Mevalonate pathway analysis of Saccharomyces cerevisiae during bioisoprene synthesis

Isoprene, synthesized through two complementary biosynthetic routes known as the mevalonate (MVA) pathway and the deoxy-xylulose phosphate pathway, is a valuable monomer that is used for rubber and several other chemical industries. Despite the recent interest in the industrial and biomedical applications of isoprene and its derivatives, the complexity of controlling its chemical synthesis due to the formation of greenhouse gases is a significant problem. To overcome the productivity and yield challenges, in addition to generating environmental and economic benefits, this study aimed to focus on the direct fermentation of cellulosic materials into bioisoprene. In this study, bioisoprene was synthesized via a biotransformation process through enzymatic hydrolysis of cassava peel using Aspergillus niger 11JK and Saccharomyces cerevisiae 19KB strain. The mevalonate (MVA) pathway (synthetic route) exploited during bioisoprene production by S. cerevisiae 19KB strain was investigated using the hydrolyzed cassava peel broth. The obtained crude extract was analyzed for bioisoprene yield and enzymatic activities using Gas chromatography. Furthermore, results of the size exclusion chromatography revealed the presence of polysaccharide hydrolyzing enzymes (e.g., amylase and cellulase), and mevalonate pathway enzymes, including isoprene synthase, mevalonate-5-diphosphate decarboxylase, and isopentyl phosphate kinase, in addition to isoprene, mevalonic acid (MVA), and its isomer dimethylallyl diphosphate (DMAPP). Based on the results obtained in this study, bioisoprene synthesis via direct fermentation of cheap and abundant carbon sources such as cassava peel using the S. cerevisiae 19KB strain will overcome the high production costs and low yield challenges of bioisoprene, thus generating significant environmental and economic benefits

Thyrotropin-releasing hormone (TRH) promotes wound re-epithelialisation in frog and human skin

There remains a critical need for new therapeutics that promote wound healing in patients suffering from chronic skin wounds. This is, in part, due to a shortage of simple, physiologically and clinically relevant test systems for investigating candidate agents. The skin of amphibians possesses a remarkable regenerative capacity, which remains insufficiently explored for clinical purposes. Combining comparative biology with a translational medicine approach, we report the development and application of a simple ex vivo frog (Xenopus tropicalis) skin organ culture system that permits exploration of the effects of amphibian skin-derived agents on re-epithelialisation in both frog and human skin. Using this amphibian model, we identify thyrotropin-releasing hormone (TRH) as a novel stimulant of epidermal regeneration. Moving to a complementary human ex vivo wounded skin assay, we demonstrate that the effects of TRH are conserved across the amphibian-mammalian divide: TRH stimulates wound closure and formation of neo-epidermis in organ-cultured human skin, accompanied by increased keratinocyte proliferation and wound healing-associated differentiation (cytokeratin 6 expression). Thus, TRH represents a novel, clinically relevant neuroendocrine wound repair promoter that deserves further exploration. These complementary frog and human skin ex vivo assays encourage a comparative biology approach in future wound healing research so as to facilitate the rapid identification and preclinical testing of novel, evolutionarily conserved, and clinically relevant wound healing promoters

Fast, automated measurement of nematode swimming (thrashing) without morphometry

Background: The "thrashing assay", in which nematodes are placed in liquid and the frequency of lateral swimming ("thrashing") movements estimated, is a well-established method for measuring motility in the genetic model organism Caenorhabditis elegans as well as in parasitic nematodes. It is used as an index of the effects of drugs, chemicals or mutations on motility and has proved useful in identifying mutants affecting behaviour. However, the method is laborious, subject to experimenter error, and therefore does not permit high-throughput applications. Existing automation methods usually involve analysis of worm shape, but this is computationally demanding and error-prone. Here we present a novel, robust and rapid method of automatically counting the thrashing frequency of worms that avoids morphometry but nonetheless gives a direct measure of thrashing frequency. Our method uses principal components analysis to remove the background, followed by computation of a covariance matrix of the remaining image frames from which the interval between statistically-similar frames is estimated. Results: We tested the performance of our covariance method in measuring thrashing rates of worms using mutations that affect motility and found that it accurately substituted for laborious, manual measurements over a wide range of thrashing rates. The algorithm used also enabled us to determine a dose-dependent inhibition of thrashing frequency by the anthelmintic drug, levamisole, illustrating the suitability of the system for assaying the effects of drugs and chemicals on motility. Furthermore, the algorithm successfully measured the actions of levamisole on a parasitic nematode, Haemonchus contortus, which undergoes complex contorted shapes whilst swimming, without alterations in the code or of any parameters, indicating that it is applicable to different nematode species, including parasitic nematodes. Our method is capable of analyzing a 30 s movie in less than 30 s and can therefore be deployed in rapid screens. Conclusion: We demonstrate that a covariance-based method yields a fast, reliable, automated measurement of C. elegans motility which can replace the far more time-consuming, manual method. The absence of a morphometry step means that the method can be applied to any nematode that swims in liquid and, together with its speed, this simplicity lends itself to deployment in large-scale chemical and genetic screens. </p

Sensory Transduction Channel Subunits, tax-4 and tax-2, Modify Presynaptic Molecular Architecture in C. elegans

During development, neural activity is important for forming proper connections in neural networks. The effect of activity on the gross morphology and synaptic strength of neurons has been well documented, but little is known about how activity affects different molecular components during development. Here, we examine the localization of four fluorescently-tagged presynaptic proteins, RAB-3, SNG-1/synaptogyrin, SYD-2/Liprin-α, and SAD-1/SAD kinase, in the C. elegans thermosensory neuron AFD. We show that tax-4 and tax-2, two genes that encode the cyclic nucleotide-gated channel necessary for sensory transduction in AFD, disrupt the localization of all four proteins. In wild-type animals, the synaptic vesicle (SV) markers RAB-3 and SNG-1 and the active zone markers SYD-2 and SAD-1 localize in a stereotyped, punctate pattern in the AFD axon. In tax-4 and tax-2 mutants, SV and SYD-2 puncta are more numerous and less intense. Interestingly, SAD-1 puncta are also less intense but do not increase in number. The change in puncta number can be rescued cell-autonomously in AFD. These results suggest that sensory transduction genes tax-4 and tax-2 are necessary for the proper assembly of presynapses

Stress Hormones Receptors in the Amygdala Mediate the Effects of Stress on the Consolidation, but Not the Retrieval, of a Non Aversive Spatial Task

This study examined the effects of the arousal level of the rat and exposure to a behavioral stressor on acquisition, consolidation and retrieval of a non-aversive hippocampal-dependent learning paradigm, the object location task. Learning was tested under two arousal conditions: no previous habituation to the experimental context (high novelty stress/arousal level) or extensive prior habituation (reduced novelty stress/arousal level). Results indicated that in the habituated rats, exposure to an out-of-context stressor (i.e, elevated platform stress) impaired consolidation and retrieval, but not acquisition, of the task. Non-habituated animals under both stressed and control conditions did not show retention of the task. In habituated rats, RU-486 (10 ng/side), a glucocorticoid receptor (GR) antagonist, or propranolol (0.75 µg/side), a beta-adrenergic antagonist, injected into the basolateral amygdala (BLA), prevented the impairing effects of the stressor on consolidation, but not on retrieval. The CB1/CB2 receptor agonist WIN55,212-2 (WIN, 5 µg/side) microinjected into the BLA did not prevent the effects of stress on either consolidation or retrieval. Taken together the results suggest that: (i) GR and β-adrenergic receptors in the BLA mediate the impairing effects of stress on the consolidation, but not the retrieval, of a neutral, non-aversive hippocampal-dependent task, (ii) the impairing effects of stress on hippocampal consolidation and retrieval are mediated by different neural mechanisms (i.e., different neurotransmitters or different brain areas), and (iii) the effects of stress on memory depend on the interaction between several main factors such as the stage of memory processing under investigation, the animal's level of arousal and the nature of the task (neutral or aversive)

Reduction in Inter-Hemispheric Connectivity in Disorders of Consciousness

Clinical diagnosis of disorders of consciousness (DOC) caused by brain injury poses great challenges since patients are often behaviorally unresponsive. A promising new approach towards objective DOC diagnosis may be offered by the analysis of ultra-slow (<0.1 Hz) spontaneous brain activity fluctuations measured with functional magnetic resonance imaging (fMRI) during the resting-state. Previous work has shown reduced functional connectivity within the “default network”, a subset of regions known to be deactivated during engaging tasks, which correlated with the degree of consciousness impairment. However, it remains unclear whether the breakdown of connectivity is restricted to the “default network”, and to what degree changes in functional connectivity can be observed at the single subject level. Here, we analyzed resting-state inter-hemispheric connectivity in three homotopic regions of interest, which could reliably be identified based on distinct anatomical landmarks, and were part of the “Extrinsic” (externally oriented, task positive) network (pre- and postcentral gyrus, and intraparietal sulcus). Resting-state fMRI data were acquired for a group of 11 healthy subjects and 8 DOC patients. At the group level, our results indicate decreased inter-hemispheric functional connectivity in subjects with impaired awareness as compared to subjects with intact awareness. Individual connectivity scores significantly correlated with the degree of consciousness. Furthermore, a single-case statistic indicated a significant deviation from the healthy sample in 5/8 patients. Importantly, of the three patients whose connectivity indices were comparable to the healthy sample, one was diagnosed as locked-in. Taken together, our results further highlight the clinical potential of resting-state connectivity analysis and might guide the way towards a connectivity measure complementing existing DOC diagnosis

The Thyroid Hormone Receptors Modulate the Skin Response to Retinoids

[Background]: Retinoids play an important role in skin homeostasis and when administered topically cause skin hyperplasia, abnormal epidermal differentiation and inflammation. Thyroidal status in humans also influences skin morphology and function and we have recently shown that the thyroid hormone receptors (TRs) are required for a normal proliferative response to 12-O-tetradecanolyphorbol-13-acetate (TPA) in mice. [Methodology/Principal Findings]: We have compared the epidermal response of mice lacking the thyroid hormone receptor binding isoforms TRα1 and TRβ to retinoids and TPA. Reduced hyperplasia and a decreased number of proliferating cells in the basal layer in response to 9-cis-RA and TPA were found in the epidermis of TR-deficient mice. Nuclear levels of proteins important for cell proliferation were altered, and expression of keratins 5 and 6 was also reduced, concomitantly with the decreased number of epidermal cell layers. In control mice the retinoid (but not TPA) induced parakeratosis and diminished expression of keratin 10 and loricrin, markers of early and terminal epidermal differentiation, respectively. This reduction was more accentuated in the TR deficient animals, whereas they did not present parakeratosis. Therefore, TRs modulate both the proliferative response to retinoids and their inhibitory effects on skin differentiation. Reduced proliferation, which was reversed upon thyroxine treatment, was also found in hypothyroid mice, demonstrating that thyroid hormone binding to TRs is required for the normal response to retinoids. In addition, the mRNA levels of the pro-inflammatory cytokines TNFα and IL-6 and the chemotactic proteins S1008A and S1008B were significantly elevated in the skin of TR knock-out mice after TPA or 9-cis-RA treatment and immune cell infiltration was also enhanced. [Conclusions/significance]: Since retinoids are commonly used for the treatment of skin disorders, these results demonstrating that TRs regulate skin proliferation, differentiation and inflammation in response to these compounds could have not only physiological but also therapeutic implications.This work was supported by grants BFU2007-62402 and SAF2008-00121 from Ministerio de Ciencia e Innovación, RD06/0020/0036 and RD06/0020/0029 from the Fondo de Investigaciones Sanitarias and by the European Grant CRESCENDO (FP-018652).Peer reviewe

Nuclear cGMP-Dependent Kinase Regulates Gene Expression via Activity-Dependent Recruitment of a Conserved Histone Deacetylase Complex

Elevation of the second messenger cGMP by nitric oxide (NO) activates the cGMP-dependent protein kinase PKG, which is key in regulating cardiovascular, intestinal, and neuronal functions in mammals. The NO-cGMP-PKG signaling pathway is also a major therapeutic target for cardiovascular and male reproductive diseases. Despite widespread effects of PKG activation, few molecular targets of PKG are known. We study how EGL-4, the Caenorhabditis elegans PKG ortholog, modulates foraging behavior and egg-laying and seeks the downstream effectors of EGL-4 activity. Using a combination of unbiased forward genetic screen and proteomic analysis, we have identified a conserved SAEG-1/SAEG-2/HDA-2 histone deacetylase complex that is specifically recruited by activated nuclear EGL-4. Gene expression profiling by microarrays revealed >40 genes that are sensitive to EGL-4 activity in a SAEG-1–dependent manner. We present evidence that EGL-4 controls egg laying via one of these genes, Y45F10C.2, which encodes a novel protein that is expressed exclusively in the uterine epithelium. Our results indicate that, in addition to cytoplasmic functions, active EGL-4/PKG acts in the nucleus via a conserved Class I histone deacetylase complex to regulate gene expression pertinent to behavioral and physiological responses to cGMP. We also identify transcriptional targets of EGL-4 that carry out discrete components of the physiological response

Biology of human hair: Know your hair to control it

Hair can be engineered at different levels—its structure and surface—through modification of its constituent molecules, in particular proteins, but also the hair follicle (HF) can be genetically altered, in particular with the advent of siRNA-based applications. General aspects of hair biology are reviewed, as well as the most recent contributions to understanding hair pigmentation and the regulation of hair development. Focus will also be placed on the techniques developed specifically for delivering compounds of varying chemical nature to the HF, indicating methods for genetic/biochemical modulation of HF components for the treatment of hair diseases. Finally, hair fiber structure and chemical characteristics will be discussed as targets for keratin surface functionalization

The biology and genetics of curly hair

YesHair fibres show wide diversity across and within all human populations, suggesting that hair fibre form and colour have been subject to much adaptive pressure over thousands of years. All human hair fibres typically have the same basic structure. However, the three-dimensional shape of the entire fibre varies considerably depending on ethnicity and geography, with examples from very straight hair with no rotational turn about the long axis, to the tightly sprung coils of African races. The creation of the highly complex biomaterials in hair follicle and how these confer mechanical functions on the fibre so formed is a topic that remains relatively unexplained thus far. We review the current understanding on how hair fibres are formed into a nonlinear coiled form and which genetic and biological factors are thought to be responsible for hair shape. We report on a new GWAS comparing low and high curl individuals in South Africa, revealing strong links to polymorphic variation in trichohyalin, a copper transporter protein CUTC and the inner root sheath component keratin 74. This builds onto the growing knowledge base describing the control of curly hair formation.Unilever R&