Synchrotron X-ray micro-computed tomography as a tool for in situ elucidation of insect bacteriomes

Obligate bacterial endosymbionts are common, influential associates of arthropods, and are often found in specific organs termed bacteriomes. Three dimensional images of bacteriomes of the leafhopper Orosius albicinctus (Hemiptera: Cicadellidae) were reconstructed from synchrotron-based X-ray micro-computed tomography (CT). Results show that bilateral bacteriomes are located between the first and second abdominal tergites, are mushroom-shaped and consist two different types of tissue. Fluorescence in situ hybridization reveals that the primary bacterial symbiont Sulcia muelleri is in the ‘cap’ part of the of organ. The technique allows a noninvasive, in situ, means of visualizing bacteriomes and will facilitate understanding their form and function.     

Fusion genes: A promising tool combating against cancer

The driving roles of fusion genes during tumorigenesis have been recognized for decades, with efficacies demonstrated in clinical diagnosis and targeted therapy. With advances in sequencing technologies and computational biology, a surge in the identification of fusion genes has been witnessed during the past decade. The discovery and presence of splicing based fusions in normal tissues have challenged our canonical conceptions on fusion genes and offered us novel medical opportunities. The specificity of fusion genes to neoplastic tissues and their diverse functionalities during carcinogenesis foster them as promising tools in the battle against cancer. It is time to re-visit and comb through our cutting-edge knowledge on fusion genes to accelerate clinical translation of these internal markers. Urged as such, we are encouraged to categorize fusion events according to mechanisms leading to their generation, oncological consequences and clinical implications, offer insights on fusion occurrence across tumors from the system level, highlight feasible practices in fusion-related pharmaceutical development, and identify understudied yet important niches that may lead future research trend in this field.     

Near-infrared spectroscopy: promising diagnostic tool for viral infections

Although several methods, including enzyme-linked immunosorbent assay, polymerase chain reaction, immunofluorescent assay, and Western blotting, have been used for the diagnosis of viral infections, none of them is ideal in terms of cost-effectiveness, speed, and accuracy. Currently, the rate of outbreak of emerging viruses is increasing and therefore the development and establishment of analytical methods for such viral infections are becoming more important. Near-infrared (NIR) spectroscopy is a fast, multicomponent assay that enables non-invasive, non-destructive analysis. Recently, the diagnosis of viral infections using NIR spectroscopy has been attempted. In this review, the potential of the NIR method in the medical and virological fields is discussed.     

BMIT facility at the Canadian Light Source: Advances in X-ray phase-sensitive imaging

The BioMedical Imaging and Therapy (BMIT) facility [1,2] located at the Canadian Light Source, provides synchrotron-specific imaging and radiation therapy capabilities. There are two separate beamlines used for experiments: the bending magnet (05B1-1) and the insertion device (05ID-2) beamline.The bending magnet beamline provides access to monochromatic beam spanning a spectral range of 15–40 keV, and the beam is 240 mm wide in the POE-2 experimental hutch. Users can also perform experiments with polychromatic (pink) beam.The insertion device beamline was officially opened for general user program in 2015. The source for the ID beamline is a multi-pole, superconducting 4.3 T wiggler. The high field gives a critical energy over 20 keV. The optics hutches prepare a beam that is 220 mm wide in the last experimental hutch SOE-1. The monochromatic spectral range spans 25–150+ keV. Several different X-ray detectors are available for both beamlines, with resolutions ranging from 2 μm to 200 μm.BMIT provides a number of imaging techniques including standard absorption X-ray imaging, K-edge subtraction imaging (KES), in-line phase contrast imaging (also known as propagation based imaging, PBI) and Diffraction Enhanced Imaging/Analyzer Based Imaging (DEI/ABI), all in either projection or CT mode. PBI and DEI/ABI are particularly important tools for BMIT users since these techniques enable visualization of soft tissue and allow for low dose imaging.     

Pseudophosphorylation of cardiac myosin regulatory light chain: a promising new tool for treatment of cardiomyopathy

Many genetic mutations in sarcomeric proteins, including the cardiac myosin regulatory light chain (RLC) encoded by the MYL2 gene, have been implicated in familial cardiomyopathies. Yet, the molecular mechanisms by which these mutant proteins regulate cardiac muscle mechanics in health and disease remain poorly understood. Evidence has been accumulating that RLC phosphorylation has an influential role in striated muscle contraction and, in addition to the conventional modulation via Ca²⁺ binding to troponin C, it can regulate cardiac muscle function. In this review, we focus on RLC mutations that have been reported to cause cardiomyopathy phenotypes via compromised RLC phosphorylation and elaborate on pseudo-phosphorylation rescue mechanisms. This new methodology has been discussed as an emerging exploratory tool to understand the role of phosphorylation as well as a genetic modality to prevent/rescue cardiomyopathy phenotypes. Finally, we summarize structural effects post-phosphorylation, a phenomenon that leads to an ordered shift in the myosin S1 and RLC conformational equilibrium between two distinct states.     

Novel KV7 ion channel openers for the treatment of epilepsy and implications for detrusor tissue contraction

Neuronal voltage-gated potassium channels, K_V7s, are the molecular mediators of the M current and regulate membrane excitability in the central and peripheral neuronal systems. Herein, we report novel small molecule K_V7 openers that demonstrate anti-seizure activities in electroshock and pentylenetetrazol-induced seizure models without influencing Rotarod readouts in mice. The anti-seizure activity was determined to be proportional to the unbound concentration in the brain. K_V7 channels are also expressed in the bladder smooth muscle (detrusor) and activation of these channels may cause localized undesired effects. Therefore, the impact of individual K_V7 isoforms was investigated in human detrusor tissue using a panel of K_V7 openers with distinct activity profiles among K_V7 isoforms. KCNQ4 and KCNQ5 mRNA were highly expressed in detrusor tissue, yet a compound that has significantly reduced activity on homomeric K_V7.4 did not reduce detrusor contraction. This may suggest that the homomeric K_V7.4 channel plays a less significant role in bladder contraction and further investigation is needed.     

X-ray computed tomography: A promising tool to investigate the brachiopod shell interior. Effects on 3D modelling and taxonomy

Barremian and Cenomanian rhynchonelliform brachiopods, one Santonian and one Holocene species (Rhynchonellida and Terebratulida), have been observed using X-ray Computed Tomography in order to investigate the shell interior. Compared to transverse serial sections used formerly, X-ray CT is a promising tool. It permits 3D modelling of the brachidium, which is important for brachiopod classification. Four types of brachidium present during the Cretaceous have been observed (crura for the Rhynchonellida, short loop for Terebratulidina–Terebratulidae, ring-loop for Terebratulidina–Cancellothyrididae and long-loop for Terebratellidina), as well as relationships lophophore/brachidium in a Holocene species. Limitations are discussed, including the nature of the sediments enclosed between the valves, diagenesis, and the necessity to observe several age groups concerning the Terebratellidina. Finally, this non-destructive tool facilitates 3D reconstruction of inaccessible brachidia to improve brachiopod taxonomy and as an aid in curating collections.     

Mass spectrometry: A tool for on-line monitoring of animal cell cultures

The magnetic sector mass spectrometer is able to detect oxygen uptake and carbon dioxide production rates from animal cell cultivations performed in 101 biorectors. Such data have not been available with the use of classic exhaust gas analysis applying paramagnetic analyzers and infra-red sensors due to the insensitivity of the apparatus available. In the course of the present work we were able to demonstrate, that the oxygen uptake rate correlates to the number of viable cells. Additionally oxygen uptake rates supplied on-line information about the actual physiology of the cells: When the rates changed during the cultivation process, this immediately indicated the occurrence of limitations of components in the medium. The information could be useful in timing key events, such as performing splits or harvesting the bioreactor.Abbreviations OUR oxygen uptake rate - CDPR carbon dioxide production rate - RQ respiratory quotient This publication is dedicated to the 65 ^th birthday of Prof. Dr. F. Wagner, University of Braunschweig.     

Embryonic stem cells: a promising tool for cell replacement therapy

Embryonic stem (ES) cells are revolutionizing the field of developmental biology as a potential tool to understand the molecular mechanisms occurring during the process of differentiation from the embryonic stage to the adult phenotype. ES cells harvested from the inner cell mass (ICM) of the early embryo can proliferate indefinitely in vitro while retaining the ability to differentiate into all somatic cells. Emerging results from mice models with ES cells are promising and raising tremendous hope among the scientific community for the ES-cell based cell replacement therapy (CRT) of various severe diseases. ES cells could potentially revolutionize medicine by providing an unlimited renewable source of cells capable of replacing or repairing tissues that have been damaged in almost all degenerative diseases such as diabetes, myocardial infarction and Parkinson's disease. This review updates the progress of ES cell research in CRT, discusses about the problems encountered in the practical utility of ES cells in CRT and evaluates how far this approach is successful experimentally.     

Complete microscale profiling of tumor microangiogenesis: A microradiological methodology reveals fundamental aspects of tumor angiogenesis and yields an array of quantitative parameters for its characterization

Complete profiling would substantially facilitate the fundamental understanding of tumor angiogenesis and of possible anti-angiogenesis cancer treatments. We developed an integrated synchrotron-based methodology with excellent performances: detection of very small vessels by high spatial resolution (~ 1 μm) and nanoparticle contrast enhancement, in vivo dynamics investigations with high temporal resolution (~ 1 ms), and three-dimensional quantitative morphology parametrization by computer tracing. The smallest (3–10 μm) microvessels were found to constitute > 80% of the tumor vasculature and exhibit many structural anomalies. Practical applications are presented, including vessel microanalysis in xenografted tumors, monitoring the effects of anti-angiogenetic agents and in vivo detection of tumor vascular rheological properties.     

Joint refinement as a tool for thorough comparison between NMR and X-ray data and structures of HU protein

Joint refinement, i.e., the simultaneous refinement of a structure against both nuclear magnetic resonance (NMR) spectroscopic and X-ray crystallographic data, was performed on the HU protein from Bacillus stearothermophilus (HUBst). The procedure was aimed at investigating the compatibility of the two data sets and at identifying conflicting information. Wherever important differences were found, such as peptide flips in the main-chain conformation, the data were further analyzed to find the cause. The NMR data showed some errors arising either from the manual interpretation of the spectra or from the incorrect account for spin diffusion. The most important artefact inherent to the X-ray data is the crystal packing of the molecules: the effects range from the limitation of the freedom of the flexible parts of the HUBst molecule to possibly one of the peptide flips.     

A Comparison of Methodologies for the Study of Functional Transmitter Neurochemistry in Human Brain

A number of different approaches to the study of functional neurochemistry in human brain are discussed. The advantages and disadvantages of three main techniques are contrasted: (i) using animal tissue preparations as models of the human brain; (ii) using human peripheral tissue preparations as models of dynamic CNS processes; and (iii) studying human tissue, obtained postmortem, directly. Animal models are often readily obtained and reliable, and the high degree of inbreeding of common laboratory animals ensures that they usually yield consistent results. However, there are a number of human disorders for which animal models are either poor or unavailable, and species differences make extrapolation from the animal to the human case difficult. Human peripheral tissue models rely on a degree of homology between peripheral and CNS processes; in most cases, the evidence for such homologies derives from animal, rather than human, studies. Moreover, several examples are known where a peripheral process mimics the equivalent glial cell activity more closely than the neuronal, which can be a serious drawback for studies of neurotransmission. The use of postmortem human brain tissue presents a number of obvious difficulties, resulting from variations in the patient's age, agonal state, sex, preterminal medication, postmortem delay, etc. Human beings are genetically and nutritionally heterogeneous, so that data variability is usually greater here than when using tissue from laboratory animals. However, it is possible to control for a number of these factors, for example, by matching samples for basal metabolic rate and tissue integrity, and recently developed tissue freezing and storage techniques permit the use of within-subject experimental designs to help reduce experimental variation. A range of neurotransmitter functions are well retained in such tissue samples, so that regional variations, differential transmitter activities, drug effects, etc., can be studied in normal tissue samples, as well as in samples taken from cases of neurological and psychiatric disease. This allows, for example, changes in neuroanatomical indices to be correlated with localised alterations in a specific neurotransmitter function. A systematic approach to the analysis and matching of tissue samples is advocated. The three approaches should be considered to be complementary, especially for the study of human brain diseases.     

A computational tool for the simulation and optimization of microbial strains accounting integrated metabolic/regulatory information

Background and scope

Recently, a number of methods and tools have been proposed to allow the use of genome-scale metabolic models for the phenotype simulation and optimization of microbial strains, within the field of Metabolic Engineering (ME). One of the limitations of most of these algorithms and tools is the fact that only metabolic information is taken into account, disregarding knowledge on regulatory events.

Implementation and performances

This work proposes a novel software tool that implements methods for the phenotype simulation and optimization of microbial strains using integrated models, encompassing both metabolic and regulatory information. This tool is developed as a plug-in that runs over OptFlux, a computational platform that aims to be a reference tool for the ME community.

Availability

The plug-in is made available in the OptFlux web site (www.optflux.org) together with examples and documentation.     

Functional morphology of Tethya species (Porifera): 1. Quantitative 3D-analysis of Tethya wilhelma by synchrotron radiation based X-ray microtomography

Rhythmic body contraction is a phenomenon in the Porifera, which is only partly understood. As a foundation for the understanding of the functional morphology of the highly contractile Tethya wilhelma, we performed a qualitative and quantitative volumetric 3D-analysis of the morphology of a complete non-contracted specimen at resolutions of 5.2 and 6.9 μm, using synchrotron radiation based X-ray computed microtomography (SR-μCT). For the first time, we were able to visualize all three major body structures of a complete poriferan without dissection of the shock-frozen, fixed and contrasted specimen in a near-to-life confirmation: poriferan tissue, mineral skeleton and aquiferous system. Applying a ‘virtual cast’ technique allowed us to analyze the structural details of the complete canal structure. Our results imply an extensive re-circulation of water inside the poriferan due to well-developed by-pass-canals, connecting excurrent and incurrent system. Nevertheless, the oscule region is strictly separated from the incurrent system. Based on our data, we developed a hypothetical flow regime for T. wilhelma, which explains the necessity of by-pass canals to minimize pressure boosts in the canal system during contraction. Additionally, re-circulation optimizes nutrient uptake, within small-sized poriferans, like T. wilhelma. Quantitative analysis allowed us to measure volumes and surfaces, displaying remarkable organizational differences between choanosome and cortex, by means of distribution of morphological elements. The surface-to-volume ratio proved to be very high, underlining the importance of the poriferan pinacoderm. We support a pinacoderm-contraction hypothesis.Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .Dedicated to Prof. Dr. Michele Sarà (Genova, Italy), in honour of his 80th birthday in 2006.     

The insulin-mimetic effect of Morin: A promising molecule in diabetes treatment

Background

Type-2 diabetes is a worldwidely diffuse disease characterized by insulin resistance that arises from alterations of receptor and/or post-receptor events of insulin signalling. Studies performed with PTP1B-deficent mice demonstrated that PTP1B is the main negative regulator of insulin signalling. Inhibition or down regulation of this enzyme causes enhanced insulin sensitivity. Hence this enzyme represents the most attractive target for development of innovative anti-diabetic drugs.

Methods

Selection of new PTP1B inhibitors among an in house library of polyphenolic compounds was carried out screening their activity. The inhibition mechanism of Morin was determined by kinetic analyses. The cellular action of Morin was assayed on HepG2 cells. Analyses of the insulin signalling pathways was carried out by Western blot methods, glycogen synthesis was estimated by measuring the incorporation of [³H]-glucose, gluconeogenesis rate was assayed by measuring the glucose release in the cell medium. Cell growth was estimated by cell count. Docking analysis was conducted with SwissDock program.

Results

We demonstrated that Morin: i) is a non-competitive inhibitor of PTP1B displaying a K_i in the μM range; ii) increases the phosphorylation of the insulin receptor and Akt; iii) inhibits gluconeogenesis and enhances glycogen synthesis. Morin does not enhance cell growth.

Conclusions

We have identified Morin as a new small molecular non-competitive inhibitor of PTP1B, which behaves as an activator and sensitizer of the insulin receptor stimulating the metabolic pathways only.

General significance

Our study suggests that Morin is a useful lead for development of new low Mr compounds potentially active as antidiabetic drugs.     

X-ray microanalysis of chromatin-bound period IV metals inGlenodinium foliaceum: A binucleate dinoflagellate

Summary Each vegetative cell of the dinoflagellateGlenodinium foliaceum possesses two distinct types of nucleus, both of which have high levels of chromatin-bound Period IV (Periodic Table) metal elements.The typical dinoflagellate (dinocaryotic) nucleus has chromatin which differs from the atypical (supernumerary) nucleus in its high degree of condensation and in the related high levels of P, Ca, and Transition metals Fe, Ni, Cu, and Zn. The complete absence of detectable Fe and Ni in the supernumerary chromatin represents a major difference which may relate to differences in phyllogenetic origin of the two nuclei.The two types of chromatin show close similarities at the molecular level, including the possession of 40 atoms of Period IV elements per 100 atoms of P—of which approximately half are Ca atoms, and half Transition metals. In both cases, the levels of Ca and Zn show a high correlation with the level of P, suggesting a direct association of these particular metal atoms with nucleic acid phosphate groups. The close similarity in metal binding at the molecular level suggests that the association of Period IV elements with the two types of chromatin is unrelated to any differences in chromatin proteins—such as the presence or absence of histones.     

Gum ghatti: A promising polysaccharide for pharmaceutical applications

Gum ghatti (Anogeissus latifolia) or Indian gum is a complex non-starch polysaccharide. It has been widely employed in food, pharmaceuticals, paper and other industries primarily due to its excellent emulsification and thickening property. Other applications of gum ghatti are inadequately investigated owing to lack of information on it. Researchers in the recent years have shown a great interest in exploring its molecular structure and functional properties. This article is aimed at discussing the structural features, functional properties and applications of gum ghatti with an emphasis on its pharmaceutical potential.