Antibodies and their derivatives radiolabelled with positron- and gamma-emitting radiometals enable sensitive and quantitative molecular Positron Emission Tomography (PET) and Single Photon Emission Computed Tomography (SPECT) imaging of antibody distribution in vivo. Chelators that are covalently attached to antibodies allow radiolabelling with metallic PET and SPECT radioisotopes. Conventional strategies for chelator-protein conjugation generate heterogeneous mixtures of bioconjugates that can exhibit reduced affinity for their receptor targets, and undesirable biodistribution and pharmacokinetics. Recent advances in bioconjugation technology enable site-specific modification to generate well-defined constructs with superior properties. Herein we survey existing site-specific chelator-protein conjugation methods. These include chelator attachment to cysteines/disulfide bonds or the glycan region of the antibody, enzyme-mediated chelator conjugation, and incorporation of sequences of amino acids that chelate the radiometal. Such technology will allow better use of PET and SPECT imaging in the development of antibody-based therapies. Monoclonal antibodies (mAbs) have demonstrated exquisite sensitivity and selectivity for their target cell surface receptors in vivo Antibodies labelled with a gamma- or positron-emitting radionuclide can be used to quantitatively image the biodistribution of the radiolabelled-antibody using whole body Single Photon Emission Computed Tomography (SPECT) or Positron Emission Tomography (PET) respectively. Such radiolabelled mAbs are extremely useful for both preclinical and clinical development of antibody-based therapies, enabling (i) non-invasive detection of the target receptors’ expression, including any potential heterogeneity in expression, (ii) estimation of an antibody’s biodistribution, therapeutic index and pharmacokinetics by quantification of antibody distribution in target and normal tissues, and (iii) prediction and assessment of a patient’s response to a specific mAb therapy by imaging with the radiolabelled antibody Radioactive metal ions are well-suited to radiolabelling antibodies for PET and SPECT imaging. Compared to non-metallic radionuclides, radiometals allow for simple radiolabelling procedures: typically, a chelator is firstly covalently attached to the antibody, and once conjugated, the chelator binds the radiometal. The half-lives of many of the metallic radionuclides, including zirconium-89 Immunoglobulin type 1 antibodies  (IgGs) aMetallic radioisotopes are incorporated into an antibody via a chelator. Many factors influence the choice of a metallic radioisotope, including the imaging modality (PET or SPECT/γ-scintigraphy imaging), matching of the half-life of the radioisotope to the pharmacokinetics of the vector, and the availability of the radioisotope itself. The chelator binds the radiometal, and the resulting radiometal–chelator complex will ideally possess both high thermodynamic and kinetic stability. This high stability is essential to ensure that the radiometal remains bound to the antibody in vivo. We (i)mild conjugation (and subsequent radiolabelling) reaction conditions are essential to preserve the tertiary and quaternary structure of the antibody;(ii)the new covalent link between the chelator and protein must be stable under physiological conditions; and(iii)the covalent modification must not compromise the binding affinity and specificity of the protein.In chelator-antibody conjugation reactions, the antibody and chelator contain complementary reactive functional groups for attachment to each other. There are several requirements for this covalent attachment:N-hydroxysuccinimide esters  to antibodies non-specific lysine modification, as is often the case, site-selectivity is not actually achieved “Click chemistry” is one such approach both optical and radionuclide imaging labels, and site-selective methods to incorporate radiotherapeutic isotopes Here, we capture an overview of site-selective conjugation methods used to prepare radiometal-labelled antibodies and antibody derivatives for PET and SPECT imaging. This includes site-directed modification of cysteines/disulfide bonds and the glycan region of the antibody, enzyme-mediated conjugation, and incorporation of sequences of amino acids that coordinate the radiometal. Others have also recently reviewed this area, including an elegant and detailed survey of site-selective antibody conjugation methods used for molecular imaging with (i)cysteine has a low abundance (1–2%) in living organisms (ii)Ka of ∼ 8.3) exceeds the reactivity of other nucleophilic groups of amino acids in proteins the nucleophilicity of the cysteine deprotonated thiol group (p(iii)single, solvent-exposed cysteine residues can be engineered into antibodies and their derivatives Cysteines are useful for selective protein modification for several reasons:The most widely employed method of conjugating antibodies via cysteines involves a Michael addition reaction of a thiol with a maleimide to form a succinimidyl thioether adduct a.111In, and their biodistribution assessed in mice bearing colorectal carcinoma tumours. The  thioether-linked radiotracer enabled efficient tumour targeting, whereas the (pyridyldithiopropionate-derived) disulfide-bridged analogue showed poor biodistribution with high kidney uptake and poor tumour targeting, due to in vivo cleavage of the S—S linkage.Radionuclide imaging studies have demonstrated that succinimidyl thioether linkages have superior stability compared to disulfide linkages. For example, maleimide and pyridyldithiopropionate a/b groupMaleimide derivatives have been widely used to incorporate chelators, via cysteine thiols, into antibody derivatives and proteins 89Zr-DFO-mAbs and radiotherapeutic 177Lu-DTPA-mAbs that demonstrated higher C—S bond stability in serum than maleimide derivatives. Furthermore, methylsulfonyl phenyloxadiazole-derived 89Zr-DFO-huA33 antibody demonstrated superior in vivo targeting behaviour compared to its maleimide-derived analogue: the former resulted in higher tumour-to-background activity ratios in a murine model bearing huA33 antigen-expressing colorectal cancer xenografts.Several new cysteine-reactive reagents that provide enhanced conjugate stability have been developed Monobromo maleimide d [67], [Single cysteine-targeted conjugation strategies are well-suited for site-specific modification of Fab or scFv fragments that contain single, exposed cysteine residues, however, they are not ideal for generating well-defined conjugates of IgG antibodies. IgG proteins contain four solvent-accessible interchain disulfide bridges in the protein hinge region, and their reduction creates eight reactive thiols. Conjugation to these reduced species results in heterogeneous mixtures of conjugates, and the cleavage and modification of these disulfide bridges (with up to eight copies of cargo) can lead to adverse pharmacokinetics, and reduce the metabolic stability of IgG antibodies in plasma and re-bridging of two cysteines. It is important that the re-bridging motif reacts rapidly with both disulfide-derived reduced thiols, to avoid incorrect re-bridging, and thus preserve structure and function of the protein Recent research has produced functional species that react with two reduced thiol groups of antibodies, thus enabling concomitant attachment of functional groups  (i)modification at these sites will not compromise antigen binding properties as they are distal to the Fab region;(ii)there are two attachment sites available per antibody; and(iii)there are several chemoselective/orthogonal reactions enabling modification of glycans and hexose groups.IgG proteins contain two conserved post-translational modification glycosylation sites  that canThis method is not suitable for smaller antibody fragments that lack glycans, or for IgG1 antibodies whose function requires the presence of the native (unmodified) Fc regions for binding to Fc-receptors (such as those involved in immune responses).111In into the satumomab antibody via a DTPA chelator at the glycan region (see below). 111In-DTPA-satumomab targets a tumour-associated glycoprotein, TAG-72, expressed in several cancers, including colorectal and ovarian cancers 111In-DTPA-satumomab’s utility in detecting colorectal and ovarian cancer lesions, and in combination with other diagnostic tests, informing clinical decision-making, including treatment and surgery.Glycan-based modification chemistry has been critically important in the clinical development of molecular imaging with antibodies in nuclear medicine 111In-capromab pendetide , similarly incorporates a DTPA chelator using this glycan technology 111In-capromab pendetide targets the prostate specific membrane antigen (PSMA) expressed in prostate cancer, and has demonstrated potential in assisting in (i) the staging of prostate cancer, particularly in identifying soft tissue metastases, and (ii) locating prostate cancer tumours when diagnostic blood tests indicate disease recurrence 111In-capromab pendetide has not demonstrated high positive predictive value and specificity for clinical management of prostate cancer, it has been fundamentally important in the development of radionuclide molecular PET and SPECT imaging of its target, PSMA. Several PSMA-targeted PET and SPECT imaging agents are currently being clinically developed Another FDA-approved antibody imaging agent, cis-glycol groups of terminal hexoses , results in increased flexibility of this antibody region. This gives rise to the increased BTG-catalysed reactivity of a glutamine residue, Q295, in close proximity to N297, and chelators bearing 5-aminopentyl groups can be site-selectively introduced into this position in the presence of BTG The bacterial transglutaminase (BTG) enzyme catalyses the formation of a new amide bond between the primary amine of a lysine side chain and the γ-carboxamide group of a glutamine side chain. Many 5-aminopentyl derivatives can act as lysine side chain surrogates and are recognized by BTG, however, BTG is only reactive towards glutamine side chains located in flexible regions of proteins/biomolecules. Glutamine side chains of IgG antibodies are thus normally unreactive to BTG. Removal of glycans from position N297 of an IgG antibody, using the enzyme SrtA) catalyses a transpeptidase reaction between an N-terminal glycine, and a specific amino acid sequence, -Lys-Pro-X-Thr-Gly- (where X is any amino acid) 64Cu has been site-selectively appended to a scFv that targets GPIIb/IIIa glycoprotein receptors overexpressed in thrombosis, atherosclerosis and inflammation. Two different conjugation strategies have been described. In the first SrtA-catalysed conjugation, the scFv, engineered to contain a -Lys-Pro-X-Thr-Gly- sequence at the C-terminus, was reacted with an N-terminal glycine residue attached to a sarcophagine chelator 3+), into antibody derivatives 6 tag is commonly incorporated at the C-terminus of targeting proteins, although N-terminal incorporation is also possible. Many 99mTc-labelled proteins have been radiolabelled using this strategy, with recent examples including a HER2-targeted sdAb vβ6 integrin-targeted diabody 99mTc(CO)3+ coordinates via two imidazole groups of a His6 tag 6 tag have improved protein radiolabelling and biodistribution. For example, the inclusion of a thiol-containing cysteine residue to a His6-containing sequence can increase metallolabelling efficiency 6 amino acid sequence can increase hydrophilicity and negative charge of a radiolabelled protein, which in turn, can decrease radioactivity retention in non-target tissue in vivo, leading to improved image contrast. For example, substituting the sequence HHHHHH for HEHEHE does not adversely affect 99mTc-radiolabelling of a HER2-targeted protein, but it does decrease radioactivity retention in non-target tissue in vivo The hexahistidine sequence, or His99mTc radiotracers, including 99mTc-labelled compounds used in renal imaging and 99mTc-labelled peptides that target cell surface receptors 99mTcV. Peptide sequences incorporating these features can be engineered into proteins for efficient binding of the 99mTcVO3+ motif. The first example of this demonstrated that Gly4Cys, engineered into the C terminus of a scFv protein, could be applied to SPECT imaging of a scFv disease target 99mTcV(O)-Gly3Cys sequence has more favourable biodistribution properties (faster clearance and lower off-target organ retention) than a C-terminal 99mTcI(CO)3-His6 homologue 99mTcV(O) binding abilities x directly at the N-terminus (instead of Glyx-Cys directly at the C-terminus) can lead to release of 99mTc radionuclide in vivo, compromising imaging ability Several low molecular weight Advances in conjugation chemistry and protein engineering have enabled development of homogenous radiolabelled antibodies. New site-specific antibody modification strategies that have not yet been applied to PET/SPECT imaging with radiolabelled antibodies could similarly be adapted to generate well-defined chelator-antibody constructs. Several preclinical studies have highlighted that site-specifically modified antibodies have improved in vivo behaviour  relative to those modified using conventional, less specific methods. Site-specifically radiolabelled antibody-based radiopharmaceuticals will deliver new clinically-useful contrast agents for molecular PET/SPECT imaging, by (i) providing clinicians with better molecular imaging tools to predict whether a patient will respond to a particular treatment or intervention, and (ii) providing scientists with reliable tools to quantitatively map the in vivo behaviour of new antibody-based therapies and/or newly-discovered receptors that are drug targets. Such technology will also be critically important in providing antibody conjugates with precisely defined structures and stoichiometries that are acceptable to regulatory authorities.


The development of science and technology of advanced materials using nanoscale units can be conducted by a novel concept involving combination of nanotechnology methodology with various research disciplines, especially supramolecular chemistry. The novel concept is called ‘nanoarchitectonics’ where self-assembly processes are crucial in many cases involving a wide range of component materials. This review of self-assembly processes re-examines recent progress in materials nanoarchitectonics. It is composed of three main sections: (1) the first short section describes typical examples of self-assembly research to outline the matters discussed in this review; (2) the second section summarizes self-assemblies at interfaces from general viewpoints; and (3) the final section is focused on self-assembly processes at interfaces. The examples presented demonstrate the strikingly wide range of possibilities and future potential of self-assembly processes and their important contribution to materials nanoarchitectonics. The research examples described in this review cover variously structured objects including molecular machines, molecular receptors, molecular pliers, molecular rotors, nanoparticles, nanosheets, nanotubes, nanowires, nanoflakes, nanocubes, nanodisks, nanoring, block copolymers, hyperbranched polymers, supramolecular polymers, supramolecular gels, liquid crystals, Langmuir monolayers, Langmuir–Blodgett films, self-assembled monolayers, thin films, layer-by-layer structures, breath figure motif structures, two-dimensional molecular patterns, fullerene crystals, metal–organic frameworks, coordination polymers, coordination capsules, porous carbon spheres, mesoporous materials, polynuclear catalysts, DNA origamis, transmembrane channels, peptide conjugates, and vesicles, as well as functional materials for sensing, surface-enhanced Raman spectroscopy, photovoltaics, charge transport, excitation energy transfer, light-harvesting, photocatalysts, field effect transistors, logic gates, organic semiconductors, thin-film-based devices, drug delivery, cell culture, supramolecular differentiation, molecular recognition, molecular tuning, and hand-operating (hand-operated) nanotechnology.  By analogy with computational optimizations of efficiency and function of novel cyber technologies such as machine learning –5, the oFurther to historical efforts in organic synthesis and materials’ fabrication, functional optimization of materials involving structural regulation at the atomic/molecular scales and nanometre scales has been widely investigated and has been accompanied by the corresponding developments in observational techniques and instrumentation on those length scales –39. WhilEmergence of the nanoarchitectonics concept can be seen as an inevitable event in the development of science and technology and its historical background can be traced in the scientific discourse . The impAccording to Masakazu Aono, the guidelines of nanoarchitectonic strategies can be summarized as follows  63]:  dyes . Hard ta3+ ion. A similar strategy can be applied to the other heavy metal ions .For site-selective modification of inorganic nanostructures, Lvov and co-workers demonstrated site-selective modifications of clay nanotube through self-assembly-type nanoarchitectonics using Schiff base binding . Clay naSelf-assembly processes can be usually conducted under ambient and mild conditions, which makes the processes appropriate for nanoarchitectonics of biological materials –118. MatIn their recent review article, Sawada and Serizawa discuss the use of M13 phage, one of the filamentous viruses, as soft matter units . Upon geSelf-assembly processes are highly influenced by the surrounding media. Ichikawa and co-workers reported lyotropic liquid crystalline behaviours of amphiphiles in dication-type amino acid ionic liquids . Nature Langmuir monolayers on water would be powerful media to regulate two-dimensional self-assembly –128. KraNote that unexplored research issues still remain in self-assembly sciences. Theoretical investigations together with experimental demonstrations have been made for further understanding on the regulations and controls of self-assembly processes and resulting complexes. Hiraoka and co-workers have been systematically investigating the importance of complementary hydrophobic molecular surfaces for precise molecular association to form discrete self-assembled complexes upon quantitative analysis of self-assembly process . Even thRecently, self-assembly processes have been re-evaluated for their dynamics. Formation of supramolecular polymers –133, pol3.Following the above-mentioned brief summary of recent trends of self-assembly research, various topics of self-assembly systems are explained below according to certain classifications. In the first category, recent examples of self-assembly systems in three-dimensional media are described.3.1.3.1.1.60 alkyl ammonium cations. Binding of fluoride anion to the oxoporphyrinogen core through hydrogen bonding efficiently promotes the ultrafast charge separation from the singlet excited state of oxoporphyrinogen to C60. The charge separation can be maintained through conversion of electron-accepting oxoporphyrinogen unit to a donor by binding with fluoride anion. This nanoarchitected complex can act as a molecular transistor.Self-assembly is a highly versatile methodology to nanoarchitect functional systems from various small units. Sizes of the nanoarchitected systems vary from molecular-level complex to material-size objects. As a small self-assembled functional unit, Hill, D’Souza, and co-workers reported self-assembled molecular complex where photoinduced charge separation is achieved through fluoride-ion binding  137]. I. I137]. Similarly, self-assembled structures often provide unusual physical effects such as regulation of electronic properties. Intramolecular structure of a single molecule can be developed into intermolecular holes through self-assembly as seen in an example demonstrated by Rathore and co-workers . Two-dimCook and co-workers prepared a self-assembled cofacial cobalt porphyrin prism that acted as a catalyst for the oxygen reduction reaction . The cobDynamic conformational changes upon self-assembly can tune chiral sensitivity around transition metal complexes as demonstrated by Shinoda and co-workers . In thisn-dodecyl)--tetrabenzo-5,6,7,12,13,14-hexaazapentacene as a small molecular unit -metallopeptide in water . This moPhoto-controllable formation and deformation of supramolecular polymer was reported by Chen, Wang, and co-workers , who useN-terminal hydrophobic groups were synthesized and self-assembled into transmembrane channel structures within lipid bilayer membranes. Selective transmembrane transport properties of Cl ions were confirmed by planar bilayer conductance measurements. Cell-membrane-like assembled structures such as vesicles and structured micelles can be nanoarchitected from various small molecules. As an interesting example, Knight, Hawker, and co-workers reported morphologically shifting spherical assemblies upon metal coordination to assemblies of hybrid peptide–polymer conjugates  photodimerization upon metal–organic assembly with Ag(I) ions. Through this one-dimensional coordination polymer route, cycloaddition reaction to pyridyl-functionalized cyclobutane can be stereo-selectively achieved with quantitative yield. Precise structure formation upon coordination-based self-assembly can be used for selective encapsulation of guest molecules. Selective capture of C60-indene or C60-anthracene bisadducts by cubic cage assembled from 4-fold-symmetric porphyrins with Fe(II) or Zn(II) was reported by Nitschke and co-workers . B. B165]. ω-norbornenyl macromonomer and norbornenyl comonomers as diluents including polystyrene, poly, and polydimethylsiloxane macromonomers. On the basis of differences of reactivity ratios between macromonomer and diluent controls, the backbone sequences as blocky, gradient, or random as well as graft diblock polymer structures with tapered, uniform, and inverse-tapered molecular shapes. Self-assembly of these polymers creates nanoarchitectonic structures with different lamellar periods and domain thicknesses. Because of general applicability of the proposed method, it can be applied for various types of polymer materials and their assemblies possibly for future extensive customization.Grubbs and co-workers reported nanoarchitectonics strategy for the self-assembly of polymers having tailored distributions of grafting sites by grafting in ring-opening metathesis polymerization . SequencHigh capability in flexible structural shifts of polymer and oligomer self-assemblies can be used for various applications such as stimuli-responsive functions. Chu and Ravoo demonstrated preparation of hierarchical supramolecular hydrogels through peptide self-assembly with photo-controlled guest release functions, using Fmoc-RGDS peptide functionalized with photo-switchable arylazopyrazole . Self-asSite-specific arrangements of responsive polymer self-assembly can be used for macroscopic actions as seen in actuator functions of surface microscopic gel arrays reported by Morin and co-workers . They pr3.3.2 . M. M2 nano3.4.2.Although pairing specificity of recognition may be slightly inferior to DNA, peptides and proteins surely have high capability of self-assembly mainly through hydrogen bonding. Accumulation of hydrogen bonding upon well-designed three-dimensional nanoarchitectures often creates surprisingly precise recognition sites –209. PepPreparation of protein–metal–organic frameworks (protein–MOFs) was demonstrated by Tezcan and co-workers who could self-assemble the metal ions presented at the vertices of the ferritin nodes in the presence of dihydroxamate linkers in synthesis of protein–MOF structures . The forWell-designed peptide segments are also good components for self-assembly. Kimura and co-workers investigated self-assembled morphologies of various types of amphiphilic block polypeptides having aromatic groups at the C-terminal of a Leu-based hydrophobic helical block and actually confirmed self-assemblies into various structures such as distorted sheet, saddle-like sheet, micelle, and so on . Upon heCoacervate microdroplets are expected to be used for synthetic cell applications but they have to be stabilized with an appropriate coating. Williams, van Hest, and co-workers proposed the use of biodegradable triblock copolymer (terpolymer) with poly(ethylene glycol), poly-(caprolactone-gradient-trimethylene carbonate), and poly-(glutamic acid) segments . The des4.Interfaces such as solid surfaces, liquid–liquid interfaces, and gas–liquid interfaces are the media that cause various interesting effects in molecular interactions and self-assembly behaviours as compared with those observed in bulk solution media –217. In 4.1.2 groups was selectively covalently modified with fluorescent dye having a succinimidyl ester moiety, 5-(and-6)-carboxytetramethylrhodamine succinimidyl ester. The latter modification of fluorescent dye can be performed without significantly damaging the preformed Ag patterns. The reported technique would be useful for printed electronics. The same research group has also demonstrated photo-reactive SAM structures to create carboxyl groups upon photo irradiation . I. IN-substert-butyl-4-hydroxyphenyl)porphyrin by deposition under ultrahigh vacuum condition [Hill et al. also reported fabrication of binary molecular monolayers composed of tetrakis(4-pyridyl)porphyrin and tetrakis  232]. T. T232]. N-[(2-hydroxy-3-methyl-5-dodec-ylphenyl)methyl]-N-(carboxymethyl)-glycine disodium salt ligand on a highly oriented pyrolytic graphite . T. Tπ-gelaO-diacetyl-6-O-pheophorbide-cellulose at the air–water interface as demonstrated by Sakakibara et al. [0−S1 transition dipole moments of the chlorin Qy bands. Photocurrent activities of the rods self-assembled at the air–water interface were also confirmed.Light-harvesting cellulose-based nanorods were fabricated through the self-assembly of a regioselectively pheophorbide-carrying cellulose acetate, 2,3-a et al. . The nann-dodecylmellitic triimide was first spread at the air–water interface, which was interacted through hydrogen bonding with macrocyclic oligoamine, 1,4,7,10-tetraazacyclododecane in subphase. Transfer of the monolayer from water surface to a mica surface through simple one-touch contact resulted in the formation of regularly sized nanodisks with a defined thickness of ca. 3 nm and tuneable diameter in the range of tens of nanometres. The formation processes to give nanosized and micro-sized regular objects are composed of a series of simple procedures including monolayer spreading, mechanical compression, and surface touching where any highly clean processes such as ultrahigh vacuum are not required.Mori et al. reported conventional methods to fabricate regularly sized nanodisks through self-assembly at the air–water interface and one-touch transfer onto a solid substrate  283,284284. A moN-isopropylacrylamide) microgels reported by Buzza, Vogel, and co-workers [Colloidal objects can be self-assembled at the air–water interface as seen in anisotropic self-assembly of isotropic colloidal microspheres of polystyrene within poly ,298. Nan4.5.2.If molecular machines and nanocars are shifted to the air–water interface, direct observation of a single molecule becomes virtually impossible. Meanwhile, synchronized motions of laterally self-assembled molecular machines would be expected. The highly anisotropic nature of dynamic interfaces, such as the air–water interface, enables us to connect macroscopic motions with molecular functions  304–307307. LateOne typical example of molecular machine controlled by macroscopic mechanical motions is the mechanical regulation of capture and release of a target guest molecule by a molecular machine self-assembled at the air–water interface  310,311311. The 4.5.3.+ complex of a cholesterol-substituted cyclen, has the helicity originated by the chirality of the side arms, which is twisted to induce modified helicity and chirality upon gradual compression of their monolayer by mechanical lateral compression (d-valine is preferential to l-valine at lower pressure regions. However, recognition selectivity is reversed to l-valine preferential at higher pressure regions. Therefore, we can select the desirable chiral selectivity by mechanical receptor tuning just by simple mechanical actions. Such flexible tuning of chiral selection cannot be attained by standard method of molecular recognition and its analyses such as crystallographic characterization of host–guest complex.The same strategy can be applied to the fine tuning of molecular receptors self-assembled at the air–water interface. Enantioselective molecular receptor, octacoordinate Napression  [312,313−1 and LiCl concentration of 10 mM, binding constant for uracil derivative is 64 times larger than that for the thymine derivative. Mechanical tuning of molecular receptor within their self-assembled structures at dynamic interface can compensate huge efforts in organic synthesis to covalently construct complicated molecular receptors capable of such fine molecular discrimination.Efficient discrimination between uracil and thymine derivatives can be accomplished by molecular receptor tuning at the air–water interface although these resembling nucleic acid bases with only one methyl group difference cannot be usually discriminated even by naturally occurring DNA and RNA. In this case, cholesterol-substituted triazacyclononane molecules self-assembled at the air–water interface were used as molecular receptors  314,315315. MechThe above-mentioned methodology for molecular recognition, molecular tuning, can be regarded as a novel category of recognition mode  316–318318. As tMechanical tuning of molecular receptors at dynamic interfaces can be coupled with photochemical processes between chromophores for colorimetric reporting. For this purpose, molecular receptor having a phenylboronic acid as a recognition moiety and a carboxyfluorescein as an indicator part was designed and spread as a monolayer at the air–water interface . Prior tThe natures of molecular tuning at the air–water interface were investigated with a simple model system. Because dihedral angles of binaphthyl unit can be quantitatively monitored, an amphiphilic binaphthyl molecule, molecular pliers, as a model molecule was mechanically deformed by lateral pressure at the air–water interface . Changes4.5.4.In contrast to the above-mentioned highly dynamic functions of molecular machines and molecular receptors at interfacial environments, highly organized static and regular molecular assemblies at interfacial media are useful for another advanced functional system such as device fabrications –332. Owi5.This review article summarizes recent research examples of nanoarchitectonics of functional materials and systems. The earlier parts of this review illustrate self-assembling systems involving various components including small molecules, polymers, inorganic materials, and biomaterials. The almost unlimited variety of these self-assembled systems confirms the great versatility of self-assembling processes for the construction of functional nanoarchitectures. Then we provided examples of self-assembly research conducted using interfacial media, including self-SAMs, thin films on solid surfaces, assemblies at liquid–liquid interfaces, and Langmuir and LB films at the air–water interfaces. Specific characteristics and improvements in functionality were obtained depending on identity of the interface. Self-assembled materials share some basic characteristics. In contrast with the widely known three-dimensional media such as highly divided solution states, molecular motility and orientation in interfacial environments are restricted to two-dimensions often resulting in unusual organizations of self-assembled objects. Interfaces also provide interaction points for materials in different phases. Molecular interactions can be significantly modulated by the heterogeneous dielectric nature of some interfacial media. In cases of interfacial self-assembled systems, lateral macroscopic action can be coupled with molecular functionalities in a medium of molecular-level thickness. Molecular organizations and materials nanoarchitected using self-assembly at interfacial media, including aqueous surfaces and electrodes, are anticipated to be applied in advanced functional systems such as molecular machine controls, sensors, and devices. Therefore, the increasing contributions of interfacial environments and their structural characteristics will have a significant influence on the future research direction of self-assembly-coupled nanoarchitectonics.Aspects of self-assembly-based nanoarchitectonics can be found in biomolecular evolutionary processes. It is obvious that interfacial environments such as cell surfaces, protein interior surfaces, and bio-macromolecular interfaces play critical roles in the functional expression of self-assembled bio-systems. Therefore, an idea of what might be achieved utilizing self-assembly nanoarchitectonics in artificial systems can be found in current biological systems. Several characteristics of highly evolved biological systems have not yet been fully accomplished using self-assembled nanoarchitectonics; for example, establishing highly sophisticated functional relays in self-assembled systems. As can be found in photosynthetic systems and signal transduction systems, highly efficient and directionally specified relays for function, signal, information, energy, and electron transfers operate as conventional actions in biological systems on the basis of highly evolved molecular dispositions in biological self-assemblies. Such high levels of organization have not been fully realized in artificial self-assembly nanoarchitectonics. Nanoarchitecting such high-level functional organizations is one of the future goals. Another potentially useful feature of biological systems is the selection and molecular ‘decision-making’ based on harmonization between multiple sometimes ambiguous responses. Beyond the precise relay of information, summation of ambiguity can result in circumstance-considered selection of outputs. This feature has not been accomplished in artificial self-assembled systems and it is a significant target to be accomplished using self-assembly nanoarchitectonics in the near future.Biological systems and nanoarchitectonics, in turn, have crucial common features . On a scNanoarchitectonics based on self-assembly relies both on exploration of chemical designs ,336 and


Tumor-targeting antibodies were initially defined as a group of therapeutic monoclonal antibodies (mAb) that recognize tumor-specific membrane proteins, block cell signaling, and induce tumor-killing through Fc-driven innate immune responses. However, in the past decade, ample evidence has shown that tumor-targeting mAb (TTmAb) eradicates tumor cells via activation of cytotoxic T cells (CTLs). In this review, we specifically focus on how TTmAbs induce adaptive anti-tumor immunity and its potential in combination therapy with immune cytokines, checkpoint blockade, radiation, and enzyme-targeted small molecule drugs. Exploring the mechanisms of these preclinical studies and retrospective clinical data will significantly benefit the development of highly efficient and specific TTmAb-oriented anti-tumor remedies. Along with HER1 (EGFR), HER3 and HER4, these proteins belong to the type I growth receptor family  and Ab-resistant EGFR-transduced B16 mouse tumor models  is another member of the type I growth receptor family. Although HER2 is characterized by lacking an identified ligand, EGFR has been found to have several other ligands besides EGF, such as transforming growth factor alpha (TGF-α)  and activates Dendritic Cells (DCs) to better process tumor antigens for cross-priming T cells in the DLN; tumor-specific CTLs travel back to the tumor site for tumor control. They further demonstrated the role of CTLA-4 in Tregs within advanced B-cell lymphoma in limiting anti-CD20-mediated tumor regression. Thus, anti-CTLA-4 and anti-CD20 combined treatment is a possible new clinical strategy in overcoming adaptive resistance and preventing relapse of B-cell lymphoma.Rituximab is a murine–human chimeric antibody that recognizes the human B-cell CD20 antigen featuring primary response rates up to 70% -associated systemic toxicity in the host -positive lymphoma  targeting into EGFR+ but anti-PD-L1-resistant tumor tissues , also known as tumor necrosis factor superfamily member 14 (TNFSF14), is one of the co-stimulatory molecules for T-cell activation  is a pleiotropic cytokine that promotes proliferation of NK and T cells induced by antigen stimulation  and programmed death-1 (PD1) are two of the major co-inhibitory immune checkpoint molecules expressed on T cells. PD1 ligand (PDL1) is another immune inhibitory molecule expressed on dendritic cells, activated T cells, and tumor cells. Anti-CTLA4 and anti-PD1/PD-L1 monoclonal antibodies have been developed as checkpoint blockades to inhibit the suppressive function of CTLA4 or PD-1/PD-L1 Pardoll . Physiolet al.et al.Studies have shown that checkpoint blockades can reverse T-cell suppression and improve the therapeutic effects. However, only a small number of patients are sensitive to such therapy. There is a pressing need for mechanism studies on this subject and potential mechanisms for synergistic effects. In the past 10 years, we and other laboratories have shown that anti-EGFR, anti-HER2, anti-CD20, and anti-CD47 in combination with immune checkpoint blockade could have synergistic effects in host adaptive anti-immune responses and tumor eradication. The mechanisms of such synergistic effect may be complicated. Antibodies targeting to tumor cells can not only reduce tumor burden but also change tumor microenvironment. Some antibodies, such as anti-Her2/neu, can increase IFN through MyD88, while others, such as anti-CD47, induce IFN through STING pathway. Our study also demonstrates that CTLA4 is a major immune suppressor in A20 tumors and leads to anti-CD20 resistance. Anti-CTLA-4 and anti-CD20 combined treatment overcomes adaptive resistance and prevents relapse in the mouse B-cell lymphoma model  is a cancer treatment that employs high dose of radiation to kill tumor cells and reduce tumor burden. Not only tumor cells but also stromal cells at the tumor site can be affected by radiation. Radiation-damaged cancer cells release tumor antigens, DNA and RNA that are captured by antigen-presenting cells to promote the priming and activation of cytotoxic T cells, and to facilitate further infiltration of immune cells. Radiation-induced inflammatory response may result in multiple IR-resistant signaling that facilitate tumor relapse (Barcellos-Hoff et al.Our previous study shows that upregulation of the PD-L1/PD-1 in tumor after IR inhibits radiation-induced anti-tumor immune responses and facilitates tumor relapse. Combination of IR with PD-L1 blockade results in the elimination of MDSCs by T-cell-derived TNF (Deng et al.et al.et al.et al.et al.et al.et al.Antibody treatment in combination with multiple chemo-therapeutic agents has been investigated in a number of tumor models (Pegram Molecular targeted therapy functions through blocking the growth of cancer cells by interfering with tumor-specific targeted molecules needed for oncogenesis and tumor growth. Tyrosine kinase inhibitors that target EGFR family members are among the most successful targeted cancer therapies (Arteaga and Engelman et al.Immunotherapy, such as nivolumab or pembrolizumab, has been approved as the standard second-line or third-line treatment for TKI-resistant patients with high-level PD-L1 expression in tumors. Unfortunately, recent clinical trials were prematurely terminated due to significant side effects from checkpoint blockade combined with prolonged treatment of EGFR TKIs, which has achieved higher response rates (Ahn Recent work exploring the role of the tumor-targeting Ab in cancer therapy has yielded significant progress. Our laboratory’s research and the work of other research groups show that tumor-targeting Ab can be effectively combined with traditional small molecule chemo- and targeted therapy as well as newly developed checkpoint blockades, thus providing new potential for creating highly specific therapies. By targeting tumors, these new therapies would address the fundamental mechanism behind tumor growth and metastasis, namely breaking immune tolerance. It is the TTmAb that bridges the innate and adaptive immunity to modulate tumor microenvironment, overcomes drug resistance, and induces effective anti-tumor response to achieve tumor regression. This is the underlying principle behind the ongoing interest in combining targeting Ab with radiation and small molecule chemo- and targeted therapies.Although this review mainly focuses on the functional studies and the clinical application of tumor-targeting antibodies, recent evidence has shown further development of such antibody fusion proteins armed with inflammation cytokines, including IFN, LIGHT, and IL-2. Moreover, immune checkpoints have been deemed critical for the cancer immune tolerance. As such, tumor-targeting checkpoint blockade would be a worthy direction for extensive future study. Exploring the synergistic treatment effect between targeting antibodies, immune cytokines, checkpoint blockade, radiotherapy, and small molecule chemo- and targeted therapies holds great promise for elucidating the underlying immunology mechanisms as well as developing effective therapies for a broad range of malignant diseases.


Drosophila melanogaster models, we investigate TE activation in association with Tau pathology in Alzheimer’s disease (AD). Leveraging RNA sequencing from 636 human brains, we discover differential expression for several retrotransposons in association with neurofibrillary tangle burden and highlight evidence for global TE transcriptional activation among the long interspersed nuclear element 1 and endogenous retrovirus clades. In addition, we detect Tau-associated, active chromatin signatures at multiple HERV-Fc1 genomic loci. To determine whether Tau is sufficient to induce TE activation, we profile retrotransposons in Drosophila expressing human wild-type or mutant Tau throughout the brain. We discover heterogeneous response profiles, including both age- and genotype-dependent activation of TE expression by Tau. Our results implicate TE activation and associated genomic instability in Tau-mediated AD mechanisms.Aging and neurodegenerative disease are characterized by genomic instability in neurons, including aberrant activation and mobilization of transposable elements (TEs). Integrating studies of human postmortem brain tissue and  Drosophila melanogaster models, Guo et al. show that Alzheimer’s disease Tau neurofibrillary tangle pathology activates transcription of transposable element loci. An altered retrotransposon transcriptional landscape and associated genomic instability are implicated in Tau-mediated neurodegenerative mechanisms.Integrating studies of human postmortem brain tissue and   Drosophila, Tau induces global nuclear chromatin relaxation (Alzheimer’s disease (AD) is the most common neurodegenerative disorder and the leading cause of dementia, with more than 13 million individuals projected to be affected in the United States by 2050 . At autolaxation , abnormalaxation . ImportaHERV-K) has been demonstrated in human cortical and spinal neurons in ALS, and the encoded Envelope (Env) protein is neurotoxic (Transposable elements (TEs) are mobile genetic sequences present in all eukaryotic genomes examined to date . Althougurotoxic .Drosophila transgenic models, highlighting global TE activation in AD and implicating Tau-mediated mechanisms.Despite the evidence for genomic instability, retrotransposon expression has not been systematically evaluated in AD. In one small study, no differences in L1 genomic copy number were detected based on targeted PCR . Here, wTo examine whether AD neurofibrillary tangle pathology is associated with TE activation, we first leveraged data from 2 prospective human clinical-pathologic studies, the Religious Orders Study and Rush Memory and Aging Project (ROSMAP). Our analyses included 636 deceased subjects with completed brain autopsies along with transcriptomic profiling of the dorsolateral prefrontal cortex based on RNA sequencing (RNA-seq). Clinical and demographic characteristics of our study cohort are detailed in HERV-Fc1, which is unique for being present at low genomic copy number . Indeed, positive associations , indicating enhanced H3K9Ac, were detected at 4 out of 13 regions with available data; 3 regions remained significant after adjustment for multiple testing  or a mutant form associated with familial FTD (TauR406W) causes age-dependent neuronal loss in association with hyperphosphorylated, misfolded Tau protein in the adult fly brain. We initially profiled 12 Drosophila TEs, including 8 LTR retrotransposons and 4 non-LTR retrotransposons, which have been previously demonstrated to be active in the fly nervous system, either following aging and/or manipulation of TE surveillance mechanisms  and L1-like, non-LTR retrotransposons (het-a). Notably, the gypsy TE is an endogenous insect retrovirus , aging, and the specific element examined. These findings were confirmed using an available RNA-seq dataset, permitting comprehensive assessments of TE expression signatures in 20-day-old TauWT versus control flies  demonstrated positive changes consistent with Tau-dependent activation . As a further control, the TauWT and TauR406W strains were each independently backcrossed to w1118 controls for 5 generations, ensuring a homogeneous genetic background. qPCR confirmed Tau-dependent increases in het-a, copia, and gypsy expression in aged animals  with reverse transcriptase and endonuclease activity. Notably, most L1 retrotransposition in the human genome is believed to be due to the evolutionary young L1H subfamily, and such elements were absent from our top hits , atm, p53, and Ago3, are potent modulators of Tau-induced neurodegenerative phenotypes in Drosophila ; (2) ELAV-GAL4/+; UAS-TauWT/+; and (3) ELAV-GAL4/+; UASTauR406W/+. qPCR was performed from total RNA prepared from fly heads in triplicate samples, using RpL32 as an internal control. Expression  was normalized to that of 1-day-old control animals. One-way ANOVA was performed to detect differences between group mean expression values, considering each TE and time point separately. Subsetted t tests (two-tailed) were subsequently performed for post hoc comparisons of each Tau genotype with control animals. For copia, two-way ANOVA was secondarily performed to differentiate genotype and age effects. Error bars in all analyses represent the SEM. RNA-seq data from ELAV-GAL4/+; UAS-TauWT/+ or control (ELAV-GAL4/+) fly heads were analyzed using SalmonTE. Statistical significance was set to FDR < 0.1. For analyses of TE copy number, genomic DNA was extracted from heads of 10-day-old flies prior to qPCR.The escribed . The fol1234


Sleep disturbance can aggravate pain, and we recently found that insomnia treatment improved osteoarthritis (OA) pain, lowered inflammation, and improved quality of life in middle-to-older aged adults. Inadequate slow wave sleep (SWS), known as deep or restorative sleep, can decline with aging and is linked to pain and inflammation. We examined how insomnia treatment affects SWS, and the relationship between SWS and pain. In a pilot trial, 33 adults, ages 51 to 74 years with OA-related knee pain and insomnia, were randomized to 6-session CBTi (n=16) or a weekly phone contact control group (n=17). The CBT-I group showed significantly more laboratory-measured SWS across a study night than controls after controlling for baseline SWS. Greater SWS intensity was associated with lower OA-related pain among the CBT-I group, but not among controls. These preliminary data suggest that behavioral sleep treatment may strengthen the beneficial influence of restorative sleep on pain.


Mesenchymal stem cells (MSCs) have been reported to possess regulatory functions on immune cells which make them alternative therapeutics for the treatment of inflammatory and autoimmune diseases. The interaction between MSCs and immune cells through paracrine factors might be crucial for these immunomodulatory effects of MSCs. Extracellular vesicles (EVs) are defined as bilayer membrane structures including exosomes and microvesicles which contain bioactive paracrine molecules affecting the characteristics of target cells. Recently, several studies have revealed that EVs derived from MSCs (MSC-EVs) can reproduce similar therapeutic impacts of parent MSCs; MSC-EVs could regulate proliferation, maturation, polarization, and migration of various immune effector cells and modulate the immune microenvironment depending on the context by delivering inflammatory cytokines, transcription factors, and microRNAs. Therefore, MSC-EVs can be applied as novel and promising tools for the treatment of immune-related disorders to overcome the limitations of conventional cell therapy regarding efficacy and toxicity issues. In this review, we will discuss current insights regarding the major outcomes in the evaluation of MSC-EV function against inflammatory disease models, as well as immune cells. MSCs (IL-) 10 , 10–15. (IL-) 10 –22. More(IL-) 10 –30.Extracellular vesicles (EVs) are bilayer membrane structures transferring bioactive components, including proteins, lipids, and coding and noncoding RNAs –33. The in vivo administration [in vivo compared to other foreign particles. Moreover, it is relatively easy to modify and/or improve the EV contents and surface property for enhancing the therapeutic potential or for utilizing as a drug delivery system [Although therapeutic potential of MSCs has been proven in preclinical studies and clinical trials for myriad diseases, conventional MSC therapy has several critical limitations to overcome; since MSCs act as a “living material” derived from different individuals, quality control is one of the major hurdles for their therapeutic use. Isolation procedure, culture condition, storage methods, and administration of MSCs can significantly affect cell viability as well as efficacy, leading to high cost and low reproducibility , 41. Furstration . In addistration , 43. Thestration , 45. Duey system –48.in vitro and in vivo models mainly focusing on their immunomodulatory properties to provide up-to-date information in EVs and MSC therapeutic fields.In this review, we will summarize and discuss the major studies investigating the efficacy of MSC-EVs in both in vivo observations, therapeutic potential of MSC-EVs has been proven against various animal models of diseases accompanied by excessive inflammation (In a number of ammation .In a rat model of inflammatory bowel disease (IBD) induced by trinitrobenzene sulfonic acid (TNBS), intravenous injection of EVs derived from rat bone marrow MSCs improved the gross and histological symptoms of colitis, including body weight loss, disease activity index, destruction of the colonic structure, and immune cell infiltration via attenuation of colonic inflammation, oxidative stress, and apoptosis . In a siα transcripts within joints treated with EVs [Cosenza et al. reported the therapeutic efficacy of MSC-EVs against inflammatory arthritis using murine models of delayed-type hypersensitivity (DTH) and collagen-induced arthritis (CIA). MSC-EVs exerted the dose-dependent anti-inflammatory effects in both models through the inhibition of B cell maturation, as well as the induction of regulatory B cells in lymph nodes . In a siwith EVs .β exerted higher therapeutic efficacy against murine sepsis models than EVs from naïve MSCs. They demonstrated that EVs from primed MSCs effectively polarized macrophages into the M2 type, which is the anti-inflammatory phenotype of macrophages. Importantly, miR-146a, a well-known anti-inflammatory miRNA, was significantly upregulated in IL-1β-treated MSCs and packed into EVs. miR-146a packed in EVs was transferred into macrophages to polarize them into the M2 type [Sepsis, a systemic inflammatory response against microbial infection, is one of the targets for MSC-based therapy, because the mortality rate of sepsis remains high in intensive care units despite advanced development of antibiotics. Recently, MSC-EVs have been evaluated for their efficacy in rodent models of sepsis induced by cecal ligation. In a rat model of sepsis syndrome, treatment of adipose MSC-derived EVs alleviated the systemic inflammatory response, organ damage, and subsequent lethality. In the study, the potency of EVs derived from healthy  or apoptotic (induced by hypoxia and serum starvation) MSCs was compared. Although no significant difference was observed in the mortality rate, healthy MSC-derived EVs more efficiently downregulated the levels of inflammatory mediators compared to apoptotic MSC-derived EVs . In anot M2 type .α, and interferon- (IFN-) γ. On the contrary, the serum IL-10 level was elevated by EV treatment [In murine models of acute graft-versus-host disease (GVHD) induced by allogeneic transplantation of hematopoietic stem cells, EVs released from human umbilical cord MSCs (hUC-MSC-EVs) were assessed for prophylactic effects. hUC-MSC-EVs ameliorated the symptoms and histopathology of GVHD, leading to the increased survival rate. An absolute number of cytotoxic T cells were significantly decreased in the EV-treated group along with the downregulated serum levels of IL-2, TNF-reatment .γ were significantly decreased whereas those of IL-4, IL-10, and TGF-β were increased along with the elevation in regulatory T (Treg) cell proportion [Type-1 diabetes mellitus (T1DM) is an autoimmune disorder leading to the irreversible destruction of insulin-producing cells in pancreatic islets. A recent study revealed that EVs derived from adipose tissue-derived MSCs can reduce clinical symptoms of streptozotocin- (STZ-) induced T1DM. Intraperitoneal injection of EVs into T1DM mice prevented hyperglycemia, body weight loss, lethality, and islet degeneration by STZ. Moreover, in splenocytes from EV-treated T1DM mice, levels of IL-17 and IFN-oportion . Given toportion .Since MSCs can accelerate the healing of tissues from injury or wound through the immunomodulatory function, several groups tried to investigate whether MSC-EVs could reproduce this ability. Li et al. demonstrated that EVs from human umbilical cord MSCs could attenuate excessive inflammation induced by burn injury. In the study, miR-181c in EVs was found to be critical for immunoregulation and EVs overexpressing miR-181c more efficiently reduced inflammation in burned rats . In addiα and IL-1β, while M2 macrophages are regarded as anti-inflammatory cells producing immune-modulating factors such as IL-10 [In vitro migration assay proved that CCR2 of MSC-EVs acts as a scavenger for CCL2 and hence prevents macrophagic accumulation and further tissue damage. Indeed, EVs isolated from CCR2 siRNA-transfected MSCs failed to provide the protective effects of control siRNA EVs. Similarly, high levels of various chemokines such as chemokine (CXC motif) ligand 10 (CXCL10), monocyte chemoattractant protein 1 (MCP-1), CXCL9, and tissue inhibitor of metalloproteinase-1 (TIMP-1) led to an accumulation of cytotoxic macrophage in colitis-induced colons, while MSC-EVs could reduce these proinflammatory cytokines and macrophage-mediated tissue damage [Macrophages, one of the principal components of the innate immune system, are originated from either yolk sac during embryonic development (tissue-resident macrophages) or bone marrow-derived monocytes (circulating macrophages) and involved in inflammatory response via phagocytosis and antigen presentation as well as in tissue homeostasis . Upon acas IL-10 . Since Mas IL-10 , 64. In as IL-10 , 66 and as IL-10 . They exe damage . In addie damage . In thisα, and IL-6 from M1 macrophages while enhancing the M2 macrophage-derived immunomodulatory factor, Arginase 1 (Arg1). Others have shown that MSC-EVs can modulate tissue-specific macrophage polarization towards the tissue regenerative/repair phenotype. Notably, in vivo tracking data of fluorescence-labelled MSC-EVs after intravenous injection suggested that EVs have homing capacity to the injury site as MSC itself and macrophages, especially M2 types, are the primary target cell of EV localization in the damaged spinal cord [In addition, the therapeutic capacity of MSC-EVs targeting imbalance of M1/M2 polarization has been proven in various disease models. In an experimental murine model of bronchopulmonary dysplasia (BPD), MSC-EVs ameliorated pulmonary fibrosis and the histological lung injury score by reducing hyperoxia-induced inflammation . Authorsnal cord . Using anal cord .κB (NF-κB) inhibition. On the contrary, EV-derived Let-7b activated the STAT3 pathway, one of the transcriptional repressors of inflammatory signaling, as well as survival-related Akt signaling. Overall, LPS-pre-EVs could accelerate wound healing during diabetes compared to MSC-EVs. In another report, MSCs under hypoxic culture condition were found to secrete EVs containing wound healing process-related microRNAs such as miR-223, miR-146b, miR-126, and miR-199a and these EVs readily correct the M1/M2 balance in muscle injury models [Since EVs contain various bioactive molecules including peptides, lipids, and nucleotides, preconditioning of MSC with inflammatory stimuli could contribute to generating more immunoreactive EVs. Ti et al. reported that EVs produced by lipopolysaccharide- (LPS-) treated MSC (LPS-pre-EVs) exhibited more potent M2 induction capacity than those from control MSCs . Interesy models . Finallyy models . To harvβ and PGE2, leading to an induction of regulatory T cells during DC and naïve T cell coculture.A growing number of studies suggest that other effector cells of innate and adaptive immune systems could be regulated by MSC-EVs. Similar to macrophages, dendritic cells (DCs) can function as antigen-presenting cells and bridge innate to adaptive immune systems. Interestingly, several reports have already demonstrated that MSCs have suppressive roles in DC activation in a secretory factor-dependent manner, implying that MSC-EVs itself could regulate the fate of DCs –78. Inde+ NK cell trafficking within the lesions, although the exact contributing EV factors underlying these therapeutic outcomes have not been defined yet [γ- and TNF-α-primed MSCs were localized in CD19+ B cells and CD56+ NK cells as well as CD3+ T cells and exhibited some immunosuppressive effects by miR-155- and miR-146-dependent inhibition of cell proliferation [In a rat experimental autoimmune uveitis (EAU) model, periocular injection of MSC-EVs restored EAU damage and retinal functions by reducing CD161ined yet , 81. In feration . The immferation .in vitro and in vivo experiments. Blazquez et al. reported that EVs from human adipose tissue-derived MSCs can suppress the proliferation of T cells [2 were abundant in the supernatant with MSC compared to MSC-EVs [A large body of studies have shown that the immunomodulatory action of MSCs is partially mediated by the suppression of proliferation, differentiation, and activation of T lymphocytes , 84. Thi T cells . Moreove T cells . Another T cells . In addi T cells . In muri T cells , 59. Alt T cells , 90. Con MSC-EVs . These din vitro analysis for the evaluation of MSC-EVs was performed. In mixed leukocyte reaction using patient-derived peripheral blood mononuclear cells (PBMCs), MSC-EVs exhibited the suppressive effect on the proliferation of PBMCs secreting proinflammatory cytokines, including IL-1, TNF-α, and IFN-γ. Moreover, MSC-EV treatment in the patient resulted in significant improvement of GvHD symptoms for more than four months. In another study, the therapeutic effect of MSC-EVs was investigated in forty patients with chronic kidney disease (CKD) [β and IL-10 in peripheral blood were increased at 12 weeks and even 1 year after MSC-EV treatment, whereas the level of TNF-α was decreased.So far, two clinical studies of MSC-EVs have been performed. In the study by Kordelas et al. , a patiese (CKD) . MSC-EVsIn vivo distribution analysis of fluorescence-labelled EVs has shown that MSC-EVs might have homing capacity to the injured or tumor-bearing site as MSCs. They also evaluated the long-term toxicity and immunogenicity of repetitive EV administration using hematological examination, histopathological analysis, and immunotyping test, which all supported that MSC-EVs might not trigger any immune response or toxic reaction. Further preclinical and clinical evaluation of EVs in various disease conditions should be followed to ensure the safety and efficacy of MSC-EVs.These two clinical studies propose MSC-EVs as promising immunomodulatory therapeutics; however, the following challenges should be considered for the practical application of EVs. First of all, acquiring large scales of MSC-EVs with high purity would be a main issue in this field. Since MSC-EVs are isolated from MSC culture media, culture conditions including the seeding cell number, media volume, and EV harvest timing can influence both the quantity and quality of EVs. In addition, the most effective EV isolation method from culture media has not been established yet. Therefore, optimization of culture methods  combining with intensive evaluation of the pros and cons of the different EV isolation methods should be preceded to improve the yield of MSC-EVs and these procedures should be regulated and controlled to ensure the clinical-grade exosome production. Recently, Mendt et al. evaluated the therapeutic effects of BM-MSCs on pancreatic cancer xenograft mouse models to provide feasible directions for clinical application of MSC-EVs . In thisSince MSC-EVs theoretically contain various MSC-derived bioactive molecules, precise mechanisms of action or key therapeutic factors have not been disclosed. To define the key factors, comparative transcriptome/proteome analysis of MSC-EVs has been conducted and revealed their differential properties in terms of functional enrichment of gene analysis and microRNA expression patterns , 94. TheA number of most recent experimental evidences suggest that MSC-derived EVs might carry similar immunomodulatory properties of MSCs, which could be beneficial for the treatment of inflammatory diseases via direct immunosuppressive function, as well as for the regenerative purpose through the improvement of the inflammatory niche. As discussed in this review, EVs from human or animal MSCs mostly contributed to the attenuation of excessive inflammation to alleviate the symptoms of immune disorders or improve the efficiency of allogeneic transplantation. Because EVs possess valuable advantages in that they can overcome the reported limitations of parental cells, including safety, reproducibility, and cost effectiveness related with storage and maintenance, there is no doubt that MSC-EVs might be a novel promising therapeutics. However, although EV's modes of action in macrophage polarization and B/NK/T cell suppression have been reported as in their parental cells, a variety of further investigations are required to precisely elucidate their mechanisms regarding immunosuppression or tolerance induction, specific for each disease condition. Furthermore, the standardization and optimization of EV production should be established along with the investigation of their efficacy and underlying mechanisms to resolve the current hurdle in the development of EV-based therapy.


Fraxinus) species in North America for at least the past 25 years. The development of methods for detecting visually-hidden EAB galleries at early stages of infestation would provide a useful tool to more effectively facilitate the planning and implementation of targeted EAB pest-suppression and management activities. We tested the efficacy of using a dual-technology electronic-nose (e-nose)/gas chromatograph device as a means for detection of EAB infestations in green ash trees in different EAB-decline classes by analysis of VOC emissions in sapwood. We found significant differences in VOC profiles for trees from the four decline classes. The VOC composition, quantities, and types of volatile metabolites present in headspace volatiles varied considerably across sample types, and resulted in distinct e-nose smellprint patterns that were characteristic of each unique chemical composition. In addition, specific VOC metabolites were identified as potential healthy and EAB-infestation biomarkers, indicative of the health states of individual trees. Few significant differences in major bark phenolic compounds were found between ash decline classes using LC-MS. The e-nose was effective in discriminating between uninfested and EAB-infested trees based on sapwood VOC emissions.The emerald ash borer (EAB) has been the most destructive and costly nonnative insect to threaten the health of ash ( The early detection of invasive insect pests in living trees, prior to the occurrence of external visible signs and symptoms of damage, is essential for effective forest and urban pest-management treatments. Stem-boring insects are particularly difficult to detect and control with the absence of visual indicators, especially before significant internal damage results in tree mortality and economic losses. Developing an effective strategy for early pest detection, such as for many xylophilous insect-borer species that cause damage to trees, is often important in the production of hardwood trees grown for quality lumber or other high-valued forest wood products. Wood-boring insects frequently cause internal damage due to the production of larval galleries that contribute to tree mortality, lumber degrades and cull losses in numerous hardwood species . NonnatiAgrilus planipennis, is a nonnative invasive insect pest that originated in eastern Asian hardwood forests and most likely was accidently introduced into the U.S. in the early 1990s in southeast Michigan (near Detroit), although not officially first detected and recognized as a nonnative pest in that state until 2002 −, formide adducts ([M+CHOOH-H]-), and electrospray-induced dimers. The 192 curated ions and their retention times were used to conduct a targeted quantification in the TraceFinder 4.1 software, using ICIS peak detection algorithm with smoothing set to 5, a 30 s retention time window, and other parameters adjusted as needed for each ion to achieve acceptable peak integration that was uniform across all samples. Every peak was checked visually, and the integration adjusted manually, if necessary. For each compound, peak areas for each symptomatic crown-decline class (2–4) were compared with peak areas for healthy trees (1) using a Student’s t-test.The results of chromatographic and e-nose data presented here are limited to chemical analyses of low molecular-weight VOC emissions from sapwood cores and nonvolatile heavier molecular-weight metabolites from bark extracts.Sensor responses of the e-nose sensor array to complex gaseous mixtures of volatile metabolites, released into sampling-chamber headspace, were analyzed statistically using 3-d PCA to measure and quantify differences between e-nose aroma signatures produced in response to VOCs released from healthy and EAB-infested sapwood. A 3-d PCA aroma map data plot of uninfested  vs. the three combined EAB-decline classes, subgroups with various levels of EAB-infestations (represented by red triangles), showed the distribution of data clusters with relative chemical relatedness between aroma classes of VOC-metabolite emissions from green ash sapwood cores A. This PThe plotted e-nose aroma data cluster for VOCs from uninfested green ash sapwood cores was well separated from combined data clusters for VOCs from sapwood cores taken from trees with the three levels of EAB-infestation . The overall discrimination index was DI = 39  for this 3-d PCA plot analysis. There were two data points, discovered as outliers derived from two heavily-infested sapwood core samples, that putatively were taken in very close proximity to an EAB larval gallery from heavily EAB-infested trees. VOC emissions from cores represented by these two outliers were found to be chemically different from sapwood core VOC-emissions from all other heavy-infestation core samples, putatively taken at a greater distance from a larval gallery. VOC emissions from outlier cores contained additional induced VOCs probably derived directly from larvae and frass sources within EAB galleries. Data from GC analyses of core VOC emissions, derived from both constitutive and infestation-induced volatiles, provided additional evidence identifying two chemically-distinct VOC-emission types (groups) from heavily infested sapwood cores , provided a means for visually indicating differences in chemical relatedness between VOC-metabolite mixtures derived from sapwood headspace volatiles for each e-nose aroma class or sample type B. Data cDFA distances between centers of data clusters of the DFA aroma plot map, determined from pairwise comparisons of aroma-class data clusters, yielded indications of absolute chemical differences in VOC-metabolite mixtures of aroma classes represented by distinct, color-coded data clusters. This information provided a metric with an exact quantitative and statistical indicator, via pattern discrimination index (PDI) percentage differences, of chemical relatedness between aroma classes as presented in These significant differences of > 93% PDI, recorded for all three pairwise comparisons, suggested high levels of e-nose discrimination between VOC-metabolites of healthy and infested sapwood sample types. The lowest DFA distances were found between the light and moderate EAB-infestation sapwood samples, although still with high levels of difference and discrimination at a PDI percentage of 90.1%. Intermediate levels of chemical differences (PDI ≈ 92%) were found between VOC-metabolites of light- and severe-infestation sample types and between moderate- and severe-infestation aroma-sample classes.Additional MOS e-nose analysis of headspace VOC metabolites derived from green ash sapwood cores, based on sensor-intensity responses for the top 32-sensors within the sensor array providing the greatest discrimination between sample types, produced sensor response patterns (smellprints) that varied significantly with relative levels of EAB-infestation of trees from different crown health-class categories associated with EAB-decline A–D. SensAnalysis of smellprint signatures of VOC-metabolites from EAB-infested sapwood cores produced considerably different results from uninfested cores. Almost half  of all sensors in the selected array had no responses to VOC-metabolites from EAB-infested cores, regardless of decline rating or level of sapwood infestation. The strongest sensor intensity response (range 7.5–8.9k) to VOC emissions from cores taken from separate trees with all three levels of EAB-infestation (decline ratings 2–4) occurred with MOS sensor S-26 (1225.08-2-H). The remaining ≤ 29 sensors in the array produced relatively low sensor-intensity responses (≤ 2k) to EAB-infested sapwood cores.Variability in individual sensor responses, among the three levels of EAB-infested cores, were notably observed for differences in sensors that had no responses and in differences in relative intensity of responses of specific sensors to changes in VOC-metabolite emissions for each decline rating with associated different levels of EAB infestations.A comparison of combined smellprint signatures resulting from simultaneous analysis of VOC metabolites from sapwood cores of trees from all four crown class ratings (different aroma classes) yielded a VOC smellprint profile showing more clearly the differences in sensor-response intensities of individual sensors in the array for all EAB-decline sample types . The mosComparisons of differences in sensor-response intensities to VOC-emissions from healthy and EAB-infested cores (from trees in various stages of EAB-decline) provided data relating to changes or shifts in VOC production over time, as healthy trees become infested and infestation levels in sapwood increase proportionally to more advanced stages of decline. This comparison is possible because sensor-response intensities are semi-quantitatively correlated with concentrations of VOCs present in sapwood-core headspace. In the majority of cases (60%), sensor response intensities to VOC-emissions from EAB-infested sapwood were greater than the same sensor-responses to VOCs from healthy sapwood when sensor responses  occurred for all four sample types analyzed. The rare instances where VOC-emissions from healthy cores were greater than those from all infested cores occurred only for six sensors, including sensor S2 (422.25-1-H), S4 (508.53-1-H), S7 (588.05-1-H), S17 (494.05-2-H), S19 (627.91-2-H), and S27 (1386.82-2-H).The analysis of GC chemical data from the dual-column fast-gas GC portion of the e-nose provided information relating to the relative quantities and numbers of VOCs that comprised the complex headspace volatile emissions from green ash sapwood core sample types of each decline rating, simultaneously analyzed with the MOS e-nose sensor array. The major peaks and associated retention times (RTs) of VOCs present in volatile emissions from cores of each crown health class (decline rating) are presented with peak areas in Metabolomic clusters were defined by whether constitutive VOCs produced by both healthy and EAB infested sapwood had metabolomic changes in relative quantities of VOCs produced. The groupings of sensor clusters were based on whether the peak areas were uniform, decreasing, or increasing in quantities of VOC-emissions from cores of EAB-infected trees relative to uninfested  control cores. Four constitutive VOCs  were identified with uniform peak areas for both healthy and infested cores. Two VOCs (peaks 2 and 3) decreased in peak area for cores from infested trees  with the exception of near-gallery severely-infested cores. Two additional VOCs  generally increased  in VOC peak areas for infested tree cores (derived presumably from host-induced or insect-derived sources) compared to cores from healthy trees. Four VOCs  also had uniform peaks areas for infested cores, compared to healthy, except for putative near-gallery cores. The two cores defined as putative near-gallery cores (a subtype among the severe decline samples), showed exceptionally high VOC emissions (> 6 to 15-fold increase) compared to those of healthy trees and were considered outliers.Other VOC emissions, produced differentially by sapwood cores of the four health class (decline-rating) categories, were identified and clustered sequentially by peak number into three categories based on emission source, including the first group  that were only released from cores of healthy trees, a second group (nonspecific VOCs) not apparently constitutively produced  nor induced by infestation, and a third group of VOCs which were produced only in cores of EAB-infested trees (decline ratings 2–4). The major peaks and RTs of VOCs present in volatile emissions from cores of each crown health class are presented with peak areas, based on sample type and emission-source clusters, in The tentative identities of host-derived constitutive VOCs identified from analysis of GC peaks, previously identified from The five constitutive VOCs , identified with uniform peak areas from both healthy and infested cores, were identified as aliphatic diketones and esters, carboxylic acids, sulfhydril ketone, lactone, and bicyclic sesquiterpenes. Two VOCs (peaks 2 and 3) that showed decreased emission in cores from infested trees relative to healthy trees tentatively were identified as aliphatic aldehydes, ketones, and alkanes.Two VOCs (peaks 1 and 9) that increased in infested cores relative to healthy cores were identified as a methyl ester and bicyclic monoterpene, respectively. Four additional VOCs  which had uniform peak areas for healthy and infested cores, but much higher peak areas for putative near-EAB gallery cores, were identified as volatile, bicyclic monoterpenes, aliphatic alkanes, esters, cyclic monoterpenes, homocyclic ethyl esters, and possibly pyran monoterpenes.The most notable major constitutive VOC (peak 7), largest in abundance based on peak area, was among those uniformly produced in all infested cores, but increased significantly in putative near-EAB-gallery sapwood cores, and was tentatively identified as most probably (1S)-(-)-α-pinene (a bicyclic monoterpene). The second largest constitutive VOC (peak 2) by area which decreased in infested cores compared to healthy was identified as propanal . The third largest VOC (peak 11) was identified as limonene (a cyclic monoterpene), while the fourth largest VOC (peak 8) was identified as either 3-ethyloctane or 3-methylnonane  or amyl-propanoate . The identity of peak 12, produced in lesser quantities but still at significantly high emissions, curiously was identified as either ethyl cyclohexane-carboxylate (a homocyclic ethyl ester) or cis-rose oxide (a pyran monoterpene). Two additional constitutive VOCs, rarely produced in sapwood of healthy and EAB-infested trees, included a peak at RT = 53.47 s, tentatively identified as aliphatic aldehydes E,E-2,4-hexadienal or methional, and a peak at RT = 60.79 s, tentatively 1-methylpropyl benzene or 2-ethyl-3-methyl-pyrazine (a pyrazine derivative).The tentative chemical identities of source-specific VOCs, produced only by sapwood of healthy hosts or only from EAB-infested sapwood due to either infestation-induced host response or direct insect-derived emissions as previously identified in peak clusters specified in The most significant VOC emissions exclusively produced from EAB-infested cores were tentatively identified for peak 9 as 6-decenal  or methyl salicylate (an aromatic esters) and peak 14 as butyl salicylate (aromatic ester) or β-caryophyllene (a bicyclic sesquiterpene), peak 10 as nerol (a monoterpene), peak 11 as hexyl 2-butenoate . Finally, peak 15 was identified as α-bisabolol oxide A  or caryophyllene acetate (a bicyclic sesquiterpene ester). One additional VOC emission specific to EAB-infested sapwood was discovered at RT = 67.89 s, identified as either n-nonanal , p-menthatriene (a monocyclic diene), or (±)-cis-rose oxide (a pyran monoterpene), which was not produced in healthy sapwood. All of the VOC emissions limited to infested cores were less volatile and higher molecular weight compounds than most healthy-specific VOCs, having RTs >72 s and KRIs >1200 for a 10 m DB-5 capillary column.LC-MS analysis detected 192 nonredundant ions in the mass spectrum of green ash bark methanolic extracts, including some of the major compounds previously identified in green ash bark, such as verbascoside, ligustroside, and oleuropein . However6 ) nonvolatile, polar phenolic compounds found by LC-MS analysis of bark extracts, which are presented in There were only 16 highly abundant , including most of the lower molecular weight compounds (RTs = 1.3–9.2 min) due to the lack of matches with any known compounds in the MS-database. Two phenolics, (peaks 72 and 88) with RTs = 9.9–10.8 min, decreased in abundance in the early decline stage (class 2) and matched the m/z of the parent and MS2 fragment ions of verbascoside, a defense biochemical in ash phloem. Two other peaks (107 and 127) had the fragment ion 461 in common with verbascoside, and could be a verbascoside isomer and derivative, respectively. One phenolic (peak 149) matched the mass spectral characteristics of ligustroside. Peak 103 matched the mass spectrum of oleuropein, and another phenolic (peak 154) was consistent with oleuropein hexoside.The majority  of the 192 total compounds extracted from green ash bark were not significantly affected by EAB infestation, regardless of decline class . Of the Electronic-nose analysis of VOC-emission profiles from tree cores of uninfested and EAB-infested trees (in various stages of decline) using PCA indicated significant differences in the composition of headspace volatiles from sapwood before and after EAB attack. Two distinct PCA data clusters (representing VOC-emissions from infested and uninfested) were observed, along with a smaller subgroup of outlier samples from heavily-infested trees, which were putatively derived from cores taken near or immediately adjacent to larval galleries. VOC emissions from the outlier heavily-infested sample types contained additional induced VOC components, likely derived directly from larval and/or frass sources. The majority of other cores taken from heavily-infested trees, presumably taken further away from larval galleries, lacked these additional induced VOC emissions. Subsequent GC-chemical analysis of VOCs provided strong evidence from peak areas, indicating that VOCs derived from putative near-gallery cores occurred at significantly higher concentrations than were observed in all other infested cores from different decline classes.Analysis of VOC-emission profiles from sapwood cores of all four decline classes, previously defined as an FIA ash-crown condition rating scale modified by Smith ,39, by DSmellprint signatures, derived from e-nose sensor-array response intensity patterns, of VOC emissions from sapwood of healthy trees consisted of strongly-positive responses for most sensors across the array. By contrast, the smellprint signatures of VOC emissions from sapwood cores of lightly infested, moderately-infested, and heavily-infested trees were superficially very similar and exhibited a sparse sensor response pattern and only weakly positive or no responses for most sensors in the array. These results suggested a much wider diversity of VOC composition and higher concentrations (abundance or quantities) of VOC emissions from healthy vs. EAB-infested sapwood.The emissions of a wider range of VOC types and greater quantities in healthy sapwood compared with emissions from sapwood of infested trees is most likely explained by host-induced shifts in metabolic pathways away from some normal anabolic activities to production of chemically and structurally related compounds for wound healing and perhaps ineffective host-defenses. Previous research has suggested that trees initiate inducible defenses in four stages in response to insect and disease attacks by downregulation of gene expression and energy resources devoted to primary metabolic activities, and upregulation of secondary metabolic pathways to produce phenolics, suberin, lignin derivatives, and phytoalexins etc., for antibiosis and antixenosis host-defense strategies ,43,44,45We identified at least eight VOCs in sapwood emissions from healthy trees that were absent in the sapwood of EAB-infested trees. The compounds were tentatively identified to include aliphatic alcohols, nitriles, aldehydes, esters, ketones, alkanes, and possibly alkyl benzenes. Further research with known chemical standards, MS, and nuclear magnetic resonance (NMR) spectroscopy will be required to confirm VOC identifications. We attribute the broad, strongly-positive response of most sensors composing the smellprint signature of healthy sapwood to be due primarily to these healthy biomarker metabolites. Healthy-plant specific VOCs, considered here as one type of plant-health chemical biomarker (not found in infested trees), may provide an effective means for identifying and chemically detecting green ash trees that have not yet been attacked by EAB. We consider these compounds to be healthy-plant biomarkers of potential utility for early detection of EAB-infestation because these compounds disappear once EAB-infestation occurs. More research will be required to determine if these plant-health biomarker VOCs detected in the sapwood are specific to green ash or also occur in VOC emissions from sapwood of other ash species.Five unique VOC emissions also were found to be induced only in EAB-infested sapwood. These compounds, identified tentatively to include aliphatic aldehydes, alcohols, salicylate esters, monoterpenes, and sesquiterpenes, were all formed in response to EAB-infestation, but absent from healthy host sapwood. We consider these specific induced VOCs to be EAB-infestation biomarkers, a second type of plant-health chemical biomarker, but indicative of infestations and absent in the sapwood of healthy trees. Following insect damage, major metabolic shifts may occur to seal wound sites, produce defensive compounds, conserve carbon resources, and attract natural enemies  of invading insect herbivores ,52,53,54Crook et al.  found thWe detected 192 non-redundant ions by LC-MS in green ash bark methanolic extracts, including some major compounds previously identified in green ash bark, such as verbascoside, ligustroside, and oleuropein. The limited induction of host chemical-defense compounds within bark tissue of EAB larvae-infested bark is consistent with previous studies that found little evidence of constitutive or induced defenses against EAB in North American ash species ,56,57,58Villari et al.  suggesteElectronic-nose instruments have the potential to be useful devices for early detection of insect infestations of trees based on identification of uniquely specific VOC emissions from affected parts of the host ,65,66,67Screening tree sapwood and bark VOC emissions could be used to help facilitate selection of trees for early preemptive and prophylactic EAB-control treatments. EAB adults are attracted to trees that are already infested, which leads to intensified attacks as females continue to lay eggs in the bark of susceptible trees that are already stressed. Consequently, early treatments may help prevent the success of initial EAB attacks, reduce the attraction of infested trees, and slow down or prevent further gallery development and colonization of the tree. We have identified new potential sapwood VOC biomarkers, both specific healthy biomarkers and EAB-infested biomarkers, which may facilitate the identification of uninfested and infested ash trees in the absence of visual EAB-infestation indicators. The use of smellprint signature libraries of green ash trees in various health states could be used to help develop EAB forest pest-management strategies for minimizing established EAB infestations.Portable e-nose devices and methods could be developed for field-use with application-specific VOC-signature libraries to noninvasively detect healthy and EAB-infestation biomarker emissions from sapwood of individual trees (even at early stages of EAB infestations) to determine their current health status. Such early detections would give land managers a better opportunity to obtain an early indication of the health state of individual trees useful for planning and implementing targeted EAB pest-suppression activities, and to intervene more successfully to protect trees against infestation and subsequent attack through application of early treatments and pest management actions.


To the Editors:2 The World Health Organization recommends that FSWs retest for HIV frequently to detect early HIV infection.3 Frequent HIV testing is also important for engagement in HIV prevention interventions, including treatment as prevention5 and pre-exposure prophylaxis.7HIV testing is important among female sex workers (FSWs) because they are at increased risk of HIV acquisition compared with members of the general population.14 The benefits of HIV testing , however, rely on correct interpretation of self-test results. HIV self-testing randomized controlled trials among FSWs in Uganda13 and Zambia14 found that HIV self-testing achieved near-universal HIV testing coverage and substituted for facility-based testing. In traditional HIV testing and counseling, HIV test results are interpreted by a trained health care professional. With HIV self-testing, the tester must correctly interpret the self-test results without professional assistance and often only the aid of the manufacturer's self-test instructions.HIV self-testing is a promising new HIV testing strategy in sub-Saharan Africa (SSA) that has been shown to increase HIV testing in diverse populations.24 In most of these studies, participants received pretest training and interpreted their own self-test result.24 None of these HIV self-testing performance studies were conducted among FSWs,24 an important key population for HIV prevention interventions.A number of oral HIV self-testing performance studies conducted in SSA found high participant-interpreted HIV self-test sensitivity and specificity (≥94% sensitivity and >98% specificity).13We explore how well FSWs in Kampala, Uganda, who received pretest training and had 2 previous opportunities to HIV self-test, can interpret images of HIV self-test results.13 Eligible participants were: 18 years or older, reported exchanging sex for money or goods (past month), HIV status naive or HIV-negative and did not report recent HIV testing (past 3 months), and Kampala-based.13 For this study, we only included participants randomized to the HIV self-testing intervention arms: direct provision of an HIV self-test from a peer educator or provision of coupon exchangeable for an HIV self-test at a health care facility from a peer educator, shortly after enrollment and 3 months later.13 The trial used OraQuick Rapid HIV-1/2 Antibody Tests , which came with a written and pictorial instruction guide (available in both English and Luganda). The trial received ethical approval from Mildmay Uganda and the Harvard T.H. Chan School of Public Health.13 All participants provided written informed consent.From October to November 2016, participants were enrolled in a three-armed HIV self-testing cluster randomized controlled trial in Kampala, Uganda.We used peer educators to conduct pretest HIV self-test training in a group setting (1 peer educator and 8 participants). The training occurred shortly after enrollment during a peer educator visit that lasted approximately 45 minutes and included information on how to use an HIV self-test and interpret the results. The peer educators had a standardized guide that they were instructed to follow and were observed by research assistants to ensure the quality and consistency of information transmitted.Participants completed a quantitative assessment at 4 months after enrollment. Here, they were asked to interpret standardized images of HIV self-test results: strong HIV-negative, strong HIV-positive, inconclusive, and weak HIV-positive. Images were presented to scale, in color, on laminated cards and were identical to those included in the manufacturer's instruction guide, which participants received to aid their interpretations. Participants were first shown an image of a strong HIV-positive or strong HIV-negative result. The image presented first reflected the result of their last HIV test, self-reported at 1 month after enrollment. Inconclusive and weak HIV-positive results were next presented in a random order. At 4 months, participants were given the option to complete a rapid HIV test . We collected electronic data using CommCare .We calculated the percentage of participants who incorrectly interpreted each of the self-test results and measured FSW-interpreted HIV self-test sensitivity and specificity. We used participant interpretations of the strong HIV-positive and strong HIV-negative self-test result images to respectively calculate self-test sensitivity and specificity; the interpretation of these images specified in the manufacturer's instruction guide were used as a reference for these measurements. We measured FSW-interpreted HIV self-test negative predictive values and positive predictive values using our sensitivity and specificity measurements and the HIV prevalence of our study population measured at 4 months with rapid HIV testing. Binomial 95% confidence intervals (CIs) were estimated for all measures. We used Stata 13.1  for all analyses.At enrollment, the majority of participants were younger than 30 years , self-reported the ability to read and write , completed up to 9 years of education , and had previously tested for HIV . At 4 months, almost all participants reported using an HIV self-test at least once , and participation in rapid HIV testing was 83% (452/544).Figure 24Incorrect interpretation of HIV self-test results is common among Kampala-based FSWs, even after pretest training and 2 previous opportunities to HIV self-test. The FSW-interpreted HIV self-test sensitivity and specificity measurements in this study are far below those measured in most of the previous SSA HIV self-testing performance studies.22 The peer-led pretest training in this study represents a realistic model for FSWs because peer educators are already a common approach for providing public sector health services to FSWs in SSA.28 Early at-home pregnancy tests went through a number of redesigns to make the test results more interpretable to users .30 To reduce misinterpretation of self-test results among FSWs, more research studies should be conducted on the design of HIV self-tests, the appropriate level of pre-test training, and the usefulness of on-demand support.Our HIV self-test performance measurements may differ from those in previous studies as a result of differences in pretest training. In previous HIV self-test performance studies, the pretest training provided was often individualized, extensive, and likely unrealistic or too expensive for a scalable HIV self-testing intervention.24 Understanding how well individuals can interpret HIV self-test results without the influence of previous HIV status knowledge is important because HIV self-testing has the potential to move HIV testing outside the health care system.13 In this unregulated environment, individuals may use HIV self-tests for first-time HIV testing or to test the HIV status of other individuals, such as a child or sexual partner.Methodological differences between our study and previous HIV self-testing performance studies may additionally explain our lower HIV self-test performance measurements. In our study, participants interpreted images of HIV self-test results rather than self-tests used to test themselves. In previous studies, measurements of self-test performance may have been biased because participants' previous knowledge of their HIV status may have influenced their interpretation of self-test results.31 higher prevalence of substance use,34 and differences in educational attainment.37Unique characteristics of FSWs may also explain the lower HIV self-test performance measurements in this study. Compared with other populations, FSWs may have challenges interpreting HIV self-test results for reasons including lower levels of health literacy,22 Participant misinterpretation of strong HIV-negative and strong HIV-positive self-test results was less common, but more concerning: false perceptions of HIV-positive status may cause emotional distress,38 result in stigma and discrimination,39 and alter prevention behaviors,43 whereas false perceptions of HIV-negative status may delay linkage to care, increasing the risk of poor health outcomes44 and secondary transmission of HIV.Concerns related to incorrect interpretation of HIV self-test results vary, based on which results are misinterpreted and how they are misinterpreted. Participant misinterpretation of inconclusive and weak HIV-positive self-test results was common, but in real-world settings, these results are rare.45 Second, we did not collect self-tests used by participants and thus were unable to measure the prevalence of weak HIV-positive and inconclusive self-test results. Third, participants may have paid less careful attention when interpreting an image of a self-test result rather than their own self-test result.This study has several limitations. First, participants did not interpret self-test results in a random order and thus, exposure to previous results may have influenced interpretations of later results.46 The effect of HIV self-testing may be diminished, however, if self-testers do not correctly interpret self-test results. To avoid misinterpretation of HIV self-test results that can result in false perceptions of HIV status, policy makers should considering implementation of realistic pretest training and on-demand HIV self-test support, whereas HIV self-test manufacturers consider redesign of HIV self-tests.HIV self-testing has the potential to dramatically increase HIV testing and aid in the achievement of 90% HIV status knowledge among all individuals living with HIV by 2020.