Staphylococcus aureus binds to the N-terminal region of corneodesmosin to adhere to the stratum corneum in atopic dermatitis.

Staphylococcus aureus colonizes the skin of the majority of patients with atopic dermatitis (AD), and its presence increases disease severity. Adhesion of S. aureus to corneocytes in the stratum corneum is a key initial event in colonization, but the bacterial and host factors contributing to this process have not been defined. Here, we show that S. aureus interacts with the host protein corneodesmosin. Corneodesmosin is aberrantly displayed on the tips of villus-like projections that occur on the surface of AD corneocytes as a result of low levels of skin humectants known as natural moisturizing factor (NMF). An S. aureus mutant deficient in fibronectin binding protein B (FnBPB) and clumping factor B (ClfB) did not bind to corneodesmosin in vitro. Using surface plasmon resonance, we found that FnBPB and ClfB proteins bound with similar affinities. The S. aureus binding site was localized to the N-terminal glycine-serine-rich region of corneodesmosin. Atomic force microscopy showed that the N-terminal region was present on corneocytes containing low levels of NMF and that blocking it with an antibody inhibited binding of individual S. aureus cells to corneocytes. Finally, we found that S. aureus mutants deficient in FnBPB or ClfB have a reduced ability to adhere to low-NMF corneocytes from patients. In summary, we show that FnBPB and ClfB interact with the accessible N-terminal region of corneodesmosin on AD corneocytes, allowing S. aureus to take advantage of the aberrant display of corneodesmosin that accompanies low NMF in AD. This interaction facilitates the characteristic strong binding of S. aureus to AD corneocytes.

Localized incorporation of outer membrane components in the pathogen Brucella abortus.

The zoonotic pathogen Brucella abortus is part of the Rhizobiales, which are alpha-proteobacteria displaying unipolar growth. Here, we show that this bacterium exhibits heterogeneity in its outer membrane composition, with clusters of rough lipopolysaccharide co-localizing with the essential outer membrane porin Omp2b, which is proposed to allow facilitated diffusion of solutes through the porin. We also show that the major outer membrane protein Omp25 and peptidoglycan are incorporated at the new pole and the division site, the expected growth sites. Interestingly, lipopolysaccharide is also inserted at the same growth sites. The absence of long-range diffusion of main components of the outer membrane could explain the apparent immobility of the Omp2b clusters, as well as unipolar and mid-cell localizations of newly incorporated outer membrane proteins and lipopolysaccharide. Unipolar growth and limited mobility of surface structures also suggest that new surface variants could arise in a few generations without the need of diluting pre-existing surface antigens.

Molecular interactions and inhibition of the staphylococcal biofilm-forming protein SdrC.

Staphylococcus aureus forms biofilms on indwelling medical devices using a variety of cell-surface proteins. There is growing evidence that specific homophilic interactions between these proteins represent an important mechanism of cell accumulation during biofilm formation, but the underlying molecular mechanisms are still not well-understood. Here we report the direct measurement of homophilic binding forces by the serine-aspartate repeat protein SdrC and their inhibition by a peptide. Using single-cell and single-molecule force measurements, we find that SdrC is engaged in low-affinity homophilic bonds that promote cell-cell adhesion. Low-affinity intercellular adhesion may play a role in favoring biofilm dynamics. We show that SdrC also mediates strong cellular interactions with hydrophobic surfaces, which are likely to be involved in the initial attachment to biomaterials, the first stage of biofilm formation. Furthermore, we demonstrate that a peptide derived from ß-neurexin is a powerful competitive inhibitor capable of efficiently blocking surface attachment, homophilic adhesion, and biofilm accumulation. Molecular modeling suggests that this blocking activity may originate from binding of the peptide to a sequence of SdrC involved in homophilic interactions. Our study opens up avenues for understanding the role of homophilic interactions in staphylococcal adhesion, and for the design of new molecules to prevent biofilm formation during infection.

Nucleation and growth of a bacterial functional amyloid at single-fiber resolution.

Curli are functional amyloids produced by proteobacteria like Escherichia coli as part of the extracellular matrix that holds cells together into biofilms. The molecular events that occur during curli nucleation and fiber extension remain largely unknown. Combining observations from curli amyloidogenesis in bulk solutions with real-time in situ nanoscopic imaging at the single-fiber level, we show that curli display polar growth, and we detect two kinetic regimes of fiber elongation. Single fibers exhibit stop-and-go dynamics characterized by bursts of steady-state growth alternated with periods of stagnation. At high subunit concentrations, fibers show constant, unperturbed burst growth. Curli follow a one-step nucleation process in which monomers contemporaneously fold and oligomerize into minimal fiber units that have growth characteristics identical to those of the mature fibrils. Kinetic data and interaction studies of curli fibrillation in the presence of the natural inhibitor CsgC show that the inhibitor binds curli fibers and predominantly acts at the level of fiber elongation.

Bacterial Sexuality at the Nanoscale.

Understanding the basic mechanisms of bacterial sexuality is an important topic in current microbiology and biotechnology. While classical methods used to study gene transfer provide information on whole cell populations, nanotechnologies offer new opportunities for analyzing the behavior of individual mating partners. We introduce an innovative atomic force microscopy (AFM) platform to study and mechanically control DNA transfer between single bacteria, focusing on the large conjugative pXO16 plasmid of the Gram-positive bacterium Bacillus thuringiensis. We demonstrate that the adhesion forces between single donor and recipient cells are very strong (∼2 nN). Using a mutant plasmid, we find that these high forces are mediated by a pXO16 aggregation locus that contains two large surface protein genes. Notably, we also show that AFM can be used to mechanically induce plasmid transfer between single partners, revealing that transfer is very fast (<15 min) and triggers major cell surface changes in transconjugant cells. We anticipate that the single-cell technology developed here will enable researchers to mechanically control gene transfer among a wide range of Gram-positive and Gram-negative bacterial species and to understand the molecular forces involved. Also, the method could be useful in nanomedicine for the design of antiadhesion compounds capable of preventing intimate cell-cell contacts, therefore providing a means to control the resistance and virulence of bacterial pathogens.

Forces guiding staphylococcal adhesion.

Staphylococcus epidermidis and Staphylococcus aureus are two important nosocomial pathogens that form biofilms on indwelling medical devices. Biofilm infections are difficult to fight as cells within the biofilm show increased resistance to antibiotics. Our understanding of the molecular interactions driving bacterial adhesion, the first stage of biofilm formation, has long been hampered by the paucity of appropriate force-measuring techniques. In this minireview, we discuss how atomic force microscopy techniques have enabled to shed light on the molecular forces at play during staphylococcal adhesion. Specific highlights include the study of the binding mechanisms of adhesion molecules by means of single-molecule force spectroscopy, the measurement of the forces involved in whole cell interactions using single-cell force spectroscopy, and the probing of the nanobiophysical properties of living bacteria via multiparametric imaging. Collectively, these findings emphasize the notion that force and function are tightly connected in staphylococcal adhesion.

Clumping Factor B Promotes Adherence of Staphylococcus aureus to Corneocytes in Atopic Dermatitis.

Staphylococcus aureus skin infection is a frequent and recurrent problem in children with the common inflammatory skin disease atopic dermatitis (AD). S. aureus colonizes the skin of the majority of children with AD and exacerbates the disease. The first step during colonization and infection is bacterial adhesion to the cornified envelope of corneocytes in the outer layer, the stratum corneum. Corneocytes from AD skin are structurally different from corneocytes from normal healthy skin. The objective of this study was to identify bacterial proteins that promote the adherence of S. aureus to AD corneocytes. S. aureus strains from clonal complexes 1 and 8 were more frequently isolated from infected AD skin than from the nasal cavity of healthy children. AD strains had increased ClfB ligand binding activity compared to normal nasal carriage strains. Adherence of single S. aureus bacteria to corneocytes from AD patients ex vivo was studied using atomic force microscopy. Bacteria expressing ClfB recognized ligands distributed over the entire corneocyte surface. The ability of an isogenic ClfB-deficient mutant to adhere to AD corneocytes compared to that of its parent clonal complex 1 clinical strain was greatly reduced. ClfB from clonal complex 1 strains had a slightly higher binding affinity for its ligand than ClfB from strains from other clonal complexes. Our results provide new insights into the first step in the establishment of S. aureus colonization in AD patients. ClfB is a key adhesion molecule for the interaction of S. aureus with AD corneocytes and represents a target for intervention.

Attachment of ribonucleotides on α-alumina as a function of pH, ionic strength, and surface loading.

The interactions between nucleic acids and mineral surfaces have been the focus of many studies in environmental sciences, in biomedicine, as well as in origin of life studies for the prebiotic formation of biopolymers. However, few studies have focused on a wide range of environmental conditions and the likely modes of attachment. Here we investigated the adsorption of ribonucleotides onto α-alumina surfaces over a wide range of pH, ionic strength, and ligand-to-solid ratio, by both an experimental and a theoretical approach. The adsorption of ribonucleotides is strongly affected by pH, with a maximum adsorption at pH values around 5. Alumina adsorbs high amounts of nucleotides >2 µmol/m(2). We used the extended triple-layer model (ETLM) to predict the speciation of the surface complexes formed as well as the stoichiometry and equilibrium constants. We propose the formation of two surface species: a monodentate inner-sphere complex, dominant at pH <7, and a bidentate outer-sphere complex, dominant at higher pH. Both complexes would involve interactions between the negatively charged phosphate group and the positively charged surface of alumina. Our results provide a better understanding of how nucleic acids attach to mineral surfaces under varying environmental conditions. Moreover, the predicted configuration of nucleotide surface species, bound via the phosphate group, could have implications for the abiotic formation of nucleic acids in the context of the origin of life.

N-Formylation modifies membrane damage associated with PSMα3 interfacial fibrillation.

The virulence of Staphylococcus aureus, a multi-drug resistant pathogen, notably depends on the expression of the phenol soluble modulins α3 (PSMα3) peptides, able to self-assemble into amyloid-like cross-α fibrils. Despite remarkable advances evidencing the crucial, yet insufficient, role of fibrils in PSMα3 cytotoxic activities towards host cells, the relationship between its molecular structures, assembly propensities, and modes of action remains an open intriguing problem. In this study, combining atomic force microscopy (AFM) imaging and infrared spectroscopy, we first demonstrated in vitro that the charge provided by the N-terminal capping of PSMα3 alters its interactions with model membranes of controlled lipid composition without compromising its fibrillation kinetics or morphology. N-formylation eventually dictates PSMα3-membrane binding via electrostatic interactions with the lipid head groups. Furthermore, PSMα3 insertion within the lipid bilayer is favoured by hydrophobic interactions with the lipid acyl chains only in the fluid phase of membranes and not in the gel-like ordered domains. Strikingly, our real-time AFM imaging emphasizes how intermediate protofibrillar entities, formed along PSMα3 self-assembly and promoted at the membrane interface, likely disrupt membrane integrity via peptide accumulation and subsequent membrane thinning in a peptide concentration and lipid-dependent manner. Overall, our multiscale and multimodal approach sheds new light on the key roles of N-formylation and intermediate self-assembling entities, rather than mature fibrils, in dictating deleterious interactions of PSMα3 with membrane lipids, likely underscoring its ultimate cellular toxicity in vivo, and in turn S. aureus pathogenesis.

WITHDRAWN: Aptamer-based nanotrains and nanoflowers as quinine delivery systems.

This article has been withdrawn: please see Elsevier Policy on Article Withdrawal (http://www.elsevier.com/locate/withdrawalpolicy). This article has been withdrawn at the request of the author, editor and publisher. The publisher regrets that an error occurred during the publication of this paper, which was intended to be published in International Journal of Pharmaceutics: X (not International Journal of Pharmaceutics). This error bears no reflection on the scientific content of this article or its authors. The publisher apologizes to the readers for this unfortunate error.

Controlling differentiation of stem cells via bioactive disordered cues.

Ideal bone tissue engineering is to induce bone regeneration through the synergistic integration of biomaterial scaffolds, bone progenitor cells, and bone-forming factors. Biomimetic scaffolds imitate the native extracellular matrix (ECM) and are often utilized in vitro as analogues of the natural ECM to facilitate investigations of cell-ECM interactions and processes. In vivo, the cellular microenvironment has a crucial impact on regulating cell behavior and functions. A PET surface was activated and then functionalized with mimetic peptides to promote human mesenchymal stem cell (hMSC) adhesion and differentiation into an osteogenic lineage. Spray technology was used to randomly micropattern peptides (RGD and BMP-2 mimetic peptides) on the PET surface. The distribution of the peptides grafted on the surface, the roughness of the surfaces and the chemistry of the surfaces in each step of the treatment were ascertained by atomic force microscopy, fluorescence microscopy, time-of-flight secondary ion mass spectrometry, Toluidine Blue O assay, and X-ray photoelectron spectroscopy. Subsequently, cell lineage differentiation was evaluated by quantifying the expression of immunofluorescence markers: osteoblast markers (Runx-2, OPN) and osteocyte markers (E11, DMP1, and SOST). In this article, we hypothesized that a unique combination of bioactive micro/nanopatterns on a polymer surface improves the rate of morphology change and enhances hMSC differentiation. In DMEM, after 14 days, disordered micropatterned surfaces with RGD and BMP-2 led to a higher osteoblast marker expression than surfaces with a homogeneous dual peptide conjugation. Finally, hMSCs cultured in osteogenic differentiation medium (ODM) showed accelerated cell differentiation. In ODM, our results highlighted the expression of osteocyte markers when hMSCs were seeded on PET surfaces with random micropatterns.

Aptamer-based nanotrains and nanoflowers as quinine delivery systems.

In this study, we designed aptamer-based self-assemblies for the delivery of quinine. Two different architectures were designed by hybridizing quinine binding aptamers and aptamers targeting Plasmodium falciparum lactate dehydrogenase (PfLDH): nanotrains and nanoflowers. Nanotrains consisted in controlled assembly of quinine binding aptamers through base-pairing linkers. Nanoflowers were larger assemblies obtained by Rolling Cycle Amplification of a quinine binding aptamer template. Self-assembly was confirmed by PAGE, AFM and cryoSEM. The nanotrains preserved their affinity for quinine and exhibited a higher drug selectivity than nanoflowers. Both demonstrated serum stability, hemocompatibility, low cytotoxicity or caspase activity but nanotrains were better tolerated than nanoflowers in the presence of quinine. Flanked with locomotive aptamers, the nanotrains maintained their targeting ability to the protein PfLDH as analyzed by EMSA and SPR experiments. To summarize, nanoflowers were large assemblies with high drug loading ability, but their gelating and aggregating properties prevent from precise characterization and impaired the cell viability in the presence of quinine. On the other hand, nanotrains were assembled in a selective way. They retain their affinity and specificity for the drug quinine, and their safety profile as well as their targeting ability hold promise for their use as drug delivery systems.

The impact of lipid polyunsaturation on the physical and mechanical properties of lipid membranes.

The lipid composition of cellular membranes and the balance between the different lipid components can be impacted by aging, certain pathologies, specific diets and other factors. This is the case in a subgroup of individuals with psychiatric disorders, such as schizophrenia, where cell membranes of patients have been shown to be deprived in polyunsaturated fatty acids (PUFAs), not only in brain areas where the target receptors are expressed but also in peripheral tissues. This PUFA deprivation thus represents a biomarker of such disorders that might impact not only the interaction of antipsychotic medications with these membranes but also the activation and signaling of the targeted receptors embedded in the lipid membrane. Therefore, it is crucial to understand how PUFAs levels alterations modulate the different physical properties of membranes. In this paper, several biophysical approaches were combined (Laurdan fluorescence spectroscopy, atomic force microscopy, differential scanning calorimetry, molecular modeling) to characterize membrane properties such as fluidity, elasticity and thickness in PUFA-enriched cell membranes and lipid model systems reflecting the PUFA imbalance observed in some diseases. The impact of both the number of unsaturations and their position along the chain on the above properties was investigated. Briefly, data revealed that PUFA presence in membranes increases membrane fluidity, elasticity and flexibility and decreases its thickness and order parameter. Both the level of unsaturation and their position affect these membrane properties.

Enzyme-free detection and quantification of double-stranded nucleic acids.

We have developed a fully enzyme-free SERRS hybridization assay for specific detection of double-stranded DNA sequences. Although all DNA detection methods ranging from PCR to high-throughput sequencing rely on enzymes, this method is unique for being totally non-enzymatic. The efficiency of enzymatic processes is affected by alterations, modifications, and/or quality of DNA. For instance, a limitation of most DNA polymerases is their inability to process DNA damaged by blocking lesions. As a result, enzymatic amplification and sequencing of degraded DNA often fail. In this study we succeeded in detecting and quantifying, within a mixture, relative amounts of closely related double-stranded DNA sequences from Rupicapra rupicapra (chamois) and Capra hircus (goat). The non-enzymatic SERRS assay presented here is the corner stone of a promising approach to overcome the failure of DNA polymerase when DNA is too degraded or when the concentration of polymerase inhibitors is too high. It is the first time double-stranded DNA has been detected with a truly non-enzymatic SERRS-based method. This non-enzymatic, inexpensive, rapid assay is therefore a breakthrough in nucleic acid detection.

Interplay of matrix stiffness and stress relaxation in directing osteogenic differentiation of mesenchymal stem cells.

The aim of this study is to investigate the impact of the stiffness and stress relaxation of poly(acrylamide-co-acrylic acid) hydrogels on the osteogenic differentiation of human mesenchymal stem cells (hMSCs). Varying the amount of the crosslinker and the ratio between the monomers enabled the obtainment of hydrogels with controlled mechanical properties, as characterized using unconfined compression and atomic force microscopy (AFM). Subsequently, the surface of the hydrogels was functionalized with a mimetic peptide of the BMP-2 protein, in order to favor the osteogenic differentiation of hMSCs. Finally, hMSCs were cultured on the hydrogels with different stiffness and stress relaxation: 15 kPa - 15%, 60 kPa - 15%, 140 kPa - 15%, 100 kPa - 30%, and 140 kPa - 70%. The cells on hydrogels with stiffnesses from 60 kPa to 140 kPa presented a star-like shape, typical of osteocytes, which has only been reported by our group for two-dimensional substrates. Then, the extent of hMSC differentiation was evaluated by using immunofluorescence and by quantifying the expression of both osteoblast markers (Runx-2 and osteopontin) and osteocyte markers (E11, DMP1, and sclerostin). It was found that a stiffness of 60 kPa led to a higher expression of osteocyte markers as compared to stiffnesses of 15 and 140 kPa. Finally, the strongest expression of osteoblast and osteocyte differentiation markers was observed for the hydrogel with a high relaxation of 70% and a stiffness of 140 kPa.

Synthesis and Characterization of Conjugated Hyaluronic Acids. Application to Stability Studies of Chitosan-Hyaluronic Acid Nanogels Based on Fluorescence Resonance Energy Transfer.

Hyaluronic acid (HA) was functionalized with a series of amino synthons (octylamine, polyethylene glycol amine, trifluoropropyl amine, rhodamine). Sodium hyaluronate (HAs) was first converted into its protonated form (HAp) and the reaction was conducted in DMSO by varying the initial ratio (-NH2 (synthon)/COOH (HAp)). HA derivatives were characterized by a combination of techniques (FTIR, 1H NMR, 1D diffusion-filtered 19F NMR, DOSY experiments), and degrees of substitution (DSHA) varying from 0.3% to 47% were determined, according to the grafted synthon. Nanohydrogels were then obtained by ionic gelation between functionalized hyaluronic acids and chitosan (CS) and tripolyphosphate (TPP) as a cross-linker. Nanohydrogels for which HA and CS were respectively labeled by rhodamine and fluorescein which are a fluorescent donor-acceptor pair were subjected to FRET experiments to evaluate the stability of these nano-assemblies.

Interaction of Tau construct K18 with model lipid membranes.

One of the hallmarks of Alzheimer's disease (AD) is the formation of neurofibrillary tangles, resulting from the aggregation of the tubulin associated unit protein (Tau), which holds a vital role in maintaining neuron integrity in a healthy brain. The development of such aggregates and their deposition in the brain seem to correlate with the onset of neurodegeneration processes. The misfolding and subsequent aggregation of the protein into paired helical filaments that further form the tangles, lead to dysfunction of the protein with neuronal loss and cognitive decline. The aggregation of the protein then seems to be a causative factor of the neurodegeneration associated with AD. The hypothesis of an involvement of the membrane in modulating the misfolding and assembly of Tau into paired helical filaments attracts increasing interests. To provide more insight about how lipids can modulate the interactions with Tau, we have conducted a comprehensive Atomic Force Microscopy (AFM) study involving supported lipid bilayers of controlled compositions with the Tau microtubule-binding construct K18. Particularly, the effects of zwitterionic and negatively charged phospholipids on the interaction have been investigated. Deleterious solubilization effects have been evidenced on fluid zwitterionic membranes as well as an inability of K18 to fragment gel phases. The role of negative lipids in the aggregation of the peptide and the particular ability of phosphatidylinositol-4,5-bisphosphate (PIP2) in inducing K18 fibrillization on membranes are also reported.

Actin Bundle Nanomechanics and Organization Are Modulated by Macromolecular Crowding and Electrostatic Interactions.

The structural and mechanical properties of actin bundles are essential to eukaryotic cells, aiding in cell motility and mechanical support of the plasma membrane. Bundle formation occurs in crowded intracellular environments composed of various ions and macromolecules. Although the roles of cations and macromolecular crowding in the mechanics and organization of actin bundles have been independently established, how changing both intracellular environmental conditions influence bundle mechanics at the nanoscale has yet to be established. Here we investigate how electrostatics and depletion interactions modulate the relative Young's modulus and height of actin bundles using atomic force microscopy. Our results demonstrate that cation- and depletion-induced bundles display an overall reduction of relative Young's modulus depending on either cation or crowding concentrations. Furthermore, we directly measure changes to cation- and depletion-induced bundle height, indicating that bundles experience alterations to filament packing supporting the reduction to relative Young's modulus. Taken together, our work suggests that electrostatic and depletion interactions may act counteractively, impacting actin bundle nanomechanics and organization.

The microbial adhesive arsenal deciphered by atomic force microscopy.

Microbes employ a variety of strategies to adhere to abiotic and biotic surfaces, as well as host cells. In addition to their surface physicochemical properties (e.g. charge, hydrophobic balance), microbes produce appendages (e.g. pili, fimbriae, flagella) and express adhesion proteins embedded in the cell wall or cell membrane, with adhesive domains targeting specific ligands or chemical properties. Atomic force microscopy (AFM) is perfectly suited to deciphering the adhesive properties of microbial cells. Notably, AFM imaging has revealed the cell wall topographical organization of live cells at unprecedented resolution, and AFM has a dual capability to probe adhesion at the single-cell and single-molecule levels. AFM is thus a powerful tool for unravelling the molecular mechanisms of microbial adhesion at scales ranging from individual molecular interactions to the behaviours of entire cells. In this review, we cover some of the major breakthroughs facilitated by AFM in deciphering the microbial adhesive arsenal, including the exciting development of anti-adhesive strategies.

High Speed AFM and NanoInfrared Spectroscopy Investigation of Aß1-42 Peptide Variants and Their Interaction With POPC/SM/Chol/GM1 Model Membranes.

Due to an aging population, neurodegenerative diseases such as Alzheimer's disease (AD) have become a major health issue. In the case of AD, Aß1 - 42 peptides have been identified as one of the markers of the disease with the formation of senile plaques via their aggregation, and could play a role in memory impairment and other tragic syndromes associated with the disease. Many studies have shown that not only the morphology and structure of Aß1 - 42 peptide assembly are playing an important role in the formation of amyloid plaques, but also the interactions between Aß1 - 42 and the cellular membrane are crucial regarding the aggregation processes and toxicity of the amyloid peptides. Despite the increasing amount of information on AD associated amyloids and their toxicity, the molecular mechanisms involved still remain unclear and require in-depth investigation at the local scale to clearly decipher the role of the sequence of the amyloid peptides, of their secondary structures, of their oligomeric states, and of their interactions with lipid membranes. In this original study, through the use of Atomic Force Microscopy (AFM) related-techniques, high-speed AFM and nanoInfrared AFM, we tried to unravel at the nanoscale the link between aggregation state, structure and interaction with membranes in the amyloid/membrane interaction. Using three mutants of Aß peptides, L34T, oG37C, and WT Aß1 - 42 peptides, with differences in morphology, structure and assembly process, as well as model lipidic membranes whose composition and structure allow interactions with the peptides, our AFM study coupling high spatial and temporal resolution and nanoscale structure information clearly evidences a local correlation between the secondary structure of the peptides, their fibrillization kinetics and their interactions with model membranes. Membrane disruption is associated to small transient oligomeric entities in the early stages of aggregation that strongly interact with the membrane, and present an antiparallel ß-sheet secondary structure. The strong effect on membrane integrity that exists when these oligomeric Aß1 - 42 peptides interact with membranes of a particular composition could be a lead for therapeutic studies.