Effects of vasoactive intestinal contractor (VIC) and endothelin on intracellular calcium level in neuroblastoma NG108-15 cells

Effects on [Ca2+]i levels of endothelin-l (ET) and vasoactive intestinal contractor peptide (VIC), which is a novel member of the endothelin family, were examined in fura 2-loaded neuroblastoma NG108-15 cells. VIC was found to be a very effective stimulus for intracellular Ca2+ mobilization and to be more potent than ET. Intracellular calcium response to sequential addition of two stimulants exhibited the homologous desensitization of either ET or VIC, but no heterologous desensitization between ET and VIC. This indicates evidence suggesting that these two peptides act through distinct receptors.     

Molecular pathogenesis of malignant melanoma: a different perspective from the studies of melanocytic nevus and acral melanoma

The Clark model for melanoma progression emphasizes a series of histopathological changes beginning from benign melanocytic nevus to melanoma via dysplastic nevus. Several models of the genetic basis of melanoma development and progression are based on this Clark’s multi-step model, and predict that the acquisition of a BRAF mutation can be a founder event in melanocytic neoplasia. However, our recent investigations have challenged this view, showing the polyclonality of BRAF mutations in melanocytic nevi. Furthermore, it is suggested that many melanomas, including acral and mucosal melanomas, arise de novo, not from melanocytic nevus. While mutations of the BRAF gene are frequent in melanomas on non-chronic sun damaged skin which are prevalent in Caucasians, acral and mucosal melanomas harbor mutations of the KIT gene as well as the amplifications of cyclin D1 or cyclin-dependent kinase 4 gene. Amplifications of the cyclin D1 gene are detected in normal-looking ‘field melanocytes’, which represent a latent progression phase of acral melanoma that precedes the stage of atypical melanocyte proliferation in the epidermis. Based on these observations, we propose an alternative genetic progression model for melanoma.     

Ageing Effect Evaluation on HD-sEMG Signals Using CCA Approach

ObjectivesThe objective of the proposed study is to exploit the technology of high-density surface electromyography (HD-sEMG), in order to evaluate the muscle activation in young and elderly subjects during a daily life gesture, namely, Sit To Stand (STS), using wireless connected ambulatory equipment (TMSi©) and Blind Source Separation (BSS) approach with Canonical Correlation Analysis (CCA).Materials and methodsSixteen subjects participated (50% females) divided into two categories (‘H1’: young (30.62 yrs ±5.92, 23.95 kg/m² ±3.08), versus ‘H2’: old (61.87 yrs ±7.98, 23.4 kg/m² ±3.38)), in the recording of HD-sEMG signals, using 32-electrodes square grids (4×8), during Sit To Stand (STS) motion, three times at spontaneous speed. The studied muscle is the Rectus Femoris (RF) muscle. The recorded HD-sEMG signals were analyzed with CCA approach to extract correlation coefficient sets according to two age categories (young versus old), in order to evaluate its discriminating power with ageing. Statistical tests (t-test) were used to evaluate the discrimination for these two categories.ResultsThe calculation of CCA correlation coefficients showed a significant difference between young and old category concerning the mean CCA correlation coefficient (P<0.001***) and also the standard deviation of the CCA correlation coefficients (P<0.0001****).ConclusionThe obtained results are promising and indicate a clear difference between the obtained source variability using CCA method between the young and the old tested subjects during daily life motion. Furthermore, these estimated sources seem to be impacted by both anatomical and functional modifications with ageing.     

Supplementation of Taurine Insulates Against Oxidative Stress,Confers Neuroprotection and Attenuates Memory Impairment in Noise Stress Exposed Male Wistar Rats

Noise has always been an important environmental factor that induces health problems in the general population. Due to ever increasing noise pollution, humans are facing multiple auditory and non-auditory problems including neuropsychiatric disorders. In modern day life it is impossible to avoid noise due to the rapid industrialization of society. Continuous exposure to noise stress creates a disturbance in brain function which may lead to memory disorder. Therefore, it is necessary to find preventive measures to reduce the deleterious effects of noise exposure. Supplementation of taurine, a semi essential amino acid, is reported to alleviate psychiatric disorders. In this study noise-exposed (100 db; 3 h daily for 15 days) rats were supplemented with taurine at a dose of 100 mg/kg for 15 days. Spatial and recognition memory was assessed using the Morris water maze and novel object recognition task, respectively. Results of this study showed a reversal of noise-induced memory impairment in rats. The derangements of catecholaminergic and serotonergic levels in the hippocampus and altered brain antioxidant enzyme activity due to noise exposure were also restored by taurine administration. This study highlights the importance of taurine supplementation to mitigate noise-induced impaired memory via normalizing the neurochemical functions and reducing oxidative stress in rat brain.

     

Fifteen compelling open questions in plant cell biology

As scientists, we are at least as excited about the open questions—the things we do not know—as the discoveries. Here, we asked 15 experts to describe the most compelling open questions in plant cell biology. These are their questions: How are organelle identity, domains, and boundaries maintained under the continuous flux of vesicle trafficking and membrane remodeling? Is the plant cortical microtubule cytoskeleton a mechanosensory apparatus? How are the cellular pathways of cell wall synthesis, assembly, modification, and integrity sensing linked in plants? Why do plasmodesmata open and close? Is there retrograde signaling from vacuoles to the nucleus? How do root cells accommodate fungal endosymbionts? What is the role of cell edges in plant morphogenesis? How is the cell division site determined? What are the emergent effects of polyploidy on the biology of the cell, and how are any such “rules” conditioned by cell type? Can mechanical forces trigger new cell fates in plants? How does a single differentiated somatic cell reprogram and gain pluripotency? How does polarity develop de-novo in isolated plant cells? What is the spectrum of cellular functions for membraneless organelles and intrinsically disordered proteins? How do plants deal with internal noise? How does order emerge in cells and propagate to organs and organisms from complex dynamical processes? We hope you find the discussions of these questions thought provoking and inspiring.

We asked 15 experts to address what they consider to be the most compelling open questions in plant cell biology and these are their questions.     

Photometric Application of the Gram Stain Method To Characterize Natural Bacterial Populations in Aquatic Environments

The Gram stain method was applied to the photometric characterization of aquatic bacterial populations with a charge-coupled device camera and an image analyzer. Escherichia coli and Bacillus subtilis were used as standards of typical gram-negative and gram-positive bacteria, respectively. A mounting agent to obtain clear images of Gram-stained bacteria on Nuclepore membrane filters was developed. The bacterial stainability by the Gram stain was indicated by the Gram stain index (GSI), which was applicable not only to the dichotomous classification of bacteria but also to the characterization of cell wall structure. The GSI spectra of natural bacterial populations in water with various levels of eutrophication showed a distinct profile, suggesting possible staining specificity that indicates the presence of a particular bacterial population in the aquatic environment.Gram’s method is the most important and fundamental orthodox method for bacterial identification. It classifies bacteria into two groups, gram-negative and gram-positive. The mechanism of Gram staining is based on the fundamental structural and chemical attributes of bacterial cell walls. The cell walls of gram-positive bacteria have a high percentage of peptidoglycan, while those of gram-negative bacteria have only a thin peptidoglycan layer (1–3, 6). In Gram’s method, an insoluble dye-iodine complex is formed inside bacterial cells and is extracted by alcohol from gram-negative but not gram-positive bacteria (6, 12, 16). There are taxonomically gram-variable species, but some cells of gram-negative or gram-positive species may show gram-variable characteristics due to environmental stress, such as unsuitable nutrients, temperature, pH, or electrolytes (3).Functional differences between gram-positive and gram-negative cell walls have been studied with special emphasis on nutrient uptake from the ambient environment. Gram-negative bacteria have a periplasmic space between the lipopolysaccharide layer and the plasma membrane. In this space, binding proteins initially attach to nutrients and take them to a membrane carrier. Gram-positive bacteria lack the periplasmic space and are believed to have no binding proteins (9). Therefore, nutrient uptake from the environment is easier for gram-negative bacteria than for gram-positive bacteria. Because of this difference, the population density of gram-negative bacteria in more oligotrophic environments could be higher than that of gram-positive bacteria (20).Gram staining is commonly used only to reflect cell wall structure. If Gram staining characterizes not only simple taxonomical dichotomy but also multiple biological functions, it may also be used to correlate bacterial cell wall structure with related physiological responses to the environment. In particular, Gram staining could supply ecological information on natural bacterial populations that are difficult to culture by the present technology.Membrane filter methods are widely used for microscopy in aquatic microbiology because of the low population densities of bacteria in many aquatic environments (4, 11, 16). However, these methods sometimes have problems associated with microscopic observations, causing unclear images of bacterial cells on Nuclepore filters when used with the conventional mounting medium (immersion oil; refractive index [nd] = 1.514). Hence, a suitable mounting agent must be applied to obtain precise image analyses of Gram-stained bacteria on Nuclepore filters.In this study, we have established a distinct method to characterize photometric Gram stain images; it involves the Gram stain index (GSI) for specifying natural bacterial populations in various aquatic environments. For this purpose, we have standardized the GSI of typical gram-negative and gram-positive bacteria by using Escherichia coli and Bacillus subtilis, respectively, and compared these GSI values to those of natural bacterial populations of several freshwater environments. The natural waters we investigated were Hyoutaro-ike pond, Matsumi-ike bog, and Lake Kasumigaura, which are oligotrophic, mesotrophic, and eutrophic water bodies, respectively, as previously determined (8, 10, 13, 18, 22, 23).     

Direct Determination of Phosphatase Activity from Physiological Substrates in Cells

A direct and continuous approach to determine simultaneously protein and phosphate concentrations in cells and kinetics of phosphate release from physiological substrates by cells without any labeling has been developed. Among the enzymes having a phosphatase activity, tissue non-specific alkaline phosphatase (TNAP) performs indispensable, multiple functions in humans. It is expressed in numerous tissues with high levels detected in bones, liver and neurons. It is absolutely required for bone mineralization and also necessary for neurotransmitter synthesis. We provided the proof of concept that infrared spectroscopy is a reliable assay to determine a phosphatase activity in the osteoblasts. For the first time, an overall specific phosphatase activity in cells was determined in a single step by measuring simultaneously protein and substrate concentrations. We found specific activities in osteoblast like cells amounting to 116 ± 13 nmol min^-1 mg^-1 for PPi, to 56 ± 11 nmol min^-1 mg^-1 for AMP, to 79 ± 23 nmol min^-1 mg^-1 for beta-glycerophosphate and to 73 ± 15 nmol min^-1 mg^-1 for 1-alpha-D glucose phosphate. The assay was also effective to monitor phosphatase activity in primary osteoblasts and in matrix vesicles. The use of levamisole – a TNAP inhibitor- served to demonstrate that a part of the phosphatase activity originated from this enzyme. An IC₅₀ value of 1.16 ± 0.03 mM was obtained for the inhibition of phosphatase activity of levamisole in osteoblast like cells. The infrared assay could be extended to determine any type of phosphatase activity in other cells. It may serve as a metabolomic tool to monitor an overall phosphatase activity including acid phosphatases or other related enzymes.     

A Computational Framework for 3D Mechanical Modeling of Plant Morphogenesis with Cellular Resolution

The link between genetic regulation and the definition of form and size during morphogenesis remains largely an open question in both plant and animal biology. This is partially due to the complexity of the process, involving extensive molecular networks, multiple feedbacks between different scales of organization and physical forces operating at multiple levels. Here we present a conceptual and modeling framework aimed at generating an integrated understanding of morphogenesis in plants. This framework is based on the biophysical properties of plant cells, which are under high internal turgor pressure, and are prevented from bursting because of the presence of a rigid cell wall. To control cell growth, the underlying molecular networks must interfere locally with the elastic and/or plastic extensibility of this cell wall. We present a model in the form of a three dimensional (3D) virtual tissue, where growth depends on the local modulation of wall mechanical properties and turgor pressure. The model shows how forces generated by turgor-pressure can act both cell autonomously and non-cell autonomously to drive growth in different directions. We use simulations to explore lateral organ formation at the shoot apical meristem. Although different scenarios lead to similar shape changes, they are not equivalent and lead to different, testable predictions regarding the mechanical and geometrical properties of the growing lateral organs. Using flower development as an example, we further show how a limited number of gene activities can explain the complex shape changes that accompany organ outgrowth.     

Increase in Dickkopf-1 Serum Level in Recent Spondyloarthritis. Data from the DESIR Cohort

Objectives

To investigate DKK-1 and SOST serum levels among patients with recent inflammatory back pain (IBP) fulfilling ASAS criteria for SpA and associated factors.

Methods

The DESIR cohort is a prospective, multicenter French cohort of 708 patients with early IBP (duration >3 months and <3 years) suggestive of AxSpA. DKK-1 and SOST serum levels were assessed at baseline and were compared between the subgroup of patients fulfilling ASAS criteria for SpA (n = 486; 68.6%) and 80 healthy controls.

Results

Mean SOST serum levels were lower in ASAS+ patients than healthy controls (49.21 ± 25.9 vs. 87.8 ± 26 pmol/L; p<0.0001). In multivariate analysis, age (p = 5.4 10⁻⁹), CRP level (p<0.0001) and serum DKK-1 level (p = 0.001) were associated with SOST level. Mean DKK-1 serum levels were higher in axial SpA patients than controls (30.03 ± 15.5 vs. 11.6 ± 4.2 pmol/L; p<0.0001). In multivariate analysis, DKK-1 serum levels were associated with male gender (p = 0.03), CRP level (p = 0.006), SOST serum level (p = 0.002) and presence of sacroiliitis on radiography (p = 0.05). Genetic association testing of 10 SNPs encompassing the DKK-1 locus failed to demonstrate a significant contribution of genetics to control of DKK-1 serum levels.

Conclusions

DKK-1 serum levels were increased and SOST levels were decreased among a large cohort of patients with early axial SpA compared to healthy controls. DKK-1 serum levels were mostly associated with biological inflammation and SOST serum levels.