Structural Analysis of the Black-Legged Tick Saliva Protein Salp15.

Salp15 is one of the proteins in the saliva of the tick Ixodes scapularis. Together with other biomolecules injected into the mammalian host at the biting site, it helps the tick to sustain its blood meal for days. Salp15 interferes with the cellular immune response of the mammalian host by inhibiting the activation of CD4+ T-lymphocytes. This function is co-opted by pathogens that use the tick as a vector and invade the host when the tick bites, such as Borrelia burgdorferi, the causative agent of Lyme borreliosis. Because of the immunity-suppressing role of Salp15, it has been proposed as a candidate for therapeutic applications in disorders of the immune system. The protein is produced as a 135-residue long polypeptide and secreted without its N-terminal signal 1-21 sequence. Detailed structural studies on Salp15 are lacking because of the difficulty in producing large amounts of the folded protein. We report the production of Salp15 and its structural analysis by NMR. The protein is monomeric and contains a flexible N-terminal region followed by a folded domain with mixed α + ß secondary structures. Our results are consistent with a three-dimensional structural model derived from AlphaFold, which predicts the formation of three disulfide bridges and a free C-terminal cysteine.

Trabecular bone patterns as a fracture risk predictor: a systematic review.

BACKGROUND: The aim of this systematic review was to evaluate the assessment of trabecular bone patterns in dental radiographs, for fracture risk prediction, compared with the current diagnostic methods. METHODS: The PRISMA guidelines were followed. According to predefined inclusion criteria (PICO), literature searches were focussed on published studies with analyses of trabecular bone patterns on intraoral and/or in panoramic radiographs, compared with Dual X-ray Absorptiometry (DXA) and/or Fracture Risk Assessment Tool (FRAX), with the outcomes; fracture and/or sensitivity and specificity for osteoporosis prediction. The included studies were quality-assessed using the QUADAS-2 tool and the certainties of evidence was assessed using the GRADE approach. RESULTS: The literature searches identified 2913 articles, whereas three were found to meet the inclusion criteria. Two longitudinal cohort studies evaluated the use of trabecular bone patterns to predict bone fractures. In one of the studies, the relative risk of fracture was significantly higher for women with sparse bone pattern, identified by visual assessment of dental radiographs, and in the other study by digital software assessment. Visual assessment in the second study did not show significant results. The cross-sectional study of digital analyses of trabecular bone patterns in relation to osteoporosis reported a sensitivity of 0.70 and a specificity of 0.69. CONCLUSION: Based on low certainty of evidence, trabecular bone evaluation on dental radiographs may predict fractures in adults without a prior diagnosis of osteoporosis, and based on very low certainty of evidence, it is uncertain whether digital image analyses of trabecular bone can predict osteoporosis.

Modulation of Structural Heterogeneity Controls Phytochrome Photoswitching.

Phytochromes sense red/far-red light and control many biological processes in plants, fungi, and bacteria. Although the crystal structures of dark- and light-adapted states have been determined, the molecular mechanisms underlying photoactivation remain elusive. Here, we demonstrate that the conserved tongue region of the PHY domain of a 57-kDa photosensory module of Deinococcus radiodurans phytochrome changes from a structurally heterogeneous dark state to an ordered, light-activated state. The results were obtained in solution by utilizing a laser-triggered activation approach detected on the atomic level with high-resolution protein NMR spectroscopy. The data suggest that photosignaling of phytochromes relies on careful modulation of structural heterogeneity of the PHY tongue.

Can reducing cosmetic substances help prevent chromate contact allergy?

BACKGROUND: Allergic contact dermatitis caused by Cr(VI) is often severe and difficult to treat. Therefore, primary prevention is a main goal but, secondary prevention can be valuable to ease the symptoms or prevent relapse of Cr(VI) dermatitis when sensitization has occurred. Barrier creams have been tried for many chemical substances, but until now there is no successful barrier cream against Cr(VI). OBJECTIVES: To investigate the ability of reducing agents to transform Cr(VI) into Cr(III) in an experimental situation, in order to find suitable chemicals to investigate for possible use in a barrier cream. METHODS: The capacity to reduce the amount of Cr(VI) was analyzed in water solutions of acetylcysteine, cysteine, dihydroxyacetone, glutathione, and iron sulfate heptahydrate. Thereafter the reducing capacity of acetylcysteine, dihydroxyacetone, glutathione, and iron sulfate on the amount of Cr(VI) in cement extracts was investigated. The content of Cr(VI) in the test solutions and in the cement extracts was estimated by the diphenyl carbazide spot test. RESULTS: All of the chosen chemicals reduced the amount of Cr(VI) in the test solutions and in the cement extracts to some extent. The reducing capacity was most prominent for iron sulfate. CONCLUSION: A reducing capacity was found for all chosen chemicals.

Micrometer-Sized Magnesium Whitlockite Crystals in Micropetrosis of Bisphosphonate-Exposed Human Alveolar Bone.

Osteocytes are contained within spaces called lacunae and play a central role in bone remodelling. Administered frequently to prevent osteoporotic fractures, antiresorptive agents such as bisphosphonates suppress osteocyte apoptosis and may be localized within osteocyte lacunae. Bisphosphonates also reduce osteoclast viability and thereby hinder the repair of damaged tissue. Osteocyte lacunae contribute to toughening mechanisms. Following osteocyte apoptosis, the lacunar space undergoes mineralization, termed "micropetrosis". Hypermineralized lacunae are believed to increase bone fragility. Using nanoanalytical electron microscopy with complementary spectroscopic and crystallographic experiments, postapoptotic mineralization of osteocyte lacunae in bisphosphonate-exposed human bone was investigated. We report an unprecedented presence of ∼80 nm to ∼3 µm wide, distinctly faceted, magnesium whitlockite [Ca18Mg2(HPO4)2(PO4)12] crystals and consequently altered local nanomechanical properties. These findings have broad implications on the role of therapeutic agents in driving biomineralization and shed new insights into a possible relationship between bisphosphonate exposure, availability of intracellular magnesium, and pathological calcification inside lacunae.

Strain-induced stiffening of nanocellulose-reinforced poly(vinyl alcohol) hydrogels mimicking collagenous soft tissues.

Soft tissues possess remarkable mechanical strength for their high water content, which is hard to mimic in synthetic materials. Here, we demonstrate how strain-induced stiffening in hydrogels plays a major role in mimicking the mechanical properties of collagenous soft tissues. In particular, nanocellulose reinforced polyvinyl alcohol (PVA) hydrogels of exceptionally high water content (90-93 wt%) are shown to exhibit collagen-like mechanical behavior typical for soft tissues. High water content and co-existence of both soft and rigid domains in the gel network are the main factors responsible for strain-induced stiffening. This observed effect due to the alignment of rigid components of the hydrogel is simulated through modeling and visualized through strain-induced birefringence experiments. Design parameters such as nanocellulose aspect ratio and solvent composition are also shown to be important to control the mechanical properties. In addition, owing to their transparency (90-95% at 550 nm) and hyperelastic properties (250-350% strain), the described hydrogels are promising materials for biomedical applications, especially in ophthalmology.

Compressive fatigue properties of an acidic calcium phosphate cement-effect of phase composition.

Calcium phosphate cements (CPCs) are synthetic bone grafting materials that can be used in fracture stabilization and to fill bone voids after, e.g., bone tumour excision. Currently there are several calcium phosphate-based formulations available, but their use is partly limited by a lack of knowledge of their mechanical properties, in particular their resistance to mechanical loading over longer periods of time. Furthermore, depending on, e.g., setting conditions, the end product of acidic CPCs may be mainly brushite or monetite, which have been found to behave differently under quasi-static loading. The objectives of this study were to evaluate the compressive fatigue properties of acidic CPCs, as well as the effect of phase composition on these properties. Hence, brushite cements stored for different lengths of time and with different amounts of monetite were investigated under quasi-static and dynamic compression. Both storage and brushite-to-monetite phase transformation was found to have a pronounced effect both on quasi-static compressive strength and fatigue performance of the cements, whereby a substantial phase transformation gave rise to a lower mechanical resistance. The brushite cements investigated in this study had the potential to survive 5 million cycles at a maximum compressive stress of 13 MPa. Given the limited amount of published data on fatigue properties of CPCs, this study provides an important insight into the compressive fatigue behaviour of such materials.

Nuclear Magnetic Resonance Structure and Mutational Analysis of the Lactococcin A Immunity Protein.

The class IId bacteriocin lactococcin A and the pediocin-like bacteriocins induce membrane leakage and cell death by specifically binding the mannose phophotransferase system (man-PTS) on their target cells. The bacteriocins' cognate immunity proteins that protect the producer cell from its own bacteriocin recognize and bind to the bacteriocin-man-PTS complex and thereby block membrane leakage. In this study, we have determined the three-dimensional structure of the lactococcin A immunity protein (LciA) by the use of nuclear magnetic resonance spectroscopy. LciA forms a four-helix bundle structure with a flexible C-terminal tail. Despite the low degree of sequence similarity between LciA and the pediocin-like immunity proteins, they share the same fold. However, there are certain differences between the structures. The C-terminal helix in LciA is considerably shorter than that observed in the pediocin-like immunity proteins, and the surface potentials of the immunity proteins differ. Truncated variants of LciA in which 6 or 10 of the C-terminal residues were removed yielded a reduced degree of protection, indicating that the unstructured C-terminal tail is important for the functionality of the immunity proteins.

Solution structure of the calmodulin-like C-terminal domain of Entamoeba α-actinin2.

Cell motility is dependent on a dynamic meshwork of actin filaments that is remodelled continuously. A large number of associated proteins that are severs, cross-links, or caps the filament ends have been identified and the actin cross-linker α-actinin has been implied in several important cellular processes. In Entamoeba histolytica, the etiological agent of human amoebiasis, α-actinin is believed to be required for infection. To better understand the role of α-actinin in the infectious process we have determined the solution structure of the C-terminal calmodulin-like domain using NMR. The final structure ensemble of the apo form shows two lobes, that both resemble other pairs of calcium-binding EF-hand motifs, connected with a mobile linker.

Porosity prediction of calcium phosphate cements based on chemical composition.

The porosity of calcium phosphate cements has an impact on several important parameters, such as strength, resorbability and bioactivity. A model to predict the porosity for biomedical cements would hence be a useful tool. At the moment such a model only exists for Portland cements. The aim of this study was to develop and validate a first porosity prediction model for calcium phosphate cements. On the basis of chemical reaction, molar weight and density of components, a volume-based model was developed and validated using calcium phosphate cement as model material. 60 mol% ß-tricalcium phosphate and 40 mol% monocalcium phosphate monohydrate were mixed with deionized water, at different liquid-to-powder ratios. Samples were set for 24 h at 37°C and 100% relative humidity. Thereafter, samples were dried either under vacuum at room temperature for 24 h or in air at 37 °C for 7 days. Porosity and phase composition were determined. It was found that the two drying protocols led to the formation of brushite and monetite, respectively. The model was found to predict well the experimental values and also data reported in the literature for apatite cements, as deduced from the small absolute average residual errors (<2.0%). In conclusion, a theoretical model for porosity prediction was developed and validated for brushite, monetite and apatite cements. The model gives a good estimate of the final porosity and has the potential to be used as a porosity prediction tool in the biomedical cement field.

RNA binding of T-cell intracellular antigen-1 (TIA-1) C-terminal RNA recognition motif is modified by pH conditions.

T-cell intracellular antigen-1 (TIA-1) is a DNA/RNA-binding protein that regulates critical events in cell physiology by the regulation of pre-mRNA splicing and mRNA translation. TIA-1 is composed of three RNA recognition motifs (RRMs) and a glutamine-rich domain and binds to uridine-rich RNA sequences through its C-terminal RRM2 and RRM3 domains. Here, we show that RNA binding mediated by either isolated RRM3 or the RRM23 construct is controlled by slight environmental pH changes due to the protonation/deprotonation of TIA-1 RRM3 histidine residues. The auxiliary role of the C-terminal RRM3 domain in TIA-1 RNA recognition is poorly understood, and this work provides insight into its binding mechanisms.

The binding of TIA-1 to RNA C-rich sequences is driven by its C-terminal RRM domain.

T-cell intracellular antigen-1 (TIA-1) is a key DNA/RNA binding protein that regulates translation by sequestering target mRNAs in stress granules (SG) in response to stress conditions. TIA-1 possesses three RNA recognition motifs (RRM) along with a glutamine-rich domain, with the central domains (RRM2 and RRM3) acting as RNA binding platforms. While the RRM2 domain, which displays high affinity for U-rich RNA sequences, is primarily responsible for interaction with RNA, the contribution of RRM3 to bind RNA as well as the target RNA sequences that it binds preferentially are still unknown. Here we combined nuclear magnetic resonance (NMR) and surface plasmon resonance (SPR) techniques to elucidate the sequence specificity of TIA-1 RRM3. With a novel approach using saturation transfer difference NMR (STD-NMR) to quantify protein-nucleic acids interactions, we demonstrate that isolated RRM3 binds to both C- and U-rich stretches with micromolar affinity. In combination with RRM2 and in the context of full-length TIA-1, RRM3 significantly enhanced the binding to RNA, particularly to cytosine-rich RNA oligos, as assessed by biotinylated RNA pull-down analysis. Our findings provide new insight into the role of RRM3 in regulating TIA-1 binding to C-rich stretches, that are abundant at the 5' TOPs (5' terminal oligopyrimidine tracts) of mRNAs whose translation is repressed under stress situations.

Induced Formation of Plasma Membrane Protrusions with Porous Materials as Instructive Surfaces.

The plasma membrane is a vital component in cellular processes, and its structure has a significant impact on cellular behavior. The physical characteristics of the extracellular environment, along with the presence of surface pores, can influence the formation of membrane protrusions. Nanoporous surfaces have demonstrated their capacity to induce membrane protrusions in both adherent and non-adherent cells. This chapter presents a methodology that utilizes a nanoporous substrate with nanotopographical constraints to effectively stimulate the formation of membrane protrusions in cells.

Hydroxyapatite particle shape affects screw attachment in cancellous bone when augmented with hydroxyapatite-containing hydrogels.

Screw-bone construct failures are a true challenge in orthopaedic implant fixation, particularly in poor quality bone. Whilst augmentation with bone cement can improve the primary stability of screws, there are cements, e.g. PMMA, that may impede blood flow and nutrients and hamper bone remodelling. In this study, soft, non-setting biomaterials based on Hyalectin gels and hydroxyapatite (HA) particles with different morphological parameters were evaluated as potential augmentation materials, using a lapine ex vivo bone model. The pull-out force, stiffness, and work to fracture were considered in evaluating screw attachment. The pull-out force of constructs reinforced with Hyalectin containing irregularly shaped nano-HA and spherically shaped micro-HA particles were found to be significantly higher than the control group (no augmentation material). The pull-out stiffness increased for the micro-HA particles and the work to fracture increased for the irregular nano-HA particles. However, there were no significant augmentation effect found for the spherical shaped nano-HA particles. In conclusion, injectable Hyalectin gel loaded with hydroxyapatite particles was found to have a potentially positive effect on the primary stability of screws in trabecular bone, depending on the HA particle shape and size.

A convolutional neural network-based method for the generation of super-resolution 3D models from clinical CT images.

BACKGROUND AND OBJECTIVE: The accurate evaluation of bone mechanical properties is essential for predicting fracture risk based on clinical computed tomography (CT) images. However, blurring and noise in clinical CT images can compromise the accuracy of these predictions, leading to incorrect diagnoses. Although previous studies have explored enhancing trabecular bone CT images to super-resolution (SR), none of these studies have examined the possibility of using clinical CT images from different instruments, typically of lower resolution, as a basis for analysis. Additionally, previous studies rely on 2D SR images, which may not be sufficient for accurate mechanical property evaluation, due to the complex nature of the 3D trabecular bone structures. The objective of this study was to address these limitations. METHODS: A workflow was developed that utilizes convolutional neural networks to generate SR 3D models across different clinical CT instruments. The morphological and finite-element-derived mechanical properties of these SR models were compared with ground truth models obtained from micro-CT scans. RESULTS: A significant improvement in analysis accuracy was demonstrated, where the new SR models increased the accuracy by up to 700 % compared with the low-resolution data, i.e. clinical CT images. Additionally, we found that the mixture of different CT image datasets may improve the SR model performance. CONCLUSIONS: SR images, generated by convolutional neural networks, outperformed clinical CT images in the determination of morphological and mechanical properties. The developed workflow could be implemented for fracture risk prediction, potentially leading to improved diagnoses and subsequent clinical decision making.

Ready-to-use abobotulinumtoxinA solution versus powder botulinumtoxinA for treatment of glabellar lines: Investigators' and subjects' experience in a Phase IV study.

BACKGROUND: Botulinum neurotoxin type A (BoNT-A) is well-established for treatment of glabellar lines (GLs), and mostly formulated as powders requiring reconstitution for injection. The approved liquid formulation, ready-to-use (RTU) abobotulinumtoxinA was developed to ease injection procedures and prevent reconstitution errors. This multicenter, open-label, Phase IV study evaluated GL treatment experience using RTU abobotulinumtoxinA versus powder BoNT-A (onabotulinumtoxinA). METHODS: Females with experience of BoNT-A facial treatment were randomized 2:1 to GL treatment with 50 U RTU abobotulinumtoxinA (N = 99) or 20 U powder BoNT-A (N = 51) and followed-up for 6 months or 1 month, respectively. Assessments included: time to prepare each product for injection (primary endpoint); investigators' experience with product preparation/reconstitution; investigators' and subjects' treatment experience; safety; and for the RTU product: aesthetic improvement of GLs; subject satisfaction. RESULTS: Compared with powder BoNT-A, RTU abobotulinumtoxinA required statistically significantly less preparation time (mean 0:33 vs. 1:34 min: s; p < 0.0001). Investigators preferred RTU abobotulinumtoxinA over powder BoNT-A (81% of treatment sessions) and found it allowed more time to communicate with subjects (97%). All investigators (100%) also found it easy-to-use, easy-to-learn, and that it fulfilled their expectations. Subjects found the RTU abobotulinumtoxinA treatment comfortable (91%), and through 6 months posttreatment, most reported satisfaction with their appearance (≥88%), looking natural (≥95%) and refreshed (≥80%). At Month 1, 99% of RTU-treated subjects had investigator-assessed improved aesthetic appearance in GLs, maintained in 76% at Month 6. No serious adverse events occurred. CONCLUSION: RTU abobotulinumtoxinA for GL treatment is well-tolerated, efficacious, shows high levels of subject satisfaction throughout 6 months, saves time, and is preferred by clinicians over powder BoNT-A. GOV REGISTRY: NCT05277337.

Fabrication and characterization of sodium alginate-silicon nitride-PVA composite biomaterials with damping properties.

Silicon nitride is utilized clinically as a bioceramic for spinal fusion cages, owing to its high strength, osteoconductivity, and antibacterial effects. Nevertheless, silicon nitride exhibits suboptimal damping properties, a critical factor in mitigating traumatic bone injuries and fractures. In fact, there is a scarcity of spinal implants that simultaneously demonstrate proficient damping performance and support osteogenesis. In our study, we fabricated a novel sodium alginate-silicon nitride/poly(vinyl alcohol) (SA-SiN/PVA) composite scaffold, enabling enhanced energy absorption and rapid elastic recovery under quasi-static and impact loading scenarios. Furthermore, the study demonstrated that the incorporation of physical and chemical cross-linking significantly improved stiffness and recoverable energy dissipation. Concerning the interaction between cells and materials, our findings suggest that the addition of silicon nitride stimulated osteogenic differentiation while inhibiting Staphylococcus aureus growth. Collectively, the amalgamation of ceramics and tough hydrogels facilitates the development of advanced composites for spinal implants, manifesting superior damping, osteogenic potential, and antibacterial properties. This approach holds broader implications for applications in bone tissue engineering.

Initial DNA interactions of the binuclear threading intercalator Λ,Λ-[µ-bidppz(bipy)4Ru2]4+: an NMR study with [d(CGCGAATTCGCG)]2.

Binuclear polypyridine ruthenium compounds have been shown to slowly intercalate into DNA, following a fast initial binding on the DNA surface. For these compounds, intercalation requires threading of a bulky substituent, containing one Ru(II), through the DNA base-pair stack, and the accompanying DNA duplex distortions are much more severe than with intercalation of mononuclear compounds. Structural understanding of the process of intercalation may greatly gain from a characterisation of the initial interactions between binuclear Ru(II) compounds and DNA. We report a structural NMR study on the binuclear Ru(II) intercalator Λ,Λ-B (Λ,Λ-[µ-bidppz(bipy)4Ru2](4+); bidppz = 11,11'-bis(dipyrido[3,2-a:2',3'-c]phenazinyl, bipy = 2,2'-bipyridine) mixed with the palindromic DNA [d(CGCGAATTCGCG)]2. Threading of Λ,Λ-B depends on the presence and length of AT stretches in the DNA. Therefore, the latter was selected to promote initial binding, but due to the short stretch of AT base pairs, final intercalation is prevented. Structural calculations provide a model for the interaction: Λ,Λ-B is trapped in a well-defined surface-bound state consisting of an eccentric minor-groove binding. Most of the interaction enthalpy originates from electrostatic and van der Waals contacts, whereas intermolecular hydrogen bonds may help to define a unique position of Λ,Λ-B. Molecular dynamics simulations show that this minor-groove binding mode is stable on a nanosecond scale. To the best of our knowledge, this is the first structural study by NMR spectroscopy on a binuclear Ru compound bound to DNA. In the calculated structure, one of the positively charged Ru(2+) moieties is near the central AATT region; this is favourable in view of potential intercalation as observed by optical methods for DNA with longer AT stretches. Circular dichroism (CD) spectroscopy suggests that a similar binding geometry is formed in mixtures of Λ,Λ-B with natural calf thymus DNA. The present minor-groove binding mode is proposed to represent the initial surface interactions of binuclear Ru(II) compounds prior to intercalation into AT-rich DNA.

Sequestration of the Abeta peptide prevents toxicity and promotes degradation in vivo.

Protein aggregation, arising from the failure of the cell to regulate the synthesis or degradation of aggregation-prone proteins, underlies many neurodegenerative disorders. However, the balance between the synthesis, clearance, and assembly of misfolded proteins into neurotoxic aggregates remains poorly understood. Here we study the effects of modulating this balance for the amyloid-beta (Abeta) peptide by using a small engineered binding protein (Z(Abeta3)) that binds with nanomolar affinity to Abeta, completely sequestering the aggregation-prone regions of the peptide and preventing its aggregation. Co-expression of Z(Abeta3) in the brains of Drosophila melanogaster expressing either Abeta(42) or the aggressive familial associated E22G variant of Abeta(42) abolishes their neurotoxic effects. Biochemical analysis indicates that monomer Abeta binding results in degradation of the peptide in vivo. Complementary biophysical studies emphasize the dynamic nature of Abeta aggregation and reveal that Z(Abeta3) not only inhibits the initial association of Abeta monomers into oligomers or fibrils, but also dissociates pre-formed oligomeric aggregates and, although very slowly, amyloid fibrils. Toxic effects of peptide aggregation in vivo can therefore be eliminated by sequestration of hydrophobic regions in monomeric peptides, even when these are extremely aggregation prone. Our studies also underline how a combination of in vivo and in vitro experiments provide mechanistic insight with regard to the relationship between protein aggregation and clearance and show that engineered binding proteins may provide powerful tools with which to address the physiological and pathological consequences of protein aggregation.