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Purpose: Magnetic resonance imaging (MRI) scanner-specific geometric distortions may contribute to scanner induced variability and decrease volumetric measurement precision for multi-site studies. The purpose of this study was to determine whether geometric distortion correction increases the precision of brain volumetric measurements in a multi-site multi-scanner study., Methods: Geometric distortion variation was quantified over a one-year period at 10 sites using the distortion fields estimated from monthly 3D T1-weighted MRI geometrical phantom scans. The variability of volume and distance measurements were quantified using synthetic volumes and a standard quantitative MRI (qMRI) phantom. The effects of geometric distortion corrections on MRI derived volumetric measurements of the human brain were assessed in two subjects scanned on each of the 10 MRI scanners and in 150 subjects with cerebrovascaular disease (CVD) acquired across imaging sites., Results: Geometric distortions were found to vary substantially between different MRI scanners but were relatively stable on each scanner over a one-year interval. Geometric distortions varied spatially, increasing in severity with distance from the magnet isocenter. In measurements made with the qMRI phantom, the geometric distortion correction decreased the standard deviation of volumetric assessments by 35% and distance measurements by 42%. The average coefficient of variance decreased by 16% in gray matter and white matter volume estimates in the two subjects scanned on the 10 MRI scanners., Conclusion: Geometric distortion correction using an up-to-date correction field is recommended to increase precision in volumetric measurements made from MRI images., (Copyright © 2022 Elsevier Inc. All rights reserved.)

For the next decade, the global water crisis remains the risk of highest concern, and ranks ahead of climate change, extreme weather events, food crises and social instability. Across the globe, nearly one in ten people is without access to an improved drinking water source. Least Developed Countries (LDCs) especially in sub-Saharan Africa (SSA) are the most affected, having disproportionately more of the global population without access to clean water than other major regions. Population growth, changing lifestyles, increasing pollution and accelerating urbanization will continue to widen the gap between the demand for water and available supply especially in urban areas, and disproportionately affect informal settlements, where the majority of SSA's urban population resides. Distribution and allocation of water will be affected by climate-induced water stresses, poor institutions, ineffective governance, and weak political will to address scarcity and mediate uncertainties in future supply. While attempts have been made by many scientists to examine different dimensions of water scarcity and urban population dynamics, there are few comprehensive reviews, especially focused on the particular situation in Sub-Saharan Africa. This paper contributes to interdisciplinary understanding of urban water supply by distilling and integrating relevant empirical knowledge on urban dynamics and water issues in SSA, focusing on progress made and associated challenges. It then points out future research directions including the need to understand how alternatives to centralized water policies may help deliver sustainable water supply to cities and informal settlements in the region., (Copyright © 2017 Elsevier B.V. All rights reserved.)

To improve bioenergy production from photosynthetic microorganisms it is necessary to optimize an extensive network of highly integrated biological processes. Systematic advances in pathway engineering and culture modification have resulted in strains with increased yields of biohydrogen, lipids, and carbohydrates, three bioenergy foci. However, additional improvements in photosynthetic efficiency are necessary to establish a viable system for biofuel production. Advances in optimizing light capture, energy transfer, and carbon fixation are essential, as the efficiencies of these processes are the principal determinants of productivity. However, owing to their regulatory, catalytic, and structural complexities, manipulating these pathways poses considerable challenges. This review covers novel developments in the optimization of photosynthesis, carbon fixation, and metabolic pathways for the synthesis of targeted bioenergy carriers., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Chlamydomonas reinhardtii (Chlamydomonas throughout) encodes two [FeFe]-hydrogenases, designated HYDA1 and HYDA2. While HYDA1 is considered the dominant hydrogenase, the role of HYDA2 is unclear. To study the individual functions of each hydrogenase and provide a platform for future bioengineering, we isolated the Chlamydomonas hydA1-1, hydA2-1 single mutants and the hydA1-1 hydA2-1 double mutant. A reverse genetic screen was used to identify a mutant with an insertion in HYDA2, followed by mutagenesis of the hydA2-1 strain coupled with a H(2) chemosensor phenotypic screen to isolate the hydA1-1 hydA2-1 mutant. Genetic crosses of the hydA1-1 hydA2-1 mutant to wild-type cells allowed us to also isolate the single hydA1-1 mutant. Fermentative, photosynthetic, and in vitro hydrogenase activities were assayed in each of the mutant genotypes. Surprisingly, analyses of the hydA1-1 and hydA2-1 single mutants, as well as the HYDA1 and HYDA2 rescued hydA1-1 hydA2-1 mutant demonstrated that both hydrogenases are able to catalyze H(2) production from either fermentative or photosynthetic pathways. The physiology of both mutant and complemented strains indicate that the contribution of HYDA2 to H(2) photoproduction is approximately 25% that of HYDA1, which corresponds to similarly low levels of in vitro hydrogenase activity measured in the hydA1-1 mutant. Interestingly, enhanced in vitro and fermentative H(2) production activities were observed in the hydA1-1 hydA2-1 strain complemented with HYDA1, while maximal H(2)-photoproduction rates did not exceed those of wild-type cells., (Copyright © 2011 Elsevier Inc. All rights reserved.)

[FeFe]-hydrogenases have been claimed as the most promising catalysts of hydrogen bioproduction and several efforts have been accomplished to express and purify them. However, previous attemps to obtain a functional recombinant [FeFe]-hydrogenase in heterologous systems such as Escherichia coli failed due to the lack of the specific maturation proteins driving the assembly of its complex active site. The unique exception is that of [FeFe]-hydrogenase from Clostridium pasteurianum that has been expressed in active form in the cyanobacterium Synechococcus PCC 7942, which holds a bidirectional [NiFe]-hydrogenase with a well characterized maturation system, suggesting that the latter is flexible enough to drive the synthesis of a [FeFe]-enzyme. However, the capability of cyanobacteria to correctly fold a [FeFe]-hydrogenase in the absence of its auxiliary maturation proteins is a debated question. In this work, we expressed the [FeFe]-hydrogenase from Chlamydomonas reinhardtii as an active enzyme in the cyanobacterium Synechocystis sp. PCC 6803. Our results, using a different experimental system, confirm that cyanobacteria are able to express a functional [FeFe]-hydrogenase even in the absence of additional chaperones., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Mediators of the stress response (e.g. glucocorticoids and norepinephrine) can be immunosuppressive. Nevertheless, immune challenge leads to the release of these compounds in vertebrates. To resolve this paradox, it has been suggested that stress hormones help restore immune homeostasis, preventing self-damage. A comparative approach may provide additional hypotheses as to why an immune challenge induces the release of stress hormones/neurohormones. Octopamine, a neurohormonal mediator of the stress response in the cricket Gryllus texensis, increased in concentration in the hemolymph during an immune challenge. Therefore, the release of stress hormones during an immune response occurs in animals across phyla. Octopamine induced an increase in lipid concentration in the hemolymph. After an acute stress (flying or running) the total number of hemocytes in the hemolymph increased. Injections of octopamine had the same effect, suggesting that it may enhance hemocyte-dependent immune functions. On the other hand, octopamine decreased lysozyme-like activity in vitro, suggesting that it inhibits some immune functions. However, lysozyme-like activity was increased by the presence of heat-killed bacteria in vitro and this increase was significantly augmented by the presence of octopamine. Therefore, the effect of octopamine on immune function differed depending on the presence of pathogens. Stress hormones may help shift immune function into the most optimal configuration depending on the physiological context., (Copyright 2009 Elsevier Inc. All rights reserved.)

Neurohypophyseal peptides potently stimulate myogenic differentiation by acting through different receptors of the same family. Here, we show that L6C5 myogenic cells express, at a high density, a single class of V1a Arg8-vasopressin (AVP) receptor. The expression of the vasopressin receptor of type 1a (V1aR) is significantly higher in proliferating myoblasts than in differentiated myotubes. The differentiation-related decrease of V1aR expression was evident both at the mRNA and at the protein level as shown by the reduction of [(3)H]-AVP binding. However, in L6C5 cells transfected with a synthetic construct containing the luciferase gene driven by the 2 kb upstream region of V1aR, we observed a stimulation of the activity of the promoter when the cells were cultured in differentiative medium. The down-regulation of the V1aR correlated with a decreased half-life of its mRNA (half-life 5.86+/-0.74 hr in 10% fetal bovine serum [FBS] versus 3.53+/-0.72 hr in 1% FBS). Cyclosporine A and dexamethasone, but not 5'-azacytidine, treatments of cells in differentiation medium restored the V1aR level to that measured in proliferating L6C5 cells, thus confirming the role of post-transcriptional mechanisms in the modulation of V1aR expression. Taken together, these data show that mRNA stability plays a role in modulating protein expression during the myogenic differentiation process.

The bcr1- and bcr3- promyelocytic leukemia/retinoic acid receptor alpha (PML/RAR alpha) are the two major fusion proteins expressed in acute promyelocytic leukemia (APL) patients. These proteins, which are present in different lengths of PML (amino acids 1-552 and 1-394, respectively), contain most of the functional domains of PML and RAR alpha, bind all-trans-retinoic acid (t-RA), and act as t-RA-dependent transcription factors. T-RA is an effective inducer of clinical remission only in patients carrying the t(15;17) and expressing the PML/RAR alpha products. However, in APL patients achieving complete remission with t-RA therapy the bcr3-PML/RAR alpha product has been found associated with a poorer prognosis than bcr1-PML/RAR alpha. In the present study we have investigated the structural and functional properties of the bcr3-PML/RAR alpha in comparison to the previously characterized bcr1-PML/RAR alpha. In particular, we have measured the binding properties of the two endogenous ligands t-RA and 9-cis-RA to both of these isoforms. T-RA binding analysis of nuclear and cytosolic extracts prepared from bcr3-PML/RAR alpha APL patients and from bcr3-PML/RAR alpha COS-1 transfected cells indicates that this protein is present only as high-molecular-weight nuclear complexes. Using saturation binding assays and Scatchard analyses we found that t-RA binds with slightly less affinity to the bcr3-PML/RAR alpha receptor than to bcr1-PML/RAR alpha or RAR alpha (Kd = 0.4 nmol/L, 0.13 nmol/L or 0.09 nmol/L, respectively). Moreover, two different high-affinity 9-cis-RA binding sites (Kd = 0.45 and 0.075 nmol/L) were detectable in the bcr3-PML/RAR alpha product but not in the bcr1-PML/RAR alpha product (Kd = 0.77 nmol/L). By competition binding experiments we showed that 9-cis-RA binds with higher specificity to the bcr3-PML/RAR alpha isoform than to the bcr1-PML/RAR alpha or RAR alpha. Consistent with these data, the binding of 9-cis-RA to the bcr3-PML/RAR alpha product resulted in increased transcriptional activation of the RA-responsive element (RARE) TRE, but not of the betaRARE, in transiently transfected COS-1 cells. These results provide evidence indicating that preferential retinoid binding to the different PML/RAR alpha products can be measured.

All-trans retinoic acid (t-RA) administration leads to complete remission in acute promyelocytic leukemia (APL) patients by inducing growth arrest and differentiation of the leukemic clone. In the present study, we show that t-RA treatment dramatically induced type II transglutaminase (type II TGase) expression in cells carrying the t(15;17) translocation and expressing the PML-RARalpha product such as the APL-derived NB4 cell line and fresh leukemic cells from APL patients. This induction correlated with t-RA-induced growth arrest, granulocytic differentiation, and upregulation of the leukocyte adherence receptor beta subunit (CD18) gene expression. The increase in type II TGase was not abolished by cycloheximide treatment, suggesting that synthesis of a protein intermediate was not required for the induction. t-RA did not significantly alter the rate of growth arrest and did not stimulate differentiation and type II TGase activity in NB4.306 cells, a t-RA-resistant subclone of the NB4 cell line, or in leukemic cells derived from two patients morphologically defined as APL but lacking the t(15;17). However, in NB4.306 cells, t-RA treatment was able to increase CD18 mRNA expression in a manner similar to NB4 cells. The molecular mechanisms involved in the induction of these genes were investigated. In NB4 cells, using novel receptor-selective ligands such as 9-cis-RA, TTNPB, AM580, and SR11217, we found that RAR- and RARalpha-selective retinoids were able to induce growth arrest, granulocytic differentiation, and type II TGase, whereas the RXR-selective retinoid SR11217 was inactive. Moreover, an RAR alpha-antagonist completely inhibited the expression of type II TGase and CD18 induced by these selective retinoids in NB4 cells. In NB4.306 cells, an RARalpha-dependent signaling pathway was found involved in the modulation of CD18 expression. In addition, expression of the PML-RARalpha gene in myeloid U937 precursor cells resulted in the ability of these cells to induce type II TGase in response to t-RA. On the basis of these results we hypothesize a specific involvement of a signaling pathway involving PML-RAR alpha for the induction of growth arrest, granulocytic differentiation, and type II TGase by retinoids in APL cells.

The activity and subcellular distribution of the calcium-phospholipid dependent protein kinase (protein kinase C) were studied in normal and dystrophic muscle cells in vitro. Clonal strains of satellite cells, isolated from normal and dystrophic (C57BL/6J/dydy) mice, differentiate in vitro at a comparable level (over 80% of fusion). Differentiated myotubes were homogenized and separated into a soluble and a particulate fraction. The activity of protein kinase C was assayed in both fractions, and was found to be mainly in the cytosol of normal cells, whereas it was mainly associated to the membrane fraction of dystrophic cells. This altered distribution of the enzyme was likely consequent to alterations in the phospholipid composition of the dystrophic cell membrane, since it was possible to partially revert the situation by modifying the membranes with liposome-delivered phospholipids. Splenic lymphocytes from dystrophic mice showed an altered distribution of protein kinase C similar to that observed in muscle cells. The possible biochemical basis and the functional consequences of this altered distribution of the enzyme in the dystrophic cells are discussed.

Spontaneously transformed mouse fibroblasts (Balb/c 3T12-3 cells) displayed an increased adhesion when cultured in the presence of 10(-6) M all-trans retinol and acquired morphological characteristics of the normal phenotype. Thus it was of interest to investigate the metabolism of [15-(14)C]retinol in this system. Within 24 hours of culture, approximately 4.25% of the [(14)C]retinol was taken up by the cells. The hydrocarbon [(14)C]anhydroretinol was a major metabolic product and was identified by gas-liquid chromatography and by its typical ultraviolet absorption spectrum with maxima at 386, 364, and 346 nm. At 24 and 40 hours anhydroretinol represented 27% and 55%, respectively, of the total nonpolar metabolites or approximately 16% and 30% of the total radioactive products. Formalin-fixed fibroblasts or cultured intestinal mucosal cells did not convert retinol into anhydroretinol. A more polar product with a UV absorption maximum at 310 nm was also found. The time course of the synthesis of this product by 3T12 cells suggested a precursor-product relationship with anhydroretinol. A microsomal preparation from 3T12 cells was also active in synthesizing [(14)C]anhydroretinol and [(14)C]metabolite-310 from [(14)C]retinol. Moreover incubation of metabolite-310 with the 3T12 microsomes yielded anhydroretinol (40% conversion in 30 minutes), suggesting that metabolite-310 is an intermediate in the synthesis of anhydroretinol by these cells. Anhydroretinol appears to be an end product of the metabolism of retinol in 3T12-3 cells, as suggested by the finding that over 90% of [(14)C]anhydroretinol incubated for 30 hours with 3T12-3 cells was recovered unaltered, without the formation of detectable retroretinol, retinol, or retinoic acid.-Bhat, P. V., L. M. De Luca, S. Adamo, I. Akalovsky, C. S. Silverman-Jones, and G. L. Peck. Retinoid metabolism in spontaneously transformed mouse fibroblasts (Balb/c 3T12-3 cells): enzymatic conversion of retinol to anhydroretinol.

The tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) dramatically modifies the differentiative program of myotubes, developed in culture from chick embryo skeletal myogenic cells. In fact TPA selectively decreases the expression of differentiative parameters with a lag of 8-10 h from its administration to the cultures. We have tested whether the reported effect of TPA depends on the synthesis of specific products during the lag phase of TPA action. The data presented indicate that inhibition of protein synthesis by the use of cycloheximide prevents the appearance of TPA induced inhibition of the expression of differentiative products, such as creatine phosphokinase (CPK) activity and acetylcholine receptors (AChR). Following removal of cycloheximide and reinitiation of normal protein synthesis, the TPA induced inhibitory effect on CPK and AChR appears after a delay of about the same length as the time lag of TPA action. Our results indicate that inhibition of protein synthesis during the lag phase of TPA action prevents the effect of this tumor promoter on myotube differentiative parameters, and suggest that the expression of differentiative traits in cultured myotubes is affected by TPA via a regulatory step implying protein synthesis.

We have compared the reactivity of sera from 34 multiple sclerosis (MS) patients and 32 normal (N) individuals with lipid vesicles containing myelin basic protein (BP) and several glycolipids reconstituted into a membrane environment. The ability of the sera to cause complement-mediated lysis of lipid vesicles containing these antigens was determined by measuring the release of a water-soluble spin label, tempocholine chloride, from the height of its electron spin resonance spectrum. Only 4 MS sera caused lysis of BP-containing vesicles which was comparable to that produced by specific antibody to BP. A number of both MS and N sera caused significant lysis of vesicles containing GM1 ganglioside or digalactosyldiglyceride. A few MS and N sera also caused significant lysis of vesicles containing GM2, GT1 and GD1a gangliosides. However, in no case was there a statistically significant difference between the mean lysis produced by MS and N sera. There was some overlap between the specific MS and N sera reactive to vesicles containing BP, GM1, GM2, and DGDG while a completely different group of MS and N sera were reactive to GT1 and GD1a gangliosides. This suggested that there was either antigenic cross reactivity between the two groups of glycolipids or two different origins of the immune response to the two groups of antigens. It was concluded that antibody-dependent complement fixation by these particular antigens, in the kind of lipid environment used, is not characteristic of or specific to MS.

The role of protein kinase C (PK-C) in the early metabolic events involved in human natural killer (NK) cell activation has been studied through the action of PK-C-specific activators and inhibitors. Highly purified human large granular lymphocytes (LGL) were treated for 1 hr with the diacylglycerol analog 1-oleoyl-2-acetyl glycerol (OAG) (10(-4)-10(-5) g/ml) or with 12-O-tetradecanoylphorbol-13-acetate (TPA) (10(-8)-10(-10) g/ml), both specific activators of PK-C. Both these agents consistently increased NK activity against K562 target cells. Suboptimal doses of either OAG or TPA also synergized with Ca2+ ionophores to augment spontaneous cytotoxic activity. Pretreatment of LGL with 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrocloride (H7) (5-40 microM), a potent PK-C inhibitor, greatly reduced NK activity in a time- and dose-dependent fashion. By contrast, N-(2-guanidinoethyl)-5-isoquinolinesulfonamide hydrochloride (HA 1004), a potent cAMP- and cGMP-dependent PK inhibitor with almost no effect on PK-C, marginally reduced NK activity. Moreover, almost complete NK activity inhibition was observed when H7 (10 microM), but not HA 1004 (50 microM), was present in the NK assay. Finally, 48 hr stimulation of LGL with TPA (10(-6) g/ml), a treatment able to inactivate most of the PK-C cellular pool, almost completely abrogated NK activity. This functional evidence was supported by phosphorylation of several endogenous substrates which occurs within 5 min in TPA-treated LGL. Two proteins of 70 and 56 kDa have been identified as major PK-C substrates, together with other phosphorylated proteins with MW ranging from 177 to 43 kDa. H7, but not HA 1004, almost completely inhibited the TPA-induced phosphorylation of all of these proteins in the NK cells. These data strongly suggest that selective activation of PK-C plays an essential role in the mechanisms of NK cell activation.

In primary culture of chick embryo muscle cells myosin synthesis is detected in mononucleated cells and increased at the onset of fusion with a maximal increment of 20-fold per plate in differentiated myotube. The possibility that the myosin synthetized by duplicating myoblast could be different from that present in post-mitotic myoblast and myotube was evaluated by investigating the regulation of its synthesis and the turnover of the molecule. Following Actinomycin D treatment (0.05 microgram/ml, 8 h), myosin synthesis is partially affected (about 50% inhibition) in pre-fusion myoblast while the synthesis is more sensitive to the drug at the onset of fusion (80% inhibition). With the progress of the differentiative stage the half-life of the molecule increases from 30 h in duplicating myoblasts to 200 h in fibers. The half-life of myosin synthetized by duplicating myoblasts in the explanted embryonic muscle, is 12 h. These data show different features of myosin heavy chains related to specific stages of differentiation and suggest the possibility that modulative changes of the molecule could induce its functional maturation during myogenesis.

Fusion of myoblasts is inhibited in cultures at low Ca++ concentration (0.44 mM); yet creatine phosphokinase and myokinase activities as well as myosin synthesis and the appearance of post-mitotic myoblasts do not significantly differ from those of control cultures (grown at 1.04 mM Ca++) which undergo cell fusion. When Ca++ concentration is increased to the control value after the second day of culture, fusion occurs very rapidly and it is not inhibited by actinomycin D or cycloheximide. Treatment with 0.06 mM bromodeoxyuridine strongly inhibits creatine phosphokinase activity and myotubes formation. The study of the kinetics of reversal of cell fusion and of creatine phosphokinase activity after removal of the analog, shows that this process is slower than the decrease of the relative content of bromodeoxyuridine incorporated into DNA. The result obtained support the following conclusions: a) the expression of the differentiative characters examined does not require cell fusion; b) the process of myotube formation seems to imply two subsequent stages consisting first of a slow maturative process, which is followed by the actual fusion of cell membranes; the former is Ca++ independent, the latter is Ca++ dependent and does not require RNA or protein synthesis.