Site‐selective protein functionalization serves as an invaluable tool for investigating protein structures and functions in complicated cellular environments and accomplishing semi‐synthetic protein conjugates such as traceable therapeutics with improved features. Dual functionalization of proteins allows the incorporation of two different types of functionalities at distinct location(s), which greatly expands the features of native proteins. The attachment and crosstalk of a fluorescence donor and an acceptor dye provides fundamental insights into the folding and structural changes of proteins upon ligand binding in their native cellular environments. Moreover, the combination of drug molecules with different modes of action, imaging agents or stabilizing polymers provides new avenues to design precision protein therapeutics in a reproducible and well‐characterizable fashion. This review aims to give a timely overview of the recent advancements and a future perspective of this relatively new research area. First, the chemical toolbox for dual functionalization of proteins is discussed and compared. The strengths and limitations of each strategy are summarized in order to enable readers to select the most appropriate method for their envisaged applications. Thereafter, representative applications of these dual‐modified protein bioconjugates benefiting from the synergistic/additive properties of the two synthetic moieties are highlighted. Site‐selective dual protein functionalization serves as an invaluable tool for investigating protein structures and functions in complicated cellular environments and accomplishing semi‐synthetic protein conjugates. The methodologies, as well as the applications of dual‐functionalized protein bioconjugates are highlighted in this review. They play many essential roles in a myriad of biological processes, such as molecular transport, energy conversion, inter‐ and intramolecular signaling.1, 2 NIn this context, the past decades revealed significant progress in the development of new methodologies for site‐selective protein functionalization to install the desired functionalities at pre‐defined sites.9, 10 Nevertheless, along with the increasing demand for multifunctional bioconjugates to perform more sophisticated biological studies in vitro as well as in vivo, appending only one type of functionality to proteins is often insufficient to customize proteins for the desired applications. For example, despite the clinical success of ADCs, classical ADCs equipped with a single type of anticancer drug could still suffer from low efficacy, drug resistance, unfavorable pharmacokinetics, immunogenicity, and the inherent hydrophobicity of the drug, which greatly hampers their further in vivo applications.18, 19In this review, dual functionalization is defined as the incorporation of two different functionalities into proteins in a site‐selective fashion, which is accomplished either at two different amino acids (AA) sites or at a single AA residue at the protein surface Figure . In bothOwing to the emerging interest for addressing previously “unanswered” fundamental scientific questions as well as offering practical solutions for biomedical applications, site‐selective dual functionalization of proteins has seen rapid developments in the past ten years. In this review, we first summarize the synthetic strategies for protein dual functionalization to provide a timely overview of this burgeoning field, which serves as a guideline for the selection of the most appropriate method for the envisaged applications Figure . In addi2Site‐selective protein functionalization either relies on the functionalization of canonical AAs on the protein surface or genetically encoded noncanonical AA (ncAA) bearing the appropriate bioorthogonal groups.Alternatively, the attachment of a multifunctional linker containing two reactive orthogonal groups to a single site on the protein surface, usually an exposed cysteine or a disulfide bond, also provides straightforward access for the incorporation of two different functionalities. These bioconjugation reagents include maleimide derivatives,−1 s−1, which is about 30 times faster than the SPAAC reaction.6 M−1 s−1), high bioorthogonality, catalyst‐free conditions and good biocompatibility.The recent advances in bioorthogonal chemistry have greatly promoted the progress of site‐selective protein modification. Nowadays, various bioorthogonal reactions have been reported with optimized reaction parameters, such as reaction rate, catalyst type, and substrate stability, to impart the desired functionalities for the envisaged applications.75, 763Most synthetic strategies for protein dual modification target canonical AAs exposed at the protein surface. These residues are immediately accessible without the need for tedious genetic engineering of recombinant protein variants and also mitigate the risk of negative effects on proteins folding and function. In this section, dual functionalization at two different sites as well as at a single site is discussed.3.1Direct modification of two rare AA residues on the protein surface is a straightforward approach to achieve dual modification of proteins. The combination of two different mono‐functionalization methods requires stringent selection to ensure orthogonality, compatibility and preferably high modification efficiency. For example, Gaunt and co‐workers developed a method based on chemoselective labeling of a single methionine residue with a hypervalent iodine reagent.An alternative strategy for site‐selective protein dual modification is based on the differences in the reactivity of cysteines in their free (thiol) and oxidized (disulfide) forms.3.2Despite the simplicity of dual modification at two distinct sites, the availability of proteins with two different AA residues with orthogonal reactivities is rather limited. Therefore, alternative methods have been developed to target one specific AA residue with a multifunctional bioconjugation reagent.4, was required to retard the elimination reaction to generate a stable and bioactive conjugate for subsequent applications.Baker, Caddick, and co‐workers have demonstrated protein dual modification using mono‐ and dibromomaleimide reagents.Alternatively, vinyl sulfones are also commonly explored Michael acceptors for protein modification due to its high electrophilic properties that enable their reaction with nucleophiles on the protein surface.In addition to maleimide analogues and sulfone derivatives, other strategies have also been developed for dual‐modification. For example, Goncalves and co‐workers reported that dichloro‐1,2,4,5‐tetrazine can undergo two successive nucleophilic aromatic substitutions to introduce the thiol‐containing payload at a cysteine residue with excellent selectivity Figure .[44] TDue to the emergence of ADCs for targeted cancer therapy, dual functionalization at disulfide site has also gained growing interest because of the presence of accessible disulfide bonds in antibodies and the antigen‐binding fragment (Fab).11, 12Besides addressing cysteines and disulfide bonds, other modification strategies at less‐explored AA residues have also been reported to expand the existing protein functionalization toolkit. For example, the hypervalent iodine reagents developed by Gaunt and co‐workers can be combined with maleimide reagents to achieve dual functionalization at cysteine and methionine sites, which is described in Figure Achieving dual modification at one single AA site has less restriction in terms of the choice of reagents compared to the functionalization at two AA sites. One possible limitation of the single site strategy is that the two orthogonal groups can be sterically hindered due to close proximity on a relatively small multifunctional linker. This could prevent bulkier groups such as polymers to be attached onto the protein. In addition, modification besides cysteine residues is relatively unexplored, thus this field would greatly benefit from further investigations of modification strategies at other low abundant AA residues such as tyrosine, serine or the N‐termininus.4To expand the reactivity beyond what is offered in native proteins, reactive groups for functionalization through genetic incorporation of new AAs  has emerged as an indispensable tool for site‐selective protein dual functionalization. In this section, genetic methods to integrate new reactive canonical AAs, ncAAs or peptides tags for protein dual functionalization are summarized.4.1The expression of recombinant proteins containing one or more point mutations is a straightforward and well‐established technology.The examples mentioned above took advantage of the different local microenvironment offered by the native protein. Inspired by Nature's elegance, Pentelute and co‐workers have developed a strategy to create a specific local chemical environment for the cysteine residue by a newly developed, fine‐tuned four‐amino‐acid peptide sequence (FCPF), which is termed as “π‐clamp” Figure .[96] T4.2E. coli are applied that are not able to synthesize a certain amino acid required for its growth. The ncAAs need to structurally resemble the natural AA to allow binding to the respective endogenous aminoacyl‐tRNA synthetase and to replace the natural AA in the polypeptide sequence.E. coli strain.The introduction of point mutations has certain limitations as the available functionalities and their respective reactivities could only be selected from the pool of the 20 canonical AAs. However, the cellular biosynthetic machinery can be manipulated to incorporate ncAAs that often represent structurally similar derivatives of the canonical AAs. These ncAAs allow protein labeling with unprecedented molecular precision.CUA pair (MjTyrRS/tRNACUA pair) derived from Methanococcus jannaschi in response to the amber (UAG) stop codon to insert p‐acetylphenylalanine, a ketone bearing ncAA, into T4 lysozyme.The genetic code expansion technique has been developed as another technique that allows the insertion of a broad variety of ncAAs with spatial precision at virtually any desired site.CUA pair (PylRS/tRNACUA pair) to produce a new variant, PyIRS/tRNAUUA, which can suppress the ochre (UAA) stop codon. In combination with the evolved MjTyrRS/tRNACUA pair, these two orthogonal aaRS/tRNA pairs were capable of recognizing and inserting two ncAAs, p‐azido‐l‐phenylalanine and Nϵ‐propargyloxycarbonyl‐l‐lysine, into a glutamine‐binding protein in response to the amber UAG codon and the ochre UAA codon /tRNACUA pair and PyIRS (AcKRS)/tRNAUUA pair, to eliminate protein aggregation and oxidation induced by the copper catalyst.E. coli, Schultz and co‐workers have also successfully incorporated two different ncAAs, which contained azido and ketone groups, in mammalian cells by utilizing the two orthogonal Methanosarcina barkeri pyrrolysyl‐tRNA synthetase (MbPylRS)/Methanosarcina mazei pyrrolysyl tRNA (MbPylRS/MmtRNAUUA pair) and tyrosyl‐tRNA synthetase (EcTyrRS)/tRNACUA pair (EcTyrRS/tRNACUA pair).Despite the simplicity of introducing a cysteine mutation as one of the target sites for protein dual modification, this approach could become problematic if the protein consists of native cysteines that play important structural or functional roles. Furthermore, cysteine residues could form disulfide bonds, which could limit expression yields and the reaction reversibility of the thiol–maleimide conjugation could also cause stability problems of the resulting protein bioconjugates.n Figure .[100] UCCU (a derivative of MjTyrRS/tRNA) and PylRS/tRNACUA, two ncAAs were site‐selectively introduced into Calmodulin, forming a triazole intramolecular crosslink through the subsequent CuAAC reaction.UCCU pair in directing the corresponding ncAA incorporation. The efficiency of the original system was substantially improved based on the evolution of the PylRS/tRNACUA to obtain an optimized quadruplet decoding variant, PylRS/tRNAUACU pair.UACU pair was combined with the AzPheRS*/tRNACUA pair to site‐selectively introduce two bioorthogonal tags, norbornene and tetrazine, into Calmodulin  and a stop codon for the incorporation of two ncAAs.n Figure .[25] DGenetic code expansion has witnessed incredible achievements in recent years, which greatly promotes and revolutionizes the field of site‐selective protein dual functionalization. The advantages of this strategy lie in the small size of the ncAAs, flexible incorporation sites but high site‐selectivity, and the diverse reactive tags available for incorporation. However, some important challenges still remain in this rapidly evolving field, for example, low catalytic efficiency of engineered aaRS which require tedious evaluation and optimization to improve their performance, relative low expression yields, repetitive optimization of the protocols for effective protein engineering, the scope and compatibilities of the functional groups capable to be inserted.4.3Besides the incorporation of ncAAs bearing bioorthogonal tags for dual modification of proteins, the insertion of two artificial peptide sequences that can be recognized by two distinct enzymes for the subsequent covalent labeling with user‐defined probes also serves as a straightforward approach for dual modification of proteins. This enzyme‐mediated peptide labeling combines the advantage of high specificity of the enzyme towards the peptide tags and excellent labeling efficiency with minimal perturbation to proteins structure and function.Despite the simplicity of a one‐step introduction of the desired functionalities by two independent reactions, the labeling efficiency may be greatly influenced by the steric effects and hydrophobicity of the introduced probes. This limitation can be overcome by the incorporation of a multifunctional scaffold possessing the bioorthogonal tags. Distefano et al. used farnesyl transferase, which can catalyze the transfer of an isoprenoid group from farnesyl diphosphate to the cysteine site of a tetrapeptide , to introduce two reactive tags to the model protein, GFP Figure .[107] In some instances, the enzyme‐mediated dual labeling of proteins can also be achieved on the native protein surface in a site‐selective manner without the need to insert artificial peptide tags. For example, in Alabi and cowokers’ work, microbial transglutaminase, which catalyzes the formation of interprotein isopeptide bonds between glutamine and lysine residues, can recognize the glutamine 295 on the aglycosylated human IgGs and thus allowed the incorporation of two bioorthogonal tags, azido group and methyltetrazine group Figure .[80] T5Driven by exciting developments in the discovery of new synthetic procedures as well as scientific curiosity, achieving higher level of protein modification in a site‐selective manner is considered crucial for advancing different fields in chemistry, biology and material science. Compared to mono‐ and dual functionalization of proteins, triple functionalization strategies allow the incorporation of three different functionalities into proteins, thus offering well‐defined protein bioconjugates with further expanded structural and functional diversities that is beyond what current synthetic strategies can accomplish. These endeavors will greatly boost our capability to investigate, modulate and re‐design the chemical and physical properties of proteins.However, triple functionalization of proteins is even more demanding than mono‐ and dual functionalization as the combination of three orthogonal chemistries have to be applied in aqueous solution and in the presence of many reactive proteinogenic groups. Furthermore, by increasing the number of functionalities attached to the protein surface, it may lead to potential detrimental influence to their functional and structural integrity. In addition, the selection of the reactive groups requires more deliberate chemical design as the increase in the hydrophobicity of the bioconjugation reagents may cause protein aggregation. The labeling efficiency may also be compromised due to the steric hindrance from the insertion of three different functionalities.MjTyr, and Pyl pairs to suppress UGA, UAG and UAA codons for the incorporation of three ncAAs, 5‐hydroxytryptophan, p‐azidophenylalanine, and cyclopropene‐lysine, in the engineered E. coli strain ATMW1 carbonyl)‐l‐lysine, 3‐methyl‐l‐histidine and Nϵ‐(carbobenzyloxy)‐l‐lysine, were genetically encoded into proteins in response to the UAG, AGGA and AGUA codons. However, subsequent functionalizations have not demonstrated yet. Compared to the reassignment of three stop codons, this work utilizes the combination of a stop codon and quadruplet codons, which do not interfere with the translation termination and serve as a more general and applicable strategy to achieve higher‐level protein functionalization. Even though there is no application shown and there are still obvious limitations, for example, low encoding efficiency, these examples represent the first proof‐of‐concept for protein triple modifications. They still represent significant technological advancements for the development of multifunctional protein conjugates, which holds immense potential for drug delivery, bioimaging, and material science.Recent examples have demonstrated the successful triple functionalization of proteins. Chatterjee and co‐workers have reported the introduction of three ncAAs into proteins via genetic code expansion. In this work, they assigned the EcTrp, 1 Figure .[119] Besides increasing the number of functionalities that can be incorporated into proteins, more complex protein bioconjugates have also been reported. For example, Chudasama et al. described an efficient and modular strategy for the generation of a bispecific antibodies as well as the dual functionalization of the resulting bioconjugate Figure .[122] 6With the emergence of protein dual functionalization techniques, homogeneous protein bioconjugates with remarkable functional complexity have been achieved and studied for diverse applications. In the following, three major fields of applications are highlighted that have greatly benefited from these novel synthesis opportunities.6.1The excitation energy transfer from a donor to an acceptor chromophore, also termed Förster resonance energy transfer (FRET), allows assessing important parameters such as chromophore distances and their orientations in real time and in complex biological environments. Although cryo‐electron microscopy is becoming a popular technique to determine structural snapshots of biomacromolecules at atomic resolution, samples need to be frozen and studies cannot be carried out in native environment. In contrast, FRET bears the advantage of easy accessibility and enables the real‐time monitoring of the dynamics and conformational changes of proteins in their native environment, thus serving as an important complementary methodology for elucidating proteins structural dynamics.2A receptor in a site‐specific fashion, which, in conjunction with a CFP, generated a FRET construct (A2A‐FlAsH‐CFP) that exhibited a 5‐fold enhanced agonist‐triggered FRET signal compared to the dual‐tagged CFP and YFP conjugate , for example, cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP), can be genetically encoded into the target proteins and are widely utilized for the investigation of proteins inside living systems.e Figure .[127]2+) concentration. CaM consists of two domains, the C‐terminal and N‐terminal domain that contain two EF‐hand motifs to bind Ca2+.2+binding, the conformation of CaM changes, which affects its binding to different target enzymes.In comparison to FPs, small molecule chromophores bear the advantage of small size, excellent photostability and high quantum yield.2+ and without Ca2+ have been resolved in detail via NMR or X‐ray crystallography. However, the transition states that dynamically occur during the Ca2+ binding processes and that play an important role in modulating the interaction of CaM with its binding partners could not be resolved with conventional structure analysis by NMR and X‐Ray.2+ concentration  but with different configuration  was conjugated with the three different drug linkers via thiol–maleimide chemistry offering three different ADCs, cAC10‐4 (without PEG24), cAC10‐5 (with PEG24) and cAC10‐6 (with branched PEG24) as depicted in Figure Despite the clinical success, most ADCs are still restricted to the conjugation with a single type of payload, for example a certain anticancer drug, to target a specific type of cancer cells. In fact, this kind of single‐functionalized ADCs often suffer from the unfavorable pharmacokinetics and the inherent hydrophobicity of the chemotherapeutic drugs, which could significantly limit the in vivo therapeutic efficiency.n Figure .[132] On the other hand, the therapeutic efficacy ADCs could also suffer from drug resistance after multiple treatments due to inherent and acquired resistance or tumor heterogenicity.89Zr for theranostic applications  to anti‐prostate stem cell antigen (PSCA) cysteine mutated diabody A2 via a dual‐modality linker, offering the dual‐modified imaging probe (18F‐ sCy5‐A2cDb)  Figure .[138]n Figure .[138] n Figure . The dua7The technical breakthroughs in protein bioconjugation chemistry has served as a major driving force for the elucidation of protein trafficking and interactions in living cells as well as the emergence of protein therapeutics. With the increasing need for personalized treatments to address great challenges in biomedical research such as efficient cell or organ targeting, overcoming drug resistance and reducing systemic toxicity, there is a high demand for protein therapeutics with improved features. Multifunctional protein conjugates provide more than one functionality, and they are expected to enhance in vivo performance of therapeutic proteins by combining different functional groups possessing therapy, diagnostics and imaging properties in a single protein system that is tailored to the requirements of the patient. Nevertheless, dual functionalization of proteins remains much more challenging relative to single functionalization owing to the plethora of reactive functional groups on the protein surface and the requirement for the optimal combination of different orthogonal reactions with high efficiencies. In this review, we have summarized the remarkable progress in both synthetic and genetic engineering strategies that have overcome these significant hurdles and thus allow covalent functionalization of proteins with two different functionalities at distinct sites.Undoubtedly, dual functionalization of proteins has witnessed significant advancements over the past ten years which offers a new arsenal of functional protein conjugates with advantages over singly‐functionalized proteins in probing protein dynamics, combination therapy and bioimaging. We envision that tremendous efforts will continue to be devoted to enriching the current methodology toolkit by exploring new chemistries to improve the specificity as well as efficiency of dual functionalization or to achieve a higher level of functionalization, for example, triple functionalization. Moreover, the proper combination of different sophisticated strategies, for example, chemical methods, genetic methods, to capitalize the advantage of different approaches from the currently expanding toolbox will provide new insights for the preparation of a myriad of functional nanomaterials. Ultimately, we believe that this will pave the way towards “smart” and “intelligent” protein conjugates that can self‐adapt to the microenvironment of diseases and provide a self‐feedback loop to achieve an output or decision, for example, termination/activation. In this way, current bottlenecks and challenges in therapeutic applications can be addressed with a completely new perspective through rationale chemical design.The authors declare no conflict of interest.Lujuan Xu studied chemistry at Zhengzhou University, where she received her Bachelor's (2014) and Master's (2016) degrees. From 2014 to 2016, she conducted her Master's thesis on the synthesis of small organic molecules for optoelectronic devices in the group of Prof. Jian Pei at Peking University. In 2017 she received a CSC scholarship to undertake her PhD with Tanja Weil at the MPIP. Her research focuses on the development of new chemical methodologies for site‐selective modification of proteins.Seah Ling Kuan received her B.Sc (Hons) in 2003 and completed her PhD (Chemistry) at the NUS in 2009. She joined Prof. Tanja Weil in NUS from 2008–2010 as a research fellow. In 2011, she began her independent research as an Alexander von Humboldt fellow in UUlm. She was a group leader in the Institute of Organic Chemistry III at UUlm from 2013–2016. She joined the MPIP in 2016 as a group leader (Protein Therapeutics). Her research focuses on the development of chemical approaches for the innovation of precision protein therapeutics to address devastating diseases such as cancer, inflammation or infections.Tanja Weil received her PhD from Mainz University in 2002. She held several leading positions at Merz Pharmaceuticals GmbH . She was an Associate Professor at the National University of Singapore  and since 2010, director at the Institute of Organic Chemistry III (OC III) at Ulm University (Uulm). In 2016, she was appointed as director of the Department of Synthesis of Macromolecules at the Max Planck Institute for Polymer Research (MPIP) in Mainz, Germany. She has received an ERC Synergy Grant. Her scientific interests focus on innovative synthesis concepts to achieve functional macromolecules, hybrid materials and life‐like systems to solve current challenges in biomedicine and material science.


Facial attractiveness has been reported to be influenced by visual features such as facial shape and the colour and texture of the skin. However, no empirical studies have examined the effects of facial skin radiance on facial attractiveness. The present study investigated whether types of skin reflection  and the position of the reflection on the face influence facial attractiveness and other affective impressions.A total of 160 female participants (1) estimated the ages and (2) evaluated attractiveness and other impressions of unfamiliar female faces in a total of seven skin reflection conditions. These conditions incorporated three types of reflection  and three positions of the reflection on the face .The facial images of radiance on entire faces were rated as appearing younger than the facial images of oily shine on entire faces and the matte faces. Attractiveness ratings and other positive impressions increased in the order of the matte (ranked lowest), the oily shine on entire face, and the radiance on entire face (ranked highest) conditions. The reflection position also influenced facial attractiveness: attractiveness ratings and other positive impressions were higher in the radiance on entire face condition than in the radiant cheeks and the radiant T‐zone conditions. Interestingly, the radiant cheek faces were rated more radiant and healthier but less feminine and less bright than the radiant T‐zone faces.These results suggest that facial radiance enhances facial attractiveness and conveys a wide variety of positive impressions on the observer. The magnitude of the effects of cheek radiance and T‐zone radiance differs across various affective impressions. Nevertheless, the results demonstrate that cheek and the T‐zone radiance both contribute to higher attractiveness and other positive impressions of the radiance on entire faces. We believe that our findings can contribute as a guide to the enhancement of positive facial impressions by means of skin radiance, thereby leading to a better understanding of the value of skincare and base makeup.  What visual features affect facial attractiveness and other impressions when we see other people's faces? It is well known that the shape, colour and texture of the face influence facial attractiveness , 2, 3, 4In addition to the above‐mentioned visual features , facial skin reflection could also influence affective impressions, including attractiveness, because according to consumer cosmetics magazines, for instance, consumers seem to have a tendency to use skincare and base makeup products to enhance their facial radiance, which is a particular type of reflection , 11. SucHowever, most previous studies on facial skin radiance focused solely on quantifying it based on physical quantities, rather than examining the effects of skin radiance on facial attractiveness. This is because skin radiance is a psychologically defined quantity, despite the fact that it is a type of optical skin reflection . For instance, Baret and colleagues  proposedIn sum, most previous studies on facial radiance tried to quantify it based on physical quantities, rather than examine effects of skin radiance on facial attractiveness and other affective impressions. The main purpose of the present study was to clarify the effects of facial skin radiance on facial attractiveness, rather than to explore the definition of skin radiance based on physical quantities.Although facial radiance can create a good impression , anotherAnother type of skin reflection that has a growing awareness among cosmetic consumers is the property of matte. Matte skin makes little impression of radiance or the oily and shiny condition because of lack of specular reflection .The purpose of the present study was to elucidate whether the types of facial skin reflection  and the position of the reflection on the face influence facial attractiveness and other affective impressions. To this end, we conducted two psychological experiments with 160 female participants each. First, we examined the effects of skin reflection types and positions on estimated age. Second, we comprehensively investigated those effects on facial attractiveness and on nine other affective impressions, including the desire to compliment, looking happy, likableness, looking tired, looking feminine, looking healthy, skin radiance, oily shine and skin brightness.A total of 160 adult females between the ages of 30 and 49 years  were recruited for the study. All participants were paying more than 1,000 yen per month on skincare products and used them for more than five days a week. They all lived in Japan, with Japanese as their native language. All were healthy, had no history of neurological disorders and had normal or corrected‐to‐normal vision. All gave written informed consent before each experiment. The experimental procedures were approved by the Ethical Committee of the Shiseido Global Innovation Center.−2 and 2.2, respectively. The order of presentation of the photo images was controlled with a computer program.All visual images and questionnaires were presented to each participant on a 24.1‐in (1920 × 1200 pixels) liquid‐crystal display. Images of faces were presented at a resolution of 719 × 1078 pixels on a grey ) background. Ten displays and ten computers were used to run the experiments for ten participants simultaneously. The displays were colour calibrated with an X‐rite i1 Pro (X‐Rite Inc.) to ensure the same colour conditions for all displays. We adjusted the maximum luminance and gamma to 160 cd mImages used in this experiment featured ten adult female models between the ages of 30 and 44 years . They all lived in Japan, and all gave written informed consent before each photography session. The procedures were approved by the ethical committee of the Shiseido Global Innovation Center. Figure The process of preparing photo stimuli was composed of two phases. In the first phase, which was the photographing phase, the faces of the female models were photographed in several poses with different cosmetics applied to them under different lighting conditions. All styles of makeup, excluding skincare, were manipulated by a professional makeup artist. In the second phase, which was the retouching phase, the photographs were retouched by a computer graphics technique. The images of all ten female models were subjected to the same process. In the photography phase, we collected image materials for a base face image as well as many types of skin reflection by changing the lighting and applying different cosmetics. In the retouching phase, we first created a base face image, which was not used in the experiment but used for preparing photographs presented in the experiment. All types of face used in the experiment were created by adding and/or removing reflections from the base face image.All female models rotated their bodies to the horizontal‐right direction, 45 degrees from the frontal angle, for the gloss to appear on the skin in the photographs. Thus, the left side of the face was captured by the camera. To control for the same facial angle for all participants, a chin rest was used. All female models sat, whereas the pictures were taken by a digital single‐lens reflex Canon 5D camera (Canon Inc.) with 50 mm lens .To express the different types of gloss on a face, we prepared several lighting conditions and cosmetics, and combined the conditions. The results of previous studies , 17, 22 Diffuse reflection light is multiple reflection where the source is unclear. Skin with a higher degree of diffuse reflection showed characteristics of more moisture and fine texture . Unlike To artificially express the characteristics of diffuse reflection of light on the skin, it was necessary to produce reflected light that did not prominently convey the feeling of skin roughness. For this purpose, we used diffused illumination for photography and applied cosmetic products that could smooth facial skin. To create a radiant image of the face, we manipulated the intensity of illumination of directional reflected light to express specular reflection light, in addition to diffuse reflection light. In contrast, to generate strong specular reflection characteristics, light that was not diffused was used. A previous study on the relation between impressions of facial skin and skin biology , 26, andAll female models were photographed with neutral facial expressions and smiling faces. When the female models were smiling, there was particularly strong specular reflection because of increased convexity of the cheeks.All female models were photographed under non‐diffuse illumination conditions (intense and non‐diffuse illumination) and under diffused illumination (light diffusing illumination). Under non‐diffusion lighting conditions, in addition to a strobe light (COMET strobe light and strobe head), two strobe lights fitted with umbrella‐shaped reflectors were used. All three lamps were used at an output of 200 W. In the diffused lighting condition, one side‐light from the other side of a diffuser cloth and a strobe light equipped with a diffuser were used at an output of 112 W. Figure All female models washed their faces before being photographed, and then applied 2 mL of lotion (ELIXIR SUPERIEUR LIFTING MOISTURE LOTION W II) and 1.4 mL of emulsion (ELIXIR SUPERIEUR LIFTING MOISTURE EMULSION W II) to their faces to avoid dryness. They then rested for about ten minutes until the redness of the skin decreased. All styles of makeup were manipulated by a professional makeup artist without skincare. Following this, the artist drew eyebrows on the female models to avoid the influence of faces without eyebrows on facial impression. Actually, some models changed the shape of their eyebrow by removing some parts of their natural eyebrow shape. They were then photographed in three patterns of applied cosmetics: applying only solid foundation (cle de peau BEAUTE RADIANT STICK FOUNDATION) to the skin, applying cosmetic 0.2 mL of cosmetic oil (Carita14) to prevent dry skin over the foundation and applying cosmetic moisturizing gel (ELIXIR SUPERIEUR SLEEPING GEL PACK W) without white particles only on the cheeks, in addition to about 0.2 mL of cosmetic oil (Carita14) above the foundation.In a trial photoshoot, foundation applied to the skin was smoothed and impressions of the smooth texture of the skin were observed. The photographed images of the face with only foundation applied to it were used as the material for diffuse reflection light to generate a radiant face and the base face. The trial photoshoot confirmed that faces coated with cosmetic oil and gel yielded reflection images showing oily and shiny skin, and irregularities on the skin surface. Cosmetic oil and gel were thus used to represent specular reflection light for oily and shiny faces.Digital images of the face were retouched by computer graphics (CG) software (Adobe Photoshop CC2017). The gamma value of the display Color Edge CG243W (Eizo Corporation) used for retouching was 2.2. We created a base face for each female model and glossy images under the three conditions by transplanting various highlighted materials from photos of the same female model to the base face. The highlighted materials were transplanted on the convex part of the face, including the forehead, ridge of the nose, cheek and chin. The ranges of the transplanted highlights were specified to naturally fit the shape of the face of each female model. In addition to one person being in charge of image retouching, five people, the three authors and two members of the staff of Shiseido Company Limited, confirmed whether the range, shape and strength of the highlights were appropriate.A base face was created by removing strong highlights in the convex parts of images of faces with a neutral expression and only foundation applied under diffusion illumination light conditions. We transplanted the image of the skin of the non‐highlighted portion onto the highlighted one. However, the natural highlight was left unchanged. Skin eruptions, spots and scratches were also removed.Images of radiant faces were created by copying glossy skin parts of a female model’s face in an image and applying it to the base face of same model’s face by retouching using the CG software. Glossy parts of the skin in the images, used as materials for creating radiant faces, were generated by combining different types of reflected lights. One had the characteristics of diffuse reflection light and another those of strong specular reflection light on facial skin. Gloss with the characteristic of diffuse reflected light was cut out from the foundation applied to the skin of the face under diffuse illumination and transplanted to the base face. Furthermore, specular reflection occurring on skin with foundation applied to it  under strong non‐diffused lighting was cut out and transplanted to the base face.Images of oily and shiny faces were created by applying oily and shiny glossy skin parts of a female model’s face to her base face. Glossy skin parts as materials for creating oily and shiny faces were generated from skin that exhibited features of specular reflection, was oil‐rich, and had remarkable irregularities. Images of oily and shiny gloss materials were cut out from facial images featuring smiling faces, with foundation and oil or foundation, oil, and gel applied, that had been shot under non‐diffuse illumination, and these materials were transplanted to the base face.We removed all highlights from the base face, including lustre.Each participant estimated the age of a female model through photographs in Task 1 and evaluated various impressions evoked by the photographs in Task 2, where the Scheffe method for paired comparison tests (the modified Nakaya method) , 28 was All participants performed both tasks and answered each question on a computer. Each participant sat one metre from the computer display. The 160 participants were divided into 10 groups of 16 each. They observed all seven types of faces  Figure . The tasF = 18.59, P < 0.001). Bonferroni post‐hoc tests revealed that the faces with radiance of the entire face were estimated as the youngest in all image conditions (ps < 0.005). Faces with radiant cheeks were evaluated to be significantly younger than oily and shine on entire faces, faces with oily and shiny cheeks, and matte faces (ps < 0.03). However, there were no significant differences between assessments of faces with radiant cheeks and those with radiant T‐zones, and with oily and shiny T‐zones (ps > 0.13). Faces with radiant T‐zones were evaluated to be significantly younger than matte faces (p < 0.001) but did not show significant differences from assessment of oily shine on entire faces, faces with oily and shiny cheeks, and those with oily and shiny T‐zones (ps > 0.08). There was no significant difference among evaluations of oily shine on entire faces, faces with oily and shiny cheeks, those with oily and shiny T‐zones, and matte faces (ps = 1).Figure F = 326.6, P < 0.001). Bonferroni post‐hoc tests showed no significant differences either between the faces with radiant cheeks and radiant T‐zones (P = 1), between the faces with oily and shiny cheeks and oily and shiny T‐zones, or between the faces with oily and shiny cheeks and oily shine on entire faces (ps > 0.05). All pairs other than the above showed significant differences (ps < 0.05). These results suggest that the faces with radiance of the entire face create the most positive impression in all image conditions. The radiant cheeks and T‐zones were rated more positive than all the faces with oily shine and the matte face. The oily and shiny T‐zone was rated more negative than the oily shine on the entire face. The matte face created the most negative impression. More detailed results of each questionnaire were shown below.In order to overview the degree of positive (or negative) impressions for the seven conditions of facial stimuli, we have devised an integrated positive‐negative measure that summed up preference values of seven abstract words . We reversed the sign of the value for the impression ‘tired’, as it represents a negative impression. Figure P < 0.001). This suggests significant differences among the image conditions. The yardstick values were calculated, and significant differences were found between certain conditions. Table Figure ‘Attractive’, ‘Want to compliment’, ‘Happy’, ‘Likable’, ‘Feminine’ and ‘Healthy’ indicated similar tendencies. These impressions were felt most strongly in cases where the entire face, the cheeks and the T‐zone were radiant. Conversely, the three conditions featuring oily and shiny faces provided weaker impressions of ‘Attractive’, ‘Want to compliment’, ‘Happy’, ‘Likable’, ‘Feminine’ and ‘Healthy’, and the condition with the matte face evoked the weakest such impressions.‘Attractive’, ‘Want to compliment’ and ‘Happy’ showed no significant differences between the conditions involving images of faces with radiant cheeks and T‐zones. There was no significant difference between the conditions of images with oily and shiny cheeks and those with oily shine on entire faces in the questions. Images of faces with oily and shiny T‐zones evoked significantly weaker impressions concerning ‘Want to compliment’, ‘Happy’ and ‘Attractive’ than other conditions involving oily and shiny faces Table .‘Likable’ exhibited no significant difference between the images of faces with radiant cheeks and T‐zones. There was no significant difference between images with oily and shiny T‐zones and oily shine on entire faces. Images of faces with oily and shiny cheeks evoked stronger impressions of ‘Likable’ than other oily and shiny faces Table .‘Feminine’ and ‘Healthy’ showed no significant differences between images with oily and shiny cheeks and oily shine on entire faces. Images with oily and shiny T‐zones evoked weaker impressions of ‘Feminine’ and ‘Healthy’ than other oily and shiny faces. ‘Feminine’ was felt significantly more strongly in images of faces with radiant T‐zones than those with radiant cheeks, and ‘Healthy’ was felt significantly more strongly in images with radiant cheeks than T‐zones Table .‘Tired’ was felt most strongly in images of matte faces, and in the three conditions featuring oily and shiny faces than the other conditions. Images of faces with radiant cheeks and T‐zones evoked weaker impressions, and images with radiance on entire faces gave the weakest impressions of ‘Tired.’ When three images featuring oily and shiny faces were compared, there was a significant difference between images with oily and shiny T‐zones, and oily shine on entire faces in impressions of ‘Tired.’ Thus, images of faces with oily and shiny T‐zones gave a stronger impression of ‘Tired’ than images of faces with oily shine on entire faces Table .‘Radiant skin’ was felt most strongly when the entire face was radiant, followed by images of faces with radiant cheeks, faces that were oily shine on entire, faces with oily and shiny cheeks and radiant T‐zone, and faces with oily and shiny T‐zones. Matte faces gave the weakest impressions of ‘Radiant skin.’ There was no significant difference in impressions of ‘Radiant skin’ between faces with oily and shiny face cheeks and radiant T‐zones Table .‘Oily and shiny skin’ was felt most strongly in images of oily shine on entire face skin, followed by faces with oily and shiny cheeks and T‐zones, and the three conditions of radiant faces. Images of matte faces gave the weakest impressions of ‘Oily and shiny skin.’ There was no significant difference between images of faces with oily and shiny cheeks and T‐zones Table .‘Bright skin’ was felt most strongly in images of faces that were radiance on entire face, followed by faces with radiant T‐zones and those that were oily shine on entire faces, those with radiant cheeks, and images of faces with oily and shiny cheeks and T‐zones. Images of matte faces gave the weakest impressions of ‘Bright skin.’ There was no significant difference between facial images with oily shine on entire faces and those with radiant T‐zones or between faces with oily and shiny T‐zones and those with oily and shiny cheeks Table .The first aim of the present study was to examine whether the types of facial skin reflection  influence facial attractiveness and other affective impressions and the second aim was to examine the influence of the position of the reflection on the face. In this study, we performed two psychological experiments where we examined effects of skin reflection types and positions on estimated age. We comprehensively investigated those effects on facial attractiveness and on nine other affective impressions, including the desire to compliment, looking happy, likableness, looking tired, looking feminine, looking healthy, skin radiance, oily shine and skin brightness. We show for the first time that radiance, a specific type of facial reflection, has a positive effect on a wide variety of impressions of the face by empirical data. Furthermore, we revealed that the position of radiance on the face differentially improves impressions of health and femininity. This is important because understanding the association between facial impressions and facial radiance could provide consumer the value of facial radiance achieved by using skincare and base makeup.Images with radiance on entire faces were evaluated as the youngest. The other two conditions featuring radiance, radiant cheeks and T‐zones, were rated younger than images of the matte face. By contrast, no significant difference was found between images of the three oily and shiny faces and the matte face. Therefore, these results suggest that not all types of facial gloss give an impression of youth.With regard to the effect of position of the gloss, no significant difference was found between images of faces with radiant cheeks and T‐zones, or between those of faces with the oily and shiny cheeks and T‐zones. Therefore, the position of gloss had no effect on age estimation.Nevertheless, there was an additive effect of radiance of the cheeks and the T‐zone. As mentioned above, images of faces with radiant cheeks and T‐zones were rated as younger than those of matte faces. In addition, the faces with radiance on entire facial skin were rated younger than those with radiance either only on cheeks or only on T‐zones. These results suggest that images of radiant cheeks and T‐zones in radiance on entire faces yielded additive effects to help the faces appear younger still.By contrast, no such additive effect was found for images of oily and shiny faces as no significant difference was found among the three oily and shiny conditions. Hence, the additive effect of reducing estimated age seems to be unique to radiance.Previous research has suggested that facial information, such as skin brightness, reflection and colour , 21, facImages of radiant faces gave the participants had the most positive impressions across all questions for images of radiant faces. In addition, oily and shiny faces evoked more positive impressions than matte faces. Therefore, radiance in faces prompts positive impressions in observers.Why did facial skin radiance enhance positive impressions? One possible reason is from a biological perspective. Radiant skin is produced by combining diffused reflection and specular reflection, and skin with diffused reflection has the feature of decreasing melanin, increasing moisture, and inducing finer texture and good blood circulation . Skin inThe questionnaires on evaluating impressions contained sensory words, such as ‘Radiant skin’, ‘Oily and shiny skin’ and ‘Bright skin’. The impression of ‘Oily and shiny skin’ was felt more strongly towards faces with shiny gloss. This tendency indicates that the method of creating stimulus images of oily and shiny faces in previous studies can appropriately evoke this impression. The impression of a ‘Radiant skin’ was felt most strongly in faces that had radiance on entire face, followed by faces with radiant cheeks. However, faces with oily and shiny cheeks, and oily shine on entire faces provided the same, or stronger, feeling of ‘Radiant skin’ than faces with radiant T‐zones, even though the two image conditions featured oily and shiny faces. The impression of ‘Radiant skin’ was felt more strongly in faces with radiant cheeks than those with radiant T‐zones. This suggests that the type of gloss as well as its position affects the impression of ‘Radiant skin.’ The impression of a ‘Bright skin’ was felt more strongly when gloss was in a larger area and faces with radiant T‐zones and oily shine on entire faces evoked the same degree of impression. Therefore, both gloss type and area can influence the impression of ‘Bright skin.’ In addition, a strong impression of ‘Bright skin’ did not always accompany that of ‘Radiant skin’ to participants.The results for the three types of faces with radiance showed that facial images with radiance on entire faces evoked the most positive impressions, whereas there was nearly no difference between impressions of faces with radiant cheeks and those with radiant T‐zones. A synergetic effect, combining radiance on the cheeks with that radiance on the T‐zone, evoked a stronger impression among the participants, as shown in all abstract and sensory words used to describe their feelings for images in these categories, except for the ‘Oily and shiny skin’ impression.The responses to some questionnaires showed differences in degree between images of faces with radiant cheeks and T‐zones. Impressions of ‘Radiant skin’ and ‘Healthy’ were felt more strongly in the former than the latter Figure , Table 1Why did different positions of skin radiance improve different positive impressions of ‘Feminine’ and ‘Healthy’? A previous study  providesThe three types of oily and shiny faces were consistently evaluated more negatively than faces with radiance. Furthermore, there were differences among the conditions of images of oily and shiny faces. Images of faces with oily and shiny T‐zones did not yield more positive impressions among the participants than the other two types of oily and shiny faces. The participants tended to evaluate facial images with oily shine on entire faces more positively than faces with only oily and shiny T‐zones in terms of both sensory expressions and abstract words. The responses to questionnaires on sensory expressions indicate that facial images with oily shine on entire faces evoked stronger impressions in the participants than faces with oily and shiny cheeks. Moreover, facial images oily shine on entire faces evoked the same strength of impression as oily and shiny cheeks in terms of abstract words, and conveyed a less ‘Likable’ impression. These results suggest the effect of the position of oily and shiny gloss on impressions of the face because faces of oily and shiny T‐zones evoked more negative impressions than them of oily and shiny cheek. There was no synergy such that combining oily and shiny gloss on the cheeks and T‐zone increased positive or negative impressions.Why did faces with oily and shiny skin yield more negative impressions than the faces with radiance? One possible reason is that faces with oily and shiny skin gave a less healthy impression because such skin features low diffused reflection and high specular reflection induced by rough texture, less moisture, and poor blood circulation . In addiOne limitation of our present study is that we recruited only Japanese females as models and observers. This limitation leaves the question of cultural effects on how skin reflection influences facial impressions, which has been implied by previous research. For example, a large trial in China has identified self‐evaluated factors relevant to the prevalence of oily skin, which may be specific to the Chinese female population Nouveau‐Richard, . A crossThis study is the first to empirically show that radiance, a specific type of facial reflection, has a positive effect on a wide variety of impressions of the face. Furthermore, we revealed that the position of radiance on the face differentially improves impressions of health and femininity. Previous studies have suggested that the shape , 2, 3, 4We hope that a new perspective on the value of facial radiance can be provided by the present scientific evidence on the effects of facial radiance on facial attractiveness and other affective impressions. Consequently, understanding the link between facial impressions and facial radiance could help determine the value of facial radiance achieved by using skincare and base makeup.


Understanding is still developing about spatial risk factors for COVID‐19 infection or mortality. This is a secondary analysis of patient records in a confined area of eastern England, covering persons who tested positive for SARS‐CoV‐2 through end May 2020, including dates of death and residence area. We obtained residence area data on air quality, deprivation levels, care home bed capacity, age distribution, rurality, access to employment centers, and population density. We considered these covariates as risk factors for excess cases and excess deaths in the 28 days after confirmation of positive Covid status relative to the overall case load and death recorded for the study area as a whole. We used the conditional autoregressive Besag—York–Mollie model to investigate the spatial dependency of cases and deaths allowing for a Poisson error structure. Structural equation models were applied to clarify relationships between predictors and outcomes. Excess case counts or excess deaths were both predicted by the percentage of population age 65 years, care home bed capacity and less rurality: older population and more urban areas saw excess cases. Greater deprivation did not correlate with excess case counts but was significantly linked to higher mortality rates after infection. Neither excess cases nor excess deaths were predicted by population density, travel time to local employment centers, or air quality indicators. Only 66% of mortality was explained by locally high case counts. Higher deprivation clearly linked to higher COVID‐19 mortality separate from wider community prevalence and other spatial risk factors. COVID‐19 is a still emerging disease that was first identified in early 2020. COVID‐19 was declared a Public Health Emergency of International Concern on 30 January 2020  than the census date (May 31) was desirable because we knew that it should adequately capture case counts and mortality, rather than have results biased by incomplete data due to delayed reporting.The data set described Covid+ patients among the population of the English county of Norfolk and a single district (Waveney) in the adjacent county of Suffolk. For historical and geographical reasons, provision of health care in Norfolk and Waveney (N&W) is combined, currently under the commissioning powers held by the N&W clinical commissioning group (NWCCG). Norfolk and Waveney is a predominantly rural and coastal area in Eastern England, the United Kingdom, that extends roughly 55 by 40 miles. The population is approximately 1 million. The county is neither especially affluent nor deprived but does have areas among the 10% most and least deprived areas in England  which means that the vast majority of all detected cases were found under the Pillar 1 framework. The data set did not enable us to separate those tested for medical treatment needs from those with occupational exposure. We believe most of the records relate to persons with medical need, because 57% of the records were for persons age 65 or older  trusts serving this population. Table Four acute NHS hospital trusts regularly serve Norfolk and Waveney residents. Data available from NWCCG suggested that historically no more than 3% of NWCCG residents seek acute hospital care from NHS acute care providers not included in our analysis data set  was unique in our data set in never providing patient residence information. Omission of these WSH patients could lead to considerable underascertainment of COVID‐19 cases in areas that had many patients who visited WSH. About 14% of south Norfolk residents preferentially travel to WSH for urgent health care because of shorter driving distances compared to reaching alternative providers (presentation proportions in Appendix Home residence for each Covid+ patient was resolved to lower super output area (LSOA). LSOAs are standard census units in England for which socioeconomic and other indicators have been calculated. LSOAs are designed to be fairly consistent in population but not geographic size. LSOAs in N&W ranged in size from 12 to 10,689 ha . LSOAs typically each contain about 650 households ., the 59www.ons.gov.uk/peoplepopulationandcommunity/populationandmigration/populationestimates/datasets/lowersuperoutputareamidyearpopulationestimatesnationalstatistics). The supplied data were available in single year bands, from 0 years old to age 90+. We used these data to calculate the percentage of resident population in each N&W LSOA that was age 65 years or older.Counts of persons in specific age ranges in each LSOA was extracted using mid‐2019 estimates of persons registered by home address as recorded in general practice (GP) primary care records. These counts were available from the Office of National Statistics , which we hypothesized might correlate with local physical and social contact rates, and that these contact rates might in turn relate to risk of excess cases.https://www.geoportal.statistics.gov.uk  from 25 to 32,406. It is more conventional to use IMD2019 values as numeric values indicating relative decile. However, this was undesirable in this data set because data resolution would have been lost; most LSOAs in this study area are in IMD2019 deciles 3–7.Communities with higher social deprivation are recognized as having poorer health resilience and greater likelihood of relatively poor health outcomes  reference thresholds  and many smaller market towns . Private motor vehicles are known to be the main mode of transport in N&W, tending to account for more than 60% of journeys . These counts were each also considered as potential predictors of COVID‐19 cases or deaths.During the first wave of COVID‐19 in England, it was widely acknowledged that a high proportion of deaths were among persons living in residential care homes Burki, . We obtaNotably, our data set does not contain information about ethnic profile of the population within each LSOA. Although such data are available within LSOAs, N&W is an area that is quite low in ethnic diversity, and this is even truer among persons who are most at risk of hospitalization or death from COVID‐19 (age 65+). Note that 96.5% of all‐age Norfolk residents self‐identified as “white” in the 2011 Census  which is a conditional autoregressive model  to compare observed number of cases with those expected to generate relative risk of cases and deaths for each LSOA. The null model with no covariates represents the relative risk of cases and deaths in LSOAs. Inclusion of covariates in this modeling framework is effectively an ecological regression which models the relative risk given the covariates included.To summarize, we calculated the expected number of cases for each LSOA to May 2020, given the population of the LSOA and the population of the whole study area and the total number of cases in all LSOAs. We then used a Besag–York–Mollie model with integrated nested Laplace approximation Risk of excess casesResults were as follows:(ii)Risk of excess deathsThree variables significantly predicted excess cases of COVID‐19 in LSOAs. These were proportion of the LSOA resident population that was greater than 65 years of age  greater urbaneness  and care home capacity . These indicate that aged populations in more urban areas or areas with more care homes had an excess of cases relative to LSOAs with younger populations in more rural settings. Analysis of the proportion of variation explained by the final model was low at 0.01 . Fig. Three variables were predictors of excess risk of dying after allowing for total cases in the same LSOA; the proportion of the population over 65 , the care home bed capacity  and lower deprivation for the LSOA . This indicates that risk of dying was dependent on the proportion of the population over 65 and the extent of deprivation, with more deprived LSOAs having higher excess mortality, as well as local concentration of care home provision. The proportional contribution to the total variation explained by the spatial distribution of LSOAs was 0.02. Fig. Variance inflation factors for both models were all below 1.2, indicating collinearity did not bias model coefficients Appendix .3.1Fig. The SEM analyses show that the number of cases in an LSOA was significantly related to higher proportion of population over 65 and lack of rurality of the LSOA. However, the standardized coefficients were below 0.1 indicating that these parameters explained less than 10% of the variation in the number of cases. The number of cases was also strongly related to bed capacity in care homes in LSOAs, effectively representing the population at greatest risk. The number of deaths was strongly related to the total number of cases by end May, as well as greater proportion of the population over 65, higher deprivation and greater care home bed capacity.4Higher case counts in areas with a higher proportion of residents age 65+ probably reflects higher probability of severe disease (given that severity of disease is linked to advanced age). People tend to associate with persons close to their own age and a higher density of older adults may mean more social contacts among older adults with each other  we observe that most N&W LSOAs (334/597) did not have any active registered care homes during March–May 2020, 189 LSOAs (32%) had just one care home while just 30 LSOAs (5%) had between three and seven operational care homes. The care home capacity variable was highly indicative of exactly where within suburbs, urban, or urban fringe areas the persons most likely to become Pillar 1 cases and experience subsequent Covid mortality would be found.Care home count and rurality had separate effects for case generation in our structural equation models. Similar to our findings, analysis in the United States found that counties with large care homes had higher COVID‐19 mortality rates than counties without large facilities  in our models are useful results. Relative transport accessibility as indicated by the journey times measure did not emerge as significant predictor of cases or deaths in our models. Nor were air quality indicators predictors of case count or mortality. Population density had no separate effect on case count or deaths. These findings vary somewhat from other studies on rural populations; for instance Zhang and Schwartz  found thWe note that the spatial variation in cases and deaths was negligible. There was little spatial dependency in the number of cases in LSOAs: neighboring LSOAs did not influence numbers of cases in adjacent LSOAs. Pillar 1 cases and subsequent deaths were not highly clustered at LSOA level geography. Cases and deaths seemed to occur independently of observable spatial contiguity.4.1The findings relate very much to the types of cases that are found under the Pillar 1 testing framework. These are positive swabs found by testing health professionals (often found through surveillance rather than symptomatic presentation) and patients with urgent medical need. A more conventional sampling framework could include all symptomatic cases . Some such cases were found concurrently in May 2020 under Pillar 2 testing protocols. Possible demographic differences between Pillar 1 and Pillar 2 cases in the county of Norfolk alone are described in the data shown as Appendix Our analysis did not consider underlying health conditions. Individuals with multiple comorbidities, especially the metabolic syndrome triad  seem to be at higher risk of mortality following Covid diagnosis  would change the broad conclusions. Lack of variation in ethnic profile was both helpful and a drawback in the analysis. We cannot use data from this study region to test whether areas with larger ethnic minority populations had more cases or deaths. However, ethnicity not being an unobserved or poorly measured confounder makes it simpler to interpret the other candidate risk factors.Our findings relate to new case emergence and mortality during a period when social distancing behavior in England varied greatly. Lockdown conditions started on 20 March 2020, with restrictions imposed to reduce social contact that were enforced by civil penalties (fines). The measures imposed were school closures for most pupils, bans on socializing with persons from outside one's own household or (size‐limited) mutual support group, closures of nonessential businesses and “stay‐at‐home” orders which proscribed loitering in public spaces .Appendix A2. Demographic traits of patients who died at NNUH.Appendix A3. Emergency admissions of Norfolk & Waveney resident patients to acute care providers, aggregated by each of the five constituent clinical commissioning group areas for financial year April 2019‐ March 2020.Appendix A4. Selection procedure for eligible records.Appendix A5. Variance inflation factors for models fit as linear regressions, to test for problematic multi‐collinearity that might unduly bias 95% confidence intervals on coefficient estimates.Appendix A6. Comparison of demographics of persons in Pillar 1 and Pillar 2 testing frameworks, through 6 August 2020.Click here for additional data file.


Caltha palustris “a common wetland plant” to study whether warming affects growth and reproduction differently depending on origin of source population, potentially affecting phenotypic response to local climate. We conducted a 2‐year in situ temperature manipulation experiment using clone pairs of C. palustris in four regions, along a 1300‐km latitudinal gradient of Sweden. Open‐top chambers were used to passively increase temperature, paired with controls. Growth and reproductive traits were measured from 320 plants (four regions × five sites × two treatments × eight plants) over two consecutive seasons to assess the effect of warming over time. We found that warming increased plant height, leaf area, number of leaves, and roots. High‐latitude populations responded more strongly to warming than low‐latitude populations, especially by increasing leaf area. Warming increased number of flowers in general, but only in the second year, while number of fruits increased in low‐latitude populations the first year. Prolonged warming leads to an increase in both number of leaves and flowers over time. While reproduction shows varying and regional responses to warming, impacts on plant growth, especially in high‐latitude populations, have more profound effects. Such effects could lead to changes in plant community composition with increased abundance of fast‐growing plants with larger leaves and more clones, affecting plant competition and ecological functions such as decomposition and nutrient retention. Effects of warming were highly context dependent; thus, we encourage further use of warming experiments to predict changes in growth, reproduction, and community composition across wetland types and climate gradients targeting different plant forms.Global warming affects plant fitness through changes in functional traits and thereby ecosystem function. Wetlands are declining worldwide, and hence, ecosystem functions linked to wetlands are threatened. We use  In a warming experiment in wetlands across 1300‐km latitudinal gradient, we found that clonal pairs of Caltha palustris increased in plant height, leaf area, number of leaves, and roots. High‐latitude populations responded more strongly than low‐latitude populations. Increasing temperatures could lead to changes in plant community composition with increased abundance of fast‐growing plants with larger leaves and more clones, affecting plant competition as well as services such as flood attenuation and nutrient retention. This has already resulted in the migration of species both toward higher latitude and altitude  passive warming at the vegetation site by using open‐top chamber (OTC) or active warming by cables, (ii) space for time approach by observing species either at different latitudes or at altitudes, or (iii) reciprocal transplant experiments which move plants to a new location under novel climatic conditions  Does warming by means of OTCs lead to changes in growth  and reproduction ? (ii) Is warming affecting growth and reproduction differently depending on latitudinal position? (iii) Does extended warming over time lead to compound effects in growth and reproduction ? We hypothesized that warming will increase growth in traits related to both plant height and leaf area, together with increases in reproductive traits, such as number of flowers and number of fruits. We also hypothesized that the magnitude of the effect will be more pronounced for plants in northern latitudes, since temperature‐related constraints on growth and reproduction will be eased and that warming effects will increase cumulatively over time.To investigate the potential effect of increasing temperature on wetlands and to make predictions about change in ecosystem functions, we carried out a field experiment in four riparian wetland regions using open‐top chambers for passive warming and clones (ramets) of the common wetland plant 22.1Caltha palustris. It is a perennial herb, 10–40 cm tall and occurs over the whole of Sweden, having a circumboreal distribution ranging from 40° to 75°N and is also found in the Southern Hemisphere. Marshes or other wetlands are the predominant habitat  in 20 wetland sites in four regions (five sites in each region) of Sweden, spanning a latitudinal gradient of 1300 km Figure  with a hSites in Lund, Uppsala, and Umeå were situated in the riparian zone of 1st‐ to 3rd‐order streams and therefore had similar hydrology with shading provided by deciduous trees  and coniferous trees (Umeå). In the northernmost region, Kiruna, all sites had little to no shading provided by trees. The OTC was placed close ca 3–4 m from the stream banks in all sites except in Kiruna where they were placed 3–4 m from the mainstream flow in the wetland. The OTCs were kept in place over the whole course of the 2‐year experiment.C. palustris from each site to be included in the experiment. The plants were then carefully divided into ramets (hereafter clones) with as little damage as possible to above‐ and belowground parts. Caltha palustris forms short‐lived rhizome (epigeogenous rhizomes) and rarely also stolons (not found in this study)  from the most adjacent whether station for all four sites.2.3C. palustris in Lund was based on phenological reports of flowering reported by citizens to the Swedish National Phenological Network  and by personal observation in the actual regions close to the experiment for Uppsala, Umeå, and Kiruna. Plant height, number of leaves, and leaf area were measured non‐destructively on all three sampling occasions. Plant height was assessed for each individual by measuring the length of the longest petiole, which always exceeded the length of the stem. Leaf area was assessed by taking photographs of the leaves against graph‐paper background and later analyzed using ImageJ .To analyze reproductive traits, we counted the number of clones per individual at the end of the experiment as the number of additional clones compared with the number initially planted. Number of fruits was also measured once as the sum of fruits, including damaged or clearly missing fruits in the summer of 2014, whereas number of flowers was measured as the sum of open and closed flowers in both spring 2014 and 2015, which is the time peak flowering for 2.4To examine the effect of experimental warming on plant height and leaf area, we used a generalized linear mixed effect (GLMM model) with either plant height or leaf area as response variable, and region , treatment (Ambient and OTC), and time of observation  as explanatory variables. We further included clones nested within site as a random effect to account both for spatial autocorrelation between sites and for the genetic relatedness of the plant individuals in our study. The data for both plant height and leaf area were transformed using natural logarithm to achieve normally distributed residuals. Best model fit was assessed by starting with the full model (main factors and interactions) and then dropping subsequent non‐significant terms while comparing the fit of the models to the data using a likelihood ratio test until either only significant terms were included in the model or until dropping terms did not yield a significantly better model. The number of leaves per individual was analyzed using generalized linear mixed effect models with a Poisson distribution and a log‐link function using the same predictor variables as above . Similarly, the number of roots was also analyzed using Poisson error distribution and region and treatment as predictor variables. The predictor time was not included as root number was only assessed at the end of the experiment.To assess the effect of warming on the reproductive traits of both flower and fruit number, we used generalized linear mixed effect models (GLMM) using negative binomial distribution. Models on flower number included all predictor variables  for the analysis while fruit number only included region and treatment. Clonal production was relatively low with most individuals producing either one, a maximum of three, or no clonal offspring. Therefore, we recoded the number of clonal offspring to a binary variable as either present or absent (Yes or No) which we then analyzed using a binomial distribution with logit link function with treatment and region as predictor variables. Here again, we included site and clone as random factors with clone nested in site for all analysis on reproduction.lme4 , we analyzed data on growth and reproduction for each region and each time point separately as well. Thus, we used the same model structure outlined above, excluding region as predictor variable but instead conducting separate analyses per region  and time, only including treatment as predictor variable. All models were checked graphically if they fulfill the assumptions of normality and homoscedasticity of residuals. All statistical analyses were carried out using R (Rstudio)  and the 3The effect of warming differed between region, year, and location Table . WarmingAlthough the temperature did not increase in all OTCs during the whole period and for all regions Table , the OTCSimilar to plant height, leaf area increased in the OTCs Table . The treNumber of flowers increased by 50 percent under warming with significant differences observed between regions Table . Flower 4By dividing plants into clones, we could measure the individual trait response to experimentally increased temperatures using open‐top chambers (OTC). In general, we found that the OTC treatment led to an increase in all growth‐related traits  but only significantly affected one of the three traits associated with reproduction, namely the number of flowers. The effect of the treatment also increased with time for most of these traits and regions, except for Uppsala that showed a diverging pattern. The interaction between region and warming generally had a stronger effect on reproductive traits compared with growth‐related traits, indicating that other factors than temperature are important for these traits.The positive effect of warming on growth traits has been shown in earlier studies, either in experimental settings  showed somewhat diverging patterns concerning plant height and leaf area compared with the three other regions. It has been shown that OTCs could change several sheltering parameters like decreasing effects of surface flooding, snow, and wind depending on forest cover  which would increase the short‐term storage of carbon and nitrogen in plant tissue. Root number also increased with warming, which could help increase plant ability to store carbon ; writing–review and editing . Matti Ermold: Conceptualization (lead); data curation (lead); formal analysis (lead); investigation (lead); methodology (lead); writing–original draft (supporting). Lenka Kuglerová: Data curation (supporting); writing–review and editing (supporting). Roland Jansson: Data curation (supporting); writing–original draft (supporting). Keith W. Larson: Data curation (supporting); writing–original draft (supporting). Ann Milbau: Data curation (supporting); writing–original draft (supporting). Sara A. O. Cousins: Conceptualization ; funding acquisition (lead); project administration (lead); resources (lead); supervision (lead); writing–original draft ; writing–review and editing .Table S1‐S2Click here for additional data file.


Post‐abortion clinics located in regions with high HIV burden may ideal locations to integrate counselling and delivery of HIV pre‐exposure prophylaxis (PrEP), aligning with normative goals for integrated delivery of HIV and reproductive health care. The objective of this study was to gauge the degree to which Kenyan women seeking care for a pregnancy loss, including induced abortion, are at risk for HIV and whether women would welcome an introduction to PrEP prior to discharge from post‐abortion care.We conducted a mixed‐methods study from August 2019 to February 2020 with women ages 15 to 30 recruited sequentially as they were accessing post‐abortion care at public and private facilities in Thika and Kisumu, Kenya. Data collection was through a cross‐sectional survey and laboratory testing for common sexually transmitted infections (N = 200), and in‐depth interviews (N = 30). Descriptive statistics summarize PrEP knowledge and referrals and a multivariable log‐link binomial model estimated correlates of receiving a referral for PrEP. Qualitative data were analysed using inductive and deductive approaches.Chlamydia trachomatis was 18.2% and Neisseria gonorrhoeae was 2.0%. Half of the women scored ≥5 on a validated tool that would correspond to an expected HIV incidence of 9.5% per year. Approximately half (55.8%) of women were familiar with PrEP prior to the study and 33.3% received a referral from study staff to a clinic offering PrEP. In qualitative interviews, women expressed interest in accessing PrEP from the gynaecology ward that provided post‐abortion care but they preferred alternative locations for PrEP refills.Among 200 HIV‐negative women , the prevalence of Kenyan women accessing post‐abortion care have substantial HIV risk and were favourable about the idea of receiving support to initiate PrEP as part of care offered during post‐abortion care. These settings can be integrated into national PrEP programmes as locations providing PrEP referrals and initiation. To combat this, a priority strategy is to integrate programmes for reproductive health and HIV prevention, yielding provision of comprehensive care that can be delivered efficiently and in a cost‐saving manner . IntegraClinics providing abortion and post‐abortion care are one type of reproductive health service yet they are often overlooked for programmatic innovation due to the vulnerability of patients and the complicated legal situation of abortion. Kenya has the highest abortion rate in the East Africa region, estimated at 48/1000 women . In a 20Post‐abortion clinics serve women with spontaneous and induced abortion who often need emergency care to be medically stabilized followed by treatment for complications and psychosocial and/or behavioural counselling . Induced22.1We conducted a cross‐sectional mixed‐methods study in Thika and Kisumu, Kenya among women who were seeking care following a pregnancy loss. From August 2019 to February 2020, women seeking post‐abortion services were sequentially recruited by research staff who were contacted by nurses and clinical officers providing post‐abortion care at Thika sub‐County Hospital, KMET , and Jaramogi Oginga Odinga Teaching and Referral Hospital, when a potentially eligible women were seen at the facility. Eligibility criteria for these women included accessing care following a pregnancy loss and being 15 to 30 years. Women were excluded if living with HIV or with a social or physical condition that may make participation unsafe as determined by the study investigators. Enrolment was stopped once 200 women were accrued based on estimates of the sample size needed for moderate precision in estimates of STI prevalence.Treponema pallidum testing (with Rapid Plasma Reagin tests) and urine sample for Neisseria gonorrhoeae and Chlamydia trachomatis testing using Aptima GenProbe (for women enrolled in Thika) or GeneXpert (for women enrolled in Kisumu). Results from STI testing were returned to women via phone or in‐person based on participant preference; women with reactive results were given treatment according to national guidelines. If results from routinely conducted rapid HIV testing were available from the post‐abortion clinic, they were abstracted from clinic records and if they were not available, rapid HIV antibody testing was conducted by study staff and results were returned to women in real time. After completing the survey, women were counselled about PrEP and all women were offered a referral for PrEP services at a nearby facility of their choice. The woman’s preference for a referral/no referral was noted on the survey form. Research staff entered data from quantitative surveys and laboratory testing into a web‐based platform (CommCare).All enrolled women completed a survey to characterize demographics, reproductive and medical history, sexual behaviour, relationship characteristics, use of alcohol and/or drugs, HIV risk perception and unintended pregnancy, experiences with the recent pregnancy and pregnancy loss, and knowledge of and experience with PrEP. The Awareness, Interest, Desire, Action marketing model was applied to experiences with PrEP to form a PrEP “knowledge‐to‐use” cascade. Heavy alcohol use was measured with the Rapid Alcohol Problem Screening tool  and all A purposive sample of women who completed the survey and had a high risk for HIV was selected for in‐depth interviews. Drawing on standards for qualitative research, we aimed to enrol up to 30 women for this sub‐sample . To dete2.2p < 0.20 were included in a multivariable model. Qualitative interviews were analysed using inductive and deductive approaches  and then I remember how it [pregnancy loss] happened…for me I wouldn’t want to go there.” “They can access PrEP from there (gynae ward) but the problem would be coming back. Some fear since you may bump into someone.” Among women who did not know about PrEP during qualitative interviews, some recounted feeling too tired or overwhelmed by the experience of seeking postabortion care to remember what they had been told about PrEP by the research team. While women felt that PrEP could be initiated at a post‐abortion clinic, they would prefer to seek PrEP refills from a different location, such as a family planning clinic or pharmacy, to avoid seeing people they know or recalling the negative experience of their pregnancy loss.Numerous characteristics were different between women who were given a PrEP referral and those who were not Table . PrEP re4To our knowledge, these are the first data to explore knowledge and interest in HIV PrEP among Kenyan women accessing facility‐based care following an abortion. We found that these women are good candidates for PrEP based on their desire and potential for being exposed to HIV. Very few women had actual experience with PrEP; many learned about it for the first time from our research staff following their survey and one‐third received a referral to a PrEP clinic. In recent family planning and maternal and child health PrEP programmes in Kenya, approximately 20% of women have received a PrEP referral, and so women seeking post‐abortion care may accept a PrEP referral with similar or slightly greater frequency, an estimate that could be used for policy and programme planning . The oveWomen’s mental health varies dramatically after an induced abortion often with changes in depression, anxiety, and substance use , 27. WomWe found higher frequencies of PrEP knowledge and interest in women in Kisumu than Thika. HIV incidence in Kisumu is the highest in Kenya and this region has been scaling up PrEP and other prevention interventions very quickly in the past five years , 30, 31.Chlamydia trachomatis or Neisseria gonorrhoeae, and thus likely at substantial risk for HIV.This formative study was designed to inform a PrEP delivery programme that will be integrated into existing post‐abortion clinics in Kenya and to begin developing rapport for research to be conducted in these settings. Thus, our study is limited in size, we were not actually providing PrEP to study participants, and we do not have data on whether PrEP referrals were realized by participants. Additionally, our study is based on sequentially recruiting women accessing post‐abortion care at health facilities, which is somewhat of a convenience sample and misses a large segment of women who induce abortion at home and do not need to seek medical care. Since abortion and seeking post‐abortion care is highly stigmatized, there are no data to determine if our sample is representative of all women who seek post‐abortion care. Our efforts to talk to pharmacists that provide medical abortion pills did not yield methods to include them in this study but these are an important source to learn from and consider as PrEP providers in future work . A final5Post‐abortion care is an existing service that can be better leveraged to integrate HIV and STI services and other facets of reproductive health . An added benefit of integration is the opportunity to reduce the stigma associated with attending a post‐abortion clinic since multiple services are available. Women in this study expressed openness to receiving these types of services and engaging with comprehensive services once their immediate needs related to pregnancy loss were met. To advance the delivery of comprehensive sexual and reproductive health services, post‐abortion clinics can become a component of national programmes for PrEP and other HIV and STI services.RH, NM and TB conceived the study design. EC, LA, BK, VO, CK, FA, NW, EO, HM, CS and EB conducted data collection. KN, DT, BK, EC, VO and YZ conducted data analysis. RH and NM prepared the first draft of the paper. All authors read and approved the final manuscript.This work was supported by Children’s Investment Fund Foundation (R‐1808‐02992).