A conserved cationic motif enhances membrane binding and insertion of the chloride intracellular channel protein 1 transmembrane domain

The chloride intracellular channel protein 1 (CLIC1) is unique among eukaryotic ion channels in that it can exist as either a soluble monomer or an integral membrane channel. CLIC1 contains no known membrane-targeting signal sequences and the environmental factors which promote membrane binding of the transmembrane domain (TMD) are poorly understood. Here we report a positively charged motif at the C-terminus of the TMD and show that it enhances membrane partitioning and insertion. A 30-mer TMD peptide was synthesized in which the positively charged motif was replaced by three glutamate residues. The peptide was examined in 2,2,2-trifluoroethanol (TFE), sodium dodecyl sulfate micelles and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine liposomes using size-exclusion chromatography, far-UV CD, and fluorescence spectroscopy. The motif appears to enhance membrane interaction via electrostatic contacts and functions as an electrostatic plug to anchor the TMD in membranes. In addition, the motif is also involved in orientating the TMD with respect to the cis and trans faces of the membrane. These findings shed light on the intrinsic and environmental factors that promote the spontaneous conversion of CLIC1 from a water-soluble to a membrane-bound protein.     

Agonist-dependent phosphorylation of the formyl peptide receptor is regulated by the membrane proximal region of the cytoplasmic tail

Formyl peptide receptor (FPR) is a chemoattractant G protein-coupled receptor (GPCR) involved in the innate immune response against bacteria. Receptor activation is terminated by receptor phosphorylation of two serine- and threonine-rich regions located in the distal half of the cytoplasmic tail. In this study we show that introduction of an amino acid with a bulky side chain (leucine or glutamine) adjacent to a single leucine, L320, in the membrane-proximal half of the cytoplasmic tail, significantly enhanced receptor phosphorylation, beta-arrestin1/2 translocation, and receptor endocytosis, without affecting G(i)-mediated ERK1/2 activation and release of intracellular calcium. In addition, the point mutations resulted in diminished susceptibility to trypsin, suggesting a conformation different from that of wild type FPR. Alignment of the FPR sequence with the rhodopsin sequence showed that L320 resides immediately C-terminal of an amphipathic region that in rhodopsin forms helix 8. Deletion of seven amino acids (Delta309-315) from the predicted helix 8 of FPR (G307-S319) caused reduced cell signaling as well as defects in receptor phosphorylation, beta-arrestin1/2 translocation and endocytosis. Thus, the amino acid content in the N-terminal half of the cytoplasmic tail influences the structure and desensitization of FPR.     

The brain under the knife: serial sectioning and the development of late nineteenth-century neuroanatomy

Major changes took place during the last quarter of the nineteenth century in the ways that the brain tissue was maintained, manipulated and studied, and, consequently, in the ways that its structure, functions and pathologies were seen and represented in neurological literature. The paper exemplifies these changes by comparing German neuroanatomy in the 1860s and early 1870s (represented above all by Theodor Meynert) with the turn-of-the-century view of the brain (represented by Constantin von Monakow and others). It argues for the crucial importance of a method--serial sectioning--to the emergence of the new view of the brain. Serial sectioning in turn owes its existence to the new techniques in staining and sectioning that were introduced in the 1870s and 1880s. In particular, the paper highlights the role of a cutting device, the microtome, in enabling serial sectioning and in thereby contributing to the emergence of a new view of the brain.     

Type II Na+-Pi cotransporters in osteoblast mineral formation: regulation by inorganic phosphate.

During calcification of bone, large amounts of phosphate (P(i)) must be transported from the circulation to the osteoid. Likely candidates for osteoblast P(i) transport are the type II sodium-phosphate cotransporters NaPi-IIa and NaPi-IIb that facilitate transcellular P(i) flux in kidney and intestine, respectively. We have therefore determined the 'cotransporters' expression in osteoblast-like cells. We have also studied the 'cotransporters' regulation by P(i) and during mineralization in vitro. Phosphate uptake and cotransporter protein expression was investigated at early, late and mineralizing culture stages of mouse (MC3T3-E1) and rat (UMR-106) osteoblast-like cells. Both NaPi-IIa and NaPi-IIb were expressed by both osteoblast-like cell lines. NaPi-IIa was upregulated in both cell lines one week after confluency. After 7 days in 3mM P(i) NaPi-IIa was strongly upregulated in both cell lines. NaPi-IIb expression was unaffected by both culture stage and P(i) supplementation. The expression of both cotransporters was unaffected by P(i) deprivation. In vitro mineralization at 1.5mM P(i) was preceded by a three-fold increase in osteoblast sodium-dependent P(i) uptake and a corresponding upregulation of both NaPi-IIa and NaPi-IIb. Their expression thus seem regulated by phosphate in a manner consistent with their playing a role in transcellular P(i) flux during mineralization.     

Process simulation and gate-to-gate life cycle assessment of hydrometallurgical refractory gold concentrate processing

Purpose

Currently, almost all cyanide-free gold leaching processes are still in the development stage. Proactively investigating their environmental impacts prior to commercialization is of utmost importance. In this study, a detailed refractory gold concentrate process simulation with mass and energy balance was built for state-of-the-art technology with (i) pressure oxidation followed by cyanidation and, compared to alternative cyanide-free technology, with (ii) pressure oxidation followed by halogen leaching. Subsequently, the simulated mass balance was used as life cycle inventory data in order to evaluate the environmental impacts of the predominant cyanidation process and a cyanide-free alternative.

Methods

The environmental indicators for each scenario are based on the mass balance produced with HSC Sim steady-state simulation. The simulated mass balances were evaluated to identify the challenges in used technologies. The HSC Sim software is compatible with the GaBi LCA software, where LCI data from HSC-Sim is directly exported to. The simulation produces a consistent life cycle inventory (LCI). In GaBi LCA software, the environmental indicators of global warming potential (GWP), acidification potential (AP), terrestrial eutrophication potential (EP), and water depletion (Water) are estimated.

Results and discussion

The life cycle assessment revealed that the GWP for cyanidation was 10.1 t CO₂-e/kg Au, whereas the halogen process indicated a slightly higher GWP of 12.6 t CO₂-e/kg Au. The difference is partially explained by the fact that the footprint is calculated against produced units of Au; total recovery by the halogen leaching route for gold was only 87.3%, whereas the cyanidation route could extract as much as 98.5% of gold. The addition of a second gold recovery unit to extract gold also from the washing water in the halogen process increased gold recovery up to 98.5%, decreasing the GWP of the halogen process to 11.5 t CO₂-e/kg Au. However, both evaluated halogen processing scenarios indicated a slightly higher global warming potential when compared to the dominating cyanidation technology.

Conclusions

The estimated environmental impacts predict that the development-stage cyanide-free process still has some challenges compared to cyanidation; as in the investigated scenarios, the environmental impacts were generally higher for halogen leaching. Further process improvements, for example in the form of decreased moisture in the feed for halide leaching, and the adaptation of in situ gold recovery practices in chloride leaching may give the cyanide-free processing options a competitive edge.

     

Validation of Morphometric Analyses of Small-Intestinal Biopsy Readouts in Celiac Disease

Background

Assessment of the gluten-induced small-intestinal mucosal injury remains the cornerstone of celiac disease diagnosis. Usually the injury is evaluated using grouped classifications (e.g. Marsh groups), but this is often too imprecise and ignores minor but significant changes in the mucosa. Consequently, there is a need for validated continuous variables in everyday practice and in academic and pharmacological research.

Methods

We studied the performance of our standard operating procedure (SOP) on 93 selected biopsy specimens from adult celiac disease patients and non-celiac disease controls. The specimens, which comprised different grades of gluten-induced mucosal injury, were evaluated by morphometric measurements. Specimens with tangential cutting resulting from poorly oriented biopsies were included. Two accredited evaluators performed the measurements in blinded fashion. The intraobserver and interobserver variations for villus height and crypt depth ratio (VH:CrD) and densities of intraepithelial lymphocytes (IELs) were analyzed by the Bland-Altman method and intraclass correlation.

Results

Unevaluable biopsies according to our SOP were correctly identified. The intraobserver analysis of VH:CrD showed a mean difference of 0.087 with limits of agreement from −0.398 to 0.224; the standard deviation (SD) was 0.159. The mean difference in interobserver analysis was 0.070, limits of agreement −0.516 to 0.375, and SD 0.227. The intraclass correlation coefficient in intraobserver variation was 0.983 and that in interobserver variation 0.978. CD3⁺ IEL density countings in the paraffin-embedded and frozen biopsies showed SDs of 17.1% and 16.5%; the intraclass correlation coefficients were 0.961 and 0.956, respectively.

Conclusions

Using our SOP, quantitative, reliable and reproducible morphometric results can be obtained on duodenal biopsy specimens with different grades of gluten-induced injury. Clinically significant changes were defined according to the error margins (2SD) of the analyses in VH:CrD as 0.4 and in CD3⁺-stained IELs as 30%.     

Combining high-throughput sequencing with fruit body surveys reveals contrasting life-history strategies in fungi

Before the recent revolution in molecular biology, field studies on fungal communities were mostly confined to fruit bodies, whereas mycelial interactions were studied in the laboratory. Here we combine high-throughput sequencing with a fruit body inventory to study simultaneously mycelial and fruit body occurrences in a community of fungi inhabiting dead wood of Norway spruce. We studied mycelial occurrence by extracting DNA from wood samples followed by 454-sequencing of the ITS1 and ITS2 regions and an automated procedure for species identification. In total, we detected 198 species as mycelia and 137 species as fruit bodies. The correlation between mycelial and fruit body occurrences was high for the majority of the species, suggesting that high-throughput sequencing can successfully characterize the dominating fungal communities, despite possible biases related to sampling, PCR, sequencing and molecular identification. We used the fruit body and molecular data to test hypothesized links between life history and population dynamic parameters. We show that the species that have on average a high mycelial abundance also have a high fruiting rate and produce large fruit bodies, leading to a positive feedback loop in their population dynamics. Earlier studies have shown that species with specialized resource requirements are rarely seen fruiting, for which reason they are often classified as red-listed. We show with the help of high-throughput sequencing that some of these species are more abundant as mycelium in wood than what could be expected from their occurrence as fruit bodies.