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This study demonstrates that a 4.25:1 length to diameter ratio (L:D) granulate screw extruder can be used for the fused granulate fabrication (FGF) of polyether ether ketone (PEEK) objects. PEEK is a biocompatible, high-performance thermoplastic with good mechanical, thermal and electrical properties. This study focused on fused granulate fabrication (FGF) which employs granulates rather than filament as the raw material, which enables great freedom in material selection at a much lower cost. The short L:D ratio screw developed and used allows for a smaller extruder than the ones found in literature. The extruder was calibrated for additive manufacturing at different speeds using a 1 mm nozzle. The results demonstrate that there is a non-linear relationship between screw speed and extrusion rate, therefore, to use fused filament fabrication systems (FFF) slicing software for FGF, the 3D printing speed for the whole printing cycle has to be kept constant. This study was conducted in an open chamber which resulted in an ultimate tensile/yield strength of 49 MPa which is around 48% of injection moulded PEEK as stated in the material datasheet. The quality of the prints was evaluated using optical profilometry which reported a surface roughness for top and side faces of 9.8 and 31 µm Ra, respectively. These results are similar to ones found in literature which used a 0.8 mm nozzle. This showed that FGF systems can be miniaturised even for additive manufacturing of high-temperature, high-viscous, high-performance polymers, such as PEEK.

This study determined the requisite process parameters for good-quality screw extrusion additive manufacturing (AM) of thermoplastic polyolefin (TPO) using fused granulate fabrication (FGF). TPO is a non-hygroscopic, cheaper, and less dense alternative to the well-established thermoplastic polyurethane (TPU). TPO was found to extrude correctly at 170 °C, on a glass build plate at 80 °C with Magigoo PP adhesive. A water uptake test on TPO reported a mass gain plateau of 0.25%, which is significantly lower than that of TPU, which suggests that TPO may not require drying before 3D printing. Tensile testing on FGF TPO specimens achieved similar stress at yield as well as stress and strain at break as indicated by the data sheet for the XY and YZ orientations. The Z direction is significantly weaker than the X and Y orientations, reaching only 30% of the stress at break. TPO achieved the best average stress at yield of 6.36 MPa using the 0.4 mm nozzle with XY printing orientation and stress and strain at break of 13.8 MPa and 1300% at YZ orientation and 1 mm nozzle. The setup achieved relatively high-quality prints of complex geometries, including the popular torture-test Benchy and a child-sized orthotic insole.

RTIM refers to the integration of rapid tooling (RT) using additive manufacturing (AM) with injection molding (IM). Due to the use of raw material (pellet), screw extrusion, fused granulate fabrication (FGF) allows for a cost-effective and versatile production of RTIM inserts especially compared to other material extrusion (MEX) AM processes, such as fused filament fabrication (FFF). This study 3D printed RTIM inserts out of high-performance thermoplastic polyetherimide (PEI) (ULTEM 9085) in granular form, using an in-house developed FGF system. A cosmetic compact was used as a case study part with a simplified design. A dynamic mechanical analysis (DMA) conducted on FGF ULTEM 9085 demonstrated that the strength of the inserts is sustained up to 140 °C, which suits injection molding using polypropylene (PP). Optical profilometry of the FGF 3D printed RTIM inserts demonstrated that when using a 0.4 mm nozzle, the flat surfaces produced had microscopic gaps larger than 10–1mm. These gaps are sufficiently large so that melt of low viscosity polymer is able to flow through, which leads to undesired part flash. The IM experiments confirmed that the inserts were capable of producing PP parts but with the predicted flash. Simulated part deflection differed both on geometry and magnitude from the actual deflection measured by optical profilometry. A total of 36 prototype parts were produced before the inserts failed due to poor inter-layer bond strength. FGF RTIM using ULTEM 9085 is deemed suitable for prototype part production. Improving the inter-layer bond strength and decreasing part complexity could increase the number of parts produced.

Additive manufacturing or 3D printing is the process of depositing material layer-by-layer to create 3-dimensional products. When creating 3D-printed products from two or more materials, multi-material additive manufacturing processes are used which eliminate the need for assembly operations. Fused filament fabrication multi-material additive manufacturing permits the production of a single printed item employing multiple materials in fused filament fabrication. This work studied the factors affecting fused filament fabrication multi-material additive manufacturing, by reviewing existing works, designing part(s), conducting design of experiments, and carrying out parts’ performance test. An E3D multi-material filament 3D printer was utilised throughout this study. The chosen polymer combination was polycarbonate (PC) and poly(methyl methacrylate) (PMMA), whilst the design and testing of the multi-material parts was limited to lap-shear testing. Results showed that the interface bonding of the PMMA/PC (PMMA printed first and followed by PC) specimens was stronger than the one of the PC/PMMA specimens. Furthermore, to investigate the effect of the contact or overlapping area on the interface bonding strength, PMMA/PC specimens with varying dimensions were designed, printed, and tested. When the contact area was reduced, a strong interface bonding between the PMMA and PC layers was still maintained.

Pellet additive manufacturing (PAM) is in terms of printing process very similar to the widely used fused deposition modelling (FDM) systems. The main difference is the use of pellets instead of filament. In this study, a pellet, single screw extruder is developed. A screw design with variable pitch and depth is modelled analytically to predict the melting behavior of acrylonitrile butadiene styrene (ABS) during steady state extrusion. The extruder screw was designed unconventionally short with a length of 85 mm and a diameter of 20 mm, giving an L:D ratio of 4.25:1. The model predicted the melting profile for ABS being extruder at 235°C at 10 RPM, the result of which was confirmed to a degree by experimentation. The extrusion rate of the screw extruder was measured at barrel temperatures of 225°C to 245°C with 5°C increments and at 5 RPM and 10 RPM. The extrusion was found to increase non-linearly with barrel temperature and screw speeds. The extruder printed adequately on an FDM style motion system with minor upgrades.

The excellent properties of polyether ether ketone (PEEK) have made it applicable for high-performance components in aerospace, electrical, chemical, and biomedical industries. Material extrusion (MEX) additive manufacturing (AM) of PEEK using fused filament fabrication (FFF) predominantly uses modified PEEK filament. Fused granulate fabrication (FGF) systems conventionally employ injection moulding and extrusion grades which are not specifically formulated for MEX AM. There is a limited amount of research on the use of these grades for MEX AM using PEEK. This study evaluated different viscosity PEEK grades at various extruder temperatures to optimise interlayer strength and therefore determine the best grades of PEEK for FGF. Compression shear testing (CST) was used to study this property as it represents a mostly shear loading condition i.e., without bending, compared to other conventional shear testing methods, whilst also being easier to fabricate. PEEK crystallinity is corelated to the interlayer strength and was evaluated using differential scanning calorimetry (DSC), Raman spectroscopy and X-ray diffraction (XRD). The highest interlayer strength of 17.70±0.58 MPa and the lowest crystallisation degree of 29.7±1.3% (Raman) was achieved by VESTAKEEP L4000G PEEK, printed at the highest extruder temperature (420°C). This grade had the highest viscosity or the lowest melt volume-flow rate (MVR). In contrast with lower MVR grades, the high MVR grade VESTAKEEP L2000G, achieved a higher increase in bond strength and crystallinity with higher extruder temperatures. At lower MVR, the crystallinity was found to decrease from 33% to 30% with increasing extruder temperatures, along with an increase in melting temperature. This behaviour was attributed to a change in crystalline morphology possibly caused by the long residence time and high processing temperature of FGF.

The authors examined the research and activism of 10th graders (ages 15–16) involved in a youth participatory action research project in a course in qualitative research at their small public school in New York City, New York. The authors, a classroom researcher and a high school teacher, looked closely at how the youth researchers used photography, collage, and videography to transform their school hallways into a space for critical conversations about race and gender. The authors examined how the hallways became a civic space shaped by collective youth resistance, multimodal counterstories, and negotiated civic engagement.

PurposeWe aimed to identify the genetic cause to a clinical syndrome encompassing hypohidrosis, electrolyte imbalance, lacrimal gland dysfunction, ichthyosis, and xerostomia (HELIX syndrome), and to comprehensively delineate the phenotype.MethodsWe performed homozygosity mapping, whole-genome sequencing, gene sequencing, expression studies, functional tests, protein bioinformatics, and histological characterization in two unrelated families with HELIX syndrome.ResultsWe identified biallelic missense mutations (c.386C>T, p.S131L and c.2T>C, p.M1T) in CLDN10B in six patients from two unrelated families. CLDN10B encodes Claudin-10b, an integral tight junction (TJ) membrane-spanning protein expressed in the kidney, skin, and salivary glands. All patients had hypohidrosis, renal loss of NaCl with secondary hyperaldosteronism and hypokalemia, as well as hypolacrymia, ichthyosis, xerostomia, and severe enamel wear. Functional testing revealed that patients had a decreased NaCl absorption in the thick ascending limb of the loop of Henle and a severely decreased secretion of saliva. Both mutations resulted in reduced or absent Claudin-10 at the plasma membrane of epithelial cells.ConclusionCLDN10 mutations cause a dysfunction in TJs in several tissues and, subsequently, abnormalities in renal ion transport, ectodermal gland homeostasis, and epidermal integrity.

We present preliminary results showing the potential of diamond nanoparticles with size < 50 nm as photoluminescent nanoprobes (i) for biological applications like biomolecule tracking in living cell and (ii) for serving as stable point-like emitters attached at the tip apex of a near-field optical microscope to achieve enhanced spatial resolution.

Chloride intracellular channel protein 1 (CLIC1) is very unusual as it adopts a soluble glutathione S-transferase-like canonical fold but can also autoinsert into lipid bilayers to form an ion channel. The conversion between these forms involves a large, but reversible, structural rearrangement of the CLIC1 module. The only identified environmental triggers controlling the metamorphic transition of CLIC1 are pH and oxidation. Until now, there have been no high-resolution structural data available for the CLIC1 integral membrane state, and consequently, a limited understanding of how CLIC1 unfolds and refolds across the bilayer to form a membrane protein with ion channel activity exists. Here we show that fluorescence spectroscopy can be used to establish the interaction and position of CLIC1 in a lipid bilayer. Our method employs a fluorescence energy transfer (FRET) approach between CLIC1 and a dansyl-labeled lipid analogue to probe the CLIC1–lipid interface. Under oxidizing conditions, a strong FRET signal between the single tryptophan residue of CLIC1 (Trp35) and the dansyl-lipid analogue was detected. When considering the proportion of CLIC1 interacting with the lipid bilayer, as estimated by fluorescence quenching experiments, the FRET distance between Trp35 and the dansyl moiety on the membrane surface was determined to be ∼15 Å. This FRET-detected interaction provides direct structural evidence that CLIC1 associates with membranes. The results presented support the current model of an oxidation-driven interaction of CLIC1 with lipid bilayers and also propose a membrane anchoring role for Trp35.

Recent studies show that current trends in yield improvement will not be sufficient to meet projected global food demand in 2050, and suggest that a further expansion of agricultural area will be required. However, agriculture is the main driver of losses of biodiversity and a major contributor to climate change and pollution, and so further expansion is undesirable. The usual proposed alternative—intensification with increased resource use—also has negative effects. It is therefore imperative to find ways to achieve global food security without expanding crop or pastureland and without increasing greenhouse gas emissions. Some authors have emphasized a role for sustainable intensification in closing global ‘yield gaps’ between the currently realized and potentially achievable yields. However, in this paper we use a transparent, data-driven model, to show that even if yield gaps are closed, the projected demand will drive further agricultural expansion. There are, however, options for reduction on the demand side that are rarely considered. In the second part of this paper we quantify the potential for demand-side mitigation options, and show that improved diets and decreases in food waste are essential to deliver emissions reductions, and to provide global food security in 2050.

Neurotensin (NT) is a tridecapeptide hormone in the periphery and neurotransmitter in the brain that principally activates three receptor subtypes, named NTS1, NTS2, and NTS3. Since little is known about its structure in the presence of its principal receptor NTS1, we determined it using the key domain of the receptor, i.e. the third extracellular loop. We conclude the following: (i) for the receptor fragment, NT binding modifies its central part, underlying the great flexibility and adaptability of this region; (ii) for bound NT, the extended conformation of its C-terminus is confirmed for the first time in experimental conditions and in the presence of a part of the receptor; and (iii) despite some substitutions, the human receptor residues that are involved in the interaction with NT could be similar to those of the rat receptor which play an important role in NT binding.

Polyamines are essential for mammalian cell growth and proliferation, and their synthesis in cells or transport from the extracellular environment has attracted much attention, especially in cancer research. Here it is shown for the first time that polyamines can be transferred from cell to cell via gap junctions to coordinate cell growth.

Mutations in SPG4, encoding the microtubule-severing protein spastin, are responsible for the most frequent form of hereditary spastic paraplegia (HSP), a heterogeneous group of genetic diseases characterized by degeneration of the corticospinal tracts. We previously reported that mice harboring a deletion in Spg4, generating a premature stop codon, develop progressive axonal degeneration characterized by focal axonal swellings associated with impaired axonal transport. To further characterize the molecular and cellular mechanisms underlying this mutant phenotype, we have assessed microtubule dynamics and axonal transport in primary cultures of cortical neurons from spastin-mutant mice. We show an early and marked impairment of microtubule dynamics all along the axons of spastin-deficient cortical neurons, which is likely to be responsible for the occurrence of axonal swellings and cargo stalling. Our analysis also reveals that a modulation of microtubule dynamics by microtubule-targeting drugs rescues the mutant phenotype of cortical neurons. Together, these results contribute to a better understanding of the pathogenesis of SPG4-linked HSP and ascertain the influence of microtubule-targeted drugs on the early axonal phenotype in a mouse model of the disease.

We report a detailed description of the energy migration dynamics in the phycocyanin 645 (PC645) antenna complex from the photosynthetic alga ChroomonasCCMP270. Many of the cryptophyceae are known to populate greater depths than most other algal families, having developed a 99.5% efficient light-harvesting system. In this study, we used femtosecond time-resolved spectroscopy and global analysis to characterize the excited-state dynamics of PC645. Several different pump colors were selected to excite different fractions of the four phycobiliprotein pairs present in the complex. Measurements were also performed at cryogenic temperature to enhance spectral resolution and selectively promote downhill energy transfers. Upon excitation of the highest-energy bilins (dihydrobiliverdins), energy is transferred from the core of the complex to the periphery within 0.82 ps. Four bilins (mesobiliverdin (MBV) A/B and phycocyanobilins (PCB) 158C/D), which are responsible for the central band of the absorption spectrum, show concerted spectral dynamics. These chromophores show a biphasic decay with lifetimes of 0.6 ps (MBV) and 5–7 ps (PCB 158) to the lowest bilin pair (PCB 82C/D) absorbing around 650–657 nm. Within this lifetime of several picoseconds, the excitations reach the PCB 82 bilins on the two poles at the smaller sides of PC645. A slow 44–46 ps energy transfer step to the lowest-energy PCB 82 bilin concludes the dynamics.

Microtubules (MTs) are cylindrical cytoskeleton polymers composed of α-βtubulin heterodimers whose dynamic properties are essential to fulfill their numerous cellular functions. In response to spatial confinement, dynamic MTs, even in the absence of protein partners, were shown to self-organize into higher order structures (spindle or striped structures) which lead to interesting dynamical properties (MT oscillations). In this study, we considered the assembly and sensitivity of dynamic MTs when in bundles. To perform this study, spermine, a natural tetravalent polyamine present at high concentrations in all eukaryote cells, was used to trigger MT bundling while preserving MT dynamics. Interestingly, we first show that, near physiological ionic strengths, spermine promotes the bundling of MTs whereas it does not lead to aggregation of free tubulin, which would have been detrimental to MT polymerization. Experimental and theoretical results also indicate that, to obtain a high rate of bundle assembly, bundling should take place at the beginning of assembly when rapid rotational movements of short and newly nucleated MTs are still possible. On the other hand, the bundling process is significantly slowed down for long MTs. Finally, we found that short MT bundles exhibit a higher sensitivity to cold exposure than do isolated MTs. To account for this phenomenon, we suggest that a collective behavior takes place within MT bundles because an MT entering into a phase of shortening could increase the probability of the other MTs in the same bundle to enter into shortening phase due to their close proximity. We then elaborate on some putative applications of our findings to in vivo conditions including neurons.

Diamond nanoparticles (nanodiamonds) have been recently proposed as new labels for cellular imaging. For small nanodiamonds (size <40 nm), resonant laser scattering and Raman scattering cross sections are too small to allow single nanoparticle observation. Nanodiamonds can, however, be rendered photoluminescent with a perfect photostability at room temperature. Such a remarkable property allows easier single-particle tracking over long time scales. In this work, we use photoluminescent nanodiamonds of size <50 nm for intracellular labeling and investigate the mechanism of their uptake by living cells. By blocking selectively different uptake processes, we show that nanodiamonds enter cells mainly by endocytosis, and converging data indicate that it is clathrin-mediated. We also examine nanodiamond intracellular localization in endocytic vesicles using immunofluorescence and transmission electron microscopy. We find a high degree of colocalization between vesicles and the biggest nanoparticles or aggregates, while the smallest particles appear free in the cytosol. Our results pave the way for the use of photoluminescent nanodiamonds in targeted intracellular labeling or biomolecule delivery.

This article reports stable photoluminescence and high-contrast optically detected electron spin resonance (ODESR) from single nitrogen-vacancy (NV) defect centers created within ultrasmall, disperse nanodiamonds of radius less than 4 nm. Unexpectedly, the efficiency for the production of NV fluorescent defects by electron irradiation is found to be independent of the size of the nanocrystals. Fluorescence lifetime imaging shows lifetimes with a mean value of around 17 ns, only slightly longer than the bulk value of the defects. After proper surface cleaning, the dephasing times of the electron spin resonance in the nanocrystals approach values of some microseconds, which is typical for the type Ib diamond from which the nanoparticle is made. We conclude that despite the tiny size of these nanodiamonds the photoactive nitrogen-vacancy color centers retain their bulk properties to the benefit of numerous exciting potential applications in photonics, biomedical labeling, and imaging.

Biomolecular motors have inspired the design and construction of artificial nanoscale motors and machines based on nucleic acids, small molecules, and inorganic nanostructures. However, the high degree of sophistication and efficiency of biomolecular motors, as well as their specific biological function, derives from the complexity afforded by protein building blocks. Here, we discuss a novel bottom‐up approach to understanding biological motors by considering the construction of synthetic protein motors. Specifically, we present a design for a synthetic protein motor that moves along a linear track, dubbed the “Tumbleweed.” This concept uses three discrete ligand‐dependent DNA‐binding domains to perform cyclically ligand‐gated, rectified diffusion along a synthesized DNA molecule. Here we describe how de novopeptide design and molecular biology could be used to produce the Tumbleweed, and we explore the fundamental motor operation of such a design using numerical simulations. The construction of this and more sophisticated protein motors is an exciting challenge that is likely to enhance our understanding of the structure‐function relationship in biological motors.

This article presents an investigation of the energy migration dynamics in intact cells of the unicellular photosynthetic cryptophyte Chroomonas CCMP270 by steady-state and time-resolved fluorescence measurements. By kinetic modeling of the fluorescence data on chlorophyll and phycocyanin 645 excitation (at 400 and 582nm respectively), it has been possible to show the excited state energy distribution in the photosynthetic antenna of this alga. Excitation energy from phycocyanin 645 is distributed nearly equally between photosystem I and photosystem II with very high efficiency on a 100-ps timescale. The excitation energy trapping times for both photosystem I (∼30ps) and photosystem I (200 and ∼540ps) correspond well to those obtained from experiments on isolated photosystems. The results are compared with previous results for another cryptophyte species, Rhodomonas CS24, and suggest a similar membrane organization for the cryptophytes with the phycobiliproteins tightly packed in the thylakoid lumen around the periphery of the photosystems.

We present preliminary results showing the potential of diamond nanoparticles with size

Using liquid chromatography−mass spectrometry, 528 proteins were identified that are expressed during growth at 4 °C in the cold adapted archaeon, Methanococcoides burtonii. Of those, 135 were annotated previously as unique or conserved hypothetical proteins. We have performed a comprehensive, integrated analysis of the latter proteins using threading, InterProScan, predicted subcellular localization and visualization of conserved gene context across multiple prokaryotic genomes. Functional information was obtained for 55 proteins, providing new insight into the physiology of M. burtonii. Many of the proteins were predicted to be involved in DNA/RNA binding or modification and cell signaling, suggesting a complex, uncharacterized regulatory network controlling cellular processes during growth at low-temperature. Novel enzymatic functions were predicted for several proteins, including a putative candidate gene for the posttranslational modification of the key methanogenesis enzyme coenzyme M methyl reductase. A bacterial-like CRISPR locus was identified as a strong candidate for archaeal-bacterial lateral gene transfer. Gene context analysis proved a valuable augmentation to the other predictive methods in several cases, by revealing conserved gene associations and annotations in other microbial genomes. Our results underscore the importance of addressing the “hypothetical protein problem” for a complete understanding of cell physiology. Keywords: proteome • LC/LC−MS/MS • archaea • methanogenesis • psychrophile • CRISPR locus • hypothetical proteins • conserved gene context

ABSTRACTThe muscle protein myosin binding protein C (MyBPC) is a large multi-domain protein whose role in the sarcomere is complex and not yet fully understood. Mutations in MyBPC are strongly associated with the heart disease familial hypertrophic cardiomyopathy (FHC) and these experiments of nature have provided some insight into the intricate workings of this protein in the heart. While some regions of the MyBPC molecule have been assigned a function in the regulation of muscle contraction, the interaction of other regions with various parts of the myosin molecule and the sarcomeric proteins, actin and titin, remain obscure. In addition, several intra-domain interactions between adjacent MyBPC molecules have been identified. Although the basic structure of the molecule (a series of immunoglobulin and fibronectin domains) has been elucidated, the assembly of MyBPC in the sarcomere is a topic for debate. By analysing the MyBPC sequence with respect to FHC-causing mutations it is possible to identify individual residues or regions of each domain that may be important either for binding or regulation. This review looks at the current literature, in concert with alignments and the structural models of MyBPC, in an attempt to understand how FHC mutations may lead to the disease state.

Stathmin is a ubiquitous 17 kDa cytosolic phosphoprotein proposed to play a general role in the integration and relay of intracellular signalling pathways. It is believed to regulate microtubule dynamics by sequestering tubulin in a complex made of two tubulin heterodimers per stathmin molecule (T2S complex). The other proteins of the stathmin family can also bind two tubulin heterodimers through their SLD (stathmin-like domain), but the different tubulin:SLD complexes display varying stabilities. In this study, we analysed the relative influence of three regions of SLDs on the interaction with tubulin and the mechanistic processes that lead to its sequestration. Tubulin-binding properties of fragments and chimaeras of stathmin and RB3SLD were studied in vitro by tubulin polymerization, size-exclusion chromatography and surface plasmon resonance assays. Our results show that the N-terminal region of SLDs favours the binding of the first tubulin heterodimer and that the second C-terminal tubulinbinding site confers the specific stability of a given tubulin:SLD complex. Our results highlight the molecular processes by which tubulin co-operatively interacts with the SLDs. This knowledge may contribute to drug development aimed at disturbing microtubules that could be used for the treatment of cancer.

Soil spatial distribution, i.e. the spatial distribution of soils within landscapes, is difficult to predict because numerous processes operate simultaneously, but variably, over time. Quantifications of large areas with an acceptable degree of precision and low in cost require the development of specific methods making the best possible use of existing soil data and auxiliary information such as soil-forming factors. The quantification of the influence of soil-forming factors on soil spatial distribution is seldom performed over large areas such as regions. This study aimed to quantify the relationship between soil spatial distribution and the soil-forming factors of geology, topography, climate, and tectonic regime in order to predict soil spatial distribution over a wide region (30,000 km2). The Armorican Massif (western France), a complex basement of Proterozoic and Paleozoic rocks affected by recent tectonic activity and characterized by variations in topography and climate, was chosen as the study site. Detailed soil maps (125,000) were used to describe soil spatial distribution along transects. An ANOVA performed on 314 transects showed a high correlation between the occurrence of soils with particular features (namely redoximorphic, leached, glossic, and albic) and geological substrate, uplift ratio, mean slope gradient, and net rainfall. No such correlation was found with fluvic soils. These soil-forming factors seem to act through saprolite quality and erosion processes, which in turn control the development of soil features. A quantification of the relationship between soil features and soil-forming factors was performed by regression analysis in order to allow further prediction of the soil spatial distribution over the entire Armorican Massif. These results revealed and quantified the hitherto unrecognized role of tectonism on soil distribution and its relative importance in respect to other soil-forming factors. Finally, such an analysis, which is based on existing maps, can help to describe, quantify, and predict detailed soil spatial distribution at smaller scales.

This study compared soil physical, chemical, and biological characteristics between natural grassland and recently abandoned rice fields in order to identify those variables that might explain the observed increase of Camponotus punctulatus anthills in abandoned rice paddy fields from Northern Argentina. Mainly due to a reduction of macropores and mesopores, overall porosity decreased by around 6% and bulk density was about 7% greater, in the 0- to 10- and 10- to 20-cm layers of the abandoned rice fields. Carbon and nitrogen content from organic matter increased (29% and 41% respectively for the 0- to 20-cm horizon) during cultivation but decreased (38% and 24%) 2 years after the last rice harvest. Forty percent of natural grassland-organic matter and 30% of abandoned rice-organic matter mineralized in less than 2 years. There was a different community structure between the abandoned rice fields and the undisturbed natural grassland and only a 20.6% (i.e. only 19 species from a total of 92) overlap in species composition. The abundance of macrofauna was greater in abandoned rice fields (2,208 individuals m –2) in comparison to natural grasslands (288 ind m –2) due to higher densities of small earthworms and Camponotus punctulatus ants; however, the Shannon index showed lower values in comparison to natural grasslands. Earthworms and C. punctulatus in the abandoned rice fields showed a change in their d 13C signature indicating a switch in diet from natural grassland organic matter (C4) to organic matter from rice (C3). Our results indicate that the effects of rice cultivation practices did not seem to produce any physical or trophic limitations to recolonization by the macrofauna. It seems that changes in overall soil conditions have favored a change in the construction behavior of C. punctulatus which, in combination with population increases, could explain the explosion in number of anthills.

Quantitative soil-landscape models, based on topographic attributes, make possible the characterization of large areas because of the widespread availability of digital elevation models (DEMs). However, these soil-landscapes models, which are usually generated and validated on the same detailed, single research site, such as a hillslope or an elementary catchment, may show high prediction errors when applied to other areas of a region. The effect of the regional variations of topography, climate, parent material, land-use, and/or soil has seldom been analyzed. The objective of this study was to test multiple-regression relationships between the soil hydromorphic index (HI) and topographic attributes on different catchments of the Armorican Massif (30,000 km2) in western France. Regression models were validated using 565 data points collected from four sites along hillslopes. These four sites, located throughout this region, were included in three catchments with surface areas of 78, 82, and 120 ha, respectively, and differing in topography (mean elevation from 39 to 202 m and slope gradient from 3.4 to 7.9), parent material (granite and schist), and precipitation (700 to 900 mm y−1). The existing models were multiple regression equations between the HI and the elevation above the stream bank, the compound topographic index (regression 1, r20.84), or the upslope drained area (regression 2, r20.86). At each validation point, systematic soil observation for HI estimation was compared with estimations from terrain attributes derived from DEMs at a 30-m resolution. Results showed small prediction errors for all study sites, with mean absolute errors between 5 and 15 of the HI range. Errors were not spatially correlated. Minimum prediction errors were encountered in the catchment for which the models were generated and also in one other that differed only in the parent material. In the other validation site, the models systematically overestimated HI. At the site of model generation, both regressions were accurate. However on the other sites, prediction errors using regression 2 were systematically higher than for regression 1, which uses a topographic index with a physical basis. These results revealed that soil-landscape models may be useful for predicting soil hydromorphy over a region but only when validated under several environmental conditions.

CLIC1 (NCC27) is an unusual, largely intracellular, ion channel that exists in both soluble and membrane-associated forms. The soluble recombinant protein can be expressed in Escherichia coli, a property that has made possible both detailed electrophysiological studies in lipid bilayers and an examination of the mechanism of membrane integration. SolubleE. coli-derived CLIC1 moves from solution into artificial bilayers and forms chloride-selective ion channels with essentially identical conductance, pharmacology, and opening and closing kinetics to those observed in CLIC1-transfected Chinese hamster ovary cells. The process of membrane integration of CLIC1 is pH-dependent. Following addition of protein to the trans solution, small conductance channels with slow kinetics (SCSK) appear in the bilayer. These SCSK modules then appear to undergo a transition to form a high conductance channel with fast kinetics. This has four times the conductance of the SCSK and fast kinetics that characterize the native channel. This suggests that the CLIC1 ion channel is likely to consist of a tetrameric assembly of subunits and indicates that despite its size and unusual properties, it is able to form a completely functional ion channel in the absence of any other ancillary proteins.

A synthetic Synechocystissp. PCC6803 DnaB split mini-intein gene was constructed for the in vivocyclization of recombinant proteins expressed in Escherichia coli. The system was used to cyclize the NH2-terminal domain of E. coliDnaB, the structure of which had been determined previously by NMR spectroscopy. Cyclization was found to proceed efficiently, with little accumulation of precursor, and the product was purified in high yield. The solution structure of cyclic DnaB-N is not significantly different from that of linear DnaB-N and it unfolds reversibly at temperatures ∼14 °C higher. Improved hydrogen bonding was observed in the first and last helices, and the length of the last helix was increased, while the 9-amino acid linker used to join the NH2and COOH termini was found to be highly mobile. The measured thermodynamic stabilization of the structure (ΔΔG≈ 2 kcal/mol) agrees well with the value estimated from the reduced conformational entropy in the unfolded form. Simple polymer theory can be used to predict likely free energy changes resulting from protein cyclization and how the stabilization depends on the size of the protein and the length of the linker used to connect the termini.

CLIC1 (NCC27) is a member of the highly conserved class of chloride ion channels that exists in both soluble and integral membrane forms. Purified CLIC1 can integrate into synthetic lipid bilayers forming a chloride channel with similar properties to those observed in vivo. The structure of the soluble form of CLIC1 has been determined at 1.4-A resolution. The protein is monomeric and structurally homologous to the glutathione S-transferase superfamily, and it has a redox-active site resembling glutaredoxin. The structure of the complex of CLIC1 with glutathione shows that glutathione occupies the redox-active site, which is adjacent to an open, elongated slot lined by basic residues. Integration of CLIC1 into the membrane is likely to require a major structural rearrangement, probably of the N-domain (residues 1-90), with the putative transmembrane helix arising from residues in the vicinity of the redox-active site. The structure indicates that CLIC1 is likely to be controlled by redox-dependent processes.

The molecular interactions driving reactive center loop (RCL) insertion are of considerable interest in gaining a better understanding of the serpin inhibitory mechanism. Previous studies have suggested that interactions in the proximal hinge/breach region may be critical determinants of RCL insertion in serpins. In this study, conformational and functional changes in plasminogen activator inhibitor-2 (PAI-2) following incubation with a panel of synthetic RCL peptides indicated that the P14 residue is critical for RCL insertion, and hence inhibitory activity, in PAI-2. Only RCL peptides with a P14 threonine were able to induce the stressed to relaxed transition and abolish inhibitory activity in PAI-2, indicating that RCL insertion into beta-sheet A of PAI-2 is dependent upon this residue. The recently solved crystal structure of relaxed PAI-2 (PAI-2.RCL peptide complex) allowed detailed analysis of molecular interactions involving P14 related to RCL insertion. Of most interest is the rearrangement of hydrogen bonding around the breach region that accompanies the stressed to relaxed transition, in particular the formation of a side chain hydrogen bond between the threonine at P14 and an adjacent tyrosine on strand 2 of beta-sheet B in relaxed PAI-2. Structural alignment of known serpin sequences showed that this pairing (or the equivalent serine/threonine pairing) is highly conserved ( approximately 87%) in inhibitory serpins and may represent a general structural basis for serpin inhibitory activity.

The structure of the serpin, plasminogen activator inhibitor type-2 (PAI-2), in a complex with a peptide mimicking its reactive center loop (RCL) has been determined at 1.6-A resolution. The structure shows the relaxed state serpin structure with a prominent six-stranded beta-sheet. Clear electron density is seen for all residues in the peptide. The P1 residue of the peptide binds to a well defined pocket at the base of PAI-2 that may be important in determining the specificity of protease inhibition. The stressed-to-relaxed state (S --> R) transition in PAI-2 can be modeled as the relative motion between a quasirigid core domain and a smaller segment comprising helix hF and beta-strands s1A, s2A, and s3A. A comparison of the Ramachandran plots of the stressed and relaxed state PAI-2 structures reveals the location of several hinge regions connecting these two domains. The hinge regions cluster in three locations on the structure, ensuring a cooperative S --> R transition. We hypothesize that the hinge formed by the conserved Gly(206) on beta-strand s3A in the breach region of PAI-2 effects the S --> R transition by altering its backbone torsion angles. This torsional change is due to the binding of the P14 threonine of the RCL to the open breach region of PAI-2.

The Sm/Lsm proteins associate with small nuclear RNA to form the core of small nuclear ribonucleoproteins, required for processes as diverse as pre-mRNA splicing, mRNA degradation and telomere formation. The Lsm proteins from archaea are likely to represent the ancestral Sm/Lsm domain. Here, we present the crystal structure of the Lsmα protein from the thermophilic archaeon Methanobacterium thermoautotrophicum at 2.0 A˚ resolution. The Lsmα protein crystallizes as a heptameric ring comprised of seven identical subunits interacting via β-strand pairing and hydrophobic interactions. The heptamer can be viewed as a propeller-like structure in which each blade consists of a seven-stranded antiparallel β-sheet formed from neighbouring subunits. There are seven slots on the inner surface of the heptamer ring, each of which is lined by Asp, Asn and Arg residues that are highly conserved in the Sm/Lsm sequences. These conserved slots are likely to form the RNA-binding site. In archaea, the gene encoding Lsmα is located next to the L37e ribosomal protein gene in a putative operon, suggesting a role for the Lsmα complex in ribosome function or biogenesis.

Stathmin family phosphoproteins (stathmin, SCG10, SCLIP, and RB3/RB3′/RB3“) are involved in signal transduction and regulation of microtubule dynamics. With the exception of stathmin, they are expressed exclusively in the nervous system, where they display different spatio-temporal and functional regulations and hence play at least partially distinct and possibly complementary roles in relation to the control of development, plasticity, and neuronal activities. At the molecular level, each possesses a specific “stathmin-like domain” and, with the exception of stathmin, various combinations of N-terminal extensions involved in their association with intracellular membrane compartments. We show here that each stathmin-like domain also displays specific biochemical and tubulin interaction properties. They are all able to sequester two α/β tubulin heterodimers as revealed by their inhibitory action on tubulin polymerization and by gel filtration. However, they differ in the stabilities of the complexes formed as well as in their interaction kinetics with tubulin followed by surface plasmon resonance as follows: strong stability and slow kinetics for RB3; medium for SCG10, SCLIP, and stathmin; and weak stability and rapid kinetics for RB3′. These results suggest that the fine-tuning of their stathmin-like domains contributes to the specific functional roles of stathmin family proteins in the regulation of microtubule dynamics within the various cell types and subcellular compartments of the developing or mature nervous system.

Because soils are both a source and a sink for atmospheric CO2, there is an increasing need to characterize the spatial distribution of soil C pools. Large amounts of organic carbon (OC) accumulate in hydric bottom-lands soils. In the Armorican Massif (Western France) where these soils represent 20% of the total surface area, the spatial characterization of OC pools is difficult to assess due to methodological problems such as high spatial variability. Soil color indexes, which combine various characteristics of soil horizons or profiles, are an alternative approach for quantifying the differences in OC storage. In addition, terrain attributes derived from Digital Elevation Models (DEM) may be useful in characterizing the distribution of soil color indexes over large areas. Thus, the overall goal of this work was the development and application of a model for use in predicting the organic carbon (OC) content of soil areas. To accomplish this, extensive examination of soil morphology combined with selected terrain attributes measured in the field and calculated from a digital elevation model (DEM) were used. Soil samples were collected in Western France from a 2-ha agricultural parcel that forms the major part of a hillslope. The results indicate that OC stocks of the entire profile were correlated highly to a soil hydromorphic index (HI) (r2= 0.80). HI is a function of the percent of the total soil profile depth constituted by horizons with some degree of hydromorphic feature development and the moist color of the surface A horizon. Using a stepwise regression technique, we constructed a prediction model of HI distribution by using the relations between HI and (i) the elevation above the stream bank (ES) (r2= 0.80); (ii) the downslope gradient (DG) (r2= 0.55); and (iii) the upslope contributing area (AMU) (r2= 0.60). Validation of this model on a second site showed that topographical attributes explained up to 75% of the profile OC stock variability. These results confirmed that the integration of a soil index and topographical information is a useful tool for prediction of OC distribution. In addition, the use of soil morphologic indexes could significantly improved the construction and the validation of soil-landscape models because it would minimize laboratory measurements of OC reservoirs.

Soil-landscape models have prediction errors that can be reduced by using auxiliary soil data. However, standard soil surveys using auger hole and laboratory analysis encounter both methodological and economical constraints because of, for example, the short-range variability of soils and the expensive field work. In the present study, the objective was to test the use of auxiliary geophysical data to improve a soil-landscape model for two Digital Elevation Model (DEM) resolutions (10 and 50 m). The study site was an agricultural parcel comprising an entire hillslope of a small (0.6 km2) catchment in Normandy, France. Prediction models based on multiple regression and co-kriging techniques were established using topographic, soil and geophysical data. For high DEM resolution (10 m), soil-landscape models based only on surface features seemed to be efficient. The use of additional geophysical or soil data improved the prediction quality slightly. For coarser DEM (50-m resolution), the prediction quality of models established using only terrain attributes was faulty. The soil hydromorphic prediction could be improved greatly by the use of auxiliary geophysical data. In this case, the improved accuracy was similar to that obtained by high soil density data (50 observations/ha). Finally, we discuss the use of both geophysical and topographical data in order to describe better the spatial distribution of soils.

Advances in molecular biology may mean that almost any protein sequence can be synthesised, but perhaps this has served to highlight the inadequacy of theoretical work. For a given protein fold, it is probably not possible to reliably predict an ideal sequence. We identify and survey several aspects of the problem. Firstly, it is not clear what is the best way to score a sequence-structure pair. Secondly, there is no consensus as to what the score function should represent (free energy or some abstract measure of sequence-structure compatibility). Finally, the number of possible sequences is astronomical and searching this space poses a daunting optimisation problem. These problems are discussed in the light of recent experimental successes.

The advantages of the organism Dictyostelium discoideumas an expression host for recombinant glycoproteins have been exploited for the production of an isotopically labeled cell surface protein for NMR structure studies. Growth medium containing [15N]NH4Cl and [13C]glycerol was used to generate isotopically labeled Escherichia coli,which was subsequently introduced to D. discoideumcells in simple Mes buffer. A variety of growth conditions were screened to establish minimal amounts of nitrogen and carbon metabolites for a cost-effective protocol. Following single-step purification by anion-exchange chromatography, 8 mg of uniformly 13C,15N-labeled protein secreted by approximately 1010D. discoideumcells was isolated from 3.3 liters of supernatant. Mass spectrometry showed the recombinant protein of 16 kDa to have incorporated greater than 99.9% isotopic label. The two-dimensional 1H-13C HSQC spectrum confirms 13C labeling of both glycan and amino acid residues of the glycoprotein. All heteronuclear NMR spectra showed a good dispersion of cross-peaks essential for high-quality structure determination.

d-Ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) catalyses the central CO2-fixing reaction of photosynthesis in a complex, multiple-step process. Several structures of rubisco complexed with substrate analogues, inhibitors and products have been determined by X-ray crystallography. The structures fall into two well-defined and distinct states. The active site is either “open” or “closed”. The timing and mechanism of the transition between these two states have been uncertain. We solved the crystal structure of unactivated (metal-free) rubisco from tobacco with only inorganic phosphate bound and conclude that phosphate binding per sedoes not trigger closure, as it does in the similarly structured enzyme, triosephosphate isomerase. Comparison of all available rubisco structures suggests that, instead, the distance between the terminal phosphates (P1 and P2) of the bisphosphate ligand is the trigger: if that distance is less than 9.1 Å, then the active site closes; if it is greater than 9.4 Å then the enzyme remains open. Shortening of the inter-phosphate distance results from the ligand binding in a more curved conformation when O atoms of the ligand’s sugar backbone interact either with the metal, if it is present, or with charged groups in the metal-binding site, if the metal is absent. This shortening brings the P1 phosphate into hydrogen bonding contact with Thr65. Thr65 exists in two discrete states related by a rotation of the backbone ψ torsion angle. This rotation is coupled to domain rotation and hence to active site closure. Rotation of the side-chain of Thr65 also affects the C-terminal strand of large subunit which packs against Loop 6 after closure. The position of the C-terminal strand in the closed state is stabilised by multiple polar interactions with a distinctive highly-charged latch site involving the side-chain of Asp473. In the open state, this latch site may be occupied instead by phosphorylated anions.

Stathmin is a cytosoluble phosphoprotein proposed to be a regulatory relay integrating diverse intracellular signaling pathway. Its interaction with tubulin modulates microtubule dynamics by destabilization of assembled microtubules or inhibition of their polymerization from free tubulin. The aim of this study was to probe the native structure of stathmin and to delineate its minimal region able to interact with tubulin. Limited proteolysis of stathmin revealed four structured domains within the native protein, corresponding to amino acid sequences 22-81 (I), 95-113 (II), 113-128 (III), and 128-149 (IV), which allows us to propose stathmin folding hypotheses. Furthermore, stathmin proteolytic fragments were mixed to interact with tubulin, and those that retained affinity for tubulin were isolated by size exclusion chromatography and identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The results indicate that, to interact with tubulin, a stathmin fragment must span a minimal core region from residues 42 to 126, which interestingly corresponds to the predicted alpha-helical "interaction region" of stathmin. In addition, an interacting stathmin fragment must include a short N- or C-terminal extension. The functional significance of these interaction constrains is further validated by tubulin polymerization inhibition assays with fragments designed on the basis of the tubulin binding results. The present results will help to optimize further stathmin structural studies and to develop molecular tools to target its interaction with tubulin.

Stathmin is a cytosoluble phosphoprotein proposed to be a regulatory relay integrating diverse intracellular signaling pathway. Its interaction with tubulin modulates microtubule dynamics by destabilization of assembled microtubules or inhibition of their polymerization from free tubulin. The aim of this study was to probe the native structure of stathmin and to delineate its minimal region able to interact with tubulin. Limited proteolysis of stathmin revealed four structured domains within the native protein, corresponding to amino acid sequences 22–81 (I), 95–113 (II), 113–128 (III), and 128–149 (IV), which allows us to propose stathmin folding hypotheses. Furthermore, stathmin proteolytic fragments were mixed to interact with tubulin, and those that retained affinity for tubulin were isolated by size exclusion chromatography and identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The results indicate that, to interact with tubulin, a stathmin fragment must span a minimal core region from residues 42 to 126, which interestingly corresponds to the predicted α-helical “interaction region” of stathmin. In addition, an interacting stathmin fragment must include a short N- or C-terminal extension. The functional significance of these interaction constrains is further validated by tubulin polymerization inhibition assays with fragments designed on the basis of the tubulin binding results. The present results will help to optimize further stathmin structural studies and to develop molecular tools to target its interaction with tubulin.