DNA barcoding, as it is currently employed, enhances use of marine benthic macrofauna as environmental condition indicators by improving the speed and accuracy of the underlying taxonomic identifications. The next generation of barcoding applications, processing bulk environmental samples, will likely only provide presence information. However, macrofauna indices presently used to interpret these data are based on species abundances. To assess the importance of this difference, we evaluated the performance of the Southern California Benthic Response Index (BRI) and the AZTI Marine Biotic Index (AMBI) when species abundance data were removed from their calculation. Presence only versions of these two indices were created by eliminating abundance weighting while preserving species identity. Associations between the presence and abundance BRI, and the presence and abundance AMBI were highly significant, with correlation coefficients of 0.99 and 0.81, respectively. The presence versions validated almost equally to the abundance-based indices when applied to the spatial and the temporal monitoring data used to validate the original indices. Simulations in which taxa were systematically removed from calculation of the indices were also conducted to assess how large the barcode library must be for the indices to be effective. Correlation between the BRI-P and BRI remained above 0.9 with only 370 species in the library and reducing the number of species to 450 had almost no effect on correlation between the presence and abundance versions of the AMBI. Marine benthic macrofauna are frequently used as indicators of environmental condition because they reside in sediments where contaminants accumulate and their immobility allows them to integrate exposure at a site Challenges in using benthos as indicators are the cost, time and error associated with identifying the biota. Organisms must be manually separated from the sediment, which can take more than a day and is subject to underestimation as some remain hidden among the debris http://www.boldsystems.org) reference library.DNA barcoding has the potential to increase the speed, accuracy and resolution of species identification Traditional DNA barcoding is based on Sanger sequencing in which each specimen is processed individually. Next-generation sequencing has the potential to further reduce the time and cost for processing through bulk processing The two indices used to compare performance between abundance and presence-only derivations were the Benthic Response Index (BRI) et al.The presence BRI (BRI-P) was calculated using the tolerance scores of Smith et al.The presence AMBI (AMBI-P) calculations followed Borja Performance of the BRI-P and AMBI-P were assessed using the two southern California data sets used to validate the original BRI, both of which were independent of the calibration data used to develop the indices. The first tested whether the BRI-P and AMBI-P reproduced known temporal gradients of benthic conditions over several years near a southern California waste-water outfall using data from two Los Angeles County Sanitation Districts monitoring sites which were sampled monthly since 1972. The first site, Station 6C  was severely impacted in the early 1970 s and has improved since that time The second data set was used to test whether the presence indices reproduced a known spatial gradient between two stations on the 60 m isobath from the Orange County Sanitation Districts outfall. Previous studies have shown that Station 0 located near the outfall has altered species composition in comparison to reference Station Con, which is located 7840 m from the outfall The first data set was also used to confirm that the observed benthic index patterns were likely responses to pollution. Sediment contaminant effects were tested by comparing benthic index values to the combined effects of eight metals , and eutrophication effects were tested using organic nitrogen concentrations. The effects of the eight metals were combined by calculating mean effects range median ERM  quotientTo assess how large the barcode library must be for an effective index, changes in Pearson correlation coefficients between the BRI-P and the BRI and the AMBI-P and AMBI were determined as taxa were systematically removed from calculation of the indices. To accomplish this, taxa were ranked according to their tolerance scores (BRI) or ecological groups and abundance (AMBI) and selected for removal at even intervals. This process was repeated with increasing percentages of taxa removed from the calculation.There was a strong linear relationship between the BRI-P and BRI  with a PThe relationship between the AMBI-P and AMBI  was alsoApplication of the BRI-P to the validation data resulted in patterns almost identical to the original abundance BRI results for both validation data sets, and AMBI-P patterns were similar to AMBI results. For the Los Angeles County outfall temporal data, the BRI-P  and the The BRI-P  and AMBIThe BRI-P and AMBI-P index values for the Los Angeles County outfall temporal data were significantly correlated with mean ERM quotients and sediment organic nitrogen concentrations , indicatReducing the number of species included in BRI-P calculations resulted in a correlation greater than 0.9 relative to the abundance BRI even when 20% of the species with tolerance scores were removed . For theThe benthic community gradients used to validate the original BRI were reproduced closely by the BRI-P and AMBI-P. BRI-P and AMBI-P values at the two temporal validation stations were also highly correlated with sediment metal and organic nitrogen concentrations and patterns over time closely matched, indicating that the benthic indices were likely responding to anthropogenic pollution effects. There were only minor reductions in classification efficiency using presence-only information and no changes in the patterns or magnitudes of difference among sites. Reductions in index performance were small relative to previous attempts to reduce processing cost by identifying taxa only to genus or family level et al.et al.et al.et al.Similarity in performance between the BRI-P, AMBI-P and the traditional BRI results because the indices rely on composition of the whole community, not just the dominant taxa. Smith While the presence based approach worked well for the BRI and AMBI, it is unclear whether it will be equally successful in all situations. For instance, this study was conducted in euhaline water where there was an average of 67 taxa per sample; the presence-only index may be less sensitive in oligohaline waters where index development is more challenging because there are typically fewer than ten taxa per sample, even at reference sites Development of a locally relevant DNA reference library has been suggested as a potential impediment to incorporation of molecular methods in routine bioassessment, but our results suggest this should not be problematic. There are DNA barcode sequences for 159 southern California taxa with index tolerance scores already cataloged in BOLD and we found that reliable indices can be produced with a reference barcode library of less than 400 taxa. Moreover, the 159 taxa are relatively evenly distributed across the disturbance gradient . Of 225 There are several aspects of molecular methods that need to be investigated before they can be adopted for biological assessments, such as improved understanding of intraspecific genetic variation within groups currently considered as single species. There are also issues associated with sample handling. For instance, formalin, the presently used preservative for benthic studies, does not preserve DNA and the typical methods for preserving DNA are not conducive to field collections. However, the lack of quantification in species identification does not seem to be an impediment to adoption of molecular methods in biological assessments.


The secretory pathway delivers proteins synthesized at the rough endoplasmic reticulum (RER) to various subcellular locations via the Golgi apparatus. Currently, efforts are focused on understanding the molecular machineries driving individual processes at the RER and Golgi that package, modify and transport proteins. However, studies are routinely performed using non-dividing cells. This obscures the critical issue of how the secretory pathway is affected by cell division. Indeed, several studies have indicated that protein trafficking is down-regulated during mitosis. Moreover, the RER and Golgi apparatus exhibit gross reorganization in mitosis. Here I provide a relatively neglected perspective of how the mitotic cyclin-dependent kinase (CDK1) could regulate various stages of the secretory pathway. I highlight several aspects of the mitotic control of protein trafficking that remain unresolved and suggest that further studies on how the mitotic CDK1 influences the secretory pathway are necessary to obtain a deeper understanding of protein transport. The timely localization of intracellular proteins is required for the survival of cells. A key pathway needed for the proper subcellular localization of proteins is the secretory pathway that transports proteins from the rough endoplasmic reticulum (RER) via the Golgi to the lysosome, plasma membrane, or the exterior of the cell Whereas the secretory pathway functions in the forward trafficking of proteins, endocytosis pathways also exist that contribute to the internalization of plasma membrane and plasma membrane proteins, and in the uptake of nutrients Protein trafficking through the secretory pathway is no less complicated than endocytosis and occurs in a highly regulated manner Cell division itself is a complex process with the main regulators being the cyclin-dependent kinases (CDKs) and their associated cyclins. Essentially, at each cell cycle phase, specific cyclins bind to their respective CDKs to form cyclin-CDK complexes that promote phase-specific events Given the scant attention paid to studying the secretory pathway with respect to the cell division cycle, I describe here several key findings indicating that protein trafficking is down-regulated in mitosis. I highlight interesting but relatively unexplored data showing functional interactions between various components of the protein trafficking machinery and the major kinase in mitosis, the mitotic cyclin-CDK1 complex . I end wAs protein export from the RER has been reviewed recently The COPII coat forces the curvature of the RER membrane  17 on thIn mammalian cells, a peripheral RER membrane protein Sec16A localized at the ERES COPII coat proteins help select cargoes for incorporation into COPII vesicles cis-Golgi or an ER-to-Golgi intermediate compartment (ERGIC) in mammalian cells. Rab GTPases are recruited to the COPII vesicles and upon activation in the presence of GEFs, exchange GDP to GTP Upon budding from the RER, the COPII-coated vesicles either uncoat fully or partially Various other proteins known as tethering factors are required for establishing the initial association between vesicles and the destination membrane N-ethylmaleimide-sensitive fusion protein (NSF)-attachment protein receptors on the vesicle membrane (vSNAREs) and target (t)SNAREs on the ERGIC membrane bring the membranes closer to each other so as to enable membrane fusion and delivery of the vesicle contents to the destination cis-Golgi  Subsequent to the actions of Rab GTPases and tethering proteins, soluble is-Golgi  or undercis-, medial-, and trans-Golgi. The proposed progression/maturation models are based on the idea that cargoes move through the Golgi stack in a non-vesicular manner. A pathway known as the retrograde pathway, involving the COPI vesicles, retrieves proteins and membranes from the Golgi to the RER and within the Golgi apparatus trans-Golgi to the cis-Golgi, leading to the maturation of the cis-Golgi. A generally accepted consensus is the post-translational modification of cargoes by glycosylation or disulphide bond formation during passage through the cis-, medial-, and trans-Golgi network , have been suggested ork TGN; .A COPI-independent pathway also functions to recycle proteins and lipids from the Golgi to the RER Upon reaching the TGN, specific cargoes are sorted for delivery to different subcellular locations such as the lysosomes, plasma membrane, and endosomes In mammalian cells, TGN-to-plasma membrane transport carriers ranging from several hundred nanometers to 1.7 µm in length are formed when membranes encompassing cargoes are pulled off from the TGN  39. BothTransport of lysosomal proteins from the TGN goes through the endosomes  towards the cell cortex The transport of vesicular carriers could further depend upon actin filaments and myosin motors. For instance, the movement of specialized transport carriers such as secretory granules (SGs) from the TGN to the cortical region in neuroendocrine PC12 cells requires microtubules and kinesin-1. Upon reaching the cortical region, the SGs rely upon actin filaments and the myosin motor, Myo5a, for delivery to the plasma membrane, resulting eventually in the fusion of the SGs to the plasma membrane and release of the neuropeptides out of the cell That microtubules and actin filaments are necessary for protein trafficking has also been reported for the glucose transporter Glut-4 in cells responding to insulin Studies on the secretory pathway have thus far revealed the complex processes needed to ensure the successful stepwise delivery of cargoes from one compartment to the next. As these studies have largely been conducted in non-dividing cells, details pertaining to how the secretory pathway is altered during cell division are unclear. Interestingly, several reports have described data showing the down-regulation of protein trafficking during mitosis. From the data discussed below, it is clear that understanding how protein transport is regulated in mitosis is an important step towards building a more comprehensive model of protein trafficking in the cell.The notion that protein trafficking is negatively regulated during mitosis is supported by studies using the membrane-spanning G protein of the vesicular stomatitis virus (VSV-G) as a protein cargo. In normal rat kidney The down-regulation of the secretory pathway in mitosis also extends to soluble proteins, as a truncated VSV-G lacking the transmembrane domain, which behaves like a luminal protein, was found at the RER in mitotic cells O-glycosylation and phosphorylation of Sec24 have previously been noted in mitotic but not interphase cells It is unclear what the mechanism is that underlies the retention of cargoes at the RER in mitosis The down-regulation of the secretory pathway might stem from a disruption to the initial steps of cargo export at the RER. In interphase CHO and HeLa cells, presumptive ERES are marked by COPII coat proteins such as Sec13 Metaphase cells have been shown to exhibit shortened buds protruding at the ERES as compared to those in interphase It is unknown what the targets of the mitotic cyclin-CDK1 are that play a role in ERES integrity. Intriguingly, an essential co-factor of Golgi biogenesis, p47, was phosphorylated by the mitotic cyclin-CDK1 The level of cargoes in the RER has in turn been shown to affect the number of ERES Using an in vitro Golgi reconstitution assay that examines the status of glycosylation of VSV-G as an indication of passage through the Golgi cis- to the trans-Golgi 3H]serine to various intermediates of GSLs through the Golgi apparatus was examined as an indication of transport through the Golgi apparatus. A late intermediate, GA2LacCer, was detected in interphase but not mitotic cells. The authors excluded the possibility that the enzyme needed for the synthesis of GA2LacCer, UDP-GalNAc:lactosylceramide(βl-4)N-acetylgalactosaminyltransferase , was absent or inactive during mitosis, and hence suggested that the absence of the GSL intermediate was indeed due to a block in intra-Golgi transport.The effect of the mitotic phase on trafficking through the Golgi apparatus was corroborated by a study that examined the transport of glycosphingolipids (GSLs) from the A likely factor contributing to the interruption of intra-Golgi transport could be the fragmentation of the Golgi apparatus during mitosis  68–71. TThe TGN-to-plasma membrane trafficking depends upon transport carriers such as SGs that carry specialized cargoes from the TGN Contrary to this observation, glycosaminoglycan chains (GACs) synthesized in CHO cells were transported from the TGN to the plasma membrane at the same levels in interphase and mitotic cells Given that the mammalian cells undergo an open mitosis, the disruption of the protein transport might be a consequence of the gross structural reorganization of the RER. During interphase, the RER in mammalian cells is continuous with the nuclear envelope and extends to the rest of the cell as peripheral tubules and sheets There are disagreements over how the structure of the RER changes during mitosis. In HeLa cells, the mitotic RER assumes an extended cisternal arrangement, with a few tubular structures juxtaposed to the mitotic spindles Saccharomyces cerevisiae goes through a closed mitosis with an intact nuclear envelope Interestingly, Similar to the RER, the Golgi apparatus is also altered structurally in mitosis. During interphase, the maintenance of the Golgi structure depends upon both the arrival of COPII vesicles from the RER fusing with the Golgi as well as the COPI vesicles that recycle membranes and proteins during intra-Golgi transport In addition, a tethering factor associated with the Golgi membrane known as GM130 is phosphorylated Other targets of the mitotic CDK1 include the Golgi reassembly stacking proteins (GRASPs), GRASP55 and GRASP65 The structural organization of the Golgi further depends upon the AAA ATPase, p97 together with its associated proteins, p47 GM130 in HeLa cells did not affect VSV-G trafficking GM130 siRNA cells with streptolysin-O and incubating the permeabilized cells with or without mitotic lysates and examining the transport of VSV-G.While the mitotic CDK1 induces Golgi disassembly, the significance of Golgi fragmentation in suspending protein trafficking has not been fully investigated. In fact, the finding that breaking down the Golgi apparatus by the small interfering RNA (siRNA)-mediated knock-down of Cytoskeletal tracks upon which the secretory vesicles travel are highly altered in mitosis. The microtubules undergo dynamic organization not only in interphase cells The maintenance of the Golgi structure depends upon both microtubules and actin Exit from mitosis, characterized by a reduction in the mitotic kinase activity, is largely due to the destruction of the mitotic cyclins Recently, it was shown that the down-regulation of Myt1 late in mitosis led to abnormal RER and Golgi morphologies in HeLa cells Another noteworthy issue is the effect of CDK1 activity on the movement of vesicles from the post-Golgi to the cleavage furrow at the cytokinesis site at the end of mitosis In mammalian cells, endocytic markers have been observed to concentrate at the cleavage furrow, which is the site of cytokinesis towards the end of mitosis That the mitotic regulator CDK1 promotes not only events such as chromosome segregation but also down-regulates protein trafficking in mitosis highlights its central role in coordinating diverse processes in the cell. This is critical for ensuring proper progression through mitosis to produce viable progeny cells. Specific details as to how the CDK1 might affect the different stages of the secretory pathway warrant further studies using synchronized cells as well as in vitro assays to provide a more complete description of how the protein trafficking process is regulated in mitosis and also in interphase. In addition to endogenous cargoes, exogenously expressed cargoes, such as green fluorescent protein (GFP) fusions, could be used to dissect how the interactions between the CDK1 and protein trafficking components lead to dynamic changes in protein transport in live cells, which is not possible when examining endogenously expressed proteins.Currently, several key questions about protein trafficking in mitosis remain unanswered, such as how the COPII coats are affected by the mitotic CDK1. Studies using in vitro RER budding assays e.g.  or permeThe effect of alterations in RER structure per se on protein export needs to be verified. For instance, structural reorganization of the RER in G1 cells devoid of CDK1 activity could be induced by altering the levels of proteins such as reticulons that modulate RER structure The effects of the mitotic CDK1 activity on the reorganization of cytoskeletal structures It would also be relevant to study whether the inhibition of the secretory pathway at the end of mitosis is due merely to relieving the inhibitory effects of the mitotic CDK1 activity, as other kinases such as Wee1 and Myt1 are needed to re-establish the RER and Golgi Finally, using high-content screening methods incorporating GFP-fused cargoes, it might be possible to screen substrates of CDK1


Halophiles are extremophilic microorganisms growing optimally at high salt concentrations. There are two strategies used by halophiles to maintain proper osmotic pressure in their cytoplasm: accumulation of molar concentrations of potassium and chloride with extensive adaptation of the intracellular macromolecules  or biosynthesis and/or accumulation of organic osmotic solutes ("osmolyte" strategy). Our work was aimed at contributing to the understanding of the shared molecular mechanisms of protein haloadaptation through a detailed and systematic comparison of a sample of several three-dimensional structures of halophilic and non-halophilic proteins. Structural differences observed between the "salt-in" and the mesophilic homologous proteins were contrasted to those observed between the "osmolyte" and mesophilic pairs.The results suggest that haloadaptation strategy in the presence of molar salt concentration, but not of osmolytes, necessitates a weakening of the hydrophobic interactions, in particular at the level of conserved hydrophobic contacts. Weakening of these interactions counterbalances their strengthening by the presence of salts in solution and may help the structure preventing aggregation and/or loss of function in hypersaline environments.Considering the significant increase of biotechnology applications of halophiles, the understanding of halophilicity can provide the theoretical basis for the engineering of proteins of great interest because stable at concentrations of salts that cause the denaturation or aggregation of the majority of macromolecules. Organisms thriving in "extreme environments", such as thermophiles, alkalophiles, acidophiles, halophiles, piezophiles and psychrophiles, have drawn much interest in the scientific community because of the molecular adaptation they underwent during evolution and for their biotechnological potential -4. The eHalophilic microorganisms are salt-loving extremophilic organisms that grow optimally at high salt concentrations. They were found  mainly iArchaea kingdoms. Eukaryotic halophilic microorganisms, such as fungi and algae, are also known [Distribution of halophilic microorganisms within the tree of life shows that they are widespread in the bacterial and so known .Archaea. The other strategy of haloadaptation is based on the biosynthesis and/or accumulation of organic osmotic solutes (osmolytes) such as ectoin, glycine betaine or others [All halophilic microorganisms share a basic property: their cytoplasm must be at least isoosmotic with their surrounding medium. There are two strategies used by halophilic microorganisms to maintain proper osmotic pressure in their cytoplasm. The first involves accumulation of molar concentrations of potassium and chloride. This strategy requires extensive adaptation of the intracellular enzymatic machinery to the presence of salts, as the proteins should maintain their proper conformation and activity at near-saturating salt concentrations. The proteome of such organisms is highly acidic, and most proteins denature when suspended in low salt. Generally, the microorganisms relying on such "salt-in" strategy are obligate halophilic r others . Cells rr others .Halophilic proteins from "salt-in" organisms are distinguished from their non-halophilic homologous proteins by exhibiting remarkable instability in low salt concentration and by maintaining soluble and active conformations in an environment generally detrimental to other proteins. Indeed, hypersaline conditions favor protein aggregation and collapse, interfere with the electrostatic interactions between protein residues, and are responsible for a general decrease in the availability of water molecules . HalophiArchaea that adopt the "salt-in" strategy (SALTIN), and 7 from Eubacteria that adopt the "osmolytes" strategy (OSMOL). In this paper, the entire analysis was carried out considering the two groups separately.A set of 15 halophilic enzymes, 9 of which from "extreme" halophiles and 6 from "halotolerant" organisms, was collected along with their non-halophilic structural homologues  have a quaternary structure different from their non-halophilic homologs (Table 552 from Marinobacter hydrocarbonoclasticus (PDB ID: 1CNO) and from Pseudomonas stutzeri (PDB ID: 1ETP) has been included in the analysis, for the reasons explained in Methods section.Four pairs of halophilic enzymes  and the magnitude of the relative polar and apolar components, protein structures were used in their quaternary form. The surface polar area formed by oxygen and nitrogen atoms was calculated. The area subset formed only by side-chain oxygen or nitrogen atoms  was also considered.ΔApA in Table In SALTIN enzymes, the fractional apolar component of the relative solvent accessible surface is significantly smaller than the corresponding area in non-halophilic counterparts  between residues was calculated for three different structural regions namely the core, the interface between the monomers and the protein surface. Residues were assigned to the various structural regions according to the alternative accessibility thresholds described in the Methods section.ACA in the core of SALTIN halophilic proteins is not significantly different, according to the statistical tests, from the ACA of their homologous counterparts. However, it is consistently smaller Figure . This trΔACAs were negative except for the pair 3IFV-1RWZ. The analysis at interface for OSMOL enzymes is reduced to two pairs and is therefore of no statistical interest, however, the ACA formed by interfacial residues in halophiles is comparable to that of non-halophilic organisms.Likewise, ACA in the SALTIN monomer interface is not significantly dissimilar from the ACA of their non-halophilic counterparts. However, even in this case, all the The surface ACA in SALTIN and OSMOL halophilic enzymes is not significantly different from the corresponding surface in the non-halophilic counterparts Figure .ΔΔApAU-F) between each halophilic and non-halophilic pair, have been calculated. According to the Wilcoxon and the t-test, none of the average values were significantly different from 0. Distribution of the differences in the SALTIN and OSMOL samples are reported in additional file Differences in apolar area buried upon folding  were calculated. The conserved contacts between hydrophobic residues (CHC) were identified for each pair of structures, and were selected only those formed by evolutionary conserved residues. Then for each CHC, the apolar contact area formed by the pair of residues was calculated. The number of CHCs selected varies for each enzymatic family depending on the relative degree of conservation of residues in that family Table .In SALTIN sample the average ACA in CHCs is significantly smaller in halophilic enzymes than non-halophilic counterparts Table . At vari→ halophile with a Z-score equal to 4.34. At variance, the most frequent exchange in the OSMOL CHCs is Leu → Ile with Z-score 3.74.Statistics on the preferred amino acid exchanges at the SALTIN CHCs indicate that Ile → Val is the most frequent replacement found in the direction mesophile Salinibacter ruber recently characterized [Haloferax volcanii [Halophilic proteins have been extensively studied since a long time  and verycterized  shares cvolcanii  displayeArchaea) that utilize the "salt-in" strategy to cope with the strong environmental osmotic pressure, and from halophilic microorganisms using the intracellular accumulation of osmolytes and low cytoplasmatic salt concentration to counterbalance the external osmotic pressure. Proteins synthesized by the "salt-in" organisms are surrounded by a high-salt concentration environment and are in contact with it. At variance, intracellular proteins from the "osmolyte" cells are surrounded by a high concentration solution of compatible solutes which apparently do not interfere significantly with protein solubility, stability and activity [t-test and the non-parametric Wilcoxon test) were used to assess the significance of observed differences of structural parameters. Only differences defined significant according to both tests were deemed bona fide significant signals.Proteins were taken both from microorganisms  that halophilicity of these proteins is directly related to a decrease in the solvent accessible surface. Authors stated that reduction of the solvent accessible area introduced upon mutation causes a progressive destabilization of the molecule, probably due to a reduction in the protein's hydrophobic effect. As a consequence, mutations increasing salt induced stabilization also destabilize the protein in the absence of salt, converting a mesophilic protein into an obligate halophilic form, a trend found in natural halophilic proteins.We noticed that a significant decrease of apolar surface area in the protein synthesized by the halophilic organism can be observed in the OSMOL sample as well, paralleled by an ostensible tendency to increase the presence of side chain oxygen atoms Table . Most ofalinarum  or in anismortui . Thermalismortui . The picismortui  by extenHowever, we did not observe any significant reduction of the accessibility surface (data not shown) in the SALTIN and OSMOL halophilic proteins whereas we noticed the tendency, in the halophilic proteins of the SALTIN samples, to reduce the apolar contact area of the residues exposing more than 25% of their surface. Therefore one could argue that the reduction of hydrophobic effect be a key component of the haloadaptation mechanisms of surfaces.However, surface property variations are not the only feature relevant to protein stability and solubility in high salt environments. Indeed, proper folding of the protein in these conditions requires the formation of appropriate hydrophobic interactions in the interior of the macromolecule. This is supposedly even more important during the early step of protein folding of the nascent polypeptide chain. In this perspective view, we analyzed the variations of apolar contact area in the hydrophobic core of the halophilic SALTIN and OSMOL surface compared to their mesophilic counterparts. No statistically significant difference could be found. Nonetheless, it should be noted that a consistent decrease of the apolar contact area in the halophilic proteins of the SALTIN sample can be observed for core residues at 0% relative accessibility Table . We beliΔΔApAU-F) between each halophilic and non-halophilic pairs display overall no significant difference. This may suggest that, globally, the fraction of apolar area lost during folding is similar in the two cases  although the apolar surface available to the halophilic protein is smaller. This observation is reflected and supported by the significant reduction of exposed apolar area  can provide the theoretical basis for the engineering of proteins of great interest because stable at concentrations of salts that cause the denaturation or aggregation of the majority of macromolecules.http://www.xbase.ac.uk/ and in the literature. The selected organisms have optimal growth conditions that support the presence of genuine adaptation to high salt concentration environments. In fact, those organisms requiring for optimum growth concentration of NaCl < 0.5 M and/or temperatures < 20°C or > 55°C, were excluded from the data sample. Moreover, the organisms were divided into two groups corresponding to different survival strategies: in the first group are the archaeal halophiles using the "salt-in" strategy (which will be referred to as SALTIN sample); in the latter the Eubacteria using the "osmolytes" strategy (OSMOL sample) [The crystallographic structures of the available high salt concentration active enzymes were found in PDB . The sea sample) .http://dunbrack.fccc.edu/PISCES.php[To select only halophilic structures matching specific criteria and to eliminate redundancy, we used the PISCES server ISCES.php, which eISCES.php. To ensuThe apolar contact area is the area of contact between coupled non-polar atoms in a protein. It was calculated for different structural environments: nucleus, interface between monomers and protein surface. The program utilized was Pdb_np_cont , which cIndividual protein chains were used for analysis of the protein core, while analyses of the protein subunit interfaces were carried out with structures in their quaternary assembly. Only the 9 pairs of proteins possessing an oligomeric biological unit were used in this case. Protein interfaces consists of those residues making apolar contacts to another protein chain, as defined by Pdb_np_cont .A residue was assigned to the structural environment "nucleus" if its relative solvent accessibility, calculated with the program NACCESS , is lessFor each structural environment considered the per-residue mean apolar contact area was calculated by dividing the total apolar contact area by the number of residues belonging to that structural environment.i.e. belonging to the classes 7, 8 and 9. These three classes contain the most conserved amino acid positions from a total of nine equally sized categories of relative degree of conservation.Conserved hydrophobic contacts (CHCs) were identified in each pair of homologous enzymes through the combined use of the web tools CE-MC  for calcX, belonging to a non-halophilic protein and involved in making CHCs, aaX→Y was defined as the number of times X is substituted by the residue Y of the halophilic protein. Likewise, aaY→X is defined. Therefore, a substitution matrix can be obtained by computing the difference between aaX→Y and aaY→X over the whole dataset of protein pairs k, according to:Amino acid substitutions of residues involved in the formation of structurally and evolutionarily conserved hydrophobic contacts between halophilic and non-halophilic proteins were determined by analysing the alignment of the SCRs of each pair. For each residue S Cis the element of the substitution matrix.where XY Rof the exchange X→Y was then calculated by dividing the difference between SC, and the overall matrix mean The mean and standard deviation of the overall exchange matrix were determined; the significance XY Rvalues ≥ 3.0 standard deviations (corresponding to a probability P ≤ 0.01 that the observed difference was obtained by chance) from the mean value were considered statistically significant [nificant .http://curie.utmb.edu/getarea.html with default settings [The ASA is the area of the protein surface which can be in contact with the solvent. The total apolar (and the complementary polar) component of the entire accessible surface area of each protein in its quaternary form was calculated with the server GETAREA settings . The conΔApAU-F) were calculated through the web server http://roselab.jhu.edu/utils/unfolded.html[ΔApAU-F in each halophilic protein and in its corresponding homolog (ΔΔApAU-F) were calculated and statistically tested.The differences between the fraction of apolar accessible surface area in the unfolded and folded form of each protein considered  in the SALTIN and OSMOL samples for different class of atoms. The differences are between the surface areas calculated in the halophilic protein and the corresponding areas in the non-halophilic counterpart. The calculations were performed considering the proteins in their quaternary structure. Only residues identically conserved in the two proteins were considered in the calculations.Click here for fileΔΔApAU-FAdditional Figure 1 - . Histograms reporting the ΔΔApAU-F in the SALTIN and OSMOL samples. The ΔΔApAU-F values were calculated as the difference between the fraction of exposed apolar area lost during folding of the halophilic protein and the fraction lost by the corresponding non-halophilic homolog. Further details are reported in the "Methods" section of the main text.Click here for fileΔACA and pairwise percentage identityAdditional Figure 2 - Correlation between single . Graph reporting the difference between the area of each halophilic CHC and that of the corresponding non-halophilic CHC (ΔACA) versus the pairwise sequence percentage identity (%id) for the SALTIN and OSMOL samples.Click here for file


The coeruleospinal inhibitory pathway (CSIP), the descending pathway from the nucleus locus coeruleus (LC) and the nucleus subcoeruleus (SC), is one of the centrifugal pain control systems. This review answers two questions regarding the role coeruleospinal inhibition plays in the mammalian brain. First is related to an abnormal pain state, such as inflammation. Peripheral inflammation activated the CSIP, and activation of this pathway resulted in a decrease in the extent of the development of inflammatory hyperalgesia. During inflammation, the responses of the dorsal horn neurons to graded heat stimuli in the LC/SC-lesioned rats did not produce a further increase with the increase of stimulus intensity in the higher range temperatures. These results suggest that the function of CSIP is to maintain the accuracy of intensity coding in the dorsal horn because the plateauing of the heat-evoked response in the LC/SC-lesioned rats during inflammation is due to a response saturation that results from the lack of coeruleospinal inhibition. The second concerns attention and vigilance. During freezing behavior induced by air-puff stimulation, nociceptive signals were inhibited by the CSIP. The result implies that the CSIP suppresses pain system to extract other sensory information that is essential for circumstantial judgment. It is a general principle that the brain regulates its sensory inputs. This principle applies to all of the somatosensory pathways that have been investigated. The inhibitory regulation of nociceptive inputs is of particular clinical interest because this regulation may lead to a reduction of pain. Inhibitory action on nociceptive processing is accomplished via descending or ascending inhibitory pathways  and the nucleus subcoeruleus (SC) is one of centrifugal pain control systems. The LC/SC provides noradrenergic innervation of the spinal cord Guyenet, . ActivatDuring the first decade of the twenty-first century, we were particularly interested in the role of the CSIP in the everyday life of mammals, including its roles in normal and abnormal pain condition. Based on our experimental results that characterized coeruleospinal inhibition of nociceptive processing in the spinal cord, this review provides an answer to the question regarding the role of coeruleospinal inhibition in the mammalian brain. We hope that our inferences will aid in a better understanding of the role of centrifugal control of sensation.In a series of our study, inflammatory pain, but not neuropathic pain, was used as an abnormal pain state. Pain can divide three groups  on the basis of the source, nociceptive, neuropathic, and psychogenic pain. Inflammatory pain is nociceptive pain via nociceptor, neuropathic pain is a morbid pain induced by dysfunction of the peripheral or central nervous system, and psychogenic pain results from the psychological reasons. We adopted inflammatory pain because peripheral inflammation is a matter of frequent occurrence in compared to other pain in everyday life.We compared the development of peripheral hyperalgesia between rats that received bilateral lesions to the LC/SC and sham-operated, control animals for 4 weeks after administration of carrageenan (an inflammatory agent) Figure . Four hoWe examined, whether coeruleospinal inhibition of nociceptive processing depends on an interaction with other inhibitory systems that involve opioid peptides Figures , 3. In tOpioid inhibitory mechanisms were inactive in both the LC-lesioned rats and the sham-operated rats at 7 days, whereas edema and hyperalgesia were still present in the inflamed paws. Comparable data have been obtained in rats with unilateral inflammation in which naloxone induced no significant effect at 1 week, whereas this drug reduced the paw-pressure threshold 24 h after the induction of inflammation  in the relation between the DIP period and the startle magnitude, which suggests that the DIP period is not influenced by the startle magnitude. This finding generates the notion that the DIP is an independent component of the startle response, although the startle reaction and the DIP are continuous behavioral responses in the air-puff startle. In this context, the DIP period might be used as another endpoint for assessing the air-puff startle.Regression analysis showed a low correlation (The startle magnitude in the LC/SC-lesioned rats was significantly less than that before lesions, although the startle magnitude only slightly decreased. This result is consistent with preceding investigations in which an LC lesion decreased the effect of the startle reaction in rats (Adams and Geyer, LC/SC neurons have been implicated in regulation of attention and vigilance (Foote et al., Because stimulation of the CSIP produces inhibition of nociceptive transmission in the spinal dorsal horn (Tsuruoka et al., Air-puff stimulation induces the DIP, which is a defensive movement, following the startle reaction. It is known that the DIP is mediated by cortical areas, as well as by the LC/SC, in the brain (Cooke and Graziano, In addition, as mentioned in the section “Activation of the CSIP by Peripheral Inflammation,” the CSIP is active during an abnormal pain state in the peripheral tissues, such as peripheral inflammation (Tsuruoka and Willis, Noxious stimuli also induce nociceptive behavior mediated by a higher center of the brain (e.g., Woolfe and MacDonald, Bite latencies with air-puff stimulation were significantly prolonged when compared to values without air-puff stimulation (Tsuruoka et al., Regarding air-puff stimulation-induced nociceptive inhibition, there is a possibility that the air-puff stimulation-induced prolongation of the bite latency is due to decreased interactions in motor response/behavioral output systems, but not in decreased sensory processing. It seems that air-puff stimulation suppresses motor response/behavioral output systems so that the DIP is induced. However, as seen in Figure In the LC/SC-lesioned rats, air-puff stimulation-induced prolongation of bite latencies was not observed in post-lesions, whereas air-puff stimulation significantly prolonged bite latencies in pre-lesions Figure . In contIt has been reported that a connection from the dorsomedial hypothalamus through the rostral ventromedial medulla takes part in the circuitry of air-puff stress (Zaretsky et al., At this time, as shown in Figure Our results may support the finding reported by Bushnell et al.  that paiThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


To determine the standard of reporting of harms-related data, in randomised controlled trials (RCTs) according to the Consolidated Standards of Reporting Trials (CONSORT) statement extension for harms.Systematic review.The Cochrane library, Ovid MEDLINE, Scopus and ISI Web of Knowledge were searched for relevant literature.We included publications of studies that used the CONSORT harms extension to assess the reporting of harms in RCTs.We identified 7 studies which included between 10 and 205 RCTs. The clinical areas of the 7 studies were: hypertension (1), urology (1), epilepsy (1), complimentary medicine (2) and two not restricted to a clinical topic. Quality of the 7 studies was assessed by a risk of bias tool and was found to be variable. Adherence to the CONSORT harms criteria reported in the 7 studies was inadequate and variable across the items in the checklist. Adverse events are poorly defined, with 6 studies failing to exceed 50% adherence to the items in the checklist.Readers of RCT publications need to be able to balance the trade-offs between benefits and harms of interventions. This systematic review suggests that this is compromised due to poor reporting of harms which is evident across a range of clinical areas. Improvements in quality could be achieved by wider adoption of the CONSORT harms criteria by journals reporting RCTs. This is the first study to systematically review empirical studies assessing the quality of reporting according to the CONSORT-harms guideline.The review was strengthened by its assessment of quality of the included studies across four key domains.This study should be regarded as a reflection of reporting standards in general rather than an assessment of adherence to the CONSORT-harms extension.Some included studies contained trials reported prior to the publication of the CONSORT harms guideline; we did not extract these results.We have not assessed changes in reporting over time.Every healthcare intervention is associated with a risk of harmful or adverse events, that must be balanced against the potential favourable outcomes.1The Consolidated Standards of Reporting Trials (CONSORT) statement aims to improve the quality of published reports of randomised controlled trials (RCTs) and has been widely endorsed by healthcare journals, leading to improvements in quality when used by manuscript authors and peer reviewers.5–7The standard CONSORT statementA protocol for the systematic review was developed by AH, CTS, CG and JJK.We included published and unpublished research that evaluated the quality of harms reporting in RCTs against the CONSORT-harms recommendations.AH, CTS and CG developed the search strategy with support from an information specialist which was then implemented by AH in the following databases: the Cochrane Methodology register, Database of Abstracts of Reviews of Effects (DARE), Ovid MEDLINE, Scopus and ISI Web of Knowledge. Conference abstracts were searched for in the Web of Knowledge Conference Proceedings Citation Indexes (CPCI-S or CPCI-SSH) and the Zetoc database.Titles and abstracts of reports identified by the search were screened by AH and full articles obtained for all potentially eligible studies. Each full article was assessed independently by two reviewers (AH and CTS) to determine eligibility.Were the trials included in the study a representative sample, for example, unselected journals, and reasonable time scale?Low risk of bias: studies included trials from a primary search of all the available literature.High risk of bias: studies were highly selective of the trials included, for example, high-impact journals or specialised-journals only.Unclear risk of bias: not stated how studies were selected.During the data extraction of CONSORT-harms criteria, were reviewers blinded to study authors, institution, journal name and sponsors?Low risk of bias: reviewers were blinded.High risk of bias: reviewers were not blinded.Unclear risk of bias: not stated.Is there evidence of selective outcome reporting in the study ?Low risk of bias: studies that considered all CONSORT-harms criteria or reasons for excluding specific criteria were transparent and justified.High risk of bias: studies did not consider all CONSORT-harms criteria.Unclear risk of bias: unclear whether all CONSORT-harms criteria were considered.Did more than one reviewer assess the CONSORT-harms criteria for each primary RCT, with a description of how agreement was achieved?Low risk of bias: data extraction was completed independently by two people or reasonable attempts were made to maximise data extraction reliability.High risk of bias: data extraction not completed independently by two people.Unclear risk of bias: not stated.Two reviewers (AH and JJK) independently assessed the methodological quality of each study using the Cochrane Risk of Bias (RoB) toolStudy characteristics: inclusion criteria including clinical area, types of interventions, databases or journals searched within the study and any search date restrictions.Sample size .Reporting quality: inclusion of any of the 10 recommendations from the 2004 CONSORT-harms checklist (Two reviewers (AH and JJK) independently extracted the data and any discrepancies were resolved through a consensus discussion with a third reviewer (CTS). Data extraction includedLead authors were contacted through email with any queries relating to the quality assessment or data extraction.2 statistic . We refrained from statistically combining results from the different studies due to the differences in their study characteristics. In accordance with the Cochrane Handbook, I2 statistics were interpreted as .13For each study, the percentage of included RCTs that satisfied each CONSORT-harms recommendation is presented with 95% CIs. Some studies had presented data for individual items described within each of the 10 criteria rather than overall data. These are presented as such in tables with footnotes to provide further explanation. Forest plots were used to graphically depict the levels of adherence to the CONSORT harms recommendations so that readers can easily discern the extent of compliance and heterogeneity between studies with the IThe search strategy identified 5083 potentially eligible study cohorts from which seven studies assessing the quality of reporting across almost 800 RCTs were included .Five studiesLead authors were contacted by email with any queries relating to the quality of their study, or CONSORT criteria; however, two authors failed to respond.2 values of over 85% are represented for all recommendations, denoting considerable heterogeneity.Results extracted for the CONSORT-harms criteria  demonstrOf the six studies that assess inclusion of harms in the title and abstract of their included RCTs, threeThe definition of adverse events in reports is unsatisfactory with most studiesWhen providing the denominators within trial reports, the results were also varied across studies, with three21Four studiesThis is the first study to systematically review empirical studies assessing the quality of reporting according to the CONSORT-harms guideline.The standard CONSORT is well established in health research with building evidence to support the use of the guideline.In this study we have focused on assessing reporting according to the CONSORT-harms criteria only. The included studies contained trials reported prior to the publication of the CONSORT-harms guideline. However, we have not assessed changes in reporting over time. Nevertheless, our results support those from previous studiesThis review was strengthened by its assessment of quality of the included studies across four key domains. With the guidance of the Cochrane reviewsupplementary online tables to help summarise key results on harms.Complete and accurate reporting is essential to guide decisions on advances in medical interventions. The responsibility to ensure greater balance between reporting of both benefits and harms lies with authors of research and journals publishing that research. We recognised that journals have limited space for the reporting of all outcomes which can lead to selective outcomes reporting.Further dissemination strategies should be used to ensure that trial journal editors and trial investigators are aware of the importance of adequate reporting of harms-related data in RCTs. As it stands, it is unclear as to whether the problem of the poor reporting of harms data in trial publications is a result of the lack of awareness of the CONSORT for harms statement, or journals and peer reviewers not implementing this guideline. The most effective strategy would follow that of the CONSORT statement with the extension for harms comprehensively incorporated in journal requirements along with clear instructions to peer reviewers for guidelines of acceptance.


Liver fibrosis, and its end stage cirrhosis are a major cause of morbidity and mortality and therapeutic options are limited. However, the traditional view of liver disease as an irreversible process is obsolete and it is now evident that the development of liver fibrosis is a dynamic and potentially bidirectional process. Spontaneous resolution of scarring is seen in animal models of liver fibrosis and in human trials in which the stimuli responsible for chronic or repeated hepatic inflammation is successfully removed. Key players in the process are hepatic stellate cells, macrophages, MMPs and their inhibitors Timps. It is also evident that in advanced fibrotic liver disease, specific histological features define what is currently described as "irreversible" fibrosis. This includes the development of paucicellular scars enriched in extensively cross-linked matrix components, such as fibrillar collagen and elastin. Our recent work has focused on the role of macrophage metalloelastase (MMP-12) in the turnover of elastin in reversible and irreversible models of fibrosis. We have shown that elastin turnover in liver injury and fibrosis is regulated by macrophages via Mmp-12 expression, activity and ratio to its inhibitor Timp-1. Failure of elastin degradation, together with increased deposition leads to accumulation of elastin in the fibrotic scars. The complications of liver fibrosis represent a major health burden to our society. World-wide, chronic viral hepatitis is the predominant cause of liver fibrosis, whereas in the Western Hemisphere alcoholic liver disease, chronic viral hepatitis and increasingly non-alcoholic fatty liver disease are the main causes. Furthermore the prevalence of liver cirrhosis in the UK has increased dramatically in the past two decades  and it iExperimental models have confirmed the possibility of reversion of established liver fibrosis -10. Rats4 model, TIMP-1 over-espressing rat develop the same level of fibrosis as wild type, but are unable to resolve after removal of the injury. In addition, in these animals hepatic stellate cell apoptosis is decreased [One of the first events in liver fibrosis is the activation of resident innate inflammatory cells and the recruitment of additional inflammatory cells. These include recruited monocytes/macrophages, with further contributions from other cell lineages in specific disease states. Many inflammatory mediators are produced by damaged hepatocytes, cholangiocytes, endothelial and inflammatory cells. The activation of hepatic myofibroblasts, including hepatic stellate cells (HSCs) is a critical step in the interlinked processes of tissue injury and regeneration. HSCs normally reside in the perisinusoidal space where their principal function is the storage of retinoids ,12. Follecreased . In our ecreased . These pWe have recently shown that the accumulation of elastin is, rather than being only the result of excessive secretion, also results from a failure of elastin degradation . In a raThe role of the ECM in promoting HSC survival should not be understated. Cell matrix components including collagen-1  and inteResident and recruited inflammatory cells have complex effects on both the development of fibrosis and its subsequent resolution . PromineMacrophages are known to be an important source of pro-fibrotic chemokines. Macrophage-conditioned media has been shown to promote HSC activation in vitro. This effect is mediated by transforming growth factor-beta (TGFβ) and the induction of platelet-derived-growth-factor (PDGF) receptors on HSCs . ConversThe role of other cells of the innate and adaptive immune system in the development of liver fibrosis is less well defined and has been reviewed in detail elsewhere . Recent Current evidence indicates that the development of liver fibrosis is a dynamic and potentially bidirectional process. Spontaneous resolution of scarring is readily seen in animal models of liver fibrosis and in human trials in which the stimuli responsible for chronic or repeated hepatic inflammation is successfully removed. However, it is also evident that in advanced fibrotic liver disease, specific histological features define what our group has described as "irreversible" fibrosis. This includes the development of paucicellular scars enriched in extensively cross-linked elastin and fibrillar collagen. In the decades to come a key priority must be the development of strategies to facilitate the repopulation and effective remodelling of areas of advanced scarring. It is likely that this will require a combination of cellular therapies and the development of pharmaceutical agents addressing multiple targets in pathways involved in fibrogenesis.The authors declare that they have no competing interests.


Medicago sativa  exposed to 3 experimental conditions of water limitation. The whole biofilm-forming ability (WBFA) for rhizospheric communities exposed to desiccation was higher than that of communities exposed to saline or nonstressful conditions. A culture-dependent ribotyping analysis indicated that communities exposed to desiccation or saline conditions were more diverse than those under the nonstressful condition. 16S rRNA gene sequencing of selected strains showed that the rhizospheric communities consisted primarily of members of the Actinobacteria and α- and γ-Proteobacteria, regardless of the water-limiting condition. Our findings contribute to improved understanding of the effects of environmental stress factors on plant-bacteria interaction processes and have potential application to agricultural management practices.Biofilms are microbial communities that adhere to biotic or abiotic surfaces and are enclosed in a protective matrix of extracellular compounds. An important advantage of the biofilm lifestyle for soil bacteria (rhizobacteria) is protection against water deprivation (desiccation or osmotic effect). The rhizosphere is a crucial microhabitat for ecological, interactive, and agricultural production processes. The composition and functions of bacterial biofilms in soil microniches are poorly understood. We studied multibacterial communities established as biofilm-like structures in the rhizosphere of  Bacteria living in terrestrial environments are normally organized as multicellular aggregates that develop on a variety of surfaces. These aggregates are highly complex communities, and this lifestyle (termed "biofilm") facilitates survival and resource optimization in hostile environments . Potera  The rhizosphere is the soil niche influenced by plant roots . It is a Terrestrial bacterial communities are exposed to various environmental stressors, of which limited water availability is typically the most critical and has the greatest effect on survival and activity of these communities . The avaPseudomonas putida were shown to undergo changes in architecture and exopolysaccharide (EPS) composition to create a more hydrated microenvironment in response to water-limiting conditions . Various physiological processes related to BFA were evaluated for selected strains from each of the 3 alfalfa rhizospheric soils. Autoaggregation assays were performed as described by Sorroche et al. (2010) . Each st Motility (swimming assay) of each strain was evaluated by inoculation (center puncture) of a plate containing reduced 1/10 TY medium with 0.3% agar  through  EPS production was assessed qualitatively based on fluorescence under UV exposure of EPS-producing strains grown on LB medium containing 0.02% calcofluor white .Chromobacterium violaceum CV026 for detection of acyl homoserine lactones (AHLs) with short acyl chains [Agrobacterium tumefaciens NTL4 (pZLR4) for detection of AHLs with long acyl chains [ The production of quorum-sensing molecules was studied qualitatively using the biosensor l chains  and the l chains .post hoc Fisher’s Least Significant Difference (LSD) test. To evaluate the overall relationships of treatments, subpopulations, ribotypes, phylogenetic affiliations, and BFA-related traits, we performed a multivariate study with principal components analysis (PCA). Statistical analyses were performed using the InfoStat software program, version 2.0 . The values presented are means of replicate experiments; the number of replicates varied depending on the experiment. The data were subjected to one-way analysis of variance (ANOVA) followed by comparison of multiple treatment levels using a posteriori development of a biofilm [ The primary lifestyle of most soil bacteria is as part of a multispecies biofilm. Soil ecosystems provide various microenvironments in which bacteria can become established as biofilms. The rhizosphere is a highly suitable location for development of the biofilm lifestyle because of the presence of root-derived nutrient sources and a biotic surface with which bacteria can actively interact ,54. In c biofilm .10 CFU per g field soil have been reported in previous studies [10 CFU per g original bulk soil (data not shown), which is reasonable in view of the low organic matter content (1.03%) of the sampled field soil and the fact that we counted only the cultivable fraction of total bacteria. Our bacterial counts in bulk soil are comparable to those of cultivable bacteria obtained in previous studies on various types of soil exposed to various agricultural practices [ Bacterial cell counts greater than 9.0 log studies . We obtaractices . Interesractices ; i.e., iractices ,59.10 CFU cultivable bacteria [10 CFU per g dry rhizospheric soil  than nonstress condition (CRS) . Exposure to water-limiting conditions clearly affected alfalfa plant development , althougMedicago truncatula and Lotus japonicus indicate that a large proportion of the genome is involved in responses to high salinity and desiccation [ Rhizodeposition of alfalfa plants exposed to saline stress differs from that of nonstressed plants because increased salinity induces changes in plant hormonal balances. Studies on the effects of salinity in iccation . The rhizospheric niche is a dynamic microenvironment harboring a polymicrobial community affected by its own metabolic processes and by the variable composition of root exudates, which depends on the plant species and their stage of growth ,70. We d Many previous studies have assessed the effects of saline stress or desiccation stress on the BFA of particular bacterial strains ,71,72. H We obtained bacterial communities from rhizospheric soils under 3 experimental treatment , as described in Materials and Methods. Each of these communities consisted of ~95 strains isolated from the count plates of greater dilution, thus representing the most numerous members of each community. The WBFA was evaluated for each community as the mean value of 5 replicates of the BFA for each microplate, containing 95 strains per treatment. Whole growth , WBFA F, and rel The WBFA  and rela These findings indicate that a decrease in soil water potential at the expense of matric potential (drying) leads to the establishment of a bacterial community in the alfalfa rhizosphere consisting of members having a greater capacity for growth under desiccation stress. This phenomenon may be related to a greater ability to exploit nutrient resources under such stress. Similarly, desiccation stress may lead to the selection of bacterial strains with greater BFA, resulting in increased survival of the strains. Previous studies have shown relationships between such an effect and the role of EPSs in the biofilm matrix; EPSs may reduce the effects of desiccation on both the survival of bacterial communities and plant growth ,6,73,74.Phaseolus vulgaris roots was altered by saline stress [ It is remarkable that the SRS community showed smaller whole growth and WBFA than the CRS or DRS communities. The reduction of water potential resulting from increased soil solute content evidently operates differently from the effect of desiccation. Either osmotic or matric stress may have a different effect on the composition of root exudates and thus on the bacterial community associated with the roots. For example, the composition of flavonoid exudates from e stress . Both soil conditions and the amount and composition of rhizodeposits regulate the specificity of plant-bacteria interactions . In the  In terms of bacterial physiology, bacteria living under water-limiting conditions (matric or solute stress) must integrate their responses to create a hydrated microenvironment to protect themselves. The association of bacterial populations as a multispecies biofilm in the rhizospheric microenvironment represents a lifestyle strategy for increasing survival under stress conditions. Desiccation stress may lead to selection of strains with higher BFA that have a survival advantage because of their ability to colonize root surfaces, whereas other mechanisms not necessarily related to BFA may play important roles in bacterial survival under saline stress conditions ,25,76. O We studied the composition of the bacterial communities associated with each of the 3 rhizospheric soils in order to elucidate (i) the structure, diversity, and identities of the predominant bacteria in biofilms established as multispecies microconsortia in the alfalfa rhizosphere, and (ii) the effects of exposure to stress conditions on such communities. To simplify the analysis, we divided the isolates from each treatment into 2 groups of 15 strains with the highest or the lowest BFA . Mean BFA values for each subpopulation were compared with those of the other subpopulations and simultaneously with the WBFA value for the whole community . The val Regardless of the treatment, BFA values were smaller for the LBFA subpopulations  and invo The coexistence of LBFA and HBFA strains within a community may alternatively be explained by a cooperative mechanism whereby particular bacterial strains that lack BFA in isolation gain some BFA in the presence of other strains (even low-BFA strains). This scenario could lead to the establishment of mixed biofilms that allow coexistence of strains, avoid competition ,90, and  To study the composition of microbial communities, we used a cultivation-dependent technique based on comparison of fingerprinting by ARDRA with restriction enzyme HaeIII. The rRNA genes are essential, and their sequences are highly conserved in bacteria. The rRNA genes also have nonconserved sequences that vary among species and families . ARDRA i16S rRNA gene library) because it allowed us to obtain bacterial strains for further characterization of their biofilm properties and studies of mixed biofilm mechanisms, coexistence processes , and bacterial traits beneficial to the host plant under stress conditions. We performed a PCR-RFLP study of the most abundant strains of the HBFA and LBFA subpopulations for each treatment . We decided to conduct this laborious method  ,101, whi16S rRNA genes by HaeIII. Three to 6 fragments ranging in size from 80 to 750 bp were generated among the 35 ribotypes obtained from the 90 strains evaluated, indicating a ratio of ~2.5 between the number of strains analyzed and the number of ribotypes obtained. Regardless of the experimental treatment, the bacterial strains that colonized the alfalfa rhizosphere were highly diverse, similarly to results in studies of tobacco [ The ribotypic variability of HBFA and LBFA subpopulations present in each type of rhizospheric soil was investigated following digestion of purified PCR products of  tobacco .Brassica napus), in which a correlation was found between bacterial diversity and monthly rainfall [ In regard to the relationships between bacterial community structure, exposure to water-limiting conditions, and BFA of the strains, we observed clear differences among the ribotypic compositions of the cultivable bacterial communities in the 3 rhizospheric soils  . In generainfall . However ARDRA revealed the presence of groups that are likely to compete with each other for establishment in the alfalfa rhizosphere because they are present under all 3 experimental conditions . Many previous studies have made comparisons of soil bacterial communities in relation to agricultural practices ,103, soi Interestingly, the numbers of unique restriction profiles were larger for the stressful treatments . GenBank accession numbers and identities of the strains studied are shown in  Representative bacterial strains were identified by complete nucleotide sequencing of the Pseudomonas [ Interestingly, most of the strains from the HBFA subpopulations were identified as α-Proteobacteria or γ-Proteobacteria, whereas those from the LBFA subpopulations were identified as Actinobacteria. In contrast, some previous studies found that the dominant species in soil biofilms were Gram-negative ,55,111. udomonas , and varudomonas . Our resudomonas . These aArthrobacter. These findings suggest that the presence of shared ribotypes with similar identities among the 3 treatments was ascribable to bacterial strains capable of establishing a mixed biofilm in the alfalfa rhizosphere, based on the ability to colonize the root environment (in the case of HBFA strains) or the opportunistic ability to become associated with mixed microconsortia (in the case of LBFA strains). Gram-negative bacteria may be primarily responsible for niche colonization and biofilm formation, whereas Actinobacteria simply take advantage of the biofilm structure to live in a protected microenvironment. The analysis of shared ribotypes demonstrated that certain members of both the HBFA and LBFA subpopulations are capable of colonizing the rhizospheric microenvironment of alfalfa regardless of stress condition. Ribotype 3 was present in the HBFA subpopulations for all 3 conditions and was associated primarily with α-Proteobacteria  and γ-Proteobacteria . Ribotype 7 included members of the LBFA subpopulations belonging to the Actinobacteria, particularly genus Agrobacterium and Rhizobium, S2 (R19) with Pseudomonas, and S37 (R25) with Shinella. The identity of rhizospheric strains with members of the Rhizobiaceae, e.g., Rhizobium and Shinella [M. sativa by the method described in our previous study [E. meliloti strain Rm 1021 (positive control) (data not shown). Regardless of these findings, it is remarkable that strains phylogenetically related to the rhizobia were present as part of mixed biofilms in the bacterial community associated with the alfalfa rhizosphere. These strains were found in the CRS and SRS groups, suggesting a tolerance to water-limiting conditions resulting from salinity but not from desiccation. In regard to the affiliations of unique majority ribotypes, we found identity of C3 (R1) with the genera Shinella , is inteus study . ResultsMicrobacterium, indicating that some Actinobacteria have good BFA. We examined the identity of strains with the R4 ribotype  that is a majority ribotype shared by the HBFA subpopulations of CRS and DRS. These strains were identified as members of the genus  Comparison of our findings for strain identity  and riboPseudomonas and rhizobia; (iii) our knowledge of community structure and diversity of Actinobacteria in alfalfa rhizospheric soils is limited; (iv) Actinobacteria may play important ecological roles in the rhizosphere microenvironment. The presence of members of the Actinobacteria (particularly Actinomycetes) in biofilm microconsortia established in the alfalfa rhizosphere is interesting for several reasons: (i) the formation of mixed biofilms including Gram-positive (Actinobacteria) and Gram-negative (Proteobacteria) bacteria provides an interesting research model of biofilm development by bacteria of different phyla; (ii) the potential role of Actinomycetes as plant growth-promoting rhizobacteria (PGPR) ,116,117  Because of the small number of strains sequenced, our results and interpretations as presented in this article represent only a restricted and partial view of a very complex and dynamic microenvironment. However, this study is the first to assess alfalfa rhizospheric communities as mixed biofilms in the context of water-limiting conditions, and our findings provide an important basis for more extensive studies in the future. Biofilm formation is a multistep process that requires the integration of various bacterial physiological processes, including quorum sensing ,119, mot We evaluated the BFA-related traits of the selected cultivable bacterial strains in relation to ribotype . In gene Our evaluation of QS signal production showed that all of the tested strains were incapable of synthesizing short-chain AHLs (data not shown). Nine of the 13 isolates were able to produce long-chain AHLs according to our methodology, and no direct relationship was found between BFA and AHL production. Three of the 4 AHL-negative strains belonged to the LBFA groups and the other (M1) belonged to an HBFA group and was positive for the remaining BFA-related traits. These findings suggest that "cross-talk" information exchange may occur in the rhizospheric microenvironment, reflecting the complexity of interactions at the biofilm microconsortium level. As suggested previously , QS sign Our motility (swimming) assays indicated the presence of 3 groups: (i) non-motile strains, which were associated with LBFA groups identified as Actinobacteria, (ii) low-motility strains, regardless of phylogenetic affiliation or BFA, and (iii) high-motility strains, which were associated with HBFA strains identified as Proteobacteria . To further integrate and provide explanations for the relationships among BFA-related traits, ribotypes, phylogenetic affiliations, and experimental treatments, we performed a multivariate PCA. This analysis provides a graph that facilitates visualization and interpretation of the data set and the variables. For the PCA, the observations (cases) were the ribotypes (R) and phylogenetic affiliations  associated with different treatments  and subpopulations ; the variables were the BFA, autoaggregation, motility, AHL production, and EPS production of each strain belonging to a given observation. The PCA applied to all data in 3 dimensions  explained ~86% of the total variability in the study. The graph generated from PC1 and PC2 (which explained ~70% of the variability)  indicate In regard to the associations among the variables, we found a strong positive correlation  plants exposed to differing water-limiting conditions harbor distinct bacterial communities (microconsortia) with differing abilities to develop biofilms and thus to establish themselves in this microenvironment. Judging by observed changes in colony sizes (counts), WBFA, and community structures, the ecological functions of rhizospheric biofilm microconsortia vary depending on exposure to stressful conditions, presumably to enhance bacterial community survival, plant growth, and protection from the stress conditions. We found that rhizospheric soils exposed to desiccation conditions (DRS) contained bacterial communities with higher WBFA in comparison to those exposed to saline stress (SRS) or no water limitation (CRS). Our results indicate that water-limitation stress led to selection of bacterial strains in the alfalfa rhizospheric niche that employed the protected biofilm microenvironment as a strategy to survive in the dry soil. The findings presented here demonstrate that the rhizospheres of  Ribotyping analysis based on ARDRA showed that bacterial communities present in the 3 types of soils  were strongly differentiated. Such heterogeneity was present between the HBFA vs. LBFA subpopulations in a given type of soil, accentuating the separation of the communities. The phylogenetic affiliation analysis of the selected strains showed that Actinobacteria and Proteobacteria are the predominant members of alfalfa rhizospheric microconsortia. Certain bacterial ribotypes were present in the alfalfa rhizosphere regardless of exposure to stressful conditions. On the other hand, exposure to the 2 water-limiting conditions  resulted in the appearance or disappearance of other ribotypes, suggesting that such stress is a key factor that modulates the physiology of the host plant, bacterial species, and rhizospheric community. The composition of alfalfa rhizospheric microconsortia appears to be strongly affected by interaction phenomena such as synergism, antagonism, cooperation, and opportunism, as evidenced by the presence in this edaphic niche of bacterial strains with differing BFA. In summary, the present findings improve our understanding of the structure and physiology of bacterial communities established as biofilm microconsortia in the rhizosphere of alfalfa, an important forage legume worldwide, exposed to differing water-limiting environments. These data provide a basis for further studies of the members of these communities at many levels  that will lead to effective new tools for improved management of agricultural practices and increased understanding of these crucial microorganisms.


This invited paper provides a summary of a keynote lecture delivered at the 2011 Australasian Podiatry Conference. Gout is the most prevalent inflammatory arthropathy. It displays a striking predilection to affect the first metatarsophalangeal joint as well as joints within the mid-foot and ankle. A number of factors are known to reduce urate solubility and enhance nucleation of monosodium urate crystals including decreased temperature, lower pH and physical shock, all of which may be particularly relevant to crystal deposition in the foot. An association has also been proposed between monosodium urate crystal deposition and osteoarthritis, which also targets the first metatarsophalangeal joint. Cadaveric, clinical and radiographic studies indicate that monosodium urate crystals more readily deposit in osteoarthritic cartilage. Transient intra-articular hyperuricaemia and precipitation of monosodium urate crystals is thought to follow overnight resolution of synovial effusion within the osteoarthritic first metatarsophalangeal joint. The proclivity of gout for the first metatarsophalangeal joint is likely to be multi-factorial in origin, arising from the unique combination of the susceptibility of the joint to osteoarthritis and other determinants of urate solubility and crystal nucleation such as temperature and minor physical trauma which are particularly relevant to the foot. Gout is a true crystal deposition disease in which all clinical manifestations are considered to be directly attributable to the presence of monosodium urate (MSU) crystals. It is one of the most prevalent inflammatory arthropathies with a prevalence of approximately 1.4%, and is the most common inflammatory arthropathy in men . The prigutta or "drop", podagra being thought to arise as a result of the bodily humours falling to the affected body part. Although our current understanding of the pathogenesis of gout is dramatically distant from humoral theory, these observations concerning the intimate relationship between gout and the foot have been reinforced over the centuries and continue today. This review will consider the ways in which gout affects the foot and discuss potential mechanisms underlying this relationship.The propensity of gout for the foot was recognised by the ancient Greeks who referred to it as podagra, literally "foot-grabber" . The namAfter an often prolonged period of asymptomatic hyperuricaemia, the initial manifestation of gout is usually an acute attack of synovitis affecting a single peripheral joint, most commonly the first metatarsophalangeal joint (MTPJ). Other commonly affected joints include the mid-tarsal joints, ankles, knees, fingers, wrists and elbows Figure . Such atGout displays a striking tendency to affect the foot, in particular the first MTPJ. The initial attack of gout affects the first MTPJ in 56-78% of patients -7 and thSub-clinical involvement in the foot also appears to be common-place. MSU crystal deposits have been observed in synovial fluid aspirated from first MTPJs that have never been affected by an acute attack of gout ,12. FurtGout has a number of chronic manifestations which are easily recognisable as such including tophaceous deposits and a characteristic erosive arthropathy. However, it is also associated with a number of other less specific foot problems. Perhaps not surprisingly given the frequency of first MTPJ involvement, hallux valgus is a common finding. In a community-based case-control study, hallux valgus was found in 41% of gout suffers compared to 25% of age- and gender-matched control subjects (odds ratio (OR) 2.10, 95% confidence interval (CI) 1.39 to 3.18, adjusted for body mass index (BMI) and use of diuretics) . Big toeGout is one of the best understood inflammatory arthropathies. Clinical features can be easily understood and interpreted in the context of a clearly elucidated pathogenetic process. Specific risk factors such as genetics, dietary factors, co-morbidity and its treatment lead to hyperuricaemia and subsequently MSU crystal formation occurs ,17. CrysAs described above, gout tends to affect distal peripheral joints, not only in the foot but also in the upper limb, with central axial joints such as the shoulders, hips and spine only rarely affected. The solubility of urate decreases with reducing temperature ,21 enhanin vitro following mechanical agitation of solutions supersaturated with sodium urate [A further well-recognised clinical feature of gout is the tendency of an acute attack to be precipitated by physical trauma such as stubbing the toe or following physical activity. Enhanced MSU crystal nucleation has been reported um urate . The samum urate . Hence, More recently, the deposition of MSU and calcium pyrophosphate dihydrate (CPPD) crystals in areas of cartilage damage has been described in a cadaveric study which examined 7855 adult human tali from 4007 donors . CrystalIn support of these observations, clinical and radiographic evidence exists of an association between gout and OA. Several case reports and small case series describe the occurrence of acute attacks of gout and/or tophi at first MTPJs and finger distal interphalangeal (DIP) joints also affected by OA -30. A PoThe observations outlined above that MSU crystals tend to deposit at sites of cartilage damage and that clinical and radiographic evidence exists of an association between gout and OA lead to the important question of the mechanism by which gout and OA might be associated. There are three possible explanations for this association.Firstly, does an association exist between the disease states of gout and nodal generalised OA? These two conditions share the common risk factor of obesity ,33. In aThe second and third explanations are related and concern the hypothesis that the association of gout and OA occurs at local joint sites and relates to the co-location of MSU crystal deposits and cartilage lesions. Specifically, they question the direction of this association, namely, does the presence of osteoarthritic cartilage predispose to the local formation and deposition of MSU crystals or do MSU crystals themselves initiate and progress cartilage damage? Evidence to support the deposition of MSU crystals in osteoarthritic cartilage rather than MSU crystals leading to cartilage damage arises from two sources. Although the primary care study described above was cross-sectional, making it difficult to infer causality, the strength of the association between involvement of gout and OA at individual joint sites did not increase with longer duration of gout . A furthThe studies discussed above provide clear evidence of an association between MSU crystal deposition and OA. Whilst further studies are required to definitively answer the questions of direction of association and causality, it appears that MSU crystals more readily deposit in osteoarthritic cartilage and that the presence of OA influences the distribution of joints affected by gout. However, OA cannot solely explain the typical distribution of joints affected by gout, as many joints commonly affected by OA such as the knees, finger IP joints, and hips are less frequently affected by gout than the first MTPJ, and other target joints for gout such as the ankle, wrist and elbow are infrequent sites for primary OA. Is it plausible therefore that the relationship between MSU crystal deposition and OA is of more relevance for the first MTPJ than other joint sites?The first MTPJ is certainly targeted by OA although foot OA is under-studied in comparison to other commonly affected sites such as the hand and knee. A recent systematic review of population-based epidemiological studies found that the estimated prevalence of radiographic OA at the first MTPJ may be as high as 39% in middle-aged to older adults . Simkin The striking predilection of gout for the first MTPJ appears to be multi-factorial in origin and arises from the unique combination of the susceptibility of the joint to OA and local anatomical considerations of temperature, minor physical trauma and biomechanical stress, leading to ideal conditions for MSU crystal formation and deposition in predisposed hyperuricaemic individuals, manifesting as clinical gout.The author declares that they have no competing interests.