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.

Scabies in the developing world--its prevalence, complications, and management.

Scabies remains one of the commonest of skin diseases seen in developing countries. Although its distribution is subject to a cycle of infection, with peaks and troughs of disease prevalence, this periodicity is often less obvious in poor communities. Scabies is a condition that affects families, particularly the most vulnerable; it also has the greatest impact on young children. Largely through the association with secondary bacterial infection caused by group A streptococci and Staphylococcus aureus, the burden of disease is compounded by nephritis, rheumatic fever and sepsis in developing countries. However, with a few notable exceptions, it remains largely neglected as an important public health problem. The purpose of this review is to provide an update on the current position of scabies with regard to its complications and control in resource-poor countries.

Image analysis of soft-tissue in-growth and attachment into highly porous alumina ceramic foam metals.

The detailed quantitative characterization of soft-tissue in-growth into highly porous artificial implants is critical to understanding the biophysical processes that will lead to the best structural scaffolding construct. Previous studies have performed mechanical peel tests and mostly qualitative histological analyses of soft-tissue. The goal of this paper is to report the results obtained from applying two image analysis algorithms to quantify the morphological structure found in histological images of stained soft-tissue in-growth into alumina ceramic foam metal implants using a canine model. Three different pore sizes were used and three different post-operative time points were considered. Using the 2D Wavelet Transform Modulus Maxima method and 2D Fourier Transform analysis, a strong anisotropic signature (directional preference) is detected in early (4-week) histological samples. The direction of preference is towards the center of the implants. The strength of the anisotropy at later time points (8 and 16 weeks) becomes gradually weaker. Our interpretation is that after a short period of time, the main tissue growth activity has been concentrated on filling the artificial implant by growing towards its center. The weaker anisotropic signature found at later time points is interpreted as the tissue growth activity strengthening its structure by growing in more random directions.

Amphipols: polymeric surfactants for membrane biology research.

Membrane proteins classically are handled in aqueous solutions as complexes with detergents. The dissociating character of detergents, combined with the need to maintain an excess of them, frequently results in more or less rapid inactivation of the protein under study. Over the past few years, we have endeavored to develop a novel family of surfactants, dubbed amphipols (APs). APs are amphiphilic polymers that bind to the transmembrane surface of the protein in a noncovalent but, in the absence of a competing surfactant, quasi-irreversible manner. Membrane proteins complexed by APs are in their native state, stable, and they remain water-soluble in the absence of detergent or free APs. An update is presented of the current knowledge about these compounds and their demonstrated or putative uses in membrane biology.

Human papillomavirus infection and p53 protein expression in vulvar intraepithelial neoplasia and invasive squamous cell carcinoma

The etiopathogenesis of vulvar intraepithelial neoplasia (VIN III) and invasive squamous cell carcinoma are largely unknown. Since there are few studies on Brazilian patients, our purpose was to determine the frequency of human papillomavirus (HPV) infection and the expression of p53 in these lesions, and associate them with other factors such as age, morphological subtypes, multicentric and multifocal disease. Thirty-eight cases of VIN III, nine of superficially invasive carcinoma, and 55 of invasive vulvar carcinoma were retrospectively evaluated from 1983 to 1995 for the presence of HPV by immunohistochemistry and in situ hybridization, and for p53 protein expression by immunohistochemistry on paraffin sections. All cases for whom material (slides and paraffin blocks) and clinical data were available were included. HPV and p53 were detected in 57.9 and 21.1 percent of the VIN III lesions, 33.3 and 66.7 percent of superficially invasive carcinomas, and 7.3 and 58.2 percent of invasive squamous cell carcinomas, respectively. HPV infection was associated with younger age in the VIN III and invasive carcinoma groups. In the latter, HPV infection was associated with the basaloid variant. p53 expression rate was higher in superficially invasive and invasive lesions and was not related to HPV infection. Our findings are similar to others and support the hypothesis that there are two separate entities of the disease, one associated with HPV and the other unrelated, with p53 inactivation possibly being implicated in some of the cases

Human papillomavirus infection and p53 protein expression in vulvar intraepithelial neoplasia and invasive squamous cell carcinoma.

The etiopathogenesis of vulvar intraepithelial neoplasia (VIN III) and invasive squamous cell carcinoma are largely unknown. Since there are few studies on Brazilian patients, our purpose was to determine the frequency of human papillomavirus (HPV) infection and the expression of p53 in these lesions, and associate them with other factors such as age, morphological subtypes, multicentric and multifocal disease. Thirty-eight cases of VIN III, nine of superficially invasive carcinoma, and 55 of invasive vulvar carcinoma were retrospectively evaluated from 1983 to 1995 for the presence of HPV by immunohistochemistry and in situ hybridization, and for p53 protein expression by immunohistochemistry on paraffin sections. All cases for whom material (slides and paraffin blocks) and clinical data were available were included. HPV and p53 were detected in 57.9 and 21.1% of the VIN III lesions, 33.3 and 66.7% of superficially invasive carcinomas, and 7.3 and 58.2% of invasive squamous cell carcinomas, respectively. HPV infection was associated with younger age in the VIN III and invasive carcinoma groups. In the latter, HPV infection was associated with the basaloid variant. p53 expression rate was higher in superficially invasive and invasive lesions and was not related to HPV infection. Our findings are similar to others and support the hypothesis that there are two separate entities of the disease, one associated with HPV and the other unrelated, with p53 inactivation possibly being implicated in some of the cases.

Computation and mutagenesis suggest a right-handed structure for the synaptobrevin transmembrane dimer.

Biological membrane fusion involves a highly precise and ordered set of protein-protein interactions. Synaptobrevin is a key player in this process. Mutagenesis studies of its single transmembrane segment suggest that it dimerizes in a sequence specific manner. Using the computational methods developed for the successful structure prediction of the glycophorin A transmembrane dimer, we have calculated a structural model for the synaptobrevin dimer. Our computational search yields a well-populated cluster of right-handed structures consistent with the experimentally determined dimerization motif. The three-dimensional structure contains an interface formed primarily by leucine and isoleucine side-chain atoms and has no interhelical hydrogen bonds. The model is the first three-dimensional picture of the synaptobrevin transmembrane dimer and provides a basis for further focused experimentation on its structure and association thermodynamics.

Specificity in transmembrane helix-helix interactions can define a hierarchy of stability for sequence variants.

The folding, stability, and oligomerization of helical membrane proteins depend in part on a precise set of packing interactions between transmembrane helices. To understand the energetic principles of these helix-helix interactions, we have used alanine-scanning mutagenesis and sedimentation equilibrium analytical ultracentrifugation to quantitatively examine the sequence dependence of the glycophorin A transmembrane helix dimerization. In all cases, we found that mutations to alanine at interface positions cost free energy of association. In contrast, mutations to alanine away from the dimer interface showed free energies of association that are insignificantly different from wild-type or are slightly stabilizing. Our study further revealed that the energy of association is not evenly distributed across the interface, but that there are several "hot spots" for interaction including both glycines participating in a GxxxG motif. Inspection of the NMR structure indicates that simple principles of protein-protein interactions can explain the changes in energy that are observed. A comparison of the dimer stability between different hydrophobic environments suggested that the hierarchy of stability for sequence variants is conserved. Together, these findings imply that the protein-protein interaction portion of the overall association energy may be separable from the contributions arising from protein-lipid and lipid-lipid energy terms. This idea is a conceptual simplification of the membrane protein folding problem and has implications for prediction and design.

Genetic selection for and molecular dynamic modeling of a protein transmembrane domain multimerization motif from a random Escherichia coli genomic library.

In order to identify new transmembrane helix packing motifs in naturally occurring proteins, we have selected transmembrane domains from a library of random Escherichia coli genomic DNA fragments and screened them for homomultimerization via their abilities to dimerize the bacteriophage lambda cI repressor DNA-binding domain. Sequences were isolated using a modified lambda cI headpiece dimerization assay system, which was shown previously to measure transmembrane helix-helix association in the E. coli inner membrane. Screening resulted in the identification of several novel sequences that appear to mediate helix-helix interactions. One sequence, representing the predicted sixth transmembrane domain (TM6) of the E. coli protein YjiO, was chosen for further analysis. Using site-directed mutagenesis and molecular dynamics, a small set of models for YjiO TM6 multimerization interface interactions were generated. This work demonstrates the utility of combining in vivo genetic tools with computational systems for understanding membrane protein structure and assembly.

The Calpha ---H...O hydrogen bond: a determinant of stability and specificity in transmembrane helix interactions.

The Calpha---H...O hydrogen bond has been given little attention as a determinant of transmembrane helix association. Stimulated by recent calculations suggesting that such bonds can be much stronger than has been supposed, we have analyzed 11 known membrane protein structures and found that apparent carbon alpha hydrogen bonds cluster frequently at glycine-, serine-, and threonine-rich packing interfaces between transmembrane helices. Parallel right-handed helix-helix interactions appear to favor Calpha---H...O bond formation. In particular, Calpha---H...O interactions are frequent between helices having the structural motif of the glycophorin A dimer and the GxxxG pair. We suggest that Calpha---H...O hydrogen bonds are important determinants of stability and, depending on packing, specificity in membrane protein folding.

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.

Conversion of phospholamban into a soluble pentameric helical bundle.

Although membrane proteins and soluble proteins may achieve their final folded states through different pathways, it has been suggested that the packing inside a membrane protein could maintain a similar fold if the lipid-exposed surface were redesigned for solubility in an aqueous environment. To test this idea, the surface of the transmembrane domain of phospholamban (PLB), a protein that forms a stable helical homopentamer within the sarcoplasmic reticulum membrane, has been redesigned by replacing its lipid-exposed hydrophobic residues with charged and polar residues. CD spectra indicate that the full-length soluble PLB is highly alpha-helical. Small-angle X-ray scattering and multiangle laser light scattering experiments reveal that this soluble variant of PLB associates as a pentamer, preserving the oligomeric state of the natural protein. Mutations that destabilize native PLB also disrupt the pentamer. However, NMR experiments suggest that the redesigned protein exhibits molten globule-like properties, possibly because the redesign of the surface of this membrane protein may have altered some native contacts at the core of the protein or possibly because the core is not rigidly packed in wild-type PLB. Nonetheless, our success in converting the membrane protein PLB into a specific soluble helical pentamer indicates that the interior of a membrane protein contains at least some of the determinants necessary to dictate folding in an aqueous environment. The design we successfully used was based on one of the two models in the literature; the alternative design did not give stable, soluble pentamers. This suggests that surface redesign can be employed in gaining insights into the structures of membrane proteins.

High-yield synthesis and purification of an alpha-helical transmembrane domain.

Polypeptides corresponding to hydrophobic transmembrane alpha-helices, such as residues 69-101 of glycophorin A, are notoriously difficult to prepare in quantities sufficient for biophysical experiments. Simple synthetic and purification approaches reported here have been developed by combining a few modifications to standard procedures, without resorting to elevated temperatures, expensive activation strategies, or complex hydrophobic solvent mixtures. The cost of screening projects, preparing labeled peptides, and examining sequence variations is thereby significantly reduced. The quality of the peptide synthesized by this small-scale 9-fluorenylmethoxycarbonyl (Fmoc) strategy is comparable to that of the peptide synthesized by an experienced resource facility using a large-scale tert-butyloxycarbonyl strategy. Using reverse-phase HPLC, the desired peptide was separated from the primary side product (a Leu or Ile deletion) and quantitatively recovered at greater than 98% purity. Baseline resolution was achieved using a water:acetonitrile gradient to elute the peptides from a cyanopropyl column at ambient temperature. Combining these approaches readily yields 10 to 20 mg of pure transmembrane peptide from a small-scale Fmoc synthesis. The approaches are readily transferable to transmembrane sequences not previously synthesized and do not require setting up a specialized facility. The time and start-up expense required to launch new studies are thereby reduced expanding the range and detail with which questions in membrane protein biophysics can be explored.

Polar residues drive association of polyleucine transmembrane helices.

Although many polar residues are directly involved in transmembrane protein functions, the extent to which they contribute to more general structural features is still unclear. Previous studies have demonstrated that asparagine residues can drive transmembrane helix association through interhelical hydrogen bonding [Choma, C., Gratkowski, H., Lear, J. D. & DeGrado, W. F. (2000) Nat. Struct. Biol. 7, 161-166; and Zhou, F. X., Cocco, M. J., Russ, W. P., Brunger, A. T. & Engelman, D. M. (2000) Nat. Struct. Biol. 7, 154-160]. We have studied the ability of other polar residues to promote helix association in detergent micelles and in biological membranes. Our results show that polyleucine sequences with Asn, Asp, Gln, Glu, and His, residues capable of being simultaneously hydrogen bond donors and acceptors, form homo- or heterooligomers. In contrast, polyleucine sequences with Ser, Thr, and Tyr do not associate more than the polyleucine sequence alone. The results therefore provide experimental evidence that interactions between polar residues in the helices of transmembrane proteins may serve to provide structural stability and oligomerization specificity. Furthermore, such interactions can allow structural flexibility required for the function of some membrane proteins.

Preconditioning of swine heart with monophosphoryl lipid A improves myocardial preservation.

BACKGROUND: Ischemic preconditioning has been proven to be a powerful tool for myocardial protection in the setting of ischemia and reperfusion. A new drug to provide pharmacologic preconditioning, monophosphoryl lipid A (MLA), was administered 24 hours before an acute coronary occlusion in pigs to determine the effect on pharmacologic preconditioning. METHODS: Two studies were completed. In the first, swine were distributed into five groups: group I, control; group II,. aminoguanidine (AMG) (30 mg/kg), a selective inducible nitric oxide synthase (iNOS) blocker; group III, MLA (10 microg/kg); group IV, MLA (35 microg/kg); and group V, MLA and AMG (35 microg/kg and 30 mg/kg, respectively). Twenty-four hours after administration of the MLA, AMG, or both, regional left anterior descending coronary artery ischemia was induced for 15 minutes followed by one hour of global normothermic cardioplegic arrest and three hour reperfusion. Left ventricular function, tissue injury, and percentage of myocardial infarction were measured. Left ventricular myocardium in the left anterior descending coronary artery region was sampled for iNOS messenger RNA (mRNA) during ischemia and reperfusion. In the second study, pigs were sacrificed 0, 4, 6, 8, and 24 hrs after MLA/AMG administration for iNOS mRNA determination in nonischemic myocardium. RESULTS: Use of MLA significantly improved postischemic ventricular function, and reduced creatinine kinase release and percentage of infarction. Monophosphoryl lipid A induced expression of iNOS mRNA in nonischemic myocardium within four hours of administration which returned to base line by 24 hours. Normothermic regional ischemia then induced expression of iNOS mRNA, which returned to base line during reperfusion. Aminoguanidine completely abolished both MLA-induced and ischemia-induced iNOS mRNA and blocked the beneficial effects of MLA. CONCLUSIONS: Use of MLA can provide myocardial preservation through enhanced expression of iNOS mRNA.

Modulation of glycophorin A transmembrane helix interactions by lipid bilayers: molecular dynamics calculations.

Starting from the glycophorin A dimer structure determined by NMR, we performed simulations of both dimer and monomer forms in explicit lipid bilayers with constant normal pressure, lateral area, and temperature using the CHARMM potential. Analysis of the trajectories in four different lipids reveals how lipid chain length and saturation modulate the structural and energetic properties of transmembrane helices. Helix tilt, helix-helix crossing angle, and helix accessible volume depend on lipid type in a manner consistent with hydrophobic matching concepts: the most relevant lipid property appears to be the bilayer thickness. Although the net helix-helix interaction enthalpy is strongly attractive, analysis of residue-residue interactions reveals significant unfavorable electrostatic repulsion between interfacial glycine residues previously shown to be critical for dimerization. Peptide volume is nearly conserved upon dimerization in all lipid types, indicating that the monomeric helices pack equally well with lipid as dimer helices do with one another. Enthalpy calculations indicate that the helix-environment interaction energy is lower in the dimer than in the monomer form, when solvated by unsaturated lipids. In all lipid environments there is a marked preference for lipids to interact with peptide predominantly through one rather than both acyl chains. Although our trajectories are not long enough to allow a full thermodynamic treatment, these results demonstrate that molecular dynamics simulations are a powerful method for investigating the protein-protein, protein-lipid, and lipid-lipid interactions that determine the structure, stability and dynamics of transmembrane alpha-helices in membranes.

Helical membrane protein folding, stability, and evolution.

Helical membrane protein folding and oligomerization can be usefully conceptualized as involving two energetically distinct stages-the formation and subsequent side-to-side association of independently stable transbilayer helices. The interactions of helices with the bilayer, with prosthetic groups, and with each other are examined in the context of recent evidence. We conclude that the two-stage concept remains useful as an approach to simplifying discussions of stability, as a framework for folding concepts, and as a basis for understanding membrane protein evolution.