Variable Importance of Macrofaunal Functional Biodiversity for Biogeochemical Cycling in Temperate Coastal Sediments

Coastal marine systems are currently subject to a variety of anthropogenic and climate-change-induced pressures. An important challenge is to predict how marine sediment communities and benthic biogeochemical cycling will be affected by these ongoing changes. To this end, it is of paramount importance to first better understand the natural variability in coastal benthic biogeochemical cycling and how this is influenced by local environmental conditions and faunal biodiversity. Here, we studied sedimentary biogeochemical cycling at ten coastal stations in the Southern North Sea on a monthly basis from February to October 2011. We explored the spatio-temporal variability in oxygen consumption, dissolved inorganic nitrogen and alkalinity fluxes, and estimated rates of nitrification and denitrification from a mass budget. In a next step, we statistically modeled their relation with environmental variables and structural and functional macrobenthic community characteristics. Our results show that the cohesive, muddy sediments were poor in functional macrobenthic diversity and displayed intermediate oxygen consumption rates, but the highest ammonium effluxes. These muddy sites also showed an elevated alkalinity release from the sediment, which can be explained by the elevated rate of anaerobic processes taking place. Fine sandy sediments were rich in functional macrobenthic diversity and had the maximum oxygen consumption and estimated denitrification rates. Permeable sediments were also poor in macrobenthic functional diversity and showed the lowest oxygen consumption rates and only small fluxes of ammonium and alkalinity. Macrobenthic functional biodiversity as estimated from bioturbation potential appeared a better variable than macrobenthic density in explaining oxygen consumption, ammonium and alkalinity fluxes, and estimated denitrification. However, this importance of functional biodiversity was manifested particularly in fine sandy sediments, to a lesser account in permeable sediments, but not in muddy sediments. The strong relationship between macrobenthic functional biodiversity and biogeochemical cycling in fine sandy sediments implies that a future loss of macrobenthic functional diversity will have important repercussions for benthic ecosystem functioning.     

A comparison between neurally induced bone marrow derived mesenchymal stem cells and olfactory ensheathing glial cells to repair spinal cord injuries in rat

Cell therapy has proven to be a highly promising method in clinical applications, raising so much hope for the treatment of injured tissues with low, if any, self regeneration potential such as central and peripheral nervous system. Neurally induced bone marrow derived mesenchymal stem cells (NIMSCs) as well as olfactory ensheathing cells (OECs) were transplanted in a rat model of sub-acute spinal cord injury and the behavioral and histological analyses were conducted. A balloon-compression technique was used to produce an injury at T8-T9 level of spinal cord. After a week post injury, rats were injected with either NIMSCs or OECs at the center of developing lesion cavity, 3 mm cranial and 3 mm caudal to the cavity. Weekly behavioral assessment using BBB score was done over five-week period post transplantation and finally histological assessment was performed to locate labeled cells in the tissue in order to evaluate the reduction of cavity formation and axonal regeneration. Evaluation of locomotor performance showed significant behavioral improvement in NIMSC group over OEC and control groups. The histological analyses revealed the presence of transplanted cells in the spinal cord parenchyma. Volume of injured area that was occupied with syrinx cavity in NIMSC group was significantly less than control group. In addition, meanwhile neurofilament-positive axons significantly showed higher expression in rats receiving NIMSC compared to the other two groups. In conclusion NIMSC caused both behavioral and histological improvement that potentially makes them a promising candidate for cell therapy approaches of spinal cord injuries.     

Detection of a Plasmodium vivax erythrocyte binding protein by flow cytometry.

BACKGROUND: The malaria parasite Plasmodium vivax preferentially invades reticulocytes. It is therefore relevant for vaccine development purposes to identify and characterize P. vivax proteins that bind specifically to the surface of reticulocytes. We have developed a two-color flow cytometric erythrocyte binding assay (F-EBA) that has several advantages over traditional erythrocyte binding assays (T-EBAs) used in malaria research. We demonstrate the use of F-EBA using the P. vivax Duffy binding protein region II (PvDBP-RII) recombinant protein as a model. This protein binds to all erythrocytes that express the Duffy receptor (Fy) and discriminates binding between normocytes and reticulocytes. METHODS: F-EBAs were performed by incubating freshly isolated Aotus nancymai, Macaca mulatta, Saimiri boliviensis, and human erythrocytes with PvDBP-RII, a fluorescent anti-His tag detection antibody, and thiazole orange before flow cytometric analysis. T-EBAs employing immunoblot detection with an anti-His antibody were performed concomitantly. RESULTS: PvDBP-RII bound to A. nancymai, M. mulatta, and human Fy+ erythrocytes, but not human Fy- erythrocytes, by F-EBAs and T-EBAs. However, F-EBAs exhibited higher sensitivity and better concordance between experiments compared with T-EBAs. CONCLUSIONS: F-EBA is a rapid, simple, and reliable method for quantifying the ability of malaria proteins to bind to the surface of erythrocytes. F-EBA can discriminate binding between erythrocyte subpopulations without enrichment protocols and may be more reliable and sensitive than T-EBAs in identifying novel erythrocyte binding proteins.     

Fermentative Utilization of Glycerol by Escherichia coli and Its Implications for the Production of Fuels and Chemicals

Availability, low prices, and a high degree of reduction make glycerol an ideal feedstock to produce reduced chemicals and fuels via anaerobic fermentation. Although glycerol metabolism in Escherichia coli had been thought to be restricted to respiratory conditions, we report here the utilization of this carbon source in the absence of electron acceptors. Cells grew fermentatively on glycerol and exhibited exponential growth at a maximum specific growth rate of 0.040 ± 0.003 h⁻¹. The fermentative nature of glycerol metabolism was demonstrated through studies in which cell growth and glycerol utilization were observed despite blocking several respiratory processes. The incorporation of glycerol in cellular biomass was also investigated via nuclear magnetic resonance analysis of cultures in which either 50% U-¹³C-labeled or 100% unlabeled glycerol was used. These studies demonstrated that about 20% of the carbon incorporated into the protein fraction of biomass originated from glycerol. The use of U-¹³C-labeled glycerol also allowed the unambiguous identification of ethanol and succinic, acetic, and formic acids as the products of glycerol fermentation. The synthesis of ethanol was identified as a metabolic determinant of glycerol fermentation; this pathway fulfills energy requirements by generating, in a redox-balanced manner, 1 mol of ATP per mol of glycerol converted to ethanol. A fermentation balance analysis revealed an excellent closure of both carbon (~95%) and redox (~96%) balances. On the other hand, cultivation conditions that prevent H₂ accumulation were shown to be an environmental determinant of glycerol fermentation. The negative effect of H₂ is related to its metabolic recycling, which in turn generates an unfavorable internal redox state. The implications of our findings for the production of reduced chemicals and fuels were illustrated by coproducing ethanol plus formic acid and ethanol plus hydrogen from glycerol at yields approaching their theoretical maximum.     

A lipopolysaccharide mutant of Bradyrhizobium japonicum that uncouples plant from bacterial differentiation.

The Tn5-containing fragment from a non-nodulating mutant of Bradyrhizobium japonicum, strain ML142, was introduced into B. japonicum strain 61A101c by marker exchange to construct strain JS314. Strain JS314 failed to nodulate several soybean varieties tested. However, on a few varieties nodulelike structures were induced to a frequency of 54% of the plants inoculated. The ultrastructure of these nodules was studied in detail by light and electron microscopy. The nodules were devoid of internal bacteria, possessed central vascular tissue (unlike the lateral vascular tissue of a normal nodule), and exhibited localized cell death of epidermal cells. Study of the cell surface polysaccharides of strain JS314 revealed that the exopolysaccharide of this strain was identical to that of the wild type. However, the lipopolysaccharide (LPS) of strain JS314 showed gross differences from that isolated from the wild-type strain. Specifically, the LPS of strain JS314 appeared to lack the high molecular weight LPS I form, strongly suggesting that the LPS lacks the O-chain. Glycosyl-composition analysis showed that the LPS of mutant JS314 lacked 2,3-di-O-methylrhamnose, 3-O-methylrhamnose, fucose, and quinovosamine. These results indicate that LPS I in B. japonicum is essential for bacterial infection of soybean, but is not required to initiate plant cortical cell division, an early plant response to infection.     

Metabolism of cationized lipoproteins by human fibroblasts: biochemical and morphologic correlations

Human plasma low density lipoprotein (LDL) that had been rendered polycationic by coupling with N, N-dimethyl-1, 3-propanediamine (DMPA) was shown by electron microscopy to bind in clusters to the surface of human fibroblasts. The clusters resembled those formed by polycationic ferritin (DMPA-feritin), a visual probe that binds to anionic site on the plasma membrane. Biochemical studies with (125)I-labeled DMPA-LDL showed that the membrane-bound lipoprotein was internalized and hydrolyzed in lysosomes. The turnover time for cell bound (125)I-DMPA-LDL, i.e., the time in which the amount of (125)I-DMPA-LDL degraded was equal to the steady-state cellular content of the lipoprotein, was about 50 h. Because the DMPA-LDL gained access to fibroblasts by binding nonspecifically to anionic sites on the cell surface rather than by binding to the physiologic LDL receptor, its uptake failed to be regulated under conditions in which the uptake of native LDL was reduced by feedback suppression of the LDL receptor. As a result, unlike the case with native LDL, the DMPA-LDL accumulated progressively within the cell, and this led to a massive increase in the cellular content of both free and esterified cholesterol. Studies with (14)C-oleate showed that at least 20 percent of the accumulated cholesteryl esters represented cholesterol that had been esterified within the cell. After 4 days of incubation with 10 μg/ml of DMPA-LDL, fibroblasts had accumulated so much cholesteryl ester that neutral lipid droplets were visible at the light microscope level with Oil Red O staining. By electron microscopy, these intracellular lipid droplets were observed to lack a tripartite limiting membrane. The ability to cause the overaccumulation of cholesteryl esters within cells by using DMPA-LDL provides a model system for study of the pathologic consequences at the cellular level of massive deposition of cholesteryl ester.     

Mapping binding residues in the Plasmodium vivax domain that binds Duffy antigen during red cell invasion

Summary Plasmodium vivax depends on interaction with the Duffy antigen/receptor for chemokines (DARC) for invasion of human erythrocytes. The 140 kDa P. vivax Duffy-binding protein (PvDBP) mediates interaction with DARC. The receptor-binding domain of PvDBP maps to its N-terminal, cysteine-rich region, region II (PvRII), which contains approximately 300 amino acid residues including 12 conserved cysteines. Using surface plasmon resonance, we show that binding of PvRII to DARC is a high-affinity interaction with a binding constant (K(D)) of 8.7 nM. The minimal binding domain of PvRII has been previously mapped to a central 170-amino-acid stretch that includes cysteines 5-8. Here, we have used site-directed mutagenesis and quantitative binding assays to map amino acid residues within PvRII that make contact with DARC. Of the seven alanine replacement mutations that had an effect on binding, five were mutations in hydrophobic residues suggesting that hydrophobic interactions play a major role in the interaction of PvDBP with DARC. Genetic diversity studies have shown that six of the seven binding residues identified in PvRII are conserved in P. vivax field isolates, which provides support for their role in interaction with DARC.     

T-cell subsets that harbor human immunodeficiency virus (HIV) in vivo: implications for HIV pathogenesis

Identification of T-cell subsets that are infected in vivo is essential to understanding the pathogenesis of human immunodeficiency virus (HIV) disease; however, this goal has been beset with technical challenges. Here, we used polychromatic flow cytometry to sort multiple T-cell subsets to 99.8% purity, followed by quantitative PCR to quantify HIV gag DNA directly ex vivo. We show that resting memory CD4(+) T cells are the predominantly infected cells but that terminally differentiated memory CD4(+) T cells contain 10-fold fewer copies of HIV DNA. Memory CD8(+) T cells can also be infected upon upregulation of CD4; however, this is infrequent and HIV-specific CD8(+) T cells are not infected preferentially. Na?ve CD4(+) T-cell infection is rare and principally confined to those peripheral T cells that have proliferated. Furthermore, the virus is essentially absent from na?ve CD8(+) T cells, suggesting that the thymus is not a major source of HIV-infected T cells in the periphery. These data illuminate the underlying mechanisms that distort T-cell homeostasis in HIV infection.