Pedigree files are ubiquitously used within bioinformatics and genetics studies to convey critical information about relatedness, sex and affected status of study samples. While the text based format of ped files is efficient for computational methods, it is not immediately intuitive to a bioinformatician or geneticist trying to understand family structures, many of which encode the affected status of individuals across multiple generations. The visualization of pedigrees into connected nodes with descriptive shapes and shading provides a far more interpretable format to recognize visual patterns and intuit family structures. Despite these advantages of a visual pedigree, it remains difficult to quickly and accurately visualize a pedigree given a pedigree text file.ped_draw a command line and web tool as a simple and easy solution to pedigree visualization. Ped_draw is capable of drawing complex multi-generational pedigrees and conforms to the accepted standards for depicting pedigrees visually. The command line tool can be used as a simple one liner command, utilizing graphviz to generate an image file. The web tool, https://peddraw.github.io, allows the user to either: paste a pedigree file, type to construct a pedigree file in the text box or upload a pedigree file. Users can save the generated image file in various formats.Here we describe ped_draw is a useful pedigree drawing tool that improves on current methods due to its ease of use and approachability. Ped_draw allows users with various levels of expertise to quickly and easily visualize pedigrees.We believe  Pedigree files are text files that convey important information about the samples within a genetics or bioinformatics study . This inped_draw addresses many of the deficiencies in current tools and provides numerous distinct advantages. Pedigreejs is perhaps the most fully featured and well powered pedigree drawing tool currently available [pedigreejs is not approachable to many users unfamiliar with Javascript or specific web programming visualization libraries such as d3. Pedigreejs requires the Javascript package manager npm (including grunt-cli) within a specific deployment and hosted environment. To deploy pedigreejs, an extensive knowledge of Javascript is required and numerous customization and visualization decisions need to be hard-coded by the user. Kinship2 provides pedigree visualizations as a package written in the R programming language [Kinship2 is also merely an R package, it requires users to write R code  even in the most simple pedigree drawing usages. Madeline 2.0 is another pedigree drawing tool, and boasts support across multiple operating systems [Madeline 2.0 has numerous dependencies in all installation scenarios, requiring Cygwin on Windows and apt, cmake, libssl-dev, libcurl-dev, libxml2-dev on Linux. On Linux in particular, these dependencies require sudo level user installation permissions that are often inaccessible on academic or shared computing resources.While a few pedigree drawing tools have been published previously, vailable . It is wlanguage . Similar systems . HoweverPed_draw was designed to occupy a specific niche within this set of available software. All of ped_draw’s dependencies are installed by default in a typical Linux environment and a Docker image provides additional portability. Ped_draw’s simplified approach removes the need for customization and allows users to easily and rapidly visualize pedigrees directly from a provided ped file.Importantly, all of these published tools have significant installation dependencies, have substantial programming learning curves and lack a simple one command input–output approach. Ped_draw is solely implemented in Python and requires Python 2.7.15 or greater. Converting the dot output of ped_draw to an image is achieved using graphviz (not provided). Image outputs from graphviz can be visualized in any graphics viewing application.ped_draw is a ped file and the output is an image file (generated by graphviz). Ped_draw generates a dot file that can be passed directly as stdout to graphviz to generate an image file. Ped_draw is capable of visualizing complex ped files including multi-generation (3 or more) parent-pairs, multiple affected individuals, large numbers of children (8 or more per parent-pair) and multiple distinct families/kindreds in the same input ped file into a single image output.The input for Ped_draw can be used as a simple one-liner command as follows (where example.ped is an example pedigree file):Generate a dot formatted file (written to stdout):$ python ~ /bin/ped_draw/ped_to_dot.py example.pedped_draw output to graphviz/dot:Generate a png image of the pedigree by piping the $ python ~ /bin/ped_draw/ped_to_dot.py example.ped | dot -T png -o example.pngped_draw in multiple examples in Fig. ped_draw and two distinct pedigrees are drawn  and users with little or no computational expertise (using the web tool).Project name: ped_draw.https://github.com/mvelinder/ped_draw and http://peddraw.github.ioProject home page: Operating system(s): UNIX (command line tool); platform-independent (web tool)Programming languages: Python, Javascript, HTMLOther requirements: Python 2.7.15 or greater (command line tool), conversion of ped_draw output dot files to pngs or other image types requires graphviz, visualization of graphical outputs can be done by any number of graphics viewing applications; Chrome 80.0.3987.149 or greater (web tool)License: MIT-licenseAny restrictions to use by non-academics: MIT-license


This scoping review focuses on the assessment of commensality in research and attempts to identify used methods for performing research on commensality. It reflects a multidisciplinary research field and draws on findings from Web of Science Core Collection, up to April 2019. The empirical material consisted of 61 studies, whereof most were qualitative research, and some were of quantitative character, including very few dietary surveys. The findings show nine papers categorized as using quantitative approaches, 52 papers were categorized as qualitative. The results show a wide variety of different ways to try to find and understand how commensality can be understood and identified. There seems to be a shift in the very concept of commensality as well as some variations around the concept. This paper argues the need to further investigate the importance of commensality for health and wellbeing, as well as the need to gather data on health and health-related behaviors, living conditions and sociodemographic data in parallel. The review shows the broad-ranging areas where commensality is researched, from cultural and historical areas to ethnographic or anthropological areas over to dietary assessment. To complement large dietary surveys with methods of assessing who you are eating with in what environment should be a simple way to further our knowledge on the circumstances of meal intake and the importance of commensality. To add 24-h dietary recall to any study of commensality is another way of identifying the importance of commensality for dietary quality. The use of mixed methods research was encouraged by several authors as a good way forward in the assessment of commensality and its importance. Researchers that find an interest in commensality research will discover that there is no easily identified common research methodology. A number of studies dealing with family meals or social eating, studied as a background for this scoping review revealed that the overlap of studies using the search term “family meal” or “eating together” with the term “commensality” was often inclusive. A recent meta-analysis of family meals and health showed that there is convincing evidence showing that family meals have an important impact on the future meal frequency and nutritional health of children . The defFor the nutritionally interested—data on commensality are almost never gathered in connection with large dietary surveys. In dietary assessment tool portals like the Diet, Anthropometry, Physical Activity (DAPA) Measurement Toolkit from the Medical Research Council in the UK, all different types of dietary assessment, subjective and objective methods, are listed for easy access. However, the only reference given to commensality is the following: “Individuals can record the time, location and whether they consumed meals alone or with others for each eating occasion, providing information on eating patterns and the eating environment” . No addiIn sociological or anthropological studies of eating situations, commensality is sometimes more thoroughly discussed and assessed . In thisA reasonable way forward, for the scholar with an interest in how eating together affects our wellbeing as well as what we eat, with whom, where, and how, would be to try to identify different ways of assessment of commensality. This could possibly lead to a broader understanding of the level of importance of eating together over research boundaries and lead to an increased comparability of studies .The aim of this paper is to make a scoping review of quantitative as well as qualitative methods of studying commensality, in nutritional surveys as well as in surveys of other character, such as cultural, sociological, or anthropological studies. We also wanted to identify in which disciplines papers were published in the area as well as how the number of papers using the term commensality has evolved over time.A scoping review mapping the body of a specific topic was used to summarize and disseminate relevant literature . This scPublished up to April 2019.Published in English.Somehow assessing commensalityDescribing commensality among humans.The database searched was Web of Science Core Collection, up to April 2019. Search term used: commensality. The following criteria had to be met for a study to be included:We included 61 studies, whereof most used qualitative research methods and some were of quantitative character, including very few dietary surveys.In the following, we have gone through the quantitative as well as the qualitative papers in some detail, and we produced a number of tables including all papers, listed under six categories.The papers we identified were roughly categorized into quantitative and different categories of qualitative papers, such as observations, interviews, and field studies.Nine papers  12,13,1,18,19,20A common problem in most studies was that the response rates were low and in some cases clearly skewed, with non-responders of lower education level. Some suggestions that could be read out from the scoping review regarding the use of quantitative methods to assess commensality were recommending the use of mixed methods approaches, applying rigorous sampling techniques, including type of venue in the investigation  and whether the venue facilitated commensality or not. Mailed or online questionnaires were used in several studies. Many authors suggested inclusion of with whom, where, how, when, questions. In all studies using questionnaires, socio-demographic characteristics were collected, in a more or less rigorous fashion and of course, ethical approval was sought, with informed consent collected from participants as soon as issues of sensitive nature were collected.Qualitative studies are described as those involving the systematic collection, organization, and interpretation of textual material derived from talk or observation . QualitaAmong these qualitative papers, two approaches were identified. The first one studying commensality to understand a phenomenon within meals, and the second to examine how to understand commensality from a meal point of view. Several papers revealed aspects of different social meanings attached to food and commensality, as well as how meaning seems to have changed over time ,36,51. TMoreover, the diversity of the applicability of the method differed within qualitative methods, ranging from more or less rather standardized questionnaires ,26, withAccording to different application of theories, theories surrounding “practices” were observed as the most commonly used in interview studies ,27,30,34One of the most common themes on the qualitative papers was in regards to family eating ,56,57,62The final papers showing up in our review turned out to be mostly from the nutrition area (28 papers); 20 of them were published in Appetite. Clinical or medical journals including pediatrics published another large part (6 papers), social science (20 papers), ethnology, anthropology (6 papers), tourism studies (1 paper), urban studies (1 paper). It seems as if the search term commensality was used in several disciplines. We did not put a limit on how old the paper in WoS could be, yet the first paper using the term commensality and fitting the inclusion criteria was identified in the year 2000. From 2000–2014, the number of papers was fairly low, only ten papers were found during this fourteen year time span. In 2015, 7 papers were found, 2016 14 papers, 2017 17 papers, 2018 10 papers and 2019 up to April 4 papers all including some type of description of assessment of commensality. Countries involved in commensality studies according to our search were mostly European, with some originating from New Zealand, Japan, Korea, African countries, USA, Canada and South America. Sometimes authors studied other cultures than their own.The diversity of disciplines identified in this review is broad and the methods used to describe or to assess commensality show the true multidisciplinarity of the subject. An important result is also that the term commensality as search term seems to be widely spread over disciplines over the last five years, while earlier research is scarce. A large proportion of the commensality research identified in this review was European, possibly indicating that the term commensality has been more used in the European arena.A great deal of the identified research is focused on health and nutrition, aiming to investigate the importance of commensality for health and wellbeing ,65,67,69There are a couple of questionnaires that could be used for authors only interested in commensality as such ,19, whilIt is very surprising that so few of regular dietary surveys that are undertaken actually look at commensality, venue or eating alone ,16,17. CThe identified qualitative studies showed that the notion of commensality to a great deal had raised interest among researchers studying meals as a central part of health or wellbeing ,55,65,67We could not identify any particular differences in assessment methods between western and eastern studies. Differences in methodology between studies of elderly and the general population could not be established.This scoping review used only one database (Web of Science Core Collection) and did not include other search terms than commensality. It could certainly have been made more comprehensive by using other search terms such as eating together, family meal, or other terms and using several databases. The concept of commensality seemed to have been more used over time, especially during the last five years. This could also mean that we missed some early studies performed with a different choice of words for commensal eating, such as “family meals,” “eating together,” or “social eating”. A full systematic review should include more complete search terms and other databases ,11. HoweThe strengths were that the high-quality database Web of Science Core Collection was used, which only includes papers published in peer-reviewed journals. The results of the search were of a magnitude that made it possible to review in a relatively rapid manner and the papers identified represented widely different disciplines. We wanted to highlight the multidisciplinary character of commensality and the included papers point at the developing field of commensality research, with commonalities and diversities in research methods. Using a quick and easy method of a scoping review provided us with a good overview of the papers using the specific term “commensality,” as a part of a project collection on diet, nutrition, and health with a focus on eating together . LearninWe conclude that in order to gather information on the importance of commensality for health and wellbeing, we should combine valid dietary survey methodology with valid estimates of commensality. Adding simple questions on where and with whom should be easy to integrate to large-scale dietary surveys. For all studies of commensality, a mix of qualitative and quantitative methods is recommended. Use of the term commensality in research papers should be encouraged, as well as providing more solid definitions of commensality and other search terms of eating together.


Aedes aegypti, Anopheles minimus and An. dirus, and two field populations of An. harrisoni and Ae. aegypti, respectively. Established TFT sublethal (LC50 and LC75), lethal concentrations (LC99) and discriminating concentrations (DCs) were selected corresponding to each mosquito test species. Spatial repellency and contact excitation (‘irritancy’) responses on adult mosquitoes to TFT were assessed using an excito-repellency assay system. At LC50, TFT exhibited strong avoidance with An. minimus (60.1% escape) and An. dirus (80% escape) laboratory strains, showing between 12 and 16x greater escape response than Ae. aegypti (5% escape). Repellency responses for field collected Ae. aegypti and An. harrisoni were 54.9 and 47.1% escape, respectively. After adjusting the initial contact escape response (a measure of combined irritancy and repellency) to estimate only escape due to contact, the LC50 and LC99 showed moderate escape irritancy with laboratory Ae. aegypti (41.4% escape) and no contact activity against the field population. Adjustment showed only weak contact activity (16.1% escape) in laboratory An. minimus at LC50. Spatial repellency is the predominant mode of action of TFT among colonized and field mosquitoes used in this study. Established baseline (susceptible) dose-response curves assist in optimizing SR products for mosquito control and pathogen transmission prevention.Airborne spatial repellency (SR) is characterized and distinguished from other chemical actions including contact locomotor excitation and toxicity. The use of volatile spatial repellents is a potential new intervention class for combatting mosquito-borne pathogen transmission; therefore, continuing investigations on the actions of these chemicals that modify mosquito host‐seeking behavior  is needed. The objective of this study is to characterize the key behavioral avoidance actions of transfluthrin (TFT) to advance spatial repellent development into practical products. Behavioral avoidance responses were observed for adult laboratory strains of  Aedes-borne viruses  are projected to expand and pose a greater risk, both geographically and demographically, in the decades ahead [Plasmodium vivax infections, a latent relapse form of parasite complicating elimination goals in the country [More than 4 billion people, primarily residing in tropical and sub-tropical countries, are at risk of infection from mosquito-borne diseases. For example, globally, an estimated 390 million dengue infections occur and around 200 million malaria cases are reported annually , 2. Denges ahead . While mMosquito vector control through the use of insecticide-treated bed nets (ITNs) and indoor residual spraying (IRS) are considered to have made major contributions towards the reduction in the global malaria burden since 2000 . Among tAedes aegypti (L.) and Aedes albopictus (Skuse) are the two most important vectors of dengue as well as chikungunya and Zika viruses [Aedes aegypti, in particular, is a formidable mosquito to control using current vector abatement methods. It is a strongly synanthropic/anthropophilic species and predominantly found in and near human dwellings with a high tendency of adults to rest inside houses [ viruses . In gene viruses . Aedes ae houses .Anopheles malaria vector species in Thailand, Anopheles dirus Peyton & Harrison and An. minimus Theobald are among the most important [Among the primary mportant , 15. Botmportant . Althougmportant , 17. Premportant , 14, 18 mportant .Ae. aegypti (37.5% repellency) at 0.001% and Ae. albopictus (45% repellency) at 0.01%. However, to our knowledge, there have been no published studies with TFT distinguishing the two types of behavioral avoidance responses (irritancy and repellency). Information on TFT and mosquito responses are limited; therefore, investigations are required on this AI for development of innovative spatial repellent products.Therefore, there has been increased interest in the spatial repellent properties of certain AIs as a possible means of arresting outdoor biting –22. By dA better understanding of the functional effects of chemical insecticides and mosquito responses is, therefore, critical in designing practical interventions of TFT-based spatial repellent products , 26. TheAedes aegypti (L.) (USDA strain), obtained from the U.S. Department of Agriculture, was maintained at the Department of Entomology, Faculty of Agriculture, Kasetsart University (KU) for over 20 years. 2) Anopheles minimus s.s. Theobald (DDC strain), from the Malaria Division, Department of Disease Control, Ministry of Public Health, Nonthaburi, Thailand, originally collected in Rong Klang District, Prae Province, northern Thailand in 1993 and maintained at the Kasetsart University laboratory shortly thereafter. 3) Anopheles dirus s.s. Peyton & Harrison (TMMU strain) was originally collected in Khao Mai Kaeo Sub-district, Bang Lamung District, Chonburi Province, eastern Thailand in 1981 and obtained from the Department of Medical Entomology, Faculty of Tropical Medicine, Mahidol University in 2016. This strain has been maintained continuously at KU insectary since 2016. Mosquito colonies were reared following standard handling procedures and conditions  at the KU laboratory [ad libitum with 10% sucrose (w/v) solution as sustenance. An artificial membrane feeding technique [An. dirus; all other species were self-mating in holding cages.Three pyrethroid-susceptible laboratory strains and two recently collected field populations of adult mosquitoes were used. Laboratory strains: 1) boratory , 30. Larechnique  using huechnique  was requAedes aegypti field population was collected as immature stages from Muang District, Nonthaburi Province, central Thailand  in 2016 and maintained at KU insectary; 2) Anopheles harrisoni Harbach & Manguin (a species member of the Minimus Complex) field population was collected from Pu Teuy Village, Sai Yok District, Kanchanaburi Province  according to Sungvornyothin et al. [1 to F3 generation mosquitoes.Field populations: 1) n et al. . The assn et al.  prior ton et al. . All ER R,3S)-3--2,2-dimethylcyclopropanecarboxylate; 97.90% purity) was used. Transfluthrin stock solutions were prepared using analytical grade acetone  as an organic solvent and silicone oil (Dow Corning® 556 cosmetic grade) as a carrier at a ratio of 1.91:0.95.Technical grade TFT  and a discriminating concentration (DC) based on doubling the LC99 x 2 for Ae. aegypti, An. minimus and An. dirus [Whatman No. 1 filter papers, measuring 14.7 x 17.5 cm, were uniformly impregnated with 2.8 mL of TFT solution using a calibrated micropipette. Papers were treated with established 50%, 75% and 99% lethal concentrations  were introduced into each of four chambers . Following a 3-min ‘acclimation’ period, the exit portal was opened for each chamber to initiate observations (escape). The number of mosquitoes that escaped from each exposure chamber into the attached receiving cage were recorded at 1-min intervals for a period of 30 min. At post-exposure, all mosquitoes were transferred to holding cups labeled by the chamber and movement response (escape or non-escape). The number of dead or knockdown (moribund) mosquitoes were recorded separately from inside (those remaining) and outside (having escaped) each chamber. All live and moribund mosquitoes in the chamber were held separately and provided with cotton-soaked 10% sugar solution for 24-h before recording the final mortality determination. The ambient air temperatures and relative humidity were recorded at the beginning of each 30-min trial period. All trials were performed during daytime hours (09:00–16:30 h) and each trial series were replicated four times. All assays were carried out in the same laboratory space, under identical conditions and ER assay systems for both colonized and field-collected mosquitoes.The percent ER escape responses and 24-h mortality were adjusted using Abbott’s formula when escape or mortality in the paired controls (untreated chambers) was between 5 and 20% of the test sample . The num25), 50% (ET50) and 75% (ET75) of test population to escape was estimated. A log-rank test [P < 0.05).Using initial escape data (Abbott’s formula adjusted only), Kaplan-Meier survival analysis was used to estimate rate of mosquito escape in contact and noncontact test formats, and for comparing differences in mosquito escape between the four concentrations . The timank test  comparedAe. aegypti, An. minimus and An. dirus using different species-specific concentrations are shown in Tables P < 0.05) greater percent escape in both noncontact and contact trials compared to the controls, except for Ae. aegypti in noncontact at LC50 (P = 0.0807) (P > 0.05) (Ae. aegypti (USDA), LC50 to LC99 produced percent escape in contact tests (Abbott’s adjusted percent only) ranging between 48 and 66% (99 (0.03412%) and the DC (0.06824%) due to the higher knockdown (KD) effect inside the chamber  compared to those that escaped (4.8–24% KD). Conversely, a positive relationship was found in noncontact test as percent escape is increasec as TFT concentration increased (Tables The contact excitation and noncontact repellency effects of TFT on laboratory-reared  0.0807) . No sign > 0.05) . For Ae. and 66% . The per(99 0.0342% and thAn. minimus (DDC), LC50 and LC75 TFT exhibited the highest mean percent escape in both contact and noncontact tests, ranging from 85.7–80.8% (contact) and 60.1–66.5% (noncontact), respectively (50 and 43.4% at the highest concentration (DC). Relatively high repellent activity in noncontact test was observed with An. dirus (TMMU) ranging from 46.4% escape (LC75) to 85.9% escape (LC99), while the contact test showed a final percent escape ranging between 54.2% (DC) and 74.1% (LC50) across all four concentrations  and contact (ET75 = 25 min) tests. Overall, at all concentrations, at least 50% of An. minimus and An. dirus escaped TFT-treated chambers in both trials within 6–12 and 3–15 min (ET50), respectively. In contrast, Ae. aegypti showed the least excito-repellency, both in final lesser percent escape and longer length of time required to escape the chamber.A wide range of escape time (ET) activity was observed between species and concentrations in noncontact and contact tests . In onlyAe. aegypti, unadjusted contact escape was significantly stronger than noncontact repellency between LC50 and LC99, but not the DC  in non-escape mosquito, while no knockdown (0%) and low to moderate %KD (4.8–36.8%) was found in escape group for all concentration in both contact and noncontact tests  and escape (63.6–83.3%) mosquito, respectively (An. dirus in both ER assays (High percentage of knockdown (82.5–94.7%) was only observed against ct tests . For An.ectively . WhereasR assays . Howeverape 62.5–00% and e %KD 4.8–6.8% was Ae. aegypti had relatively lower escape rates in noncontact tests at all concentrations, and more moderate responses in the contact tests  for escape data, the survival curves at 1-min intervals in contact and noncontact tests were generated Figs . Comaprect tests . Conversats Figs  and 3.Ae. aegypti exhibited only moderate behavioral avoidance (between 26.5–43.3% escape) with contact exposure at LC50—LC99, and only contributed 9.5% of the escape at the highest concentration (DC)  . However minimus  and 10% n. dirus . These fAe. aegypti showed no significant differences in escape responses except for LC50- LC99 and LC99-DC pairings. For noncontact tests, all LC50 pairings with the other three higher concentration were significantly different. For An. minimus, significant differences in contact escape were found in four pairwise comparisons (excluding LC50-LC75 and LC99-DC); whereas there was no difference in escape between noncontact comparisons. For An. dirus, no significant differences were observed in contact escape pairings and in only one instance was a difference detected in the noncontact assays (LC75-LC99).Within species, log-rank multiple paired comparisons between TFT concentrations and escape responses in contact and noncontact tests were performed . In contAe. aegypti (NON) were found resistant to TFT  using the previously established DC (0.06824%); whereas, An. harrisoni (KAN) was fully susceptible to TFT using the DC 0.06382% [WHO insecticide susceptibility tests showed that field-collected 0.06382% .50) established for Ae. aegypti (USDA) (0.00852%) and An. minimus (DDC) (0.00423%) [Ae aegypti (NON) and An. harrisoni (KAN), respectively. In noncontact tests, Ae. aegypti (NON) had significantly higher escape response compared to the USDA colony . There was a significantly higher percent escape in An. minimus (DDC) compared to An. harrisoni (KAN) in both noncontact (P = 0.0048) and contact (P < 0.0001) tests  (P < 0.0001) reductions in estimated percent escape due to contact only was also seen in the colonized strains. High % knockdown (>93.6%) were also observed in non-escape An. harrisoni KAN for both contact and noncontact tests, while 68–74% knockdown was found for non-escape Ae. aegypti NON mosquito. Both field populations recovered  after 24-h post exposure with reduced mortality ranging from 0% to 20%   were useP < 0.000; 1 and 63%  were contact) . Followi% to 20% .Ae. aegypti, An. minimus and An. dirus. When allowing mosquitoes a free flight choice, TFT was most effective in eliciting substantial spatial repellency, particularly Anopheles mosquitoes. Relatively high escape reactions in both contact and noncontact tests with all concentrations, lethal and sublethal, occurred with An. minimus and An. dirus. Aedes aegypti showed a more reduced and delayed escape responses compared to the anophelines. Transfluthrin at LC50, LC75, and LC99 also induced a moderate knockdown effect and 24 h mortality against Ae. aegypti, but higher KD and mortality on anophilines, particularly An. dirus. At the final concentrations used, the lower post-exposure (24 h) mortality compared with the higher initial percentage of knockdown mosquitoes at 30 min , for those not escaping (remaining in the exposure chamber) and those successfully escaping the chambers, suggests that the overall behavioral avoidance was the primary mode of action of TFT.The key feature of the excito-repellency (ER) assay system is the separation between contact excitation (‘irritancy’) and noncontact spatial repellency of a target compound that result in movement away or non movement from the physical source of the active compound (i.e. impregnated papers) ,44. UsinPreviously, the standard WHO susceptibility assay, using lethal and discriminating concentrations of TFT on cellulose filter papers, was used to measure phenotypic response of mosquitoes with survival or mortality as endpoints . Given tThe relatively high vapor pressure chemistry of TFT compared to other pyrethroids, e.g., deltamethrin and permethrin, may require the use of treated papers in tests much closer to time for treatment. Alternatively, if wanting to link actual dose with response, when repeating similar experimentation, the preferred method is to analyze the amount of AI present in the papers at the time of testing. The optimum time-lag between treatment and testing procedures needs to be explored further and verified for comparing test standardization and interpretation of findings with other compounds with high vapor pressure properties . Additionally, the physical/chemical effects of silicon oil (diluent) on the rate of vaporization needs clarification. Until there are a determination and agreement on preferred testing procedures, we provide a cautionary note when using treated papers in testing systems for the evaluation of TFT and similar compounds against insects.Ae. aegypti [Culex quinquefasciatus Say [An. minimus with increasing concentration when allowed direct contact with TFT, due to the stronger knockdown effect inside the chamber (non-escaping mosquitoes). A notable decrease in escape was also observed in Ae. aegypti between the three lower and highest concentrations, while An. dirus had the highest percent escape at the lowest concentration. The reduced escape is attributed to the stronger knockdown effect inside the chamber (non-escaping mosquitoes). In noncontact tests, colonized Ae. aegypti escape was greater at concentrations above the LC50. Although the repellency escape in DDC and TMMU anopheline strains tended to decline with increased concentration, all but one pairing (LC75 vs. LC99) found differences were not significant. Overall, both Anopheles species showed significantly stronger escape responses compared to Ae. aegypti (statistics not shown). Nentwig et al. [Ae. aegypti. Estrada et al. [Ae. aegypti and Ae. albopictus with increasing TFT concentrations ranging from 0.0005% to 10% using the high-throughput screening system.The degree of contact irritability exhibited by mosquitoes can vary according to species and the type and dose of insecticide used , 25. Hig aegypti  and Culeatus Say . Contrasg et al.  reportedAn. minimus and An. dirus. Contact excitation played a much larger role with colonized Ae. aegypti (USDA) with LC50, more comparable with repellency produced by LC75 and LC99, and a reduced excitation response for the DC. Conversely, using the LC50, the Ae. aegypti (NON) field population showed no contact response. Nentwig et al. [Ae. aegypti using three difference tests systems . Wagman et al. [The ER contact test design potentially measures the combined contact and noncontact mechansims of behavioral avoidance. Unlike the noncontact test design that measures spatial repellency only, the contact test cannot definitively separate between excitation and repellency responses. To estimate the effect of physical contact with a treated surface, mean contact escape responses were adjusted a second time see  using thg et al.  also shon et al.  also repAedes, Anopheles and Culex mosquitoes [The adjusted contact data showed colonized and field population anophelines produced relatively poor contact excitation compared to marked repellent activity. The robust repellency response seen with TFT is in contrast to other less volatile pyrethroids. Using the same ER assay system and a range of different pyrethroids, including deltamethrin, bifenthrin, permethrin, α-cypermethrin and λ-cyhalothrin, contact excitation appears the predominant mode of action for squitoes , 35, 36.squitoes .An. minimus was more susceptible to KD and mortality than An. dirus when exposed to 2% TFT for 60-min in a large semi-field enclosure . However, in this study, An. dirus showed much greater KD and mortality compared to An. minimus, while Ae. aegypti was less sensitive to KD (except for the DC). Exposing to TFT for 30-min or less (mosquito escaped from the treated chamber) may result in variable KD response for each mosquito species. Additionally, mosquitoes recovered within 24-h with decreased mortality in all treatment (except for Ae. agypti at LC50). In this study, spatial repellency of TFT occurred more quickly than a toxic response at test concentrations by causing mosquitoes to escape from the chamber. However, as shown in the recovery of the non-escape groups from knockdown, the test concentrations may not be high enough to completely kill all mosquitoes. As exposure time is one of the important criteria for evaluating the chemical action [Beside ER responses, mosquitoes encountering TFT can be knocked down (KD) or can be killed, depending on the exposure and species , 48. Marl action , furtherAn. minimus in the noncontact tests. This finding suggests that lower sublethal concentrations  TFT may be sufficient in producing strong spatial protection with low knockdown in ceratin mosquitoes . Grieco et al. [50 of deltamethrin, cyphenothrin, d-tetramethrin and tetramethrin were optimal concentrations for repelling Ae. aegypti. Therefore, this was the rationale for using the single TFT LC50 for field populations of Ae. aegypti and An. harrisoni in the ER assay system.Regardless of concentration tested, there was no significant difference in escape responses in o et al.  suggesteo et al. . Mongkalo et al.  also docAe. aegypti from Nonthaburi Province was resistant to TFT, while An. harrisoni from Kanchanaburi Province was fully susceptible at the discriminating concentration [Ae. aegypti NON population compared to the susceptible USDA strain. The USDA strain had a very low repellency response at LC50 compared to contact excitation, while the opposite was observed for the NON population. Similarly, Kongmee et al. [Ae. aegypti escaped the deltamethrin-treated ER chamber faster than a susceptible population. Conversely, Boonyuan et al. [Cx. quinquefasciatus escaped compared to four resistant field populations. Thanispong et al. [Ae. aegypti from various locations in Thailand had similar behavioral responses regardless of degree of background resistance to DDT and α-cypermethrin. It has also been suggested that if the chemical (i.e. TFT) can elicit the behavioral responses through spatial repellency and contact excitation actions, then the degree of resistance in certain mosquitoes to a toxic action would be minimal to no effect in disrupting human-mosquito contact [At time of testing, the field-collected ntration . Signifie et al.  found thn et al.  reportedg et al.  observed contact .An. minimus DDC laboratory strain and recent field An. harrisoni KAN population, genetically closely related ‘sibling’ species, are susceptible to TFT, a significantly  greater escape response was observed among DDC than KAN mosquitoes. Similarly, DDC had more rapid escape response to bifenthrin (pyrethroid) than recently field-collected An. minimus [An. harrisoni from same location (Pu Teuy Village) found weak behavioral responses to pyrethroids compared to An. minimus collected further north in Mae Sot District (Tak Province). Conversely, long-established laboratory strains of An. albimanus  [An. dirus  [Although the long-standing  minimus . Potikas minimus  using Anlle, FL)  and An. Bangkok)  showed rBangkok) , given oAn. arabiensis compared to houses in the unprotected village. They concluded diversion of An. arabiensis from repellent users to non-users. Therefore, it is important to be aware of possible diversion when implementing a spatial repellent as a transmission control measure. However, if applying a spatial repellent that able to protect multiple persons over wide areas , this could minimize the risk of possible diversionary effects [One possible outcome of using a spatial repellent is the diversion of repelled mosquitoes to other areas, thereby possibly increasing transmission risk elsewhere , 51. We  effects .Understanding the behavioral responses of mosquitoes to insecticides is relevant for supporting current mosquito control tools and development of novel ones. We encourage those investigating chemical properties for the control of mosquito-borne pathogens to include detailed observations on behavioral responses as well as toxicity. The use of varying concentrations of insecticide is preferred to observe the sublethal avoidance responses to each, especially so in spatial repellancy assays. In our view, even in the presence of significant physiological resistance, if an insecticide remains effective for controlling disease transmission by preventing human bite exposure via altered behavior it should continue to be part of the abatement arsenal. The ability to evoke strong mosquito avoidance behavior for reducing human-vector contact is invaluable for understanding a chemical’s properties to control transmission.vs. recent field collection) to TFT. Further studies on mosquitoes from different locations can help optimize currently available chemical-based intervention tools and spur the development of spatial repellency products using TFT and similar vapor-active compounds. Furthermore, both indoor and outdoor evaluations of TFT action protecting against mosquito bites using different delivery formats is required under natural settings.This study assessed adult female mosquito behavioral responses with exposure to TFT using an ER assay system. These findings demonstrate clear ER responses but also differences in avoidance behavior between mosquito species and conditions (colony S1 Table(*) significant difference at 95% CI; LC, lethal concentration; DC, discriminating concentration; NC; noncontact control, NT; noncontact treatment, CC; contact control, CT; contact treatment.(DOCX)Click here for additional data file.S2 TableLC, lethal concentration; DC, discriminating concentration.(DOCX)Click here for additional data file.


Microvascular dysfunction plays an important role in the pathogenesis of heart failure with preserved ejection fraction (HFpEF). However, no mechanistic link between systemic microvasculature and congestion, a central feature of the syndrome, has yet been investigated.+ excess.This study aimed to investigate capillary–interstitium fluid exchange in HFpEF, including lymphatic drainage and the potential osmotic forces exerted by any hypertonic tissue Na+, and K+) analyses; and 2) venous occlusion plethysmography to assess peripheral microvascular filtration coefficient (measuring capillary fluid extravasation) and isovolumetric pressure (above which lymphatic drainage cannot compensate for fluid extravasation).Patients with HFpEF and healthy control subjects of similar age and sex distributions (n = 16 per group) underwent: 1) a skin biopsy for vascular immunohistochemistry, gene expression, and chemical , in keeping with blood vascular rarefaction and the lack of any observed hypertonic skin Na+ excess, but the lymphatic drainage was impaired .Skin biopsies in patients with HFpEF showed rarefaction of small blood and lymphatic vessels ; residual skin lymphatics showed a larger diameter (p = 0.007) and lower expression of lymphatic differentiation and function markers  compared with control subjects. In patients with HFpEF, microvascular filtration coefficient was lower : 2.33 to 3.88] l × 100 ml of tissuePeripheral lymphatic vessels in patients with HFpEF exhibit structural and molecular alterations and cannot effectively compensate for fluid extravasation and interstitial accumulation by commensurate drainage. Reduced lymphatic reserve may represent a novel therapeutic target.  Heart failure (HF) is a leading cause of morbidity and mortality . Current2 extraction paralleling microvascular rarefaction, were reported as determinants of exercise intolerance in patients with HFpEF  accumulation. This phenomenon was proposed to be water-independent   , 17, 18,+ levels could impact microvessels and result in excess fluid extravasation and edema in patients with HFpEF.Therefore, the HAPPIFY  study was designed to investigate capillary–interstitium fluid exchange and to test the hypothesis that an osmotic effect secondary to high interstitial NaSubjects with stable HFpEF, identified from outpatient HF clinics in Glasgow, United Kingdom (n = 16), and volunteers of similar age and sex distributions, with no history of cardiovascular or renal disease, hypertension, or diabetes , were recruited between August 2017 and December 2018. HFpEF diagnosis was per the 2016 European Society of Cardiology guideline definition: 1) signs or symptoms of HF; 2) elevated B-type natriuretic peptide (BNP) >35 pg/ml; 3) ejection fraction ≥50%; and 4) evidence of structural heart disease  or diastolic dysfunction . ExclusiEligibility and main visits were conducted in the morning, in a temperature-controlled room. Subjects did not consume caffeine, alcohol, or food; did not smoke; and avoided vasoactive or diuretic medications for >4 h prior to assessments. The study procedures included history and physical examination; full blood count, BNP, hemoglobin A1c, and renal, liver, and thyroid function; urinary albumin-to creatinine ratio (morning spot sample); simultaneous bilateral brachial and calf blood pressure (BP) measurement; pulsed wave velocity; echocardiography; flow-mediated dilatation; venous occlusion strain gauge plethysmography; and a gluteal skin punch biopsy. Participants who were not on anticoagulants and who had no history of severe obstructive iliac artery disease were offered to participate in an optional substudy, involving a larger surgical excision of skin and subcutaneous fat tissue around the site of the punch biopsy, for dissection of resistance arteries and molecular biology. This study conforms to the Declaration of Helsinki and the protocol was approved by the West of Scotland Research Ethics Committee 3 (ref. 17/WS/0091) and Greater Glasgow and Clyde NHS Research and Development (ref. GN17CA152).+-free lidocaine cream  as described  underwent a 4-mm skin punch biopsy on a gluteal external upper quadrant, after topical numbing with Naescribed . One-halA skin portion of the larger biopsies obtained from participants eligible and consenting also to the substudy was immediately frozen for molecular biology (7 patients with HFpEF and 10 HC subjects); the remaining tissue was used to dissect small resistance arteries for ex vivo functional testing of endothelium-dependent and endothelium-independent relaxation by wire myography.+ and K+ were measured in the HNO3-digested dry samples by flame photometry  and expressed as absolute content (mmol/g DW) or concentration .As reported , frozen + lymphatic vessels and blood endothelial cells, respectively. Tiled images of the entire epidermal line and the subpapillary dermis were acquired at 20× magnification. Blinded automated image analysis for microvessels quantification in the 600-μm-thick dermal area starting from the epidermis–dermis junction and spanning the entire length of the biopsy was performed with ImageJ version 1.52q , by pre-defined homogeneous thresholding criteria and the “analyze particles” function (Paraffin-embedded skin sections (5-μm thick) including both epidermis and dermis were stained with anti-human LYVE-1  antibody , after overnight low-temperature antigen retrieval  and nonspecific signal blocking. Alexa fluor 488–conjugated secondary antibody  and Ulex europaeus agglutinin I lectin  were used to stain LYVE-1function .RNA was extracted from gluteal surgical biopsies. After preamplification  of target complementary DNA, a Custom TaqMan Array Card  was used to perform quantitative gene expression of markers or growth and transcription factors specific to blood or lymphatic vessels. Gene expression levels were compared and presented as ΔCt values, with β-actin as housekeeping gene.V)  and net fluid extravasation toward the interstitium at increasing venous occluding pressures, as described previously , measuring limb volume changes as ml/100 ml of tissue, was used to assess forearm and calf arterial blood flow, peripheral venous pressure , as described previously (V) / blood flow . The time course  of this interstitial fluid accumulation was calculated as the averaged first derivative of the plethysmographic tracing for each capillary hydrostatic pressure (= pressure applied to the cuff) step, in portions devoid of motion artifacts and excluding the venous filling phase ; these slopes are proportional to hydrostatic pressure by a coefficient called microvascular filtration coefficient (Kf) (μl × 100 ml of tissue–1 × min–1 × mm Hg–1) (i), were determined for each limb and participant, blindly to group allocation, by least-squares regression . Limbs with <3 points free of motion artifacts for regression fitting   21). Th. ThV, up fitting  or with f in patients with HFpEF compared with HC subjects. All statistical analyses were performed using Prism and SPSS version 25 . Categorical variables were compared by chi-square test. Continuous variables were tested for normality of distribution by graphical plotting and Kolmogorov-Smirnov test. Parametric or nonparametric unpaired unadjusted tests were used for comparison of primary (microvascular) and secondary endpoints between groups, accordingly. Least-squares fit was used for both nonlinear regression and linear regression. The α level was set at 0.05, and all statistical tests were 2-tailed.Recruitment was stopped after 16 of 20 initially estimated subjects per group, when interim analysis showed futility in relation to the primary hypothesis of a higher KAn extended description of the study methods is available in the Clinical characteristics of the 2 study groups are provided in Classical assessment of peripheral vascular function also revealed higher carotid-femoral pulsed wave velocity, as a measure of stiffness of large arteries, and lower brachial flow-mediated dilatation, which was accompanied by lower post-ischemic reactive hyperemia . Consist+ concentration (mmol/l of water), and total Na+ content (mmol/g DW) in the ESD were similar between patients with HFpEF and HC subjects (+ content were lower in patients with HFpEF than in HC subjects (+ and (Na+ + K+) concentrations  were similar between groups, as in ESD. Na+ + K+ concentration consistently fell within a physiological range (140 to 155 mmol/l) (+ content did not differ by group (p = 0.810) , Nasubjects . In the subjects , in keepsubjects , which l mmol/l)  in both = 0.810)  or tissuPatients with HFpEF had dermal rarefaction of blood microvessels compared with HC subjects (p = 0.003) , indepenLymphatic vessels were similarly reduced in number (p = 0.012) but were larger (p = 0.007) in patients with HFpEF compared with HC subjects . NeitherV was higher, although not to values suggestive of overt decompensation , and arterial resistance similar to HC subjects; at variance, their rest Pensation .Table 2M–1 × min–1), induced with progressively higher PV by stepped inflations of proximal cuffs, was not increased in patients with HFpEF above HC subjects as was initially predicted. In fact, the slope of the regression line between interstitial fluid accumulation and cuff pressure was lower in patients with HFpEF compared with HC subjects in the calf ; a similar nonsignificant trend was observed in the forearm (5.16 [IQR: 3.86 to 5.43] μl × 100 ml of tissue–1 × min–1 × mm Hg–1 vs. 5.66 [IQR: 4.69 to 8.38] μl × 100 ml of tissue–1 × min–1 × mm Hg–1) : in our in vivo experimental setting, this reflects the critical pressure up to which lymphatic drainage can fully compensate for the continuous extravasation of fluid at even low PV, and no net interstitial fluid accumulation occurs   and 4 calf  tracings were excluded from the analysis of microfiltration; 1 additional calf tracing from a patient with HFpEF was excluded as per procedure-specific exclusion criteria . The net capillary fluid extravasation toward the interstitium (ml × 100 ml of tissuemm Hg–1) . However x-axis) .Figure 3In summary, the clearance of interstitial fluid fails early to meet demands imposed by the capillary extravasation induced by hydraulic (venous) pressure in HFpEF.+ hypertonic excess to facilitate edema, we showed that the lymphatic vessels of patients with HFpEF exhibit structural and molecular alterations in the skin and cannot effectively compensate for fluid extravasation and interstitial accumulation by commensurate drainage. Along with direct ex vivo evidence of primary dysfunctional small subcutaneous resistance arteries and rarefaction of skin blood vessels, as already described in myocardium  is central to HF syndrome , but to + has been reported in the skin and skeletal muscle of aged hypertensive patients  concentration between study groups rules out any hypertonic Na+ excess. Accordingly, and contrarily to our initial expectations, the rate of tissue fluid accumulation induced by increasing hydraulic pressures  was not higher in patients with HFpEF compared with HC subjects. In keeping with the dependence of Kf on the vascular surface available for fluid exchange and with the rarefaction of blood vessels observed in HFpEF, it actually was lower. It is possible that blood flow in HFpEF, similar to HC subjects when normalized by limb volume, was partially diverted from exchanging capillaries to functional shunting  was lower in patients with HFpEF compared with HC subjects in both arms and legs. A lower Pi and a higher CVP , and that drainage, up to 8 l/day, is provided by lymphatics . Under tLymphatic vessels are organized into a series of coordinated functional units, each called lymphangion, separated by intraluminal 1-way valves and capable of spontaneous pumping activity. Their physiology has striking similarities with heart function, namely a systole-diastole cycle and a contractility modulated by pre- and afterload, conferring remarkable plasticity in response to increased requirements . The comThe morphological changes observed in skin lymphatics, with rarefaction of terminal capillaries and larger residual vessels, as well as the reduced gene expression of factors important for lymphangiogenesis and valve and vascular integrity maintenance ,28, offeRegardless of ‘which comes first’ considerations, reduced capacity of the lymphatic system to prevent or minimize development of edema could mark the clinical and prognostic distinction between patients with uncomplicated comorbidity and those presenting as HFpEF . MoreoveIn addition to the aforementioned lack of causality demonstration, which warrants additional dedicated studies, other limitations deserve to be mentioned. Healthy subjects were chosen as control subjects and comparison of HFpEF with comorbidity-matched patients is missing; similarly, a comparison between patients with HFpEF with and without obesity  would fuPerspectivesCOMPETENCY IN MEDICAL KNOWLEDGE: In patients with HFpEF, systemic microvascular dysfunction, including vascular rarefaction, involves both capillary and lymphatic vessels, reducing lymphatic reserve and dysregulating vascular–interstitial fluid homeostasis.TRANSLATIONAL OUTLOOK: Better understanding of the structural and molecular mechanisms responsible for reduced lymphatic reserve may facilitate the development of pharmacological interventions that improve interstitial fluid drainage in patients with HFpEF fluid drainage.This study provides the first description of morphological and functional changes in the lymphatic vasculature in HFpEF, resulting in reduced clearance of extravasated fluid. Along with demonstration of a reduced blood microvascular density in the skin, such findings draw attention to a globally and systemically defective microcirculation. This definition extends beyond the traditional arterial dysfunction and calls for better understanding of the role of arteries, veins, lymphatics, and their mutual crosstalk for tissue fluid (and likely inflammatory) homeostasis in the pathogenesis of this multifaceted, syndromic disease.This study was supported by British Heart Foundation Centre of Research Excellence Awards to Drs. Touyz, Delles, Petrie, and Rossitto (RE/13/5/30177 and RE/18/6/34217+). Dr. Touyz is supported by a British Heart Foundation Chair award (CH/12/29762). The authors have reported that they have no relationships relevant to the contents of this paper to disclose.


MYBL2 expression was a robust marker of poor overall survival and disease-free survival outcomes, regardless of disease stage. Clinically, elevated MYBL2 expression identified patients with aggressive early onset disease, increased lymph node involvement, and increased incidence of distant metastases. Analysis of genomic sequencing data demonstrated that MYBL2 High lung adenocarcinomas had elevated somatic mutation burden, widespread chromosomal alterations, and alterations in single-strand DNA break repair pathways. In this study, we provide evidence that impaired single-strand break repair, combined with a loss of cell cycle regulators TP53 and RB1, give rise to MYBL2-mediated transcriptional programs. Omics data supports a model wherein tumors with significant genomic instability upregulate MYBL2 to drive genes that control replication stress responses, promote error-prone DNA repair, and antagonize faithful homologous recombination repair. Our study supports the use of checkpoint kinase 1 (CHK1) pharmacological inhibitors, in targeted MYBL2 High patient cohorts, as a future therapy to improve lung adenocarcinoma patient outcomes.It has long been recognized that defects in cell cycle checkpoint and DNA repair pathways give rise to genomic instability, tumor heterogeneity, and metastasis. Despite this knowledge, the transcription factor-mediated gene expression programs that enable survival and proliferation in the face of enormous replication stress and DNA damage have remained elusive. Using robust omics data from two independent studies, we provide evidence that a large cohort of lung adenocarcinomas exhibit significant genome instability and overexpress the DNA damage responsive transcription factor MYB proto-oncogene like 2 (MYBL2). Across two studies, elevated  RAD51, an ATPase central to HR repair . Lung adenocarcinoma is the most prevalent subtype of NSCLC and has a five-year overall survival rate of less than 18% . The pooMYBL2 High)  (L2 High) . FunctioL2 High) –24. Whil elusive . Here, whttp://tardis.cgu.edu/tw/msignaturedb/)  values, etc.) were excluded on a test by test basis for statistical evaluation purposes. Exact patient numbers are reported for both MYBL2 High and Low cohorts in each figure legend. Access to a novel lung adenocarcinoma cohort was provided by the ORIEN consortium. Data in the July 2019 ORIEN private cBioPortal instance was analyzed in this study.We sought to identify drivers of novel genomic instability phenotypes in lung adenocarcinomas with wildtype HR effectors using omics data available from The Cancer Genome Atlas (TCGA) and the Oncology Research Information Exchange Network (ORIEN) consortium. TCGA Firehose Legacy (Lung Adenocarcinoma) data was obtained from cBioPortal . Differeturedb/) . TCGA MYRAD51 High and Low cohorts using a quartile-based approach; the top 25% of samples expressing RAD51 were called RAD51 High and the bottom 25% of samples were called RAD51 Low  values following RNA-sequencing (RNA-seq) and RPPA differential expression analyses. This fits well given, when stratifying on expression of a transcription factor, more stringent upper thresholds maximize transcription-factor specific biologic signal. To validate our findings, we applied the same cutoffs when analyzing a novel, independent lung adenocarcinoma cohort provided by ORIEN. Here, ORIEN lung adenocarcinomas with RNA-seq data were stratified into MYBL2 High and Low cohorts using our modified quartile-based approach; the top 21% of ORIEN samples expressing MYBL2 were called MYBL2 High and the bottom 27% were called MYBL2 Low.TCGA samples with RNA-sequencing data were stratified into AD51 Low Figure 1The Kaplan-Meier product limit estimator was used to estimate time-to-event distributions for OS and DFS. The log-rank test was used to test for differences in time-to-event distributions with a two-sided test. For both TCGA and ORIEN, OS refers to the time between initial diagnosis and time of death. DFS refers to the time between initial therapy and disease progression or death. Patients who did not experience an event or were lost to follow-up were considered censored at the time of last follow-up/contact. Cox proportional hazard models were used to assess the prognostic value of individual risk factors for TCGA patient OS and DFS outcomes. For both OS and DFS Cox proportional hazards models, patient smoking history and tumor (T) stage variables were dichotomized ahead of analyses. Kaplan-Meier survival analyses and Cox proportional hazards modeling were conducted using survminer and survival R packages .MYBL2 High and Low cohorts. Tumor size was manually extracted from digital pathology reports accompanying TCGA tumors. For ORIEN tumors, we analyzed potential differences in OS, DFS, disease-stage, and metastatic disease sites between MYBL2 High and Low cohorts.Clinical data accompanying TCGA and ORIEN tumors were analyzed for several specific endpoints. For TCGA tumors, we investigated potential differences in overall survival (OS), disease-free survival (DFS), tumor (T) stage, lymph node (N) involvement, metastatic (M) disease codes, age at diagnosis, patient smoking history, and tumor size when comparing MYBL2 High and MYBL2 Low tumors (sign of Log Ratio) * ).WEB-based GEne SeT AnaLysis Toolkit  was used to analyze a pre-ranked list of differentially expressed genes between TCGA w tumors . The preMYBL2 High and MYBL2 Low TCGA (N = 248) and ORIEN (N = 79) patient cohorts . ChIP-seq data for histone specific modifications were downloaded for H3K27Ac (GEO : GSM733743) and H3K4me3 (GEO : GSM733737). ChIP-seq reads were aligned to the human hg19 reference genome and visualized using the Integrated Genome Viewer tool . To idenME suite . IdentifLung adenocarcinoma cell lines A549, NCI-H23, NCI-H1568, and NCI-H1651 were obtained from American Type Culture Collection . A549 and H23 cells were maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), penicillin, and streptomycin. H1651 were cultured in DMEM:F12 medium, as recommended by ATCC, with the following modifications. DMEM:F12 culture media was supplemented with EGF , 1% FBS, and penicillin/streptomycin. H1568 cells were cultured using the same DMEM:F12 supplemented media as described for H1651.Cell extracts were isolated using RIPA lysis method and westerns were performed as described previously . Primary5 cells per well (~60% confluency) on Day 0. On Day 1, inhibitors were added to culture media and mixed thoroughly. On Day 3, culture media was aspirated, cells were washed with 1 mL of PBS, and subsequently incubated with fresh media and PrestoBlue HS cell viability dye (ThermoFisher P50200); PrestoBlue HS cell viability dye was added at a 1:10 (volume:volume) ratio according to manufacturer instructions. PrestoBlue HS dye was also incubated with cell-free, media only controls to account for background signal from culture media. After PrestoBlue HS addition, culture plates were incubated at 37°C for two hours. Following incubation, PrestoBlue HS fluorescence signal was quantified using a SpectraMax M2 microplate reader. Resulting signal was background corrected and is reported as a ratio of 560/590 nm fluorescence.NSCLC cells were seeded onto 24 well culture plates at a density of 1.3 x 10p and q  values < 0.05 were considered statistically significant.Statistical tests used throughout this study are indicated within figure legends. For all boxplots, data is displayed as minimum, first quartile, median, third quartile, and maximum. For all bar graphs, data is presented as mean +/- standard deviation. For all analyses, RAD51 mRNA expression using a quartile-based approach . Since elevated RAD51 gene expression is commonly associated with cancers with defective HR repair pathways, we stratified tumors based on RAD51 mRNA expression to identify lung adenocarcinomas with elevated genomic instability  scores, compared to RAD51 Low  on tability –16. Kaplrcinomas –12. UsinAD51 Low . The comAD51 Low , 35. HigAD51 Low . Using wRAD51 High lung adenocarcinomas. To do this, we systematically screened all known human transcription factors against a list of significantly differentially expressed (q < 0.05) genes between RAD51 High and RAD51 Low tumors  driving w tumors . MYB prorcinomas –24. OtheRAD51 and MYBL2 expression, we examined whether stratifying lung adenocarcinomas on MYBL2 mRNA expression alone could predict OS and DFS outcomes. TCGA lung adenocarcinomas were stratified into MYBL2 High and MYBL2 Low cohorts using a modified quartile-based method . Subsequent Kaplan-Meier analyses revealed that patients with MYBL2 High lung adenocarcinomas had significantly worse OS rates, compared to patients with MYBL2 Low lung adenocarcinomas   . In this independent cohort, patients with MYBL2 High tumors again had significantly worse OS and DFS rates    and ORIEN (p = 5.7e–4) cohorts (MYBL2 High tumors were significantly larger at diagnosis (p = 0.016) and presented with increased regional lymph node involvement  at diagnosis, while 64% of MYBL2 Low patients were either lifelong non-smokers or reformed for >15 years   stage . For this analysis, tumor (T) stage was selected because it captures both local invasion and tumor size datapoints , age at diagnosis , and high tumor (T) stage  were all significantly associated with diminished OS. Patient smoking history did not have a significant effect on OS . For DFS outcomes, we also found that high MYBL2 expression , age at diagnosis , and high tumor (T) stage  were significantly associated with diminished DFS. As with OS, patient smoking history did not have a significant effect on DFS outcomes . Together, this data confirmed that MYBL2 expression is an important prognostic variable for both OS and DFS patient outcomes, after adjusting for other key clinical factors such as age at diagnosis, tumor (T) stage, and patient smoking history.Given that elevated tapoints . When anMYBL2 High tumors had significantly higher somatic mutation load (p = 1.4e–4) and increased genomic alterations (p = 9.8e–14), compared to MYBL2 Low (MYBL2 High lung adenocarcinomas had significantly higher combined HRD scores (p = 2.22e–30)   . Moreover, we found that BRCA1 and BRCA2 transcripts were significantly overexpressed in MYBL2 High tumors  genes to identify any potential defects in DNA damage sensing (checkpoint), single-strand break repair, or double-strand break repair pathways  repair pathways. While ATM transcript was significantly under-expressed in MYBL2 High tumors, ATM was not significantly suppressed at the protein level  was significantly impaired in MYBL2 High tumors due to the loss of irreplaceable effectors XPA and XPC  repair, and inhibition of HR through Holliday junction rejection (HJ-rejection)  genes sense and respond to replication stress , indicative of a chronic, ATR-mediated intra-S checkpoint response due to replication stress ,  ,  was used to identify MYBL2 High and MYBL2 Low cell lines. Importantly, cell lines with elevated MYBL2 transcript showed increased MYBL2 and CHK1 protein expression by western analysis , compared to MYBL2 Low cells (A549)  , replication stress , error-prone DNA repair , and lung differentiation (SFTPB). Elevated expression of each panel gene tracked independently with significantly poorer OS and DFS outcomes when tested using TCGA data , which helps recruit POLQ for MMEJ repair , promote error-prone MMEJ repair , and mediate HJ-rejection   insertions and deletions with overlapping microhomology at breakpoint junctions ..MYBL2 HiMYBL2 High lung adenocarcinoma. Consistent with previous findings in other carcinomas, MYBL2 High tumors frequently carry inactivating alterations in TP53 and RB1 tumor suppressor genes   or MMEJ repair (POLQ). Given the increased likelihood for disease recurrence with MYBL2 High tumors, promising new inhibitors need to be explored following disease relapse or in combination with current standard-of-care regimens  . Much litability , 49. Leetoxicity . Since er models . Additioeveloped , 51–53. regimens .MYBL2 High disease not only constitutes one of the most aggressive subtypes of lung adenocarcinoma but it also encompasses a large cohort of patients . Based on current cancer statistics, MYBL2 High lung adenocarcinoma is estimated to represent 21,067 new cases this year alone. Therefore, the identification and development of novel therapeutic strategies, including CHK1/CHK2 inhibitors, for the treatment of MYBL2 High disease will provide significant clinical benefit.Collectively, our study highlights the importance of MYBL2 in coordinating replication stress responses and error-prone repair in lung adenocarcinomas with proficient HR pathways. https://www.oriencancer.org/request-an-account. Publicly available datasets were analyzed in this study. These data can be found here: TCGA Firehose Legacy data can be found in cBioPortal  (https://www.ncbi.nlm.nih.gov/geo/) . COSMIC signature data can be found in the mSignatureDB database (http://tardis.cgu.edu.tw/msignaturedb/).The data analyzed in this study is subject to the following licenses/restrictions: Access to ORIEN data is controlled by M2Gen and the ORIEN consortium. Requests to access these datasets should be directed to al.org/) . GenomicBM conceptualized the study, contributed to the investigation, formal analysis, writing the original draft, and writing, reviewing, and editing the manuscript. NW contributed to the formal analysis and wrote, reviewed, and edited the manuscript. PG wrote, reviewed, and edited the manuscript. PS wrote, reviewed, and edited the manuscript. RH, RG, WA, TV, SA, TW, and VC provided the resources, and wrote, reviewed, and edited the manuscript. DJ and DA wrote, reviewed, and edited the manuscript. MM conceptualized the study, acquired funding, supervised the study, wrote the original draft, and wrote, reviewed, and edited the manuscript. All authors contributed to the article and approved the submitted version.This work was supported by the National Cancer Institute  and the National Institutes of Health (NIH R01 GN118798 to PS and NIH R01 GM111911 to PG). Patient consent, specimen procurement, specimen processing, data abstraction, and access to molecular and clinical data were supported in part by the UVA Cancer Center Support Grant, P30CA044579. Funding sources listed were not involved in the design of this study, the analysis or interpretation of the data, the writing of this manuscript, or the decision to submit for publication.RG has received research support from Pfizer, Merck, Takeda, Jounce Therapeutics, Helsinn, Bristol Myers Squibb, and Celgene as well as personal fees from AstraZeneca, Pfizer, Merck, Bristol Myers Squibb, and Ariad. RH has received research support from Merck, AstraZeneca, Mirati Therapeutics, and Abbvie as well as personal fees from Pfizer and Takeda. SA has received research funding from AstraZeneca, Amgen, Genentech, Merck Sharp & Dohme, Nektar Therapeutics, Exelixis Inc., and Kura Oncology. DJ serves as a senior medical advisor for Diffusion Pharmaceuticals and as a consultant for Merck and AstraZeneca. BM and MM have a provisional patent Serial No. 62/928,018.The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


TNF receptor (TNFR) 1/2-deficient bone marrow-derived macrophages yielded significantly reduced TNF expression following low levels of PAMP stimulation, suggesting that signaling through the TNFRs in immune cells drives a feed-forward regulatory mechanism wherein low levels of TNF allow sustained enhancement of TNF expression in an iRhom2/ADAM17-dependent manner. Thus, we demonstrate that inflammatory expression of TNF and IL1β is differentially regulated following high or low doses of PAMP stimulation, invoking the activation of a previously unknown regulatory mechanism of inflammation.A well-controlled innate immune response is characterized by a rapid yet self-limiting inflammatory response. Although much is known about the range of inflammatory stimuli capable of triggering an innate immune response, the mechanisms which govern the degree of inflammation induced by inflammatory insults and the mechanisms in place to reset or maintain homeostasis are poorly understood. Tumor necrosis factor (TNF) is a potent early response pro-inflammatory cytokine produced by immune cells following a broad range of insults spanning autoimmunity and metabolic diseases to pathogenic infections. Previous studies have shown that a disintegrin and metalloproteinase (ADAM) 17 controls the release of soluble TNF and epidermal growth factor receptor signaling. Utilizing a genetic model of ADAM17 deficiency through the deletion of its regulator, the inactive rhomboid 2 (iRhom2), we show that loss of ADAM17 activity in innate immune cells leads to decreased expression of various cytokines in response to low levels of pathogen-associated molecular pattern (PAMP) stimulation but not at high-dose stimulation. In addition,  TNF sighallenge . Additioactivity . iRhom2 activity . Howeverin vivo  1/2-/- (TNFRSF1A/TNFRSF1B-/-) and TNF-/- bone marrow-derived macrophages (BMDMs), highlighting a critical role for soluble TNF as a coordinator of the inflammatory response under low PAMP challenge. Taken together, our studies indicate the presence of a novel, previously uncharacterized inflammatory pathway wherein iRhom2 mediates the innate immune response through the expression of inflammatory cytokines in a dose-dependent manner.The present study aimed to investigate the potential role of iRhom2 in the regulation of various pro-inflammatory cytokines in murine macrophages upon pattern recognition receptor stimulation. Given that iRhom2 controls the ADAM17-mediated release of several soluble factors from innate immune cells, including TNF and interleukin 6 receptor (IL6R) that modulate the progression of inflammation in vivo , we askeiRhom2-/-, TNF-/-, TNFRSF1A-/-, and TNFRSF1A/TNFRSF1B-/- mice were used for all experiments. iRhom2-/- mice obtained from Dr. Tak Mak’s lab at the Campbell Family Institute for Breast Cancer Research were described previously  Guidelines.Age-matched C57BL/6 wild type (WT), eviously . TNF-/-Limulus amoebocyte lysate-Pyrochrome kit, Associates of Cape Cod, Inc., East Falmouth, MA, USA). CL264 was from EMD Millipore , and Escherichia coli lipopolysaccharide (LPS) was from Chemicon International . The metalloproteinase inhibitor DPC333 was kindly provided by the Hospital for Special Surgery, New York, NY  from femoral and tibial bone marrow were propagated in the presence of macrophage colony-stimulating factor (M-CSF). This macrophage growth factor was secreted by L929 cells and used in the form of L929 cell conditioned medium. BMDMs were cultured in DMEM containing 10% heat-inactivated fetal bovine serum (FBS), 30% L929 conditioned medium, 1% non-essential amino acids, and 1% penicillin-streptomycin. Fresh medium was added on days 3, 5, and 7 prior to all experiments.6 cells per well in 1 ml of L929 conditioned medium overnight. BMDMs were then starved of L929 in 900 μl of culture medium containing RMPI 1640 with 10% FBS and penicillin/streptomycin for 4 h. BMDMs were then treated with 10 μl s of LPS diluted in culture medium at a final concentration of 0.002, 0.02, 0.2, 2, 200, or 2,000 ng/ml. Treatment was over the course of up to 4 h. An equivalent volume of culture medium was included as a negative control.Isolated murine BMDMs were seeded into 6-well plates at a density of 3.0×10TNF, IL1β, IL13, CXCL2, IL10, IL4, ACTB, and TBP were purchased from MilliporeSigma . TBP and ACTB were used as reference genes for all RT-qPCR experiments, and gene expression values were calculated using the 2-ΔΔCT method.Isolated BMDMs were subjected to mechanical homogenization in RLT buffer, and mRNA was isolated using a RNeasy Mini Kit according to the manufacturer’s protocol . mRNA was then reverse transcribed into cDNA using RevertAidM-MuLV , reverse transcriptase, which was utilized for RT-qPCR using a SYBR green master mix on an Eppendorf  RealPlex thermocycler. KiCqStart primer sets for Soluble TNF levels were quantified in cell culture medium using a sandwich ELISA . Soluble IL10 levels were quantified in cell culture medium by simplex sandwich ELISA according to the manufacturer’s protocols . Absorbance values from the IL10 analysis were normalized to the highest recorded values (100%) to present changes in relative protein production.Monoclonal antibodies were purchased from BioLegend. Clones were anti-CD120a , anti-CD120b , anti-CD284 , Rat IgG2a (isotype control 1:200), and Armenian Hamster IgG (isotype control 1:200). Cell viability dye BV Ghost 150 (1:300) was used to exclude dead cells. Flow cytometry was performed using a CtyoFLEX  and data were analyzed with FlowJo v10.7.t test. Multiple comparisons between experimental groups were analyzed by two‐way ANOVA. Multiple parametric statistical comparisons between experimental groups versus a control group were accomplished by Dunnett’s method. Pairwise multiple comparison procedures were performed via the Bonferroni’s multiple comparison’s test. P values of ≤0.05 were considered statistically significant.Values are expressed as means ± standard error of the mean (s.e.m.). The standard error values indicate the variation between mean values obtained from three independent samples per group. Statistical analyses were performed using an unpaired, two‐tailed student’s TNF or IL1β at 2,000 ng/ml of LPS stimulation -/- mice. -/-TNFR1/2 BMDMs yielded significantly less TNF expression than WT BMDMs for up to 4 h, suggesting that TNFR signaling is necessary for TNF expression early in the inflammatory response  of CL264  of CL264, further suggesting that low levels of inflammatory stimulus are regulated in an iRhom2/ADAM17-dependent manner  ligands. We treated WT and iRhom2-/- BMDMs with low doses of LPS in the presence of a low concentration of recombinant TNF. We observed that the significant reduction in TNF expression in iRhom2-/- BMDMs could be rescued by the addition of recombinant TNF  polarization to reduce localized inflammation in tissues  and by a Carver Trust Collaborative Pilot Grant through the University of Iowa to TM as well as by a T32 AI007485 to JS.CB and the Hospital for Special Surgery have identified iRhom2 inhibitors and have co-founded the start-up company SciRhom in Munich to commercialize these inhibitors. CB and TM hold a patent on a method of identifying agents for combination with inhibitors of iRhoms.The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


This special issue includes seven articles. We have a brief review of mental health issues among Asian and Pacific Island (API) communities. Two papers examine non-biological factors that may be associated with mood disorders, using a nationally representative sample of Asian Americans (AA). These papers are particularly informative because they report disaggregated results across four AA subgroups. Another two articles examine different interventions and their efficacy to improve mood and quality of life among APIs. Finally, the case of a clinical encounter during COVID-19 was examined through the lens of Asian critical theory. Although these papers differ in their focuses and methodologies, collectively, they provide valuable information about mental health issues among the API community.


Tumor necrosis factor (TNF) is a pleiotropic cytokine powerfully influencing diverse processes of the central nervous system (CNS) under both physiological and pathological conditions. Here, we analyze current literature describing the molecular processes involved in TNF synthesis and release from microglia, the resident immune cells of the CNS and the main source of this cytokine both in brain development and neurodegenerative diseases. A special attention has been given to the unconventional vesicular pathway of TNF, based on the emerging role of microglia-derived extracellular vesicles (EVs) in the propagation of inflammatory signals and in mediating cell-to-cell communication. Moreover, we describe the contribution of microglial TNF in regulating important CNS functions, including the neuroinflammatory response following brain injury, the neuronal circuit formation and synaptic plasticity, and the processes of myelin damage and repair. Specifically, the available data on the functions mediated by microglial EVs carrying TNF have been scrutinized to gain insights on possible novel therapeutic strategies targeting TNF to foster CNS repair. Microglia represent the resident immune cells of the central nervous system (CNS). In physiological conditions they are involved in sensing the environment and regulating brain homeostasis by performing different functions. Upon brain injury, they transform from immunosurveillant into reactive cells and acquire a variety of immune-phenotypes committed to both detrimental and protective functions ,2. ReactAmong the different factors through which microglia exert their regulatory activity on brain functions, TNF is the one that probably best reflects the dichotomous nature of these immune cells, being able to induce harmful or beneficial effects, depending on the specific isoform and receptor subtypes it activates. Indeed, while TNF has been historically defined as a potent inflammatory mediator driving tissue degeneration in disease conditions, growing evidence provides concrete clues of a parallel role of the cytokine in regenerative processes , thus coHere, we review current literature on the mechanisms regulating TNF production, intercellular trafficking and release from microglial cells, with a special attention to the role of microglia-derived extracellular vesicles (EVs) in these processes. Furthermore, we describe the variety of biological effects exerted by microglia-released TNF on different brain functions, which could be particularly relevant for the development of specific therapeutic strategies aimed at containing the progression of brain damage or promoting tissue repair.TNF is a pleiotropic cytokine produced as a precursor membrane-anchored form of 26 kDa, called transmembrane TNF (tmTNF), by immune cells, including microglia, astrocytes as well as other different cell types . After cUpon TNF binding, TNFR1 recruits the TNFR1-associated death domain protein (TRADD) that induces cell apoptosis by activating Fas-associated death domain protein (FADD) and caspase 8 . HoweverThe intracellular signaling pathways activated by TNFR2 are less characterized compared with those of TNFR1. Given that TNFR2 lacks death domain, the receptor only recruits TRAF2 and activates PI3K-Akt/PKB and NF-κB pathways for mediating pro-survival signals ,21. AccoSeveral studies showed that TNF is upregulated in activated microglia at transcriptional and translational level , in partIncreased release of TNF by microglia can enhance the activation of the entire population of microglia in a positive feedback mechanism ,31,32, sIn line with these findings, a large body of evidence indicated that microglial TNF is involved in brain damage following acute injury or chronic neuroinflammation . High le2+ influx in microglia [Under pathological events, microglia and damaged cells release at the sites of CNS injury a large amount of ATP, a danger signal that activates specific receptors, called P2 purinoceptors, ubiquitously expressed on neurons and glia ,41. ATP icroglia  that evoicroglia . Howevericroglia .MAP kinase cascades  are involved in TNF production downstream of P2X7 activation . SpecifiOnce synthetized, TNF is released via conventional or non-conventional pathways . The conThe non-conventional secretory pathways typically mediate release of proteins lacking leader sequences, through an ER-Golgi transport-independent mechanism. Unconventional secretion allows cells to secrete cytokines without the requirement for post-translational modification, which can influence their functions, and may protect the host when conventional mechanisms are compromised, for example under ER stress . SeveralATP is a major signal inducing unconventional TNF secretion via EVs . In macrAs described above, TNF synthesis in microglia strictly depends on the activation state of these cells and it is finely regulated by signals released in the extracellular milieu in response to brain damage, including ATP . SpecifiEVs are small particles formed by a double layer of phospholipids, which are gaining increasing interest as fundamental mediators of intercellular communications in the brain. EVs signals to adjacent cells but they can travel very long distances and deliver complex messages to distant cells . EVs canEVs can be classified based on site of origin in microvesicles (100 nm to 1 µm in diameter), directly shedding from the cell membrane, and exosomes (30–100 nm in diameter), originating from multivesicular bodies in the endosomal compartment . AnotherGiven all the properties described above, EVs hold great potential for several research and clinical applications, ranging from the basic study of pathophysiological mechanisms , to the Nonetheless, EVs represent ideal carriers for the delivery of transmembrane proteins such as tmTNF. Intriguingly, EV production by microglia is massively induced by the same mechanism driving TNF expression, namely activation of the P2X7 receptor by ATP . This suImportantly, EVs can also carry solTNF among their cargoes and the TNF isoform, soluble versus membrane-bound, carried by EVs greatly impacts the response of recipient cells . TmTNF iBesides TNF itself, other important players of the TNF signaling pathway have been reported in EVs, including TNFR1, associated signaling proteins and TACE, the enzyme responsible for tmTNF cleavage and release of solTNF from the plasma membrane. Indeed, TNFR1-containing small EVs have been detected both in vitro ,81 and iThe presence of enzymatically active TACE within EVs has been reported in blood samples of HIV-infected subjects , whose rAs already mentioned, EVs containing TNF and related signaling molecules have been detected in the CSF  and in tCollectively, the evidence described above suggests that microglia-derived EVs may represent ideal vectors for the delivery of TNF molecules, capable to promote both deleterious and reparative responses in target cells.As already discussed, inflammatory stimuli trigger a complex cellular and molecular response in microglial cells, which includes the release of soluble and transmembrane TNF and other related factors. In the following paragraphs, we will discuss the double-edged role of TNF deriving from microglial cells in shaping brain functions, including the neuroinflammatory response to tissue damage, neuronal circuit formation and synaptic plasticity, and myelin degeneration and repair .Neuroinflammation can be considered as the first line of defense to preserve or restore the CNS homeostasis in response to harmful stimuli, degenerative conditions and traumatic injuries. This process mainly involves the synchronized action of different cell types, primarily including microglia and astrocytes . The proMicroglia-derived TNF has been implicated in sustaining the pro-inflammatory activation of microglial cells through a positive feedback mechanism mediated by TNFR1 activation . AccordiIn addition to the aforementioned effects on microglial cells, TNF released by inflamed microglia, together with IL-1α and C1q, has been demonstrated to drive detrimental A1 astrocyte activation, leading to a final toxic effect on neurons and oligodendrocytes underlying the pathological mechanisms of several neurodegenerative disorders . NotablySimilar to microglia, activated astrocytes themselves can produce and release TNF, giving rise to an autocrine/paracrine loop of activation and cytokine release . HoweverIn parallel to pro-inflammatory effects, tmTNF-mediated activation of microglial TNFR2 has been demonstrated to be essential for preserving the protective functions of these cells. Consistently, microglia-specific TNFR2 knockout mice display early onset of EAE and increased infiltration of pro-inflammatory immune cells from the periphery . In thisThe best documented role of microglia in controlling neuronal functions comes from the analysis of TNF effects on synaptic connectivity . HoweverTNF released by microglia has an important role in regulating synaptic plasticity . SpecifiTNF not only controls homeostatic synaptic activity, but also induces neurotoxicity via autocrine/paracrine loops involving other endogenous mediators. First, TNF activates TNFR1 on microglia, amplifying its production and release . Second,Furthermore, TNF also induces neuronal death by caspase activation and apoptosis induction, as demonstrated in cultured cerebellar neurons exposed to microglia conditioned medium, containing both solTNF and TNF-carrying vesicles . The samA receptors [Another mechanism by which TNF promotes neurotoxicity is the inhibition of GABAergic transmission. In vitro studies in cultured rat and mouse hippocampal neurons indicate that TNF treatment induces a rapid and persistent decrease of inhibitory synaptic strength as well as a downregulation of cell surface levels of GABAeceptors . This reeceptors . This deSimilar alterations of synaptic transmission are induced by microglial EVs, which potentiate excitatory transmission while inhibiting release of the inhibitory transmitter GABA in cultures, brain slices and in the visual cortex ,127. HowSo far, only a single study implicated the TNF cargo of microglial EVs in neuronal dysfunction. Specifically, it has been shown that exposure of murine microglial cell line BV2 to α-synuclein, to mimic the neurodegenerative environment associated to PD, increases the secretion of EVs enriched in TNF and major histocompatibility complex II (MHCII) molecules and promotes neuronal apoptosis . In lineBesides PD, important roles of microglial TNF have been described in several other neuropathological contexts, such as AD, stress, ischemia, schizophrenia, and bipolar disorder . Like inElevated levels of TNF were found in hippocampal microglia after acute stress and were shown to correlate with working memory deficits. Interestingly, treatment of stressed mice with the TNF inhibitor etanercept rescued such defects in accordance with a reduction in hippocampal TNF .A dramatic upregulation of microglial TNF has been found in animal models of cerebral ischemia , specifiThe action of TNF can be influenced by environmental factors, such as exercise. Indeed, chronic exercise ameliorates cognitive impairment in wild-type mice by reducing the expression of hippocampal TNF and increasing the expression of TNFR1 and TNFR2. These benefits included reduced errors and improvements in spatial learning and spatial memory deficits in the Morris water maze . HoweverGiven “beneficial” and “deleterious” functions of microglia and TNF in neurodegenerative diseases, the clinical application of anti-TNF therapies remains very difficult. However, an in-depth understanding of the effects of microglial TNF on synaptic functions and of the mechanisms underlying its actions may provide important insights for the development of novel therapeutic strategies aimed to limit neuronal loss and to improve cognitive deficits in patients.A common feature of a diverse group of neurodegenerative diseases, from MS to psychiatric disorders and brain injuries, is demyelination, namely the damage to the myelin sheath. Myelin is the lipidic layer produced by oligodendrocytes which, enwrapping neuronal axons, allows the efficient action potential propagation by saltatory conduction within the CNS and provides mechanical and trophic support to neurons . Myelin Microglia heavily influence the pro-remyelinating attempts promoted by OPCs surrounding myelin lesions . Indeed,A very recent paper showed that TNF released by microglial cells, besides being fundamental for efficient clearance and degradation of myelin debris by phagocytes, it is also required for the generation of new myelinating cells within demyelinated areas, suggesting direct effects of this cytokine on OPCs . This isApparently in contrast with the aforementioned results, genetic ablation of TNF resulted in exacerbation of neurological deficits and augmentation of neuroinflammation and demyelination in the EAE mouse model. Of note, these deleterious effects were rescued by exogenous TNF administration , highligThe understanding of such opposing effects of TNF on oligodendrocytes  has incrOf note, TNF has been proved to be a key component of microglia-derived EVs involved in the astrocyte harmful conversion into oligo-toxic cells both in vitro and following lysolecithin-induced focal demyelination in vivo . The impIntriguingly, TNF levels are dramatically decreased, but still detectable, in EVs derived from inflammatory microglia co-cultured with immunosuppressive mesenchymal stem cells (MSC-EVs), which do not cause harmful astrocyte transformation and instead promote oligodendrocyte maturation . Based oActivated microglial cells can release TNF through conventional ER-Golgi dependent or unconventional EVs-mediated secretory pathways in response to different stimuli. Accumulating evidence highlights the powerful but often controversial effects exerted by this cytokine on CNS cells. The dichotomous role of TNF in orchestrating different CNS processes, ranging from neuroinflammation and neurodegeneration to myelin repair, probably represents the main reason why TNF-targeting drugs have been proved to be largely unsuccessful for the treatment of neurological disorders ,168,169.Importantly, recent findings provide new insights on the mechanisms of TNF release and signaling that may be helpful for the development of novel therapeutic approaches. In this respect, the first thing to consider is the existence of two different isoforms, solTNF and tmTNF, which generally induce opposing effects by activating distinct TNFR1 and TNFR2 receptors . A generBesides directly targeting TNF signaling pathways with specific drugs, the mechanism of cytokine release may also represent an important target for an upstream modulation of TNF levels. Of note, unconventional release of TNF via EVs in response to ATP stimulation appears to be the prevalent mechanism of TNF secretion from activated microglia under disease conditions. Thus, blocking the shedding of microglial EVs by using pharmacological inhibitors  might reFinally, the phenotype of TNF-donor cells seems to pivotally determine TNF levels packaged into EVs, which in turn impact the final effects of EVs on target cells. Accordingly, EVs produced by inflammatory microglia contain high amounts of TNF and induce detrimental astrocyte conversion and demyelination ,76. On t