WHF Position Statement on COVID Vaccination.

The current COVID-19 pandemic has challenged health systems and communities globally. As such, several countries have embarked on national COVID-19 vaccination programmes in order to curb spread of the disease. However, at present, there isn't yet enough dosages to enable vaccination of the general population. Different vaccine prioritization strategies are thus being implemented in different communities in order to permit for a systematic vaccination of individuals. Here, on behalf of the World Heart Federation, we emphasize the need for individuals with Cardiovascular disease to be prioritized in national vaccine prioritization programmes as these are high risk individuals.

Ensuring the global availability of high-quality dental restorative materials.

The Minamata Convention, a global legally binding instrument (treaty) on mercury, has been the catalyst for the emerging agenda on global dental materials research. If the current and future challenges of oral health maintenance and healing on a global scale are to be met, a logical and effective research agenda for the discovery and introduction of new, environmentally sustainable, dental materials must be developed through a coordinated effort involving materials scientists, dental clinicians, representatives of industry, members of regional and national regulatory bodies, and advocacy from research organizations. For universal impact, this agenda should be created with awareness of several important ongoing initiatives, such as the WHO non-communicable diseases action plan, the UN sustainable development agenda, and the IADR Global Oral Health In Inequalities Research Agenda (GOHIRA). A significant contributor to this cause is the FDI and its membership, who, through their Vision 2020 initiative, acknowledge their role and responsibility in globally preventing and managing dental disease and providing leadership to the profession in terms of information dissemination and affecting change. Dental researchers also have an obligation to advocate for appropriate funding to match the identified research needs, thus enhancing the possibility that key decision-makers will provide the needed support to achieve the research agenda agreed upon by this diverse group of stakeholders.

FDI Global Caries Initiative; implementing a paradigm shift in dental practice and the global policy context.

The implementation of a new paradigm for caries management is necessary for the profession to respond effectively to changing population health needs. The FDI Global Caries Initiative (GCI) is a 10 year programme aimed at developing and implementing a new paradigm for caries management, one that would contribute to a common vision of health. The article reviews the global health policy landscape and examines how it might influence and shape the implementation of the GCI.

Ethanol inhibition of nicotinic acetylcholine type alpha 7 receptors involves the amino-terminal domain of the receptor.

Recent studies have suggested that alcohols can affect the function of neurotransmitter-gated ion channels by a direct interaction with the receptor protein. However, the molecular region of the receptor protein that mediates the alcohol action is not known. To address this question, we studied the effect of ethanol on the function of recombinant nicotinic acetylcholine type alpha 7 (nACh alpha 7) receptors, 5-hydroxytryptamine (serotonin) type 3 (5-HT3) receptors, and a chimeric receptor constructed from these two receptors. The receptors were expressed in Xenopus oocytes and their function was studied using the two-electrode voltage-clamp technique. Ethanol inhibited the response of nACh alpha 7 receptors in a concentration-dependent manner over the concentration range of 5-100 mM; the EC50 for this inhibition was 33 mM ethanol. Ethanol decreased the maximal amplitude (Emax) of the nACh alpha 7 receptor agonist concentration-response curve, without significantly affecting the EC50. In contrast, ethanol potentiated 5-HT3 receptor-mediated responses at low agonist concentrations. The potentiation was concentration-dependent over the concentration range of 10-100 mM; the EC50 for this potentiation was 57 mM ethanol. The magnitude of the ethanol potentiation of 5-HT3 receptor-mediated responses decreased with increasing agonist concentration. The chimeric receptor had the amino-terminal domain from the nACh alpha 7 receptor and the transmembrane and carboxyl-terminal domains from the 5-HT3 receptor. Ethanol was found to inhibit the function of this chimeric receptor in a manner similar to that of nACh alpha 7 receptors. Because the inhibition transfers with the amino-terminal domain of the receptor, the observations suggest that the amino-terminal domain of the receptor is involved in the inhibition.

Identification of a new component of the agonist binding site of the nicotinic alpha 7 homooligomeric receptor.

Tryptophan 54 of the alpha 7 neuronal nicotinic homooligomeric receptor is homologous to gamma-Trp-55 and delta-Trp-57 of non-alpha subunits of Torpedo receptor labeled by d-tubocurarine. This residue was mutated on the alpha 7-V201-5-hydroxytryptamine (5HT)3 homooligomeric chimera, which displays alpha 7 nicotinic pharmacology, and for which both equilibrium binding studies and electrophysiological recordings could be carried out in parallel. Replacement of Trp-54 by a Phe, Ala, or His causes a progressive decrease both in binding affinity and in responses (EC50 or IC50) for acetylcholine, nicotine, and dihydro-beta-erythroidine, without significant modification in alpha-Bgtx binding. Except for Gln-56, comparatively small effects are observed when the other residues of the 52-58 region are mutated into alanine. These data support the participation of Trp-54 to ligand binding, and provide evidence for a new "complementary component" of the alpha 7 nicotinic binding site, distinct from its three-loop "principal component," and homologous to the "non-alpha component" present on gamma and delta subunits.

Three-dimensional structures of the free and the antigen-complexed Fab from monoclonal anti-lysozyme antibody D44.1.

The three-dimensional structures of the free and antigen-complexed Fabs from the mouse monoclonal anti-hen egg white lysozyme antibody D44.1 have been solved and refined by X-ray crystallographic techniques. The crystals of the free and lysozyme-bound Fabs were grown under identical conditions and their X-ray diffraction data were collected to 2.1 and 2.5 A, respectively. Two molecules of the Fab-lysozyme complex in the asymmetric unit of the crystals show nearly identical conformations and thus confirm the essential structural features of the antigen-antibody interface. Three buried water molecules enhance the surface complementarity at the interface and provide hydrogen bonds to stabilize the complex. Two hydrophobic buried holes are present at the interface which, although large enough to accommodate solvent molecules, are void. The combining site residues of the complexed FabD44.1 exhibit reduced temperature factors compared with those of the free Fab. Furthermore, small perturbations in atomic positions and rearrangements of side-chains at the combining site, and a relative rearrangement of the variable domains of the light (VL) and the heavy (VH) chains, detail a Fab accommodation of the bound lysozyme. The amino acid sequence of the VH domain, as well as the epitope of lysozyme recognized by D44.1 are very close to those previously reported for the monoclonal antibody HyHEL-5. A feature central to the FabD44.1 and FabHyHEL-5 complexes with lysozyme are three salt bridges between VH glutamate residues 35 and 50 and lysozyme arginine residues 45 and 68. The presence of the three salt bridges in the D44.1-lysozyme interface indicates that these bonds are not responsible for the 1000-fold increase in affinity for lysozyme that HyHEL-5 exhibits relative to D44.1.

Crystallization and preliminary X-ray diffraction study of an idiotope-anti-idiotope Fv-Fv complex.

A complex between the Fv fragment of an anti-hen eggwhite lysozyme antibody (D1.3) and the Fv fragment of an antibody specific for an idiotypic determinant of D1.3 has been crystallized in a form suitable for X-ray diffraction analysis. Both Fv fragments were expressed in soluble form in Escherichia coli and purified by affinity chromatography; diffraction-quality crystals were only obtained following separation of each Fv into distinct isoelectric forms. The crystals belong to space group C2, have unit cell dimensions a = 152.8 A, b = 79.4 A, c = 51.5 A, beta = 100.2 degrees, and diffract to better than 2.2 A resolution. The solvent content of the crystals is approximately 60% (v/v) with one Fv-Fv complex in the asymmetric unit. The ability to readily express both components of an antigen-antibody system in bacteria will allow us to rigorously assess the energetic contribution of individual amino acids to complex formation through pairwise mutagenesis of interacting residues.

Chimaeric nicotinic-serotonergic receptor combines distinct ligand binding and channel specificities.

The neuronal nicotinic alpha 7 (nAChR) and 5-hydroxytryptamine (5HT3) receptors are ligand-gated ion channels with a homologous topological organization and have activation and desensitization reactions in common. Yet these homo-oligomeric receptors differ in the pharmacology of their binding sites for agonists and competitive antagonists, and in their sensitivity to Ca2+ ions. The alpha 7 channel is highly permeable to Ca2+ ions and external Ca2+ ions potentiate, in an allosteric manner, the permeability response to acetylcholine, as shown for other neuronal nAChRs. The 5HT3 channel, in contrast, is not permeable to Ca2+ ions, but blocked by them. To assign these properties to delimited domains of the primary structure, we constructed several recombinant chimaeric alpha 7-5HT3 receptors. We report here that one of the constructs expresses a functional receptor that contains the serotonergic channel still blocked by Ca2+ ions, but is activated by nicotinic ligands and potentiated by external Ca2+ ions.

Three-dimensional structure of a heteroclitic antigen-antibody cross-reaction complex.

Although antibodies are highly specific, cross-reactions are frequently observed. To understand the molecular basis of this phenomenon, we studied the anti-hen egg lysozyme (HEL) monoclonal antibody (mAb) D11.15, which cross-reacts with several avian lysozymes, in some cases with a higher affinity (heteroclitic binding) than for HEL. We have determined the crystal structure of the Fv fragment of D11.15 complexed with pheasant egg lysozyme (PHL). In addition, we have determined the structure of PHL, Guinea fowl egg lysozyme, and Japanese quail egg lysozyme. Differences in the affinity of D11.15 for the lysozymes appear to result from sequence substitutions in these antigens at the interface with the antibody. More generally, cross-reactivity is seen to require a stereochemically permissive environment for the variant antigen residues at the antibody-antigen interface.

Crystallization and preliminary crystallographic analysis of a tetrameric isolectin from Vicia villosa, specific for the Tn antigen.

Isolectin B4 isolated from Vicia villosa seeds is specific for the Tn antigen, a carcinoma-associated molecular marker. Crystals of the isolectin grown in the presence of carbohydrate are tetragonal, space group P4(1) (or P4(3), with a = 91.3 A, c = 151.7 A and one tetramer in the asymmetric unit. The crystals diffract X-rays to 2.8 A resolution and are suitable for high-resolution structural analysis.

Determination of detergent critical micellar concentration by solubilization of a colored dye.

The critical micellar concentration of detergent was determined by the solubilization of a colored dye found in standard waterproof pen. The amount of solubilized dye is directly proportional to the concentration of micelles in the detergent solution. The method has been validated with a series of standard detergents.

Short chain phospholipids in membrane protein crystallization: a 31P-NMR study of colloidal properties of dihexanoyl phosphatidylcholine.

The colloidal features of short chain phospholipids can be deduced from 31P-NMR analysis by comparison with available data on phospholipid aqueous dispersion. In this study with dihexanoyl phosphatidylcholine, detergent phase separation was obtained by temperature shift and by addition of the precipitating agent polyethylene glycol. The 31P-NMR spectra indicate that the detergent micelles fuse to enter the hexagonal HII and lamellar phases. Consequences for the crystallization of membrane proteins are discussed.

Three-dimensional structure and antigen binding specificity of antibodies.

A number of specific Fab and Fv fragments and their complexes with antigens (avian lysozymes), haptens, and anti-idiotopic Fabs have been studied by immunochemical and crystallographic techniques. Antigen and antibody interact through closely complementary contacting surfaces, without major conformational changes. An idiotopic determinant of a monoclonal antibody is shown to include parts of most of its complementarity determining regions. The specificity of antigen recognition resides in the close complementarity of the antigenic determinant with the antibody combining site.

Crystallization and preliminary X-ray diffraction study of the bacterially expressed Fv from the monoclonal anti-lysozyme antibody D1.3 and of its complex with the antigen, lysozyme.

The associated heavy (VH) and light (VL) chain variable domains (Fv) of the monoclonal anti-lysozyme antibody D1.3, secreted from Escherichia coli, have been crystallized in their antigen-bound and free forms. FvD1.3 gives tetragonal crystals, space group P4(1)2(1)2 (or P4(3)2(1)2), with a = 90.6 A, c = 56.4 A. The FvD1.3-lysozyme complex crystallizes in space group C2, with a = 129.2 A, b = 60.8 A, c = 56.9 A and beta = 119.3 degrees. The crystals contain one molecule of Fv or of the Fv-lysozyme complex in their asymmetric units and diffract X-rays to high resolution, making them suitable for X-ray crystallographic studies.

In vitro folding and oligomerization of a membrane protein. Transition of bacterial porin from random coil to native conformation.

Porin, a channel-forming protein spanning bacterial outer membranes, was denatured in 6 M guanidinium hydrochloride or, alternatively, in sodium dodecyl sulfate at 95 degrees C. Circular dichroism spectra revealed that this protein, which in its native state consist of beta-pleated sheets as the sole detectable secondary structure, is transformed into random coil configuration in the chaotropic agent, or into alpha-helical structure in the detergent. From either state, the mature protein refolds in presence of amphiphilic molecules, attaining full structural and functional competence. As structural criteria, the native trimeric state was assayed by analytical ultracentrifugation, gel electrophoresis in sodium dodecyl sulfate, protease resistance, and circular dichroism spectroscopy. Channel formation in planar lipid bilayers reveals that the refolded protein is also functionally competent. It is concluded that the information required for the complete folding of porin is contained within the primary sequence of the mature polypeptide. The study of rapid refolding clearly reveals that this process occurs in the time range of seconds and that preexisting bilayers are not a prerequisite.

Crystallization of porin using short chain phospholipids.

A protein from outer membranes of Escherichia coli K-12, porin OmpF was crystallized either with short acyl chain phospolipids or with various detergents as amphiphiles. In dihexanoyl phosphatidylcholine, crystals greater than 0.3 mm in all dimensions were obtained that diffract X-rays beyond 3.5 A. Crystal morphology and unit cell dimensions were indistinguishable from those of detergent-grown crystals, indicating that the role of the phospholipids used is similar to those of many conventional detergents.