viability of dry blood spots to be used as a sample transport system. Our results show that 16S rRNA PCR is the approach with a lowest detection limit, 5 CFU/µL, and thus, the best diagnostic PCR tool studied. Dry blood spots diminish the sensitivity of the assay. CONCLUSIONS/SIGNIFICANCE: From the tested PCRs, the 16S rRNA PCR-approach is the best to be used in the direct blood detection of acute cases of Carrion's disease. However its use in samples from dry blood spots results in easier management of transport samples in rural areas, a slight decrease in the sensitivity was observed. The usefulness to detect by PCR the presence of low-bacteriemic or asymptomatic carriers is doubtful, showing the need to search for new more sensible techniques.

Possible vertical transmission of Bartonella bacilliformis in Peru.

A 22-day-old male was admitted with a 2-day history of irritability, dyspnea, jaundice, fever, and gastrointestinal bleeding. A thin blood smear was performed, which showed the presence of intraerythrocyte bacteria identified as Bartonella bacilliformis, and subsequently, the child was diagnosed with Carrion's disease. The diagnosis was confirmed by specific polymerase chain reaction. The child was born in a non-endemic B. bacilliformis area and had not traveled to such an area before hospitalization. However, the mother was from an endemic B. bacilliformis area, and posterior physical examination showed the presence of a wart compatible with B. bacilliformis in semi-immune subjects. These data support vertical transmission of B. bacilliformis.

Digestive flexibility in females of the subterranean rodent ctenomys talarum in their natural habitat.

We studied the occurrence of digestive strategies at different levels in females of the subterranean herbivorous rodent Ctenomys talarum living in their natural habitat. We determined the dimensions of different parts of the gastrointestinal tract and organs along as the activity of key digestive enzymes(sucrase, maltase and N-aminopeptidase) in small intestine in females seasonally caught. Females of C. talarum did not show seasonal variations in the mass of the different parts of the gastrointestinal tract. In nonreproductive females large intestine was longer in autumn, whereas reproductive females did not show seasonal variations in the length of the different parts of the gut. Females of C. talarum exhibited a high sucrose, maltase and N-aminopeptidase activity in small intestine, although these activities were higher in small intestine of females caught in autumn (nonreproductive) than in females caught in winter (reproductive). The results show that C. talarum females exhibit characteristics in the gut at the morphological and biochemical level, which could represent digestive strategies to face the constraints imposed by their costly particular way of life.

Seasonal changes in body composition of Ctenomys talarum (Rodentia: Octodontidae): an herbivore subterranean rodent.

Ctenomys talarum is a subterranean herbivorous rodent whose burrow systems exhibit particular characteristics, distinct from other subterranean environments. We studied seasonal variation in body composition of C. talarum in relation to energetic requirements. Body lipid content seasonally changed in C. talarum, related to reproductive cycle and thermorregulatory mechanisms. A decrease in protein body content was found only in spring. Ash content of females was lowest when most of them are in post partum estro. Observed variations in water body content could be associated with plant water content and/or metabolic regulation. Our results show the occurrence of seasonal variations in body composition in C. talarum, which could be related to the high cost of reproduction and the subterranean life style of this species.

Phenotypic plasticity in response to low quality diet in the South American omnivorous rodent Akodon azarae (Rodentia: Sigmodontinae).

We studied the responses in the omnivorous rodent A. azarae submitted to a low quality diet at morphological, physiological and biochemical levels. At short term, a decrease in body mass occurred. A later increase in food consumption constituted a strategy that allowed a temporal recovery of physical condition. However, hyperphagia appeared not to be enough to maintain physical condition after 30 days of low quality diet consumption. At the morphological level, an increase in length (9%) of the anterior portion of the gut occurred, the part of the gut where digestion and absorption take place. A decrease in small intestine weight could be related with the long-term impairment of body condition. Inhibition of sucrase specific activity in small intestine would indicate a down-regulation of sucrase-isomaltase complex. Total maltase specific activity in small intestine was not affected suggesting an up-regulation of sucrase-independent maltase specific activity. A down-regulation of protease specific activity in small intestine occurred in response to low quality diet. The specific activity of disaccharidases in caecum and large intestine was down-regulated. The strategies and constraints at different levels of A. azarae upon low quality diet are discussed.

Identification of Grb2 as a novel binding partner of the signaling lymphocytic activation molecule-associated protein binding receptor CD229.

Ag recognition by the TCR determines the subsequent fate of the T cell and is regulated by the involvement of other cell surface molecules, termed coreceptors. CD229 is a lymphocyte cell surface molecule that belongs to the CD150 family of receptors. Upon tyrosine phosphorylation, CD229 recruits various signaling molecules to the membrane. One of these molecules is the signaling lymphocytic activation molecule-associated protein, of which a deficiency leads to the X-linked lymphoproliferative syndrome. We report that CD229 interacts in a phosphorylation-dependent manner with Grb2. We mapped this interaction showing that the Src homology 2 domain of Grb2 and the tyrosine residue Y606 in CD229 are required for CD229-Grb2 complex formation. The Grb2 motif in the cytoplasmic tail of CD229 is distinct and independent from the two tyrosines required for efficient signaling lymphocytic activation molecule-associated protein recruitment. CD229, but not other members of the CD150 family, directly bound Grb2. We also demonstrate that CD229 precipitates with Grb2 in T lymphocytes after pervanadate treatment, as well as CD229 or TCR ligation. Interestingly, the CD229 mutant lacking the Grb2 binding site is not internalized after CD229 engagement with specific Abs. Moreover, a dominant negative form of Grb2 (containing only Src homology 2 domain) impaired CD229 endocytosis. Unexpectedly, Erk phosphorylation was partially inhibited after activation of CD229 plus CD3. Consistent with this, CD229 ligation partially inhibited TCR signaling in peripheral blood cells and CD229-Jurkat cells transfected with the 3XNFAT-luciferase reporter construct. Altogether, the data suggest a model whereby CD229 ligation attenuates TCR signaling and Grb2 recruitment to CD229 controls its rate of internalization.

The adaptor protein 3BP2 binds human CD244 and links this receptor to Vav signaling, ERK activation, and NK cell killing.

Adaptor proteins, molecules that mediate intermolecular interactions, are crucial for cellular activation. The adaptor 3BP2 has been shown to positively regulate NK cell-mediated cytotoxicity. In this study we present evidence for a physical interaction between 3BP2 and the CD244 receptor. CD244, a member of the CD150 family, is a cell surface protein expressed on NK, CD8+ T, and myeloid cells. CD244 interacts via its Src homology 2 domain with the X-linked lymphoproliferative disease gene product signaling lymphocytic activation molecule-associated protein (SAP)/SH2 domain protein 1A. 3BP2 interacts with human but not murine CD244. CD244-3BP2 interaction was direct and regulated by phosphorylation, as shown by a three-hybrid analysis in yeast and NK cells. Tyr337 on CD244, part of a consensus motif for SAP/SH2 domain protein 1A binding, was critical for the 3BP2 interaction. Although mutation of Tyr337 to phenylalanine abrogated human 3BP2 binding, we still observed SAP association, indicating that this motif is not essential for SAP recruitment. CD244 ligation induced 3BP2 phosphorylation and Vav-1 recruitment. Overexpression of 3BP2 led to an increase in the magnitude and duration of ERK activation, after CD244 triggering. This enhancement was concomitant with an increase in cytotoxicity due to CD244 ligation. However, no differences in IFN-gamma secretion were found when normal and 3BP2-transfected cells were compared. These results indicate that CD244-3BP2 association regulates cytolytic function but not IFN-gamma release, reinforcing the hypothesis that, in humans, CD244-mediated cytotoxicity and IFN-gamma release involve distinct NK pathways.

The cell surface expression of SAP-binding receptor CD229 is regulated via its interaction with clathrin-associated adaptor complex 2 (AP-2).

CD229 (Ly9) is a cell surface receptor selectively expressed on T and B lymphocytes, and it belongs to the CD150 receptor family. Like other receptors of this family, CD229 interacts with SAP/SH2D1a protein, mutation of which is responsible for the fatal X-linked lymphoproliferative disease. Receptors of the CD150 family function as costimulatory molecules, regulating cytokine production and cytotoxicity. Thus, their signaling and regulation in lymphocytes may be critical to an understanding of the pathogenesis of the X-linked lymphoproliferative disease. Here we show that CD229 interacts with the mu(2) chain of the AP-2 adaptor complex that links transmembrane proteins to clathrin-coated pits. CD229 was the only member of the CD150 family associated with AP-2. We also show that the mu(2) chain interacts with the Y(470)EKL motif of CD229. The integrity of this site was necessary for CD229 internalization, but it was not involved in SAP recruitment. Moreover, CD229 binds to the AP-2 complex in T and B cell lines, and it is internalized rapidly from the cell surface on T cells after antibody ligation. In contrast, cross-linking of CD229 receptors with intact antibody inhibited CD229 internalization on B cells. However, when F(ab')(2) antibodies were used, CD229 internalization was similar on T and B cells, suggesting that Fcgamma receptors control CD229 cell surface expression. Furthermore, CD229 was regulated by T cell receptor and B cell receptor signaling because coligation with antibodies against anti-CD3 and anti-IgM increased the rate of CD229 endocytosis. These data suggest that CD229 cell surface expression on lymphocytes surface is strongly and differentially regulated within the CD150 family members.

Phenotypic flexibility of digestive morphology and physiology of the South American omnivorous rodent Akodon azarae (Rodentia: Sigmodontinae).

We have determined the occurrence of responses at different levels (morphological, physiological and biochemical) in the omnivorous rodent Akodon azarae upon cold acclimation (15 degrees C). A short-term enhancement in food consumption appeared to account for the maintenance of both mass and body composition. At the morphological level, the main response was an increase in the dimensions of small intestine, which constitutes the section of the gut where absorption and secretion take place. An increase in sucrase specific activity was only found in small intestine. Sucrose independent maltase activity was very low since 99.8% of total maltase activity was due to sucrase-isomaltase (SI) complex. Protease specific activities were not affected. The fact that resting metabolic rates determined at 15 and 23 degrees C were similar in cold acclimated animals suggests a change in lower critical temperature. In conclusion, our results show that A. azarae exhibits different strategies to support cold environment that could lead to an enhancement in digestion and absorption efficiency. Furthermore, this work suggests that low temperature is an independent cue of other environmental factors to trigger the strategies allowing the maintenance of body condition in A. azarae.

Mouse novel Ly9: a new member of the expanding CD150 (SLAM) family of leukocyte cell-surface receptors.

Human CS1, also known as novel Ly9, 19A24, or CRACC, is a member of the immunoglobulin gene superfamily (IgSF) expressed on natural killer cells and other leukocytes. Here we describe the cloning of the mouse homologue of this gene. The mouse novel Ly9 gene is shown to encode a transmembrane protein composed of two extracellular immunoglobulin-like domains, a transmembrane region and an 88-amino acid cytoplasmic domain. Mouse novel Ly9 is structurally similar to the extracellular domains of CD84 and CD229 (Ly9). Both mouse and human novel Ly9 genes mapped close to the CD229gene in a region where other members of the CD150 family have also been mapped, and analysis of their genomic sequences showed that they have an identical intron/exon organization. Northern blot analysis revealed that the expression of mouse and human novel Ly9 was predominantly restricted to hematopoietic tissues, with the exception of testis. Here we show that SAP (SH2D1A), an adapter protein responsible for the X-linked lymphoproliferative disease, binds to the phosphorylated cytoplasmic tail of human but not mouse novel Ly9. Taken together, these data indicate that mouse novel Ly9 is a new member of the expanding CD150 family of cell surface receptors.