It was the aim to establish and validate in vivo confocal Raman spectroscopy for characterization of the lip barrier in conjunction with transepidermal water loss (TEWL) and skin capacitance assessments. For the first time in vivo, barrier‐relevant components of the lip  and ceramides are described.In 32 healthy volunteers, a dental tongue fixation device was inserted to prevent both voluntary and involuntary lip moisturization during measurements. Seventeen individual parameters relating to water, ceramide, and NMF content were assessed via Raman spectroscopy. Additionally, corneometry and TEWL were measured. To give a guidance for the required volunteer group size of future lip barrier studies for all test parameters, coefficients of variation (CV) were calculated and plots showing the required sample size for a given percentage treatment effect.Raman spectroscopy assessed parameters on the lower lip comprehensively characterized the state of the lip barrier. Parameter variability was sufficiently low to corroborate changes in most parameters using relatively small study populations.Lip skin is comparatively well hydrated. Biophysical measurement of the lip barrier function is a challenge, as unconscious licking of the lower lip has to be prevented. In vivo confocal Raman spectroscopy provides insightful parameters for the characterization of the lip barrier and sufficiently low inter‐individual variability to assess relatively small parameter changes employing relatively few study subjects. Differences at the molecular level and at a high spatial resolution are detectable, and these insights might provide a breakthrough in the evaluation of lip barrier function and developing solutions for lip care. These vermilion borders are the only part of the facial anatomy where the oral mucosa is permanently exposed to the environment.Lips are covered by a thin layer of stratum corneum with their red coloration believed to result from a combination of decreased density of keratin and translucency of the tissue allowing visualization of the underlying capillaries.A number of studies have used corneometry and transepidermal water loss (TEWL) as methods for examining the hydration and dryness of lips.As a sound investigative and widely accepted method,For further understanding, we wanted to investigate Raman parameters of spatially resolved lip water content, NMF, and ceramide content in comparison with established advanced in vivo methods for evaluating and characterizing both stratum corneum capacitance and transepidermal water loss (TEWL) of the lip skin. Our intention was to establish consistency and validity of these combined methods for meaningful lip product(s) evaluation. Lip treatment products often incorporate oils, waxes, and paraffins in their formulations, with the desired outcome of preventing skin barrier dryness and roughness as well repairing this condition. Well‐established capacitance methods measure the degree of hydration including the widely used Corneometer.On the other hand, TEWL is often used to measure “moisturization,” by the degree which water evaporates from skin.2This non‐invasive study was executed according to the principles of good clinical practice (GCP), with healthy volunteers providing their written informed consent for inclusion.® , and transepidermal water loss (TEWL) was measured by Aquaflux®  on 32 subjects providing valid data.Thirty‐four healthy female volunteers were recruited with a mean age of 48.4 ± 13.0 years (mean ± standard deviation). Measurements of ceramide content and water profiles were performed by in vivo confocal Raman spectrometer  on a sub‐panel of 23 of the recruited subjects. Skin moisturization was measured by Corneometer2.1Inclusion required volunteers willing to actively participate in the study and abide by its requirements. Written informed consent was required for participation. The study was open to female participants aged 18‐65 years with a BMI of <30 and Fitzpatrick skin types I‐III with uniform skin color and absence of erythema or dark pigmentation within the test area.The volunteers were required to have normal to dry lips of average size (particularly the lower lip).2.2Exclusion criteria required were pregnancy or lactation, drug/alcohol disorders, and infectious diseases; cancer and those undergoing chemotherapy and/or radiotherapy; topical medication at the test site within 7 days prior to study commencement; history of any acute or chronic disease that could interfere with or increase the risk on study participation; active (flaring) disease or chronic skin allergies (atopic dermatitis/eczema), or recently treated skin cancer (within the last 12 months); visually dry, chapped, fissured, or cracked lips at the baseline visit; planned surgeries and/or invasive medical procedures during the course of the study; medical procedures, such as laser resurfacing, or plastic surgery to the test sites (lips) within the last 2 months; history of hypersensitivity to any cosmetics and/or personal care products; history of immunosuppression/immune deficiency disorders (including [HIV infection or AIDS]) or currently using immunosuppressive medications; sunburn, abrasions, scar tissue, tattoos, or diseases of the skin that might interfere with the evaluation made in this study, or that expose study participants to unacceptable risks as determined by a technician; uncontrolled diseases such as asthma, diabetes, hypertension, hyperthyroidism, or hypothyroidism regular use of tanning beds.33.1Instrumental measurements took place in an air‐conditioned room at a temperature of 21 ± 1°C and at 50 ± 5% relative humidity. Subjects remained climatized for at least 30 minutes beforehand. To avoid undesirable moisture accumulation of the lower lip during acclimatization and measurements, a dental plastic device was fixed to the subject's tongue . A small amount of the scattered light is found at wavelengths higher than the incident laser light. This part of the scattered light provides information about the molecular composition of the skin. Approximately 8 profiles per test area and assessment time were captured in the center part of the lower lip.3.2.1The concentration profiles (approximately 8) were calculated from Raman spectra (wavenumber ranged from 2600 to 3800 cm‐1) that were taken at different depths. Profiles were defined by water content at skin depths of 0, 2, 4, 6, up to 24 μm, measured from the surface of the skin, in steps of 2 μm. The following parameters were assessed from obtained water profiles: water content at a depth of 0 μm; water content within stratum corneum including stratum disjunctum ; water contents below the stratum corneum (water content in deeper layers); water gradient within and below the stratum corneum; and thickness of stratum corneum including stratum disjunctum  and total NMF  were calculated from Raman spectra (wavenumber ranged from 400 to 1800 cm‐1) that were taken at different depths. Profiles were defined by total ceramide content at skin depths of 5 and 10 μm, measured from the surface of the skin.3.3Transepidermal water loss is a non‐invasive method to measure the barrier function of the skin and is regarded as a sensitive parameter to quantify skin barrier function. TEWL was measured with a closed chamber system using an Aquaflux AF200  as described elsewhere.3.4The measurement of stratum corneum hydration was performed by the electrical capacitance method with a Corneometer CM 825 (Courage & Khazaka). Measurement principles are described elsewhere [24]. Five measurements per test area and assessment time were performed Figure .4Stratum corneum and stratum disjunctum thickness and water gradients were calculated from curves of total water content according to an appropriate non‐linear model. Additionally, total water contents within stratum corneum including stratum disjunctum and total water contents below the stratum corneum  were calculated as AUC (area under curves). Furthermore, the stratum corneum thicknesses were divided into three equally spaced areas. For each of these areas the AUC was calculated Figure .Results of water concentration profiles by Raman spectroscopy for all measured depths 0 to 24 μm) were summarized to mean concentration profiles (curves). Accordingly, ceramide and NMF in the two measured depths (5 and 10 μm) were summarized to mean concentrations over the repeated measurements per depth for both areas. For each instrumental parameter (TEWL and skin hydration) and each Raman water and ceramide/ NMF parameters, pairwise comparisons of treatments were performed with t test. Parameter distributions were assumed to be normal. For all test parameters means, standard deviations and confidence intervals were calculated. Plots with required sample size numbers for hypothetical percentual lip treatment effects were displayed .5Table As expected, water content assessed via Raman was lowest on the surface (parameter 1) and increased gradually (parameters 3‐5) until reaching a plateau (parameter 6). Accordingly, two water gradients were identified: a steep one within the SC (parameter 7) and a level one below SC (parameter 8). SC thickness was calculated at the point of intersect of these tangents (parameter 9). Furthermore, water content normalized to SC thickness (parameters 10‐13) was observed to increase with increasing depth. Both, NMF and ceramide content slightly decreased at greater depths (parameters 14‐17).The required difference in parameter for significant distinction is a measure proportionate to the parameters' variability. From the three groups of Raman parameters assessed  the largest variability was observed in the amount of NMF (parameters 14 and 15). The lowest variability was noted in the water content including increased skin depth (parameter 6). Notably, Raman parameters calculated by normalizing water content to stratum corneum thickness (parameters 10‐13) varied less than the values they have been calculated from.Greater inter‐group variances were seen between the TEWL data and corneometry data (parameter 18 and 19), and these need to be taken into consideration when measuring these parameters. This is also corroborated by the variances and water gradient fluctuations seen in the Raman spectra (parameters 7 and 8). When evaluating the three methods used in this study, there was more precise less scattering of data with the Raman measurements as compared to TEWL and capacitance measurements.For each parameter, the necessary number of subjects to detect a statistically significant change (power ≥0.8) was calculated. As expected, this number is proportionate to parameter variability and indirectly proportionate to the magnitude of change to be observed. In general, investigators seeking to verify smaller changes will therefore require the recruitment of higher subject numbers. This relationship for all parameters is illustrated in Figure 6Prior studies have demonstrated the combination of TEWL (open‐chamber) and corneometryThis study was therefore undertaken to establish consistency and validity of three combined methods for more purposeful lip skin barrier evaluation. To this end, for the first time, Raman spectroscopy in conjunction with well‐established methods of TEWL and capacitance measurements has been applied on the lip. In our study, we also found that a prerequisite for reproducible measurements was to fix the tongue with a dental device to prevent the involuntary reflex of lip moisturization.Stable measurements enabled the assessment of a plethora of parameters relevant for in vivo characterization of the lip skin barrier. Our own unpublished results on forearm skin for example indicate that SC thickness tends to decrease in well‐kept skin and water content in the deepest part of the SC tends to increase with improved water barrier. Furthermore, we saw that an increased water gradient can be interpreted as a direct measure for skin barrier quality, as it signifies improved barrier properties achieved by thinner SC layers. The relevance of each parameter will vary in focus depending on the context of an investigation—ranging from fundamental skin physiology research to evaluation of cosmetic efficacy, as well as those parameters identified during the course of clinical studies.In this study, the values obtained are not very different from those measurements undertaken on the volar forearm Table  with theAlthough measured parameter variability may have a number of physiological causes, this variability is an important indicator for establishing method reproducibility and hence highly relevant for future experimental design(s). For instance, the observed NMF variability could be explained as follows: Firstly, NMF is a collective term for several cutaneous water attracting components  with different metabolic regulatory pathways. Thus the joint variability is not surprisingly larger. Secondly, filaggrin as a major component of NMF is formed gradually during the course of terminal keratinocyte differentiation. Hence, NMF forms a decreasing gradient actually increasing toward the SC surface. Relatively low amounts of NMF were found in lip SC. The values were approximately the half of what was found in volar forearm SC. Lower amounts of a compound necessarily correlate with higher variability as seen comparing the coefficients of the variability of NMF at 5 and 10 µm. Likewise, the high variability of the water gradient below the SC can be attributed to the low value of the gradient itself, illustrating the hydration capacity reaching a plateau value below the SC. Notably, the variability of water content normalized to SC thickness exhibits a lower variability than constituent values. This indicates a strong correlation between water content and SC thickness across the study population. Clearly, the water‐holding capacity of skin is a remarkably stable physiological parameter.The required number of subjects for a statistically significant corroboration of a defined parameter change varies markedly between parameters. This number is proportionate to the variability of the parameter itself. Hence, determination of changes in water content and derived parameters Figure A—with thImportantly, parameters exhibiting a low inter‐individual variability and thus requiring low subject numbers for statistically sound statements may be particularly difficult to alter through external intervention by cosmetic ingredients or pharmaceutical drugs. Water content below the SC, for example, exhibits remarkably low variability Table  resultin7This in vivo confocal Raman spectroscopy led tri‐method approach in the evaluation of the lip barrier and skin lip hydration can be considered both relevant and valid. It offers a complimentary assessment of lip skin barrier functioning. For the first time in vivo, barrier‐relevant components of the lip (NMFs and Ceramide) are described. Inter‐subject variations also are considered for making statistical calculations required for such studies to ensure statistically significant distinction (power ≥0.8) between test groups for product evaluation. In vivo confocal Raman spectroscopy is able to detect differences at the molecular level and at a high spatial resolution, and these insights might provide a breakthrough in the evaluation of lip barrier function and developing solutions for lip care.


Neoantigens are tumor‐specific mutated proteins that are exempt from central tolerance and are therefore capable of efficiently eliciting effective T‐cell responses. The identification of immunogenic neoantigens in tumor‐specific mutated proteins has promising clinical implications for cancer immunotherapy. However, the factors that may contribute to neoantigen immunogenicity are not yet fully understood. Through molecular mimicry of antigens arising during cancer progression, the gut microbiota and previously encountered pathogens potentially have profound impacts on T‐cell responses to previously unencountered tumor neoantigens. Here, we review the characteristics of immunogenic neoantigens and how host exposure to microbes may affect T‐cell responses to neoantigens. We address the hypothesis that pre‐existing heterologous memory T‐cell immunity is a major factor that influences neoantigen immunogenicity in individual cancer patients. Accumulating data suggest that differences in individual histories of microbial exposure should be taken into account during the optimisation of algorithms that predict neoantigen immunogenicity. In this review, we discuss pre‐existing heterologous memory T‐cell immunity, which could be a major factor that influences neoantigen immunogenicity in individual cancer patients. Differences in individuals' histories of microbial exposure should be taken into account during the optimisation of algorithms that predict neoantigen immunogenicity. Most tumor‐associated antigens (TAAs) targeted in clinical applications are overexpressed self‐proteins, namely self‐antigens. Self‐antigens have low immunogenicity because the high‐affinity T cells that recognise these antigens are deleted through the mechanism of central tolerance during thymic development to avoid triggering autoimmunity. Low immunogenicity is one of the key reasons for the ineffectiveness of TAA‐based therapeutic antitumor vaccines. In contrast to low‐immunogenicity TAAs, neoantigens, which are generated during the degradation of mutated proteins, are promising anticancer vaccines. As they are considered to be foreign peptides by the immune system, they may be highly immunogenic and capable of eliciting strong T‐cell‐mediated immune responses.Previously, a therapeutic MHC I‐restricted neoantigen vaccine was shown to elicit effective T‐cell immunity that led to tumor rejection in mice.in silico human leucocyte antigen (HLA)‐binding prediction algorithms after pinpointing missense mutations in patient tumor tissue by exome sequencing. These neoantigen prediction algorithms are effective for the majority of HLA class I alleles but perform rather poorly for HLA class II alleles.Currently, the identification of candidate neoantigens is performed mainly with In this review, we discuss the characteristics of immunogenic peptides and then elucidate how the gut microbiota and other environmental microbes may affect neoantigen immunogenicity via cross‐reactive heterologous memory T‐cell immunity as a result of molecular mimicry among antigens. We propose that pre‐existing memory T‐cell immunity to these microbes is likely to be an immunoediting factor that sculpts tumors in individual patients by influencing neoantigen‐specific T‐cell responses.Although the factors that affect neoantigen immunogenicity have not been specifically addressed thus far and remain largely unknown, the features of neoantigens correlated with immunotherapeutic efficacy or disease prognosis have been described previously.Nevertheless, as the host TCR repertoire evolutionarily adjusts itself to detect pathogenic antigens, it is believed that neoantigens homologous to pathogenic antigens are more likely to be immunogenic than nonhomologous neoantigens.One of the key features of T cells is their antigen specificity determined by the TCR. Although T‐cell recognition is very sensitive to a specific antigen,However, studies have shown that homology among TCR‐contacting residues contributes to TCR cross‐reactivity between self‐peptides and microbial or environmental peptides.Neoantigens are generated from mutated self‐antigens; thus, the degree of similarity in three‐dimensional shape between the TCR contact residues of a neoantigen and those of a self‐antigen determines the foreignness of the neoantigen. In other words, neoantigen immunogenicity is set by the degree of similarity between the TCR contacts of a neoantigen and those of both self‐antigens and foreign antigens Figure a.Mammals have trillions of gut microorganisms, including predominantly bacteria but also fungi, archaea, viruses and protozoans. The microbiota coexists with the host and is highly related to both the development and function of the immune system. The interactions of defined microorganisms with their host can be highly contextual with the same microbe developing as a mutualist or parasite according to the nutritional, coinfection or genetic landscape of the host. As pathogens, the microorganisms of the microbiota can lead to infection and inflammation; as mutualists, they help the host digest food, act as a barrier for pathogen defence and regulate a variety of mucosal immune responses.More deeply, commensal microorganisms are required for the maturation of the immune system, which ‘learns’ to distinguish commensal bacteria from pathogenic bacteria and tolerate these commensal microorganisms. The gut microbiota continues to influence the immune system in different ways. It has been shown to modulate neutrophil migration and function and to affect the differentiation of T‐cell populations into different types of helper cells (Th) or into regulatory T cells. Additionally, the gastrointestinal tract is one of the main entry sites for pathogens. In gastrointestinal infections, pathogens are captured by Peyer's patch‐resident M cells that allow pathogen‐derived antigens to be transferred across the epithelial cell layer and delivered to the peripheral immune system, specifically to antigen‐presenting cells such as dendritic cells (DCs), leading to the activation of T cells, and B cells, leading to the secretion of IgA antibodies. Interestingly, commensal antigens also use this mechanism to induce the production of low amounts of IgA antibodies through the modulation of their immunodominant epitopes to facilitate colonisation.Through exposure to various complex bacterial antigens, the immune system accurately adapts its innate and adaptive responses against self‐antigens and nonself antigens. Accumulating evidence shows the function of the gut microbiota in modulating both host carcinogenesis and antitumor immunity. The previously mentioned study of long‐term survivors of pancreatic cancer found that neoantigen‐specific T cells existed in the peripheral blood and that tumor‐infiltrating leucocytes cross‐reacted with both neoantigens and homologous microbial antigens.et al. showed that memory‐phenotype, human immunodeficiency virus (HIV) antigen‐specific T cells existed in the peripheral blood of HIV‐seronegative adults. In contrast, these memory‐phenotype cells were absent in umbilical cord blood at birth. Interestingly, these HIV‐specific T cells produced IFN‐γ and IL‐2 when stimulated with peptides from gut microbes, including human gut commensals such as Ruminococcus, Lachnospiraceae and Bifidobacterium species. Therefore, one plausible conclusion is that host exposure to the cross‐reactive antigens of diverse environmental or gut microbes results in the generation of memory T cells that can respond to unencountered antigens,Gut commensal microbes that coexist innocuously with the host express the foreign antigens that the host is most frequently exposed to. Exposure to microbial antigens not only generates memory T cells specific for these antigens but also increases the number of T cells that cross‐react with previously unencountered antigens. Therefore, the gut microbiota potentially has a profound impact on the T‐cell response to previously unencountered tumor neoantigens arising during cancer progression or viral antigens acquired in a relatively late stage. A study by Su Fusobacterium is mostly related to inflammatory influences, and Bacteroides is mostly related to tolerogenic influences in humans.Bifidobacterium was found to be associated with the antitumor effects of programmed cell death protein 1 ligand 1 (PD‐L1)‐specific antibody therapy in a mouse model of melanoma. Oral administration of Bifidobacterium to recipient mice with an established tumor was shown to increase the tumor‐specific CD8 T‐cell response and reduce tumor growth.Bacteroides fragilis and Burkholderia cepacia, which both belong to the genus Bacteroides, enhance the antitumor effect of anti‐CTLA‐4 antibodies in mice,Bifidobacterium species ameliorate the gut immunopathology associated with CTLA‐4 blockade.Akkermansia muciniphila has been identified to be associated with the best clinical response in non‐small‐cell lung cancer (NSCLC) patients.The gut microbiota greatly influences both innate and adaptive immune responses. Recently, it was revealed that microbiome sequence similarity can increase or decrease the immunogenicity of MHC II‐restricted CD4 T‐cell epitopes.However, sequence similarity with a microbiota‐derived epitope does not guarantee that an epitope will have higher immunogenicity than an epitope without sequence similarity. It has been reported that CD4 T‐cell epitope immunogenicity tends to be associated with the nature of the microbiota that shares sequence similarity with the epitope.Unlike mice that live in specific‐pathogen‐free environments, humans are constantly exposed to various microbes after birth and receive various microbial vaccines. A serological profiling study of human populations revealed that every adult was exposed to an average of 10 viral species and some adults were even exposed to 84 species.It has been shown that prior exposure to a related or completely unrelated pathogen can alter the host immune response to a second heterologous pathogen. This phenomenon is named heterologous immunity. Memory T cells play a critical role in long‐term heterologous immunity.et al.How pre‐existing heterologous immunity to viral and microbial pathogens affects neoantigen‐specific T‐cell responses has not yet been fully investigated. However, the previously mentioned study by Balachandran In all, evidence supports the possibility that microbial exposure can shape neoantigen‐specific T‐cell responses through TCR‐mediated cross‐reactivity Table . It remaGiven that gut microbiota and environmental bacterial exposure histories vary among individuals in different countries, pre‐existing heterologous memory T‐cell immunity may also vary among individual cancer patients. The T‐cell immune repertoire and function shaped by regional differences in the gut microbiota, environmental bacteria and endemic infectious agents should be investigated in the process of determining the quality or immunogenicity of a neoantigen. In general, we propose that the degree of similarity between microbial antigens and self‐antigens can be considered to be an immunogenicity parameter for neoantigen prediction.The authors declare no conflict of interest.


To the Editor,We read with interest the recent article by Yu et al, which reported asymptomatic transmission of coronavirus disease 2019 (COVID‐19).So far, asymptomatic carriers of COVID‐19 may be found by the following ways.Since April 1, 2020, the number of asymptomatic infectors has been reported online by National Health Commission of the People's Republic of China on a daily basis, and the following management measures were required to be carried out to minimize the risk of their transmission in China.At present, persons who are significant epidemiological associations with COVID‐19 patients  will be put under 14‐day centralized medical observation in China.In future, further definition of high‐risk populations and development of effective screening strategies and programs will support rapid identification and management of asymptomatic carrier transmission of COVID‐19.In conclusion, asymptomatic carriers of COVID‐19 can be contagious. Identification and management of these asymptomatic infectors has been strengthened in China. These measures may also help other countries to combat the COVID‐19 epidemic.The authors declare that they have no competing interests.Jianhui Peng: Formal analysis; Writing‐original draft; Writing‐review & editing . Dongwei Su: Formal analysis; Writing‐original draft; Writing‐review & editing . Ziwei Zhang: Writing‐review & editing . Mingke Wang: Conceptualization (lead); Formal analysis ; Project administration (lead); Supervision (lead); Validation (lead); Writing‐original draft ; Writing‐review & editing (lead).The article does not contain the participation of any human being and animal.


Cenozoic mammal evolution and faunal turnover are considered to have been influenced and triggered by global climate change. Teeth of large terrestrial ungulates are reliable proxies to trace long‐term climatic changes due to their morphological and physicochemical properties; however, the role of premolar molarization in ungulate evolution and related climatic change has rarely been investigated. Recently, three patterns of premolar molarization among perissodactyls have been recognized: endoprotocrista‐derived hypocone (type I); paraconule–protocone separation (type II); and metaconule‐derived pseudohypocone (type III). These three patterns of premolar molarization play an important role in perissodactyl diversity coupled with global climate change during the Cenozoic in Asia. Those groups with a relatively higher degree of premolar molarization, initiated by the formation of the hypocone, survived into Neogene, whereas those with a lesser degree of molarization, initiated by the deformation of existing ridges and cusps, went extinct by the end of the Oligocene. In addition, the hypothesis of the “Ulan Gochu Decline” is proposed here to designate the most conspicuous decrease of perissodactyl diversity that occurred in the latest middle Eocene rather than at the Eocene–Oligocene transition in Asia, as conventionally thought; this event was likely comparable to the contemporaneous post‐Uintan decline of the North American land fauna. Three patterns of premolar molarization play an important role in perissodactyl diversity coupled with global climate change during the Cenozoic in Asia. In addition, the hypothesis of the “Ulan Gochu Decline” is proposed to designate the most conspicuous decrease of perissodactyl diversity that occurred in the latest middle Eocene rather than at the Eocene–Oligocene transition in Asia, as conventionally thought. The second one (the “Mongolian Remodelling”), comparable to the European “Grande Coupure,” was at the EOT and may have been a response to the sudden global drop of temperature. It is noteworthy that the endemic Asian taxa sporadically dispersed to North America during the Paleogene, but there was apparently little dispersal in the opposite direction Beard, . A very Hispanotherium with hypsodont teeth in the middle Miocene indicates a more abrasive diet and a slightly more open and drier habitat, which is enhanced in the late Miocene with the spread of Old World savanna palaeobiome  occurred during the latest middle Eocene rather than at the Eocene–Oligocene boundary in Asia, as conventionally thought. However, whether this event also impacted other fossil mammals in Asia needs further investigation.None declared.Bin Bai: Data curation ; formal analysis ; funding acquisition ; investigation ; methodology ; project administration ; software (lead); visualization (lead); writing–original draft (lead); writing–review and editing (lead). Jin Meng: Methodology ; project administration ; supervision ; writing–original draft (supporting). Christine M. Janis: Formal analysis ; investigation ; methodology ; writing–original draft (supporting); writing–review and editing (supporting). Zhao‐Qun Zhang: Data curation ; formal analysis ; funding acquisition ; methodology . Yuan‐Qing Wang: Funding acquisition ; project administration ; supervision .


This study reveals that softness of boiled cassava roots and in‐ground storage‐ability as key attributes influencing varietal preference in addition to high yield, non‐bitter roots, disease resistance, early maturity and drought resistance This study aimed to identify cassava quality attributes preferred by users along the food chain, in order to provide breeders with criteria for prioritisation. Survey and consumer‐testing studies were conducted within Apac and Luwero districts in Uganda. Additionally, sensory evaluation by trained panellists was conducted to determine descriptors for assessing quality of boiled roots. Results revealed softness of boiled roots and in‐ground storability as key attributes influencing varietal preference besides high yield, non‐bitter roots, disease resistance, early maturity and drought resistance. For some attributes like in‐ground storability, preference differed significantly between locations and showed differentiation by gender. Local varieties were found to be superior in quality attributes. From sensory evaluation, twenty‐one descriptors associated with appearance, texture, taste and aroma of boiled roots were determined. Findings from this study are vital for breeders to adopt gender‐responsive approaches in order to develop varieties that meet the needs and preferences of end users. Manihot esculenta Crantz) ranks as the sixth major crop after wheat, rice, maize, potato and barley and is an important source of food and income for over 800 million people globally  to determine the cassava quality attributes preferred by men and women end users; and (ii) to determine key descriptors that can be used in sensory evaluation of boiled cassava roots and then applied by breeders to improve consumer acceptability of new varieties.This section is divided into two related objectives, namely (i) determination of qualities of cassava preferred by men and women based on survey and consumer‐testing and (ii) determination of sensory descriptors for sensory evaluation of boiled cassava, outlined hereafter.A descriptive survey employing mixed methods of data collection was conducted to understand the perceptions of men and women on food‐value attribute qualities of boiled cassava. Mixed methods of data collection were used to provide a more comprehensive approach to the research question Almalki, . Accordiet al.  and then covered with two to three pairs of banana leaves. Similar to what people typically do, another saucepan was placed on top as a cover and then roots are cooked on a firewood stove for 1 h. In Apac, the sectioned cassava roots were placed in a saucepan prepared with water at about a quarter volume of the saucepan. Another saucepan was used as a cover and the roots were cooked for 45 min on a firewood stove. Adopting local cassava preparation and cooking methods, that is, steaming and boiling were considered to enable informed assessment of preferred quality attributes.In preparation for consumer‐testing, caution was taken to select appropriate places which would be prepared with chairs and tables for comfort during testing and which allowed easy recruitment of men, women and youth. Upon seeking consent to participate, samples from all the four to five varieties were presented at ago since we were interested in the consumer's comment on the appearance and texture before tasting and evaluation. The enumerator administered the questionnaire to the participant as they tasted and evaluated the samples. To ensure content validity, the tools were reviewed by social science research professionals from National Crops Resources Research Institute (NaCRRI), the University of Greenwich, and the French Agricultural Research Centre for International Development to verify the representativeness and suitability of the questions. Ethical considerations were met by seeking the participants' consent to participate before commencing with the interview. Participants signed a consent form upon understanding the objective of the research and allowing to participate. In addition, the respondents were assured before the interviews began that any information linking results to individuals would be kept confidential.Having learnt from consumer‐testing and surveys, there was need to establish standard descriptors that can be routinely used by sensory panellists to assess quality attributes during varietal development process by breeders. To this end, seven randomly selected plants per plot of a given variety were harvested and then randomly selected ten marketable roots taking care of root representation of the whole sample set. Soil and debri were removed from the roots before packing them in labelled bags prior to being transported to the laboratory for processing.In the laboratory, the roots were washed to remove dirt and/or adhering soil debri. The distal and proximal ends of the roots were cut‐off and discarded, and thereafter, the remaining central portions of the roots were peeled and cut into equal root sections each measuring 6 cm long. Thereafter, fourteen to fifteen root sections were randomly sampled per clone, washed in clean tap water and wrapped in a banana leaf which was later tied with a banana fibre and a coloured string for ease of identification at serving point. The wrapped root sections were then cooked in a saucepan measuring at least 57.5 cm in diameter and height of 21.0 cm whose base had been laid with banana sheath and about 5000 mL of water added to it. The banana sheath is to stop water from getting in contact with the wrapped root sections to ensure cooking is only by steam.Cooking was done on a gas cooker with four burners all set at maximum for 55 min. It was important to maintain the same water temperature and cooking time across all test samples. At the end of cooking, we turned off the gas cooker and allowed for 10 min of cooling at room temperature (about 23 °C) before we started serving the cooked samples to trained panellists who included seven males and six females for sensory evaluation. The panellists were purposively selected because they had been previously trained on how to undertake sensory evaluation of various food products from root, tuber and banana crops. Each panellist was provided a cup of drinking water (Rwenzori brand). At the time of serving boiled cassava to panellists, the plastic plates onto which the boiled cassava was served were labelled with unique labels corresponding to the test cassava clones.Principally, one clone was served to the panellist at a time and requested to evaluate the sample based on their prior knowledge regarding appearance and texture both in mouth and touch; taste and aroma were also assessed. The panellists were given plain paper to write descriptors, after which a discussion was held with all panellists and the team facilitating the activity. The panellists were requested to highlight the key terminologies they had used to evaluate the attributes of cooked cassava. The highlighted descriptors were written on flip charts that were pinned on the wall of the training room so that everybody could easily follow the discussion. The highlighted descriptors were carefully grouped into categories such as texture by hand, aroma, appearance, taste and texture by mouth. In the end, after a series of discussions and evaluating a number of cassava clones, a catalogue of descriptors for boiled cassava was developed Table Content analysis was employed to analyse qualitative data collected from the surveys and consumer‐testing  between men and women, and across different locations  than that of women except for in‐ground storage ability attribute for which men had a significantly higher  percentage than women  between Apac and Luwero, there were no significant differences  between the two districts for the other attributes  in Apac than in Luwero, while the percentage of women who prefer early maturing and drought‐resistant varieties is significantly higher  in Luwero than Apac , an activity mainly done by women who claim that NAROCASS 1 produce tasty and much brew compared to other varieties. Despite Bao being the most preferred variety in Apac, it was not mentioned as one of the preferred varieties in Luwero. Previously, a study by Iragaba et al. , cassava flour, kalo (cassava thick paste), and produces a lot of local brew’ women FGD in Apac. Information collected from a women FGD in Apac district indicated that NAROCASS 1 was preferred: ‘It is high yielding, the stems have ready market and makes very strong local brew which has a nice aroma’.End users in Apac prefer Bao, NAROCASS 1 and NASE 14, varieties for boiled form of consumption, whereas in Luwero the most preferred varieties are NASE 14 and TME 14 Table . Informaa et al.  also fouet al., et al., Consumer‐testing results indicate that most of the attributes preferred by users are the same as those we identified during the survey Fig. . The facet al., et al., Participants in the consumer‐testing exercise were also asked how often they consumed boiled cassava. Results indicated that most consumed cassava on a daily basis or several times a week Table . This reet al., et al., et al., In addition to the survey and consumer‐testing, sensory evaluation was conducted to develop descriptors which could be used in routine assessment of quality boiled cassava of new cassava clones developed by the breeding programmes. For each of the major preferred attributes of boiled cassava identified by end users, we developed descriptors and a standardised methodology by which they can be assessed by panellists Table . AccordiP‐value > 0.05) between men and women both within a given district and across different districts, whereas in‐ground storability of cassava roots was more preferred by men in Luwero district. In designing breeding programmes, there is a need to consider the differentiated preferences in order to develop varieties that meet the needs, aspirations and preferences of both men and women. Most consumers indicated that they eat cassava on a daily basis. This points out the importance of the crop in the livelihoods of men and women in study areas. End users in the food chain for boiled cassava indicated an array of preferred attributes. This information is critical for breeders in developing new varieties that meet the needs and preferences of users along the food chain. Breeding for multiple attributes calls for systematic attribute prioritisation during breeding work. Use of selection indices can optimise the weight given to each of the attributes and increase the likelihood of adoption of new varieties. The varieties found to have desirable attributes preferred by end users could be used to enrich breeders' germplasm. Secondly, in this paper, the twenty‐one sensory descriptors determined could be used by breeders in assessing the quality of the cassava breeding lines before they are released to the end users.Data sets presented in this paper have two major findings. Firstly, that men and women ranked softness of boiled roots and in‐ground storability in addition to high fresh yield, non‐bitter roots, disease resistance, early maturity and drought resistance as the most preferred quality attributes about cassava. Exceptionally, the preference for soft boiled cassava roots was not significantly different ; Data curation ; Formal analysis (lead); Methodology ; Writing‐original draft (lead); Writing‐review & editing (lead). Sophia Hamba: Conceptualization ; Data curation ; Formal analysis ; Methodology ; Writing‐original draft ; Writing‐review & editing . Ritah Ann Nanyonjo: Conceptualization ; Data curation ; Formal analysis ; Methodology ; Writing‐original draft ; Writing‐review & editing . Michael Kanaabi: Conceptualization ; Data curation ; Formal analysis ; Methodology ; Writing‐original draft ; Writing‐review & editing . Ephraim Nuwamanya: Conceptualization ; Data curation ; Formal analysis ; Methodology ; Writing‐original draft ; Writing‐review & editing . Doreen Mpamire: Data curation ; Formal analysis ; Methodology ; Writing‐original draft ; Writing‐review & editing . Nicholas Muhumuza: Data curation ; Formal analysis ; Methodology ; Writing‐original draft ; Writing‐review & editing . Elizabeth Khakasa: Conceptualization (supporting); Data curation ; Formal analysis ; Methodology ; Writing‐original draft ; Writing‐review & editing . Hale Ann Tufan: Conceptualization ; Funding acquisition ; Investigation ; Methodology ; Supervision ; Writing‐review & editing . Robert Sezi Kawuki: Conceptualization ; Data curation ; Formal analysis (supporting); Funding acquisition ; Investigation ; Methodology ; Project administration (lead); Resources ; Supervision (lead); Validation ; Visualization ; Writing‐original draft (supporting); Writing‐review & editing (supporting).This study was approved by the National Research Ethics Committee. Research teams obtained ethical approval prior to the fieldwork. Participants were informed about the study objectives, they could stop the interview at any point. Consent from sensory panellists and from consumers participating in this study was obtained and the research respected the rules of voluntary participation and anonymity. Food samples were prepared according to good hygiene and manufacturing practices.


Prior to the implementation of social distancing measures, we monitored the close family contacts of the first 400 cases of COVID‐19 in Singapore for SARS‐CoV‐2 infection to determine the risk of infection with age. Adjusting for gender and household size, the risk of COVID‐19 infection in household contacts was found to increase with age. The outcome from this analysis is important to guide containment measures for the ongoing pandemic such as school closures and healthcare resource allocation.2Close household contacts of a laboratory‐confirmed COVID‐19‐infected case in Singapore, a city‐state in South‐East Asia with a population of 5.7 million, were contact traced and interviewed for demographic characteristics. We limit our analysis to contacts of the first 400 COVID‐19‐infected cases in Singapore, that is those identified before 21 March 2020 as the number of cases in the population with unidentified source of infection was less than 40 at that point and the likelihood of household cases being exposed to the population was low. This is also prior to the tightening of precautionary measures introduced by the government to minimise the spread of COVID‐19We tested household contacts for SARS‐CoV‐2 when (a) they exhibit recent or active symptoms compatible with COVID‐19 or (b) they are aged 12 and below (with effect from March 2020), regardless of their symptoms, as this age group may not report symptoms. Those testing negative and other well household contacts were quarantined at home or in government quarantine facilities and monitored daily for development of symptoms for 14 days from their last exposure to a case.Multivariate logistic‐regression model was used to examine the relationship between infection outcome of a household contact and age, adjusted by the duration of exposure. Exposure between each case and household contact is assumed to be independent and measured from the case's symptom onset to last date of exposure with the household contact or to symptoms onset in the infected household contact, whichever comes first. The duration of exposure for each household contact is the sum of their exposures to all cases in the household . Sensitivity analysis was performed to adjust for potential demographic confounders such as gender and household size. Household contacts with the same exposure outside of the household as the household index case and cases who practice self‐isolation from their household contacts were omitted from the analysis.We computed the 95% confidence intervals (CI) for secondary clinical attack rate (SCAR) among household contacts.3Of the 400 cases identified before 21 March 2020, 46 cases and their households were omitted due to common exposure between cases and household contacts or cases reported to self‐isolate from their household contacts. Of the remaining cases, 34 had no household contacts, 277 were primary or co‐primary cases in their household and 43 household contacts were tested positive for SARS‐CoV‐2. A total of 265 households and 875 household contacts were identified, and the overall median household size was 4 (IQR 3‐6).After adjusting for the duration of exposure, risk of infection was estimated to increase with age Figure . This as4Our study suggests that the risk of COVID‐19 infection in the household setting increases with age. This finding corroborates with other studies on the demographic characteristics of COVID‐19‐infected cases in the population.Outcomes of this study were not affected by censoring of the data as contacts were monitored until the end of their quarantine period. The mean SCAR of 3.8% was similar to 2009 influenza A H1N1 pandemicWhile symptomatic contacts and asymptomatic children aged 12 and below were referred for testing, respiratory samples were not collected from other healthy household contacts. Thus, we were unable to ascertain the true proportion of asymptomatic cases and hence secondary infection rate. Furthermore, we did not interview the household contacts for clinical risk factors for COVID‐19 and this could confound the analysis. With ongoing clinical trials for post‐exposure prophylaxis of SARS‐CoV‐2 infection, the findings from the current study would serve as a comparison to other household studies with the administration of such intervention in the future.Rachael Pung: Conceptualization ; Formal analysis (lead); Investigation (lead); Methodology (lead); Writing‐original draft (lead); Writing‐review & editing (lead). Minah Park: Conceptualization ; Investigation (supporting); Methodology (supporting); Writing‐review & editing (supporting). Alex R. Cook: Conceptualization ; Methodology (supporting); Supervision ; Writing‐original draft (supporting); Writing‐review & editing (supporting). Vernon J. Lee: Conceptualization ; Supervision ; Writing‐original draft (supporting); Writing‐review & editing (supporting).Appendix S1Click here for additional data file.


Eptesicus serotinus) that were positive for European bat 1 lyssavirus in the brain. We also performed immunohistochemical and virological analyses on digestive tract samples of these bats to determine potential sources of lyssavirus in the faeces. We found that lyssavirus detection by RT‐qPCR was nearly as sensitive in faecal pellets  as in oral swabs . The likely source of lyssavirus in the faeces was virus excreted into the oral cavity from the salivary glands (5/6 bats positive by immunohistochemistry and RT‐qPCR) or tongue (3/4 bats positive by immunohistochemistry) and swallowed with saliva. Virus could not be isolated from any of the seven faecal pellets, suggesting the lyssavirus detected in faeces is not infectious. Lyssavirus detection in the majority of faecal pellets of infected bats shows that this novel material should be further explored for lyssavirus prevalence studies in bats.Rabies is caused by infection with a lyssavirus. Bat rabies is of concern for both public health and bat conservation. The current method for lyssavirus prevalence studies in bat populations is by oral swabbing, which is invasive for the bats, dangerous for handlers, time‐consuming and expensive. In many situations, such sampling is not feasible, and hence, our understanding of epidemiology of bat rabies is limited. Faeces are usually easy to collect from bat colonies without disturbing the bats and thus could be a practical and feasible material for lyssavirus prevalence studies. To further explore this idea, we performed virological analysis on faecal pellets and oral swabs of seven serotine bats ( People can acquire rabies by contact with rabid bats. This makes bat rabies of concern for both public health and bat conservation.There is limited knowledge on the epidemiology of bat rabies hampering the application of preventive measures. Therefore, we should improve our strategies to investigate free‐ranging bat populations for rabies prevalence.Our finding of lyssavirus RNA in faecal pellets of six out of seven confirmed rabid bats suggests testing faeces should be further explored as a strategy for epidemiologic studies.1Rabies is a fatal neurologic disease caused by an infection with a lyssavirus. People usually acquire the infection from bites by infected carnivores or bats. The pathogenesis is similar in all species, including bats. Typically, virus enters a new host via a bite from an infected host and infects nerves in the area of the bite. Once in the nervous system, the virus spreads to the brain, and from there reaches nerves of the salivary glands and the tongue. Once it is in these organs, virus is excreted into the oral cavity, from where it can be transmitted to the next host , that died in the Netherlands between December 2016 and December 2018, extensive autopsies were performed. All bats were tested for European bat lyssavirus (EBLV‐1) RNA in the brain. Eight of these 21 tested positive, and of seven of the eight bats, a faecal sample was available for further testing, and these were thus selected for our study. One serotine bat from the same series, which tested negative for EBLV‐1 RNA in the brain, and for which a faeces sample was available, was selected as negative control. Autopsies of these eight bats took place after storage of the carcasses at −20°C variably up to 17 months. Faecal pellets were taken from the rectum of all eight bats at autopsy, with one exception. The exception was an EBLV‐1‐positive bat whose rectum was empty at autopsy. Instead, faecal pellets collected at 3 and 2 days before death of this bat from its cage in a rehabilitation centre were used. In addition to faecal pellets, samples collected at autopsy included oral swabs and tissue samples of brain, salivary gland and intestine. Faecal pellets and oral swabs were stored in virus transport medium at −80°C directly after sampling. Tissue samples of brain, salivary gland and intestine were stored −80°C. These samples remained at −80°C for 14 months before testing took place. Duplicate tissue samples of salivary gland and intestine, as well as samples of tongue, were fixed in 10% neutral‐buffered formalin, embedded in paraffin wax and cut in 4‐μm‐thick sections within 3 weeks after autopsy.We received bat carcasses from already existing collections of bat rehabilitators who gave us their consent to use the carcasses for this investigation. These bats either had been found dead or had been euthanized by the bat rehabilitators because of bad prognosis for recovery. The bat carcasses were transported to our facility and investigated under permit FF/75A/2015/036 from the Dutch Ministry of Economic Affairs. On 21 serotine bat carcasses, Cq) values were inversely correlated with the amount of specific RNA that was detected in the original sample. On RT‐qPCR positive faecal samples RT‐PCR was performed, and products were sequenced according to the protocol of Heaton et al.,   of the RT‐PCR product. One of the six RT‐qPCR positive faecal pellets was the sample taken from a live bat that was being cared for at a rehabilitation centre, and that died 4 days later of rabies. This shows it is possible to detect virus in faeces of live bats. Despite detection of lyssavirus RNA, virus could not be cultured from faecal pellets of any of the seven bats, suggesting it did not contain infectious lyssavirus. In contrast, lyssavirus was cultured from brains of five (71.4%) of the seven bats, indicating storage conditions still allowed successful virus culture.Detection of lyssavirus infection was nearly as sensitive in faecal pellet samples (6/7 bats [86%] positive by RT‐qPCR) as in oral swabs  and/or tongue  suggested that these tissues were the likely sources of lyssavirus RNA in the six positive faeces samples. It cannot be ruled out that lyssavirus originated from the intestinal wall . For a population of 1,000 bats or more, required sample size is 148. Because faeces are often so easy to collect under bat roosts, these are feasible sample numbers  (Mørk & Prestrud, We realize that further validation is needed before it can be determined whether faecal pellets can be used for lyssavirus prevalence studies in free‐living bat populations. Questions remaining include how many days prior to disease or death lyssavirus RNA can be detected in faeces of infected bats, and how long lyssavirus RNA can be detected in bat faeces after defaecation, and how to relate the number of sampled faecal pellets at a roosting site to the number of bats present at that site. However, we wish to share our idea and these preliminary results so that other researchers investigating lyssavirus infections in bats have the opportunity to explore this sampling strategy. The circulation of lyssaviruses in bats is a concern for both public health and bat conservation (Banyard et al., The authors declare no conflict of interest.


Delineating common and separable neural alterations in substance use disorders (SUD) is imperative to understand the neurobiological basis of the addictive process and to inform substance‐specific treatment strategies. Given numerous functional MRI (fMRI) studies in different SUDs, a meta‐analysis could provide an opportunity to determine robust shared and substance‐specific alterations. The present study employed a coordinate‐based meta‐analysis covering fMRI studies in individuals with addictive cocaine, cannabis, alcohol, and nicotine use. The primary meta‐analysis demonstrated common alterations in primary dorsal striatal, and frontal circuits engaged in reward/salience processing, habit formation, and executive control across different substances and task‐paradigms. Subsequent sub‐analyses revealed substance‐specific alterations in frontal and limbic regions, with marked frontal and insula‐thalamic alterations in alcohol and nicotine use disorders respectively. Examining task‐specific alterations across substances revealed pronounced frontal alterations during cognitive processes yet stronger striatal alterations during reward‐related processes. Finally, an exploratory meta‐analysis revealed that neurofunctional alterations in striatal and frontal reward processing regions can already be determined with a high probability in studies with subjects with comparably short durations of use. Together the findings emphasize the role of dysregulations in frontostriatal circuits and dissociable contributions of these systems in the domains of reward‐related and cognitive processes which may contribute to substance‐specific behavioral alterations. The present results emphasize the role of dysregulations in striato‐frontal circuits as shared pathological substrate across substance use disorders. In addition, the meta‐analytic findings revealed for the first time evidence for robust substance‐specific alterations in limbic‐thalamo‐frontal circuits that may contribute to substance‐specific behavioral dysregulations and furthermore suggest that dysregulations in the domains of motivational and cognitive processes are mediated by separable brain systems. The CBMA approach was preferred to other methods like image‐based meta‐analysis because it takes advantage of the published coordinates, and quantitatively provides a summary of the presented results under the specific research question, while the latter approach is limited by the availability of whole‐brain images . We first conducted a main ALE analysis to determine core regions that neurally underpin substance use disorders across substances. This was followed by sub‐meta‐analyses employing substance‐specific subtraction and conjunction analysis to further specify common and substance‐specific neural alterations as well as functional domain‐specific alterations for reward and cognitive processes. Based on previous animal models and human imaging research, we hypothesized common alterations in striatal systems engaged in reward/motivation  and habit formation  as well as partly dissociable effects on frontal systems engaged in executive control and behavioral regulation. Moreover, we examined whether the observed substance‐specific alterations are driven by an interaction between the substance used and the class of task paradigms employed.22.1We obtained articles including four kinds of substances that are regularly abused namely cocaine, cannabis, alcohol, and nicotine (cigarettes or tobacco). Utilizing Scopus, PubMed, and Web of Science, peer‐reviewed studies published between January 1, 2000 and November 1, 2019 were collected using the following search terms; “Alcohol” or “Cocaine” or “Cannabis” or “Nicotine/Tobacco/cigarette” and “Functional magnetic resonance imaging” or “fMRI.” The reference list of the selected articles was inspected separately. We targeted articles that reported: whole‐brain coordinates either in the main paper or supplementary material with stereoscope coordinates in either Talairach or MNI  space, comparisons between healthy controls and patients with substance dependency or heavy usage. The exclusion criteria were as follows: (a) Articles reporting only region‐of‐interest (ROI) results , (b) Articles with poly‐drug users and high comorbidities with psychiatric or somatic disorders , (c) Articles focusing on parental exposure, and (d) Articles reporting results from the exact same data set from previous studies. The breakdown of article screening and exclusion for the main and sub‐meta‐analysis is shown in Figure 2.2In initial meta‐analyses, the brain functional alterations for each substance use disorders (SUD) were computed relative to their respective control groups. Next, we used a three‐step approach (a) to establish brain functional alterations across all SUDs in comparison to their control groups, (b) to establish common and distinguishable alterations between the SUDs by employing conjunction and differential contrasts that compare the SUD‐specific activation likelihood estimation (ALE) maps  between the SUD categories by means of conjunction and subtraction analysis, and, (c) to investigate whether common neurofunctional alterations across the SUDs are related to the classes of experimental task paradigms employed or by the effects of the drug use in the study populations.p < .05 on the cluster‐level was applied to control for multiple comparisons. In line with recent recommendations for the application of cluster‐based correction methods  activation into two categories of task paradigms that reflected distinct functional domains; that is, “Reward” comprising the processing of reward, motivation, or anticipation and “Cognition” with cognitive‐control, behavior, and emotion tasks. Despite conceptual frameworks proposing differential alterations with respect to the processing of drug‐associated rewards (particularly drug cue reactivity) and natural rewards, specifically exaggerated reactivity to drug‐associated rewards and attenuated reactivity to nondrug rewards we decided to pool these studies under the domain reward processing. This decision increased the power of the corresponding meta‐analyses and additionally adhered to the main aim of the study which was to determine neurofunctional alterations in SUD while not further disentangling hyper‐ from hypo‐activations on the analytic level. The examination of the different paradigm domains serves the primary purpose to investigate whether the conjunctions were influenced by the experimental design or solely by the substances abused. To this end we computed whole‐brain ALE firstly for all studies based on the experimental task, secondly, we grouped the foci into patient and control per each SUD category and computed the cluster ALE to ascertain the group interactions under the two categories of experimental paradigms. We also extracted the foci generated by the probabilities of functional change from each cluster including the task associated, using the Kruskal‐Willies test we computed the dependency test to ensure homogeneity between the two categories of tasks forming the conjunction. Significant threshold was set to 33.1SD) age, 33.9 (11.7)) and 2,564 control subjects (mean (SD) age, 31.3 (11.6)), with no significant difference between the four SUD groups p = 0.7.Out of the 99 studies included in the meta‐analysis, cocaine studies contributed to 30% (828) of the total foci, while cannabis, alcohol, and nicotine contributed to 23.01% (637), 27.45% (760), and 19.44% (538), respectively of the total foci analyzed, indicating no bias toward a single substance for the ALE computations. Table 3.2We initially conducted a main meta‐analysis that incorporated the ALE maps from all SUDs versus the respective control groups (Step 1). This analysis aimed at determining neurofunctional alterations across all SUDs and revealed neurofunctional alterations primarily located in the dorsal striatum, including the caudate and putamen as well as the prefrontal, limbic and insular cortex, including inferior, superior, and medial frontal regions as well as the anterior cingulate cortex (ACC) and the anterior insula Figure , Table 23.3The subtraction (direct voxel‐wise subtraction of ALE images) analyses aimed at determining differential neurofunctional alterations between the SUDs and revealed primarily differential neurofunctional alterations in frontal regions . Findings from this exploratory analysis revealed that striatal and medial frontal regions involved in reward and value processing showed a higher probability to be identified in studies with participants with shorter duration of use while frontal regions engaged in regulatory and executive control such as the inferior, superior and precentral gyrus exhibited a higher probability to be identified in studies conducted in SUD samples with a longer duration of use  . Consequently, a total of 39 reward‐related and 55 cognitive processing studies entered this analysis. Detailed results of this analysis are provided in Figures 4The present meta‐analytic approach employed a whole‐brain coordinate‐based meta‐analysis to determine shared and substance‐specific alterations in the most prevalent substance use disorders  as determined in previous fMRI studies. Given that most previous studies could be classified in examining reward‐related functions or cognitive functions, we additionally examined domain‐specific alterations to determine whether dysregulations in distinct behavioral domains are neurally mediated by separable brain systems. In line with most overarching translational addiction models  varies as a function of the duration of use. Specifically, striatal and frontal regions engaged in reward processing showed a high probability of being identified in studies conducted in SUD subjects with a comparably short duration of use, while frontal regions engaged in regulatory and executive control such as the inferior and superior frontal gyrus exhibited a higher probability of being identified in studies conducted in SUD subjects with a longer duration of use. Together these findings may suggest that reward‐related processing regions may become compromised during earlier stages of the addictive process while cognitive control regions become compromised during later stages. Moreover, the findings may suggest that the observed alterations—at least partly—represent consequences of continued substance use rather than stable predisposing alterations that precede the onset of drug use.Although this meta‐analysis revealed task‐specific alterations between the various groups of studies, we could not conduct substance‐specific sub‐analyses comparing the different tasks within each substance group. This was due to the limited number of substance‐specific studies. The within‐group task‐based analysis would have thrown more light on the substance‐specific alterations for the individual substance use disorders. Furthermore, the reproducibility of common models for substance abuse in this meta‐analysis may also be related to selection and publication bias of studies. Moreover, despite our a priori hypothesis rooted in extensive animal models and human imaging studies the meta‐analysis was not pre‐registered. Finally, our analyses did not examine the direction of activation alterations between SUD subjects and controls and thus it cannot interfere whether the observed alterations represent hyper‐ or hypo‐activations in SUD.5Summarizing, our analysis reveals consistent results with convergent models from animal and human studies demonstrating that addiction is characterized by neural dysregulations in systems subserving salience/reward processes, habit learning, and executive control, including decision‐making and response inhibition, specifically the dorsal striatum and the prefrontal cortex. On the other hand, the present meta‐analytic approach allowed us to determine substance‐specific alterations in frontal, limbic, as well as insular regions, pointing to specific pathological alterations in addition to shared pathological pathways.The authors report no conflicts of interest.Appendix S1: Supporting infomationClick here for additional data file.Figure S1 Subtraction analysis between pairs of studies.“>” the symbol indicates where ALE peaks are greater in one study compared to the other. Cluster forming (p < .001) and cluster‐level threshold (p < .05)Click here for additional data file.Figure S2 Contributions to the conjunction between the two category of task paradigm, A) the Percentage distribution of each task paradigm to the conjunctions, B) the percentage distribution of foci to the conjunction. N; the number of foci per paradigmClick here for additional data file.Figure S3 Comparing conjunction based on studies and tasks. Each row Figure label shows conjunction based on studies  and row label 2 shows conjunction based on task respectivelyClick here for additional data file.


FGFR3/2 genetic alterations who progressed during or after ≥1 line of prior platinum‐containing chemotherapy (PCC), including within 12 months of neoadjuvant or adjuvant PCC. Concordance between the clinical trial assay (CTA) used in a phase 2 study and QIAGEN's therascreen® FGFR kit , the FDA‐approved companion diagnostic (CDx) with erdafitinib, was evaluated in this bridging study. Study samples included 100 CTA‐confirmed FGFR‐positive samples from 100 erdafitinib‐treated mUC patients, plus 200 CTA‐confirmed FGFR‐negative samples from the phase 2 study. The primary objective was met if the lower bound of 95% CI of objective response rate (ORR) in CDx‐confirmed patients with FGFR alterations was >25%. Demographics were similar between the bridging study and CTA‐screened patients. In total, 292 of 300 samples (97.3%) with valid CDx results showed high analytical concordance versus CTA . Investigator‐assessed ORR in the 81 CDx‐identified, erdafitinib‐treated patients who tested positive for both assays was 45.7%  versus 40.4%  for CTA and met the criteria for primary objective. High ORR and clinical concordance to CTA suggest that QIAGEN's CDx can reliably select mUC patients who would potentially benefit from erdafitinib treatment.Erdafitinib, a pan‐fibroblast growth factor receptor (FGFR) inhibitor received accelerated approval from the US Food and Drug Administration (FDA) for locally advanced or metastatic urothelial carcinoma (mUC) in adult patients with specific  FGFR3 mutations, FGFR3 fusion and an upregulation of FGFR mRNA and protein expression Treatment of metastatic urothelial carcinoma (mUC) in patients who are cisplatin‐ineligible or cisplatin‐refractory represents a significant unmet medical need. Gene expression and sequencing studies have improved our understanding of molecular subtypes of UC and revealed signature genomic alterations that are potentially targetable, paving the way for personalized medicine FGFR mutations/fusions FGFR3/2 genetic alterations, whose disease progressed during or following ≥1 line of platinum‐containing chemotherapy (PCC), including within 12 months of neoadjuvant or adjuvant PCC therascreen® FGFR RGQ (Rotor‐Gene Q MDx instrument) Reverse transcription (RT)‐polymerase chain reaction (PCR) kit, developed by QIAGEN Manchester Ltd., for use as a companion diagnostic (CDx) with erdafitinib to detect FGFR genetic alterations and guide patient selection for the approved therapeutic indication Erdafitinib, a potent pan‐FGFR (FGFR1–4) tyrosine kinase inhibitor, has shown antitumor activity in several cancer cell lines that was associated with inhibition of downstream FGFR signaling FGFR alteration status in patients enrolled for the erdafitinib phase 2 study. Given the differences in assay design and instrument platform between the CTA and QIAGEN's CDx assay, a bridging study was designed to compare the clinical performance of the CDx versus the CTA, establish agreement between two assays and evaluate clinical efficacy outcome of erdafitinib in patients with locally advanced or mUC identified with FGFR alteration using the CDx.CDxs are critical for the implementation of personalized medicine and enable appropriate use of the paired therapy by accurately identifying patients who would most likely benefit from the targeted treatment therascreen® FGFR kit (CDx) developed by QIAGEN is a qualitative in vitro diagnostic test for the detection of four‐point mutations  and five fusions  in the FGFR2/3 genes. It is a two‐step, multiplex, real‐time RT‐PCR test designed to detect FGFR alterations in RNA derived from formalin fixed paraffin embedded (FFPE) UC tissue samples archived during the BLC2001 study The FGFR3 mutations or FGFR2/3 fusions based on evaluation of appropriate tumor tissue samples. Molecular screening was performed centrally at Almac Diagnostics using the CTA, a two‐step RNA‐based PCR assay designed to detect FGFR alterations in FFPE tissue. All FFPE samples were banked by Almac during the entirety of the BLC2001 study Details of study design and eligibility criteria of the BLC2001 study have been reported previously FGFR‐negative (CTA−) patients screened during the BLC2001 study were used for this bridging study  and CTA‐confirmed The relevant independent ethics committee or institutional review board approved the study protocol, including the use of archived tumor tissue. Informed consent forms were collected at the Drug Study Investigator sites and were not provided to the device investigator but were available for verification if requested by study monitors.FGFR2/3 alterations in RNA extracted from FFPE UC specimens using the RGQ system.The FGFR kit comprised the RNeasy DSP FFPE kit, the QIAGEN RGQ and the Rotor‐Gene Assay Manager (RGAM) software package for automatic analysis and reporting of the results. The detection was based on selective amplification of nine 2. Macrodissection was performed if necessary to attain a minimum of 80% tumor area. All steps outlined in the RNeasy DSP FFPE kit protocol were performed at room temperature (15 to 25 °C). Once extracted the RNA samples could either be tested immediately with the RT‐PCR kit or stored at −90 to −65 °C and a maximum of five freeze–thaw cycles were permissible.RNA extraction was performed using the RNeasy DSP FFPE kit on 4 to 5 μm thick FFPE sections with a tumor area between 100 and 500 mmFor the bridging study, two independent RNA extractions were performed for each patient sample. If sufficient tissue was not available, residual CTA‐derived RNA was used. After extraction, the two RNA samples from the same patient were pooled and the RNA quantified by spectrophotometry using the absorbance at 260 nm. Each pooled RNA sample was divided into two equal aliquots (A1 and A2). The first aliquot (A1) was tested using the FGFR kit at Almac Diagnostics. The second aliquot (A2) was stored at −100 to −65 °C and shipped to Horizon Discovery  for use in the accuracy validation study. Samples with RNA quantity below minimum requirements (18 ng/μL) were not used in the accuracy study and were stored at Almac Diagnostics.The FGFR RGQ RT‐PCR kit provides high cDNA yields for sensitive detection of all target transcripts. It uses an RNA template and a blend of primers complementary to 3′ and 5′ ends of the RNA to produce the cDNA. The reverse transcriptase enzyme synthesizes the first strand of cDNA, which is used as input into the PCR. The process of RT was performed by incubating the normalized RNA (250 ng) sample (A1) with a master mix containing the primer mix and reverse transcriptase enzyme provided with the FGFR kit. The resulting cDNA sample was then subjected to real‐time PCR using the four real‐time PCR multiplex reaction mixes.FGFR mutations and fusions. For each of the two mutation mixes, a blocking oligonucleotide specific for the wild‐type region of each target was included to increase amplification specificity. Independent mutation‐specific TaqMan oligonucleotide probes detected the specific FGFR targets. The RGQ and associated software were used to control the real‐time PCR and to determine the FGFR alteration status of the clinical samples. The RGAM software provided general functionality including PCR run setup and thermal cycling control, as well as management of data, results, assay profiles, and system configuration.The FGFR RGQ RT‐PCR kit contained four ready‐to‐use PCR mixes that include HotStarTaq DNA Polymerase and PCR buffer. A specific set of oligonucleotide primers within each of four reaction mix reagents was used to amplify target CT value: the PCR cycle at which the fluorescence from a particular reaction crosses a threshold value), the RGAM software qualitatively determined the FGFR alteration status of the samples and reported which samples were positive for the indicated FGFR alteration. The run controls (positive and negative template controls) were assessed to ensure that CT values were within the internal control acceptance criteria. Samples were classified as target positive if the CT was less than or equal to the cut‐off for the target assay.Based on predetermined analytical cycle threshold value  samples given that the samples were CTA‐confirmed FGFR‐positive (CTA+); NPA was calculated as the proportion of CDx‐confirmed FGFR‐negative (CDx−) samples given that the samples were CTA−; and OPA was calculated as the proportion of agreement between CDx and CTA among all samples tested. In addition, to determine the overall accuracy of the CDx, discordant and concordant samples were tested and compared with a validated reference standard, droplet digital PCR (ddPCR). An orthogonal method was developed based on ddPCR technology. The ddPCR FGFR system constituted a two‐step nucleic acid RT and multiplex ddPCR test on the QX200TM Droplet Digital™ PCR System. The assay was developed and validated by Horizon Discoveries for the specific detection of the nine FGFR targets of interest. A contrived model of in vitro transcription RNA for each target spiked into a wild‐type universal RNA background was initially used to develop and optimize the assay. As described previously, the second aliquot (A2) was tested at Horizon Diagnostics using the RT‐ddPCR assay.Assessment of analytical concordance between the CDx and CTA was based on estimation of positive percent agreement (PPA), negative percent agreement (NPA), and overall percent agreement (OPA). PPA was calculated as the proportion of CDx‐confirmed The primary clinical efficacy objective was to estimate the ORR  of regimen 3 in patients who were CDx+. The secondary objective was to estimate the ORR in a subgroup of erdafitinib‐treated chemotherapy‐relapsed/refractory patients who were CDx+.P values were calculated based on two group t‐test for continuous measures and chi‐square test for category data. In the analysis of concordance, the PPA, NPA, and OPA, along with the two‐sided 95% confidence interval (CI), were calculated using the CTA as the reference method. For the purpose of power analysis in evaluation of concordance, 90% PPA and 95% NPA were expected. The accuracy study acceptance criteria were met if the lower limit of the two‐sided exact 95% CI for PPA for overall mutation status was ≥85% and the lower limit of the two‐sided exact 95% CI for the NPA for overall mutation status was ≥90%.Demographics and baseline characteristics were summarized descriptively. For representativeness analysis, the Percentage ORR was summarized along with 95% CI. The primary objective was met if the lower bound of the 95% CI for the observed ORR in CDx+ patients was >25%. The overall ORR in all patients was calculated by taking the weighted average of ORR for positive discordant (CDx+/CTA−) and positive concordant (CDx+/CTA+) patients; however, since CTA− patients were not included in the BLC2001 study, a range of hypothetical ORR values  were used. Bootstrapping was performed to calculate 95% CI of the weighted ORR.n = 99 erdafitinib‐treated) and 200 CTA− samples from the BLC2001 study were eligible for analysis in the bridging study  difference between patients tested and not tested in the bridging study, except for ethnicity and percent viable cells.Demographics between the bridging study patients and CTA‐screened patients were similar Table . The meaEstimation of percent agreement (95% CI) between CDx and CTA, with CTA as the reference method, demonstrated good analytical concordance for the 292 samples with valid CDx results   that met the criteria for the accuracy study  to 63.6% for the FGFR3‐Y373C mutation (n = 11).Investigator‐assessed ORR in the 99 erdafitinib‐treated patients who were CTA+ in the regimen 3 of the BLC2001 study was 40.4%. Overall, 81 of these 99 patients also tested positive on the CDx assay. The investigator‐assessed ORR (95% CI) was 45.7% , meeting the criterion for primary objective  and met the primary efficacy objective . Sixty‐nine of these patients tested CDx+ in the bridging analysis and 32 of 69 had an investigator assessed ORR .therascreen® FGFR RGQ RT‐PCR kit developed by QIAGEN could reliably identify patients with FGFR3 point mutations and FGFR2/3 fusions who are eligible for erdafitinib treatment. Overall, the bridging study population was demographically similar to the CTA‐screened BLC2001 study population FGFR gene events identified by the CDx and the primary objective of >25% ORR was met, consistent with the BLC2001 study. This conclusion is further supported by imputation analysis which demonstrated that, when all patients with CDx+/CTA− were assumed to be nonresponsive to erdafitinib, there was no negative impact to the weighted ORR.The FGFR3 alterations in UC determines sensitivity to specific targeted chemotherapy and therefore it is important to employ accurate identification tests to detect these driver mutations and to maximize potential benefits of FGFR‐targeted therapies FGFR is an attractive target for several solid malignancies including UC, the sixth most prevalent type of cancer in the United States Overall, the performance measures validated in this bridging study should be interpreted in the context of the study population and design.FGFR alterations in patients with locally advanced or mUC. Several FGFR inhibitors such as FGF401 (FGFR4 inhibitor), AZD4547, and BGJ398 (both FGFR1–3 inhibitors) have been investigated in phase 1 and 2 studies in situ hybridization (FISH), immunohistochemistry, quantitative real‐time PCR, and next‐generation sequencing (NGS) have been used in these studies for the detection of FGFR mutations for screening and selection of patients FGFR3 mutations were assessed using a custom‐designed SNaPshot assay  for all common mutations in FGFR3 coding exons including exons 7, 10, and 15. The assay was performed on representative transurethral resection of bladder tumor specimens FGFR alterations FGFR mutations, with more specific FGFR inhibitors such as erdafitinib coming into clinical practice, targeted techniques and multiplex testing such as NGS would increase the likelihood of identifying actionable FGFR mutations. However, concordance of NGS with PCR‐based methods must be established before using NGS in routine practice. Thus, the availability of a validated CDx for the approved therapeutic indication could potentially increase access to the test and assist clinicians to efficiently select suitable patients who would benefit the most from erdafitinib therapy. In conclusion, the findings of high analytical concordance, accuracy of diagnosis and consistent detection of clinical response benefit to erdafitinib in the intended patient population from the BLC2001 study supports the clinical performance of QIAGEN's therascreen® FGFR RGQ RT‐PCR kit as a CDx for erdafitinib.After receiving breakthrough therapy designation in March 2018, erdafitinib was granted accelerated approval by the US FDA in April 2019 SW, MB, CM, AE, MS, and AH were involved in the study design, data collection, analysis, and interpretation. SW and AE were the project statisticians and oversaw data analysis and interpretation. All authors met the ICMJE criteria and those who fulfilled the criteria are listed as authors. All authors had access to the study data, provided direction and formal review of the manuscript, and made the final decision about where to publish these data. All authors contributed toward drafting and revising the paper and agreed to be accountable for all aspects of the work.


Cells utilize different means of inter‐cellular communication to function properly. Here, we review the crosstalk between cancer cells and their surrounding environment through microRNA (miRNA)‐containing extracellular vesicles (EVs). The current findings suggest that the export of miRNAs and uptake of miRNA‐containing EVs might be an active process. As post‐transcriptional regulators of gene expression, cancer‐derived miRNAs that are taken up by normal cells can change the translational profile of the recipient cell towards a transformed proteome. Stromal cells can also deliver miRNAs via EVs to cancer cells to support tumour growth and cancer progression. Therefore, gaining a better understanding of EV‐mediated inter‐cellular communication in the tumour microenvironment might lead to the development of novel diagnostic and therapeutic strategies. Cancer cells communicate mutually with tumour microenvironment (TME) cells via extracellular vesicles (EVs). Cancer cells secrete miRNAs into EVs that are subsequently taken up by TME cells in which miRNAs promote a tumour‐supportive phenotype. Cancer cells also receive exogenous miRNAs from TME cell–derived EVs to support tumour growth. This bidirectional signalling results in tumour progression and metastasis. These include membrane proteins, enzymes, signalling molecules and RNAs including both coding and non‐coding RNAs. MicroRNAs (miRNAs) are a class of small single‐stranded non‐coding RNAs (of ~22 nt in length) that play an important role in the regulation of gene expression in eukaryotes.The expression of miRNA is tightly regulated.Although miRNAs are mainly localized in the cytosol, they are also secreted in extracellular complexes such as EVs. Cell‐free miRNAs have been identified in different body fluids such as serum/plasma,2Stable circulating miRNAs have been found to complex with lipids,Two processes govern the presence of a specific miRNA in EVs; these are loading and sorting. Loading refers to mechanisms in which EV cargoes are recruited to the site of vesicle formation, whereas sorting is an active selection process in which some specific miRNAs are exclusively destined for secretion. These processes happen simultaneously following vesicle biogenesis.2.1Various attempts have been made to analyse the loading and sorting of miRNA into exosomes. Exosome biogenesis includes either the recruitment of the endosomal sorting complex required for transport system (ESCRT‐dependent) or the synthesis of ceramide (ESCRT‐independent).Some miRNAs are known to be selectively sorted into EVs, although the exact mechanisms are mostly unknown. Villarroya‐Beltri et alGenomic position might also play a role in miRNA secretion. miRNAs often cluster together in the genome and are often transcribed together. By comparing the miRNAs of the cells and exosomes of related individuals, Tsang et alAs miRNA can be selectively sorted into EVs, the concentration of miRNA in EVs (a quantitative aspect of miRNA sorting) can provide new information regarding miRNA secretion. Squadrito et al2.22+‐) and membrane‐binding proteins, play an important role in forming a curved structure on the membrane for blebbing and folding.Not much is known about the loading and sorting of miRNAs into microvesicles. Although the biogenesis of microvesicles and exosomes is regulated by overlapping sets of molecules, the former still utilizes a distinct collection of regulatory proteins.Annexin A2 is also reported to regulate the levels of miRNAs in EVs Figure .47 It 3Upon secretion, EVs are taken up by their parental cells or by other (either adjacent or distant) cells via autocrine or paracrine signalling.3.1Since EVs are carriers of proteins and other polynucleotides as well as miRNAs, the endocytic pathway is likely to be common for all EVs, whatever they carry. EVs are taken up into cells by a number of different mechanisms, including endocytosis, phagocytosis, macropinocytosis, and plasma membrane fusion, all of which utilize different sets of proteins for binding and internalization.Glycosylation also plays a role in the specific uptake of EVs. For example, the uptake of EVs by recipient cells is, in some cases, regulated by D‐mannose, D‐glucosamine and β‐galactosides, and the underlying mechanism involves sugar‐binding proteins.3.2After internalization into the cell, most EVs remain in endosomes. It is therefore challenging for EV‐derived miRNAs to get into the cytosol so that they can be loaded onto RISCs and perform their physiological role. Montecalvo et alLittle is known about the fate of EVs after being endocytosed. However, Heusermann et al3.3The functionality of EV‐derived miRNAs in recipient cells is another key question in the field. In order to perform translational silencing, single‐stranded miRNAs must bind to AGO2 and other proteins to form the RISC complex during their maturation.Interestingly, annexin A1 is also capable of binding to both DNA and RNA upon formation of an A1‐S100 heterotetramer complex; this suggests an additional possible role for annexin A1 in regulating RNA sorting in EVs.In an attempt to elucidate the underlying mechanism, Melo et al4With the ability to suppress the expression of multiple target mRNAs, miRNAs transferred via EVs support tumour growth at each stage of cancer progression. This has become a robust means of communication between cancer cells and the tumour microenvironment (TME). Here, we summarize our current understanding of the mutual communication both among cancer cells and between cancer cells and other cells of the TME.4.1TP53 and TP53INP1, and the fibroblasts differentiate into cancer‐associated fibroblasts (CAFs). A similar mechanism is observed in pancreatic cancer–derived microvesicles, which contain an elevated level of miR‐155  in the surrounding immune cells, especially macrophages Figure .77 ThiDendritic cells also play a part in the tumour modulation of the immune system.In addition to macrophages and dendritic cells, natural killer (NK) cells are also critical effectors in the innate immune response.miRNA‐containing EVs from tumour cells also modulate and suppress T‐lymphocyte function. The delivery of miR‐214 from Lewis lung cancer cell–derived exosomes to recipient T cells was shown to significantly downregulate PTEN and promote a regulatory T cell (Treg) phenotype Figure .84 TheSMAD4 and STAT6 are downregulated  targets SOCS5 in endothelial cells  and GLUT1 genes in recipient lung fibroblasts and brain astrocytes in a pre‐metastatic niche, resulting in decreased glucose uptake and metabolism in these cells and thereby promoting breast cancer metastasis.5 Figure .103These findings demonstrate that the transfer of miRNAs from cancer cells to cells of the TME is an active and dynamic process. The same miRNA  can have different effects on the recipient cells by targeting different mRNAs or interacting with different proteins. Of note, the miRNAs being studied here are usually the most abundance miRNAs in the cancer‐derived EVs. The role of miRNAs with low abundant in EVs is more difficult to assess. These miRNAs are usually excluded from functional analysis because their addition to EV‐recipient cells is likely insufficient to make any significant functional impact. Further studies are desirable to investigate the combinatory effects of multiple miRNAs in EVs on their recipient cells.4.2APAF1) to promote chemoresistance and suppress apoptosis of recipient cancer cells  state. Meanwhile, these activated cells support the growth and progression of the tumour by transferring miRNAs into the cancer cells. These findings provide a better understanding of the crosstalk between cancer cells and their microenvironment and facilitate the development of novel therapeutic treatments and early diagnosis.5Further investigations are still required to fully understand the crosstalk between cancer cells and their microenvironment via EVs. Among the various cargos (ie proteins and other RNAs), miRNAs play an important role as signalling molecules. However, not enough research has been conducted to determine the synergistic contribution of different molecules on the signal transduction between cancer cells and the microenvironment. As RNA‐profiling, proteome analysis and bioinformatics are advancing at a rapid rate, the role of EV‐miRNAs can be elucidated further through a comparison of the EV contents and network mapping of the genome and proteome.Additional problems in the field include both the nomenclature and the purification of EVs.Different mechanisms might govern the export of miRNAs. However, it is still unclear which pathways contribute the most to the level of circulating miRNAs for each cancer type. Elucidating the export mechanism of miRNAs would help to support their application as biomarkers for cancer. This would be highly advantageous as the detection of miRNAs is both fast and robust, and thus, they would be a valuable tool for cancer diagnosis and prognosis.LTV, GJ and TTP prepared the manuscript. TTP and YK provided illustrations. MTL obtained funding, trained and wrote the manuscript. The authors declare no competing interests.