VOCAL INDIVIDUALITY OF IN-AIR WEDDELL SEAL (LEPTONYCHOTES WEDDELLII) PUP "PRIMARY" CALLS

As a result of selective pressures faced during lactation, vocal recognition may play a crucial role in maintaining the phocid mother–pup bond during the period of dependence. To investigate this possibility, we examined whether Weddell seal ( Leptonychotes weddellii ) pups produce individually distinctive "primary" calls. One temporal, nine fundamental frequency features, and two spectral characteristics were measured. A discriminant function analysis (DFA) of 15 Vestfold Hills pups correctly classified 52% of calls, while the cross-validation procedure classified 29% of calls to the correct pup. A second DFA of 10 known-age McMurdo Sound pups correctly classified 44% of "test" calls. For novel calls, the probabilities of attaining such classification rates by chance are low. The relationship between age and call stereotypy indicated that pups 2 wk and older may be more vocally distinctive. Overall, findings suggest that Weddell seal pup "primary" calls are moderately distinctive and only exhibit sufficient stereotypy to aid maternal recognition by approximately two weeks of age.     

A new endogenous form of PYY isolated from canine ileum: Gly-extended PYY(1-36)

We purified and identified the peptide YY (PYY) forms present and determined their levels from a portion of the canine ileum directly adjacent to the cecum by a new extraction method designed to prevent and evaluate degradation of endogenous peptides. We used three reverse phase chromatography steps with radioimmunoassay of fractions for PYY-like-immunoreactivity (PYY-LI). The purified fractions underwent intact protein/peptide mass spectrometry identification and sequencing (i.e. "top-down" MS analysis). This analysis confirmed the identity of a new form of PYY, PYY(1-36)-Gly, which co-elutes with PYY(1-36)-NH(2) through all three of separation steps used. The PYY(1-36)-Gly form represents approximately 20% of the total PYY found in this region of the canine intestine. In addition, we also found that the PYY(3-36)-NH(2) form represents 6% of the total PYY in the canine ileo-cecal junction. The physiological implication of the Gly-extended form of PYY(1-36) warrants further investigation.     

N-Acetyl Aspartic Acid (NAA) and N-Acetyl Aspartylglutamic Acid (NAAG) in Human Ventricular,Subarachnoid, and Lumbar Cerebrospinal Fluid

N-Acetylaspartic and N-acetylaspartylglutamic acid concentrations in human ventricular, subarachnoid and lumbar cerebrospinal fluid were measured by combined gas chromatography-mass spectrometry using selected ion monitoring with deuterated internal standards. N-Acetylaspartate concentrations were in the range 55, 9, and 1 M, respectively; N-acetylaspartylglutamate concentrations in the same fluids were in the range 8, 3 and 4 M, respectively. There did not appear to be any difference in lumbar fluid concentrations of either compound between control subjects, schizophrenic patients, Alzheimer's disease patients and a pooled group of patients with neurological degeneration. Ventricular concentrations of both compounds were greatly increased in deceased patients suggesting that maintenance of their intracellular concentrations is probably energy dependent. The concentrations of these compounds in lumbar cerebrospinal fluid from living, and ventricular cerebrospinal fluid from deceased subjects were weakly correlated with one another. In lumbar fluid neither compound appeared to be correlated with age. Analysis of serially collected lumbar samples from two subjects showed a weak concentration gradient for both compounds. Neither antipsychotic medication nor the acid transport inhibitor probenecid had any effect on lumbar concentrations of either compound. Attempts to use anion exchange high pressure liquid chromatography with UV detection for measurement of the low concentrations of N-acetylaspartate found in cerebrospinal fluid from living subjects were unsuccessful.     

Relationship Between Plasma and Cerebrospinal Fluid Norepinephrine and Dopamine Metabolites in a Nonhuman Primate

Major and minor pathways of metabolism in the mammalian CNS result in the formation of 3-methoxy-4-hydroxyphenylethylene glycol (MHPG) and normetanephrine (NMN) from norepinephrine (NE), and homovanillic acid (HVA) and 3-methoxytyramine (3-MT) from dopamine (DA), respectively. The correlational relationships between HVA and 3-MT and between MHPG and NMN in primate CSF and plasma have not been described. These relationships may help to elucidate the usefulness of CSF and plasma metabolites as indices of CNS NE and DA activity. In addition, because NMN is unlikely to cross the blood-brain barrier. CSF NMN concentrations would not be confounded by contributions from plasma, which is a major issue with CSF MHPG. We have obtained repeated samples of plasma and CSF from drug-naive male squirrel monkeys and have measured the concentrations of MHPG, HVA, NMN, and 3-MT to define their correlational relationships. For the NE metabolites, significant correlations were obtained for CSF MHPG and NMN (r = 0.806, p less than 0.001), plasma MHPG and CSF NMN (r = 0.753, p less than 0.001), and plasma and CSF MHPG (r = 0.776, p less than 0.001). These results suggest that CSF and plasma MHPG and CSF NMN may reflect gross changes in whole brain steady-state noradrenergic metabolism. Only a single significant relationship was demonstrated for the DA metabolites, with CSF 3-MT correlating with plasma HVA (r = 0.301, p less than 0.025). The results for the DA metabolites probably reflect regional differences in steady-state brain dopaminergic metabolism.     

Identification of Differentially Expressed Proteins in Murine Embryonic and Postnatal Cortical Neural Progenitors

Background

The central nervous system (CNS) develops from a heterogeneous pool of neural stem and progenitor cells (NSPC), the underlying differences among which are poorly understood. The study of NSPC would be greatly facilitated by the identification of additional proteins that mediate their function and that would distinguish amongst different progenitor populations.

Methodology/Principal Findings

To identify membrane and membrane-associated proteins expressed by NSPC, we used a proteomics approach to profile NSPC cultured as neurospheres (NS) isolated from the murine cortex during a period of neurogenesis (embryonic day 11.5, E11.5), as compared to NSPC isolated at a peak of gliogenesis (postnatal day 1, P0) and to differentiated E11.5 NS. 54 proteins were identified with high expression in E11.5 NS, including the TrkC receptor, several heterotrimeric G proteins, and the Neogenin receptor. 24 proteins were identified with similar expression in E11.5 and P0 NS over differentiated E11.5 NS, and 13 proteins were identified with high expression specifically in P0 NS compared to E11.5 NS. To illustrate the potential relevance of these identified proteins to neural stem cell biology, the function of Neogenin was further studied. Using Fluorescence Activated Cell Sorting (FACS) analysis, expression of Neogenin was associated with a self-renewing population present in both E11.5 and adult subventricular zone (SVZ) NS but not in P0 NS. E11.5 NS expressed a putative Neogenin ligand, RGMa, and underwent apoptosis when exposed to a ligand-blocking antibody.

Conclusions/Significance

There are fundamental differences between the continuously self-renewing and more limited progenitors of the developing cortex. We identified a subset of differentially expressed proteins that serve not only as a set of functionally important proteins, but as a useful set of markers for the subsequent analysis of NSPC. Neogenin is associated with the continuously self-renewing and neurogenic cells present in E11.5 cortical and adult SVZ NS, and the Neogenin/RGMa receptor/ligand pair may regulate cell survival during development.     

The Lck inhibitor,AMG-47a,blocks necroptosis and implicates RIPK1 in signalling downstream of MLKL

Necroptosis is a form of caspase-independent programmed cell death that arises from disruption of cell membranes by the mixed lineage kinase domain-like (MLKL) pseudokinase after its activation by the upstream kinases, receptor interacting protein kinase (RIPK)-1 and RIPK3, within a complex known as the necrosome. Dysregulated necroptosis has been implicated in numerous inflammatory pathologies. As such, new small molecule necroptosis inhibitors are of great interest, particularly ones that operate downstream of MLKL activation, where the pathway is less well defined. To better understand the mechanisms involved in necroptosis downstream of MLKL activation, and potentially uncover new targets for inhibition, we screened known kinase inhibitors against an activated mouse MLKL mutant, leading us to identify the lymphocyte-specific protein tyrosine kinase (Lck) inhibitor AMG-47a as an inhibitor of necroptosis. We show that AMG-47a interacts with both RIPK1 and RIPK3, that its ability to protect from cell death is dependent on the strength of the necroptotic stimulus, and that it blocks necroptosis most effectively in human cells. Moreover, in human cell lines, we demonstrate that AMG-47a can protect against cell death caused by forced dimerisation of MLKL truncation mutants in the absence of any upstream signalling, validating that it targets a process downstream of MLKL activation. Surprisingly, however, we also found that the cell death driven by activated MLKL in this model was completely dependent on the presence of RIPK1, and to a lesser extent RIPK3, although it was not affected by known inhibitors of these kinases. Together, these results suggest an additional role for RIPK1, or the necrosome, in mediating human necroptosis after MLKL is phosphorylated by RIPK3 and provide further insight into reported differences in the progression of necroptosis between mouse and human cells.Subject terms: Kinases, Necroptosis     

Diverse dengue type 2 virus populations contain recombinant and both parental viruses in a single mosquito host

Envelope (E) protein genes sampled from populations of dengue 2 (DEN-2) virus in individual Aedes aegypti mosquitoes and in serum from dengue patients were copied to cDNA, cloned, and sequenced. The nucleotide sequences of the E genes in more than 70% of the clones differed from the consensus sequence for the corresponding virus population at up to 11 sites, and 24 of the 94 clones contained at least one stop codon. Virus populations recovered up to 2 years apart yielded clones with similar polymorphisms in the E gene. For one mosquito, the clones obtained fell into two genotypes. One group of sequences was closely related to those of viruses recovered from dengue patients in the same locality (Yangon, Myanmar) since 1995 and were classified as Asian 1 genotype. The second group were Cosmopolitan genotype viruses which were also circulating in Yangon in 2000 and which were related to DEN-2 viruses sampled from southern China in 1999. Finally, one clone was identified as a recombinant genome composed of portions of these two "parental" genotypes. This is the first report of recombinant and parental dengue viruses in a single host.     

The relative contributions of seed and pollen dispersal to gene flow and genetic diversity in seedlings of a tropical palm

Seed and pollen dispersal shape patterns of gene flow and genetic diversity in plants. Pollen is generally thought to travel longer distances than seeds, but seeds determine the ultimate location of gametes. Resolving how interactions between these two dispersal processes shape microevolutionary processes is a long‐standing research priority. We unambiguously isolated the separate and combined contributions of these two dispersal processes in seedlings of the animal‐dispersed palm Oenocarpus bataua to address two questions. First, what is the spatial extent of pollen versus seed movement in a system characterized by long‐distance seed dispersal? Second, how does seed dispersal mediate seedling genetic diversity? Despite evidence of frequent long‐distance seed dispersal, we found that pollen moves much further than seeds. Nonetheless, seed dispersal ultimately mediates genetic diversity and fine‐scale spatial genetic structure. Compared to undispersed seedlings, seedlings dispersed by vertebrates were characterized by higher female gametic and diploid seedling diversity and weaker fine‐scale spatial genetic structure for female gametes, male gametes and diploid seedlings. Interestingly, the diversity of maternal seed sources at seed deposition sites (N _em) was associated with higher effective number of pollen sources (N _ep), higher effective number of parents (N _e) and weaker spatial genetic structure, whereas seed dispersal distance had little impact on these or other parameters we measured. These findings highlight the importance maternal seed source diversity (N _em) at frugivore seed deposition sites in driving emergent patterns of fine‐scale genetic diversity and structure.     

N-Acetylglucosaminylation of Serine-Aspartate Repeat Proteins Promotes Staphylococcus aureus Bloodstream Infection

Staphylococcus aureus secretes products that convert host fibrinogen to fibrin and promote its agglutination with fibrin fibrils, thereby shielding bacteria from immune defenses. The agglutination reaction involves ClfA (clumping factor A), a surface protein with serine-aspartate (SD) repeats that captures fibrin fibrils and fibrinogen. Pathogenic staphylococci express several different SD proteins that are modified by two glycosyltransferases, SdgA and SdgB. Here, we characterized three genes of S. aureus, aggA, aggB (sdgA), and aggC (sdgB), and show that aggA and aggC contribute to staphylococcal agglutination with fibrin fibrils in human plasma. We demonstrate that aggB (sdgA) and aggC (sdgB) are involved in GlcNAc modification of the ClfA SD repeats. However, only sdgB is essential for GlcNAc modification, and an sdgB mutant is defective in the pathogenesis of sepsis in mice. Thus, GlcNAc modification of proteins promotes S. aureus replication in the bloodstream of mammalian hosts.     

Mutations in CSPP1, Encoding a Core Centrosomal Protein,Cause a Range of Ciliopathy Phenotypes in Humans

Ciliopathies are characterized by a pattern of multisystem involvement that is consistent with the developmental role of the primary cilium. Within this biological module, mutations in genes that encode components of the cilium and its anchoring structure, the basal body, are the major contributors to both disease causality and modification. However, despite rapid advances in this field, the majority of the genes that drive ciliopathies and the mechanisms that govern the pronounced phenotypic variability of this group of disorders remain poorly understood. Here, we show that mutations in CSPP1, which encodes a core centrosomal protein, are disease causing on the basis of the independent identification of two homozygous truncating mutations in three consanguineous families (one Arab and two Hutterite) affected by variable ciliopathy phenotypes ranging from Joubert syndrome to the more severe Meckel-Gruber syndrome with perinatal lethality and occipital encephalocele. Consistent with the recently described role of CSPP1 in ciliogenesis, we show that mutant fibroblasts from one affected individual have severely impaired ciliogenesis with concomitant defects in sonic hedgehog (SHH) signaling. Our results expand the list of centrosomal proteins implicated in human ciliopathies.