nils-herrmann/clean_scirepeval huggingface dataset:
From sporadic single genes to a broader transcriptomic approach: insights into the formation of the biomineralized exoskeleton in decapod crustaceans.
Venue: Journal of structural biology

One fundamental character common to pancrustaceans (Crustacea and Hexapoda) is a mineralized rigid exoskeleton whose principal organic components are chitin and proteins. In contrast to traditional research in the field that has been devoted to the structural and physicochemical aspects of biomineralization, the present study explores transcriptomic aspects of biomineralization as a first step towards adding a complementary molecular layer to this field. The rigidity of the exoskeleton in pancrustaceans dictates essential molt cycles enabling morphological changes and growth. Thus, formation and mineralization of the exoskeleton are concomitant to the timeline of the molt cycle. Skeletal proteinaceous toolkit elements have been discovered in previous studies using innovative molt-related binary gene expression patterns derived from transcriptomic libraries representing the major stages comprising the molt cycle of the decapod crustacean Cherax quadricarinatus. Here, we revisited some prominent exoskeleton-related structural proteins encoding and, using the above molt-related binary pattern methodology, enlarged the transcriptomic database of C.quadricarinatus. The latter was done by establishing a new transcriptomic library of the cuticle forming epithelium and molar tooth at four different molt stages (i.e., inter-molt, early pre-molt, late pre-molt and post-molt) and incorporating it to a previous transcriptome derived from the gastroliths and mandible. The wider multigenic approach facilitated by the newly expanded transcriptomic database not only revisited single genes of the molecular toolkit, but also provided both scattered and specific information that broaden the overview of proteins and gene clusters which are involved in the construction and biomineralization of the exoskeleton in decapod crustaceans.
A novel approach to investigate the evolution of structured tandem repeat protein families by exon duplication.
Venue: Journal of structural biology

Tandem Repeat Proteins (TRPs) are ubiquitous in cells and are enriched in eukaryotes. They contributed to the evolution of organism complexity, specializing for functions that require quick adaptability such as immunity-related functions. To investigate the hypothesis of repeat protein evolution through exon duplication and rearrangement, we designed a tool to analyze the relationships between exon/intron patterns and structural symmetries. The tool allows comparison of the structure fragments as defined by exon/intron boundaries from Ensembl against the structural element repetitions from RepeatsDB. The all-against-all pairwise structural alignment between fragments and comparison of the two definitions (structural units and exons) are visualized in a single matrix, the "repeat/exon plot". An analysis of different repeat protein families, including the solenoids Leucine-Rich, Ankyrin, Pumilio, HEAT repeats and the β propellers Kelch-like, WD40 and RCC1, shows different behaviors, illustrated here through examples. For each example, the analysis of the exon mapping in homologous proteins supports the conservation of their exon patterns. We propose that when a clear-cut relationship between exon and structural boundaries can be identified, it is possible to infer a specific "evolutionary pattern" which may improve TRPs detection and classification.
Intracellular calcium phosphate deposits contribute to transcellular calcium transport within the hepatopancreas of Porcellio scaber.
Venue: Journal of structural biology

Like in most Crustacea, the cuticle of terrestrial isopods is hardened by a calcareous mineral phase. This rigid cuticle is frequently shed during a process called moulting. To reduce calcium loss, most terrestrial isopod species eat the shed cuticle, the exuviae, and absorb the calcium from it through large tubular diverticula of the intestine, called the mid gut glands or hepatopancreas. After moulting the absorbed calcium should be transported immediately into the hemolymph from which it is used to rapidly mineralize the new cuticle. This suggests that the hepatopancreas epithelium transports calcium from the lumen to the hemolymph. We used TEM, energy-filtered TEM and electron-probe X-ray microanalysis to analyse the distribution of elevated calcium within the hepatopancreas cells of P. scaber. We used animals in the postmoult stage that have eaten their exuviae and, as a control, those that have not ingested the exuviae. To minimize calcium loss within the samples, we used high pressure frozen and freeze substituted samples and propane-1-3-diol as floatation medium for thin-sectioning. The results reveal intracellular dense deposits containing calcium, phosphorus and oxygen at the apical microvillus membrane, within the cytoplasm, attached to vesicles and to the basolateral membrane, as well as extracellular between cells and the basal lamina. Control animals were devoid of these deposits. The results indicate that calcium from the exuviae is absorbed and transported across the epithelium into the hemolymph. We propose that during transport, intracellular calcium is bound to phosphate avoiding toxic effects of high concentrations of ionized calcium.
Crystal structure of the periplasmic sensor domain of histidine kinase VbrK suggests indirect sensing of β-lactam antibiotics.
Venue: Journal of structural biology

Bacterial two-component regulatory systems (TCS) play important roles in sensing environmental stimuli and responding to them by regulating gene expression. VbrK/VbrR, a TCS in Vibrio parahaemolyticus, confers resistance to β-lactam antibiotics through activating a β-lactamase gene. Its periplasmic sensor domain was previously suggested to detect β-lactam antibiotics by direct binding. Here, we report a crystal structure of the periplasmic sensing domain of VbrK (VbrKSD) using sulfur-based single-wavelength anomalous diffraction (S-SAD) phasing. Contrary to most bacterial sensor domains which form dimers, we show that VbrKSD is a monomer using size exclusion chromatography coupled with multi-angle light scattering. This observation is also supported by molecular dynamics simulations. To quantify the binding affinity of β-lactam antibiotics to VbrKSD, we performed isothermal titration calorimetry and other biophysical analyses. Unexpectedly, VbrKSD did not show any significant binding to β-lactam antibiotics. Therefore, we propose that the detection of β-lactam antibiotics by VbrK is likely to be indirect via an as yet unidentified mechanism.
On the structural organization of the bacillary band of Trichuris muris under cryopreparation protocols and three-dimensional electron microscopy.
Venue: Journal of structural biology

Whipworms of the genus Trichuris are nematode parasites that infect mammals and can lead to various intestinal diseases of human and veterinary interest. The most intimate interaction between the parasite and the host intestine occurs through the anterior region of the nematode body, inserted into the intestinal mucosa during infection. One of the most prominent structures of the nematode surface found at the infection site is the bacillary band, a surface domain formed by a number of cells, mostly stichocytes and bacillary glands, whose structure and function are still under debate. Here, we used confocal microscopy, field emission scanning electron microscopy, helium ion microscopy, transmission electron microscopy and FIB-SEM tomography to unveil the functional role of the bacillary gland cell. We analyzed the surface organization as well as the intracellular milieu of the bacillary glands of Trichuris muris in high pressure frozen/freeze-substituted samples. Results showed that the secretory content is preserved in all gland openings, presenting a projected pattern. FIB-SEM analysis showed that the lamellar zone within the bacillary gland chamber is formed by a set of lacunar structures that may exhibit secretory or absorptive functions. In addition, incubation of parasites with the fluid phase endocytosis marker sulforhodamine B showed a time-dependent uptake by the parasite mouth, followed by perfusion through different tissues with ultimate secretion through the bacillary gland. Taken together, the results show that the bacillary gland possess structural characteristics of secretory and absorptive cells and unequivocally demonstrate that the bacillary gland cell functions as a secretory structure.
Comparison of carbonate precipitation induced by Curvibacter sp. HJ-1 and Arthrobacter sp. MF-2: Further insight into the biomineralization process.
Venue: Journal of structural biology

Microorganisms are generally involved in the nucleation, growth and phase transformation of carbonate minerals, and influence the development of their morphology and polymorphism. However, understanding of the process of microbially induced carbonate precipitation (MICP) remains limited. Herein, MICP experiments were carried out using Curvibacter sp. HJ-1 and Arthrobacter sp. MF-2 in M2 medium, and the processes of MICP were monitored. Bacterial cells induced the precipitation of carbonate by creating favorable physicochemical conditions and acting as nucleation templates for carbonate particles and thereby, markedly influenced the morphology and growth of the carbonate structure. The extracellular polymeric substance (EPS) secreted by the bacteria was readily absorbed by the precipitated carbonate, which modified its crystal growth orientation. The MgCO3 content of Mg-calcite, induced by MF-2, was dramatically higher than that induced by HJ-1; HJ-1 promoted the formation and stability of aragonite. Multiple formation mechanisms coexisted during the evolution process of the mineral morphologies in the presence of the bacteria. The spherulites observed mainly evolved from dumbbell-like precursors in the presence of MF-2, whereas aggregate growth was the main formation mechanism of radial spherulites in the presence of HJ-1.
Phosphatidylserine controls calcium phosphate nucleation and growth on lipid monolayers: a physicochemical understanding of matrix vesicles-driven biomineralization.
Venue: Journal of structural biology

Bone biomineralization is an exquisite process by which a hierarchically organized mineral matrix is formed. Growing evidence has uncovered the involvement of one class of extracellular vesicles, named matrix vesicles (MVs), in the formation and delivery of the first mineral nuclei to direct collagen mineralization. MVs are released by mineralization-competent cells equipped with a specific biochemical machinery to initiate mineral formation. However, little is known about the mechanisms by which MVs can trigger this process. Here, we present a combination of in situ investigations and ex vivo analysis of MVs extracted from growing-femurs of chicken embryos to investigate the role played by phosphatidylserine (PS) in the formation of mineral nuclei. By using self-assembled Langmuir monolayers, we reconstructed the nucleation core - a PS-enriched motif thought to trigger mineral formation in the lumen of MVs. In situ infrared spectroscopy of Langmuir monolayers and ex situ analysis by transmission electron microscopy evidenced that mineralization was achieved in supersaturated solutions only when PS was present. PS nucleated amorphous calcium phosphate that converted into biomimetic apatite. By using monolayers containing lipids extracted from native MVs, mineral formation was also evidenced in a manner that resembles the artificial PS-enriched monolayers. PS-enrichment in lipid monolayers creates nanodomains for local increase of supersaturation, leading to the nucleation of ACP at the interface through a multistep process. We posited that PS-mediated nucleation could be a predominant mechanism to produce the very first mineral nuclei during MV-driven bone/cartilage biomineralization.
Crossfibrillar mineral tessellation in normal and Hyp mouse bone as revealed by 3D FIB-SEM microscopy.
Venue: Journal of structural biology

In bone, structural components such as mineral extend across length scales to provide essential biomechanical functions. Using X-ray micro-computed tomography (µCT), and focused-ion beam scanning electron microscopy (FIB-SEM) in serial-surface-view mode, together with 3D reconstruction, entire mouse skeletons and small bone tissue volumes were examined in normal wildtype (WT) and mutant Hyp mice (an animal model for X-linked hypophosphatemia/XLH, a disease with severe hypomineralization of bone). 3D thickness maps of the skeletons showed pronounced irregular thickening and abnormalities of many skeletal elements in Hyp mice compared to WT mice. At the micro- and nanoscale, near the mineralization front in WT tibial bone volumes, mineralization foci grow as expanding prolate ellipsoids to abut and pack against one another to form a congruent and contiguous mineral tessellation pattern within collagen bundles that contributes to lamellar periodicity. In the osteomalacic Hyp mouse bone, mineralization foci form and begin initial ellipsoid growth within normally organized collagen assembly, but their growth trajectory aborts. Mineralization-inhibiting events in XLH/Hyp (low circulating serum phosphate, and increased matrix osteopontin) combine to result in decreased mineral ellipsoid tessellation - a defective mineral-packing organization that leaves discrete mineral volumes isolated in the extracellular matrix such that ellipsoid packing/tessellation is not achieved. Such a severely altered mineralization pattern invariably leads to abnormal compliance, other aberrant biomechanical properties, and altered remodeling of bone, all of which indubitably lead to macroscopic bone deformities and anomalous mechanical performance in XLH/Hyp. Also, we show the relationship of osteocytes and their cell processes to this mineralization pattern.
Amyloidogenicity as a driving force for the formation of functional oligomers.
Venue: Journal of structural biology

Insoluble amyloid fibrils formed by self-assembly of amyloidogenic regions of proteins have a cross-β-structure. In this work, by using targeted molecular dynamics and rigid body simulation, we demonstrate that if a protein consists of an amyloidogenic region and a globular domain(s) and if the linker between them is short enough, such molecules cannot assemble into amyloid fibrils, instead, they form oligomers with a defined and limited number of β-strands in the cross-β core. We show that this blockage of the amyloid growth is due to the steric repulsion of globular structures linked to amyloidogenic regions. Furthermore, we establish the relationship between the linker length and the number of monomers in such nanoparticles. We hypothesise that such oligomerisation can be a yet unrecognised way to form natural protein complexes involved in biological processes. Our results can also be used in protein engineering for designing soluble nanoparticles carrying different functional domains.
The crystal structure of protein-transporting chaperone BCP1 from Saccharomyces cerevisiae.
Venue: Journal of structural biology

BCP1 is a protein enriched in the nucleus that is required for Mss4 nuclear export and identified as the chaperone of ribosomal protein Rpl23 in Saccharomyces cerevisiae. According to sequence homology, BCP1 is related to the mammalian BRCA2-interacting protein BCCIP and belongs to the BCIP protein family (PF13862) in the Pfam database. However, the BCIP family has no discernible similarity to proteins with known structure. Here, we report the crystal structure of BCP1, presenting an α/β fold in which the central antiparallel β-sheet is flanked by helices. Protein structural classification revealed that BCP1 has similarity to the GNAT superfamily but no conserved substrate-binding residues. Further modeling and protein-protein docking work provide a plausible model to explain the interaction between BCP1 and Rpl23. Our structural analysis presents the first structure of BCIP family and provides a foundation for understanding the molecular basis of BCP1 as a chaperone of Rpl23 for ribosome biosynthesis.
Structure and topology of the linkers in the conserved lepidosaur β-keratin chain with four 34-residue repeats support an interfilament role for the central linker.
Venue: Journal of structural biology

The β-keratin chain with four 34-residue repeats that is conserved across the lepidosaurs (lizards, snakes and tuatara) contains three linker regions as well as a short, conserved N-terminal domain and a longer, more variable C-terminal domain. Earlier modelling had shown that only six classes of structure involving the four 34-residue repeats were possible. In three of these the 34-residue repeats were confined to a single filament (Classes 1, 2 and 3) whereas in the remaining three classes the repeats lay in two, three or four filaments, with some of the linkers forming interfilament connections (Classes 4, 5 and 6). In this work the members of each class of structure (a total of 20 arrangements) have been described and a comparison has been made of the topologies of each of the linker regions. This provides new constraints on the structure of the chain as a whole. Also, analysis of the sequences of the three linker regions has revealed that the central linker (and only the central linker) contains four short regions displaying a distinctive dipeptide repeat of the form (S-X)2,3 separated by short regions containing proline and cysteine residues. By analogy with silk fibroin proteins this has the capability of forming a β-sheet-like conformation. Using the topology and sequence data the evidence suggests that the four 34-residue repeat chain adopts a Class 4a structure with a β-sandwich in filament 1 connected through the central linker to a β-sandwich in filament 2.
Insights into DEPTOR regulation from in silico analysis of DEPTOR complexes.
Venue: Journal of structural biology

DEPTOR is an inhibitor of the mTOR kinase which controls cell growth. DEPTOR consists of two DEP domains and a PDZ domain connected by an unstructured linker, and its stability is tightly regulated through post-translational modifications of its linker region that contains the 286SSGYFS291 degron. Based on the mTORC1 complex, our modelling suggests a possible spatial arrangement of DEPTOR which is characterised to form a dimer. Our model shows that DEPTOR's two PDZ domains bind separately to the dimeric mTOR's FAT domains ∼13 Å apart, while the extended linkers are sufficiently long to span from the FAT domain to the kinase domain and beyond to join a shared dimer of the DEP domains. This places S299 closest to the kinase's catalytic site, indicating that phosphorylation would start with it and successively upstream towards the degron. The CK1α kinase is responsible for phosphorylation of DEPTOR's degron, and our docking analysis further reveals that CK1α contains sites to bind DEPTOR's pS286, pS287 and pT295, which may act as priming phosphates for the phosphorylation of S291 of DEPTOR's degron. DEPTOR's linker can also be ubiquitylated by the UbcH5A-SCFβ-TrCP complex without its PDZ dissociating from mTOR according to the modelling. As the catalytic cleft of mTOR's kinase is restricted, interactions between the kinase's unstructured segment surrounding the cleft and DEPTOR's linker, which may involve S293 and S299, may be critical in controlling DEPTOR's access to the catalytic cleft and hence its phosphorylation by mTOR in a manner dependent on mTOR's activation.
An optimized approach using cryofixation for high-resolution 3D analysis by FIB-SEM.
Venue: Journal of structural biology

Compared with conventional two-dimensional transmission electron microscopy (TEM), focused ion beam scanning electron microscopy (FIB-SEM) can provide more comprehensive 3D information on cell substructures at the nanometer scale. Biological samples prepared by cryofixation using high-pressure freezing demonstrate optimal preservation of the morphology of cellular structures, as these are arrested instantly in their near-native states. However, samples from cryofixation often show a weak back-scatter electron signal and bad image contrast in FIB-SEM imaging. In addition, it is impossible to do large amounts of heavy metal staining. This is commonly achieved via established osmium impregnation (OTO) en bloc staining protocols. Here, we compared the FIB-SEM image quality of brain tissues prepared using several common freeze-substitution media, and we developed an approach that overcomes these limitations through a combination of osmium tetroxide, uranyl acetate, tannic acid, and potassium permanganate at proper concentrations, respectively. Using this optimized sample preparation protocol for high-pressure freezing and freeze-substitution, perfect smooth membrane morphology, even of the lipid bilayers of the cell membrane, was readily obtained using FIB-SEM. In addition, our protocol is broadly applicable and we demonstrated successful application to brain tissues, plant tissues, Caenorhabditis elegans, Candida albicans, and chlorella. This approach combines the potential of cryofixation for 3D large volume analysis of subcellular structures with the high-resolution capabilities of FIB-SEM.
Deep learning for 3D imaging and image analysis in biomineralization research.
Venue: Journal of structural biology

Biomineralization research examines structure-function relations in all types of exo- and endo-skeletons and other hard tissues of living organisms, and it relies heavily on 3D imaging. Segmentation of 3D renderings of biomineralized structures has long been a bottleneck because of human limitations such as our available time, attention span, eye-hand coordination, cognitive biases, and attainable precision, amongst other limitations. Since recently, some of these routine limitations appear to be surmountable thanks to the development of deep-learning algorithms for biological imagery in general, and for 3D image segmentation in particular. Many components of deep learning often appear too abstract for a life scientist. Despite this, the basic principles underlying deep learning have many easy-to-grasp commonalities with human learning and universal logic. This primer presents these basic principles in what we feel is an intuitive manner, without relying on prerequisite knowledge of informatics and computer science, and with the aim of improving the reader's general literacy in artificial intelligence and deep learning. Here, biomineralization case studies are presented to illustrate the application of deep learning for solving segmentation and analysis problems of 3D images ridden by various artifacts, and/or which are plainly difficult to interpret. The presented portfolio of case studies includes three examples of imaging using micro-computed tomography (µCT), and three examples using focused-ion beam scanning electron microscopy (FIB-SEM), all on mineralized tissues. We believe this primer will expand the circle of users of deep learning amongst biomineralization researchers and other life scientists involved with 3D imaging, and will encourage incorporation of this powerful tool into their professional skillsets and to explore it further.