Typical Endpoint Research Articles

Fortification of low-fat yogurt with melloco flour (Ullucus tuberosus): Physicochemical and rheological effects

Yogurt is a fermented milk product containing Lactic Acid Bacteria (BAL) (Lactobacillus bulgaricus and Streptococcus thermophilus). Although the application of additives is not mandatory, the use of gums or dairy extenders have been studied to establish the effect on the product. Among the additives most used in yogurt, production is focalized on natural or artificial sweeteners, stabilizing agents, flavors and colorants. Nowadays, consumers are looking for products that seem less artificial and more natural. In this sense, melloco, an Andean crop that, thanks to its mucilaginous content, could become an excellent additive that provides stability and viscosity. This study aimed to evaluate the effect of melloco flour (Ullucus tuberorus) yellow variety (INIAP-Quillu) on low-fat yogurt's quality properties. In this study, semi-skimmed milk (2% fat) was supplemented with 0.9% (w / v) of melloco flour, fermented and stored at 4°C for 20 days. Sensory attributes such as color, texture, taste, smell and overall acceptability were evaluated; the proximal analysis was performed using official methods of the Association of Official Analytical Chemists (AOAC). The physical and rheological properties and microbiological analysis were evaluated during 20 days of refrigerated storage. The use of melloco flour to enrich the reduced-fat yogurt improved acid production during fermentation. The average pH of the samples decreased from 4.8 to 4.18 after 20 days of storage. The color parameters did not differ significantly in all samples and remained constant during storage. The viscosity showed a rheological behavior of the pseudoplastic fluid. This study evidenced that the acid production in the yogurt enriched with melloco flour was higher during fermentation than the control sample, the typical end-point of pH (4.5) was reached by enriched yogurt in 5.5 hours compared to 7 hours for control. Sensory attributes also allowed establishing that the sample with 0.9% of flour presents the best overall acceptability. In general, the results suggest that melloco flour can be considered as good gelling and nutritional agent for the production of yogurt. Key words: enriched, fermentation, Melloco, microbial growth, yogurt, physicochemical properties, rheology, sensorial properties

Abstract 1301: Real-time single-cell drug response assay in metastatic breast cancer cell lines using quantitative phase imaging

Abstract Introduction: The ability for oncologists to predict a cancer's response to therapy is limited to a few biomarkers used for histologic diagnosis and targeted therapy. Often, in advanced and metastatic disease these biomarkers provide no alternative options for next step systemic treatments. There is a need in oncology for functional assays that can determine a tumor's response to a drug, regardless of its tissue of origin, previous treatments, or mutation status. Quantitative phase imaging (QPI) can measure changes in single cell mass in response to drug treatment in vitro and ex vivo. This platform offers advantages over other functional/metabolic assays in that it monitors changes in real-time and on a single cell basis, revealing heterogeneity in drug response. Methods: Here, we describe the validation of QPI for the measurement of breast cancer cell response to therapy versus CellTiterGlo (CTG), an endpoint ATP assay. We ran a series of 3-day drug response assays using QPI alongside CTG. We used a 96 well plate with a 6-point dose response between 1.6 nM and 20 μM for multiple cell lines spanning a range of receptor statuses (MCF7, MDA-MB-231, BT-474) with two controls in triplicate. We analyzed single-cell data to measure the heterogeneity of response and assessed how cell-to-cell heterogeneity is affected by dose. Our response data were fitted to a four-parameter Hill equation to compute the IC50 and depth of response. Results: We found that QPI can determine IC50s for effective treatments as validated by concordance to CTG. As measured by QPI, doxorubicin has a substantial depth of response, indicating cytotoxic effects. Doxorubicin data also show a tighter range of growth rates at high concentration than control, which implies low heterogeneity of response. As expected, ER positive MCF7 cells responded to hydroxy-tamoxifen. This response shows a similar reduction in heterogeneity to doxorubicin but with a reduced depth of response indicating a cytostatic effect. MDA-MB-231 response to palbociclib exhibits a wide range of growth rates, indicating an increase in heterogeneity as measured by QPI. Fluorouracil response shows no significant difference in heterogeneity from control. Conclusion: In summary, QPI is a useful tool for functional assays that can capture IC50, depth of response, and single-cell heterogeneity of response. In particular, this additional information about single-cell behavior and heterogeneity cannot be measured using a typical endpoint assay. Future work is needed to prove the clinical utility of functional assays from QPI. Citation Format: Tarek E. Moustafa, Edward R. Polanco, Andrew Butterfield, Sandra D. Scherer, Bryan E. Welm, Philip S. Bernard, Thomas A. Zangle. Real-time single-cell drug response assay in metastatic breast cancer cell lines using quantitative phase imaging [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1301.

Major Improvements in Robustness and Efficiency during the Screening of Novel Enzyme Effectors by the 3-Point Kinetics Assay.

The throughput level currently reached by automatic liquid handling and assay monitoring techniques is expected to facilitate the discovery of new modulators of enzyme activity. Judicious and dependable ways to interpret vast amounts of information are, however, required to effectively answer this challenge. Here, the 3-point method of kinetic analysis is proposed as a means to significantly increase the hit success rates and decrease the number of falsely identified compounds (false positives). In this post-Michaelis-Menten approach, each screened reaction is probed in three different occasions, none of which necessarily coincide with the initial period of constant velocity. Enzymology principles rather than subjective criteria are applied to identify unwanted outliers such as assay artifacts, and then to accurately distinguish true enzyme modulation effects from false positives. The exclusion and selection criteria are defined based on the 3-point reaction coordinates, whose relative positions along the time-courses may change from well to well or from plate to plate, if necessary. The robustness and efficiency of the new method is illustrated during a small drug repurposing screening of potential modulators of the deubiquinating activity of ataxin-3, a protein implicated in Machado-Joseph disease. Apparently, intractable Z factors are drastically enhanced after (1) eliminating spurious results, (2) improving the normalization method, and (3) increasing the assay resilience to systematic and random variability. Numerical simulations further demonstrate that the 3-point analysis is highly sensitive to specific, catalytic, and slow-onset modulation effects that are particularly difficult to detect by typical endpoint assays.

The Role of an IL-10/Hyaluronan Axis in Dermal Wound Healing.

Scar formation is the typical endpoint of postnatal dermal wound healing, which affects more than 100 million individuals annually. Not only do scars cause a functional burden by reducing the biomechanical strength of skin at the site of injury, but they also significantly increase healthcare costs and impose psychosocial challenges. Though the mechanisms that dictate how dermal wounds heal are still not completely understood, they are regulated by extracellular matrix (ECM) remodeling, neovascularization, and inflammatory responses. The cytokine interleukin (IL)-10 has emerged as a key mediator of the pro- to anti-inflammatory transition that counters collagen deposition in scarring. In parallel, the high molecular weight (HMW) glycosaminoglycan hyaluronan (HA) is present in the ECM and acts in concert with IL-10 to block pro-inflammatory signals and attenuate fibrotic responses. Notably, high concentrations of both IL-10 and HMW HA are produced in early gestational fetal skin, which heals scarlessly. Since fibroblasts are responsible for collagen deposition, it is critical to determine how the concerted actions of IL-10 and HA drive their function to potentially control fibrogenesis. Beyond their independent actions, an auto-regulatory IL-10/HA axis may exist to modulate the magnitude of CD4+ effector T lymphocyte activation and enhance T regulatory cell function in order to reduce scarring. This review underscores the pathophysiological impact of the IL-10/HA axis as a multifaceted molecular mechanism to direct primary cell responders and regulators toward either regenerative dermal tissue repair or scarring.

Induction of Fibrogenic Phenotype in Human Mesenchymal Stem Cells by Connective Tissue Growth Factor in a Hydrogel Model of Soft Connective Tissue.

Scar formation is the typical endpoint of wound healing in adult mammalian tissues. An overactive or prolonged fibrogenic response following injury leads to excessive deposition of fibrotic proteins that promote tissue contraction and scar formation. Although well-defined in the dermal tissue, the progression of fibrosis is less explored in other connective tissues, such as the vocal fold. To establish a physiologically relevant 3D model of loose connective tissue fibrosis, we have developed a synthetic extracellular matrix using hyaluronic acid (HA) and peptidic building blocks carrying complementary functional groups. The resultant network was cell adhesive and protease degradable, exhibiting viscoelastic properties similar to the human vocal fold. Human mesenchymal stem cells (hMSCs) were encapsulated in the HA matrix as single cells or multicellular aggregates and cultured in pro-fibrotic media containing connective tissue growth factor (CTGF) for up to 21 days. hMSCs treated with CTGF-supplemented media exhibited an increased expression of fibrogenic markers and ECM proteins associated with scarring. Incorporation of α-smooth muscle actin into F-actin stress fibers was also observed. Furthermore, CTGF treatment increased the migratory capacity of hMSCs as compared to the CTGF-free control groups, indicative of the development of a myofibroblast phenotype. Addition of an inhibitor of the mitogen-activated protein kinase (MAPK) pathway attenuated cellular expression of fibrotic markers and related ECM proteins. Overall, this study demonstrates that CTGF promotes the development of a fibrogenic phenotype in hMSCs encapsulated within an HA matrix and that the MAPK pathway is a potential target for future therapeutic endeavors towards limiting scar formation in loose connective tissues.

Abstract 374: Direct measurements of cellular ATP levels in tumor cell lines using real-time, quantitative live-cell analysis

Abstract Metabolic reprogramming is a hallmark of tumor cells, owing to the increased energetic and biosynthetic demands associated with maintaining high rates of proliferation. Therapeutic strategies targeting unique, critical metabolic pathways of tumor cells are under investigation and in clinical trials. Standard approaches to monitoring drug induced metabolic perturbations are limited to endpoint assays that provide population-based measurements and limited kinetic information. We developed a genetically encoded ATP sensor to enable direct, automated analysis of cellular ATP levels using IncuCyte® S3. In this study, our live cell imaging approach was utilized to evaluate the effect of cancer therapeutics on ATP levels in tumor cell lines, focusing primarily on compounds that target metabolic vulnerabilities. Cell lines stably expressing a genetically encoded, fluorescent ATP sensor or a control (non-ATP binding) sensor were generated. ATP levels were monitored and analyzed using an IncuCyte® S3 equipped with a specialized filter set and data acquisition module. Cellular ATP levels were measured over the course of hours to days following compound treatment. Reductions in ATP levels could be observed within an hour of administration, highlighting the sensitivity and temporal resolution of the IncuCyte® S3 ATP sensor assay. Transient reductions in ATP followed by recovery, which would have been missed by typical endpoint assays, were noted using our live cell analysis approach. Sustained decreases in ATP were associated with enhanced antiproliferative efficacy compared to conditions under which recovery of ATP levels were observed. For example, triple-negative breast cancer (TNBC) cell lines have been shown to be more dependent on activity of glutaminase 1 (GLS1), which catalyzes the first step in utilization of glutamine to fuel mitochondrial metabolism, than their receptor-positive counterparts. Using our live cell analysis approach, we observed a rapid drop in ATP upon glutamine deprivation or inhibition of GLS1 by CB-839 in the TNBC cell line MDA-MB-231. ATP levels remained below that of vehicle-treated cells for the duration of the three-day time course. In contrast, the estrogen receptor-positive cell line MCF-7 displayed a more modest decrease in ATP and full recovery within 48 hours. Quantification of phase confluence confirmed that CB-839 had a stronger antiproliferative effect in MDA-MB-231 cells compared to MCF-7 cells. Visualization of morphology in tandem with automated ATP sensor analysis provide additional insight into the heterogeneity of cellular responses to compound addition. Overall, these data highlight the ability of IncuCyte® S3 to provide direct, kinetic measurement of ATP by live-cell analysis. Citation Format: Cicely L. Schramm, Grigory S. Filonov, Michael L. Bowe, Yong X. Chen, Laura A. Skerlos, Dyke P. McEwen, Daniel M. Appledorn. Direct measurements of cellular ATP levels in tumor cell lines using real-time, quantitative live-cell analysis [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 374.

Relationships of Changes in Pharmacokinetic Parameters of Substrate Drugs in Drug-Drug Interactions on Metabolizing Enzymes and Transporters.

A general objective of drug-drug interaction (DDI) studies is to determine whether potential interactions of new molecular entities with concomitantly administered other drugs exist and, if DDIs occur, whether dosage adjustments are required. A typical end point for DDI evaluations is the ratio of area under the plasma concentration-time curve (AUC) of substrate drugs (AUCR), whereas the ratios of maximal plasma concentration (Cmax ) and terminal half-life (t1/2 ) are also important to understand DDI mechanisms (Cmax R and t1/2 R, respectively). Because changes in substrate AUC by precipitant drugs ultimately result from alterations of Cmax and t1/2 , AUCR can be considered a hybrid parameter of Cmax R and t1/2 R, for example, AUCR≈Cmax R × t1/2 R. The primary objective of this study was to investigate the relationships between AUCR, Cmax R, and t1/2 R in physiologically based pharmacokinetic model-predicted and clinically observed DDI results. First, the model-predicted results showed the excellent proportional relationship between AUCR and (Cmax R×t1/2 R) in DDI results of virtual substrates having a wide range of oral bioavailability with coadministration of ketoconazole, ritonavir, and rifampin. Second, the reasonable proportional relationships were also observed in the clinically observed DDI results of midazolam and statins (atorvastatin, cerivastatin, fluvastatin, lovastatin, pitavastatin, pravastatin, rosuvastatin, and simvastatin) with various inhibitors and inducers. Finally, these results suggest that utilization of the proportional relationship between AUCR and (Cmax R ×t1/2 R) can provide an additional framework to further interpret DDI results reasonably and clearly. Furthermore, the proportional relationship can be purposely used to assess study design and pharmacokinetic analyses in DDI studies.

Abstract B150: Identifying tolerable schedules for targeted anti-cancer agents by applying ordered logistic regression modeling to empirical pathology scores in mice

Abstract Even targeted oncology agents may have significant toxicity, either target-mediated or otherwise. Some targets are simultaneously drivers of cancer yet essential for normal turnover in certain tissues. We sought to develop a predictive model for gut toxicity, to enable model-based selection of the optimal dose and schedule in mice. Mice were treated under several schedules (twice daily, once every one, two or three days), at various doses (6.25-100 mg/kg) of the inhibitor AZ17, and for various durations. Target inhibition is expected to disregulate normal turnover of the gut lining. Epithelial histopathology findings collected from individual animals at various timepoints after the final dose were manually scored on an empirical five-point scale. An ordered logistic regression model was fit to all animals individually across all regimens simultaneously, to describe the probability of observing toxicity at a particular level on the five-point scale as a function of dose and time. It has recently been applied to clinical data, but this is a novel approach in the pre-clinical space. This model-based approach allows integration of data collected at various timepoints and from different studies. It differs from the standard PK/PD approach in that it attempts to predict not the toxicity score itself (which can only be an integer), but the probability of observing each score on the scale. It allows for recovery once dosing stops. The predicted probabilities will thus vary with dose, schedule, duration of treatment and time since last dose. We illustrate with a study of 30 mice, showing that the model was able to capture a number of essential features of any such model: (i) the steep nature of the dose-response relationship; (ii) robustness to outliers (e.g., when a single animal experienced severe tox at a generally tolerated dose); (iii) robustness to non-exposure-driven tox (e.g., finding in vehicle-treated animal). Since staining/reading of pathology slides is not resource intensive, this approach is suitable even for lead-optimization projects. Such data could be collected at the end of a standard tolerability study, in which body-weight is the typical endpoint. It could also be collected from tumor-bearing animals, either a pharmacodynamic or efficacy study. Both applications conform with the 3R's imperative to ‘reduce, refine, replace’ use of animals. Since the model, by design, makes no assumptions about mechanism, it could easily be extended to assess synergistic tox when combined with chemotherapy. Citation Format: Francis Gibbons, Shenghua Wen, Prasad Nadella. Identifying tolerable schedules for targeted anti-cancer agents by applying ordered logistic regression modeling to empirical pathology scores in mice. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B150.

DISTRIBUTION AND ISOMERIZATION OF TERPANES IN PYROLYZATES OF LIGNITE AT HIGH PRESSURES AND TEMPERATURES

Experimental pyrolysis of Eocene lignite in the presence of water was conducted in a closed system for a heating duration of 120 min at temperatures of 400, 500, 600 and 700 °C and pressures of 1 to 3 GPa (equivalent to burial depths of about 30 to 100 km). The distribution of terpanes in the resulting pyrolysates was analyzed in order to investigate the effects of pressure and temperature on maturation of organic matter for this heating time at mantle depths. The results indicate that terpanes show more mature characteristics with increasing temperature at a given pressure. However, high pressures significantly retarded the thermal evolution of organic matter. Also, high pressures had a non‐linear effect on maturation, resulting in anomalous variations of maturity parameters and ratios (tricyclics / hopanes, Ts/Tm, C3122S / (22S+22R), and βα‐moretane /αβ‐hopanes). In addition, the isomerization of hopanes at the C‐22 position did not reach typical end‐point values, even at 700°C. These results are a preliminary step towards a better understanding of organic matter maturation and oil and gas exploration in high temperature and pressure regimes.

Settlement, ADR, and Class Action Superiority

When a defendant sets up a private voluntary compensation scheme or includes an arbitration clause in its form contracts, the resulting alternative dispute resolution (ADR) system bears striking resemblance to the typical endpoint of class action litigation—a settlement that sets up an administrative claims resolution scheme as an alternative to litigation on a mass basis. This article addresses the question of when the existence of one of these ADR systems—offering a shortcut to the same endpoint—should block future class action litigation. It examines important differences in timing, bargaining dynamics, and agency costs in these three contexts. And it argues that a court asked to certify a class in the face of an existing private resolution scheme should undertake a functional and essentially comparative inquiry, considering not only the potential savings in transaction and agency costs but also the underlying bargaining dynamics that may make these ADR shortcuts more vulnerable to unilateral manipulation by defendants than class action settlements.

Population pharmacodynamic model of the longitudinal FEV1 response to an inhaled long-acting anti-muscarinic in COPD patients

The precise assessment of the dose-response to bronchodilators in the treatment of chronic obstructive pulmonary disease is hindered by the low signal to noise ratio of the typical clinical endpoint FEV(1). Kinetic-pharmacodynamic (K-PD) models which use time course of response over a range of doses are in principle suited for the assessment of the dose response relationship of pulmonary administered drugs. A K-PD model was successfully developed using the longitudinal FEV1 data collected in the clinical study for a novel bronchodilator X. A superposition of two cosine functions was selected to describe the circadian variability in FEV(1) at baseline. The onset (ka) and offset (kde) of drug action were described with first-order rate constants of 0.214/h and 0.141/h, respectively. Drug potency, EKD(50,) was estimated as 6.56 μg/h, and the maximum response, Emax as 0.631 L. Between-subject variability for kde, EKD(50) and Emax were 65, 84.7 or 34.4% (expressed as coefficient variation). The model-based simulation predicted that for the same total daily dose of once-daily and twice-daily regimens, the trough FEV(1) response to a twice-daily regimen was higher, and the maximum FEV(1) response to once-daily regimen was higher, while the predicted average FEV(1) response was about the same.

Pathology Issues Related to Neoadjuvant Treatment and the Evaluation of Pathological Response

Tumor response to neoadjuvant therapy for breast carcinoma has been validated as an excellent intermediate endpoint for disease-free survival and overall survival. Pathological complete response (pCR) is the typical endpoint to assess tumor response, and most studies define pCR as absence of invasive carcinoma in the breast. The assessment of pCR poses a challenge to the pathologist. Patients with a complete clinical response may not have a grossly identifiable lesion in the breast, and the delineation of the tumor bed in consultation with radiologist/surgeon is essential in these cases for appropriate sampling; determination of size of microscopic foci of residual carcinoma also requires careful mapping of the tumor bed; modified Bloom and Richardson system may be difficult to apply on the residual carcinoma due to paucity of cells and therapy effect in neoplastic cells. Some authors have included absence of metastases to axillary lymph nodes in the definition of pCR. The biological significance of microscopic foci of residual invasive carcinoma in the breast, lymphovascular invasion, and isolated tumor cells in lymph nodes is unclear. AJCC TNM system can be used to assess tumor response after neoadjuvant therapy and has been shown to be superior than other response classification systems in predicting survival. The relative simplicity, ease of use, and familiarity by the practicing pathologist with this system make this system both user-friendly and reproducible. A modification of the AJCC TNM system is proposed to assess pCR.

Comparative susceptibilities of insect cell lines to infection by the occlusion-body derived phenotype of baculoviruses

Twelve insect cell lines from six species were tested for susceptibility to baculovirus infection by occlusion-derived virus (ODV) phenotype through the use of a typical endpoint assay procedure. ODV from three nucleopolyhedroviruses were prepared by alkali treatment (sodium carbonate) of occlusion bodies (OBs) and the virus preparations were titered on various cell lines. More than a four-log difference was realized for each of theses viruses between the various cell lines. The TN368 line from Trichoplusia ni was only marginally susceptible to ODV from each virus, showing only 3–6 infectious units (IU) per million OBs while the gypsy moth line, LdEp was most susceptible, realizing more than 100,000 IU/million OBs. The other lines tested showed various levels of susceptibility between these two extremes and also varied between the three viruses tested. In additional tests, the ODV were treated with trypsin prior to application to the cells. With most cell lines, this treatment increased the infectivity of each virus by 2–10-fold. Exceptions to this trend included the gypsy moth LdEp line, on which the trypsinized ODV from two of the viruses were slightly less infectious than each virus without trypsin, and the TN-368 line, on which the trypsinized ODV was 5000–75,000 times more infectious. The variable results of trypsinized virus on the different lines are probably due to the levels of endogenous protease activity in the various lines, but the mode of action of the trypsin has not been elucidated. Ultimately, the variable response of cell lines to ODV of different viruses, and the variable effects of trypsin on the ODV may lead to an improved understanding of the infection process of this virus phenotype as well as factors relating to baculovirus host range.

DNA uptake and repair enzyme access to transfected DNA is under reported by gene expression

Gene expression is the typical biological end point of interest following transfection. However, transcription may not accurately assess DNA uptake, or the ability of transfected DNA to be acted on by other enzymatic pathways. We have compared DNA uptake to gene expression and the unrelated enzymatic process of DNA double strand break (DSB) repair. Transfection efficiency (at limiting DNA concentration) was assessed as a function of DNA uptake and gene expression in the DSB repair proficient WI38VA13 and MO59K cell lines and the DSB repair defective cell line MO59J, by comparing eGFP expression from the pHygEGFP expression vector with uptake of rhodamine labeled linear pSP189 plasmid (3:1). Repair proficient cells expressed eGFP most efficiently, but never approached DNA uptake levels (⩾90%). Although transfected DNAs were stable in repair proficient cells and degraded in MO59J cells, most cells did not express eGFP, but in the repair proficient cells linear DNA did undergo DSB repair.

Effects of temperature on the susceptibility of insect cells to infection by baculoviruses.

Three insect cell lines were tested for susceptibility to baculovirus infection by use of a typical endpoint assay procedure. Cell lines from Spodoptera frugiperda (IPLB-Sf21AE), Lymantria dispar (IPLB-LdEIta), and Heliothis virescens (IPLB-HvE6s) in 96-well tissue culture plates were each infected with dilutions of extra cellular virus suspensions of the Autographa californica nucleopolyhedrovirus (AcMNPV). In addition, the L. dispar and H. virescens cells were also infected with L. dispar nucleopolyhedrovirus, and Helicoverpa zea nucleopolyhedrovirus, respectively. Each cell/virus combination was incubated at three temperatures: 22, 27 and 32 degrees C and wells were scored for positive infection (presence of occlusion bodies in cell nuclei) at 2 to 4 day intervals for up to 4 weeks. The resulting data were analyzed by the Spearman-Kärber method, providing virus titers for each combination of virus, cell line, and temperature. The results were categorized by accuracy (assuming the highest titer achieved was the most accurate) and by rapidity of maximum titer. AcMNPV reached the highest titer in each line at 22 degrees C although equivalent titers were reached with both AcMNPV and HzSNPV in the HvE6a line at all three temperatures. This line actually reported about 100-fold less AcMNPV than the other two lines with the same virus sample. Alternatively, the Sf21AE and LdEIta lines reached 10-fold higher titers at the lowest temperature as compared with the higher temperatures, although also at a slower rate.

A Darwinian Theory of Ethnicity, "Race," and Nationality

If we could first know where we are, and whither we are tending, we could better judge what to do and how to do it. Abraham Lincoln (1971:40) Introduction The figure summarizes this paper. The theory it proposes views large-scale human sociocultural evolution (aka world history) as a Darwinian speciation cycle that started with the origin of Homo sapiens somewhere in the Great Rift Valley of east Africa, proceeded to the worldwide dispersion of small populations of that species, and to their ethnic and racial diversification. But then, instead of driving straight ahead to the typical endpoint of the cycle (and the start of a new one) via the evolution of new hominid species from these populations -- i.e., speciation -- Homo sapiens suddenly reversed field and began fostering closer and closer contact and interchange among them all. Nations and nationalities, major instrumentalities in this process, remain dominant (though declining) features of our life today. This much is history -- past and present (solid lines in the figure). The rest is future (dashed lines), and no future is predictable with certainty. The theory offered here, then, proposes only a conditional and probabilistic, not an inviolably deterministic or prophetic, prediction: If the population contact just mentioned continues on its present course, it will very likely produce a global consolidation of Homo sapiens into a single population consisting of one ethnicity, one race, and one nationality. Darwin's explanatory mechanism for the evolution of all species of life -- namely, toward maximum species survival-fitness (Darwin [1859] 1968; see Klein 1999:3) -- is the basic theoretical support for this prediction (there is empirical support too, as we shall see). That is to say, insofar as global species consolidation would advance the survival-fitness of Homo sapiens far beyond that which any ethnic, racial, or nationality population could ever hope for as a separate entity, D arwinian natural selection will favor such consolidation. The theory that has just been outlined will be referred to hereinafter, for brevity's sake, as OD2C2 (with all due apologies to R2D2 and Y2K). The next section describes, and cites appropriate evidential grounds for, the five turning-points of OD2C2. Then, in the sections that follow, the discussion will focus on explaining the transitions from one turning-point to the next. Five Turning-Points Origin There is now a general consensus among paleoanthropologists and geneticists that the species Homo sapiens (latest product of the 2.5 million year-old evolution of the genus Homo), evolved roughly 200 thousand years ago, somewhere in the southern limb of the Great Rift Valley that stretches some 1500 miles through eastern Africa from the southern end of the Red Sea into present-day Tanzania (see Klein 1999:146, 257, 511-514; Ayala 1995:1935; Tattersall 1995:243-246; Marks 1995:17; Fagan 1989:81-120; 1990:15, 20; Wilford 1995; compare Wolpoff, Zhi, and Thorpe 1984:464-467; Norris 1999).'''''' Homo sapiens seems to have spent its first 150 thousand years eking out a precarious but mostly peaceful existence in small, nomadic, food-foraging bands that were slowly growing and dispersing locally -- that is, up and down the African Rift Valley -- and coexisting there with other hominid species that have not survived (see Tattersall 2000). Dispersion: Local and Non-Local However, about 50 thousand years ago (see Klein 1999:494; Stringer and McKie 1996:143-169) -- and almost surely under pressure of competition -- some of these bands began foraging beyond the valley itself, thereby taking the first steps in what would turn out to be a two-thousand-generations-long, leapfrog, push-me-pull-you, journey to the ends of the Earth. In the course of this journey, many bands would be killed off by one thing or another, but others would survive to establish settlements on all the major landmasses of the Earth except Antarctica: south, west, and north Africa, Arabia and the Middle East, Eurasia generally, Australia and Polynesia, Siberia, the Arctic, the Americas, and, less than 2 thousand years ago, New Zealand (see Fagan 1990:234; Molnar 1992:190). …

Microbial Endpoints: The Rationale for their Exclusion as Ecological Assessment Endpoints

The functional importance of bacteria and fungi in terrestrial systems is recognized widely. However, microbial population, community, and functional measurement endpoints change rapidly and across very short spatial scales. Measurement endpoints of microbes tend to be highly responsive to typical fluxes of temperature, moisture, oxygen, and many other noncontaminant factors. Functional redundancy across broad taxonomic groups enables wild swings in community composition without remarkable change in rates of decomposition or community respiration. Consequently, it is exceedingly difficult to relate specific microbial activities with indications of adverse and unacceptable environmental conditions. Moreover, changes in microbial processes do not necessarily result in consequences to plant and animal populations or communities, which in the end are the resources most commonly identified as those to be protected. Therefore, unless more definitive linkages are made between specific microbial effects and an adverse condition for typical assessment endpoint species, microbial endpoints will continue to have limited use in risk assessments; they will not drive the process as primary assessment endpoints.

Focused attention during selective adaptation along a place of articulation continuum.

The current investigation manipulated subjects' attention to adaptor tokens in five selective adaptation experiments. All stimuli were synthetic consonant-vowel syllables, with the consonant varying from [b] to [d] by formant frequency transitions. Two distractor conditions (auditory and visual) were compared to a more typical endpoint-[d alpha] adaptor condition. Distraction from endpoint-[d alpha] adaptors to phonetically distinct [si] and [integral of i] was used to observe whether smaller adaptation effects would result when attention was not focused on adaptor stimuli. In contrast, a focused attention condition required subjects to whisper [b alpha] adaptors right after they were heard. Performance in the focused attention condition was compared to a more typical endpoint-[b alpha] adaptation condition. Results indicated that focused attention did not affect the size of the adaptation effect. Asymmetrical adaptation results for [d alpha] vs [b alpha] adaptors, and a larger amount of adaptation with the presence of contralateral "distractor" syllables, resembled findings in psychoacoustic studies of discrimination and loudness adaptation. These results suggest that two levels of auditory processing (not special to speech perception) were responsible for the observed adaptation effects.