Biogeography in deep time - What do phylogenetics, geology, and paleoclimate tell us about early platyrrhine evolution?

Molecular data have converged on a consensus about the genus-level phylogeny of extant platyrrhine monkeys, but for most extinct taxa and certainly for those older than the Pleistocene we must rely upon morphological evidence from fossils. This raises the question as to how well anatomical data mirror molecular phylogenies and how best to deal with discrepancies between the molecular and morphological data as we seek to extend our phylogenies to the placement of fossil taxa. Here I present parsimony-based phylogenetic analyses of extant and fossil platyrrhines based on an anatomical dataset of 399 dental characters and osteological features of the cranium and postcranium. I sample 16 extant taxa (one from each platyrrhine genus) and 20 extinct taxa of platyrrhines. The tree structure is constrained with a "molecular scaffold" of extant species as implemented in maximum parsimony using PAUP with the molecular-based 'backbone' approach. The data set encompasses most of the known extinct species of platyrrhines, ranging in age from latest Oligocene (∼26 Ma) to the Recent. The tree is rooted with extant catarrhines, and Late Eocene and Early Oligocene African anthropoids. Among the more interesting patterns to emerge are: (1) known early platyrrhines from the Late Oligocene through Early Miocene (26-16.5Ma) represent only stem platyrrhine taxa; (2) representatives of the three living platyrrhine families first occur between 15.7 Ma and 13.5 Ma; and (3) recently extinct primates from the Greater Antilles (Cuba, Jamaica, Hispaniola) are sister to the clade of extant platyrrhines and may have diverged in the Early Miocene. It is probable that the crown platyrrhine clade did not originate before about 20-24 Ma, a conclusion consistent with the phylogenetic analysis of fossil taxa presented here and with recent molecular clock estimates. The following biogeographic scenario is consistent with the phylogenetic findings and climatic and geologic evidence: Tropical South America has been a center for platyrrhine diversification since platyrrhines arrived on the continent in the middle Cenozoic. Platyrrhines dispersed from tropical South America to Patagonia at ∼25-24 Ma via a "Paraná Portal" through eastern South America across a retreating Paranense Sea. Phylogenetic bracketing suggests Antillean primates arrived via a sweepstakes route or island chain from northern South America in the Early Miocene, not via a proposed land bridge or island chain (GAARlandia) in the Early Oligocene (∼34 Ma). Patagonian and Antillean platyrrhines went extinct without leaving living descendants, the former at the end of the Early Miocene and the latter within the past six thousand years. Molecular evidence suggests crown platyrrhines arrived in Central America by crossing an intermittent connection through the Isthmus of Panama at or after 3.5Ma. Any more ancient Central American primates, should they be discovered, are unlikely to have given rise to the extant Central American taxa in situ.

MRP3: a molecular target for human glioblastoma multiforme immunotherapy.

BACKGROUND: Glioblastoma multiforme (GBM) is refractory to conventional therapies. To overcome the problem of heterogeneity, more brain tumor markers are required for prognosis and targeted therapy. We have identified and validated a promising molecular therapeutic target that is expressed by GBM: human multidrug-resistance protein 3 (MRP3). METHODS: We investigated MRP3 by genetic and immunohistochemical (IHC) analysis of human gliomas to determine the incidence, distribution, and localization of MRP3 antigens in GBM and their potential correlation with survival. To determine MRP3 mRNA transcript and protein expression levels, we performed quantitative RT-PCR, raising MRP3-specific antibodies, and IHC analysis with biopsies of newly diagnosed GBM patients. We used univariate and multivariate analyses to assess the correlation of RNA expression and IHC of MRP3 with patient survival, with and without adjustment for age, extent of resection, and KPS. RESULTS: Real-time PCR results from 67 GBM biopsies indicated that 59/67 (88%) samples highly expressed MRP3 mRNA transcripts, in contrast with minimal expression in normal brain samples. Rabbit polyvalent and murine monoclonal antibodies generated against an extracellular span of MRP3 protein demonstrated reactivity with defined MRP3-expressing cell lines and GBM patient biopsies by Western blotting and FACS analyses, the latter establishing cell surface MRP3 protein expression. IHC evaluation of 46 GBM biopsy samples with anti-MRP3 IgG revealed MRP3 in a primarily membranous and cytoplasmic pattern in 42 (91%) of the 46 samples. Relative RNA expression was a strong predictor of survival for newly diagnosed GBM patients. Hazard of death for GBM patients with high levels of MRP3 RNA expression was 2.71 (95% CI: 1.54-4.80) times that of patients with low/moderate levels (p = 0.002). CONCLUSIONS: Human GBMs overexpress MRP3 at both mRNA and protein levels, and elevated MRP3 mRNA levels in GBM biopsy samples correlated with a higher risk of death. These data suggest that the tumor-associated antigen MRP3 has potential use for prognosis and as a target for malignant glioma immunotherapy.

Molecular cloning and expression of the cDNA for the hamster alpha 1-adrenergic receptor.

The cDNA for the Syrian hamster alpha 1-adrenergic receptor has been cloned with oligonucleotides corresponding to the partial amino acid sequence of the receptor protein purified from DDT1MF-2 smooth muscle cells. The deduced amino acid sequence encodes a 515-residue polypeptide that shows the most sequence identity with the other adrenergic receptors and the putative protein product of the related clone G-21. Similarities with the muscarinic cholinergic receptors are also evident. Expression studies in COS-7 cells confirm that we have cloned the alpha 1-adrenergic receptor that couples to inositol phospholipid metabolism.

Low Molecular Weight Opioid Peptide Esters Could be Developed as a New Class of Analgesics.

Low molecular weight opioid peptide esters (OPE) could become a class of analgesics with different side effect profiles than current opiates. OPE may have sufficient plasma stability to cross the blood brain barrier (BBB), undergo ester hydrolysis and produce analgesia. OPE of dipeptides, tyr-pro and tyr-gly conjugated to ethanol have a structure similar to the anesthestic agent, etomidate. Based upon the analgesic activity of dipeptide opioids, Lipinski's criteria, and permeability of select GABA esters to cross the BBB, opioid peptides (OP) conjugated to ethanol, cholesterol or 3-glucose are lead recommendations. Preliminary animal data suggests that tyr-pro-ethyl ester crosses the BBB and unexpectedly produces hyperalgesia. Currently, there are no approved OP analgesics available for clinical use. Clinical trials of good manufacturing practice OP administered to patients suffering from chronic pain with indwelling intrathecal pumps could resolve the issue that OP may be superior to opiates and may redirect research.

Temporal Stability of Molecular Diversity Measures in Natural Populations of Drosophila pseudoobscura and Drosophila persimilis

Many molecular ecological and evolutionary studies sample wild populations at a single point in time, failing to consider that data they collect represents genetic variation from a potentially unrepresentative snapshot in time. Variation across time in genetic parameters may occur quickly in species that produce multiple generations of offspring per year. However, many studies of rapid contemporary microevolution examine phenotypic trait divergence as opposed to molecular evolutionary divergence. Here, we compare genetic diversity in wild caught populations of Drosophila persimilis and D. pseudoobscura collected 16 years apart at the same time of year and same site at four X-linked and two mitochondrial loci to assess genetic stability. We found no major changes in nucleotide diversity in either species, but we observed a drastic shift in Tajima’s D between D. pseudoobscura timepoints at one locus associated with the increased abundance of a set of related haplotypes. Our data also suggests that D. persimilis may have recently accelerated its demographic expansion. While the changes we observed were modest, this study reinforces the importance of considering potential temporal variation in genetic parameters within single populations over short evolutionary timescales.

Interrogation of individual intratumoral B lymphocytes from lung cancer patients for molecular target discovery.

Intratumoral B lymphocytes are an integral part of the lung tumor microenvironment. Interrogation of the antibodies they express may improve our understanding of the host response to cancer and could be useful in elucidating novel molecular targets. We used two strategies to explore the repertoire of intratumoral B cell antibodies. First, we cloned VH and VL genes from single intratumoral B lymphocytes isolated from one lung tumor, expressed the genes as recombinant mAbs, and used the mAbs to identify the cognate tumor antigens. The Igs derived from intratumoral B cells demonstrated class switching, with a mean VH mutation frequency of 4%. Although there was no evidence for clonal expansion, these data are consistent with antigen-driven somatic hypermutation. Individual recombinant antibodies were polyreactive, although one clone demonstrated preferential immunoreactivity with tropomyosin 4 (TPM4). We found that higher levels of TPM4 antibodies were more common in cancer patients, but measurement of TPM4 antibody levels was not a sensitive test for detecting cancer. Second, in an effort to focus our recombinant antibody expression efforts on those B cells that displayed evidence of clonal expansion driven by antigen stimulation, we performed deep sequencing of the Ig genes of B cells collected from seven different tumors. Deep sequencing demonstrated somatic hypermutation but no dominant clones. These strategies may be useful for the study of B cell antibody expression, although identification of a dominant clone and unique therapeutic targets may require extensive investigation.