Phylogeography of Bufo marinus from its natural and introduced ranges.

The marine toad, Bufo marinus, has a broad natural distribution extending from the south-west of the USA to southern Peru and the central Amazon. It was introduced to several localities in the Caribbean and Pacific Oceans to control sugar cane pests. We sequenced 468 bp of mitochondrial DNA (mtDNA) containing the ND3 gene, and flanking tRNA genes from toads spanning the broad natural and introduced ranges. Consistent with the known history of introductions and expected effects of serial bottlenecks, mtDNA within introduced populations in Hawaii and Australia was uniform and most closely related to samples from eastern Venezuela and French Guiana. However, mtDNA nucleotide diversity in the geographic region spanning the source areas is also relative low (0.18-0.46%) and the absence of variation in the introduced populations precludes quantitative assessment of the reduction in genetic diversity. Unexpectedly, there was a large phylogeographic break (5.4% sequence divergence) within the natural range separating populations east and west of the Venezuelan Andes. We hypothesize that the two major lineages of B. marinus were isolated by the uplift of the eastern Andean cordillera which was completed approximately 2.7 Ma. Another species of the marinus group, B. paracnemis, had mtDNA paraphyletic, with marinus, being nested within the eastern lineage. Thus, at least one speciation event within the marinus group postdates the split within marinus. These findings suggest that the taxonomy of B. marinus should be re-evaluated and that the search for pathogens to control Australian populations should be conducted in populations from both lineages in the natural range.     

A Comparative pO2 Probe and [18F]-Fluoro-Azomycinarabino-Furanoside ([18F]FAZA) PET Study Reveals Anesthesia-Induced Impairment of Oxygenation and Perfusion in Tumor and Muscle

Tumor hypoxia can be identified by [¹⁸F]FAZA positron emission tomography, or invasively using oxygen probes. The impact of anesthetics on tumor hypoxia remains controversial. The aim of this comprehensive study was to investigate the impact of isoflurane and ketamine/xylazine anesthesia on [¹⁸F]FAZA uptake and partial oxygen pressure (pO₂) in carcinoma and muscle tissue of air- and oxygen-breathing mice.

Methods

CT26 colon carcinoma-bearing mice were anesthetized with isoflurane (IF) or ketamine/xylazine (KX) while breathing air or oxygen (O₂). We performed 10 min static PET scans 1 h, 2 h and 3 h after [¹⁸F]FAZA injection and calculated the [¹⁸F]FAZA-uptake and tumor-to-muscle ratios (T/M). In another experimental group, we placed a pO₂ probe in the tumor as well as in the gastrocnemius muscle to measure the pO₂ and perfusion.

Results

Ketamine/xylazine-anesthetized mice yielded up to 3.5-fold higher T/M-ratios compared to their isoflurane-anesthetized littermates 1 h, 2 h and 3 h after [¹⁸F]FAZA injection regardless of whether the mice breathed air or oxygen (3 h, KX-air: 7.1 vs. IF-air: 1.8, p = 0.0001, KX-O₂: 4.4 vs. IF-O₂: 1.4, p < 0.0001). The enhanced T/M-ratios in ketamine/xylazine-anesthetized mice were mainly caused by an increased [¹⁸F]FAZA uptake in the carcinomas. Invasive pO₂ probe measurements yielded enhanced intra-tumoral pO₂ values in air- and oxygen-breathing ketamine/xylazine-anesthetized mice compared to isoflurane-anesthetized mice (KX-air: 1.01 mmHg, IF-air: 0.45 mmHg; KX-O₂ 9.73 mmHg, IF-O₂: 6.25 mmHg). Muscle oxygenation was significantly higher in air-breathing isoflurane-anesthetized (56.9 mmHg) than in ketamine/xylazine-anesthetized mice (33.8 mmHg, p = 0.0003).

Conclusion

[¹⁸F]FAZA tumor uptake was highest in ketamine/xylazine-anesthetized mice regardless of whether the mice breathed air or oxygen. The generally lower [¹⁸F]FAZA whole-body uptake in isoflurane-anesthetized mice could be due to the higher muscle pO₂-values in these mice compared to ketamine/xylazine-anesthetized mice. When performing preclinical in vivo hypoxia PET studies, oxygen should be avoided, and ketamine/xylazine-anesthesia might alleviate the identification of tumor hypoxia areals.     

The 2012/2013 ABRF Proteomic Research Group Study: Assessing Longitudinal Intralaboratory Variability in Routine Peptide Liquid Chromatography Tandem Mass Spectrometry Analyses*

Questions concerning longitudinal data quality and reproducibility of proteomic laboratories spurred the Protein Research Group of the Association of Biomolecular Resource Facilities (ABRF-PRG) to design a study to systematically assess the reproducibility of proteomic laboratories over an extended period of time. Developed as an open study, initially 64 participants were recruited from the broader mass spectrometry community to analyze provided aliquots of a six bovine protein tryptic digest mixture every month for a period of nine months. Data were uploaded to a central repository, and the operators answered an accompanying survey. Ultimately, 45 laboratories submitted a minimum of eight LC-MSMS raw data files collected in data-dependent acquisition (DDA) mode. No standard operating procedures were enforced; rather the participants were encouraged to analyze the samples according to usual practices in the laboratory. Unlike previous studies, this investigation was not designed to compare laboratories or instrument configuration, but rather to assess the temporal intralaboratory reproducibility. The outcome of the study was reassuring with 80% of the participating laboratories performing analyses at a medium to high level of reproducibility and quality over the 9-month period. For the groups that had one or more outlying experiments, the major contributing factor that correlated to the survey data was the performance of preventative maintenance prior to the LC-MSMS analyses. Thus, the Protein Research Group of the Association of Biomolecular Resource Facilities recommends that laboratories closely scrutinize the quality control data following such events. Additionally, improved quality control recording is imperative. This longitudinal study provides evidence that mass spectrometry-based proteomics is reproducible. When quality control measures are strictly adhered to, such reproducibility is comparable among many disparate groups. Data from the study are available via ProteomeXchange under the accession code PXD002114.The broad-reaching use and application of mass spectrometry-based proteomics in the international research community continues to exponentially grow and expand. As the technology has developed and practitioners have become skilled in performing complex workflows, the community has not only gained interest in assessing data across laboratories but also in maintaining consistent quality control within a laboratory. Koecher et al. raised the issue of quality control measures and how this aspect of mass spectrometry-based proteomics is generally neglected in scientific publications (1). Fortunately, studies characterizing the stability of liquid chromatography-tandem MS (LC-MSMS)¹ quality control performance among numerous laboratories are emerging. The relationship between sample preparation schemes, data acquisition and reduction strategies, and bioinformatic analyses have been comprehensively reviewed by Tabb (2).Several studies exist where intra- and interlaboratory reproducibility between multiple sites has been assessed under different settings. Perhaps the most systematic and detailed of these investigations are from the Human Proteome Organization (HuPO) test sample working group (3); the National Cancer Institute Clinical Proteomic Tumor Analysis Consortium (NCI CPTAC) (4); and the ProteoRed Consortium (5, 6). The HuPO group utilized an equimolar mixture of 20 highly purified recombinant human proteins (5 pmol per protein) distributed to 27 different laboratories and analyzed without constraint according to optimized LCMS and database search protocols from each of the laboratories (3). The study was not an assessment of instrument performance for highly sensitive detection of proteins, as all participating laboratories had acquired raw data of sufficient quality to identify all 20 proteins (and a specific subset of tryptic peptides). The study revealed, however, that discrepancies in peptide identification and protein assignment were the result of differences in data analysis strategies rather than data collection.The NCI CPTAC group used a standardized Saccharomyces cerevisiae proteome digest that was analyzed on ion-trap-based LCMS platforms in five independent laboratories according to both an established standard operating procedure (SOP) and with no SOP constraint (4). All data analysis was centralized, and thus, any observed variations were entirely because of the LCMS platform. By applying the performance metrics developed by Rudnick et al. (7), several key points emerged: (1) as expected, intralaboratory variation was less than interlaboratory variation; and (2) overall, the interlaboratory variation in peptide identifications and some of the other performance metrics were comparable between instruments, although there were large differences in the average values for some metrics (e.g. MS1 signal intensity, dynamic sampling).The ProteoRed Consortium initiated the ProteoRed Multicenter Experiment for Quality Control (PMEQC) (5, 6). This longitudinal QC multicenter study involved 12 institutes, and was designed to assess: (1) intralaboratory repeatability of LC-MSMS proteomic data; (2) interlaboratory reproducibility; and (3) reproducibility across multiple instrument platforms. Participants received samples of undigested or tryptically digested yeast proteins and were requested to follow strict analytical guidelines. Data analysis was centralized and performed under standard procedures using a common workflow. The study revealed that the overall performance with respect to metrics such as reproducibility, sensitivity, dynamic range etc. was directly related to the degree of operator expertise, and less dependent on instrumentation.Several studies not specifically focused on quality control have also yielded insight into proteomic reproducibility. The HuPO plasma proteome project (HuPO PPP) distributed 20 human samples (five serum plus 3 × 5 plasma samples treated with three different anticoagulants) to 35 laboratories spanning 13 countries (8). The purpose of this large-scale study was not to assess reproducibility per se, but rather to generate the largest and most comprehensive data set on the protein composition of human plasma/serum. On a smaller scale, the ISB standard 18 protein mixture (purified proteins from cow, horse, rabbit, chicken, E. coli, and B. licheniformus) was also assessed between laboratories on eight different LCMS platforms (9). These data reside in a comprehensive, multiplatform database as a resource for the proteomic community. Additional interlaboratory assessments have consisted of multiple reaction monitoring-based measurements of peptides/proteins in plasma (10, 11) and protein–protein interactions at both the biochemical and proteomic level (12).For team leaders/directors of proteomic laboratories and any researcher collaborating with such groups, major questions that may arise concerning data consistency are: how well are quality controls being implemented in the daily operations? Do the quality control measures effectively support data reproducibility? To address this, the Protein Research Group of the Association of Biomolecular Resource Facilities (ABRF-PRG) designed a study whereby LC-MSMS data obtained from the analysis of a commercially available bovine protein mixture predigested with trypsin were collected at routine intervals over a period of 9 months. Raw MS data files from a total of 64 participating laboratories were accumulated, and HPLC and MS performance were evaluated through QC metrics (13). The main impetus of the study was to recognize key sources of variability in HPLC and MS analyses under extended and routine operating conditions for each laboratory and to catalog the state of quality control in a diverse set of proteomic laboratories.No standard operating protocol was imposed on the participants; instead, contributors were encouraged to employ the methods that were typically applied in individual laboratories. Optimization of instrument methods on the provided sample was discouraged. A survey was conducted with each sample submission to catalog individual laboratory practices, instrument configurations, acquisition settings, including routine and nonroutine maintenance procedures. Unlike previous investigations where emphasis was placed on the preparation, distribution, and evaluation of protein standards to appraise and/or standardize LCMS platforms between laboratories, the key interest in this study was purely to determine the intralaboratory performance, reproducibility, and consistency of participating laboratories over an extended period of time.The rapidly expanding number of proteomic laboratories have incorporated divergent HPLC systems, mass spectrometers, solvent systems, columns etc. As a result, analyzing data from a large number of laboratories necessitates tools that can accommodate data from a broad range of platforms. For example, to expect a small laboratory with a decade-old three-dimensional ion trap mass spectrometer to achieve the same sensitivity as a laboratory with a high-resolution hybrid instrument would be unfair. Correspondingly, the data analysis needs to include axes beyond simple peptide-level sensitivity. Nevertheless, the laboratory with the older instrumentation may be consistently better at maximizing performance from the chosen instrument platform compared with a laboratory with the latest high-end equipment.The focus of this study was to estimate the degree of variability in intralaboratory performance over a 9-month period. This goal was achieved using quality metrics that are applicable to most LC-MSMS workflows. The inclusion of data from many laboratories will enable the proteomic community to determine the current state of quality control within a typical laboratory. The survey data enabled the mapping of some alterations in instrument performance to documented laboratory events, e.g. mass spectrometer calibration. The study was designed neither to compare one laboratory with another, nor to discriminate between classes of instrumentation.Questions of data quality and performance in the proteomic community are appropriately aligned with the heightened awareness of a perceived lack of reproducibility of scientific findings in general (1). This community has endeavored to provide tools to assess proteomic data quality, and this study provides additional insight into the application of such tools and the quality of data within respective laboratories.     

Suppression of a Natural Killer Cell Response by Simian Immunodeficiency Virus Peptides

Natural killer (NK) cell responses in primates are regulated in part through interactions between two highly polymorphic molecules, the killer-cell immunoglobulin-like receptors (KIRs) on NK cells and their major histocompatibility complex (MHC) class I ligands on target cells. We previously reported that the binding of a common MHC class I molecule in the rhesus macaque, Mamu-A1*002, to the inhibitory receptor Mamu-KIR3DL05 is stabilized by certain simian immunodeficiency virus (SIV) peptides, but not by others. Here we investigated the functional implications of these interactions by testing SIV peptides bound by Mamu-A1*002 for the ability to modulate Mamu-KIR3DL05⁺ NK cell responses. Twenty-eight of 75 SIV peptides bound by Mamu-A1*002 suppressed the cytolytic activity of primary Mamu-KIR3DL05⁺ NK cells, including three immunodominant CD8⁺ T cell epitopes previously shown to stabilize Mamu-A1*002 tetramer binding to Mamu-KIR3DL05. Substitutions at C-terminal positions changed inhibitory peptides into disinhibitory peptides, and vice versa, without altering binding to Mamu-A1*002. The functional effects of these peptide variants on NK cell responses also corresponded to their effects on Mamu-A1*002 tetramer binding to Mamu-KIR3DL05. In assays with mixtures of inhibitory and disinhibitory peptides, low concentrations of inhibitory peptides dominated to suppress NK cell responses. Consistent with the inhibition of Mamu-KIR3DL05⁺ NK cells by viral epitopes presented by Mamu-A1*002, SIV replication was significantly higher in Mamu-A1*002⁺ CD4⁺ lymphocytes co-cultured with Mamu-KIR3DL05⁺ NK cells than with Mamu-KIR3DL05^- NK cells. These results demonstrate that viral peptides can differentially affect NK cell responses by modulating MHC class I interactions with inhibitory KIRs, and provide a mechanism by which immunodeficiency viruses may evade NK cell responses.     

Correlation of Perfusion MRI and 18F-FDG PET Imaging Biomarkers for Monitoring Regorafenib Therapy in Experimental Colon Carcinomas with Immunohistochemical Validation

ObjectivesTo investigate a multimodal, multiparametric perfusion MRI / ¹⁸F-fluoro-deoxyglucose-(¹⁸F-FDG)-PET imaging protocol for monitoring regorafenib therapy effects on experimental colorectal adenocarcinomas in rats with immunohistochemical validation.ResultsRegorafenib significantly (p<0.01) suppressed PF (81.1±7.5 to 50.6±16.0 mL/100mL/min), PV (12.1±3.6 to 7.5±1.6%) and PS (13.6±3.2 to 7.9±2.3 mL/100mL/min) as well as TTB (3.4±0.6 to 1.9±1.1) between baseline and day 7. Immunohistochemistry revealed significantly (p<0.03) lower tumor microvascular density (CD-31, 7.0±2.4 vs. 16.1±5.9) and tumor cell proliferation (Ki-67, 434.0 ± 62.9 vs. 663.0 ± 98.3) in the therapy group. Perfusion MRI parameters ΔPF, ΔPV and ΔPS showed strong and significant (r = 0.67-0.78; p<0.01) correlations to the PET parameter ΔTTB and significant correlations (r = 0.57-0.67; p<0.03) to immunohistochemical Ki-67 as well as to CD-31-stainings (r = 0.49-0.55; p<0.05).ConclusionsA multimodal, multiparametric perfusion MRI/PET imaging protocol allowed for non-invasive monitoring of regorafenib therapy effects on experimental colorectal adenocarcinomas in vivo with significant correlations between perfusion MRI parameters and ¹⁸F-FDG-PET validated by immunohistochemistry.     

Technical Note: Calcium and carbon stable isotope ratios as paleodietary indicators

Calcium stable isotope ratios are hypothesized to vary as a function of trophic level. This premise raises the possibility of using calcium stable isotope ratios to study the dietary behaviors of fossil taxa and to test competing hypotheses on the adaptive origins of euprimates. To explore this concept, we measured the stable isotope composition of contemporary mammals in northern Borneo and northwestern Costa Rica, two communities with functional or phylogenetic relevance to primate origins. We found that bone collagen δ¹³C and δ¹⁵N values could differentiate trophic levels in each assemblage, a result that justifies the use of these systems to test the predicted inverse relationship between bioapatite δ¹³C and δ⁴⁴Ca values. As expected, taxonomic carnivores (felids) showed a combination of high δ¹³C and low δ⁴⁴Ca values; however, the δ⁴⁴Ca values of other faunivores were indistinguishable from those of primary consumers. We suggest that the trophic insensitivity of most bioapatite δ⁴⁴Ca values is attributable to the negligible calcium content of arthropod prey. Although the present results are inconclusive, the tandem analysis of δ⁴⁴Ca and δ¹³C values in fossils continues to hold promise for informing paleodietary studies and we highlight this potential by drawing attention to the stable isotope composition of the Early Eocene primate Cantius. Am J Phys Anthropol 154:633–643, 2014. © 2014 Wiley Periodicals, Inc.     

Kinetochore Biorientation in Saccharomyces cerevisiae Requires a Tightly Folded Conformation of the Ndc80 Complex

Accurate transmission of genetic material relies on the coupling of chromosomes to spindle microtubules by kinetochores. These linkages are regulated by the conserved Aurora B/Ipl1 kinase to ensure that sister chromatids are properly attached to spindle microtubules. Kinetochore–microtubule attachments require the essential Ndc80 complex, which contains two globular ends linked by large coiled-coil domains. In this study, we isolated a novel ndc80 mutant in Saccharomyces cerevisiae that contains mutations in the coiled-coil domain. This ndc80 mutant accumulates erroneous kinetochore–microtubule attachments, resulting in misalignment of kinetochores on the mitotic spindle. Genetic analyses with suppressors of the ndc80 mutant and in vitro cross-linking experiments suggest that the kinetochore misalignment in vivo stems from a defect in the ability of the Ndc80 complex to stably fold at a hinge in the coiled coil. Previous studies proposed that the Ndc80 complex can exist in multiple conformations: elongated during metaphase and bent during anaphase. However, the distinct functions of individual conformations in vivo are unknown. Here, our analysis revealed a tightly folded conformation of the Ndc80 complex that is likely required early in mitosis. This conformation is mediated by a direct, intracomplex interaction and involves a greater degree of folding than the bent form of the complex at anaphase. Furthermore, our results suggest that this conformation is functionally important in vivo for efficient error correction by Aurora B/Ipl1 and, consequently, to ensure proper kinetochore alignment early in mitosis.