Estimating the global burden of scabies: what else do we need?

Scabies is one of the most common disorders identified in any estimate of global skin disease prevalence. Furthermore, quantifying its impact on individuals and societies has been problematic. There has been a lack of clear case definitions and laboratory tests. There have been few epidemiological studies, particularly those focusing on low-income countries, variation in prevalence within high-income countries, or estimates of the effect of scabies on health beyond the skin, such as renal disease or mental wellbeing. Economic studies are also lacking. However, the new strategy of integrating surveillance for skin Neglected Tropical Diseases may well produce advancements on these issues, in addition to providing an overarching structure for health improvement and disease control.

The 2020 International Alliance for the Control of Scabies Consensus Criteria for the Diagnosis of Scabies.

BACKGROUND: Scabies is a common parasitic skin condition that causes considerable morbidity globally. Clinical and epidemiological research for scabies has been limited by a lack of standardization of diagnostic methods. OBJECTIVES: To develop consensus criteria for the diagnosis of common scabies that could be implemented in a variety of settings. METHODS: Consensus diagnostic criteria were developed through a Delphi study with international experts. Detailed recommendations were collected from the expert panel to define the criteria features and guide their implementation. These comments were then combined with a comprehensive review of the available literature and the opinion of an expanded group of international experts to develop detailed, evidence-based definitions and diagnostic methods. RESULTS: The 2020 International Alliance for the Control of Scabies (IACS) Consensus Criteria for the Diagnosis of Scabies include three levels of diagnostic certainty and eight subcategories. Confirmed scabies (level A) requires direct visualization of the mite or its products. Clinical scabies (level B) and suspected scabies (level C) rely on clinical assessment of signs and symptoms. Evidence-based, consensus methods for microscopy, visualization and clinical symptoms and signs were developed, along with a media library. CONCLUSIONS: The 2020 IACS Criteria represent a pragmatic yet robust set of diagnostic features and methods. The criteria may be implemented in a range of research, public health and clinical settings by selecting the appropriate diagnostic levels and subcategories. These criteria may provide greater consistency and standardization for scabies diagnosis. Validation studies, development of training materials and development of survey methods are now required. What is already known about this topic? The diagnosis of scabies is limited by the lack of accurate, objective tests. Microscopy of skin scrapings can confirm the diagnosis, but it is insensitive, invasive and often impractical. Diagnosis usually relies on clinical assessment, although visualization using dermoscopy is becoming increasingly common. These diagnostic methods have not been standardized, hampering the interpretation of findings from clinical research and epidemiological surveys, and the development of scabies control strategies. What does this study add? International consensus diagnostic criteria for common scabies were developed through a Delphi study with global experts. The 2020 International Alliance for the Control of Scabies (IACS) Criteria categorize diagnosis at three levels of diagnostic certainty (confirmed, clinical and suspected scabies) and eight subcategories, and can be adapted to a range of research and public health settings. Detailed definitions and figures are included to aid training and implementation. The 2020 IACS Criteria may facilitate the standardization of scabies diagnosis.

Systematic review of the diagnosis of scabies in therapeutic trials.

Human scabies (infestation with the mite Sarcoptes scabiei var hominis) causes a significant disease burden worldwide, yet there are no agreed diagnostic guidelines. We aimed to determine whether a consistent approach to diagnosing scabies has been used for published scabies therapeutic trials. The data sources used were the MEDLINE, Embase and Cochrane databases, from 1946 to 29 August 2013. Eligible studies were trials of therapeutic interventions against scabies in human subjects, published in English, enrolling patients with scabies, and using various therapeutic interventions. Language was a limitation of this study as some relevant trials published in languages other than English may have been excluded. Each study was reviewed by two independent authors, who assessed the clinical examination and testing approaches used for scabies diagnosis in the included studies. We found that of 71 included trials, 40 (56%) specified which clinical findings were used for diagnosis, which were predominantly rash, rash distribution, pruritus and mite burrows. Parasitological testing was used in 63% of trials (n = 45) and was used more frequently in clinic-based than in field studies. Nearly one-quarter of trials (24%, n = 17) did not define the diagnostic method used. Overall, the diagnostic approaches were poorly described, prohibiting accurate comparison of existing studies. This review further supports the need for consensus diagnostic guidelines for scabies.

A transmembrane helix dimer: structure and implications.

The three-dimensional structure of the dimeric transmembrane domain of glycophorin A (GpA) was determined by solution nuclear magnetic resonance spectroscopy of a 40-residue peptide solubilized in aqueous detergent micelles. The GpA membrane-spanning alpha helices cross at an angle of -40 degrees and form a small but well-packed interface that lacks intermonomer hydrogen bonds. The structure provides an explanation for the previously characterized sequence dependence of GpA dimerization and demonstrates that van der Waals interactions alone can mediate stable and specific associations between transmembrane helices.

A complete mapping of the proteins in the small ribosomal subunit of Escherichia coli.

The relative positions of the centers of mass of the 21 proteins of the 30S ribosomal subunit from Escherichia coli have been determined by triangulation using neutron scattering data. The resulting map of the quaternary structure of the small ribosomal subunit is presented, and comparisons are made with structural data from other sources.

Compositional mapping of cholesteryl ester droplets in the fatty streaks of human aorta.

Frozen sections prepared from human aortic tissue containing fatty streak lesions were examined on a thermally controlled stage with a polarizing light microscope. Distinct birefringent droplets, 0.5-5 mum in diameter, were observed, many apparently aggregated into clusters. The clusters were about 20 X 20 mum in diameter (the approximate size of foam cells). Upon being heated, each smectic droplet exhibited a sudden change of birefringence, indicating a change of state. The transition temperatures were compared to assess compositional distributions in the tissue. We found that for 52% of the clusters the standard deviation of the cluster's droplet melting point distribution was less than half that observed in the surrounding microscopic field. If clusters were intracellular lipid inclusions, this observation indicates that the lipid composition within a foam cell is more homogeneous than that of the overall field. However, using statistical methods, we compared droplet melting populations from cluster to cluster and found significant heterogeneity. The observations can be interpreted to suggest that many foam cells modify the cholesteryl ester fatty acid composition of their accumulations be selective uptake, temporal sampling, or chemical reaction. Furthermore, the intercellular heterogeneity suggests that different cells in the lesion may have different metabolic and transport enzyme affinities or be in different states.

Molecular organization of the cholesteryl ester droplets in the fatty streaks of human aorta.

X-ray diffraction patterns from human arterial specimens containing atherosclerotic fatty streak lesions exhibited a single sharp reflection, corresponding to a structural spacing of about 35 A. Specimens without lesions did not. When specimens with fatty streaks were heated, an order-to-disorder phase transition was revealed by the disappearance of the sharp reflection. The transition was thermally reversible and its temperature varied from aorta to aorta over a range from 28 degrees to 42 degrees C. Since cholesteryl ester droplets are a major component of fatty streaks, comparison studies were made of the diffraction behavior from pure cholesteryl esters. We found that the diffraction patterns of the fatty streak material could be accounted for by the organization of the cholesteryl esters into a liquid-crystalline smectic phase that melts from the smectic to a less ordered phase upon heating. When combined with the conclusions of others from polarized light microscopy, our study shows that a droplet in the smectic phase has well-defined concentric layers of lipid molecules. In each layer, the long axes of the molecules have a net radial orientation with respect to the droplet, but the side-to-side organization is disordered. We suggest that the accessibility of portions of the lipids for specific binding to enzymes or transport proteins may be restricted when they are in the smectic state, and that exchange of lipids with surrounding membranes or other potential binding sites may likewise be inhibited. The restriction in the smectic phase should be greater than in the less ordered phases that exist at higher temperatures.

Helical membrane proteins: diversity of functions in the context of simple architecture.

During the past year, research on helical membrane proteins has brought insights into the use of deviations from canonical alpha-helical conformation to support function and the further investigation of the sequestration of protein regions from the lipid bilayer to enhance these structural alternatives. Also, the structural roles of polar sidechains, the identification of motifs in helix interactions and the significance of certain topologies on a genome-wide scale have been further explored.

Dimerization of the p185neu transmembrane domain is necessary but not sufficient for transformation.

The neu proto-oncogene encodes a receptor tyrosine kinase (RTK). The oncogenic allele neu* (p185*) bears a glutamic acid for valine substitution at position 664 within the predicted transmembrane domain. We have used this mutant to explore the role of the transmembrane domain in signal transduction by RTKs. Analysis of a panel of neu* proteins with second-site mutations in the transmembrane domain revealed a strong correlation of dimerization with transformation. Both dimerization and transformation are dependent on a domain formed by the amino acids Val663-Glu664-Gly665 (VEG). However, movement of the VEG elsewhere within the transmembrane domain promoted weak dimerization but not transformation. Epidermal growth factor receptor (EGFR)/neu chimeras were used to determine if mutations that disrupt activation by Glu664 affect hormone-regulated signal transduction as well. These mutations (of Val663 and Gly665) did not affect regulation by EGF. Introduction of the known transmembrane dimerization domain from Glycophorin A (GpA) stimulated dimerization, but was not sufficient for transformation. These results indicate that dimerization is necessary but not sufficient for transforming activity. The homologous wild-type domain, VVG, is not required for hormone-regulated signaling.

The planar distributions of surface proteins and intramembrane particles in Acholeplasma laidlawii are differentially affected by the physical state of membrane lipids.

We have studied the influence of changes in lipid organization on the planar distribution of two classes of membrane proteins: integral proteins which have amino groups exposed to labelling at the membrane surface by the biotinavidin-ferritin procedure, and those proteins which penetrate the lipid bilayer sufficiently to be seen as intramembranous particles by freeze-fracture electron-microscopy. When the membranes are examined at temperatures below the lipid phase transition, the first class is dispersed and the second patched. At temperatures in the middle of the transition range, both classes are patched. At temperatures just above the phase transition the first class is dispersed and the second patched, and at temperatures well above the transition both classes are dispersed. Freeze-etch studies of avidin-ferritin-labeled membranes confirmed that the distribution seen by the labeling and the freeze-fracture techniques coexist in single membranes. Thus, there exist two distinct classes of membrane proteins with differential organizational responses to the lipid state.

Bacteriorhodopsin remains dispersed in fluid phospholipid bilayers over a wide range of bilayer thicknesses.

We have used vesicles made from delipidated bacteriorhodopsin and synthetic lecithins to address the following questions. If the transmembrane dimension of a protein hydrophobic surface differs from the equilibrium thickness of its lipid bilayer environment, will protein monomers aggregate to decrease the protein-lipid contact surface area? If so, how large must the difference be to induce aggregation? Using lecithins with acyl chains from di-10:0 to di-24:1, the thickness of the bilayer hydrocarbon region above the lipid phase transition temperature (tm) was varied from 14.5 A less than to 7.5 A more than the transmembrane dimension of the bacteriorhodopsin hydrophobic region. Bacteriorhodopsin remains dispersed when the surrounding bilayer hydrophobic region is 4 A thicker or 10 A thinner than the bacteriorhodopsin hydrophobic surface. Only the thin- (10:0) and thick- (24:1) bilayer samples showed any bacteriorhodopsin aggregation above tm. Thus a surprisingly large difference between protein and lipid hydrophobic thicknesses can be accommodated without protein aggregation. The lipid bilayer can evidently sustain large local distortions with a small change in free energy.

Lipid bilayer thickness varies linearly with acyl chain length in fluid phosphatidylcholine vesicles.

The thickness of the lipid bilayer in vesicles made of pure phosphatidylcholines, with acyl chain lengths ranging from 10 to 24 carbons, has been determined by analysis of continuous X-ray scattering data from vesicle pellets at temperatures above the lipid phase transition temperature. Bilayer thickness was found to vary linearly with the number of carbons per acyl chain. The lines for saturated and monounsaturated acyl chains were slightly displaced but had similar slopes. For the saturated species di-12:0, di-14:0, di-16:0, and di-18:0 phosphatidylcholine the surface areas per molecule were all 65.7 to 66.5 A2, while the monounsaturated species and di-10:0 phosphatidylcholine all occupied 67.7 to 70.1 A2 per molecule.

The GxxxG motif: a framework for transmembrane helix-helix association.

In order to identify strong transmembrane helix packing motifs, we have selected transmembrane domains exhibiting high-affinity homo-oligomerization from a randomized sequence library based on the right-handed dimerization motif of glycophorin A. Sequences were isolated using the TOXCAT system, which measures transmembrane helix-helix association in the Escherichia coli inner membrane. Strong selection was applied to a large range of sequences ( approximately 10(7) possibilities) and resulted in the identification of sequence patterns that mediate high-affinity helix-helix association. The most frequent motif isolated, GxxxG, occurs in over 80% of the isolates. Additional correlations suggest that flanking residues act in concert with the GxxxG motif, and that size complementarity is maintained at the interface, consistent with the idea that the identified sequence patterns represent packing motifs. The convergent identification of similar sequence patterns from an analysis of the transmembrane domains in the SwissProt sequence database suggests that these packing motifs are frequently utilized in naturally occurring helical membrane proteins.

Statistical analysis of amino acid patterns in transmembrane helices: the GxxxG motif occurs frequently and in association with beta-branched residues at neighboring positions.

To find motifs that mediate helix-helix interactions in membrane proteins, we have analyzed frequently occurring combinations of residues in a database of transmembrane domains. Our analysis was performed with a novel formalism, which we call TMSTAT, for exactly calculating the expectancies of all pairs and triplets of residues in individual sequences, taking into account differential sequence composition and the substantial effect of finite length in short segments. We found that the number of significantly over and under-represented pairs and triplets was much greater than the random expectation. Isoleucine, glycine and valine were the most common residues in these extreme cases. The main theme observed is patterns of small residues (Gly, Ala and Ser) at i and i+4 found in association with large aliphatic residues (Ile, Val and Leu) at neighboring positions (i.e. i+/-1 and i+/-2). The most over-represented pair is formed by two glycine residues at i and i+4 (GxxxG, 31.6 % above expectation, p<1x10(-33)) and it is strongly associated with the neighboring beta-branched residues Ile and Val. In fact, the GxxxG pair has been described as part of the strong interaction motif in the glycophorin A transmembrane dimer, in which the pair is associated with two Val residues (GVxxGV). GxxxG is also the major motif identified using TOXCAT, an in vivo selection system for transmembrane oligomerization motifs. In conjunction with these experimental observations, our results highlight the importance of the GxxxG+beta-branched motif in transmembrane helix-helix interactions. In addition, the special role for the beta-branched residues Ile and Val suggested here is consistent with the hypothesis that residues with constrained rotameric freedom in helical conformation might reduce the entropic cost of folding in transmembrane proteins. Additional material is available at http://engelman.csb.yale. edu/tmstat and http://bioinfo.mbb.yale. edu/tmstat.

Positions of S2, S13, S16, S17, S19 and S21 in the 30 S ribosomal subunit of Escherichia coli.

Neutron scattering distance data are presented for 33 protein pairs in the 30 S ribosomal subunit from Escherichia coli, along with the methods used for measuring distances between its exchangeable components. When combined with prior data, these new results permit the positioning of S2, S13, S16, S17, S19 and S21 in the 30 S ribosomal subunit, completing the mapping of its proteins by neutron scattering. Comparisons with other data suggest that the neutron map is a reliable guide to the quaternary structure of the 30 S subunit.

Refolding of bacteriorhodopsin in lipid bilayers. A thermodynamically controlled two-stage process.

Possible steps in the folding of bacteriorhodopsin are revealed by studying the refolding and interaction of two fragments of the molecule reconstituted in lipid vesicles. (1) Two denatured bacteriorhodopsin fragments have been purified starting from chymotryptically cleaved bacteriorhodopsin. Cleaved bacteriorhodopsin has been renatured from a mixture of the fragments in Halobacterium lipids/retinal/dodecyl sulfate solution following removal of dodecyl sulfate by precipitation with potassium. The renatured molecules have the same absorption spectrum and extinction coefficient as native cleaved bacteriorhodopsin. They are integrated into small lipid vesicles as a mixture of monomers and aggregates. Extended lattices form during the partial dehydration process used to orient samples for X-ray and neutron crystallography. (2) Correct refolding of cleaved bacterioopsin occurs upon renaturation in the absence of retinal. Regeneration of the chromophore and reformation of the purple membrane lattice are observed following subsequent addition of all-trans retinal. (3) The two chymotryptic fragments have been reinserted separately into lipid vesicles and refolded in the absence of retinal. Circular dichroism spectra of the polypeptide backbone transitions indicate that they have regained a highly alpha-helical structure. The kinetics of chromophore regeneration following reassociation have been studied by absorption spectroscopy. Upon vesicle fusion, the refolded fragments first reassociate, then bind retinal and finally regenerate cleaved bacteriorhodopsin. The complex formed in the absence of retinal is kinetically indistinguishable from cleaved bacterioopsin. The refolded fragments in lipid vesicles are stable for months, both as separate entities and after reassociation. These observations provide further evidence that the native folded structure of bacteriorhodopsin lies at a free energy minimum. They are interpreted in terms of a two-stage folding mechanism for membrane proteins in which stable transmembrane helices are first formed. They subsequently pack without major rearrangement to produce the tertiary structure.

Tertiary structure of bacteriorhodopsin. Positions and orientations of helices A and B in the structural map determined by neutron diffraction.

Positions and rotations of two helices in the tertiary structure of bacteriorhodopsin have been studied by neutron diffraction using reconstituted, hybrid purple membrane samples. Purple membrane was biosynthetically 2H-labeled at non-exchangeable hydrogen positions of leucine and tryptophan residues. Two chymotryptic fragments were purified, encompassing either the first two or the last five of the seven putative transmembrane segments identified in the amino acid sequence of bacteriorhodopsin. The 2H-labeled fragments, diluted to variable extents with the identical, unlabeled fragment, were mixed with their unlabeled counterpart; bacteriorhodopsin was then renatured and reconstituted. The crystalline purple membrane samples thus obtained contained hybrid bacteriorhodopsin molecules in which certain transmembrane segments had been selectively 2H-labeled to various degrees. Neutron diffraction powder patterns were recorded and analyzed both by calculating difference Fourier maps and by model building. The two analyses yielded consistent results. The first and second transmembrane segments in the sequence correspond to helices 1 and 7 of the three-dimensional structure, respectively. Rotational orientations of these two helices were identified using best fits to the observed diffraction intensities. The data also put restrictions on the position of the third transmembrane segment. These observations are discussed in the context of folding models for bacteriorhodopsin, the environment of the retinal Schiff base, and site-directed mutagenesis experiments.

Assessment of the aggregation state of integral membrane proteins in reconstituted phospholipid vesicles using small angle neutron scattering.

The assessment of the physical size of integral membrane protein complexes has generally been limited to samples solubilized in non-ionic detergent, a process which may introduce artifacts of unknown scope and severity. A system has been developed that allows observation of the small angle scattering profile of an integral membrane protein while incorporated in small unilamellar phospholipid vesicles. Contrast matching of isotopically substituted phospholipid eliminates the contribution of the bilayer to the observed scattering, resulting in a profile dependent only on the structure of the individual membrane protein complexes and their spatial arrangement in the vesicle. After appropriate compensation for their spatial arrangement, information about the molecular mass and radius of gyration of the individual complexes can be obtained. The validity of the approach has been established using monomeric bacteriorhodopsin as a model system.

The effect of point mutations on the free energy of transmembrane alpha-helix dimerization.

Glycophorin A forms homodimers through interaction of the single, helical transmembrane domains of the monomers. The dimers are stable in sodium dodecylsulfate (SDS), permitting a number of studies that have identified a critical motif of residues that mediates dimer formation. We have used analytical ultracentrifugation to measure the energy of dimerization in a non-denaturing detergent solution and have observed the changes in energy arising from two of the mutants previously studied. Use of the detergent pentaoxyethylene octyl ether (C8E5) is a great advantage, since its micelles are neutrally buoyant and the detergent allows a reversible association to occur between monomer and dimer states of the glycophorin A transmembrane helices during the time-scale of sedimentation equilibrium. Use of this detergent in analytical ultracentrifugation may enable a wide range of studies of molecular association events in membrane proteins. We find that the glycophorin A transmembrane helix dimerizes with a dissociation constant of 240(+/-50) nM, corresponding to a free energy of dissociation of 9.0(+/-0.1) kcal mol-1. Point mutants that were found to be disruptive in SDS (L75A, I76A) reduced the dimer affinity in the C8E5 detergent environment (Kd=1.7(+/-0.2) microM and 4.2(+/-0.9) microM, respectively). Thus, the earlier findings are placed on a quantitative, relative energy scale of association by our measurements. Molecular modeling and simulations suggest that the energy differences can be accounted for as changes in van der Waals interactions between helices.