Presence Of Subpopulations Research Articles

Arctic char Salvelinus alpinus movement dynamics relative to ice breakup in a high Arctic embayment

Arctic waters are warming rapidly due to climate change, which is altering the timing of seasonal sea ice dynamics. Summer ice breakup provides a critical productivity bloom that consumers depend on. Arctic char Salvelinus alpinus undertake seasonal migrations to marine waters presumably to exploit this prey pulse. However, specific migratory timing and residency of char relative to breakup is largely unknown. To investigate char movement, individuals were tagged with acoustic transmitters in 2017 (n = 58) and 2018 (n = 52) within Tremblay Sound, Nunavut, Canada, resulting in 625482 detections. Generalized linear models were used to relate multiple movement metrics to ice conditions as well as biological and temporal factors. Char exhibited multiple migratory patterns (chronologies), by which fish either migrated to/from rivers within Tremblay Sound (n = 67), rivers in external systems (n = 35), or a combination of both (n = 8). Fish that migrated exclusively from external systems were longer (~14 cm) than fish exhibiting other chronologies. Migration timing was related to chronology and preempted the specific ice-off date. Fish migrating from rivers within Tremblay Sound entered the marine environment ~7.5 d before the start of ice-off, while external migrants entered ~21 d after ice-off start. Differing migratory chronologies highlight the complexity of char migrations and may indicate the presence of subpopulations or potential ontogenetic shifts in migration behavior. Our study underscores the importance of the brief ice-free season, with potential implications for climate change resilience and management of this important resource in a changing Arctic.

Abstract 2105: Single cell RNA sequencing reveals multiple adaptive resistance mechanisms to regorafenib in colon cancer

Abstract Relapses in cancer therapy occur due to the presence of sub-populations that exhibit intrinsic or acquired resistance to treatment. Acquired resistance can arise via non-mutational adaptive mechanisms in response to a perturbation. Identification of these heterogenous mechanisms mediated via stochastic changes in cellular signaling is challenging using traditional bulk methods that average signals at a cellular population level. We harnessed the power of single-cell RNA sequencing (scRNA-seq) to identify the adaptive resistance mechanisms to regorafenib, a clinically approved multi-tyrosine kinase inhibitor, in a colon cancer model. HCT116 cells were treated with an IC50 dose of regorafenib or vehicle control for 1 hour, 3 days, 7 days or 12 days and subjected to scRNA-seq using the 10X Genomics platform. Cells were sequenced at an average depth of 50000 reads/cell using Illumina sequencing. We controlled for batch effects by incorporating independent biological replicates and multiplexing using cell “hashing” with barcoded antibodies (BD Biosciences). Graph-based clustering, differential expression analysis and cell cycle phase assignment was performed using the Seurat algorithm. Proliferation was assessed using PI staining for cell cycle status, CellTiter Blue viability assay and clonogenic assays. We analyzed a total of 19,879 single cells following a time-course of regorafenib or vehicle treatment. Regorafenib treatment at one hour resulted in no significant transcriptional changes. At all further time points, we could confirm the down-regulation of MAPK signaling, reflecting the mechanism of action of regorafenib. At 72 hours, we detected sub-populations with varying activation levels of ER stress response and increased lipid metabolism, together with upregulation of transcription factors SOX4 and ID3. These features continued to be detected till 7 and 12 days after treatment. By 12 days, we observed upregulation of keratins 7, 8, 10, 18, 19 together with markers of G2M/S phase progression. Survival of clonal cell populations at 12 days was confirmed by crystal violet staining. Small molecule-based inhibition of ER stress (azoramide) and lipid synthesis (fatostain) was synergistic with regorafenib at day 3 and successfully inhibited the emergence of these resistant clones by day 12. Our results reveal multiple signaling events that lead to a resistance phenotype characterized by increased proliferation and keratin upregulation that can be counteracted by combination therapies. For future studies we will continue to dissect the molecular networks underlying these events by gene silencing and protein over-expression studies. Citation Format: Anuja Sathe, Billy T. Lau, Sue Grimes, Stephanie Greer, Hanlee Ji. Single cell RNA sequencing reveals multiple adaptive resistance mechanisms to regorafenib in colon cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2105.

Utility of effect size to define populations with durable clinical outcomes across trials of metastatic colorectal cancer.

e15104 Background: ASCO defined clinically meaningful trial endpoints in colorectal cancer (CRC) as absolute overall survival (OS) of 3-5 months and OS hazard ratio (HR) ≤0.67. Few tools predict clinically distinct subgroups from heterogeneous populations. Effect size (Glass’s delta) calculates the absolute difference in clinical outcomes relative to the standard deviation of control group. We hypothesized that effect sizes ≥1 would be useful in predicting subgroup populations of clinical significance for trials with an indeterminate HR. Methods: Prospective phase II-III trials in metastatic CRC were queried from clinicaltrials.gov and cataloged by clinical outcomes of PFS and OS. Effect size was back-calculated from trials reporting 95% confidence intervals and compared with absolute difference in clinical outcome and hazard ratio. Results: 46 prospective trials were evaluable including 49% with biomarker selection, 57% in the first-line setting, and more commonly studied with targeted therapy over chemotherapy (70 v. 30%; p < 0.001). EGFR inhibitors were studied in 34% and VEGF inhibitors in 23%. Both effect size and HR correlated similarly with PFS (correlation coefficients, R = 0.65 vs. 0.76) and OS (R = 0.83 vs. 0.84) across these studies. Of studies with an indeterminate HR (0.69-0.86; n = 24), 10 studies had a significant effect size (Glass’s delta > 1), indicating the potential for sub-populations with improved clinical benefit within the experimental treatment group. These included 4 trials with planned biomarker analyses. Remaining trials were enriched for studies investigating anti-angiogenic therapies (83% vs. 23%, p = 0.001), including bevacizumab, aflibercept, regorafenib, and ramucirumab. Conclusions: Effect size holds potential as a measure to indicate the presence of subpopulations of patients benefiting in clinical trials. Those trials with a significant effect size despite an indeterminate HR should be examined closely for such populations. In CRC, effect size indicates the potential of a subgroup of patients who benefit significantly from anti-angiogenic agents. Further investigations are needed to validate effect size as a tool to delineate improved clinical outcomes from heterogeneous populations.

Abstract 4584: High-definition single-cell analysis of liquid and solid biopsies reveals heterogeneity of circulating tumor cells in metastatic colorectal cancer

Abstract Treatment of cancer patients is transitioning to personalized strategies using targeted drugs with the required companion biomarkers. Many current biomarkers are tissue-based, requiring a solid biopsy and limiting sampling frequency. Given this, there is significant interest in determining whether noninvasive, blood-based liquid biopsies, utilizing measurements of factors that exist in the blood such as circulating tumor cells (CTCs), reflect solid tumors and can serve as substitutes for solid tumor biopsies. Colorectal cancer with hepatic metastases (mCRC) is one of very few scenarios where metastatic patients routinely undergo surgical resection, providing an opportunity for accessible tissue and simultaneous blood sampling. We collected blood and tumor touch preparations from patients undergoing resection of CRC with the purpose to determine the correlation of solid biopsies and the liquid phase of solid tumors in the mCRC setting. Samples were processed using the previously described HD-SCA (High-Definition Single-Cell Analysis) workflow for high-resolution imaging and characterization of tumor cells at a single-cell level. Briefly, tumor cells were identified by presence of nuclear stain, pan-cytokeratin, and absence of CD45. The colon-specific marker CDX2 was included as a fourth color for further characterization. Subsets of cells were subjected to copy number variation (CNV) profiling. The comparison of pre- and post-surgery blood samples showed that the concentration of CTCs decreased significantly after surgery. The concentration was markedly higher in patients with synchronous disease undergoing simultaneous resection of the primary tumor, indicating that the presence of CTCs is related to tumor burden. Principal component analysis demonstrated that the CTCs were more similar to the metastatic cells than to the primary colon cells. While protein levels varied between the liquid and solid biopsy cell populations, morphologic parameters including, e.g., cell and nuclear area and roundness, were similar in the two. The intrapatient analysis showed heterogeneous CTC populations, in general with a small subset of cells bearing characteristics more similar to the solid tumor cells than the bulk of CTCs. The presence of subpopulations was further indicated by the CNV analysis, which showed that only a minority of CTCs had genomic rearrangements and that, in contrast to the solid biopsy cells, the rearranged cells were largely nonclonal. Based on the high-content HD-SCA data, the CTCs were further grouped into different subtypes. In conclusion, we introduce an approach for high-content comparison of liquid and solid biopsies at a single-cell level. We show that the concentration of CTCs is related to tumor burden, and present a subclassification of rare circulating cells in mCRC. Citation Format: Anna S. Gerdtsson, Jana-Aletta Thiele, Randolph Schaffer, Kelly Bethel, Steven Curley, Heinz-Josef Lenz, Diana L. Hanna, Jorge Nieva, Anand Kolatkar, Mariam Rodriguez Lee, James Hicks, Peter Kuhn. High-definition single-cell analysis of liquid and solid biopsies reveals heterogeneity of circulating tumor cells in metastatic colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4584.

Geographic substructure in craniometric estimates of admixture for contemporary American populations.

This study investigates heterogeneity in craniometrically-derived estimates of admixture in order to reveal population substructure in a sample of Black, White, Hispanic, and Native American individuals from the FDB. It reports evidence of spatial trends in population-specific patterns of admixture and contextualizes its results in terms of demographic diversity in the United States. The FDB was sampled to capture the population variation within forensic casework, skeletal collections, and the U.S. population-at-large. Individuals were selected for the availability of population identifier, sex, and geographic information. Variation in inferred admixture proportions was evaluated, per population and by sex, for evidence of geographic substructure. Comparative data was sourced from the U.S. Census. This analysis identifies significant associations between the estimated Black, Native American and White component memberships and place of birth and recovery. The sampled populations differ significantly in admixture proportions, in a systematic way. Admixture patterns vary in accordance with the densities and relative proportions of the U.S. census populations. There is considerable variation in admixture estimates, not just between, but notably within, all four of the populations. This substructure can be explained by differences in geography, including regions, divisions, and states. This article's findings agree with census trends and speak broadly to admixture dynamics and ancestral diversity among contemporary Americans. They are also specifically relevant to those cases in the FDB. The presence of subpopulations has implications for cranial research, forensic identification, and studies of biological variation in the United States.

Production of Homogeneous Cultured Human Corneal Endothelial Cells Indispensable for Innovative Cell Therapy.

Cultured human corneal endothelial cells (cHCECs) are anticipated to become an alternative to donor corneas for the treatment of corneal endothelial dysfunction. The aim of this study was to establish proper culture protocols to successfully obtain a reproducibly homogeneous subpopulation (SP) with matured cHCEC functions and devoid of cell-state transition suitable for cell-injection therapy. The presence of SPs in cHCECs was investigated in terms of surface cluster-of-differentiation (CD) marker expression level by flow cytometry, as combined analysis of CD markers can definitively specify the SP (effector cells) conceivably the most suitable for cell therapy among diverse SPs. The culture conditions were evaluated by flow cytometry in terms of the proportion (E-ratio) of effector cells designated by CD markers. Flow cytometry analysis identifying CD44-CD166+CD133-CD105-CD24-CD26- effector cells proved convenient and reliable for standardizing the culture procedures. To ascertain the reproducible production of cHCECs with E-ratios of more than 90% and with no karyotype abnormality, the preferred donor age was younger than 29 years. The continuous presence of Rho-associated protein kinase (ROCK)-inhibitor Y-27632 greatly increased the E-ratios, whereas the presence of transforming growth factor-beta/Smad-inhibitor SB431542 greatly reduced the number of recovered cHCECs. The seeding cell density during culture passages proved vital for maintaining a high E-ratio for extended passages. The continuous presence of ROCK-inhibitor Y-27632 throughout the cultures greatly improved the E-ratio. Our findings elucidated the culture conditions needed to obtain reproducible cHCECs with high E-ratios, thus ensuring homogeneous cHCECs with matured functions for the treatment of corneal endothelial dysfunction.

Verbal response time and duration indices of deception in humans interviewed by a computer-generated agent

To examine the relationship between verbal response time, response duration, and deception during an interview with a computer-generated agent, we developed a model using logistic regression conducted on a training group (n=90) and cross-validated the model on an additional 127 participants who either did or did not engage in a simulated crime at a mock security checkpoint. Verbal responses during the interviews required simple “yes” or “no” utterances, which examinees were instructed to produce “promptly” but not in a speeded manner. The results showed that, overall, 75 of 127 (59.1%) participants in the cross-validation group were correctly classified (p<.05). This result was due to the ability to correctly classify nondeceptive participants (specificity), and we interpreted this finding as being due to the presence of subpopulations of deceptive participants. Group analyses also revealed that response durations to mock crime-relevant questions were shorter when participants believed that a human was controlling the interview (p<.05), and that relative changes in response durations among different types of questions during the interview were related to deceptive status. The response duration shortening observed in the present study was attributed to variations in social dominance, motivational systems, or some combination of these factors.

Improving Psychological Measurement: Does It Make a Difference? A Comment on Nesselroade and Molenaar (2016)

ABSTRACTNesselroade and Molenaar advocate the use of an idiographic filter approach. This is a fixed-effects approach, which may limit the number of individuals that can be simultaneously modeled, and it is not clear how to model the presence of subpopulations. Most important, Nesselroade and Molenaar's proposal appears to be best suited for modeling long time series on a few variables for a few individuals. Long time series are not common in psychological applications. Can it be applied to the usual longitudinal data we face? These are characterized by short time series (four to five points in time), hundreds of individuals, and dozens of variables. If so, what do we gain? Applied settings most often involve between-individual decisions. I conjecture that their approach will not outperform common, simpler, methods. However, when intraindividual decisions are involved, their approach may have an edge.

Surviving the heat: heterogeneity of response in Saccharomyces cerevisiae provides insight into thermal damage to the membrane.

Environmental heat stress impacts on the physiology and viability of microbial cells with concomitant implications for microbial activity and diversity. Previously, it has been demonstrated that gradual heating of Saccharomyces cerevisiae induces a degree of thermal resistance, whereas a heat shock results in a high level of cell death. Here, we show that the impact of exogenous nutrients on acquisition of thermal resistance differs between strains.Using single-cell methods, we demonstrate the extent of heterogeneity of the heat-stress response within populations of yeast cells and the presence of subpopulations that are reversibly damaged by heat stress. Such cells represent potential for recovery of entire populations once stresses are removed. The results show that plasma membrane permeability and potential are key factors involved in cell survival, but thermal resistance is not related to homeoviscous adaptation of the plasma membrane. These results have implications for growth and regrowth of populations experiencing environmental heat stress and our understanding of impacts at the level of the single cell. Given the important role of microbes in biofuel production and bioremediation, a thorough understanding of the impact of stress responses of populations and individuals is highly desirable.

Subpopulations in aMPV vaccines are unlikely to be the only cause of reversion to virulence

Avian metapneumovirus (aMPV) infects respiratory and reproductive tracts of domestic poultry, often involving secondary infections, and leads to serious economic losses in most parts of the world. While in general disease is effectively controlled by live vaccines, reversion to virulence of those vaccines has been demonstrated on several occasions. Consensus sequence mutations involved in the process have been identified in more than one instance. In one previous subtype A aMPV candidate vaccine study, small subpopulations were implicated. In the current study, the presence of subpopulations in a subtype B vaccine was investigated by deep sequencing. Of the 19 positions where vaccine (strain VCO3/50) and progenitor (strain VCO3/60616) consensus sequences differed, subpopulations were found to have sequence matching progenitor sequence in 4 positions. However none of these mutations occurred in a virulent revertant of that vaccine, thereby demonstrating that the majority progenitor virus population had not survived the attenuation process, hence was not obviously involved in any return to virulence. However within the vaccine, a single nucleotide variation was found which agreed with consensus sequence of a derived virulent revertant virus, hence this and other undetected, potentially virulent subpopulations, can be involved in reversion. Much deeper sequencing of progenitor, vaccine and revertant may clarify whether problematic virulent subpopulations are present and therefore whether these need to be routinely removed during aMPV vaccine preparation prior to registration and release.

Kinematics and subpopulations' structure definition of blue fox (Alopex lagopus) sperm motility using the ISAS® V1 CASA system.

Over recent years, technological advances have brought innovation in assisted reproduction to the agriculture. Fox species are of great economical interest in some countries, but their semen characteristics have not been studied enough. To advance the knowledge of function of fox spermatozoa, five samples were obtained by masturbation, in the breeding season. Kinetic analysis was performed using ISAS® v1 system. Usual kinematic parameters (VCL, VSL, VAP, LIN, STR, WOB, ALH and BCF) were considered. To establish the standardization for the analysis of samples, the minimum number of cells to analyse and the minimum number of fields to capture were defined. In the second step, the presence of subpopulations in blue fox semen was analysed. The minimum number of cells to test was 30, because kinematic parameters remained constant along the groups of analysis. Also, the effectiveness of ISAS® D4C20 counting chamber was studied, showing that the first five squares presented equivalent results, while in the squares six and seven, the kinematic parameters showed a reduction in all of them, but not in the concentration or motility percentage. Kinematic variables were grouped into two principal components (PC). A linear movement characterized PC1, while PC2 showed an oscillatory movement. Three subpopulations were found, varying in structure among different animals.

Moroccan Leishmania infantum: Genetic Diversity and Population Structure as Revealed by Multi-Locus Microsatellite Typing

Leishmania infantum causes Visceral and cutaneous leishmaniasis in northern Morocco. It predominantly affects children under 5 years with incidence of 150 cases/year. Genetic variability and population structure have been investigated for 33 strains isolated from infected dogs and humans in Morocco. A multilocus microsatellite typing (MLMT) approach was used in which a MLMtype based on size variation in 14 independent microsatellite markers was compiled for each strain. MLMT profiles of 10 Tunisian, 10 Algerian and 21 European strains which belonged to zymodeme MON-1 and non-MON-1 according to multilocus enzyme electrophoresis (MLEE) were included for comparison. A Bayesian model-based approach and phylogenetic analysis inferred two L.infantum sub-populations; Sub-population A consists of 13 Moroccan strains grouped with all European strains of MON-1 type; and sub-population B consists of 15 Moroccan strains grouped with the Tunisian and Algerian MON-1 strains. Theses sub-populations were significantly different from each other and from the Tunisian, Algerian and European non MON-1 strains which constructed one separate population. The presence of these two sub-populations co-existing in Moroccan endemics suggests multiple introduction of L. infantum from/to Morocco; (1) Introduction from/to the neighboring North African countries, (2) Introduction from/to the Europe. These scenarios are supported by the presence of sub-population B and sub-population A respectively. Gene flow was noticed between sub-populations A and B. Five strains showed mixed A/B genotypes indicating possible recombination between the two populations. MLMT has proven to be a powerful tool for eco-epidemiological and population genetic investigations of Leishmania.

Sensitivity of Bacillus weihenstephanensis to acidic changes of the medium is not dependant on physiological state

This study aims to quantify the effect of salt and acid preliminary exposure on acid resistance of vegetative cells of Bacillus weihenstephanensis. The psychrotolerant strain KBAB4 was cultured until the mid-exponentially phase (i) in BHI, (ii) in BHI supplemented with 2.5% salt or (iii) in BHI acidified at pH 5.5 with HCl. The growing cells were subsequently inactivated in lethal acid conditions ranging from 4.45 to 4.70. Based on statistical criteria, a primary mixed-Weibull model was used to fit the acid inactivation kinetics. The acid resistance was enhanced for acid-adapted cells and decreased for salt-adapted cells. The secondary modelling of the bacterial resistance allowed the quantification of the change in pH leading to a ten folds variation of the bacterial resistance, i.e. cells sensitivity (zpH). This sensitivity was not significantly affected whatever the preliminary mild exposure and the presence of sub-populations with different acid resistances. These results highlighted that pre-incubation conditions influence bacterial acid resistance without affecting the sensitivity to acidic modifications, with a 10 fold reduction of Bacillus acid resistance observed for a reduction of 0.37 pH unit. Quantification of such adaptive stress response might be instrumental in quantitative risk assessment more particularly in food formulation, particularly for low-acid minimally processed foods.

Rubber Price Effect on Exchange Rate: A Bayesian Mixture Model Approach

Mixture model is a probabilistic model that denotes the presence of subpopulations within an overall population meanwhile finite mixture model is a mixture model with finite-dimensional. In this paper, finite mixture model is applied and the application of Bayesian method to fit finite mixture model is popular and these application is adopt in the present study in order to explore the relationship between rubber price and exchange rate for Malaysia, Thailand, Philippines and Indonesia. Exchange rate plays leading role for a country because it represents the development for that country. The changes of exchange rate influence the flows of investment either the import or export prices for a nation’s. While another data set that adopt is rubber price. Rubber is an economic commodity that prospers in tropical climate. It is an important raw material because the latex that extracted is the primary source of natural rubber that enables to produce many useful products such as tires, surgical gloves, industrial hoses, rubber sheeting, industrial form parts and others household rubber products. Results found that rubber price effect on the change of exchange rate for Malaysia, Thailand, Philippines and Indonesia.

Abstract P1-05-20: Fluorescent Hyaluronan Probes Distinguish Heterogeneous Breast Cancer Cell Subsets and Predict their Invasive Behavior

Abstract Background: Tumor heterogeneity is a determining factor in diagnosis and therapy of breast cancer (Bca). We investigated Bca cellular heterogeneity by a functional fluorescent hyaluronan (HA) probe, and monitored its binding characteristics in relation to the degree of Bca aggression. HA is a polysaccharide ligand for CD44 and RHAMM receptors, which are linked to cell plasticity and predict poor clinical outcome in Bca. Methods: Fluorescent HA probes, multiplexed profiling, and cell sorting strategies were developed and used to detect/capture tumor cell subpopulations with differences in HA binding (HA−/low vs. HAhigh) and HA receptors surface display. Whereas we concentrated on the most metastatic cell line, MDA-MB-231, we also monitored the presence of subpopulations in other Bca lines and showed heterogeneity in all. Results: The HA probe binding was highly heterogeneous, and the highest binding level was detected in highly invasive triple-negative basal subtypes such as MDA-MB-231. HA probes bound to the cell surface but also accumulated in cytoplasm and nucleus. Binding levels were dramatically reduced upon ‘reversion’ of highly malignant cells to a non-malignant phenotype in three-dimensional cultures, suggesting that the level of cellular binding to fluorescent HA probe provides a measure of malignant behavior. Comparison between HAhigh and HA−/low subpopulations revealed surprising differences in morphology, proliferation and invasion in culture, which were retained in two in vivo models. HAhigh subpopulations exhibited higher levels of invasion but surprisingly lower levels of proliferation compared to either unsorted parental cells or the HA−/low subpopulation. Conclusions: Querying HA binding in Bca lines reveals an unexpected heterogeneity with respect to tumor phenotype in vivo, morphology in 3D, invasion and proliferation. These results may aid in diagnosis and therapy of invasive Bca subpopulations and early sorting of cancer patients needing intensive chemotherapy. Keywords: Heterogeneity, 3D cultures, Hyaluronan, CD44, RHAMM/HMMR, Tumor phenotype, Breast cancer, Invasion. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P1-05-20.

Comparative rates of growth of the Port Jackson shark throughout its southern Australian range

Port Jackson sharks are distributed throughout southern Australia, with evidence suggesting that potential subpopulations exist. If subpopulations are evident, then phenotypic variation among groups should result in differences in life-history parameters. The present study tested for patterns of spatial variability of life-history parameters among regional Port Jackson shark populations. Rates of growth from Port Jackson sharks caught in the gulf waters of South Australia were calculated on the basis of counts of vertebral increments. Growth parameters were obtained by fitting the length-at-age data to von Bertalanffy and Gompertz growth functions. While the derived growth curves fit the length-at-age data well (r2 ranged from 0.87 to 0.91), parameters showed considerable differences between the two functions, with the von Bertalanffy function providing the more realistic estimates of growth (combined sexes: k = 0.081 year–1, L∞ = 1232 mm total length and t0 = –1.937 years). Life-history parameters for South Australian Port Jackson sharks were collated with the available data for the species, facilitating comparisons among regional populations. Growth curves among populations varied significantly; however, considerable overlap in the length ranges of size at birth and sizes at maturity among populations were evident. Overall, the data presented here do not provide definitive support for the presence of subpopulations across the distribution of the Port Jackson shark, suggesting that molecular analysis maybe required to directly test for structuring.

A GC–MS metabolic profiling study of plasma samples from mice on low- and high-fat diets

Metabolic profiling of biofluids, based on the quantitative analysis of the concentration profile of their free low molecular mass metabolites, has been playing increasing role employed as a means to gain understanding of the progression of metabolic disorders, including obesity. Chromatographic methods coupled with mass spectrometry have been established as a strategy for metabolic profiling. Among these, GC–MS, targeting mainly the primary metabolism intermediates, offers high sensitivity, good peak resolution and extensive databases. However, the derivatization step required for many involatile metabolites necessitates specific data validation, normalization and analysis protocols to ensure accurate and reproducible performance. In this study, the GC–MS metabolic profiles of plasma samples from mice maintained on 12- or 15-month long low (10 kcal%) or high (60 kcal%) fat diets were obtained. The profiles of the trimethylsilyl(TMS)-methoxime(MeOx) derivatives of the free polar metabolites were acquired through GC–(ion trap)MS, using [U– 13C]-glucose as the internal standard. After the application of a recently developed data correction and normalization/filtering protocol for GC–MS metabolomic datasets, the profiles of 48 out of the 77 detected metabolites were used in multivariate statistical analysis. Data mining suggested a decrease in the activity of the energy metabolism with age. In addition, the metabolic profiles indicated the presence of subpopulations with different physiology within the high- and low-fat diet mice, which correlated well with the difference in body weight among the animals and current knowledge about hyperglycemic conditions.

Thermostability of subpopulations of H2N3 influenza virus isolates from mallard ducks.

Maintenance of avian influenza virus in waterfowl populations requires that virions remain infectious while in the environment. Temperature has been shown to negatively correlate with persistence time, which is the duration for which virions are infectious. However, thermostability can vary between isolates regardless of subtype, and it is not known whether this variation occurs when host and geographic location of isolation are controlled. In this study, we analyzed the thermostabilities of 7 H2N3 viruses isolated from mallard ducks in Alberta, Canada. Virus samples were incubated at 37 degrees C and 55 degrees C, and infectivity titers were calculated at different time points. Based on the rate of infectivity inactivation at 37 degrees C, isolates could be grouped into either a thermosensitive or thermostable fraction for both egg- and MDCK-grown virus populations. Titers decreased more rapidly for isolates incubated at 55 degrees C, and this loss of infectivity occurred in a nonlinear, 2-step process, which is in contrast with the consensus on thermostability. This suggests that stock samples contain a mixture of subpopulations with different thermostabilities. The rate of decrease for the sensitive fraction was approximately 14 times higher than that for the stable fraction. The presence of subpopulations is further supported by selection experiments and plaque purification, both of which result in homogenous populations that exhibit linear decreases of infectivity titer. Therefore, variation of thermostability of influenza virus isolates begins at the level of the population. The presence of subpopulations with high thermostability suggests that avian viruses can persist in water longer than previously estimated, thus increasing the probability of transmission to susceptible hosts.

Abstract C8: Putative tumor-initiating cell populations in mantle cell lymphoma exhibit microenvironmentally influenced differences in stem cell-like and tumorigenic properties

Abstract The presence of subpopulations of cells possessing enhanced tumor formation capabilities has been described in several tissues. These cells have been termed tumor-initiating cells (TICs) or cancer stem cells and have been isolated using various techniques, including the side population (SP), the use of cell surface markers including CD133 and CD44 and aldehyde dehydrogenase activity. We have elected to evaluate the stem cell-like and tumor forming capabilities of SP and CD133+/CD44+ cells in Mantle Cell Lymphoma (MCL), an aggressive and relatively therapy unresponsive form of Non-Hodgkin's Lymphoma. All analyses were conducted in the presence of micro-environmental niche cells (murine stromal 5 cells) or extracellular matrix components (Matrigel) as these factors are known to play an integral role in processes associated with stem cell self-renewal and differentiation. SP cells and CD133+/CD44+ cells were present in Granta 519, an aggressive MCL cell line. The in-vitro technique of the Cobblestone Area Forming Cell (CAFC) assay was utilized to assess the self-renewal capabilities of SP and CD133+/CD44+ cells when the two cell types were cultured in the presence and absence of Matrigel, a mixture of extracellular matrix proteins derived from the Englebreth-Holm murine sarcoma. CAFC assays revealed enhanced self-renewal capabilities for Granta SP cells over non-SP cells, with the addition of Matrigel increasing the self-renewal frequency of SP and non-SP cells by 5.8 and 8.6 fold respectively. Conversely, Granta CD133+/CD44+ and CD133−/CD44− cells possessed similar self-renewal capabilities, with the addition of Matrigel having a negligible effect. When evaluated for chemotherapy resistance, the murine stromal 5 (MS5)-adherent CD133+/CD44+ cells and the non-adherent SP cells were more resistant to hydroxyurea while all MS5-adherent Granta cells were resistant to this agent. In-vivo analyses demonstrated tumor formation of Matrigel mixed SP and non-SP populations but not for Matrigel mixed CD133+/CD44+ or CD133−/CD44− cells. Although micro-metastatic tumors were not detected for SP and non-SP injected mice, flow cytometric analyses identified Granta cells, characterized by CD20 expression, in all murine tissues examined. IL-8, a proangiogenic cytokine implicated in tumor metastasis, may be involved in the metastatic spread of Granta cells as it was significantly present in the supernatants of Granta SP MS5 co-cultures, but was negligible or undetected when non-SP, CD133+/CD44+ and CD133−/CD44− cells were co-cultured with MS5 cells. These studies suggest that the SP phenotype may be a better marker for the isolation of stem-like cells in MCL. Moreover, the micro-environmental components found in Matrigel, rather than cell phenotype differences, appear to play a vital role in the initiation of tumor development by SP and non-SP cells. The tumor micro-environmental niche may therefore be an important target for development of novel therapeutic agents. Citation Information: Cancer Res 2009;69(23 Suppl):C8.

Twelve new polymorphic microsatellite loci and PCR multiplexing in the whitefly, Bemisia tabaci

Twelve new dinucleotide microsatellite loci of the whitefly, Bemisia tabaci, were obtained from enriched genomic libraries. Three polymerase chain reaction multiplex sets comprising three, five and four loci were optimized and characterized across 133 B. tabaci females from Israeli rearings and natural populations collected in four Mediterranean countries (Tunisia, France, Spain and Morocco). There were three to 24 alleles per locus and the observed heterozygosity was from 0.084 to 0.420. Deviation from Hardy-Weinberg equilibrium was detected at four loci associated with significant heterozygote deficiencies due to null alleles and presence of subpopulations that were mostly in the Tunisian sample. The 12 loci carried independent information.