Analysis of Microtubule Sliding Patterns in Chlamydomonas Flagellar Axonemes Reveals Dynein Activity on Specific Doublet Microtubules

Generating the complex waveforms characteristic of beating eukaryotic cilia and flagella requires spatial regulation of dynein-driven microtubule sliding. To generate bending, one prediction is that dynein arms alternate between active and inactive forms on specific subsets of doublet microtubules. Using an in vitro microtubule sliding assay combined with a structural approach, we determined that ATP induces sliding between specific subsets of doublet microtubules, apparently capturing one phase of the beat cycle. These studies were also conducted using high Ca2+ conditions. InChlamydomonas, high Ca2+ induces changes in waveform which are predicted to result from regulating dynein activity on specific microtubules. Our results demonstrate that microtubule sliding in high Ca2+ buffer is also induced by dynein arms on specific doublets. However, the pattern of microtubule sliding in high Ca2+ buffer significantly differs from that in low Ca2+. These results are consistent with a ‘switching hypothesis’ of axonemal bending and provide evidence to indicate that Ca2+ control of waveform includes modulation of the pattern of microtubule sliding between specific doublets. In addition, analysis of microtubule sliding in mutant axonemes reveals that the control mechanism is disrupted in some mutants

Tumour micro-environment elicits innate resistance to RAF inhibitors through HGF secretion.

Drug resistance presents a challenge to the treatment of cancer patients. Many studies have focused on cell-autonomous mechanisms of drug resistance. By contrast, we proposed that the tumour micro-environment confers innate resistance to therapy. Here we developed a co-culture system to systematically assay the ability of 23 stromal cell types to influence the innate resistance of 45 cancer cell lines to 35 anticancer drugs. We found that stroma-mediated resistance is common, particularly to targeted agents. We characterized further the stroma-mediated resistance of BRAF-mutant melanoma to RAF inhibitors because most patients with this type of cancer show some degree of innate resistance. Proteomic analysis showed that stromal cell secretion of hepatocyte growth factor (HGF) resulted in activation of the HGF receptor MET, reactivation of the mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3-OH kinase (PI(3)K)-AKT signalling pathways, and immediate resistance to RAF inhibition. Immunohistochemistry experiments confirmed stromal cell expression of HGF in patients with BRAF-mutant melanoma and showed a significant correlation between HGF expression by stromal cells and innate resistance to RAF inhibitor treatment. Dual inhibition of RAF and either HGF or MET resulted in reversal of drug resistance, suggesting RAF plus HGF or MET inhibitory combination therapy as a potential therapeutic strategy for BRAF-mutant melanoma. A similar resistance mechanism was uncovered in a subset of BRAF-mutant colorectal and glioblastoma cell lines. More generally, this study indicates that the systematic dissection of interactions between tumours and their micro-environment can uncover important mechanisms underlying drug resistance

Immune-mediated competition in rodent malaria is most likely caused by induced changes in innate immune clearance of merozoites

Malarial infections are often genetically diverse, leading to competitive interactions between parasites. A quantitative understanding of the competition between strains is essential to understand a wide range of issues, including the evolution of virulence and drug resistance. In this study, we use dynamical-model based Bayesian inference to investigate the cause of competitive suppression of an avirulent clone of Plasmodium chabaudi (AS) by a virulent clone (AJ) in immuno-deficient and competent mice. We test whether competitive suppression is caused by clone-specific differences in one or more of the following processes: adaptive immune clearance of merozoites and parasitised red blood cells (RBCs), background loss of merozoites and parasitised RBCs, RBC age preference, RBC infection rate, burst size, and within-RBC interference. These processes were parameterised in dynamical mathematical models and fitted to experimental data. We found that just one parameter μ, the ratio of background loss rate of merozoites to invasion rate of mature RBCs, needed to be clone-specific to predict the data. Interestingly, μ was found to be the same for both clones in single-clone infections, but different between the clones in mixed infections. The size of this difference was largest in immuno-competent mice and smallest in immuno-deficient mice. This explains why competitive suppression was alleviated in immuno-deficient mice. We found that competitive suppression acts early in infection, even before the day of peak parasitaemia. These results lead us to argue that the innate immune response clearing merozoites is the most likely, but not necessarily the only, mediator of competitive interactions between virulent and avirulent clones. Moreover, in mixed infections we predict there to be an interaction between the clones and the innate immune response which induces changes in the strength of its clearance of merozoites. What this interaction is unknown, but future refinement of the model, challenged with other datasets, may lead to its discovery

A melanocyte lineage program confers resistance to MAP kinase pathway inhibition

BRAFV600E-mutant malignant melanomas depend on RAF/MEK/ERK (MAPK) signaling for tumor cell growth1. RAF and MEK inhibitors show remarkable clinical efficacy in BRAFV600E melanoma2, 3; however, resistance to these agents remains a formidable challenge2, 4. Global characterization of resistance mechanisms may inform the development of more effective therapeutic combinations. Here, we performed systematic gain-of-function resistance studies by expressing >15,500 genes individually in a BRAFV600E melanoma cell line treated with RAF, MEK, ERK, or combined RAF/MEK inhibitors. These studies revealed a cyclic AMP-dependent melanocytic signaling network not previously associated with drug resistance, including G-protein coupled receptors, adenyl cyclase, protein kinase A and cAMP response element binding protein (CREB). Preliminary analysis of biopsies from BRAFV600E melanoma patients revealed that phosphorylated (active) CREB was suppressed by RAF/MEK-inhibition but restored in relapsing tumors. Expression of transcription factors activated downstream of MAP kinase and cAMP pathways also conferred resistance, including c-FOS, NR4A1, NR4A2 and MITF. Combined treatment with MAP kinase pathway and histone deacetylase inhibitors suppressed MITF expression and cAMP-mediated resistance. Collectively, these data suggest that oncogenic dysregulation of a melanocyte lineage dependency can cause resistance to RAF/MEK/ERK inhibition, which may be overcome by combining signaling- and chromatin-directed therapeutics

Network Analysis of Gut Microbiome Throughout a Whole Foods Based High Fiber Dietary Intervention Reveals Complex Community Dynamics in Melanoma Survivors

https://openworks.mdanderson.org/sumexp22/1139/thumbnail.jp

Are Mandates the Answer? Improving Palliative Care and Pain Management in Vermont

Background: The Vermont legislature (bill H.435, Sec. 19) has tasked the Vermont Board of Medical Practice (VBMP) with making a formal recommendation on improving Vermont health professionals’ knowledge and practice of Palliative Care and Pain Management (PC/PM). In collaboration with the VBMP, our group set out to answer the following questions: • How confident/competent are VT physicians in the practice of PC/PM? • What are the barriers to achieving optimal patient care in PC/PM? • Do VT physicians believe mandatory CME would improve the overall quality of care in PC/PM? • What are the best methods of providing Continuing Medical Education (CME)?https://scholarworks.uvm.edu/comphp_gallery/1040/thumbnail.jp

Impact of a Whole Foods Based High Fiber Diet on Gut Microbiome in Melanoma Survivors

https://openworks.mdanderson.org/sumexp21/1237/thumbnail.jp

Tuning the Magnetic Transport of an Induction LINAC using Emittance

The Lawrence Livermore National Laboratory Flash X-Ray (FXR) machine is a linear induction accelerator used to produce a nominal 18 MeV, 3 kA, 65 ns pulse width electron beam for hydrodynamic radiographs. A common figure of merit for this type of radiographic machine is the x-ray dose divided by the spot area on the bremsstrahlung converter where a higher FOM is desired. Several characteristics of the beam affect the minimum attainable x-ray spot size. The most significant are emittance (chaotic transverse energy), chromatic aberration (energy variation), and beam motion (transverse instabilities and corkscrew motion). FXR is in the midst of a multi-year optimization project to reduce the spot size. This paper describes the effort to reduce beam emittance by adjusting the fields of the transport solenoids and position of the cathode. If the magnetic transport is not correct, the beam will be mismatched and undergo envelope oscillations increasing the emittance. We measure the divergence and radius of the beam in a drift section after the accelerator by imaging the optical transition radiation (OTR) and beam envelope on a foil. These measurements are used to determine an emittance. Relative changes in the emittance can be quickly estimated from the foil measurements allowing for an efficient, real-time study. Once an optimized transport field is determined, the final focus can be adjusted and the new x-ray spot measured. A description of the diagnostics and analysis is presented

Challenges and Opportunities in Cancer Immunotherapy: A Society for Immunotherapy of Cancer (SITC) Strategic Vision

Cancer immunotherapy has flourished over the last 10-15 years, transforming the practice of oncology and providing long-term clinical benefit to some patients. During this time, three distinct classes of immune checkpoint inhibitors, chimeric antigen receptor-T cell therapies specific for two targets, and two distinct classes of bispecific T cell engagers, a vaccine, and an oncolytic virus have joined cytokines as a standard of cancer care. At the same time, scientific progress has delivered vast amounts of new knowledge. For example, advances in technologies such as single-cell sequencing and spatial transcriptomics have provided deep insights into the immunobiology of the tumor microenvironment. With this rapid clinical and scientific progress, the field of cancer immunotherapy is currently at a critical inflection point, with potential for exponential growth over the next decade. Recognizing this, the Society for Immunotherapy of Cancer convened a diverse group of experts in cancer immunotherapy representing academia, the pharmaceutical and biotechnology industries, patient advocacy, and the regulatory community to identify current opportunities and challenges with the goal of prioritizing areas with the highest potential for clinical impact. The consensus group identified seven high-priority areas of current opportunity for the field: mechanisms of antitumor activity and toxicity; mechanisms of drug resistance; biomarkers and biospecimens; unique aspects of novel therapeutics; host and environmental interactions; premalignant immunity, immune interception, and immunoprevention; and clinical trial design, endpoints, and conduct. Additionally, potential roadblocks to progress were discussed, and several topics were identified as cross-cutting tools for optimization, each with potential to impact multiple scientific priority areas. These cross-cutting tools include preclinical models, data curation and sharing, biopsies and biospecimens, diversification of funding sources, definitions and standards, and patient engagement. Finally, three key guiding principles were identified that will both optimize and maximize progress in the field. These include engaging the patient community; cultivating diversity, equity, inclusion, and accessibility; and leveraging the power of artificial intelligence to accelerate progress. Here, we present the outcomes of these discussions as a strategic vision to galvanize the field for the next decade of exponential progress in cancer immunotherapy

PDGFRα up-regulation mediated by sonic hedgehog pathway activation leads to BRAF inhibitor resistance in melanoma cells with BRAF mutation

Control of BRAF(V600E) metastatic melanoma by BRAF inhibitor (BRAF-I) is limited by intrinsic and acquired resistance. Growth factor receptor up-regulation is among the mechanisms underlying BRAF-I resistance of melanoma cells. Here we demonstrate for the first time that PDGFRα up-regulation causes BRAF-I resistance. PDGFRα inhibition by PDGFRα-specific short hairpin (sh)RNA and by PDGFRα inhibitors restores and increases melanoma cells' sensitivity to BRAF-I in vitro and in vivo. This effect reflects the inhibition of ERK and AKT activation which is associated with BRAF-I resistance of melanoma cells. PDGFRα up-regulation is mediated by Sonic Hedgehog Homolog (Shh) pathway activation which is induced by BRAF-I treatment. Similarly to PDGFRα inhibition, Shh inhibition by LDE225 restores and increases melanoma cells' sensitivity to BRAF-I. These effects are mediated by PDGFRα down-regulation and by ERK and AKT inhibition. The clinical relevance of these data is indicated by the association of PDGFRα up-regulation in melanoma matched biopsies of BRAF-I +/- MEK inhibitor treated patients with shorter time to disease progression and less tumor regression. These findings suggest that monitoring patients for early PDGFRα up-regulation will facilitate the identification of those who may benefit from the treatment with BRAF-I in combination with clinically approved PDGFRα or Shh inhibitors