Interactions between ploidy and resource availability shape clonal interference at initiation and recurrence of glioblastoma.

Glioblastoma (GBM) is the most aggressive form of primary brain tumor. Complete surgical resection of GBM is almost impossible due to the infiltrative nature of the cancer. While no evidence for recent selection events have been found after diagnosis, the selective forces that govern gliomagenesis are strong, shaping the tumor's cell composition during the initial progression to malignancy with late consequences for invasiveness and therapy response. We present a mathematical model that simulates the growth and invasion of a glioma, given its ploidy level and the nature of its brain tissue micro-environment (TME), and use it to make inferences about GBM initiation and response to standard-of-care treatment. We approximate the spatial distribution of resource access in the TME through integration of in-silico modelling, multi-omics data and image analysis of primary and recurrent GBM. In the pre-malignant setting, our in-silico results suggest that low ploidy cancer cells are more resistant to starvation-induced cell death. In the malignant setting, between first and second surgery, simulated tumors with different ploidy compositions progressed at different rates. Whether higher ploidy predicted fast recurrence, however, depended on the TME. Historical data supports this dependence on TME resources, as shown by a significant correlation between the median glucose uptake rates in human tissues and the median ploidy of cancer types that arise in the respective tissues (Spearman r = -0.70; P = 0.026). Taken together our findings suggest that availability of metabolic substrates in the TME drives different cell fate decisions for cancer cells with different ploidy and shapes GBM disease initiation and relapse characteristics.

Mathematical Modeling of Clonal Interference by Density-Dependent Selection in Heterogeneous Cancer Cell Lines.

Many cancer cell lines are aneuploid and heterogeneous, with multiple karyotypes co-existing within the same cell line. Karyotype heterogeneity has been shown to manifest phenotypically, thus affecting how cells respond to drugs or to minor differences in culture media. Knowing how to interpret karyotype heterogeneity phenotypically would give insights into cellular phenotypes before they unfold temporally. Here, we re-analyzed single cell RNA (scRNA) and scDNA sequencing data from eight stomach cancer cell lines by placing gene expression programs into a phenotypic context. Using live cell imaging, we quantified differences in the growth rate and contact inhibition between the eight cell lines and used these differences to prioritize the transcriptomic biomarkers of the growth rate and carrying capacity. Using these biomarkers, we found significant differences in the predicted growth rate or carrying capacity between multiple karyotypes detected within the same cell line. We used these predictions to simulate how the clonal composition of a cell line would change depending on density conditions during in-vitro experiments. Once validated, these models can aid in the design of experiments that steer evolution with density-dependent selection.

Mathematical modeling identifies LAG3 and HAVCR2 as biomarkers of T cell exhaustion in melanoma.

Cytotoxic T lymphocytes (CTLs) control tumors via lysis of antigen-presenting targets or through secretion of cytokines such as interferon-γ (IFNG), which inhibit tumor cell proliferation. Improved understanding of CTL interactions within solid tumors will aid the development of immunotherapeutic strategies against cancer. In this study, we take a systems biology approach to compare the importance of cytolytic versus IFNG-mediated cytostatic effects in a murine melanoma model (B16F10) and to dissect the contribution of immune checkpoints HAVCR2, LAG3, and PDCD1/CD274 to CTL exhaustion. We integrated multimodal data to inform an ordinary differential equation (ODE) model of CTL activities inside the tumor. Our model predicted that CTL cytotoxicity played only a minor role in tumor control relative to the cytostatic effects of IFNG. Furthermore, our analysis revealed that within B16F10 melanomas HAVCR2 and LAG3 better characterize the development of a dysfunctional CTL phenotype than does the PDCD1/CD274 axis.

Intra-tumor heterogeneity, turnover rate and karyotype space shape susceptibility to missegregation-induced extinction.

The phenotypic efficacy of somatic copy number alterations (SCNAs) stems from their incidence per base pair of the genome, which is orders of magnitudes greater than that of point mutations. One mitotic event stands out in its potential to significantly change a cell's SCNA burden-a chromosome missegregation. A stochastic model of chromosome mis-segregations has been previously developed to describe the evolution of SCNAs of a single chromosome type. Building upon this work, we derive a general deterministic framework for modeling missegregations of multiple chromosome types. The framework offers flexibility to model intra-tumor heterogeneity in the SCNAs of all chromosomes, as well as in missegregation- and turnover rates. The model can be used to test how selection acts upon coexisting karyotypes over hundreds of generations. We use the model to calculate missegregation-induced population extinction (MIE) curves, that separate viable from non-viable populations as a function of their turnover- and missegregation rates. Turnover- and missegregation rates estimated from scRNA-seq data are then compared to theoretical predictions. We find convergence of theoretical and empirical results in both the location of MIE curves and the necessary conditions for MIE. When a dependency of missegregation rate on karyotype is introduced, karyotypes associated with low missegregation rates act as a stabilizing refuge, rendering MIE impossible unless turnover rates are exceedingly high. Intra-tumor heterogeneity, including heterogeneity in missegregation rates, increases as tumors progress, rendering MIE unlikely.

Mathematical Modelling Based on In Vivo Imaging Suggests CD137-Stimulated Cytotoxic T Lymphocytes Exert Superior Tumour Control Due to an Enhanced Antimitotic Effect on Tumour Cells.

Several immunotherapeutic strategies for the treatment of cancer are under development. Two prominent strategies are adoptive cell transfer (ACT) of CTLs and modulation of CTL function with immune checkpoint inhibitors or with costimulatory antibodies. Despite some success with these approaches, there remains a lack of detailed and quantitative descriptions of the events following CTL transfer and the impact of immunomodulation. Here, we have applied ordinary differential equation models to two photon imaging data derived from a B16F10 murine melanoma. Models were parameterised with data from two different treatment conditions: either ACT-only, or ACT with intratumoural costimulation using a CD137 targeted antibody. Model dynamics and best fitting parameters were compared, in order to assess the mode of action of the CTLs and examine how the CD137 antibody influenced their activities. We found that the cytolytic activity of the transferred CTLs was minimal without CD137 costimulation, and that the CD137 targeted antibody did not enhance the per-capita killing ability of the transferred CTLs. Instead, the results of our modelling study suggest that an antiproliferative effect of CTLs exerted upon the tumour likely accounted for the majority of the reduction in tumour growth after CTL transfer. Moreover, we found that CD137 most likely improved tumour control via enhancement of this antiproliferative effect, as well as prolonging the period in which CTLs were inside the tumour, leading to a sustained duration of their antitumour effects following CD137 stimulation.

Conditions to optimise the developmental competence of immature equine oocytes.

Optimising the developmental potential of immature equine oocytes and invitro-produced (IVP) embryos was explored through modifications of established media and holding temperature. In Experiment 1, delaying spontaneous resumption of meiosis through the process of simulated physiological oocyte maturation with the addition of the adenylate cyclase activator forskolin (50µM) and the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (100µM) to overnight holding medium before maturation improved blastocyst production (P<0.05). In Experiment 2, the blastocyst production rate was increased significantly when cumulin (100ng mL-1) was added to the overnight holding or culture media (P<0.05). In Experiment 3, immature oocytes held overnight at 16°C before maturation had improved developmental competence than those held at 20°C and 5°C (P<0.05). There was no difference between maturation rates, but blastocyst formation per cleaved oocyte was significantly greater in oocytes held overnight at 16°C than at 20°C or 5°C. Furthermore, blastocyst formation per recovered oocyte and per fertilised oocyte was greater when oocytes were held before maturation at 16°C than at 5°C (P<0.05). In Experiment 4, the addition of sodium ascorbate (AC; 50µg mL-1) to the maturation and/or culture media of oocytes and IVP embryos did not improve blastocyst production, but did appear to lower cleavage rates compared with oocytes and embryos cultured without AC.

Heterogeneous, delayed-onset killing by multiple-hitting T cells: Stochastic simulations to assess methods for analysis of imaging data.

Although quantitative insights into the killing behaviour of Cytotoxic T Lymphocytes (CTLs) are necessary for the rational design of immune-based therapies, CTL killing function remains insufficiently characterised. One established model of CTL killing treats CTL cytotoxicity as a Poisson process, based on the assumption that CTLs serially kill antigen-presenting target cells via delivery of lethal hits, each lethal hit corresponding to a single injection of cytotoxic proteins into the target cell cytoplasm. Contradicting this model, a recent in vitro study of individual CTLs killing targets over a 12-hour period found significantly greater heterogeneity in CTL killing performance than predicted by Poisson-based killing. The observed killing process was dynamic and varied between CTLs, with the best performing CTLs exhibiting a marked increase in killing during the final hours of the experiments, along with a "burst killing" kinetic. Despite a search for potential differences between CTLs, no mechanistic explanation for the heterogeneous killing kinetics was found. Here we have used stochastic simulations to assess whether target cells might require multiple hits from CTLs before undergoing apoptosis, in order to verify whether multiple-hitting could explain the late onset, burst killing dynamics observed in vitro. We found that multiple-hitting from CTLs was entirely consistent with the observed killing kinetics. Moreover, the number of available targets and the spatiotemporal kinetics of CTL:target interactions influenced the realised CTL killing rate. We subsequently used realistic, spatial simulations to assess methods for estimating the hitting rate and the number of hits required for target death, to be applied to microscopy data of individual CTLs killing targets. We found that measuring the cumulative duration of individual contacts that targets have with CTLs would substantially improve accuracy when estimating the killing kinetics of CTLs.

Contact-Dependent Killing by Cytotoxic T Lymphocytes Is Insufficient for EL4 Tumor Regression In Vivo.

Immunotherapies are an emerging strategy for treatment of solid tumors. Improved understanding of the mechanisms employed by cytotoxic T lymphocytes (CTL) to control tumors will aid in the development of immunotherapies. CTLs can directly kill tumor cells in a contact-dependent manner or may exert indirect effects on tumor cells via secretion of cytokines. Here, we aim to quantify the importance of these mechanisms in murine thymoma EL4/EG7 cells. We developed an agent-based model (ABM) and an ordinary differential equation model of tumor regression after adoptive transfer of a population of CTLs. Models were parameterized based on in vivo measurements of CTL infiltration and killing rates applied to EL4/EG7 tumors and OTI T cells. We quantified whether infiltrating CTLs are capable of controlling tumors through only direct, contact-dependent killing. Both models agreed that the low measured killing rate of CTLs in vivo was insufficient to cause tumor regression. In our ABM, we also simulated CTL production of the cytokine IFNγ in order to explore how an antiproliferative effect of IFNγ might aid CTLs in tumor control. In this model, IFNγ substantially reduced tumor growth compared with direct killing alone. Collectively, these data demonstrate that contact-dependent killing is insufficient for EL4 regression in vivo and highlight the potential importance of cytokine-induced antiproliferative effects in T-cell-mediated tumor control. SIGNIFICANCE: Computational modeling highlights the importance of cytokine-induced antiproliferative effects in T-cell-mediated control of tumor progression.

Single-cell imaging of CAR T cell activity in vivo reveals extensive functional and anatomical heterogeneity.

CAR T cells represent a potentially curative strategy for B cell malignancies. However, the outcome and dynamics of CAR T cell interactions in distinct anatomical sites are poorly understood. Using intravital imaging, we tracked interactions established by anti-CD19 CAR T cells in B cell lymphoma-bearing mice. Circulating targets trapped CAR T cells in the lungs, reducing their access to lymphoid organs. In the bone marrow, tumor apoptosis was largely due to CAR T cells that engaged, killed, and detached from their targets within 25 min. Notably, not all CAR T cell contacts elicited calcium signaling or killing while interacting with tumors, uncovering extensive functional heterogeneity. Mathematical modeling revealed that direct killing was sufficient for tumor regression. Finally, antigen-loss variants emerged in the bone marrow, but not in lymph nodes, where CAR T cell cytotoxic activity was reduced. Our results identify a previously unappreciated level of diversity in the outcomes of CAR T cell interactions in vivo, with important clinical implications.

Comparison of satellite reflectance algorithms for estimating turbidity and cyanobacterial concentrations in productive freshwaters using hyperspectral aircraft imagery and dense coincident surface observations.

We analyzed 37 satellite reflectance algorithms and 321 variants for five satellites for estimating turbidity in a freshwater inland lake in Ohio using coincident real hyperspectral aircraft imagery converted to relative reflectance and dense coincident surface observations. This study is part of an effort to develop simple proxies for turbidity and algal blooms and to evaluate their performance and portability between satellite imagers for regional operational turbidity and algal bloom monitoring. Turbidity algorithms were then applied to synthetic satellite images and compared to in situ measurements of turbidity, chlorophyll-a (Chl-a), total suspended solids (TSS) and phycocyanin as an indicator of cyanobacterial/blue green algal (BGA) abundance. Several turbidity algorithms worked well with real Compact Airborne Spectrographic Imager (CASI) and synthetic WorldView-2, Sentinel-2 and Sentinel-3/MERIS/OLCI imagery. A simple red band algorithm for MODIS imagery and a new fluorescence line height algorithm for Landsat-8 imagery had limited performance with regard to turbidity estimation. Blue-Green Algae/Phycocyanin (BGA/PC) and Chl-a algorithms were the most widely applicable algorithms for turbidity estimation because strong co-variance of turbidity, TSS, Chl-a, and BGA made them mutual proxies in this experiment.

Evaluating the portability of satellite derived chlorophyll-a algorithms for temperate inland lakes using airborne hyperspectral imagery and dense surface observations.

This study evaluated the performances of twenty-nine algorithms that use satellite-based spectral imager data to derive estimates of chlorophyll-a concentrations that, in turn, can be used as an indicator of the general status of algal cell densities and the potential for a harmful algal bloom (HAB). The performance assessment was based on making relative comparisons between two temperate inland lakes: Harsha Lake (7.99 km2) in Southwest Ohio and Taylorsville Lake (11.88 km2) in central Kentucky. Of interest was identifying algorithm-imager combinations that had high correlation with coincident chlorophyll-a surface observations for both lakes, as this suggests portability for regional HAB monitoring. The spectral data utilized to estimate surface water chlorophyll-a concentrations were derived from the airborne Compact Airborne Spectral Imager (CASI) 1500 hyperspectral imager, that was then used to derive synthetic versions of currently operational satellite-based imagers using spatial resampling and spectral binning. The synthetic data mimics the configurations of spectral imagers on current satellites in earth's orbit including, WorldView-2/3, Sentinel-2, Landsat-8, Moderate-resolution Imaging Spectroradiometer (MODIS), and Medium Resolution Imaging Spectrometer (MERIS). High correlations were found between the direct measurement and the imagery-estimated chlorophyll-a concentrations at both lakes. The results determined that eleven out of the twenty-nine algorithms were considered portable, with r2 values greater than 0.5 for both lakes. Even though the two lakes are different in terms of background water quality, size and shape, with Taylorsville being generally less impaired, larger, but much narrower throughout, the results support the portability of utilizing a suite of certain algorithms across multiple sensors to detect potential algal blooms through the use of chlorophyll-a as a proxy. Furthermore, the strong performance of the Sentinel-2 algorithms is exceptionally promising, due to the recent launch of the second satellite in the constellation, which will provide higher temporal resolution for temperate inland water bodies. Additionally, scripts were written for the open-source statistical software R that automate much of the spectral data processing steps. This allows for the simultaneous consideration of numerous algorithms across multiple imagers over an expedited time frame for the near real-time monitoring required for detecting algal blooms and mitigating their adverse impacts.

A Critical Evaluation of the Factual Accuracy and Scholarly Foundations of The Witch-Hunt Narrative.

We comment on The Witch-Hunt Narrative ( TWHN) by Cheit. As its first hypothesis, TWHN argues that most of the famous ritual child abuse cases of the 1980s and 1990s were not really witch-hunts at all. In response, we criticize the TWHN definition of a witch-hunt as overly narrow and idiosyncratic. Based on the scholarly literature, we propose 10 criteria for identifying a witch-hunt. We rate four well-known ritual child abuse cases with these criteria and show they were classic witch-hunts. As its second hypothesis, TWHN argues that most defendants in child ritual abuse cases were guilty or probably guilty. In response, we point out many instances in which TWHN has omitted or mischaracterized important facts or ignored relevant scientific information running contrary to its hypotheses. We conclude that TWHN is often factually inaccurate and tends to make strong assertions without integrating relevant scholarly and scientific information. Scholars should approach the book with caution.

Simulation of lower limb axial arterial length change during locomotion.

The effect of external forces on axial arterial wall mechanics has conventionally been regarded as secondary to hemodynamic influences. However, arteries are similar to muscles in terms of the manner in which they traverse joints, and their three-dimensional geometrical requirements for joint motion. This study considers axial arterial shortening and elongation due to motion of the lower extremity during gait, ascending stairs, and sitting-to-standing motion. Arterial length change was simulated by means of a graphics based anatomic and kinematic model of the lower extremity. This model estimated the axial shortening to be as much as 23% for the femoropopliteal arterial region and as much as 21% for the iliac artery. A strong correlation was observed between femoropopliteal artery shortening and maximum knee flexion angle (r²=0.8) as well as iliac artery shortening and maximum hip angle flexion (r²=0.9). This implies a significant mechanical influence of locomotion on arterial behavior in addition to hemodynamics factors. Vascular tissue has high demands for axial compliance that should be considered in the pathology of atherosclerosis and the design of vascular implants.

Structure based drug design: development of potent and selective factor IXa (FIXa) inhibitors.

On the basis of our understanding on the binding interactions of the benzothiophene template within the FIXa active site by X-ray crystallography and molecular modeling studies, we developed our SAR strategy by targeting the 4-position of the template to access the S1 beta and S2-S4 sites. A number of highly selective and potent factor Xa (FXa) and FIXa inhibitors were identified by simple switch of functional groups with conformational changes toward the S2-S4 sites.

Studies of benzothiophene template as potent factor IXa (FIXa) inhibitors in thrombosis.

FIXa is a serine protease enzyme involved in the intrinsic pathway of the coagulation cascade. The upstream intervention of the coagulation cascade in selectively inhibiting FIXa would leave hemostasis intact via the extrinsic pathway, leading to an optimum combination of efficacy and safety with low incidence of bleeding. We have identified 2-amindinobenzothiophene template as a lead scaffold for FIXa inhibiton based on its homology with urokinase plasminogen activator (uPA). Subsequent SAR work on the template revealed a number of highly potent FIXa inhibitors, though with moderate selectivity against FXa. X-ray study with one of the analogues demonstrated active site binding interaction with the induced opening of the S1 beta pocket and a secondary binding at the S2-S4 sites, which is in direct contrast with the previous finding.

Arctic lake physical processes and regimes with implications for winter water availability and management in the National Petroleum Reserve Alaska.

Lakes are dominant landforms in the National Petroleum Reserve Alaska (NPRA) as well as important social and ecological resources. Of recent importance is the management of these freshwater ecosystems because lakes deeper than maximum ice thickness provide an important and often sole source of liquid water for aquatic biota, villages, and industry during winter. To better understand seasonal and annual hydrodynamics in the context of lake morphometry, we analyzed lakes in two adjacent areas where winter water use is expected to increase in the near future because of industrial expansion. Landsat Thematic Mapper and Enhanced Thematic Mapper Plus imagery acquired between 1985 and 2007 were analyzed and compared with climate data to understand interannual variability. Measured changes in lake area extent varied by 0.6% and were significantly correlated to total precipitation in the preceding 12 months (p < 0.05). Using this relation, the modeled lake area extent from 1985 to 2007 showed no long-term trends. In addition, high-resolution aerial photography, bathymetric surveys, water-level monitoring, and lake-ice thickness measurements and growth models were used to better understand seasonal hydrodynamics, surface area-to-volume relations, winter water availability, and more permanent changes related to geomorphic change. Together, these results describe how lakes vary seasonally and annually in two critical areas of the NPRA and provide simple models to help better predict variation in lake-water supply. Our findings suggest that both overestimation and underestimation of actual available winter water volume may occur regularly, and this understanding may help better inform management strategies as future resource use expands in the NPRA.

In the Belly of the Beast: Traumatic Countertransference.

The authors discuss their work together in a consultation process. One author led many groups that were created to facilitate recovery for persons directly impacted by the terrorist attacks on September 11, 2001; the other acted as consultant to the therapist/leader. The leader was a resident of New York City at the time of the attacks; the consultant lived in a distant Midwestern city and the two had never met prior to the work. They describe their experience of working together and the role of this collaboration in the lives of the leader and the groups. Well aware that little exists in the literature about groups led by leaders having experienced the same trauma as group members, the authors pay special attention to the countertransference that each underwent and raise questions about the effects of that on the group process.

In the belly of the beast: traumatic countertransference.

The authors discuss their work together in a consultation process. One author led many groups that were created to facilitate recovery for persons directly impacted by the terrorist attacks on September 11, 2001; the other acted as consultant to the therapist/leader. The leader was a resident of New York City at the time of the attacks; the consultant lived in a distant Midwestern city and the two had never met prior to the work. They describe their experience of working together and the role of this collaboration in the lives of the leader and the groups. Well aware that little exists in the literature about groups led by leaders having experienced the same trauma as group members, the authors pay special attention to the countertransference that each underwent and raise questions about the effects of that on the group process.