New Data on Miocene Neotropical Provinciality from Cerdas, Bolivia

We provide the first faunal report for the early/middle Miocene fauna of Cerdas, Bolivia (16.5–15.3 Ma; 20° 52′ S, 66° 19′ W), based primarily on new specimens collected in 2007. As many as twelve species of mammals in nine families are represented. Notoungulates include Palyeidodon obtusum (Toxodontidae), Protypotherium cf. attenuatum and Protypotherium sp. nov. (Interatheriidae), ‘Plesiotypotherium’ minus and possibly Microtypotherium choquecotense (Mesotheriidae), and Hegetotherium? sp. nov. (Hegetotheriidae). Xenarthrans include Stenotatus planus and Prozaedyus sp. (Cingulata: Dasypodidae), Peltephilidae gen. et sp. nov. (Cingulata), and Xyophorus cf. bondesioi (Pilosa: Nothrotheriidae). A new species of litoptern is also present (Macraucheniidae) as well as an unidentified rodent (Chinchillidae: Lagostominae). Two of these Cerdas species occur in Friasian sensu stricto/Colloncuran SALMA faunas of Patagonia, and perhaps one in Santacrucian SALMA faunas. Among middle latitude localities, Cerdas resembles Chucal, Chile (late early Miocene, Santacrucian SALMA) in community composition (e.g., abundant mesotheriids, few rodent species), but has no species in common; it shares one species with Quebrada Honda, Bolivia (middle Miocene, Laventan SALMA), and perhaps as many as three more. These observations indicate that Cerdas is not referable to the Santacrucian, and that the upper limit of this SALMA in the middle latitudes falls somewhere between 17.5 Ma (the top of Chucal) and 16.5 Ma (the base of Cerdas). Based on the range of dates proposed for the youngest Santacrucian intervals in Patagonia, a diachronous offset of up to 2.1 Ma may exist at this point in the SALMA sequence between middle and high latitude faunas.     

Microstructural abnormalities in subcortical reward circuitry of subjects with major depressive disorder

Background

Previous studies of major depressive disorder (MDD) have focused on abnormalities in the prefrontal cortex and medial temporal regions. There has been little investigation in MDD of midbrain and subcortical regions central to reward/aversion function, such as the ventral tegmental area/substantia nigra (VTA/SN), and medial forebrain bundle (MFB).

Methodology/Principal Findings

We investigated the microstructural integrity of this circuitry using diffusion tensor imaging (DTI) in 22 MDD subjects and compared them with 22 matched healthy control subjects. Fractional anisotropy (FA) values were increased in the right VT and reduced in dorsolateral prefrontal white matter in MDD subjects. Follow-up analysis suggested two distinct subgroups of MDD patients, which exhibited non-overlapping abnormalities in reward/aversion circuitry. The MDD subgroup with abnormal FA values in VT exhibited significantly greater trait anxiety than the subgroup with normal FA values in VT, but the subgroups did not differ in levels of anhedonia, sadness, or overall depression severity.

Conclusions/Significance

These findings suggest that MDD may be associated with abnormal microstructure in brain reward/aversion regions, and that there may be at least two subtypes of microstructural abnormalities which each impact core symptoms of depression.     

A New Adenovirus Based Vaccine Vector Expressing an Eimeria tenella Derived TLR Agonist Improves Cellular Immune Responses to an Antigenic Target

Background

Adenoviral based vectors remain promising vaccine platforms for use against numerous pathogens, including HIV. Recent vaccine trials utilizing Adenovirus based vaccines expressing HIV antigens confirmed induction of cellular immune responses, but these responses failed to prevent HIV infections in vaccinees. This illustrates the need to develop vaccine formulations capable of generating more potent T-cell responses to HIV antigens, such as HIV-Gag, since robust immune responses to this antigen correlate with improved outcomes in long-term non-progressor HIV infected individuals.

Methodology/Principal Findings

In this study we designed a novel vaccine strategy utilizing an Ad-based vector expressing a potent TLR agonist derived from Eimeria tenella as an adjuvant to improve immune responses from a [E1-]Ad-based HIV-Gag vaccine. Our results confirm that expression of rEA elicits significantly increased TLR mediated innate immune responses as measured by the influx of plasma cytokines and chemokines, and activation of innate immune responding cells. Furthermore, our data show that the quantity and quality of HIV-Gag specific CD8⁺ and CD8⁻ T-cell responses were significantly improved when coupled with rEA expression. These responses also correlated with a significantly increased number of HIV-Gag derived epitopes being recognized by host T cells. Finally, functional assays confirmed that rEA expression significantly improved antigen specific CTL responses, in vivo. Moreover, we show that these improved responses were dependent upon improved TLR pathway interactions.

Conclusion/Significance

The data presented in this study illustrate the potential utility of Ad-based vectors expressing TLR agonists to improve clinical outcomes dependent upon induction of robust, antigen specific immune responses.     

Polyfunctional T-cell responses are disrupted by the ovarian cancer ascites environment and only partially restored by clinically relevant cytokines

Background

Host T-cell responses are associated with favorable outcomes in epithelial ovarian cancer (EOC), but it remains unclear how best to promote these responses in patients. Toward this goal, we evaluated a panel of clinically relevant cytokines for the ability to enhance multiple T-cell effector functions (polyfunctionality) in the native tumor environment.

Methodology/Principal Findings

Experiments were performed with resident CD8+ and CD4+ T cells in bulk ascites cell preparations from high-grade serous EOC patients. T cells were stimulated with α-CD3 in the presence of 100% autologous ascites fluid with or without exogenous IL-2, IL-12, IL-18 or IL-21, alone or in combination. T-cell proliferation (Ki-67) and function (IFN-γ, TNF-α, IL-2, CCL4, and CD107a expression) were assessed by multi-parameter flow cytometry. In parallel, 27 cytokines were measured in culture supernatants. While ascites fluid had variable effects on CD8+ and CD4+ T-cell proliferation, it inhibited T-cell function in most patient samples, with CD107a, IFN-γ, and CCL4 showing the greatest inhibition. This was accompanied by reduced levels of IL-1β, IL-1ra, IL-9, IL-17, G-CSF, GM-CSF, Mip-1α, PDGF-bb, and bFGF in culture supernatants. T-cell proliferation was enhanced by exogenous IL-2, but other T-cell functions were largely unaffected by single cytokines. The combination of IL-2 with cytokines engaging complementary signaling pathways, in particular IL-12 and IL-18, enhanced expression of IFN-γ, TNF-α, and CCL4 in all patient samples by promoting polyfunctional T-cell responses. Despite this, other functional parameters generally remained inhibited.

Conclusions/Significance

The EOC ascites environment disrupts multiple T-cell functions, and exogenous cytokines engaging diverse signaling pathways only partially reverse these effects. Our results may explain the limited efficacy of cytokine therapies for EOC to date. Full restoration of T-cell function will require activation of signaling pathways beyond those engaged by IL-2, IL-12, IL-18, and IL-21.     

Identification of potent and novel small-molecule inhibitors of caspase-3

The design and synthesis of a series of novel, reversible, small molecule inhibitors of caspase-3 are described.     

Unique stabilizing interactions identified in the two-stranded alpha-helical coiled-coil: crystal structure of a cortexillin I/GCN4 hybrid coiled-coil peptide

We determined the 1.17 A resolution X-ray crystal structure of a hybrid peptide based on sequences from coiled-coil regions of the proteins GCN4 and cortexillin I. The peptide forms a parallel homodimeric coiled-coil, with C(alpha) backbone geometry similar to GCN4 (rmsd value 0.71 A). Three stabilizing interactions have been identified: a unique hydrogen bonding-electrostatic network not previously observed in coiled-coils, and two other hydrophobic interactions involving leucine residues at positions e and g from both g-a' and d-e' interchain interactions with the hydrophobic core. This is also the first report of the quantitative significance of these interactions. The GCN4/cortexillin hybrid surprisingly has two interchain Glu-Lys' ion pairs that form a hydrogen bonding network with the Asn residues in the core. This network, which was not observed for the reversed Lys-Glu' pair in GCN4, increases the combined stability contribution of each Glu-Lys' salt bridge across the central Asn15-Asn15' core to approximately 0.7 kcal/mole, compared to approximately 0.4 kcal mole(-1) from a Glu-Lys' salt bridge on its own. In addition to electrostatic and hydrogen bonding stabilization of the coiled-coil, individual leucine residues at positions e and g in the hybrid peptide also contribute to stability by 0.7 kcal/mole relative to alanine. These interactions are of critical importance to understanding the stability requirements for coiled-coil folding and in modulating the stability of de novo designed macromolecules containing this motif.     

Structure of a gametocyte protein essential for sexual development in Plasmodium falciparum

Malaria transmission is dependent on the development of sexual forms of Plasmodium falciparum, called gametocytes, in the vertebrate host. Pfg27 is an abundantly expressed sexual stage-specific protein that is essential for gametocytogenesis in P. falciparum. We describe the crystal structure of Pfg27, which reveals a novel fold composed of two pseudo dyad-related repeats of the helix-turn-helix motif. Structurally equivalent helices of each repeat either form a dimer interface or interact with RNA in vitro. One side of the dimer presents an unprecedented juxtaposition of four polyproline (PXXP) motifs. Preliminary binding data indicate that these sites are capable of binding Src homology-3 (SH3) modules. Molecular modeling suggests that the dimer can accommodate two SH3 modules simultaneously, potentially enabling molecular crosstalk between SH3-containing proteins. The structural and initial biochemical evidence suggests that Pfg27 may serve as a platform for RNA and SH3 binding.     

Plasmodium falciparum protein phosphatase type 1 functionally complements a glc7 mutant in Saccharomyces cerevisiae

We have identified a new homologue of protein phosphatase type 1 from Plasmodium falciparum, designated PfPP1, which shows 83-87% sequence identity with yeast and mammalian PP1s at the amino acid level. The PfPP1 sequence is strikingly different from all other P. falciparum Ser/Thr phosphatases cloned so far. The deduced 304 amino acid sequence revealed the signature sequence of Ser/Thr phosphatase LRGNHE, and two putative protein kinase C and five putative casein kinase II phosphorylation sites. Calyculin A, a potent inhibitor of Ser/Thr phosphatase 1 and 2A showed hyperphosphorylation of a 51kDa protein among other parasite proteins. Okadaic acid on the other hand, was without any effect suggesting that PP1 activity might predominate over PP2A activity in intra-erythrocytic P. falciparum. Complementation studies showed that PfPP1 could rescue low glycogen phenotype of Saccharomyces cerevisiae glc7 (PP1) mutant, strongly suggesting functional interaction of PfPP1 and yeast proteins involved in glycogen metabolism.     

The p11 subunit of annexin II heterotetramer is regulated by basic carboxypeptidase

The Ca(2+)-dependent phospholipid-binding protein annexin II heterotetramer (AIIt) is composed of two copies of annexin II and a p11 dimer. The interaction of the carboxyl-terminal lysine residues of the p11 subunit of AIIt with the lysine-binding kringle domains of plasminogen is believed to play a key role in plasminogen binding and stimulation of the tPA-catalyzed cleavage of plasminogen to plasmin. In the current report, we show that AIIt-stimulated plasminogen activation is regulated by basic carboxypeptidases, in vitro. The incubation of AIIt with a 1/400 molar ratio of carboxypeptidase B for periods as short as 2 min resulted in a significant loss in AIIt-stimulated plasminogen activation. Carboxypeptidase B (CpB) as well as thrombin-activated fibrinolysis inhibitor (TAFIa) and carboxypeptidase N (CpN) rapidly reduced AIIt-stimulated plasminogen activation by 80%. The molar ratio of carboxypeptidase/AIIt for half-maximal inhibition of AIIt was 1/4700, 1/700, and 1/500 for CpB, TAFIa, and CpN, respectively. Treatment of AIIt with carboxypeptidase resulted in loss of both carboxyl-terminal lysine residues from the p11 subunit, which correlated with a decrease in the k(cat) and an increase in the K(m) for plasminogen activation. The data reveal a novel mechanism for the regulation of AIIt-stimulated plasminogen activation.