Optimized CUBIC protocol for three-dimensional imaging of chicken embryos at single-cell resolution.

The CUBIC tissue-clearing protocol has been optimized to produce translucent immunostained whole chicken embryos and embryo brains. When combined with multispectral light-sheet microscopy, the validated protocol presented here provides a rapid, inexpensive and reliable method for acquiring accurate histological images that preserve three-dimensional structural relationships with single-cell resolution in whole early-stage chicken embryos and in the whole brains of late-stage embryos.

Development of an Inverted Epifluorescence Microscope for Long-Term Monitoring of Bacteria in Multiplexed Microfluidic Devices.

Developing more efficient methods for antibiotic susceptibility testing is a pressing issue in novel drug development as bacterial resistance to antibiotics becomes increasingly common. Microfluidic devices have been demonstrated to be powerful platforms that allow researchers to perform multiplexed antibiotic testing. However, the level of multiplexing within microdevices is limited, evidencing the need of creating simple, low-cost and high-resolution imaging systems that can be integrated in antibiotic development pipelines. This paper describes the design and development of an epifluorescence inverted microscope that enables long-term monitoring of bacteria inside multiplexed microfluidic devices. The goal of this work is to provide a simple microscope powerful enough to allow single-cell analysis of bacteria at a reduced cost. This facilitates increasing the number of microscopes that are simultaneously used for antibiotic testing. We prove that the designed system is able to accurately detect fluorescent beads of 100 nm, demonstrating comparable features to high-end commercial microscopes and effectively achieving the resolution required for single-cell analysis of bacteria. The proposed microscope could thus increase the efficiency in antibiotic testing while reducing cost, size, weight, and power requirements, contributing to the successful development of new antibiotic drugs.

Positron Emission Tomography: Current Challenges and Opportunities for Technological Advances in Clinical and Preclinical Imaging Systems.

Positron emission tomography (PET) imaging is based on detecting two time-coincident high-energy photons from the emission of a positron-emitting radioisotope. The physics of the emission, and the detection of the coincident photons, give PET imaging unique capabilities for both very high sensitivity and accurate estimation of the in vivo concentration of the radiotracer. PET imaging has been widely adopted as an important clinical modality for oncological, cardiovascular, and neurological applications. PET imaging has also become an important tool in preclinical studies, particularly for investigating murine models of disease and other small-animal models. However, there are several challenges to using PET imaging systems. These include the fundamental trade-offs between resolution and noise, the quantitative accuracy of the measurements, and integration with X-ray computed tomography and magnetic resonance imaging. In this article, we review how researchers and industry are addressing these challenges.

Application of the compressed sensing technique to self-gated cardiac cine sequences in small animals.

PURPOSE: Self-gated cine sequences are a common choice for cardiac MRI in preclinical applications. The aims of our work were to apply the compressed sensing technique to IntraGateFLASH cardiac MRI studies on rats and to find the maximum acceleration factor achievable with this technique. THEORY AND METHODS: Our reconstruction method extended the Split Bregman formulation to minimize the total variation in both space and time. In addition, we analyzed the influence of the undersampling pattern on the acceleration factor achievable. RESULTS: Our results show that acceleration factors of up to 15 are achievable with our technique when appropriate undersampling patterns are used. The introduction of a time-varying random sampling clearly improved the efficiency of the undersampling schemes. In terms of computational efficiency, the proposed reconstruction method has been shown to be competitive as compared with the fastest methods found in the literature. CONCLUSION: We successfully applied our compressed sensing technique to self-gated cardiac cine acquisition in small animals, obtaining an acceleration factor of up to 15 with almost unnoticeable image degradation.

Synthesis of 9,9'-[1,2-ethanediylbis(oxymethylene)]bis-2-amino-1,9-dihydro-6H-purin-6-one, an impurity of acyclovir.

The synthesis of 9,9'-[1,2-ethanediylbis(oxymethylene)]bis-2-amino-1,9-dihydro-6H-purin-6-one, a minor impurity of acyclovir, is described. Starting with commercial N-(9-acetyl-6-oxo-1H-purin-2-yl)acetamide, the process uses an acid catalysed phase transfer catalysis (PTC) process to produce the selective alkylation at the 9 position of the guanine ring.

Innovations in ex vivo Light Sheet Fluorescence Microscopy.

Light Sheet Fluorescence Microscopy (LSFM) has revolutionized how optical imaging of biological specimens can be performed as this technique allows to produce 3D fluorescence images of entire samples with a high spatiotemporal resolution. In this manuscript, we aim to provide readers with an overview of the field of LSFM on ex vivo samples. Recent advances in LSFM architectures have made the technique widely accessible and have improved its acquisition speed and resolution, among other features. These developments are strongly supported by quantitative analysis of the huge image volumes produced thanks to the boost in computational capacities, the advent of Deep Learning techniques, and by the combination of LSFM with other imaging modalities. Namely, LSFM allows for the characterization of biological structures, disease manifestations and drug effectivity studies. This information can ultimately serve to develop novel diagnostic procedures, treatments and even to model the organs physiology in healthy and pathological conditions.

Determination of the Pharmacokinetics and Pharmacodynamics of Isoniazid, Rifampicin, Pyrazinamide and Ethambutol in a Cross-Over Cynomolgus Macaque Model of Mycobacterium tuberculosis Infection.

Innovative cross-over study designs were explored in non-human primate (NHP) studies to determine the value of this approach for the evaluation of drug efficacy against tuberculosis (TB). Firstly, the pharmacokinetics (PK) of each of the drugs Isoniazid (H), Rifampicin (R), Pyrazinamide (Z) and Ethambutol (E), that are standardly used for the treatment of tuberculosis, was established in the blood of macaques after oral dosing as a monotherapy or in combination. Two studies were conducted to evaluate the pharmacokinetics and pharmacodynamics of different drug combinations using cross-over designs. The first employed a balanced, three-period Pigeon design with an extra period; this ensured that treatment by period interactions and carry-over could be detected comparing the treatments HR, HZ and HRZ using H37Rv as the challenge strain of Mycobacterium tuberculosis (M. tb). Although the design accounted for considerable variability between animals, the three regimens evaluated could not be distinguished using any of the alternative endpoints assessed. However, the degree of pathology achieved using H37Rv in the model during this study was less than expected. Based on these findings, a second experiment using a classical AB/BA design comparing HE with HRZ was conducted using the M. tb Erdman strain. More extensive pathology was observed, and differences in computerized tomography (CT) scores and bacteriology counts in the lungs were detected, although due to the small group sizes, clearer differences were not distinguished. Type 1 T helper (Th1) cell response profiles were characterized using the IFN-γ ELISPOT assay and revealed differences between drug treatments that corresponded to decreases in disease burden. Therefore, the studies performed support the utility of the NHP model for the determination of PK/PD of TB drugs, although further work is required to optimize the use of cross-over study designs.

Feasibility of U-curve method to select the regularization parameter for fluorescence diffuse optical tomography in phantom and small animal studies.

When dealing with ill-posed problems such as fluorescence diffuse optical tomography (fDOT) the choice of the regularization parameter is extremely important for computing a reliable reconstruction. Several automatic methods for the selection of the regularization parameter have been introduced over the years and their performance depends on the particular inverse problem. Herein a U-curve-based algorithm for the selection of regularization parameter has been applied for the first time to fDOT. To increase the computational efficiency for large systems an interval of the regularization parameter is desirable. The U-curve provided a suitable selection of the regularization parameter in terms of Picard's condition, image resolution and image noise. Results are shown both on phantom and mouse data.

Simplified Statistical Image Reconstruction for X-ray CT With Beam-Hardening Artifact Compensation.

CT images are often affected by beam-hardening artifacts due to the polychromatic nature of the X-ray spectra. These artifacts appear in the image as cupping in homogeneous areas and as dark bands between dense regions such as bones. This paper proposes a simplified statistical reconstruction method for X-ray CT based on Poisson statistics that accounts for the non-linearities caused by beam hardening. The main advantages of the proposed method over previous algorithms are that it avoids the preliminary segmentation step, which can be tricky, especially for low-dose scans, and it does not require knowledge of the whole source spectrum, which is often unknown. Each voxel attenuation is modeled as a mixture of bone and soft tissue by defining density-dependent tissue fractions and maintaining one unknown per voxel. We approximate the energy-dependent attenuation corresponding to different combinations of bone and soft tissues, the so-called beam-hardening function, with the 1D function corresponding to water plus two parameters that can be tuned empirically. Results on both simulated data with Poisson sinogram noise and two rodent studies acquired with the ARGUS/CT system showed a beam hardening reduction (both cupping and dark bands) similar to analytical reconstruction followed by post-processing techniques but with reduced noise and streaks in cases with a low number of projections, as expected for statistical image reconstruction.

A radiological score for the assessment of tuberculosis progression: Validation in mouse models.

The sensitivity of in vivo low-dose high-resolution micro-computed tomography imaging enables monitoring the lung damage caused by tuberculosis. Here, we propose a radiological score integrated in the experimental workflow that enables longitudinal monitoring for prospective efficacy studies in drug development programs. The score is based on an automatic measurement of total unaffected lung volume in vivo normalized for inter-subject comparison. It was validated on well-characterized progression of chronic tuberculosis in Erdman and H37Rv strains in C3HeB/FeJ-based models. We demonstrated that a decrease in the score value indicates increasing adverse effects and vice versa. The colony-forming units count confirmed the variability in the host response suggested by the score values. The correlation between changes in the mice's weight and the score is consistent with disease progression. The classification of disease extent by k-means clustering of the score values provided the definition of the lung damage severity according to the bacillus strain. The proposed score will reduce sources of bias and improve the statistical robustness of studies by the attrition of non-infected subjects or subjects with a weak immune response. Readily available quantifications allow for a fast assessment of the therapeutic potential in drug-resistant tuberculosis strains.

Automatic quantification of histological studies in allergic asthma.

The evaluation of new therapies to treat allergic asthma makes frequent use of histological studies. Some of them are based on microscope observation of stained paraffin lung sections to quantify cellular infiltrate, an effect directly related to allergic processes. Currently, there is no software tool available for doing this quantification automatically. This paper presents a methodology and a software tool for the quantification of cellular infiltrate in lung tissue images in an allergic asthma mouse model. The image is divided into regions of equal size, which are then classified by means of a segmentation algorithm based on texture analysis. The classification uses three discriminant functions, built from parameters derived from the histogram and the co-occurrence matrix. These functions were calculated by means of a stepwise discriminant analysis on 79 samples from a training set. Results provided a correct classification of 96.8% on an independent test set of 251 samples labeled manually. Regression analysis showed a good agreement between automatic and manual methods. A reliable and easy to implement method has been developed to provide an automatic method for quantifying microscopy images of lung histological studies. Results showed similar accuracy to that provided by an expert, while allowing analyzing a much larger number of fields in a repeatable way.

Automated method for small-animal PET image registration with intrinsic validation.

PURPOSE: We propose and compare different registration approaches to align small-animal PET studies and a procedure to validate the results by means of objective registration consistency measurements. PROCEDURES: We have applied a registration algorithm based on information theory, using different approaches to mask the reference image. The registration consistency allows for the detection of incorrect registrations. This methodology has been evaluated on a test dataset (FDG-PET rat brain images). RESULTS: The results show that a multiresolution two-step registration approach based on the use of the whole image at the low resolution step, while masking the brain at the high resolution step, provides the best robustness (87.5% registration success) and highest accuracy (0.67-mm average). CONCLUSIONS: The major advantages of our approach are minimal user interaction and automatic assessment of the registration error, avoiding visual inspection of the results, thus facilitating the accurate, objective, and rapid analysis of large groups of rodent PET images.

Relevance of the Fanconi anemia pathway in the response of human cells to trabectedin.

Trabectedin (Yondelis; ET-743) is a potent anticancer drug that binds to DNA by forming a covalent bond with a guanine in one strand and one or more hydrogen bonds with the opposite strand. Using a fluorescence-based melting assay, we show that one single trabectedin-DNA adduct increases the thermal stability of the double helix by >20 degrees C. As deduced from the analysis of phosphorylated H2AX and Rad51 foci, we observed that clinically relevant doses of trabectedin induce the formation of DNA double-strand breaks in human cells and activate homologous recombination repair in a manner similar to that evoked by the DNA interstrand cross-linking agent mitomycin C (MMC). Because one important characteristic of this drug is its marked cytotoxicity on cells lacking a functional Fanconi anemia (FA) pathway, we compared the response of different subtypes of FA cells to MMC and trabectedin. Our data clearly show that human cells with mutations in FANCA, FANCC, FANCF, FANCG, or FANCD1 genes are highly sensitive to both MMC and trabectedin. However, in marked contrast to MMC, trabectedin does not induce any significant accumulation of FA cells in G2-M. The critical relevance of FA proteins in the response of human cells to trabectedin reported herein, together with observations showing the role of the FA pathway in cancer suppression, strongly suggest that screening for mutations in FA genes may facilitate the identification of tumors displaying enhanced sensitivity to this novel anticancer drug.

X-ray-based virtual slicing of TB-infected lungs.

Hollow organs such as the lungs pose a considerable challenge for post-mortem imaging in preclinical research owing to their extremely low contrast and high structural complexity. The aim of our study was to enhance the contrast of tuberculosis lesions for their stratification by 3D x-ray-based virtual slicing. Organ samples were taken from five control and five tuberculosis-infected mice. Micro-Computed Tomography (CT) scans of the subjects were acquired in vivo (without contrast agent) and post-mortem (with contrast agent). The proposed contrast-enhancing technique consists of x-ray contrast agent uptake (silver nitrate and iodine) by immersion. To create the histology ground-truth, the CT scan of the paraffin block guided the sectioning towards specific planes of interest. The digitalized histological slides reveal the presence, extent, and appearance of the contrast agents in lung structures and organized aggregates of immune cells. These findings correlate with the contrast-enhanced micro-CT slice. The abnormal densities in the lungs due to tuberculosis disease are concentrated in the right tail of the lung intensity histograms. The increase in the width of the right tail (~376%) indicates a contrast enhancement of the details of the abnormal densities. Postmortem contrast agents enhance the x-ray attenuation in tuberculosis lesions to allow 3D visualization by polychromatic x-ray CT, providing an advantageous tool for virtual slicing of whole lungs. The proposed contrast-enhancing technique combined with computational methods and the diverse micro-CT modalities will open the doors to the stratification of lesion types associated with infectious diseases.

Applications of Light-Sheet Microscopy in Microdevices.

Light-sheet fluorescence microscopy (LSFM) has been present in cell biology laboratories for quite some time, mainly as custom-made systems, with imaging applications ranging from single cells (in the micrometer scale) to small organisms (in the millimeter scale). Such microscopes distinguish themselves for having very low phototoxicity levels and high spatial and temporal resolution, properties that make them ideal for a large range of applications. These include the study of cellular dynamics, in particular cellular motion which is essential to processes such as tumor metastasis and tissue development. Experimental setups make extensive use of microdevices (bioMEMS) that provide better control over the substrate environment than traditional cell culture experiments. For example, to mimic in vivo conditions, experiment biochemical dynamics, and trap, move or count cells. Microdevices provide a higher degree of empirical complexity but, so far, most have been designed to be imaged through wide-field or confocal microscopes. Nonetheless, the properties of LSFM render it ideal for 3D characterization of active cells. When working with microdevices, confocal microscopy is more widespread than LSFM even though it suffers from higher phototoxicity and slower acquisition speeds. It is sometimes possible to illuminate with a light-sheet microdevices designed for confocal microscopes. However, these bioMEMS must be redesigned to exploit the full potential of LSFM and image more frequently on a wider scale phenomena such as motion, traction, differentiation, and diffusion of molecules. The use of microdevices for LSFM has extended beyond cell tracking studies into experiments regarding cytometry, spheroid cultures and lab-on-a-chip automation. Due to light-sheet microscopy being in its early stages, a setup of these characteristics demands some degree of optical expertise; and designing three-dimensional microdevices requires facilities, ingenuity, and experience in microfabrication. In this paper, we explore different approaches where light-sheet microscopy can achieve single-cell and subcellular resolution within microdevices, and provide a few pointers on how these experiments may be improved.

NIK as a Druggable Mediator of Tissue Injury.

NF-κB-inducing kinase (NIK, MAP3K14) is best known as the apical kinase that triggers non-canonical NF-κB activation and by its role in the immune system. Recent data indicate a role for NIK expressed by non-lymphoid cells in cancer, kidney disease, liver injury, glucose homeostasis, osteosarcopenia, vascular calcification, hematopoiesis, and endothelial function. The spectrum of NIK-associated disease now ranges from immunodeficiency (when NIK is defective) to autoimmunity, cancer, sterile inflammation, fibrosis, and metabolic disease when NIK is overactive. The development of novel small-molecule NIK inhibitors has paved the way to test NIK targeting to treat disease in vivo, and may eventually lead to NIK targeting in the clinic. In addition, NIK activators are being explored for specific conditions such as myeloid leukemia.

MAP3K kinases and kidney injury.

Mitogen-activated protein kinases (MAP kinases) are functionally connected kinases that regulate key cellular process involved in kidney disease such as all survival, death, differentiation and proliferation. The typical MAP kinase module is composed by a cascade of three kinases: a MAP kinase kinase kinase (MAP3K) that phosphorylates and activates a MAP kinase kinase (MAP2K) which phosphorylates a MAP kinase (MAPK). While the role of MAPKs such as ERK, p38 and JNK has been well characterized in experimental kidney injury, much less is known about the apical kinases in the cascade, the MAP3Ks. There are 24 characterized MAP3K (MAP3K1 to MAP3K21 plus RAF1, BRAF and ARAF). We now review current knowledge on the involvement of MAP3K in non-malignant kidney disease and the therapeutic tools available. There is in vivo interventional evidence clearly supporting a role for MAP3K5 (ASK1) and MAP3K14 (NIK) in the pathogenesis of experimental kidney disease. Indeed, the ASK1 inhibitor Selonsertib has undergone clinical trials for diabetic kidney disease. Additionally, although MAP3K7 (MEKK7, TAK1) is required for kidney development, acutely targeting MAP3K7 protected from acute and chronic kidney injury; and targeting MAP3K8 (TPL2/Cot) protected from acute kidney injury. By contrast MAP3K15 (ASK3) may protect from hypertension and BRAF inhibitors in clinical use may induced acute kidney injury and nephrotic syndrome. Given their role as upstream regulators of intracellular signaling, MAP3K are potential therapeutic targets in kidney injury, as demonstrated for some of them. However, the role of most MAP3K in kidney disease remains unexplored.

Augmented acquisition of cocaine self-administration and altered brain glucose metabolism in adult female but not male rats exposed to a cannabinoid agonist during adolescence.

Marijuana consumption during adolescence has been proposed to be a stepping-stone for adult cocaine addiction. However, experimental evidence for this hypothesis is missing. In this work we chronically injected male and female Wistar rats with either the cannabinoid agonist CP 55,940 (CP; 0.4 mg/kg) or its corresponding vehicle. Adult acquisition (seven 30 min daily sessions) and maintenance (fourteen 2 h daily sessions) of cocaine self-administration (1 mg/kg), food-reinforced operant learning under conditions of normal (ad libitum access to food), and high motivation (food-restriction schedule) were measured. Additionally, brain metabolic activity was analyzed by means of [(18)F]-fluorodeoxyglucose positron emission tomography. During the acquisition phase, female CP-treated rats showed a higher rate of cocaine self-administration as compared to vehicle-treated females and males; no differences were found between both male groups. This effect disappeared in the maintenance phase. Moreover, no differences among groups were evident in the food-reinforced operant task, pointing to the cocaine-specific nature of the effect seen in self-administration rather than a general change in reward processing. Basal brain metabolic activity also changed in CP-treated females when compared to their vehicle-treated counterparts with no differences being found in the males; more specifically we observed a hyper activation of the frontal cortex and a hypo activation of the amygdalo-entorhinal cortex. Our results suggest that a chronic exposure to cannabinoids during adolescence alters the susceptibility to acquire cocaine self-administration, in a sex-specific fashion. This increased susceptibility could be related to the changes in brain metabolic activity induced by cannabinoids during adolescence.

High resolution image in bone biology II. Review of the literature.

Bone microstructure has usually been assessed by obtaining samples invasively and analyzing them with conventional histomorphometric methods. Improvements in high-resolution image acquisition systems have enabled non-invasive assessment of bone morphology and a more precise 3-D evaluation by means of "virtual biopsies", permitting bone assessment in regeneration or remodeling processes. Among other applications, this imaging technique can be used for the ultrastructural analysis of bone and for studies of regeneration techniques, biomechanics in bone physiotherapy, and periimplant bone healing. This review describes the different applications of high-resolution imaging techniques in bone biology and the morphometric results obtained with these images in mechanobiology in general and maxillary bone in particular.

Unsupervised CT Lung Image Segmentation of a Mycobacterium Tuberculosis Infection Model.

Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis that produces pulmonary damage. Radiological imaging is the preferred technique for the assessment of TB longitudinal course. Computer-assisted identification of biomarkers eases the work of the radiologist by providing a quantitative assessment of disease. Lung segmentation is the step before biomarker extraction. In this study, we present an automatic procedure that enables robust segmentation of damaged lungs that have lesions attached to the parenchyma and are affected by respiratory movement artifacts in a Mycobacterium Tuberculosis infection model. Its main steps are the extraction of the healthy lung tissue and the airway tree followed by elimination of the fuzzy boundaries. Its performance was compared with respect to a segmentation obtained using: (1) a semi-automatic tool and (2) an approach based on fuzzy connectedness. A consensus segmentation resulting from the majority voting of three experts' annotations was considered our ground truth. The proposed approach improves the overlap indicators (Dice similarity coefficient, 94% ± 4%) and the surface similarity coefficients (Hausdorff distance, 8.64 mm ± 7.36 mm) in the majority of the most difficult-to-segment slices. Results indicate that the refined lung segmentations generated could facilitate the extraction of meaningful quantitative data on disease burden.