Feeding behavior and trophic niche partitioning between co-existing river otter species

Niche partitioning occurs among coexisting populations to reduce the effects of competitive exclusion among species of similar niche. The aim of the present study is to verify the trophic niche partitioning and feeding behavior between two mustelids, the Giant otter and the Neotropical otter, through the dry and rainy season hydrologic of the Lower Xingu River. Our results suggest that the diets of both mustelids are composed primarily of fish of the family Anostomidae (Headstanders). Despite extensive niche overlap, our results indicate partitioning is facilitated by differences in niche breadth, with potential implications for conservation of both species in the case of declines in prey abundance and diversity. Both species inhabit an area recently impacted by completion of the Belo Monte Hydropower Plant, resulting in large changes to the hydrologic regime. Thus, our results provide important information for conservation efforts regarding the feeding behavior and co-occurrence of both species, as well as providing a baseline for monitoring future health of these mustelid populations. The present study is the first to test the hypothesis of niche partitioning between these two mustelids outside a protected area in the Amazon.

     

Deeper in the human cornea proteome using nanoLC-Orbitrap MS/MS: An improvement for future studies on cornea homeostasis and pathophysiology

The cornea is a transparent, avascular, and highly specialized connective tissue that provides the majority of light refraction in the optical system of the eye. The human cornea is composed of several layers interacting in a complex manner and possessing specific functions, like eye protection and optical clearness. Only few proteomic studies of mammalian cornea have been performed leading to the identification of less than 200 proteins in human corneas. The present study explores the proteome of the intact normal human cornea using a shot-gun nanoLC-MS/MS strategy and an LTQ Orbitrap mass spectrometer. A total of 2070 distinct corneal proteins were identified from five human cornea samples, which represents a 14-fold improvement in the number of proteins identified so far for human cornea. This enlarged dataset of human corneal proteins represents a valuable reference library for further studies on cornea homeostasis and pathophysiology. Network and gene ontology analyses were used to determine biological pathways specific of the human cornea. They allowed the identification of subnetworks of putative importance for corneal diseases, like a redox regulation and oxidative stress network constituted of aldehyde and alcohol dehydrogenases, most of them being described for the first time in human cornea.     

Evidence from Artificial Septal Targeting and Site-Directed Mutagenesis that Residues in the Extracytoplasmic β Domain of DivIB Mediate Its Interaction with the Divisomal Transpeptidase PBP 2B

Bacterial cytokinesis is achieved through the coordinated action of a multiprotein complex known as the divisome. The Escherichia coli divisome is comprised of at least 10 essential proteins whose individual functions are mostly unknown. Most divisomal proteins have multiple binding partners, making it difficult to pinpoint epitopes that mediate pairwise interactions between these proteins. We recently introduced an artificial septal targeting approach that allows the interaction between pairs of proteins to be studied in vivo without the complications introduced by other interacting proteins (C. Robichon, G. F. King, N. W. Goehring, and J. Beckwith, J. Bacteriol. 190:6048-6059, 2008). We have used this approach to perform a molecular dissection of the interaction between Bacillus subtilis DivIB and the divisomal transpeptidase PBP 2B, and we demonstrate that this interaction is mediated exclusively through the extracytoplasmic domains of these proteins. Artificial septal targeting in combination with mutagenesis experiments revealed that the C-terminal region of the β domain of DivIB is critical for its interaction with PBP 2B. These findings are consistent with previously defined loss-of-function point mutations in DivIB as well as the recent demonstration that the β domain of DivIB mediates its interaction with the FtsL-DivIC heterodimer. These new results have allowed us to construct a model of the DivIB/PBP 2B/FtsL/DivIC quaternary complex that strongly implicates DivIB, FtsL, and DivIC in modulating the transpeptidase activity of PBP 2B.Bacterial cytokinesis is a highly coordinated process that is carried out by a multiprotein complex known as the divisome (9, 11, 37, 39). In Escherichia coli, there are at least 10 essential divisomal proteins that carry out the division process. Divisome formation is initiated at the incipient division site by the recruitment of the FtsZ ring (1) which provides a molecular scaffold onto which the other divisional proteins are subsequently loaded (24, 33) (Fig. ​(Fig.1).1). In E. coli, the first proteins to load after FtsZ are a group of predominantly cytoplasmic proteins (FtsA, ZapA, and ZipA) that stabilize nascent FtsZ protofilaments and tether them to the membrane. The stabilized Z-ring then acts as a platform for recruitment of the remaining essential divisomal proteins, which are all single- or multipass membrane proteins (i.e., FtsE/FtsX, FtsK, FtsQ, FtsB, FtsL, FtsW, FtsI, and FtsN). With the exception of FtsI, a transpeptidase that cross-links septal murein, the biochemical function of these proteins is unknown.Open in a separate windowFIG. 1.Schema showing the hierarchical pathway of divisome assembly in E. coli and B. subtilis (adapted from reference 30). For a protein to be recruited to the divisome, all of the proteins upstream from it in the hierarchical recruitment pathway must already be present at the septum. Groups of proteins that form a subcomplex independent of other divisomal proteins, such as the ternary complex formed between E. coli FtsQ, FtsB, and FtsL, are highlighted by gray boxes. Red lines denote pairwise protein-protein interactions that have been experimentally demonstrated using genetic and/or biochemical approaches. The question mark indicates that the precise location of FtsW in the divisome assembly pathway in B. subtilis is currently unknown. (C) Possible outcomes of a heterologous septal targeting experiment in E. coli in which ZapA-DivIB is employed as the bait and GFP-PBP 2B is the prey. A direct interaction between DivIB and PBP 2B should result in a fluorescent ring at midcell (or a pair of dots when viewed in cross-section) due the recruitment of GFP-PBP 2B to the divisome (left panel). In contrast, a halo of fluorescence should be visible around the cell periphery due to the membrane-bound GFP-PBP 2B if there is no interaction between these two proteins (right panel).Divisomal protein recruitment in both Bacillus subtilis and E. coli occurs in a stepwise manner. For example, for FtsQ to be recruited to the E. coli divisome, all of the proteins upstream from it in the hierarchical recruitment pathway shown in Fig. ​Fig.1A1A must already be present at the septum. However, this pathway is not completely linear; some proteins appear to form subcomplexes prior to their recruitment to the divisome, such as the ternary complex formed between E. coli FtsQ, FtsB, and FtsL (2, 12, 14, 15). The situation in B. subtilis is more complex and less well understood. For example, B. subtilis DivIB, DivIC, FtsL, and PBP 2B appear to be recruited to the septum as an interdependent group late in the cell cycle (10) (Fig. ​(Fig.1B).1B). To further complicate matters, once these individual proteins or subcomplexes have been recruited to the divisome, they engage in a complex network of protein-protein interactions with other divisomal proteins (7, 8, 18, 23).The plethora of protein-protein interactions at the bacterial divisome makes it difficult to decipher which molecular epitopes on individual proteins mediate their interaction with other divisomal proteins. Thus, we recently introduced an artificial septal targeting (AST) technique that allowed us to examine interactions between pairs of interacting B. subtilis divisomal proteins in E. coli (30). This technique involves artificially targeting one of the B. subtilis proteins (the “bait”) to the E. coli divisome by fusing it to E. coli ZapA and then using fluorescence microscopy to determine whether it can recruit to the septum a green fluorescent protein (GFP) fusion to a putative interacting partner (the “prey”) (Fig. ​(Fig.1C).1C). The primary advantage of the AST technique is that it allows direct assessment of the interaction between two B. subtilis divisomal proteins without interference from other members of the divisome.We previously used AST to demonstrate a direct interaction between B. subtilis FtsL and DivIC and between DivIB and PBP 2B (30). The latter finding is consistent with the observation from bacterial two-hybrid studies that B. subtilis DivIB interacts directly with both PBP 2B and FtsL (5) and that the E. coli orthologs of these proteins (FtsI and FtsQ, respectively) also interact strongly (18). The extracellular domain of DivIB is divided into three subdomains, termed α, β, and γ (31). It was recently shown using a combination of nuclear magnetic resonance (NMR) spectroscopy and small-angle X-ray scattering (SAXS) that the concave face of the DivIB β domain makes direct contact with the C-terminal head of the FtsL-DivIC heterodimeric coiled coil (25), forming a stabilizing “cap” for these two intrinsically unstable proteins (32). In contrast, the α and γ regions of DivIB are not critical for formation of the DivIB/FtsL/DivIC ternary complex (25).The FtsQ/DivIB-FtsI/PBP 2B interaction appears to be widely conserved in both Gram-negative and Gram-positive bacteria, and therefore we decided to investigate the molecular details of this evolutionarily conserved interaction. By using a combination of AST and site-directed mutagenesis, we show that DivIB and PBP 2B interact exclusively through their extracytoplasmic regions and that this interaction is mediated by residues near the C terminus of DivIB. In combination with the results of previous studies, these new data have allowed us to construct a working model of the DivIB/PBP 2B/FtsL/DivIC complex.     

Exome sequencing identifies PDE4D mutations as another cause of acrodysostosis

Acrodysostosis is a rare autosomal-dominant condition characterized by facial dysostosis, severe brachydactyly with cone-shaped epiphyses, and short stature. Moderate intellectual disability and resistance to multiple hormones might also be present. Recently, a recurrent mutation (c.1102C>T [p.Arg368^∗]) in PRKAR1A has been identified in three individuals with acrodysostosis and resistance to multiple hormones. After studying ten unrelated acrodysostosis cases, we report here de novo PRKAR1A mutations in five out of the ten individuals (we found c.1102C>T [p.Arg368^∗] in four of the ten and c.1117T>C [p.Tyr373His] in one of the ten). We performed exome sequencing in two of the five remaining individuals and selected phosphodiesterase 4D (PDE4D) as a candidate gene. PDE4D encodes a class IV cyclic AMP (cAMP)-specific phosphodiesterase that regulates cAMP concentration. Exome analysis detected heterozygous PDE4D mutations (c.673C>A [p.Pro225Thr] and c.677T>C [p.Phe226Ser]) in these two individuals. Screening of PDE4D identified heterozygous mutations (c.568T>G [p.Ser190Ala] and c.1759A>C [p.Thr587Pro]) in two additional acrodysostosis cases. These mutations occurred de novo in all four cases. The four individuals with PDE4D mutations shared common clinical features, namely characteristic midface and nasal hypoplasia and moderate intellectual disability. Metabolic screening was normal in three of these four individuals. However, resistance to parathyroid hormone and thyrotropin was consistently observed in the five cases with PRKAR1A mutations. Finally, our study further supports the key role of the cAMP signaling pathway in skeletogenesis.     

IL-6-dependent PGE2 secretion by mesenchymal stem cells inhibits local inflammation in experimental arthritis

Background

Based on their capacity to suppress immune responses, multipotent mesenchymal stromal cells (MSC) are intensively studied for various clinical applications. Although it has been shown in vitro that the immunomodulatory effect of MSCs mainly occurs through the secretion of soluble mediators, the mechanism is still not completely understood. The aim of the present study was to better understand the mechanisms underlying the suppressive effect of MSCs in vivo, using cells isolated from mice deficient in the production of inducible nitric oxide synthase (iNOS) or interleukin (IL)-6 in the murine model of collagen-induced arthritis.

Principal Findings

In the present study, we show that primary murine MSCs from various strains of mice or isolated from mice deficient for iNOS or IL-6 exhibit different immunosuppressive potential. The immunomodulatory function of MSCs was mainly attributed to IL-6-dependent secretion of prostaglandin E2 (PGE2) with a minor role for NO. To address the role of these molecules in vivo, we used the collagen-induced arthritis as an experimental model of immune-mediated disorder. MSCs effectively inhibited collagen-induced inflammation during a narrow therapeutic window. In contrast to wild type MSCs, IL-6-deficient MSCs and to a lesser extent iNOS-deficient MSCs were not able to reduce the clinical signs of arthritis. Finally, we show that, independently of NO or IL-6 secretion or Treg cell induction, MSCs modulate the host response by inducing a switch to a Th2 immune response.

Significance

Our data indicate that MSCs mediate their immunosuppressive effect via two modes of action: locally, they reduce inflammation through the secretion of anti-proliferative mediators, such as NO and mainly PGE2, and systemically they switch the host response from a Th1/Th17 towards a Th2 immune profile.     

Improved Isolation of SlaA and SlaB S-layer proteins in Sulfolobus acidocaldarius

The Sulfolobus acidocaldarius S-layer is composed of two main proteins: SlaA, which forms the ordered structure of the S-layer matrix, and SlaB, which supports and anchors the S-layer into the tetraether lipid membrane. While SlaA has previously been purified by exploiting its thermotolerance and high resistance to detergents, SlaB has resisted isolation, particularly from the cell membrane. Removal of proteins other than those of the S-layer is especially difficult if large batch-scale culture volumes are unavailable. Here, we describe a benchtop-scale protocol for the purification of SlaA from S. acidocaldarius, enabling isolation of SlaB using size exclusion chromatography (gel filtration). Using this protocol, we were able to identify for the first time tetraether lipids strongly attached to SlaB via heat- and detergent-resistant interactions.

     

Identification of Symptomatic Fetuses Infected with Cytomegalovirus Using Amniotic Fluid Peptide Biomarkers

Cytomegalovirus (CMV) is the most common cause of congenital infection, and is a major cause of sensorineural hearing loss and neurological disabilities. Evaluating the risk for a CMV infected fetus to develop severe clinical symptoms after birth is crucial to provide appropriate guidance to pregnant women who might have to consider termination of pregnancy or experimental prenatal medical therapies. However, establishing the prognosis before birth remains a challenge. This evaluation is currently based upon fetal imaging and fetal biological parameters, but the positive and negative predictive values of these parameters are not optimal, leaving room for the development of new prognostic factors. Here, we compared the amniotic fluid peptidome between asymptomatic fetuses who were born as asymptomatic neonates and symptomatic fetuses who were either terminated in view of severe cerebral lesions or born as severely symptomatic neonates. This comparison allowed us to identify a 34-peptide classifier in a discovery cohort of 13 symptomatic and 13 asymptomatic neonates. This classifier further yielded 89% sensitivity, 75% specificity and an area under the curve of 0.90 to segregate 9 severely symptomatic from 12 asymptomatic neonates in a validation cohort, showing an overall better performance than that of classical fetal laboratory parameters. Pathway analysis of the 34 peptides underlined the role of viral entry in fetuses with severe brain disease as well as the potential importance of both beta-2-microglobulin and adiponectin to protect the injured fetal brain infected with CMV. The results also suggested the mechanistic implication of the T calcium channel alpha-1G (CACNA1G) protein in the development of seizures in severely CMV infected children. These results open a new field for potential therapeutic options. In conclusion, this study demonstrates that amniotic fluid peptidome analysis can effectively predict the severity of congenital CMV infection. This peptidomic classifier may therefore be used in clinical settings during pregnancy to improve prenatal counseling.     

In vivo bioluminescence imaging of Ca signalling in the brain of Drosophila

Many different cells' signalling pathways are universally regulated by Ca(2+) concentration [Ca(2+)] rises that have highly variable amplitudes and kinetic properties. Optical imaging can provide the means to characterise both the temporal and spatial aspects of Ca(2+) signals involved in neurophysiological functions. New methods for in vivo imaging of Ca(2+) signalling in the brain of Drosophila are required for probing the different dynamic aspects of this system. In studies here, whole brain Ca(2+) imaging was performed on transgenic flies with targeted expression of the bioluminescent Ca(2+) reporter GFP-aequorin (GA) in different neural structures. A photon counting based technique was used to undertake continuous recordings of cytosolic [Ca(2+)] over hours. Time integrals for reconstructing images and analysis of the data were selected offline according to the signal intensity. This approach allowed a unique Ca(2+) response associated with cholinergic transmission to be identified by whole brain imaging of specific neural structures. Notably, [Ca(2+)] transients in the Mushroom Bodies (MBs) following nicotine stimulation were accompanied by a delayed secondary [Ca(2+)] rise (up to 15 min. later) in the MB lobes. The delayed response was sensitive to thapsigargin, suggesting a role for intra-cellular Ca(2+) stores. Moreover, it was reduced in dunce mutant flies, which are impaired in learning and memory. Bioluminescence imaging is therefore useful for studying Ca(2+) signalling pathways and for functional mapping of neurophysiological processes in the fly brain.