Nitrate Reduction Functional Genes and Nitrate Reduction Potentials Persist in Deeper Estuarine Sediments. Why?

Denitrification and dissimilatory nitrate reduction to ammonium (DNRA) are processes occurring simultaneously under oxygen-limited or anaerobic conditions, where both compete for nitrate and organic carbon. Despite their ecological importance, there has been little investigation of how denitrification and DNRA potentials and related functional genes vary vertically with sediment depth. Nitrate reduction potentials measured in sediment depth profiles along the Colne estuary were in the upper range of nitrate reduction rates reported from other sediments and showed the existence of strong decreasing trends both with increasing depth and along the estuary. Denitrification potential decreased along the estuary, decreasing more rapidly with depth towards the estuary mouth. In contrast, DNRA potential increased along the estuary. Significant decreases in copy numbers of 16S rRNA and nitrate reducing genes were observed along the estuary and from surface to deeper sediments. Both metabolic potentials and functional genes persisted at sediment depths where porewater nitrate was absent. Transport of nitrate by bioturbation, based on macrofauna distributions, could only account for the upper 10 cm depth of sediment. A several fold higher combined freeze-lysable KCl-extractable nitrate pool compared to porewater nitrate was detected. We hypothesised that his could be attributed to intracellular nitrate pools from nitrate accumulating microorganisms like Thioploca or Beggiatoa. However, pyrosequencing analysis did not detect any such organisms, leaving other bacteria, microbenthic algae, or foraminiferans which have also been shown to accumulate nitrate, as possible candidates. The importance and bioavailability of a KCl-extractable nitrate sediment pool remains to be tested. The significant variation in the vertical pattern and abundance of the various nitrate reducing genes phylotypes reasonably suggests differences in their activity throughout the sediment column. This raises interesting questions as to what the alternative metabolic roles for the various nitrate reductases could be, analogous to the alternative metabolic roles found for nitrite reductases.     

The effects of aging on the intimal region of the human saphenous vein: insights from multimodal microscopy and quantitative image analysis

We hypothesized that structural remodeling associated with advancing age occurs in human saphenous veins. To address this hypothesis, we have identified structural remodeling in human saphenous veins by applying histochemistry, fluorescence staining and quantitative image analysis to specifically assess intimal area, intimal cellularity and intimal collagen content and organization. Saphenous veins were collected from patients undergoing coronary artery bypass graft surgery. Area measurements and cellularity were quantified using the image analysis software Stereo Investigator, employing planimetry and counting frames, respectively. Collagen content and organization were quantified in MetaMorph image analysis software based on measurements of color (hue, saturation, and intensity) from polarized light images. Intimal area and cellularity showed no statistically significant increases with age; in contrast, total collagen content showed a significant decrease with advancing age. Furthermore, collagen fiber types also demonstrated a statistically significant alteration with age; increases in age resulted in decreases in larger collagen fibers. No significant changes in small collagen fibers were identified. These results raise the possibility that age-associated structural alterations in total collagen content, specifically collagen fiber size, could be a factor in the etiology of age-associated venous diseases.     

Modulation of function in a minimalist heme-binding membrane protein

De novo designed heme-binding proteins have been used successfully to recapitulate features of natural hemoproteins. This approach has now been extended to membrane-soluble model proteins. Our group designed a functional hemoprotein, ME1, by engineering a bishistidine binding site into a natural membrane protein, glycophorin A (Cordova et al. in J Am Chem Soc 129:512–518, 2007). ME1 binds iron(III) protoporphyrin IX with submicromolar affinity, has a redox potential of −128 mV, and displays peroxidase activity. Here, we show the effect of aromatic residues in modulating the redox potential in the context of a membrane-soluble model system. We designed aromatic interactions with the heme through a single-point mutant, G25F, in which a phenylalanine is designed to dock against the porphyrin ring. This mutation results in roughly tenfold tighter binding to iron(III) protoporphyrin IX (K _d,app = 6.5 × 10⁻⁸ M), and lowers the redox potential of the cofactor to −172 mV. This work demonstrates that specific design features aimed at controlling the properties of bound cofactors can be introduced in a minimalist membrane hemoprotein model. The ability to modulate the redox potential of cofactors embedded in artificial membrane proteins is crucial for the design of electron transfer chains across membranes in functional photosynthetic devices.     

Modular genetic control of sexually dimorphic behaviors

Sex hormones such as estrogen and testosterone are essential for sexually dimorphic behaviors in vertebrates. However, the hormone-activated molecular mechanisms that control the development and function of the underlying neural circuits remain poorly defined. We have identified numerous sexually dimorphic gene expression patterns in the adult mouse hypothalamus and amygdala. We find that adult sex hormones regulate these expression patterns in a sex-specific, regionally restricted manner, suggesting that these genes regulate sex typical behaviors. Indeed, we find that mice with targeted disruptions of each of four of these genes (Brs3, Cckar, Irs4, Sytl4) exhibit extremely specific deficits in sex specific behaviors, with single genes controlling the pattern or extent of male sexual behavior, male aggression, maternal behavior, or female sexual behavior. Taken together, our findings demonstrate that various components of sexually dimorphic behaviors are governed by separable genetic programs.     

Increased genetic and phenotypic stability of a promising live-attenuated respiratory syncytial virus vaccine candidate by reverse genetics

Human respiratory syncytial virus (RSV) is the most important viral cause of serious pediatric respiratory illness worldwide. Currently, the most promising live-attenuated vaccine candidate is a temperature-sensitive (ts) cDNA-derived virus named rA2cp248/404/1030ΔSH, in reference to its set of attenuating mutations. In a previous clinical study, more than one-third of postvaccination nasal wash isolates exhibited partial loss of the ts phenotype. Most of this instability appeared to be due to reversion at a missense point mutation called 1030. This 1030 mutation is a single-nucleotide tyrosine-to-asparagine substitution at position 1321 (Y1321N) of the polymerase L protein that contributes to the ts and attenuation phenotypes of the vaccine candidate. The goals of the present study were to identify a reversion-resistant codon at position 1321 conferring a comparable level of attenuation and to use this to develop a genetically stable version of the vaccine virus. We modified wild-type (wt) RSV to insert each of the 20 possible amino acids at position 1321; 19 viruses were recoverable. We also investigated small deletions at or near this position, but these viruses were not recoverable. Phenotypic analysis identified alternative attenuating amino acids for position 1321. Several of these amino acids were predicted, based on the genetic code, to be refractory to deattenuation. Classical genetics, using temperature stress tests in vitro combined with nucleotide sequencing, confirmed this stability but identified a second site with a compensatory mutation at position 1313. It was possible to stabilize the 1313 site as well, providing a stable 1030 mutation. Further stress tests identified additional incidental mutations, but these did not reverse the ts/attenuation phenotype. An improved version of the vaccine candidate virus was constructed and validated in vitro by temperature stress tests and in vivo by evaluation of attenuation in seronegative chimpanzees. In addition to developing an improved version of this promising live-attenuated RSV vaccine candidate, this study demonstrated the propensity of an RNA virus to escape from attenuation but also showed that, through systematic analysis, genetics can be used to cut off the routes of escape.     

Time-Dependent Modulation of Mitogen Activated Protein Kinases and AKT in Rat Hippocampus and Cortex in the Pilocarpine Model of Epilepsy

The epileptogenesis may involve a variety of signaling events that culminate with synaptic reorganization. Mitogen-activated protein kinases (MAPKs) and AKT may be activated by diverse stimulus including neurotransmitter, oxidative stress, growth factors and cytokines and are involved in synaptic plasticity in the hippocampus and cerebral cortex. The pilocarpine model in rodents reproduces the main features of mesial temporal lobe epilepsy related to hippocampus sclerosis (MTLE-HS) in humans. We analyze the phosphorylation profile of MAPKs (ERK1/2, p38(MAPK), JNK1/2/3) and AKT by western blotting in the hippocampus (Hip) and cortex (Ctx) of male adult wistar rats in different periods, after pilocarpine induced status epilepticus (Pilo-SE) and compared with control animals. Biochemical analysis were done in the Hip and Ctx at 1, 3, 12?h (acute period), 5?days (latent period) and 50?days (chronic period) after Pilo-SE onset. Hence, the main findings include increased phosphorylation of ERK1 and p38(MAPK) in the Hip and Ctx 1 and 12?h after the Pilo-SE onset. The JNK2/3 isoform (54?kDa) phosphorylation was decreased at 3?h after the Pilo-SE onset and in the chronic period in the Hip and Ctx. The AKT phosphorylation increased only in the Hip during the latent period. Our study demonstrates, in a systematic manner, the profile of MAPKs and AKT modulation in the hippocampus and cerebral cortex in response to pilocarpine. Based in the role of each signaling enzyme is possible that these changes may be related, at least partially, to modifications in the intrinsic neuronal physiology and epileptogenic synaptic network that appears in the MTLE-HS.     

The mechanism of shared but distinct CSF-1R signaling by the non-homologous cytokines IL-34 and CSF-1

Interleukin-34 (IL-34) and colony stimulating factor-1 (CSF-1) both signal through the CSF-1R receptor tyrosine kinase, but they have no sequence homology, and their functions and signaling activities are not identical. We report the crystal structures of mouse IL-34 alone and in complex with the N-terminal three immunoglobulin-like domains (D1-D3) of mouse CSF-1R. IL-34 is structurally related to other helical hematopoietic cytokines, but contains two additional helices integrally associated with the four shared helices. The non-covalently linked IL-34 homodimer recruits two copies of CSF-1R on the sides of the helical bundles, with an overall shape similar to the CSF-1:CSF-1R complex, but the flexible linker between CSF-1R D2 and D3 allows these domains to clamp IL-34 and CSF-1 at different angles. Functional dissection of the IL-34:CSF-1R interface indicates that the hydrophobic interactions, rather than the salt bridge network, dominate the biological activity of IL-34. To degenerately recognize two ligands with completely different surfaces, CSF-1R apparently takes advantage of different subsets of a chemically inert surface that can be tuned to fit different ligand shapes. Differentiated signaling between IL-34 and CSF-1 is likely achieved by the relative thermodynamic independence of IL-34 vs. negative cooperativity of CSF-1 at the receptor-recognition sites, in combination with the difference in hydrophobicity which dictates a more stable IL-34:CSF-1R complex compared to the CSF-1:CSF-1R complex.