Elucidating lipid conformations in the ripple phase: Machine learning reveals four lipid populations.

A new mixed radial-angular, three-particle correlation function method in combination with unsupervised machine learning was applied to examine the emergence of the ripple phase in dipalmitoylphosphatidylcholine (DPPC) lipid bilayers using data from atomistic molecular dynamics simulations of system sizes ranging from 128 to 4096 lipids. Based on the acyl tail conformations, the analysis revealed the presence of four distinct conformational populations of lipids in the ripple phases of the DPPC lipid bilayers. The expected gel-like (ordered; Lo) and fluid-like (disordered; Ld) lipids are found along with their splayed tail equivalents (Lo,s and Ld,s). These lipids differ, based on their gauche distribution and tail packing. The disordered (Ld) and disordered-splayed (Ld,s) lipids spatially cluster in the ripple in the groove side, that is, in an asymmetric manner across the bilayer leaflets. The ripple phase does not contain large numbers of Ld lipids; instead they only exist on the interface of the groove side of the undulation. The bulk of the groove side is a complex coexistence of Lo,Lo,s, and Ld,s lipids. The convex side of the undulation contains predominantly Lo lipids. Thus, the structure of the ripple phase is neither a simple coexistence of ordered and disordered lipids nor a coexistence of ordered interdigitating gel-like (Lo) and ordered-splayed (Lo,s) lipids, but instead a coexistence of an ordered phase and a complex mixed phase. Principal component analysis further confirmed the existence of the four lipid groups.

The streptococcal multidomain fibrillar adhesin CshA has an elongated polymeric architecture.

The cell surfaces of many bacteria carry filamentous polypeptides termed adhesins that enable binding to both biotic and abiotic surfaces. Surface adherence is facilitated by the exquisite selectivity of the adhesins for their cognate ligands or receptors and is a key step in niche or host colonization and pathogenicity. Streptococcus gordonii is a primary colonizer of the human oral cavity and an opportunistic pathogen, as well as a leading cause of infective endocarditis in humans. The fibrillar adhesin CshA is an important determinant of S. gordonii adherence, forming peritrichous fibrils on its surface that bind host cells and other microorganisms. CshA possesses a distinctive multidomain architecture comprising an N-terminal target-binding region fused to 17 repeat domains (RDs) that are each ∼100 amino acids long. Here, using structural and biophysical methods, we demonstrate that the intact CshA repeat region (CshA_RD1-17, domains 1-17) forms an extended polymeric monomer in solution. We recombinantly produced a subset of CshA RDs and found that they differ in stability and unfolding behavior. The NMR structure of CshA_RD13 revealed a hitherto unreported all ß-fold, flanked by disordered interdomain linkers. These findings, in tandem with complementary hydrodynamic studies of CshA_RD1-17, indicate that this polypeptide possesses a highly unusual dynamic transitory structure characterized by alternating regions of order and disorder. This architecture provides flexibility for the adhesive tip of the CshA fibril to maintain bacterial attachment that withstands shear forces within the human host. It may also help mitigate deleterious folding events between neighboring RDs that share significant structural identity without compromising mechanical stability.

Hyaluronan in cancer - from the naked mole rat to nanoparticle therapy.

Hyaluronan, a glycosaminoglycan, abundant in the tumour microenvironment, is a key player in many processes associated with cancer. Recently the cancer resistance of the naked mole rat has been attributed to the presence of an ultra-high molecular weight form of this molecule. The physical properties of this multifunctional biopolymer have been extensively studied in the context of synovial joints. However, relatively little has been reported with regard to the soft matter properties of hyaluronan in relation to cancer. In this review we examine the role of hyaluronan in cancer, paying particular attention to its mechanical interactions with malignant cells and its soft matter properties. In addition we discuss the use of hyaluronan based gels to study cancer invasion as well as nanoparticle based strategies for disease treatment.

In vitro assembly of a viral envelope.

Viruses such as influenza and Ebola are enveloped in lipid bilayers annexed from host cells and containing glycoproteins essential for the infection process. At the molecular level little is known about the assembly process in terms of physical interactions between the lipids and glycoproteins. In this paper we assemble HIV glycoproteins in lipid vesicles in order to examine envelope assembly, a process that is usually only executed under control of a host cell. Using atomic force microscopy it was possible to observe fusion of individual envelope like particles, and contrast this with the behaviour of lipid vesicles without envelope glycoproteins. It was found that the inclusion of glycoproteins caused the vesicles to distort and that the subsequent fusion "footprint" with a lipid bilayer was related to the envelopes' unique morphology. This non-spherical morphology suggests that the presence of a viral capsid may be essential for the stability of an enveloped virus. Interactions between trans-membrane gp41 and gp120, the spikes protruding from a virion, were examined using supported lipid bilayers. Interactions between the gp120 and membrane-located gp41 resulted in the assembly of unusual molecular wires, one molecule in height and with a zigzag arrangement of gp120 molecules. In this work we have shown that purely physical/chemical interactions have dramatic effects on glycoprotein/lipid assembly and should be considered in the development of virus based technologies such as virosomes.

Lipid directed assembly of the HIV capsid protein.

Experimental evidence for in vivo capsid assembly suggests that capsid formation initiates from interactions between capsid (CA) proteins and lipids in the viral envelope. Various in vitro studies aiming to elucidate the detailed mechanisms of capsid self-assembly products have been carried out in conditions far removed from those, which would be encountered in a physiological environment. In this work we used lipid bilayers as a platform for studying the assembly of the CA protein with the rationale that the lipid-CA interactions play an important role in the nucleation of these structures. Observations using atomic force microscopy (AFM) have allowed a 'curling tadpole' mechanism to be suggested for the capsid self-assembly process. Stable dimeric CA proteins are able to move across the lipid bilayer to associate into trimers-of-dimers. These trimers form distinctly curved chains, which coil up to form larger features. As the feature grows additional trimers associate with the feature, giving a tadpole-like appearance. By comparing capsid assembly on mica, on single component lipid bilayers, and phase separated lipid bilayers, it was possible to determine the effect of lipid-protein interactions on capsid assembly.

Occupational therapy in pulmonary rehabilitation programs: A scoping review.

Pulmonary rehabilitation (PR) is a multidisciplinary intervention forming the cornerstone of chronic respiratory disease management, improving individuals' exercise capacities and abilities to complete activities of daily living (ADLs). Although the occupational therapy (OT) scope of practice focuses on similar outcomes as PR, the tasks/roles and benefit of including OT in PR has not been reviewed. This scoping review synthesized the i) tasks/roles, ii) recommendations of guidelines, iii) prevalence and iv) effects of OT as part of PR programs. Searching of four databases (MEDLINE, EMBASE, CINAHL and Cochrane), OT association websites, and hand searching was performed, and 51 records were included. The OT tasks/roles most reported include teaching energy conservation techniques (n = 23), addressing ADLs (n = 17), and assisting with breathlessness management (n = 10). Using the Canadian Model of Occupational Performance and Engagement these tasks/roles were grouped into person (n = 16 unique tasks/roles), occupation (n = 6 tasks/roles), environment (n = 5 tasks/roles), and other (n = 3 tasks/roles) domains and were endorsed by two available practice guidelines addressing OT in PR programs. From 13 PR surveys across four continents, 17-92% of PR programs included OT. Inclusion of OT in PR resulted in positive effects on ADLs, pulmonary function, dyspnea, quality of life, and mortality. Although an increasing number of PR programs include occupational therapists in their multidisciplinary teams, there is a paucity of original studies and guidelines reporting on the tasks/roles and benefits of OT in PR. Further research is needed to clearly define the tasks/roles of OT in multidisciplinary PR teams and contributions to enhancing patient outcomes.

The naked truth: a comprehensive clarification and classification of current 'myths' in naked mole-rat biology.

The naked mole-rat (Heterocephalus glaber) has fascinated zoologists for at least half a century. It has also generated considerable biomedical interest not only because of its extraordinary longevity, but also because of unusual protective features (e.g. its tolerance of variable oxygen availability), which may be pertinent to several human disease states, including ischemia/reperfusion injury and neurodegeneration. A recent article entitled 'Surprisingly long survival of premature conclusions about naked mole-rat biology' described 28 'myths' which, those authors claimed, are a 'perpetuation of beautiful, but falsified, hypotheses' and impede our understanding of this enigmatic mammal. Here, we re-examine each of these 'myths' based on evidence published in the scientific literature. Following Braude et al., we argue that these 'myths' fall into four main categories: (i) 'myths' that would be better described as oversimplifications, some of which persist solely in the popular press; (ii) 'myths' that are based on incomplete understanding, where more evidence is clearly needed; (iii) 'myths' where the accumulation of evidence over the years has led to a revision in interpretation, but where there is no significant disagreement among scientists currently working in the field; (iv) 'myths' where there is a genuine difference in opinion among active researchers, based on alternative interpretations of the available evidence. The term 'myth' is particularly inappropriate when applied to competing, evidence-based hypotheses, which form part of the normal evolution of scientific knowledge. Here, we provide a comprehensive critical review of naked mole-rat biology and attempt to clarify some of these misconceptions.

The Role of Occupational Therapy in Pulmonary Rehabilitation Programs: Protocol for a Scoping Review.

BACKGROUND: Chronic respiratory diseases are highly prevalent and compromise an individual's ability to perform activities of daily living (ADLs) and participate in meaningful life roles. Pulmonary rehabilitation (PR) is a well-established intervention aimed at restoring an individual's exercise capacity and improving their ability to complete their ADLs. Occupational therapists help individuals engage in meaningful "occupations," improving their health and well-being. Given the concordance in the aims of PR and the occupational therapy (OT) scope of practice, occupational therapists appear to be well suited as key players in PR programs. However, the benefits of adding OT to PR programs have been sparsely reported in the literature and the role of OT in PR has never been synthesized or reported in national and international guidelines. OBJECTIVE: The aim of this review is to explore the role of OT in PR programs, the current guideline recommendations for the inclusion of OT in PR programs, the estimated prevalence of OT in PR programs, and the reported or anticipated effects of OT interventions in PR programs. METHODS: The review will be conducted following the Joanna Briggs Institute (JBI) methodology for scoping reviews. A comprehensive search will be undertaken in the Cochrane Database of Systematic Reviews, EMBASE, MEDLINE, and CINAHL (EBSCO) to identify and retrieve relevant literature published in English, French, or Portuguese. Gray literature on international OT association websites will also be identified, including position statements and guidelines relevant to PR programs. All literature published since the establishment of the effectiveness of PR for chronic respiratory disease in 1994 that explores OT in PR programs for these patients will be included. Search results will be exported to Covidence for title, abstract, and full-text screening by two independent reviewers. Data will be extracted by two independent reviewers using a pilot-tested template including the following: the number of PR programs including OT (specifically from surveys), the purpose of the study, the study design, patient characteristics, respiratory conditions included, PR components, OT role, outcomes, and results. Findings will be presented using a narrative summary, supplemented by figures and/or tables. Key themes will be displayed in an infographic or schematic. RESULTS: The study was initiated in January 2021 and registered with the Open Science Framework (OSF) in February 2021, prior to title and abstract screening. Data collection and analysis and drafting of the manuscript will occur throughout 2021, with expected publication in 2022. CONCLUSIONS: The results of this scoping review will help health care professionals improve patient care by broadening their understanding and awareness of the role of OT in PR programs. This role clarification may help to inform program development and clinical decision making and will serve to optimize the delivery of multidisciplinary care for patients in PR programs, ultimately improving patient outcomes. TRIAL REGISTRATION: OSF Registries ZH63W; https://osf.io/zh63w. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): DERR1-10.2196/30244.

Punch resection alaplasty technique in dogs and cats with stenotic nares: 14 cases.

Punch resection alaplasty is a previously unreported surgical technique for the management of stenotic nares. Nine dogs underwent the procedure in conjunction with soft palate resection and/or laryngeal sacculectomy. Three dogs and two cats were treated with the novel punch resection alaplasty technique alone. Symmetrical resection was achieved, providing excellent cosmesis. Good to excellent results were achieved in all cases, with owners feeling that their pet's overall well being was improved following surgery.

Cholesterol-rich naked mole-rat brain lipid membranes are susceptible to amyloid beta-induced damage in vitro.

Naked mole-rats are extraordinarily long-lived rodents that offer unique opportunities to study the molecular origins of age-related neurodegenerative diseases. Remarkably, they do not accumulate amyloid plaques, even though their brains contain high concentrations of amyloid beta (Aß) peptide from a young age. Therefore, they represent a particularly favourable organism to study the mechanisms of resistance against Aß neurotoxicity. Here we examine the composition, phase behaviour, and Aß interactions of naked mole-rat brain lipids. Relative to mouse, naked mole-rat brain lipids are rich in cholesterol and contain sphingomyelin in lower amounts and of shorter chain lengths. Proteins associated with the metabolism of ceramides, sphingomyelins and sphingosine-1-phosphate receptor 1 were also found to be decreased in naked mole-rat brain lysates. Correspondingly, we find that naked mole-rat brain lipid membranes exhibit a high degree of phase separation, with the liquid ordered phase extending to 80% of the supported lipid bilayer. These observations are consistent with the 'membrane pacemaker' hypothesis of ageing, according to which long-living species have lipid membranes particularly resistant to oxidative damage. We also found that exposure to Aß disrupts naked mole-rat brain lipid membranes significantly, breaking the membrane into pieces while mouse brain derived lipids remain largely intact upon Aß exposure.

The material properties of naked mole-rat hyaluronan.

Hyaluronan (HA) is a key component of the extracellular matrix. Given the fundamental role of HA in the cancer resistance of the naked mole-rat (NMR), we undertook to explore the structural and soft matter properties of this species-specific variant, a necessary step for its development as a biomaterial. We examined HA extracted from NMR brain, lung, and skin, as well as that isolated from the medium of immortalised cells. In common with mouse HA, NMR HA forms a range of assemblies corresponding to a wide distribution of molecular weights. However, unique to the NMR, are highly folded structures, whose characteristic morphology is dependent on the tissue type. Skin HA forms tightly packed assemblies that have spring-like mechanical properties in addition to a strong affinity for water. Brain HA forms three dimensional folded structures similar to the macroscopic appearance of the gyri and sulci of the human brain. Lung HA forms an impenetrable mesh of interwoven folds in a morphology that can only be described as resembling a snowman. Unlike HA that is commercially available, NMR HA readily forms robust gels without the need for chemical cross-linking. NMR HA gels sharply transition from viscoelastic to elastic like properties upon dehydration or repeated loading. In addition, NMR HA can form ordered thin films with an underlying semi-crystalline structure. Given the role of HA in maintaining hydration in the skin it is plausible that the folded structures contribute to both the elasticity and youthfulness of NMR skin. It is also possible that such densely folded materials could present a considerable barrier to cell invasion throughout the tissues, a useful characteristic for a biomaterial.

Evaluation of cellophane banding with and without intraoperative attenuation for treatment of congenital extrahepatic portosystemic shunts in dogs.

OBJECTIVE: To evaluate the effect of intraoperative attenuation of congenital extrahepatic portosystemic shunts (CEPSSs) during cellophane banding procedures in dogs. STUDY DESIGN: Retrospective case series and prospective study. ANIMALS: 18 cases evaluated retrospectively and 14 dogs evaluated prospectively. PROCEDURES: Gradual occlusion of CEPSSs was performed via cellophane banding. Shunts were occluded to a diameter < 3.0 mm during surgery in dogs prospectively enrolled in the partial attenuation group, whereas the shunt was not attenuated during surgery in dogs prospectively enrolled in the no-attenuation group or in dogs that had previously undergone surgery and were retrospectively evaluated. Postprandial serum bile acids (PPSBA) concentrations were measured before surgery and at various time points after surgery. RESULTS: Mean +/- SD PPSBA concentrations were 26.8 +/- 24.5 micromol/L at < 2.25 months after surgery (n = 16 dogs), 22.1 +/- 14.0 micromol/L from 2.25 to 6 months after surgery (12 dogs), and 34.9 +/- 32.5 micromol/L at > 6 months after surgery (22 dogs). In the prospectively enrolled dogs, mean PPSBA concentrations increased over time in dogs in the partial attenuation group, but not in dogs in the no-attenuation group. CONCLUSIONS AND CLINICAL RELEVANCE: Cellophane banding may be used to occlude larger CEPSSs and may decrease the need for intraoperative monitoring of portal vein blood pressure. The technique may facilitate minimally invasive treatment of CEPSSs in dogs. Intraoperative attenuation of CEPSSs to a diameter < 3.0 mm is not necessary and may result in a less favorable outcome.

Role of the Three-Phase Boundary of the Platinum-Support Interface in Catalysis: A Model Catalyst Kinetic Study.

A series of microstructured, supported platinum (Pt) catalyst films (supported on single-crystal yttria-stabilized zirconia) and an appropriate Pt catalyst reference system (supported on single-crystal alumina) were fabricated using pulsed laser deposition and ion-beam etching. The thin films exhibit area-specific lengths of the three-phase boundary (length of three-phase boundary between the Pt, support, and gas phase divided by the superficial area of the sample) that vary over 4 orders of magnitude from 4.5 × 102 to 4.9 × 106 m m-2, equivalent to structural length scales of 0.2 µm to approximately 9000 µm. The catalyst films have been characterized using X-ray diffraction, atomic force microscopy, high-resolution scanning electron microscopy, and catalytic activity tests employing the carbon monoxide oxidation reaction. When Pt is supported on yttria-stabilized zirconia, the reaction rate clearly depends upon the area-specific length of the three-phase boundary, l(tpb). A similar relationship is not observed when Pt is supported on alumina. We suggest that the presence of the three-phase boundary provides an extra channel of oxygen supply to the Pt through diffusion in or on the yttria-stabilized zirconia support coupled with surface diffusion across the Pt.

Gelatin- and starch-based hydrogels. Part A: Hydrogel development, characterization and coating.

The present work aims at constructing the ideal scaffold matrix of which the physico-chemical properties can be altered according to the targeted tissue regeneration application. Ideally, this scaffold should resemble the natural extracellular matrix (ECM) as close as possible both in terms of chemical composition and mechanical properties. Therefore, hydrogel films were developed consisting of methacrylamide-modified gelatin and starch-pentenoate building blocks because the ECM can be considered as a crosslinked hydrogel network consisting of both polysaccharides and structural, signaling and cell-adhesive proteins. For the gelatin hydrogels, three different substitution degrees were evaluated including 31%, 72% and 95%. A substitution degree of 32% was applied for the starch-pentenoate building block. Pure gelatin hydrogels films as well as interpenetrating networks with gelatin and starch were developed. Subsequently, these films were characterized using gel fraction and swelling experiments, high resolution-magic angle spinning (1)H NMR spectroscopy, rheology, infrared mapping and atomic force microscopy. The results indicate that both the mechanical properties and the swelling extent of the developed hydrogel films can be controlled by varying the chemical composition and the degree of substitution of the methacrylamide-modified gelatin applied. The storage moduli of the developed materials ranged between 14 and 63kPa. Phase separation was observed for the IPNs for which separated starch domains could be distinguished located in the surrounding gelatin matrix. Furthermore, we evaluated the affinity of aggrecan for gelatin by atomic force microscopy and radiolabeling experiments. We found that aggrecan can be applied as a bioactive coating for gelatin hydrogels by a straightforward physisorption procedure. Thus, we achieved distinct fine-tuning of the physico-chemical properties of these hydrogels which render them promising candidates for tissue engineering approaches.

An initial investigation of photocurable three-dimensional lactic acid based scaffolds in a critical-sized cranial defect.

Degradable polymer networks formed by the photoinitiated polymerization of multifunctional monomers have great potential as in situ forming materials, especially for bone tissue engineering. In this study, one specific chemistry was analyzed with respect to bone formation in a critical-sized defect model with and without adsorbed osteoinductive growth factors present. The scaffolds degraded in approximately 8 months and possessed an elastic modulus similar to that of trabecular bone. A porous scaffold fabricated with approximately 80% porosity and pore diameters ranging from 45 to 150 mm was implanted in a critical-sized cranial defect in rats. When implanted alone, the scaffolds were filled primarily with fibrous tissue after 9 weeks with only mild inflammation at the defect site. When the scaffolds released osteoinductive growth factors, statistically more bone filled the scaffold. For instance, 65.8+/-9.4% (n=5) of the defects were filled with radiopaque tissue in the osteoinductive releasing scaffolds, whereas only 24.2+/-7.4% (n=5) of the defects were filled in the untreated defects 9 weeks after implantation. These results illustrate not only the benefits of delivering osteoinductive factors when developing synthetic bone grafts, but the potential of these materials for supporting the infiltration and development of bone in large defects.

Creating a bio-hybrid signal transduction pathway: opening a new channel of communication between cells and machines.

Manipulation of signal transduction pathways presents a viable mechanism to interface cells with electronics. In this work, we present a two-step signal transduction pathway involving cellular and electronic transduction elements. In order to circumvent many of the conventional difficulties encountered when harnessing chemical signalling for the purpose of electronics communication, gaseous nitric oxide (NO) was selected as the signalling molecule. By genetic engineering of the nitric oxide synthase protein eNOS and insertion of light-oxygen-voltage (LOV) domains, we have created a photoactive version of the protein. The novel chimeric eNOS was found to be capable of producing NO in response to excitation by visible light. By coupling these mutant cells to a surface modified platinum electrode, it was possible to convert an optical signal into a chemical one, followed by subsequent conversion of the chemical signal into an electrical output.

Protein directed assembly of lipids.

Highly ordered ring-like structures are formed via the directed assembly of lipid domains in supported bilayers, using the extracellular matrix protein fibronectin. The ability of biological molecules to guide nanoscale assembly suggests potential biomimetic approaches to nanoscale structures.

Nanoscale viscoelastic properties of an aligned collagen scaffold.

Localised mechanical properties for aligned collagen scaffolds derived from Type 1 collagen were determined by application of nanoindentation based techniques. It was possible to measure the modulus and hardness with nanometre control over the depth of penetration and quasi-static testing under displacement control yielded average modulus values ranging from 1.71 GPa to 3.31 GPa; a narrower range of values than obtained by other methods. Hardness values of 222 MPa to 256 MPa were recorded and showed little scatter, highlighting the potential of nanoindentation hardness values as a reproducible and accurate measure of soft material properties. Open loop Load-displacement curves for the collagen exhibited the expected shapes for a viscoelastic material and it was thus possible to apply dynamic stiffness measurement at the nano scale. As well as determining the storage modulus (0.71 GPa) and the loss modulus (0.40 GPa) at the sub-micron length and nano depth resolution it was also possible to discriminate between surface and bulk readings allowing surface effects to be discarded if necessary. In addition to being a more accurate indentation method than atomic force microscopy, the localised dynamic mechanical properties of collagen were measured for the first time. These results demonstrate that this nanoindentation technique can serve as a powerful tool for the characterisation of collagen based biomaterials that are used as scaffolds for a variety of engineered tissues, such as artificial skin, skeletal muscle, heart valves and neuroregeneration guides.

Automated robotic harvesting of protein crystals-addressing a critical bottleneck or instrumentation overkill?

One of the critical steps in high throughput crystallography that so far has evaded automation is the actual harvesting of the delicate crystals from the mother liquor in which they are growing. The late-stage operation of harvesting is presently a most risky and loss-intensive procedure, compounded by its tight integration with the critical steps of cryo-protection and cryo-quenching. Recent advances in micromanipulation robotics and micro-fabrication have made it possible to seriously consider automation of protein crystal harvesting. Based on the experience gained during the development of an operator-assisted (and now operator-assisting) universal micromanipulation robot (UMR) prototype, we discuss the challenges ahead for the design of a fully autonomous, integrated system capable of the reliable harvesting of protein microcrystals. Experience from participation in NIH structural genomics projects and feedback from bottleneck workshops indicates that genuine demand exists in the high throughput community as well as in pharmaceutical production pipelines, justifying the effort and resources to develop autonomous harvesting robotics.

Operator-assisted harvesting of protein crystals using a universal micromanipulation robot.

High-throughput crystallography has reached a level of automation where complete computer-assisted robotic crystallization pipelines are capable of cocktail preparation, crystallization plate setup, and inspection and interpretation of results. While mounting of crystal pins, data collection and structure solution are highly automated, crystal harvesting and cryocooling remain formidable challenges towards full automation. To address the final frontier in achieving fully automated high-throughput crystallography, the prototype of an anthropomorphic six-axis universal micromanipulation robot (UMR) has been designed and tested; this UMR is capable of operator-assisted harvesting and cryoquenching of protein crystals as small as 10 microm from a variety of 96-well plates. The UMR is equipped with a versatile tool exchanger providing full operational flexibility. Trypsin crystals harvested and cryoquenched using the UMR have yielded a 1.5 A structure demonstrating the feasibility of robotic protein crystal harvesting.