Reconstruction of protein backbones from the BriX collection of canonical protein fragments

As modeling of changes in backbone conformation still lacks a computationally efficient solution, we developed a discretisation of the conformational states accessible to the protein backbone similar to the successful rotamer approach in side chains. The BriX fragment database, consisting of fragments from 4 to 14 residues long, was realized through identification of recurrent backbone fragments from a non-redundant set of high-resolution protein structures. BriX contains an alphabet of more than 1,000 frequently observed conformations per peptide length for 6 different variation levels. Analysis of the performance of BriX revealed an average structural coverage of protein structures of more than 99% within a root mean square distance (RMSD) of 1 Angstrom. Globally, we are able to reconstruct protein structures with an average accuracy of 0.48 Angstrom RMSD. As expected, regular structures are well covered, but, interestingly, many loop regions that appear irregular at first glance are also found to form a recurrent structural motif, albeit with lower frequency of occurrence than regular secondary structures. Larger loop regions could be completely reconstructed from smaller recurrent elements, between 4 and 8 residues long. Finally, we observed that a significant amount of short sequences tend to display strong structural ambiguity between alpha helix and extended conformations. When the sequence length increases, this so-called sequence plasticity is no longer observed, illustrating the context dependency of polypeptide structures.     

Shewanella oneidensis MR-1 Uses Overlapping Pathways for Iron Reduction at a Distance and by Direct Contact under Conditions Relevant for Biofilms

We developed a new method to measure iron reduction at a distance based on depositing Fe(III) (hydr)oxide within nanoporous glass beads. In this “Fe-bead” system, Shewanella oneidensis reduces at least 86.5% of the iron in the absence of direct contact. Biofilm formation accompanies Fe-bead reduction and is observable both macro- and microscopically. Fe-bead reduction is catalyzed by live cells adapted to anaerobic conditions, and maximal reduction rates require sustained protein synthesis. The amount of reactive ferric iron in the Fe-bead system is available in excess such that the rate of Fe-bead reduction is directly proportional to cell density; i.e., it is diffusion limited. Addition of either lysates prepared from anaerobic cells or exogenous electron shuttles stimulates Fe-bead reduction by S. oneidensis, but iron chelators or additional Fe(II) do not. Neither dissolved Fe(III) nor electron shuttling activity was detected in culture supernatants, implying that the mediator is retained within the biofilm matrix. Strains with mutations in omcB or mtrB show about 50% of the wild-type levels of reduction, while a cymA mutant shows less than 20% of the wild-type levels of reduction and a menF mutant shows insignificant reduction. The Fe-bead reduction defect of the menF mutant can be restored by addition of menaquinone, but menaquinone itself cannot stimulate Fe-bead reduction. Because the menF gene encodes the first committed step of menaquinone biosynthesis, no intermediates of the menaquinone biosynthetic pathway are used as diffusible mediators by this organism to promote iron reduction at a distance. CymA and menaquinone are required for both direct and indirect mineral reduction, whereas MtrB and OmcB contribute to but are not absolutely required for iron reduction at a distance.     

Growth and C:N:P ratios in copepods grazing on N- or Si-limited phytoplankton blooms

Hydrobiologia - The aim of this study was to investigate how nutrient limitation in phytoplankton blooms affects growth and C:N:P ratios in marine pelagic copepods. We performed two mesocosm...     

Innovative development and validation of an HPLC/DAD method for the qualitative and quantitative determination of major cannabinoids in cannabis plant material

GC is commonly used for the analysis of cannabis samples, e.g. in forensic chemistry. However, as this method is based on heating of the sample, acidic forms of cannabinoids are decarboxylated into their neutral counterparts. Conversely, HPLC permits the determination of the original composition of plant cannabinoids by direct analysis. Several HPLC methods have been described in the literature, but most of them failed to separate efficiently all the cannabinoids or were not validated according to general guidelines. By use of an innovative methodology for modelling chromatographic responses, a simple and accurate HPLC/DAD method was developed for the quantification of major neutral and acidic cannabinoids present in cannabis plant material: Δ9-tetrahydrocannabinol (THC), THC acid (THCA), cannabidiol (CBD), CBD acid (CBDA), cannabigerol (CBG), CBG acid (CBGA) and cannabinol (CBN). Δ8-Tetrahydrocannabinol (Δ8-THC) was determined qualitatively. Following the practice of design of experiments, predictive multilinear models were developed and used in order to find optimal chromatographic analytical conditions. The method was validated following an approach using accuracy profiles based on β-expectation tolerance intervals for the total error measurement, and assessing the measurements uncertainty. This analytical method can be used for diverse applications, e.g. plant phenotype determination, evaluation of psychoactive potency and control of material quality.     

Itaconic acid production using sago starch hydrolysate by Aspergillus terreus TN484-M1

Sago starch was hydrolyzed using either chemical agents, or enzymes at various pH and concentrations. Hydrolysis using 5000 AUN/ml (0.5%, w/v) glucoamylase exhibited the highest itaconic acid yield up to 0.36 g/g sago starch, whereas hydrolysis using nitric acid at pH 2.0 yielded 0.35 g/g sago starch. The medium was optimized and the composition was (g/l) 140 sago starch, 1.8 corn steep liquor, 1.2 MgSO(4).7H(2)O and 2.9 NH(4)NO(3). When the optimal conditions of hydrolysis and medium composition were applied to itaconic acid production in a 3-l jar fermentor, the itaconic acid production was 48.2 g/l with a yield of 0.34 g/g sago starch. This was filtered from the cultured broth and 37.1g of itaconic acid was recovered with a purity of 97.2%. This result showed that sago starch could be converted to a value-added product with only a simple pretreatment.     

Human impacts on tropical forest dynamics

People living near or in tropical forest ecosystems have traditionally extracted forest products, i.e. timber, food and medicinal plants for their livelihood. Such practice does not create too much disturbance when the population is still sparse, and the product is used only for their own needs. When population pressure becomes greater, and when the motive of extraction is profit, then the disturbance become serious and created environmental problems. Major exploitation of the Indonesian rain forest for timber began in the 1960s and is continuing today. The lowland rain forests of Sumatra and Kalimantan have been particularly logged. Exploitation has often been destructive because Forest Department rules have been widely ignored. Moreover, once roads have given access to formerly inaccessible areas, farmers have often moved in after the timber companies and then cleared the relict, regenerating forest for either permanent or shifting cultivation. The traditional shifting cultivations have been practised for years, producing millions of ha of impoverished secondary types of forest, degraded lands and alang-alang (Imperata cylindrica) grasslands. Forests have also been lost through conversion of land to plantation agriculture and transmigration programmes, mining, construction roads and railways and also natural disturbances, such as drought and fire. This paper will discuss the human impact upon tropical forest dynamics in general, with examples from Indonesian and other Southeast Asia countries' tropical forests.     

Cytokeratin 20-positive hepatocellular carcinoma

The differential diagnosis between hepatocellular carcinoma (HCC), cholangiocarcinoma (CC) and metastatic colorectal adenocarcinoma (MCA) may be difficult when only based on morphology. For this purpose immunohistochemical analyses are often required, utilizing antibodies directed against CK8-18, Hep-Par1, glypican 3, CK7, CK19, CK20. Here we report a case of a 65-year-old man who presented with a clinical picture of decompensated cirrhosis. Ultrasonography revealed two nodular areas in the right liver lobe. Liver needle biopsy revealed micro-macronodular cirrhosis associated with HCC with trabecular and pseudoglandular patterns. Immunohistochemically, tumour cells were diffusely positive for CK8-18 and also diffusely immunostained by glypican 3 and Hep-Par1. Interestingly, a diffuse and strong staining for CK20 was detected in the vast majority of tumor cells, particularly in the areas showing a pseudo-glandular pattern. No immunostaining for CK7 and CK19 was found in the tumor cells. The tumor behaved aggressively, with a rapid diffusion to the whole liver. The patient died from the disease few months after presentation. These findings underline that the interpretation of the expression of CK20 alone in the differential diagnosis among HCC, CC and MCA should be done with caution because a diffuse immunoreactivity for CK20 alone may not rule out the diagnosis of HCC.Key words: hepatocellular carcinoma, cholangiocarcinoma, metastatic colorectal carcinomas, CK20.The differential diagnosis between hepatocellular carcinoma (HCC), cholangiocarcinoma (CC) and metastatic colorectal adenocarcinoma (MCA) may be difficult when only based on morphology (Terracciano et al., 2003).In fact, a subset of extrahepatic adenocarcinomas of different origin may show a solid “hepatoid” pattern virtually indistinguishable from HCC (Porcell et al., 2000). On the other hand, the undifferentiated form of HCC may mimic poorly differentiated tumors of different origin, while its tubular and adenoid variants may be indistinguishable from CC or from MCA.In these cases, immunohistochemical analyses are often required (Stroescu et al., 2006).The panel of antibodies utilized to solve this differential diagnosis includes: CK8-18 (Porcell et al., 2000) Hep-Par1 (Leong et al., 1998) (Zimmerman et al., 2001), glypican 3 (GPC3) (Yamauchi et al., 2005) (Capurro et al., 2003), CK7 (Maeda et al., 1996) (Chu et al., 2000), CK20 (Faa G et al., 1998), CK19, CEA and Alpha-fetoprotein (Onofre et al., 2007) (Lau et al., 2002).Immunoreactivity of tumour cells for CK8-18, Hep-Par 1 and GPC3 is considered suggestive of HCC; a diffuse immunoreactivity for CK7 and CK19 is in favour of the diagnosis of CC; a diffuse positivity for CK20 and negativity for CK7 are normally associated with MCA.Here we report a case of HCC with a peculiar immunohistochemical profile, characterized by the association of the typical immunoreactivity of HCC with a diffuse and strong positivity for CK20, generally considered typical of MCA.