Hendra virus (HeV) is one of the two prototypical members of the Henipavirus genus of paramyxoviruses, which are designated biosafety level 4 (BSL-4) organisms due to the high mortality rate of Nipah ...virus (NiV) and HeV in humans. Paramyxovirus cell entry is mediated by the fusion protein, F, in response to binding of a host receptor by the attachment protein. During posttranslational processing, the fusion peptide of F is released and, upon receptor-induced triggering, inserts into the host cell membrane. As F undergoes a dramatic refolding from its prefusion to postfusion conformation, the fusion peptide brings the host and viral membranes together, allowing entry of the viral RNA. Here, we present the crystal structure of the prefusion form of the HeV F ectodomain. The structure shows very high similarity to the structure of prefusion parainfluenza virus 5 (PIV5) F, with the main structural differences in the membrane distal apical loops and the fusion peptide cleavage loop. Functional assays of mutants show that the apical loop can tolerate perturbation in length and surface residues without loss of function, except for residues involved in the stability and conservation of the F protein fold. Structure-based disulfide mutants were designed to anchor the fusion peptide to conformationally invariant residues of the F head. Two mutants were identified that inhibit F-mediated fusion by stabilizing F in its prefusion conformation.
Summary
The tra operon of the prototypical F plasmid and its relatives enables transfer of a copy of the plasmid to other bacterial cells via the process of conjugation. Tra proteins assemble to form ...the transferosome, the transmembrane pore through which the DNA is transferred, and the relaxosome, a complex of DNA‐binding proteins at the origin of DNA transfer. F‐like plasmid conjugation is characterized by a high degree of plasmid specificity in the interactions of tra components, and is tightly regulated at the transcriptional, translational and post‐translational levels. Over the past decade, X‐ray crystallography of conjugative components has yielded insights into both specificity and regulatory mechanisms. Conjugation is repressed by FinO, an RNA chaperone which increases the lifetime of the small RNA, FinP. Recent work has resulted in a detailed model of FinO/FinP interactions and the discovery of a family of FinO‐like RNA chaperones. Relaxosome components include TraI, a relaxase/helicase, and TraM, which mediates signalling between the transferosome and relaxosome for transfer initiation. The structures of TraI and TraM bound to oriT DNA reveal the basis of specific recognition of DNA for their cognate plasmid. Specificity also exists in TraI and TraM interactions with the transferosome protein TraD.
Mechanical compliance has been demonstrated to be a key determinant of cell behavior, directing processes such as spreading, migration, and differentiation. Durotaxis, directional migration from ...softer to more stiff regions of a substrate, has been observed for a variety of cell types. Recent stiffness mapping experiments have shown that local changes in tissue stiffness in disease are often accompanied by an altered ECM composition in vivo. However, the importance of ECM composition in durotaxis has not yet been explored. To address this question, we have developed and characterized a polyacrylamide hydrogel culture platform featuring highly tunable gradients in mechanical stiffness. This feature, together with the ability to control ECM composition, allows us to isolate the effects of mechanical and biological signals on cell migratory behavior. Using this system, we have tracked vascular smooth muscle cell migration in vitro and quantitatively analyzed differences in cell migration as a function of ECM composition. Our results show that vascular smooth muscle cells undergo durotaxis on mechanical gradients coated with fibronectin but not on those coated with laminin. These findings indicate that the composition of the adhesion ligand is a critical determinant of a cell’s migratory response to mechanical gradients.
Spider silks have been a focus of research for almost two decades due to their outstanding mechanical and biophysical properties. Recent advances in genetic engineering have led to the synthesis of ...recombinant spider silks, thus helping to unravel a fundamental understanding of structure–function–property relationships. The relationships between molecular composition, secondary structures and mechanical properties found in different types of spider silks are described, along with a discussion of artificial spinning of these proteins and their bioapplications, including the role of silks in biomineralization and fabrication of biomaterials with controlled properties.
To successfully develop a functional tissue-engineered vascular patch, recapitulating the hierarchical structure of vessel is critical to mimic mechanical properties. Here, we use a cell sheet ...engineering strategy with micropatterning technique to control structural organization of bovine aortic vascular smooth muscle cell (VSMC) sheets. Actin filament staining and image analysis showed clear cellular alignment of VSMC sheets cultured on patterned substrates. Viability of harvested VSMC sheets was confirmed by Live/Dead® cell viability assay after 24 and 48 h of transfer. VSMC sheets stacked to generate bilayer VSMC patches exhibited strong inter-layer bonding as shown by lap shear test. Uniaxial tensile testing of monolayer VSMC sheets and bilayer VSMC patches displayed nonlinear, anisotropic stress-stretch response similar to the biomechanical characteristic of a native arterial wall. Collagen content and structure were characterized to determine the effects of patterning and stacking on extracellular matrix of VSMC sheets. Using finite-element modeling to simulate uniaxial tensile testing of bilayer VSMC patches, we found the stress-stretch response of bilayer patterned VSMC patches under uniaxial tension to be predicted using an anisotropic hyperelastic constitutive model. Thus, our cell sheet harvesting system combined with biomechanical modeling is a promising approach to generate building blocks for tissue-engineered vascular patches with structure and mechanical behavior mimicking native tissue.
Type 1 and type 2 diabetes mellitus is a serious and lifelong condition commonly characterised by abnormally elevated blood glucose levels due to a failure in insulin production or a decrease in ...insulin sensitivity and function. Over the years, prevalence of diabetes has increased globally and it is classified as one of the leading cause of high mortality and morbidity rate. Furthermore, diabetes confers a huge economic burden due to its management costs as well as its complications are skyrocketing. The conventional medications in diabetes treatment focusing on insulin secretion and insulin sensitisation cause unwanted side effects to patients and lead to incompliance as well as treatment failure. Besides insulin and oral hypoglycaemic agents, other treatments such as gene therapy and induced β-cells regeneration have not been widely introduced to manage diabetes. Therefore, this review aims to deliver an overview of the current conventional medications in diabetes, discovery of newer pharmacological drugs and gene therapy as a potential intervention of diabetes in the future.
Epigenetics regulation plays a critical role in determining cell identity by controlling the accessibility of lineage-specific regulatory regions. In muscle stem cells, epigenetic mechanisms of how ...chromatin accessibility is modulated during cell fate determination are not fully understood. Here, we identified a long noncoding RNA, LncMyoD, that functions as a chromatin modulator for myogenic lineage determination and progression. The depletion of LncMyoD in muscle stem cells led to the down-regulation of myogenic genes and defects in myogenic differentiation. LncMyoD exclusively binds with MyoD and not with other myogenic regulatory factors and promotes transactivation of target genes. The mechanistic study revealed that loss of LncMyoD prevents the establishment of a permissive chromatin environment at myogenic E-box–containing regions, therefore restricting the binding of MyoD. Furthermore, the depletion of LncMyoD strongly impairs the reprogramming of fibroblasts into the myogenic lineage. Taken together, our study shows that LncMyoD associates with MyoD and promotes myogenic gene expression through modulating MyoD accessibility to chromatin, thereby regulating myogenic lineage determination and progression.
Despite their central role in peanut allergy, human monoclonal IgE antibodies have eluded characterization.
We sought to define the sequences, affinities, clonality, and functional properties of ...human monoclonal IgE antibodies in peanut allergy.
We applied our single-cell RNA sequencing–based SEQ SIFTER discovery platform to samples from allergic individuals who varied by age, sex, ethnicity, and geographic location in order to understand commonalities in the human IgE response to peanut allergens. Select antibodies were then recombinantly expressed and characterized for their allergen and epitope specificity, affinity, and functional properties.
We found striking convergent evolution of IgE monoclonal antibodies (mAbs) from several clonal families comprising both memory B cells and plasmablasts. These antibodies bound with subnanomolar affinity to the immunodominant peanut allergen Ara h 2, specifically a linear, repetitive motif. Further characterization of these mAbs revealed their ability to single-handedly cause affinity-dependent degranulation of human mast cells and systemic anaphylaxis on peanut allergen challenge in humanized mice. Finally, we demonstrated that these mAbs, reengineered as IgGs, inhibit significant, but variable, amounts of Ara h 2– and peanut-mediated degranulation of mast cells sensitized with allergic plasma.
Convergent evolution of IgE mAbs in peanut allergy is a common phenomenon that can reveal immunodominant epitopes on major allergenic proteins. Understanding the functional properties of these molecules is key to developing therapeutics, such as competitive IgG inhibitors, that are able to stoichiometrically outcompete endogenous IgE for allergen and thereby prevent allergic cascade in cases of accidental allergen exposure.
Mechanical compliance is emerging as an important environmental cue that can influence certain cell behaviors, such as morphology and motility. Recent in vitro studies have shown that cells ...preferentially migrate from less stiff to more stiff substrates; however, much of this phenomenon, termed durotaxis, remains ill-defined. To address this problem, we studied the morphology and motility of vascular smooth muscle cells on well-defined stiffness gradients. Baselines for cell spreading, polarization, and random motility on uniform gels with moduli ranging from 5 to 80 kPa were found to increase with increasing stiffness. Subsequent analysis of the behavior of vascular smooth muscle cells on gradient substrata (0–4 kPa/100
μm, with absolute moduli of 1–80 kPa) demonstrated that the morphology on gradient gels correlated with the absolute modulus. In contrast, durotaxis (evaluated quantitatively as the tactic index for a biased persistent random walk) and cell orientation with respect to the gradient both increased with increasing magnitude of gradient, but were independent of the absolute modulus. These observations provide a foundation for establishing quantitative relationships between gradients in substrate stiffness and cell response. Moreover, these results reveal common features of phenomenological cell response to chemotactic and durotactic gradients, motivating further mechanistic studies of how cells integrate and respond to multiple complex signals.
Time Off Work in Hand Injury Patients Wong, Joyce Y.P., MMedSc
The Journal of hand surgery (American ed.),
05/2008, Letnik:
33, Številka:
5
Journal Article
Recenzirano
Purpose To evaluate the average time off work for patients with hand injuries as well as factors influencing the length of time off work for subjects who had returned to work. Methods A total of 124 ...subjects were recruited and followed up 2 weeks after being discharged from therapy or until they resumed work. Factors affecting the time off work such as the severity of injury, number of operations, educational level, physical job demand, and presence of compensation claims, complications, and posttraumatic stress were also examined by multiple regression. The relationship between the severity of injury, time off work, and length of rehabilitation time was also examined. Results In 124 subjects, 109 resumed their previous jobs, with an average of 8 weeks off work and 9 weeks in regular rehabilitation. The result shows that the severity of injury, number of operations, and presence of compensation claims were the predictors for the length of time off work in hand injury patients. In addition, the time off work and length of rehabilitation time increased with more severe injury. However, a cross-over was noted in the severe grade of severity of injury; that is, the time off work was shorter than the length of rehabilitation time for those with a less severe level of injury (minor to moderate level) and vice versa for those with major severity of injury. Conclusions To a certain extent, the result enhances our understanding of prolonged time off work after hand injury and may help to optimize rehabilitation after injury. For those who are in the high-risk group with factors that are not under control, early planning and retraining should be given to avoid prolonged time off work.
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