Thermoanaerobacter thermohydrosulfuricus WC1 Shows Protein Complement Stability during Fermentation of Key Lignocellulose-Derived Substrates

Thermoanaerobacter spp. have long been considered suitable Clostridium thermocellum coculture partners for improving lignocellulosic biofuel production through consolidated bioprocessing. However, studies using “omic”-based profiling to better understand carbon utilization and biofuel producing pathways have been limited to only a few strains thus far. To better characterize carbon and electron flux pathways in the recently isolated, xylanolytic strain, Thermoanaerobacter thermohydrosulfuricus WC1, label-free quantitative proteomic analyses were combined with metabolic profiling. SWATH-MS proteomic analysis quantified 832 proteins in each of six proteomes isolated from mid-exponential-phase cells grown on xylose, cellobiose, or a mixture of both. Despite encoding genes consistent with a carbon catabolite repression network observed in other Gram-positive organisms, simultaneous consumption of both substrates was observed. Lactate was the major end product of fermentation under all conditions despite the high expression of gene products involved with ethanol and/or acetate synthesis, suggesting that carbon flux in this strain may be controlled via metabolite-based (allosteric) regulation or is constrained by metabolic bottlenecks. Cross-species “omic” comparative analyses confirmed similar expression patterns for end-product-forming gene products across diverse Thermoanaerobacter spp. It also identified differences in cofactor metabolism, which potentially contribute to differences in end-product distribution patterns between the strains analyzed. The analyses presented here improve our understanding of T. thermohydrosulfuricus WC1 metabolism and identify important physiological limitations to be addressed in its development as a biotechnologically relevant strain in ethanologenic designer cocultures through consolidated bioprocessing.     

Conditional expression of Spry1 in neural crest causes craniofacial and cardiac defects

Background  

Growth factors and their receptors are mediators of organogenesis and must be tightly regulated in a temporal and spatial manner for proper tissue morphogenesis. Intracellular regulators of growth factor signaling pathways provide an additional level of control. Members of the Sprouty family negatively regulate receptor tyrosine kinase pathways in several developmental contexts. To gain insight into the role of Spry1 in neural crest development, we analyzed the developmental effects of conditional expression of Spry1 in neural crest-derived tissues.     

Developmental changes in the responses of O2 uptake and ventilation to acutely declining O2 tensions in larval krill Meganyctiphanes norvegica

The effect of exposure to acutely declining oxygen tensions on O₂ uptake (M_O2) and ventilation has been investigated in different larval stages of Northern krill Meganyctiphanes norvegica (calytopis III/early furcilia I, late furcilia I, furcilia III and V). An ability to regulate M_O2 during acutely declining P_O2 began to appear about furcilia III (critical O₂ tension or P_c=15.4±0.73 kPa) and had improved by furcilia V (P_c=12.6±0.39 kPa). Hypoxia-related hyperventilation was achieved by an increase in pleopod (but not thoracic limb) activity (P_c∼11 kPa), a sensitivity which also appeared at, or just before, furcilia V even though an earlier stage (furcilia III) had a full compliment of functional setose pleopods. While this regulatory ability appeared as the gills were beginning to form, furcilia V is still early in gill ontogeny compared with adults. Preexposure to very moderate hypoxia (60% and 70% O₂ saturation) of furcilia III and V resulted in substantial mortality, but where it did not (furcilia V, 80% O₂ saturation), there was no effect of keeping krill at this P_O2 on either M_O2 or ventilation, suggesting that the development of respiratory regulation in M. norvegica is not open to environmental influence in the same way as for other crustaceans. We suggest that ontogeny of pleopod control provides furcilia V+ with both a stronger means of propulsion, allowing the ontogeny of DVM but also with an ability to regulate M_O2 during exposure to acutely declining P_O2s. The onset of respiratory regulation (furcilia V) preceded the onset of DVM (furcilia VI+). As pleopod ontogeny is associated intimately with the ontogeny of DVM and respiratory regulation, in the Gullmarsfjord this co-occurrence is fortuitous as krill can be required during DVM to migrate into hypoxic water which they are not equipped to deal with, in physiological terms, before furcilia V.     

Anaerobic capacity of a crustacean sensitive to low environmental oxygen tensions,the freshwater amphipod Gammarus pulex (L.)

We investigated the accumulation of haemolymph L-lactate in response to anoxia, progressive hypoxia and activity, in a species that rarely encounters low PO₂ in its natural environment, the freshwater amphipod Gammarus pulex (L.). Individuals survived <1.5 h anoxia and accumulated L-lactate in the haemolymph at a rate of 10.26 mmol l^–1 h^–1. No lactate was excreted into the medium during this time. Despite the fact that most individuals of G. pulex were oxyconformers, exposure to acutely declining PO₂s did not result in a switch to anaerobic pathways until PO₂ < 4.4 kPa. Even then the concentrations accumulated remained very low until PO₂ < 0.46 kPa. There was no accumulation of L-lactate in the haemolymph as a result of exhaustive activity. Consequently, it was suggested that (a) it cannot be automatically assumed that anaerobic metabolism is switched on only at the point at which oxyregulation breaks down, (b) that the poor anaerobic capacity observed plays little, if any, role in the metabolic response to hypoxia by Gammarus pulex.     

Medical Innovation in a Children's Hospital: ‘Diseases desperate grown by desperate appliance are relieved,or not at all’

A balance needs to be struck between facilitating compassionate access to innovative treatments for those in desperate need, and the duty to protect such vulnerable individuals from the harms of untested/unlicensed treatments. We introduced a principle‐based framework (2009) to evaluate such requests and describe its application in the context of recently evolved UK, US and European regulatory processes. 24 referrals (20 individual; four group) were received by our quaternary children's hospital Clinical Ethics Committee (CEC) over the 5‐year period (2011‐16). The CEC‐rapid response group evaluated individual cases within 48‐hours; the main referrers being haematology/oncology, immunology or transplant services (14). Most requests were for drug/vaccine/pre‐trial access (13) or biological/cellular therapies (8). The majority of individual requests were approved (19/20); neutral or negative opinions were given in 5, including 3 group requests. Recently evolved regulatory processes share common criteria and conditions to our framework including: demonstration of clinical need; sound scientific basis with lack of viable alternative; risks‐benefit/best interests evaluation; arrangements for fully informed consent; no compromise of arrangements to test treatment for licensing purposes; consideration of resource implications. There are differences between individual processes and with our framework, with respect to procedures, scope, application format, costs and obligation to make available all outcome data. Our experience has emphasized the need for an independent, principled, consistent, fair and transparent response to the increasing demand for innovative treatment on a compassionate basis. We believe that there is a need for harmonization of the recent proliferation of regulation and legislation in this area.     

Refining CLAMP — Investigations towards improving the Climate Leaf Analysis Multivariate Program

CLAMP (Climate Leaf Analysis Multivariate Program) has been used for the past 17 years to estimate palaeoclimatic conditions. The reliability and applicability of this method, based on leaf physiognomic characters of fossil woody dicots, has been widely discussed over the same period. The present study focuses on some technical aspects of CLAMP, mainly on its robustness in the context of the theoretical unimodal requirements of Canonical Correspondence Analysis, and introduces “correction coefficients” for these aspects of the statistical approach as a new way of interpreting and improving on CLAMP estimates. This tool was tested on datasets derived from 17 European fossil floras ranging in age from the Late Oligocene to the Pliocene. Additionally, an objective statistical method for the selection of the best-suited modern vegetation dataset from 144 site (Physg3br) or 173 (Physg3ar) extant biotopes is proposed.     

Cytochemical characterization of secretory and cell surface glycoconjugates by light and electron microscopy.

Lectin methods have increased the capacity for histochemical characterization and differentiation of glycoproteins and have demonstrated, for example, greater reactivity of gastrointestinal than of respiratory tract secretions with the periodate-concanavalin A-horseradish peroxidase method for localizing mannose-rich glycoprotein. Application of a battery of ultrastructural cytochemical methods with specificity for the constituents characteristically present in the complex carbohydrates provides knowledge of the distribution of the various recognizable types of glycoconjugates in tissues and cells showing, for example, marked differences in glycoconjugates of the apical compared with the basolateral plasmalemma in a given cell type and differences between apical plasmalemmas or basement membranes of different cell types. Such information raises questions as to the biologic significance of the different complex carbohydrates in various sites and, hopefully, will lead to a clearer understanding of their physiologic roles.     

The assessing of acidophilia with Biebrich scarlet, ponceau de xylidine and woodstain scarlet.

Horobin and Bennion stated in 1973 that the bonding of Biebrich scarlet at an alkaline pH is hydrophobic as the isomers of Biebrich scarlet (ponceau de xylidine and woodstain scarlet) do not stain at that pH. If correct this would negate the use of Biebrich scarlet as proposed by Spicer and Lillie in 1961 as a measure of acidophilia. We have found all three isomers to stain similarly at an alkaline pH. Goldstein stated in 1963 that Biebrich scarlet saturated with urea stained elastic fibers only faintly or not at all. This we confirmed; we also found that the staining of other acidophilic structures (Paneth cell granules, sperm heads, etc.) was blocked. If only hydrogen bonding and not ionic bonding is blocked in an aqueous dye solution saturated with urea then the exact meaning of alkaline Biebrich scarlet staining is unknown.     

Lectin cytochemistry of cell types in human and canine pituitary

Labeled lectins specific for different sugars were employed to identify different cell types in pituitaries from six human autopsies and seven dogs. To determine the lectins bound by each cell type, fixed-paraffin embedded sections serial to those stained with lectins were immunostained for specific hormones and the serial pairs were examined in a comparison microscope. In human pituitaries corticotrophs stained selectively with lectins having affinity for alpha-L-fucose and the core region of complex type N-glycosyl-proteins. Some corticotrophs also stained for the presence of terminal beta-galactose. Thyrotrophs stained selectively with a periodate oxidation-borohydride reduction-concanavalin A sequence. Some mammotrophs evidenced content of glycoconjugate with terminal beta-galactose. Dendritic cells stained selectively for abundant glycogen with the periodate-reduction-concanavalin A sequence and a lectin from Griffonia simplicifolia. Adenohypophyseal cells of dog pituitary differed in showing absence of terminal beta-galactose in corticotrophs, presence of terminal beta-galactose in thyrotrophs, presence of glycoconjugate with N-glycosidically bound oligosaccharide in thyrotrophs and gonadotrophs and presence of terminal beta-galactose with a different lectin affinity in mammotrophs. The main contributions of lectin histochemistry applied to the pituitary include providing an additional histologic method for identification of some cell types, and localizing glycosylated prohormone or other biochemically unrecognized non-hormone glycoconjugates whose function in pituitary cells remains to be explained.     

Immunolocalization of copper-zinc superoxide dismutase. II. Rat

Copper-zinc superoxide dismutase (CuZn SOD) has been localized in formalin-fixed rat tissues. Staining with a modified immunoenzyme bridge technique using the avidin-biotin-peroxidase complex revealed abundant endogenous CuZn SOD in cells that function in transporting ions, either cellularly, as in the case of tracheal, bronchiolar, and colonic epithelial cells, gastric oxyntic cells, and cells lining the salivary ducts and proximal convoluted tubules in the nephron, or intracellularly, as exemplified by skeletal muscle and neurons. Additionally, the enzyme was consistently demonstrable in hepatocytes, endocrine cells of the islets of Langerhans, and the highly membranous oligodendrocytes in the central nervous system. Cellular processes that maintain high ionic gradients appear especially vulnerable to the superoxide anion, thus necessitating the presence of CuZn SOD to scavenge toxic free radicals of oxygen. Comparison of these observations with other immunocytochemical reports indicates that the cellular distribution of CuZn SOD varies between different species.