An improved phylogeny of the Andean tit-tyrants (Aves, Tyrannidae): more characters trump sophisticated analyses

The phylogeny of the flycatcher genus Anairetes was previously inferred using short fragments of mitochondrial DNA and parsimony and distance-based methods. The resulting topology spurred taxonomic revision and influenced understanding of Andean biogeography. More than a decade later, we revisit the phylogeny of Anairetes tit-tyrants using more mtDNA characters, seven unlinked loci (three mitochondrial genes, six nuclear loci), more closely related outgroup taxa, partitioned Bayesian analyses, and two coalescent species-tree approaches (Bayesian estimation of species trees, BEST; Bayesian evolutionary analysis by sampling trees, (*)BEAST). Of these improvements in data and analyses, the fourfold increase in mtDNA characters was both necessary and sufficient to incur a major shift in the topology and near-complete resolution. The species-tree analyses, while theoretically preferable to concatenation or single gene approaches, yielded topologies that were compatible with mtDNA but with weaker statistical resolution at nodes. The previous results that had led to taxonomic and biogeographic reappraisal were refuted, and the current results support the resurrection of the genus Uromyias as the sister clade to Anairetes. The sister relationship between these two genera corresponds to an ecological dichotomy between a depauperate humid cloud forest clade and a diverse dry-tolerant clade that has diversified along the latitudinal axis of the Andes. The species-tree results and the concatenation results each reaffirm the primacy of mtDNA to provide phylogenetic signal for avian phylogenies at the species and subspecies level. This is due in part to the abundance of informative characters in mtDNA, and in part to its lower effective population size that causes it to more faithfully track the species tree.     

N-(3-fluoro-4-(2-arylthieno[3,2-b]pyridin-7-yloxy)phenyl)-2-oxo-3-phenylimidazolidine-1-carboxamides: A novel series of dual c-Met/VEGFR2 receptor tyrosine kinase inhibitors

A series of N-(3-fluoro-4-(2-arylthieno[3,2-b]pyridin-7-yloxy)phenyl)-2-oxo-3-phenylimidazolidine-1-carboxamides targeting c-Met and VEGFR2 tyrosine kinases was designed and synthesized. The compounds were potent against these two enzymes with IC₅₀ values in the low nanomolar range in vitro, possessed favorable pharmacokinetic profiles and showed high efficacy in vivo in several human tumor xenograft models in mice.     

N-(4-(6,7-Disubstituted-quinolin-4-yloxy)-3-fluorophenyl)-2-oxo-3-phenylimidazolidine-1-carboxamides: A novel series of dual c-Met/VEGFR2 receptor tyrosine kinase inhibitors

A series of N-(4-(6,7-disubstituted-quinolin-4-yloxy)-3-fluorophenyl)-2-oxo-3-phenylimidazolidine-1-carboxamides targeting c-Met and VEGFR2 tyrosine kinases was designed and synthesized. The compounds were potent against these two enzymes with IC₅₀ values in the low nanomolar range in vitro, possessed favorable pharmacokinetic profiles and showed high efficacy in vivo in several human tumor xenograft models in mice.     

Mutations within the putative membrane-spanning domain of the simian immunodeficiency virus transmembrane glycoprotein define the minimal requirements for fusion, incorporation, and infectivity

The membrane-spanning domain (MSD) of a number of retroviral transmembrane (TM) glycoproteins, including those from the human and simian immunodeficiency viruses (HIV and SIV), have been predicted to contain a charged arginine residue. The wild-type SIV TM glycoprotein is 354 amino acids long. The entire putative cytoplasmic domain of SIV (amino acids 193 to 354) is dispensable for virus replication in vitro, and such truncation-containing viruses are capable of reaching wild-type titers after a short delay. We show here that further truncation of eight additional amino acids to TM185 results in a protein that lacks fusogenicity but is, nevertheless, efficiently incorporated into budding virions. By analyzing a series of nonsense mutations between amino acids 193 and 185 in Env expression vectors and in the SIVmac239 proviral clone, a region of the SIV TM that contains the minimum requirement for glycoprotein-mediated cell-to-cell fusion and that for virus replication was identified. Virus entry and infectivity were evident in truncations to a minimum of 189 amino acids, whereas cell-cell fusion was observed for a protein of only 187 amino acids. Glycoprotein was efficiently incorporated into budding virions in truncations up to 185 amino acids, indicating that such proteins are membrane anchored and are transported to the cell surface. However, truncation of the TM to 180 amino acids resulted in a protein that displays a transport defect and may be retained in the endoplasmic reticulum. Based on our analyses of these mutants, an alternative model for the MSD of SIV is proposed. Our model suggests that membrane-imbedded charged residues can be neutralized by side-chain interactions with lipid polar head groups. As a consequence, the membrane-spanning region can be reduced by more than a helical turn. This new model accounts for the ability of truncations within the predicted MSD to remain membrane anchored and maintain biological activity.     

A tyrosine motif in the cytoplasmic domain of mason-pfizer monkey virus is essential for the incorporation of glycoprotein into virions

Mason-Pfizer monkey virus (M-PMV) encodes a transmembrane (TM) glycoprotein with a 38-amino-acid-long cytoplasmic domain. After the release of the immature virus, a viral protease-mediated cleavage occurs within the cytoplasmic domain, resulting in the loss of 17 amino acids from the carboxy terminus. This maturational cleavage occurs between a histidine at position 21 and a tyrosine at position 22 in the cytoplasmic domain of the TM protein. We have demonstrated previously that a truncated TM glycoprotein with a 21-amino-acid-long cytoplasmic tail showed enhanced fusogenicity but could not be incorporated into virions. These results suggest that postassembly cleavage of the cytoplasmic domain removes a necessary incorporation signal and activates fusion activity. To investigate the contribution of tyrosine residues to the function of the glycoprotein complex and virus replication, we have introduced amino acid substitutions into two tyrosine residues found in the cytoplasmic domain. The effects of these mutations on glycoprotein biosynthesis and function, as well as on virus infectivity, have been examined. Mutation of tyrosine 34 to alanine had little effect on glycoprotein function. In contrast, substitutions at tyrosine 22 modulated fusion activity in either a positive or negative manner, depending on the substituting amino acid. Moreover, any nonaromatic substitution at this position blocked glycoprotein incorporation into virions and abolished infectivity. These results demonstrate that M-PMV employs a tyrosine signal for the selective incorporation of glycoprotein into budding virions. Antibody uptake studies show that tyrosine 22 is part of an efficient internalization signal in the cytoplasmic domain of the M-PMV glycoprotein that can also be positively and negatively influenced by changes at this site.     

Bacteria and nematodes in the conjunctiva of mule deer from Wyoming and Utah

Swabs of conjunctiva were collected from 44 live and 226 hunter-harvested mule deer (Odocoileus hemionus) from Wyoming and Utah (USA). We identified 29 gram negative and 22 gram positive bacterial taxonomic categories, but many isolates from hunter-harvested animals were environmental contaminants. Staphylococcus spp. and Micrococcus spp. were the most common gram positive bacteria isolated, and Enterobacter spp., Escherichia coli, and Pseudomonas spp. were common gram negative bacteria isolated. Thelazia californiensis were found in 15% of hunter-harvested deer in Utah in 1994 and in 8% in 1995. Nematodes were found in 40% of live deer in 1995 and 66% in 1996. Three live animals showed clinical signs of infectious keratoconjunctivitis (IKC) in 1996, but pathogenic bacteria were not isolated from these individuals. Hemolytic, non-piliated Moraxella ovis was isolated from two clinically normal live deer in 1996 and isolates were similar to those cultured from IKC cases from Wyoming and Utah.     

Comparison of Lactate, Base Excess, Bicarbonate, and pH as Predictors of Mortality after Severe Trauma in Rhesus Macaques (Macaca mulatta)

Social group housing of rhesus macaques at biomedical facilities is advocated to improve the psychologic wellbeing of these intelligent and social animals. An unintended outcome of social housing in this species is increased intraspecific aggression resulting in cases of severe multiple trauma and posttraumatic shock. The metabolic correlates of oxygen debt are likely important quantifiers of the severity of posttraumatic shock and may serve as useful guides in the treatment of these cases. The purpose of this retrospective study was to evaluate venous blood lactate, base excess, bicarbonate, and pH as predictors of mortality. These 4 variables were assessed in 84 monkeys with severe traumatic injury and shock. Data were available from blood samples collected prior to resuscitation therapy and the day after resuscitation therapy. The pre- and postresuscitation therapy levels of the variables then were tested for association with 6-d survival. When measured prior to resuscitation therapy, all variables were strongly correlated with each other and had a statistically significant association with survival. No single variable had both strong specificity and high sensitivity when measured prior to resuscitation therapy. Survival analysis showed that as the number of categorical indicators of acidosis increased, 6-d survival decreased. Analysis of the 4 variables after resuscitation therapy indicated that lactate was the only variable significantly associated with survival in our study.Many research facilities maintain populations of rhesus macaques in group housing that allows them to engage in social behaviors that are normal for the species, such as grooming, play, and huddling with conspecifics.²¹ An unintended consequence of social housing is intraspecific aggression. The trauma, specifically crush injuries from multiple bite wounds to the limbs and face, that may result from this aggression can be severe and often leads to significant morbidity and mortality if ignored or poorly treated.¹⁷ Major multiple trauma injuries result in sudden physical and metabolic alterations that can progress to organ dysfunction, organ failures, and even death. In addition to multiple severe skin wounds, the hemorrhage and obligatory edema that occur within the injured soft tissues have significant effect on circulating blood volume, resulting in intravascular volume depletion and hypovolemic shock. The associated decreased organ perfusion and impaired oxygen delivery result in regional hypoxia and anaerobic metabolism. The end-products of anaerobic metabolism are 2 ATP molecules and pyruvate, which is converted to lactic acid. Prolonged and severe tissue hypoperfusion results in the generation of large quantities of hydrogen ions (H⁺) from lactic acid, resulting in metabolic acidosis.^2,9Serum markers of metabolic acidosis may be measured as part of the critical care diagnostic plan to assess the severity of injury, determine treatment efficacy, and provide prognostic information. The most common of these markers include lactate, base excess (or base deficit), bicarbonate, and pH. Technologic advances in point-of-care testing have made rapid laboratory assessment of these markers accurate, practical, and affordable for veterinary medical facilities. However, no studies to date have validated markers of metabolic acidosis as predictors of mortality in rhesus macaques. Nor has the significance of these values been examined when used in combination or when they provide conflicting data.Lactate, an end product of anaerobic metabolism, can be measured directly by many point-of-care analyzers. As calculated by most point-of-care analyzers, base excess is determined by using measured carbon dioxide, pH, and serum bicarbonate values and represents the concentration of titratable base minus the concentration of titratable acid needed to normalize the pH of a liter of blood to physiologic levels. A decreased base excess is thought to represent the presence of unmeasured anions. In acute trauma cases, the primary unmeasured anion is assumed to be lactate.⁴ Therefore, base excess usually is viewed as a surrogate marker for lactic acidosis.^9,11 Bicarbonate is a buffer for serum hydrogen ions released during anaerobic metabolism. As metabolic acidosis worsens, bicarbonate levels decrease. In humans, bicarbonate and base excess are strongly correlated.^8,10 Compared with lactate, base excess, and bicarbonate, serum pH is less clinically relevant for assessing metabolic acidosis in human patients.^6,9,19 This difference most likely is due to the extensive compensatory physiologic mechanisms in place to maintain normal pH. pH is a direct measure of acidemia, whereas lactate, base excess, and bicarbonate are common means of characterizing acidosis.⁹Multiple studies have been published in the human medical literature that validate the ability of lactate, base excess, bicarbonate, and pH for predicting morbidity and mortality among trauma and surgical patients.^12,14,20,23 However, no single value has proven superior to the others in these regards,⁹ and the validity of these measurements has not been demonstrated in nonhuman primates. Several human studies have noted the predictive value of the initial lactate level.^1,3,16 Other studies have demonstrated outcome is better predicted by other variables²² or have shown no correlation between lactate level and mortality.¹³During hospitalization, the validity of these markers may be confounded by the rapid intravenous infusion of large volumes of resuscitation fluids and electrolytes administered to increase circulatory volume in shock patients. The confounding affects of shock therapy are important because these markers of metabolic acidosis frequently are used by clinicians to guide resuscitation. For example, the administration of large quantities of exogenous lactate (massive infusion of lactated Ringers solution) has been shown to increase lactate to levels significantly greater than those expected to result from the shock process alone.^7,18 However, this elevation of lactate is transient and not associated with acidosis, because the end products of lactate metabolism are bicarbonate and glucose.^7,24 In addition, the administration of sodium bicarbonate during resuscitation likely would confound the utility of bicarbonate measurements. The administration of sodium bicarbonate would similarly confound the utility of base excess as an endpoint for resuscitation, because bicarbonate is used in the calculation of base excess.For the past 7 y, serial measurements of these systemic markers of metabolic acidosis have been used to guide the need for and response to resuscitation of nonhuman primate cases at our institution, the Oregon National Primate Research Center. Although the human medical literature and our experience in practice indicate that these measurements are valuable for case management, no previous scientific studies have validated them as indicators of morbidity or mortality in nonhuman primates. The purpose of this retrospective study was to evaluate venous blood lactate, base excess, bicarbonate, and pH values, obtained before and after treatment of shock, as predictors of 6-d mortality after trauma in rhesus macaques.     

Determining prevalence of bluetongue and epizootic hemorrhagic disease viruses in mule deer in Arizona (USA) using whole blood dried on paper strips compared to serum analyses

We investigated the feasibility of using whole blood dried on paper strips as a means to collect antibody prevalence data for the epizootic hemorrhagic disease viruses (EHDV) and bluetongue viruses (BTV) from hunter-harvested male mule deer (Odocoileus hemionus) in October 2002 from Arizona, USA. We compared antibody prevalence estimates in mule deer from paired paper strip and serum samples. Prevalence data obtained from elution of dried blood on paper strips proved to be consistent with results from serum in 94% of the samples tested. The paper strip method allows easy collection of blood from dead animals, with a smaller amount of blood being needed for analyses. Also, samples do not need to be refrigerated before analyses. We also used serum samples to determine hemorrhagic disease (HD) serotype exposure status of mule deer harvested from 4 distinct areas in Arizona. Antibodies to BTV and EHDV were identified in 3 of the 4 areas, with positive results to EHDV-1, EHDV-2, BTV-10, and BTV-11 being most common. Many animals did not have antibodies against the BTV serotypes. Exposure varied geographically and potentially with elevation. Hemorrhagic disease viruses commonly infect Arizona mule deer, except on the Kaibab Plateau in northern Arizona.     

The identification of novel ovarian proteases through the use of genomic and bioinformatic methodologies

Proteolytic activities are essential for follicular growth, ovulation, as well as for luteal formation and regression. Using suppression subtractive hybridization (SSH), a novel mouse ovary-selective gene (termed protease serine 35, Prss35) was identified. Analysis of the mouse genome database using the Prss35 sequence led to the identification of a homologous protease (protease serine 23, Prss23). PRSS35 possesses general features that are characteristic of serine (Ser) proteases, but is unique in that the canonical Ser that defines this enzyme family is replaced by a threonine (Thr). In contrast, PRSS23 possesses the standard catalytic Ser typical for this family of proteases. As determined by real-time polymerase chain reaction (PCR), the Prss35 mRNA levels increased around the time of ovulation and remained elevated in the developing corpus luteum. Steroid ablation/replacement studies demonstrated progesterone-dependent regulation of Prss35 gene expression prior to follicle rupture. Prss35 gene expression was localized to the theca cells of pre-antral follicles, the theca and granulosa cells of pre-ovulatory and ovulatory follicles, as well as to the developing corpus luteum. In contrast, Prss23 mRNA levels decreased transiently after ovulation induction and again in the postovulatory period. Prss23 gene expression was noted primarily in the granulosa cells of the secondary/early antral follicles. PRSS35 and PRSS23 orthologs in the rat, human, rhesus macaque, chimpanzee, cattle, dog, and chicken were identified and found to be highly homologous to one another (75-99% homology). Collectively, these results suggest that the PRSS35 and PRSS23 genes have been conserved as critical ovarian proteases throughout the course of vertebrate evolution.     

Discovery of a novel and potent series of thieno[3,2-b]pyridine-based inhibitors of c-Met and VEGFR2 tyrosine kinases

A series of thieno[3,2-b]pyridine-based inhibitors of c-Met and VEGFR2 tyrosine kinases is described. The compounds demonstrated potency with IC₅₀ values in the low nanomolar range in vitro while the lead compound also showed in vivo activity against various human tumor xenograft models in mice. Further exploration of this class of compounds is underway.