High prevalence of long-term olfactory disorders in healthcare workers after COVID-19: A case-control study.

BACKGROUND: More than a year after recovering from COVID-19, a large proportion of individuals, many of whom work in the healthcare sector, still report olfactory dysfunctions. However, olfactory dysfunction was common already before the COVID-19 pandemic, making it necessary to also consider the existing baseline prevalence of olfactory dysfunction. To establish the adjusted prevalence of COVID-19 related olfactory dysfunction, we assessed smell function in healthcare workers who had contracted COVID-19 during the first wave of the pandemic using psychophysical testing. METHODS: Participants were continuously tested for SARS-CoV-2 IgG antibodies since the beginning of the pandemic. To assess the baseline rate of olfactory dysfunction in the population and to control for the possibility of skewed recruitment of individuals with prior olfactory dysfunction, consistent SARS-CoV-2 IgG naïve individuals were tested as a control group. RESULTS: Fifteen months after contracting COVID-19, 37% of healthcare workers demonstrated a quantitative reduction in their sense of smell, compared to only 20% of the individuals in the control group. Fifty-one percent of COVID-19-recovered individuals reported qualitative symptoms, compared to only 5% in the control group. In a follow-up study 2.6 years after COVID-19 diagnosis, 24% of all tested recovered individuals still experienced parosmia. CONCLUSIONS: In summary, 65% of healthcare workers experienced parosmia/hyposmia 15 months after contracting COVID-19. When compared to a control group, the prevalence of olfactory dysfunction in the population increased by 41 percentage points. Parosmia symptoms were still lingering two-and-a half years later in 24% of SARS-CoV-2 infected individuals. Given the amount of time between infection and testing, it is possible that the olfactory problems may not be fully reversible in a plurality of individuals.

Accessing active fragments for drug discovery utilising nitroreductase biocatalysis.

Biocatalysis has played a limited role in the early stages of drug discovery. This is often attributed to the limited substrate scope of enzymes not affording access to vast areas of novel chemical space. Here, we have shown a promiscuous nitroreductase enzyme (NR-55) can be used to produce a panel of functionalised anilines from a diverse panel of aryl nitro starting materials. After screening on analytical scale, we show that sixteen substrates could be scaled to 1 mmol scale, with several poly-functional anilines afforded with ease under the standard conditions. The aniline products were also screened for activity against several cell lines of interest, with modest activity observed for one compound. This study demonstrates the potential for nitroreductase biocatalysis to provide access to functional fragments under benign conditions.

Effect of cardiolipin on the lamellarity and elongation of liposomes hydrated in PBS.

Lamellarity and shape are important factors in the formation of vesicles and determine their role in biological systems and pharmaceutical applications. Cardiolipin (CL) is a major lipid in many biological membranes and exerts a great influence on their structural organization due to its particular structure and physico-chemical properties. Here, we used small-angle X-ray and neutron scattering to study the effects of CL with different acyl chain lengths and saturations (CL14:0, CL18:1, CL18:2) on vesicle morphology and lamellarity in membrane models containing mixtures of phosphatidylcholine and phosphatidylethanolamine with different acyl chain lengths and saturations (C14:0 and C 18:1). Measurements were performed in the presence of Phosphate Buffer Saline (PBS), at 37°C, to better reflect physiological conditions, which resulted in strong effects on vesicle morphology, depending on the type and amount of CL used. The presence of small quantities of CL (from 2.5%) reduced inter-membrane correlations and increased perturbation of the membrane, an effect which is enhanced in the presence of matched shorter saturated acyl chains, and mainly unilamellar vesicles (ULV) are formed. In extruded vesicles, employed for SANS experiments, flattened vesicles are observed partly due to the hypertonic effect of PBS, but also influenced by the type of CL added. Our experimental data from SAXS and SANS revealed a strong dependence on CL content in shaping the membrane microstructure, with an apparent optimum in the PC:CL mixture in terms of promoting reduced correlations, preferred curvature and elongation. However, the use of PBS caused distinct differences from previously published studies in water in terms of vesicle shape, and highlights the need to investigate vesicle formation under physiological conditions in order to be able to draw conclusions about membrane formation in biological systems.

Galactokinase-like protein from Leishmania donovani: Biochemical and structural characterization of a recombinant protein.

Leishmaniasis is a spectrum of conditions caused by infection with the protozoan Leishmania spp. parasites. Leishmaniasis is endemic in 98 countries around the world, and resistance to current anti-leishmanial drugs is rising. Our work has identified and characterised a previously unstudied galactokinase-like protein (GalK) in Leishmania donovani, which catalyses the MgATP-dependent phosphorylation of the C-1 hydroxyl group of d-galactose to galactose-1-phosphate. Here, we report the production of the catalytically active recombinant protein in E. coli, determination of its substrate specificity and kinetic constants, as well as analysis of its molecular envelope using in solution X-ray scattering. Our results reveal kinetic parameters in range with other galactokinases with an average apparent Km value of 76 µM for galactose, Vmax and apparent Kcat values with 4.46376 × 10-9 M/s and 0.021 s-1, respectively. Substantial substrate promiscuity was observed, with galactose being the preferred substrate, followed by mannose, fructose and GalNAc. LdGalK has a highly flexible protein structure suggestive of multiple conformational states in solution, which may be the key to its substrate promiscuity. Our data presents novel insights into the galactose salvaging pathway in Leishmania and positions this protein as a potential target for the development of pharmaceuticals seeking to interfere with parasite substrate metabolism.

Impairment of quality of life due to COVID-19-induced long-term olfactory dysfunction.

Introduction: Olfactory dysfunction is one of many long-lasting symptoms associated with COVID-19, estimated to affect approximately 60% of individuals and often lasting several months after infection. The associated daily life problems can cause a decreased quality of life. Methods: Here, we assessed the association between perceived quality of life and both qualitative and quantitative olfactory function (distorted and weakened sense of smell, respectively) in 58 individuals who had undergone confirmed SARS-CoV-2 infection and who complained about olfactory dysfunction. Results: Participants with large quantitative olfactory dysfunction experienced a greater reduction in their quality of life. Moreover, our participants had a high prevalence of qualitative olfactory dysfunction (81%) with a significant correlation between qualitative olfactory dysfunction and daily life impairment. Strong drivers of low quality of life assessments were lack of enjoyment of food as well as worries related to coping with long-term dysfunctions. Discussion: These results stress the clinical importance of assessing qualitative olfactory dysfunction and the need to develop relevant interventions. Given the poor self-rated quality of life observed, healthcare systems should consider developing support structures, dietary advice, and guidelines adapted to individuals experiencing qualitative olfactory dysfunction.

Molecular Changes in Dengue Envelope Protein Domain III upon Interaction with Glycosaminoglycans.

Dengue fever is a rapidly emerging vector-borne viral disease with a growing global burden of approximately 390 million new infections per annum. The Dengue virus (DENV) is a flavivirus spread by female mosquitos of the aedes genus, but the mechanism of viral endocytosis is poorly understood at a molecular level, preventing the development of effective transmission blocking vaccines (TBVs). Recently, glycosaminoglycans (GAGs) have been identified as playing a role during initial viral attachment through interaction with the third domain of the viral envelope protein (EDIII). Here, we report a systematic study investigating the effect of a range of biologically relevant GAGs on the structure and oligomeric state of recombinantly generated EDIII. We provide novel in situ biophysical evidence that heparin and chondroitin sulphate C induce conformational changes in EDIII at the secondary structure level. Furthermore, we report the ability of chondroitin sulphate C to bind EDIII and induce higher-order dynamic molecular changes at the tertiary and quaternary structure levels which are dependent on pH, GAG species, and the GAG sulphation state. Lastly, we conducted ab initio modelling of Small Angle Neutron Scattering (SANS) data to visualise the induced oligomeric state of EDIII caused by interaction with chondroitin sulphate C, which may aid in TBV development.

Characterization of Three Druggable Hot-Spots in the Aurora-A/TPX2 Interaction Using Biochemical, Biophysical, and Fragment-Based Approaches.

The mitotic kinase Aurora-A and its partner protein TPX2 (Targeting Protein for Xenopus kinesin-like protein 2) are overexpressed in cancers, and it has been proposed that they work together as an oncogenic holoenzyme. TPX2 is responsible for activating Aurora-A during mitosis, ensuring proper cell division. Disruption of the interface with TPX2 is therefore a potential target for novel anticancer drugs that exploit the increased sensitivity of cancer cells to mitotic stress. Here, we investigate the interface using coprecipitation assays and isothermal titration calorimetry to quantify the energetic contribution of individual residues of TPX2. Residues Tyr8, Tyr10, Phe16, and Trp34 of TPX2 are shown to be crucial for robust complex formation, suggesting that the interaction could be abrogated through blocking any of the three pockets on Aurora-A that complement these residues. Phosphorylation of Aurora-A on Thr288 is also necessary for high-affinity binding, and here we identify arginine residues that communicate the phosphorylation of Thr288 to the TPX2 binding site. With these findings in mind, we conducted a high-throughput X-ray crystallography-based screen of 1255 fragments against Aurora-A and identified 59 hits. Over three-quarters of these hits bound to the pockets described above, both validating our identification of hotspots and demonstrating the druggability of this protein-protein interaction. Our study exemplifies the potential of high-throughput crystallography facilities such as XChem to aid drug discovery. These results will accelerate the development of chemical inhibitors of the Aurora-A/TPX2 interaction.

Characterization and structural determination of a new anti-MET function-blocking antibody with binding epitope distinct from the ligand binding domain.

The growth and motility factor Hepatocyte Growth Factor/Scatter Factor (HGF/SF) and its receptor, the product of the MET proto-oncogene, promote invasion and metastasis of tumor cells and have been considered potential targets for cancer therapy. We generated a new Met-blocking antibody which binds outside the ligand-binding site, and determined the crystal structure of the Fab in complex with its target, which identifies the binding site as the Met Ig1 domain. The antibody, 107_A07, inhibited HGF/SF-induced cell migration and proliferation in vitro and inhibited growth of tumor xenografts in vivo. In biochemical assays, 107_A07 competes with both HGF/SF and its truncated splice variant NK1 for MET binding, despite the location of the antibody epitope on a domain (Ig1) not reported to bind NK1 or HGF/SF. Overlay of the Fab-MET crystal structure with the InternalinB-MET crystal structure shows that the 107_A07 Fab comes into close proximity with the HGF/SF-binding SEMA domain when MET is in the "compact", InternalinB-bound conformation, but not when MET is in the "open" conformation. These findings provide further support for the importance of the "compact" conformation of the MET extracellular domain, and the relevance of this conformation to HGF/SF binding and signaling.

A closed conformation of the Caenorhabditis elegans separase-securin complex.

The protease separase plays a key role in sister chromatid disjunction and centriole disengagement. To maintain genomic stability, separase activity is strictly regulated by binding of an inhibitory protein, securin. Despite its central role in cell division, the separase and securin complex is poorly understood at the structural level. This is partly owing to the difficulty of generating a sufficient quantity of homogeneous, stable protein. Here, we report the production of Caenorhabditis elegans separase-securin complex, and its characterization using biochemical methods and by negative staining electron microscopy. Single particle analysis generated a density map at a resolution of 21-24 Å that reveals a close, globular structure of complex connectivity harbouring two lobes. One lobe matches closely a homology model of the N-terminal HEAT repeat domain of separase, whereas the second lobe readily accommodates homology models of the separase C-terminal death and caspase-like domains. The globular structure of the C. elegans separase-securin complex contrasts with the more elongated structure previously described for the Homo sapiens complex, which could represent a different functional state of the complex, suggesting a mechanism for the regulation of separase activity through conformational change.

Developing Antagonists for the Met-HGF/SF Protein-Protein Interaction Using a Fragment-Based Approach.

In many cancers, aberrant activation of the Met receptor tyrosine kinase leads to dissociation of cells from the primary tumor, causing metastasis. Accordingly, Met is a high-profile target for the development of cancer therapies, and progress has been made through development of small molecule kinase inhibitors and antibodies. However, both approaches pose significant challenges with respect to either target specificity (kinase inhibitors) or the cost involved in treating large patient cohorts (antibodies). Here, we use a fragment-based approach in order to target the protein-protein interaction (PPI) between the α-chain of hepatocyte growth factor/scatter factor (HGF/SF; the NK1 fragment) and its high-affinity binding site located on the Met Sema domain. Surface plasmon resonance was used for initial fragment library screening and hits were developed into larger compounds using substructure (similarity) searches. We identified compounds able to interfere with NK1 binding to Met, disrupt Met signaling, and inhibit tumorsphere generation and cell migration. Using molecular docking, we concluded that some of these compounds inhibit the PPI directly, whereas others act indirectly. Our results indicate that chemical fragments can efficiently target the HGF/SF-Met interface and may be used as building blocks for generating biologically active lead compounds. This strategy may have broad application for the development of a new class of Met inhibitors, namely receptor antagonists, and in general for the development of small molecule PPI inhibitors of key therapeutic targets when structural information is not available.

Structural Insights into Separase Architecture and Substrate Recognition through Computational Modelling of Caspase-Like and Death Domains.

Separases are large proteins that mediate sister chromatid disjunction in all eukaryotes. They belong to clan CD of cysteine peptidases and contain a well-conserved C-terminal catalytic protease domain similar to caspases and gingipains. However, unlike other well-characterized groups of clan CD peptidases, there are no high-resolution structures of separases and the details of their regulation and substrate recognition are poorly understood. Here we undertook an in-depth bioinformatical analysis of separases from different species with respect to their similarity in amino acid sequence and protein fold in comparison to caspases, MALT-1 proteins (mucosa-associated lymphoidtissue lymphoma translocation protein 1) and gingipain-R. A comparative model of the single C-terminal caspase-like domain in separase from C. elegans suggests similar binding modes of substrate peptides between these protein subfamilies, and enables differences in substrate specificity of separase proteins to be rationalised. We also modelled a newly identified putative death domain, located N-terminal to the caspase-like domain. The surface features of this domain identify potential sites of protein-protein interactions. Notably, we identified a novel conserved region with the consensus sequence WWxxRxxLD predicted to be exposed on the surface of the death domain, which we termed the WR motif. We envisage that findings from our study will guide structural and functional studies of this important protein family.

Biophysical and computational fragment-based approaches to targeting protein-protein interactions: applications in structure-guided drug discovery.

Drug discovery has classically targeted the active sites of enzymes or ligand-binding sites of receptors and ion channels. In an attempt to improve selectivity of drug candidates, modulation of protein-protein interfaces (PPIs) of multiprotein complexes that mediate conformation or colocation of components of cell-regulatory pathways has become a focus of interest. However, PPIs in multiprotein systems continue to pose significant challenges, as they are generally large, flat and poor in distinguishing features, making the design of small molecule antagonists a difficult task. Nevertheless, encouragement has come from the recognition that a few amino acids - so-called hotspots - may contribute the majority of interaction-free energy. The challenges posed by protein-protein interactions have led to a wellspring of creative approaches, including proteomimetics, stapled α-helical peptides and a plethora of antibody inspired molecular designs. Here, we review a more generic approach: fragment-based drug discovery. Fragments allow novel areas of chemical space to be explored more efficiently, but the initial hits have low affinity. This means that they will not normally disrupt PPIs, unless they are tethered, an approach that has been pioneered by Wells and co-workers. An alternative fragment-based approach is to stabilise the uncomplexed components of the multiprotein system in solution and employ conventional fragment-based screening. Here, we describe the current knowledge of the structures and properties of protein-protein interactions and the small molecules that can modulate them. We then describe the use of sensitive biophysical methods - nuclear magnetic resonance, X-ray crystallography, surface plasmon resonance, differential scanning fluorimetry or isothermal calorimetry - to screen and validate fragment binding. Fragment hits can subsequently be evolved into larger molecules with higher affinity and potency. These may provide new leads for drug candidates that target protein-protein interactions and have therapeutic value.

Structural biology and drug discovery for protein-protein interactions.

Although targeting protein-protein interfaces of regulatory multiprotein complexes has become a significant focus in drug discovery, it continues to pose major challenges. Most interfaces would be classed as 'undruggable' by conventional analyses, as they tend to be large, flat and featureless. Over the past decade, encouragement has come from the discovery of hotspots that contribute much of the free energy of interaction, and this has led to the development of tethering methods that target small molecules to these sites, often inducing adaptive changes. Equally important has been the recognition that many protein-protein interactions involve a continuous epitope of one partner and a well-defined groove or series of specific small pockets. These observations have stimulated the development of stapled α-helical peptides and other proteomimetic approaches. They have also led to the realisation that fragments might gain low-affinity 'footholds' on some protein-protein interfaces, and that these fragments might be elaborated to useful modulators of the interactions.

Hookworm SCP/TAPS protein structure--A key to understanding host-parasite interactions and developing new interventions.

SCP/TAPS proteins are a diverse family of molecules in eukaryotes, including parasites. Despite their abundant occurrence in parasite secretomes, very little is known about their functions in parasitic nematodes, including blood-feeding hookworms. Current information indicates that SCP/TAPS proteins (called Ancylostoma-secreted proteins, ASPs) of the canine hookworm, Ancylostoma caninum, represent at least three distinct groups of proteins. This information, combined with comparative modelling, indicates that all known ASPs have an equatorial groove that binds extended structures, such as peptides or glycans. To elucidate structure-function relationships, we explored the three-dimensional crystal structure of an ASP (called Ac-ASP-7), which is highly up-regulated in expression in the transition of A. caninum larvae from a free-living to a parasitic stage. The topology of the N-terminal domain is consistent with pathogenesis-related proteins, and the C-terminal extension that resembles the fold of the Hinge domain. By anomalous diffraction, we identified a new metal binding site in the C-terminal extension of the protein. Ac-ASP-7 is in a monomer-dimer equilibrium, and crystal-packing analysis identified a dimeric structure which might resemble the homo-dimer in solution. The dimer interaction interface includes a novel binding site for divalent metal ions, and is proposed to serve as a binding site for proteins involved in the parasite-host interplay at the molecular level. Understanding this interplay and the integration of structural and functional data could lead to the design of new approaches for the control of parasitic diseases, with biotechnological outcomes.

Enhanced ITM2A expression inhibits chondrogenic differentiation of mesenchymal stem cells.

Mesenchymal stem cells (MSC) from bone marrow or adipose tissue (ASC) are broadly discussed as a cell population able to support cartilage regeneration and thus represent interesting candidates for cell-based tissue engineering in cartilage. ASC could represent an easily accessible and therefore particularly suitable source of cells. Their chondrogenic differentiation potential is, however, lower than that of MSC. The aim of this work was to characterise ASC in comparison to MSC in order to identify genes which may be involved in mechanisms causing the altered chondrogenic potential of ASC. Representational difference analysis was used to identify genes with higher expression in undifferentiated ASC than in MSC. Expression levels of identified genes were confirmed by real-time RT-PCR. Integral membrane protein 2A (ITM2A) was higher expressed in expanded ASC than in MSC in a donor-independent manner. During early chondrogenic differentiation in spheroid cultures ITM2A levels remained low in MSC and a transient down-regulation occurred in ASC correlating with successful chondrogenesis. Persisting ITM2A levels were found in non-differentiating ASC. Consistent with this finding, forced expression of ITM2A in the mouse mesenchymal stem cell line C3H10T1/2 prevented chondrogenic induction. In conclusion, ITM2A may in early stages of differentiation be associated with an inhibition of the initiation of chondrogenesis and elevated expression of ITM2A in ASC may therefore be linked to the poorer chondrogenic differentiation potential of these cells.

The crystal structure of calcium-bound annexin Gh1 from Gossypium hirsutum and its implications for membrane binding mechanisms of plant annexins.

Plant annexins show distinct differences in comparison with their animal orthologues. In particular, the endonexin sequence, which is responsible for coordination of calcium ions in type II binding sites, is only partially conserved in plant annexins. The crystal structure of calcium-bound cotton annexin Gh1 was solved at 2.5 A resolution and shows three metal ions coordinated in the first and fourth repeat in types II and III binding sites. Although the protein has no detectable affinity for calcium in solution, in the presence of phospholipid vesicles, we determined a stoichiometry of four calcium ions per protein molecule using isothermal titration calorimetry. Further analysis of the crystal structure showed that binding of a fourth calcium ion is structurally possible in the DE loop of the first repeat. Data from this study are in agreement with the canonical membrane binding of annexins, which is facilitated by the convex surface associating with the phospholipid bilayer by a calcium bridging mechanism. In annexin Gh1, this membrane-binding state is characterized by four calcium bridges in the I/IV module of the protein and by direct interactions of several surface-exposed basic and hydrophobic residues with the phospholipid membrane. Analysis of the protein fold stability revealed that the presence of calcium lowers the thermal stability of plant annexins. Furthermore, an additional unfolding step was detected at lower temperatures, which can be explained by the anchoring of the N-terminal domain to the C-terminal core by two conserved hydrogen bonds.

Natural MHC class I polymorphism controls the pathway of peptide dissociation from HLA-B27 complexes.

Analysis of antigen dissociation provides insight into peptide presentation modes of folded human leukocyte antigen (HLA) molecules, which consist of a heavy chain, beta2-microglobulin (beta2m), and an antigenic peptide. Here we have monitored peptide-HLA interactions and peptide dissociation kinetics of two HLA-B27 subtypes by fluorescence depolarization techniques. A single natural amino-acid substitution distinguishes the HLA-B*2705 subtype that is associated with the autoimmune disease ankylosing spondylitis from the non-disease-associated HLA-B*2709 subtype. Peptides with C-terminal Arg or Lys represent 27% of the natural B*2705 ligands. Our results show that dissociation of a model peptide with a C-terminal Lys (GRFAAAIAK) follows a two-step mechanism. Final peptide release occurs in the second step for both HLA-B27 subtypes. However, thermodynamics and kinetics of peptide-HLA interactions reveal different molecular mechanisms underlying the first step, as indicated by different activation energies of 95+/-8 kJ/mol (HLA-B*2705) and 150+/-10 kJ/mol (HLA-B*2709). In HLA-B*2709, partial peptide dissociation probably precedes fast final peptide release, while in HLA-B*2705 an allosteric mechanism based on long-range interactions between beta2m and the peptide binding groove controls the first step. The resulting peptide presentation mode lasts for days at physiological temperature, and determines the peptide-HLA-B*2705 conformation, which is recognized by cellular ligands such as T-cell receptors.

Molecular modeling of prohibitin domains.

Prohibitins comprise a family of highly conserved ubiquitous eukaryotic proteins that mainly localize to the mitochondria. They have been implicated in important cellular processes such as cellular signaling and transcriptional control, apoptosis, cellular senescence, and mitochondrial biogenesis. Using molecular modeling techniques, we have generated structural models of human prohibitins BAP32 and BAP37, which have previously been shown to exist as large ringlike oligomers in the membrane-bound state. The middle domain of prohibitins is evolutionary conserved in the family of SPFH (PHB) domain proteins. On the basis of the known structure of flotillin-2, another member of the SPFH-domain family, we have generated homology models for BAP32 and BAP37, and elucidated the implications for formation of high molecular weight oligomers. A model for the dimeric-building block of BAP32: BAP37 for such assemblies was generated and its stability scrutinized by molecular dynamics simulations. The model of BAP32 was also analyzed as to potential ligand-binding sites and the previously identified ligand melanogenin was docked into a membrane-proximal cavity. The results are discussed in the context of prohibitin interactions with mitochondrial AAA-proteases and we suggest two possible interaction interfaces between the BAP32:BAP37 building block and the protease.

Premature induction of hypertrophy during in vitro chondrogenesis of human mesenchymal stem cells correlates with calcification and vascular invasion after ectopic transplantation in SCID mice.

OBJECTIVE: Functional suitability and phenotypic stability of ectopic transplants are crucial factors in the clinical application of mesenchymal stem cells (MSCs) for articular cartilage repair, and might require a stringent control of chondrogenic differentiation. This study evaluated whether human bone marrow-derived MSCs adopt natural differentiation stages during induction of chondrogenesis in vitro, and whether they can form ectopic stable cartilage that is resistant to vascular invasion and calcification in vivo. METHODS: During in vitro chondrogenesis of MSCs, the expression of 44 cartilage-, stem cell-, and bone-related genes and the deposition of aggrecan and types II and X collagen were determined. Similarly treated, expanded articular chondrocytes served as controls. MSC pellets were allowed to differentiate in chondrogenic medium for 3-7 weeks, after which the chondrocytes were implanted subcutaneously into SCID mice; after 4 weeks in vivo, samples were evaluated by histology. RESULTS: The 3-stage chondrogenic differentiation cascade initiated in MSCs was primarily characterized by sequential up-regulation of common cartilage genes. Premature induction of hypertrophy-related molecules (type X collagen and matrix metalloproteinase 13) occurred before production of type II collagen and was followed by up-regulation of alkaline phosphatase activity. In contrast, hypertrophy-associated genes were not induced in chondrocyte controls. Whereas control chondrocyte pellets resisted calcification and vascular invasion in vivo, most MSC pellets mineralized, in spite of persisting proteoglycan and type II collagen content. CONCLUSION: An unnatural pathway of differentiation to chondrocyte-like cells was induced in MSCs by common in vitro protocols. MSC pellets transplanted to ectopic sites in SCID mice underwent alterations related to endochondral ossification rather than adopting a stable chondrogenic phenotype. Further studies are needed to evaluate whether a more stringent control of MSC differentiation to chondrocytes can be achieved during cartilage repair in a natural joint environment.

Biochemical characterization of annexin B1 from Cysticercus cellulosae.

Annexin B1 from Cysticercus cellulosae has recently been identified using immunological screening in an attempt to find novel antigens for vaccine development against cysticercosis. The protein possesses anticoagulant activity and carries significant therapeutic potential due to its thrombus-targeting and thrombolytic properties. We investigated the biochemical properties of annexin B1 using liposome and heparin Sepharose copelleting assays, as well as CD spectroscopy. The calcium-dependent binding to acidic phospholipid membranes is reminiscent of other mammalian annexins with a clear preference for high phosphatidylserine content. A unique property of annexin B1 is its ability to bind to liposomes with high phosphatidylserine content in the absence of calcium, which might be due to the presence of several basic residues on the convex protein surface that harbours the membrane-binding loops. Annexin B1 demonstrates lectin properties and binds to heparin Sepharose in a cooperative, calcium-dependent manner. Although this binding is reversible to a large extent, a small fraction of the protein remains bound to the glycosaminoglycan even in the presence of high concentrations of EDTA. Analogous to annexin A5, we propose a model of heparin wrapped around the protein thereby engaging in calcium-dependent and calcium-independent interactions. Although the calcium-independent heparin-binding sites identified in annexin A5 are not conserved, we hypothesize three possible sites in annexin B1. Results from CD spectroscopy and thermal denaturation indicate that, in solution, the protein binds calcium with a low affinity that leads to a slight increase in folding stability.