Spectral unmixing of multiply stained fluorescence samples T

The widespread use of fluorescence microscopy along with the vast library of available fluorescent stains and staining methods has been extremely beneficial to researchers in many fields, ranging from material sciences to plant biology. In clinical diagnostics, the ability to combine different markers in a given sample allows the simultaneous detection of the expression of several different molecules, which in turn provides a powerful diagnostic tool for pathologists, allowing a better classification of the sample at hand. The correct detection and separation of multiple stains in a sample is achieved not only by the biochemical and optical properties of the markers, but also by the use of appropriate hardware and software tools. In this chapter, we will review and compare these tools along with their advantages and limitations

Consistent and elastic registration of histological sections using vector-spline regularization

The final publication is available at Springer via http://dx.doi.org/10.1007/11889762_8Revised Papers on Second International ECCV Workshop, CVAMIA 2006 Graz, Austria, May 12, 2006Here we present a new image registration algorithm for the alignment of histological sections that combines the ideas of B-spline based elastic registration and consistent image registration, to allow simultaneous registration of images in two directions (direct and inverse). In principle, deformations based on B-splines are not invertible. The consistency term overcomes this limitation and allows registration of two images in a completely symmetric way. This extension of the elastic registration method simplifies the search for the optimum deformation and allows registering with no information about landmarks or deformation regularization. This approach can also be used as the first step to solve the problem of group-wise registration.Ignacio Arganda-Carreras is being supported by a predoctoral FPI-CAM fellow- ship since October 2003. Carlos Ortiz-de-Solorzano is supported by a Ramon y Cajal (Spanish Ministry of Education and Science ryc-2004-002353) and a Marie Curie International Reintegration Grant (FP6-518688). Jan Kybic was sponsored by the Czech Ministery of Education under project number MSM210000012. Par- tial support is acknowledged to Comunidad de Madrid through grant GR/SAL/0234, to Instituto de Salud Carlos III-Fondo de Investigaciones Sanitarias (FIS) through the IM3 Network and grant 040683 and to the Plan Nacional de Investigación Científica, Desarrollo e Innovación Tecnológica (I+D+I)

A hybrid physics-based and data-driven framework for cellular biological systems: Application to the morphogenesis of organoids

How cells orchestrate their cellular functions remains a crucial question to unravel how they organize in different patterns. We present a framework based on artificial intelligence to advance the understanding of how cell functions are coordinated spatially and temporally in biological systems. It consists of a hybrid physics-based model that integrates both mechanical interactions and cell functions with a data-driven model that regulates the cellular decision-making process through a deep learning algorithm trained on image data metrics. To illustrate our approach, we used data from 3D cultures of murine pancreatic ductal adenocarcinoma cells (PDAC) grown in Matrigel as tumor organoids. Our approach allowed us to find the underlying principles through which cells activate different cell processes to self-organize in different patterns according to the specific microenvironmental conditions. The framework proposed here expands the tools for simulating biological systems at the cellular level, providing a novel perspective to unravel morphogenetic patterns

Inhibition of telomerase activity preferentially targets aldehyde dehydrogenase-positive cancer stem-like cells in lung cancer

<p>Abstract</p> <p>Background</p> <p>Mortality rates for advanced lung cancer have not declined for decades, even with the implementation of novel chemotherapeutic regimens or the use of tyrosine kinase inhibitors. Cancer Stem Cells (CSCs) are thought to be responsible for resistance to chemo/radiotherapy. Therefore, targeting CSCs with novel compounds may be an effective approach to reduce lung tumor growth and metastasis. We have isolated and characterized CSCs from non-small cell lung cancer (NSCLC) cell lines and measured their telomerase activity, telomere length, and sensitivity to the novel telomerase inhibitor MST312.</p> <p>Results</p> <p>The aldehyde dehydrogenase (ALDH) positive lung cancer cell fraction is enriched in markers of stemness and endowed with stem cell properties. ALDH+ CSCs display longer telomeres than the non-CSC population. Interestingly, MST312 has a strong antiproliferative effect on lung CSCs and induces p21, p27 and apoptosis in the whole tumor population. MST312 acts through activation of the ATM/pH2AX DNA damage pathway (short-term effect) and through decrease in telomere length (long-term effect). Administration of this telomerase inhibitor (40 mg/kg) in the H460 xenograft model results in significant tumor shrinkage (70% reduction, compared to controls). Combination therapy consisting of irradiation (10Gy) plus administration of MST312 did not improve the therapeutic efficacy of the telomerase inhibitor alone. Treatment with MST312 reduces significantly the number of ALDH+ CSCs and their telomeric length <it>in vivo</it>.</p> <p>Conclusions</p> <p>We conclude that antitelomeric therapy using MST312 mainly targets lung CSCs and may represent a novel approach for effective treatment of lung cancer.</p

Automated Quantitative Analysis of a Mouse Model of Chronic Pulmonary Inflammation using Micro X-ray Computed Tomography

Micro-CT has emerged as an excellent tool for in-vivo imaging of the lungs of small laboratory animals. Several studies have shown that it can be used to assess the evolution of pulmonary lung diseases in longitudinal studies. However, most of them rely on non-automatic tools for image analysis, or are merely qualitative. In this article, we present a longitudinal, quantitative study of a mouse model of silica-induced pulmonary inflammation. To automatically assess disease progression, we have devised and validated a lung segmentation method that combines threshold-based segmentation, atlas-based segmentation and level sets. Our volume measurements, based on the automatic segmentations, point at a compensation mechanism which leads to an increase of the healthy lung volume in response to the loss of functional tissue caused by inflammation

Evaluation of bioluminescent imaging for noninvasive monitoring of colorectal cancer progression in the liver and its response to immunogene therapy

<p>Abstract</p> <p>Background</p> <p>Bioluminescent imaging (BLI) is based on the detection of light emitted by living cells expressing a luciferase gene. Stable transfection of luciferase in cancer cells and their inoculation into permissive animals allows the noninvasive monitorization of tumor progression inside internal organs. We have applied this technology for the development of a murine model of colorectal cancer involving the liver, with the aim of improving the pre-clinical evaluation of new anticancer therapies.</p> <p>Results</p> <p>A murine colon cancer cell line stably transfected with the luciferase gene (MC38Luc1) retains tumorigenicity in immunocompetent C57BL/6 animals. Intrahepatic inoculation of MC38Luc1 causes progressive liver infiltration that can be monitored by BLI. Compared with ultrasonography (US), BLI is more sensitive, but accurate estimation of tumor mass is impaired in advanced stages. We applied BLI to evaluate the efficacy of an immunogene therapy approach based on the liver-specific expression of the proinflammatory cytokine interleukin-12 (IL-12). Individualized quantification of light emission was able to determine the extent and duration of antitumor responses and to predict long-term disease-free survival.</p> <p>Conclusion</p> <p>We show that BLI is a rapid, convenient and safe technique for the individual monitorization of tumor progression in the liver. Evaluation of experimental treatments with complex mechanisms of action such as immunotherapy is possible using this technology.</p

The use of mixed collagen-Matrigel matrices of increasing complexity recapitulates the biphasic role of cell adhesion in cancer cell migration: ECM sensing, remodeling and forces at the leading edge of cancer invasion

The migration of cancer cells is highly regulated by the biomechanical properties of their local microenvironment. Using 3D scaffolds of simple composition, several aspects of cancer cell mechanosensing (signal transduction, EMC remodeling, traction forces) have been separately analyzed in the context of cell migration. However, a combined study of these factors in 3D scaffolds that more closely resemble the complex microenvironment of the cancer ECM is still missing. Here, we present a comprehensive, quantitative analysis of the role of cell-ECM interactions in cancer cell migration within a highly physiological environment consisting of mixed Matrigel-collagen hydrogel scaffolds of increasing complexity that mimic the tumor microenvironment at the leading edge of cancer invasion. We quantitatively show that the presence of Matrigel increases hydrogel stiffness, which promotes ß1 integrin expression and metalloproteinase activity in H1299 lung cancer cells. Then, we show that ECM remodeling activity causes matrix alignment and compaction that favors higher tractions exerted by the cells. However, these traction forces do not linearly translate into increased motility due to a biphasic role of cell adhesions in cell migration: at low concentration Matrigel promotes migration-effective tractions exerted through a high number of small sized focal adhesions. However, at high Matrigel concentration, traction forces are exerted through fewer, but larger focal adhesions that favor attachment yielding lower cell motility

Evaluation of monocytes as carriers for armed oncolytic adenoviruses in murine and Syrian hamster models of cancer

Replication-competent (oncolytic) adenoviruses (OAV) can be adapted as vectors for the delivery of therapeutic genes, with the aim of extending the antitumor effect beyond direct cytolysis. Transgene expression using these vectors is usually intense but short-lived, and repeated administrations are hampered by the rapid appearance of neutralizing antibodies (NAbs). We have studied the performance of monocytes as cell carriers to improve transgene expression in cancer models established in athymic mice and immunocompetent Syrian hamsters. Human and hamster monocytic cell lines (MonoMac6 and HM-1, respectively) were loaded with replication-competent adenovirus-expressing luciferase. Intravenous administration of these cells caused a modest increase in transgene expression in tumor xenografts, but this effect was virtually lost in hamsters. In contrast, intratumoral administration of HM-1 cells allowed repeated cycles of expression and achieved partial protection from NAbs in preimmunized hamsters bearing pancreatic tumors. To explore the therapeutic potential of this approach, HM-1 cells were loaded with a hypoxia-inducible OAV expressing the immunostimulatory cytokine interleukin-12 (IL-12). Three cycles of treatment achieved a significant antitumor effect in the hamster model, and transgene expression was detected following each administration, in contrast with the rapid neutralization of the free virus. We propose monocytes as carriers for multiple intratumoral administrations of armed OAVs

Muscular and tendon degeneration after achilles rupture: new insights into future repair strategies

Achilles tendon rupture is a frequent injury with an increasing incidence. After clinical surgical repair, aimed at suturing the tendon stumps back into their original position, the repaired Achilles tendon is often plastically deformed and mechanically less strong than the pre-injured tissue, with muscle fatty degeneration contributing to function loss. Despite clinical outcomes, pre-clinical research has mainly focused on tendon structural repair, with a lack of knowledge regarding injury progression from tendon to muscle and its consequences on muscle degenerative/regenerative processes and function. Here, we characterize the morphological changes in the tendon, the myotendinous junction and muscle belly in a mouse model of Achilles tendon complete rupture, finding cellular and fatty infiltration, fibrotic tissue accumulation, muscle stem cell decline and collagen fiber disorganization. We use novel imaging technologies to accurately relate structural alterations in tendon fibers to pathological changes, which further explain the loss of muscle mechanical function after tendon rupture. The treatment of tendon injuries remains a challenge for orthopedics. Thus, the main goal of this study is to bridge the gap between clinicians'' knowledge and research to address the underlying pathophysiology of ruptured Achilles tendon and its consequences in the gastrocnemius. Such studies are necessary if current practices in regenerative medicine for Achilles tendon ruptures are to be improved

Dark Matter Searches Using NaI(Tl) at the Canfranc Underground Laboratory: Past, Present and Future

Sodium Iodide Thallium doped (NaI(Tl)) scintillation detectors have been applied to the direct searches for dark matter since the 1980s and have produced one of the most challenging results in the field-the observation by the DAMA/LIBRA collaboration of an annual modulation in the detection rate for more than twenty cycles. This result is very difficult to reconcile with negative results derived from other experiments using a large variety of target materials and detection techniques. However, it has been neither confirmed nor refuted in a model independent way up to the present. Such a model independent test of the DAMA/LIBRA result is the goal of the ANAIS-112 experiment, presently in the data taking phase at the Canfranc Underground Laboratory in Spain. ANAIS-112 design and operation leans on the expertise acquired at the University of Zaragoza in direct searches for Dark Matter particles using different targets and techniques and in particular using NaI(Tl) scintillation detectors for about thirty years, which are reviewed in the first section of this manuscript. In addition to presenting the status and more recent results of the ANAIS-112 experiment, open research lines, continuing this effort, will be presented