A fish-based biotic integrity index for assessment of lowland streams in southeastern Brazil

This study was carried out to develop and apply a fish-based biotic integrity index to assess lowland streams in a highly deforested region of the Upper Paraná River basin. Fifty-six first-order segments were randomly selected for environmental and fish evaluation. Because previous analysis had identified the main type of effect on the streams of the region as physical habitat degradation, 22 qualitatively biological attributes were selected and tested over a physical condition gradient between reference and degraded sites. Sensitivity and redundancy of each attribute revealed that five metrics were adequate for discriminating higher quality from degraded sites. Of the fifty-six streams assessed, one (2%) was classified as good, four (7%) as fair, ten (18%) as poor, and forty-one (73%) as very poor, indicating that, on a regional scale, many aspects of biological integrity are altered, indicative of serious degradation. Considering that first-order segments amount to 11,000 km in total, it is noticeable that 10,000 km of the stream segments have no more than half of the expected conditions, indicative of poor or very poor biotic integrity conditions. Possible strategies of mitigating this scenario are discussed. Handling editor: S. M. Thomaz     

mTOR/S6 Kinase Pathway Contributes to Astrocyte Survival during Ischemia

Neurons are highly dependent on astrocyte survival during brain damage. To identify genes involved in astrocyte function during ischemia, we performed mRNA differential display in astrocytes after oxygen and glucose deprivation (OGD). We detected a robust down-regulation of S6 kinase 1 (S6K1) mRNA that was accompanied by a sharp decrease in protein levels and activity. OGD-induced apoptosis was increased by the combined deletion of S6K1 and S6K2 genes, as well as by treatment with rapamycin that inhibits S6K1 activity by acting on the upstream regulator mTOR (mammalian target of rapamycin). Astrocytes lacking S6K1 and S6K2 (S6K1;S6K2^−/−) displayed a defect in BAD phosphorylation and in the expression of the anti-apoptotic factors Bcl-2 and Bcl-xL. Furthermore reactive oxygen species were increased while translation recovery was impaired in S6K-deficient astrocytes following OGD. Rescue of either S6K1 or S6K2 expression by adenoviral infection revealed that protective functions were specifically mediated by S6K1, because this isoform selectively promoted resistance to OGD and reduction of ROS levels. Finally, “in vivo” effects of S6K suppression were analyzed in the permanent middle cerebral artery occlusion model of ischemia, in which absence of S6K expression increased mortality and infarct volume. In summary, this article uncovers a protective role for astrocyte S6K1 against brain ischemia, indicating a functional pathway that senses nutrient and oxygen levels and may be beneficial for neuronal survival.Astrocytes are the most abundant cells in the central nervous system. Their functions are crucial for central nervous system homeostasis, because they provide trophic, metabolic, and antioxidant support to neurons. In addition, astrocytes show the ability to modulate synaptic activity and are responsible for preserving neuronal integrity in conditions of disease and injury. In this regard, recent evidence indicates that they are protective for neurons during cerebral ischemia (1). As there is a growing consensus that astrocyte dysfunction may compromise the ability of neurons to survive, the need for studies that clarify the molecular mechanisms involved in the astrocytic response to ischemia is plainly justified.Among the intracellular pathways that integrate signals from nutrients and oxygen, the mammalian target of rapamycin (mTOR)² kinase plays an evolutionary conserved role in the regulation of cell growth, proliferation, survival, and metabolism (2). mTOR exists in the cell in at least two distinct complexes with different partners, mTORC1 and mTORC2. The activity of mTORC1 is exquisitely sensitive to the energy status of the cell and is blocked by the macrolide antibiotic rapamycin. Glucose and oxygen deprivation inhibits mTORC1 activity, respectively, through the regulation of AMP-activated kinase and REDD1/REDD2 proteins (3–5). These factors favor the action of the tuberous sclerosis proteins TSC1 and TSC2, which suppress mTORC1 by forming a complex with GTPase-activating protein (GAP) activity for the small GTPase Rheb (6).In turn, mTORC1 phosphorylates at least three distinct classes of substrates, the eIF4E-binding proteins (4EBP-1 to -3), the ribosomal protein S6 kinases (S6K1 and S6K2) and the serum- and glucocorticoid-inducible kinase 1. However, the pathophysiological role of the mTOR pathway during hypoxia-induced brain damage and the involvement of the distinct mTOR effectors remain to be established.The anabolic actions of the mTOR pathway may in part depend on the regulation of protein synthesis. mTOR associates with the translation initiation factor eIF3 (7). In turn the mTORC1 substrates 4EBPs interact with the cap-binding protein eIF4E (8), while S6Ks phosphorylate the ribosomal protein rpS6 and eIF4B (9, 10). However loss of function mouse mutants of 4EBPs and S6Ks failed to uncover a role of these effectors in global protein synthesis during resting conditions, while instead suggesting an involvement in energy homeostasis and mitochondrial function (11–13). Therefore, mTOR plays critical anabolic and energetic functions still poorly understood, raising the appealing possibility that hypoxia-induced down-regulation of the mTOR pathway could be linked to brain damage. In this regard, S6K, besides stimulating phosphorylation of the ribosomal proteins rpS6 and eIF4B, has been shown to inactivate the anti-apoptotic factor BAD and the insulin receptor substrate IRS (13, 14), demonstrating additional targets that are not directly involved in protein synthesis and may be relevant for the physiological action of the pathway. Moreover, S6K activity is decreased in in vivo paradigms of global and focal brain ischemia (15–17); whereas insulin-activated cardioprotection during ischemia/reoxygenation-induced injury is linked to S6K activation (18).Here we show that oxygen and glucose deprivation (OGD) decreases S6K1 mRNA levels in astrocyte cultures, leading to a reduction of S6K1 protein and activity. S6K loss of function leads to increased astrocyte death during ischemia, impairment of protein synthesis recovery, unbalance between mitochondrial pro- and anti-apoptotic factors and rise in ROS levels. Finally we reveal an effect of S6K suppression on mouse mortality and infarct volume following permanent middle cerebral artery occlusion. Our data indicate a novel role of S6K1 promoting astrocyte survival, protein synthesis, and brain protection in conditions of ischemic stress.     

Constitutive activation of the mTOR signaling pathway within the normal glomerulus

Fructose induces several kinds of human metabolic disorders; however, information regarding fructose-induced kidney injury is still limited. This study examined fructose-induced kidney injury in mice and clarified the differential susceptibility of three mouse strains: C57Bl/6J, CBA/JN and DBA/2N. In this study all mice were fed with an equal calorie count for sixteen weeks to remove the influence of total energy intake from metabolic effects by fructose-feeding. Only DBA/2N mice, but not C57Bl/6J and CBA/JN mice, fed with fructose displayed tubulointerstitial fibrosis localized on the outer cortex of the kidney together with the increase of mRNA expression of Kim1 and Ngal in the absence of distinct glomerular lesions and albuminuria - decidedly different from diabetic nephropathy. In time-course study of DBA/2N mice fed with fructose diet, the inflammation and fibrosis in the outer cortex of the kidney were enhancing after eight weeks, in parallel with the accumulation of oxidative stress. This progression of renal damage in DBA/2N mice was accompanied with increasing mRNA expression of GLUT5. These results suggest that the responsiveness of GLUT5 expression to fructose at the kidney is one of pivotal roles for the progression of fructose-induced kidney injury.     

Digital herbarium archives as a spatially extensive, taxonomically discriminate phenological record; a comparison to MODIS satellite imagery

This study demonstrates that phenological information included in digital herbarium archives can produce annual phenological estimates correlated to satellite-derived green wave phenology at a regional scale (R = 0.183, P = 0.03). Thus, such records may be utilized in a fashion similar to other annual phenological records and, due to their longer duration and ability to discriminate among the various components of the plant community, hold significant potential for use in future research to supplement the deficiencies of other data sources as well as address a wide array of important issues in ecology and bioclimatology that cannot be addressed easily using more traditional methods.     

A new process of IgG purification by negative chromatography: Adsorption aspects of human serum proteins onto ω-aminodecyl-agarose

The adsorbent ω-aminodecyl-agarose was evaluated as to its feasibility for the adsorption of human serum and plasma proteins, aiming at the purification of immunoglobulin G (IgG). The contribution of electrostatic and hydrophobic interactions (mixed-mode) and the effects of buffer system on the adsorption of serum proteins were also studied. The adsorption isotherm parameters of human serum albumin (HSA) and IgG were evaluated, pointing to the existence of cooperative effects in the process. A positive (n = 2.30 ± 0.38) and negative cooperativity (n = 0.63 ± 0.12) were observed for IgG and HSA binding, respectively. High purity IgG was obtained (based on total protein concentration and nephelometric analysis of HSA, transferrin, and immunoglobulins A, G, and M) with a 75% recovery in Hepes 25 mmol L⁻¹ pH 6.8 feeding human serum. These results indicate that the use of ω-aminodecyl-agarose is a potential technique for purification of IgG from human serum.     

Mutational Insights into the Roles of Amino Acid Residues in Ligand Binding for Two Closely Related Family 16 Carbohydrate Binding Modules

Carbohydrate binding modules (CBMs) are specialized proteins that bind to polysaccharides and oligosaccharides. Caldanaerobius polysaccharolyticus Man5ACBM16-1/CBM16-2 bind to glucose-, mannose-, and glucose/mannose-configured substrates. The crystal structures of the two proteins represent the only examples in CBM family 16, and studies that evaluate the roles of amino acid residues in ligand binding in this family are lacking. In this study, we probed the roles of amino acids (selected based on CBM16-1/ligand co-crystal structures) on substrate binding. Two tryptophan (Trp-20 and Trp-125) and two glutamine (Gln-81 and Gln-93) residues are shown to be critical in ligand binding. Additionally, several polar residues that flank the critical residues also contribute to ligand binding. The CBM16-1 Q121E mutation increased affinity for all substrates tested, whereas the Q21G and N97R mutants exhibited decreased substrate affinity. We solved CBM/substrate co-crystal structures to elucidate the molecular basis of the increased substrate binding by CBM16-1 Q121E. The Gln-121, Gln-21, and Asn-97 residues can be manipulated to fine-tune ligand binding by the Man5A CBMs. Surprisingly, none of the eight residues investigated was absolutely conserved in CBM family 16. Thus, the critical residues in the Man5A CBMs are either not essential for substrate binding in the other members of this family or the two CBMs are evolutionarily distinct from the members available in the current protein database. Man5A is dependent on its CBMs for robust activity, and insights from this study should serve to enhance our understanding of the interdependence of its catalytic and substrate binding modules.     

Biochemical and Domain Analyses of FSUAxe6B,a Modular Acetyl Xylan Esterase,Identify a Unique Carbohydrate Binding Module in Fibrobacter succinogenes S85

Acetyl xylan esterase (EC 3.1.1.72) is a member of a set of enzymes required to depolymerize hemicellulose, especially xylan that is composed of a main chain of β-1,4-linked xylopyranoside residues decorated with acetyl side groups. Fibrobacter succinogenes S85 Axe6B (FSUAxe6B) is an acetyl xylan esterase encoded in the genome of this rumen bacterium. The enzyme is a modular protein comprised of an esterase domain, a carbohydrate-binding module, and a region of unknown function. Sequences that are homologous to the region of unknown function are paralogously distributed, thus far, only in F. succinogenes. Therefore, the sequences were designated Fibrobacter succinogenes-specific paralogous module 1 (FPm-1). The FPm-1s are associated with at least 24 polypeptides in the genome of F. succinogenes S85. A bioinformatics search showed that most of the FPm-1-appended polypeptides are putative carbohydrate-active enzymes, suggesting a potential role in carbohydrate metabolism. Truncational analysis of FSUAxe6B, together with catalytic and substrate binding studies, has allowed us to delineate the functional modules in the polypeptide. The N-terminal half of FSUAxe6B harbors the activity that cleaves side chain acetyl groups from xylan-like substrates, and the binding of insoluble xylan was determined to originate from FPm-1. Site-directed mutagenesis studies of highly conserved active-site residues in the esterase domain suggested that the esterase activity is derived from a tetrad composed of Ser₄₄, His₂₇₃, Glu₁₉₄, and Asp₂₇₀, with both Glu₁₉₄ and Asp₂₇₀ functioning as helper acids, instead of a single carboxylate residue proposed to initiate catalysis.The development of strategies for biomass conversion to fuels (biofuels) is a subject of keen interest as we search for energy resources alternative to fossil fuels (39). Plant cell matter accounts for 150 to 200 billion tons of biomass on our planet annually (31). It is technically possible, but economically far from realization, to convert plant cell wall to biofuels (41). Thus, currently, plant cell wall utilization as a source of biofuels is mostly at the laboratory scale, although there is a great impetus to move production to the industrial scale.The main components of the plant cell wall are cellulose, hemicellulose, and lignin. These components form complex structures that provide the plant with physical strength (42). Biologically, there are two major steps in the production of alcohols from plant-based feedstock. The first step is an enzymatic hydrolysis of the plant cell wall components to fermentable sugars, and the second step is fermentation of the resultant sugars into alcohols. A major limitation of the process is the lack of highly efficient biocatalysts required for the first step. However, it is known that microbes, either as individuals or consortia, that harbor genes encoding enzymes that hydrolyze plant cell wall polysaccharides abound in nature. Research efforts directed at deepening knowledge of how multiple enzymes participate synergistically to degrade the plant cell wall will accelerate the capacity to achieve the goal of converting biomass to biofuels on a large scale (12, 27). However, improvement of “enzyme cocktails” developed for depolymerization of lignocellulosic biomass will be dependent on a better understanding of the structure/function of individual enzymes that together constitute the arsenal of enzymes (hydrolyzome) used by naturally occurring organisms known to be highly efficient in plant cell wall degradation.Ruminant animals harbor a variety of plant cell wall-degrading bacteria in their first stomach or rumen (26). These animals digest forages with the aid of a microbial consortium that is able to metabolize plant cell wall polysaccharides to short-chain fatty acids, the main energy source for the ruminant host. Fibrobacter succinogenes is a ubiquitous rumen bacterium and has been estimated in previous reports to occupy 0.1% to 1.0% of the microbial population in the cattle rumen, based on the quantification of 16S rRNA genes as a marker (25, 43). F. succinogenes is a significant cellulolytic rumen bacterium, and it has the ability to grow on crystalline cellulose as a sole source of carbon and energy (17). Additionally, it has been demonstrated that this bacterium can solubilize hemicelluloses, although it only partially utilized the constituent monosaccharides released (34). As further evidence, F. succinogenes failed to grow on xylose (33), a constituent of most hemicelluloses. Since F. succinogenes is a highly versatile microbe capable of degrading both cellulose and hemicellulose, strains of this bacterium are attractive models to study natural strategies for efficient deconstruction of plant cell wall polysaccharides.Through analysis of the genome sequence of F. succinogenes S85, a gene cluster that encodes more than 10 hemicellulose-targeting enzymes was identified. Most of the enzymes in the cluster are modular polypeptides, a common feature in many carbohydrate-active enzymes. Kam and coworkers (23) previously identified two acetyl xylan esterases (Axe6A and Axe6B) in this cluster and predicted that each gene encoded a polypeptide composed of two domains: an esterase catalytic domain and a family 6 carbohydrate-binding module (CBM6). Whereas Axe6A was fairly well characterized, difficulties in expression of recombinant Axe6B restricted its characterization (23). In this report, overproduction of recombinant F. succinogenes S85 Axe6B (FSUAxe6B) is demonstrated, and furthermore, it is shown that rather than having two domains, the polypeptide harbors three domains composed of an esterase, CBM6, and a region of unknown function. Bioinformatics analysis suggested that the unknown domain observed in FSUAxe6B is, so far, distributed only in F. succinogenes S85; thus, it was designated F. succinogenes-specific paralogous module 1 or FPm-1. Twenty-four polypeptides, with the majority containing glycoside hydrolase family motifs and CBMs, were found to harbor this peptide at the extreme C-terminal region. In addition to assigning a carbohydrate binding function to FPm-1, critical residues that confer esterase activity to the N-terminal half of FSUAxe6B were also identified through site-directed mutagenesis.     

Toxoplasma gondii: The severity of toxoplasmic encephalitis in C57BL/6 mice is associated with increased ALCAM and VCAM-1 expression in the central nervous system and higher blood-brain barrier permeability

In order to investigate the differential ALCAM, ICAM-1 and VCAM-1 adhesion molecules mRNA expression and the blood-brain barrier (BBB) permeability in C57BL/6 and BALB/c mice in Toxoplasma gondii infection, animals were infected with ME-49 strain. It was observed higher ALCAM on day 9 and VCAM-1 expression on days 9 and 14 of infection in the central nervous system (CNS) of C57BL/6 compared to BALB/c mice. The expression of ICAM-1 was high and similar in the CNS of both lineages of infected mice. In addition, C57BL/6 presented higher BBB permeability and higher IFN-γ and iNOS expression in the CNS compared to BALB/c mice. The CNS of C57BL/6 mice presented elevated tissue pathology and parasitism. In conclusion, our data suggest that the higher adhesion molecules expression and higher BBB permeability contributed to the major inflammatory cell infiltration into the CNS of C57BL/6 mice that was not efficient to control the parasite.     

Food insecurity among Inuit preschoolers: Nunavut Inuit Child Health Survey, 2007–2008

Background

Food security (i.e., a condition in which all people, at all times, have physical and economic access to sufficient, safe and nutritious food to meet their dietary needs and food preferences for an active and healthy life) has been noted to be lower in indigenous communities in Canada. We investigated the prevalence of inadequate food security, or food insecurity, among Inuit households with preschool children.

Methods

We conducted a cross-sectional survey of the health status of 388 randomly selected Inuit children aged three to five years in 16 Nunavut communities during the period from 2007 to 2008. From the survey data, we classified levels of food insecurity specifically among children. We also classified levels of overall food insecurity of the household of each child. We calculated the weighted prevalence of levels of child food insecurity and of household food insecurity.

Results

Nearly 70% of Inuit preschoolers resided in households rated as food insecure (69.6%; 95% confidence interval [CI] 64.7%–74.6%). Overall, 31.0% of children were moderately food insecure, and 25.1% were severely food insecure, with a weighted prevalence of child food insecurity of 56.1% (95% CI 51.0%–61.3%). Primary care-givers in households in which children were severely food insecure reported experiencing times in the past year when their children skipped meals (75.8%), went hungry (90.4%) or did not eat for a whole day (60.1%). Primary caregivers in households in which children were moderately food insecure reported experiencing times in the past year when they worried food would run out (85.1%), when they fed their children less expensive food (95.1%) and when their children did not eat enough because there was no money for food (64.3%).

Interpretation

We observed a high prevalence of household food insecurity, with a substantial proportion of children with severely food insecure status. Interventions are needed to ensure a healthy start in life for Inuit preschoolers.According to the 1996 World Food Summit, food security exists when “all people, at all times, have physical and economic access to sufficient, safe and nutritious food to meet their dietary needs and food preferences for an active and healthy life.”¹ Food security is essential for public health² and important for child development. Suboptimal food security, or food insecurity, has deleterious effects on children’s academic performance and psychosocial development.^3,4 In Canada, food insecurity is higher in the three territories than in the provinces.⁵ According to limited data, a high degree of food insecurity exists among Inuit in particular.^6,7 Further, in most remote communities, the cost of food is at least double that in southern Canadian cities.⁸We assessed the prevalence of food insecurity among Inuit preschool-aged children.