Clostridioides difficile infection, the potential use of FMT in treating other infectious diseases is an area of active research. In this review, we discuss key examples of this treatment based on recent findings relating to the interplay between microbiota and infection, and potential further exploitations of FMT/IMT.The intestinal microbiota is recognized to play a role in the defense against infection, but conversely also acts as a reservoir for potentially pathogenic organisms. Disruption to the microbiome can increase the risk of invasive infection from these organisms; therefore, strategies to restore the composition of the gut microbiota are a potential strategy of key interest to mitigate this risk. Fecal  Microbiota Transplantation (FMT/IMT), is the administration of minimally manipulated screened healthy donor stool to an affected recipient, and remains the major ‘whole microbiome’ therapeutic approach at present. Driven by the marked success of using FMT in the treatment of recurrent  This defensive system includes a consortium of phylogenetically diverse commensal microbes, including bacteria and other components. Colonization resistance is the term used to describe the way in which the microbiome operates both directly and indirectly to prevent colonization and invasive infection from pathogens, as well as to provide immune regulation2 As an example of such an AMP, Type VI secretion system (T6SS) is a protein translocation complex secreted by members of the Bacteroidetes that has wide-spanning functions in killing and reducing the function and colonization ability of invading pathogens.3 An alternative direct route is the ability of commensal bacteria to act in competition with pathogens for resources and niches, e.g. indigenous E. coli competing with pathogenic E. coli 0157 for the amino acid proline (which it can exploit to promote growth);4 in addition, E. coli Nissle 1917 is able to compete with Shigella and limit its ability to cause invasive disease within the gut wall.5 As an additional route, gut microbial metabolites may also directly impact upon the life cycle of pathogenic bacteria, including bile acids, tryptophan-based metabolites, and short chain fatty acids (SCFAs); SCFAs are by-products of bacterial fermentation from nondigestible carbohydrates, and can induce production of AMPs6 and inhibit growth and fitness of pathogens, both directly7 and via routes including intracellular acidification.8One such direct route by which members of the intestinal microbiome may contribute to colonization resistance is through the production of bacteriocins/antimicrobial peptides (AMPs) and other proteins by commensal bacteria that may kill pathobionts and other competitors through different mechanisms, including attack on bacterial cell walls.9 Mucins are glycoproteins which act to protect the gut barrier against inflammation and colitis.10 Pathogens such as Clostridioides difficile are recognized to decrease the level of the major intestinal mucin, muc2;11 conversely, the commensal bacterial species, Bifidobacterium longum, restores growth of mucin.12Indirect mechanisms of colonization resistance include microbiome-mediated regulation of the integrity of the gut barrier function to prevent penetration/ translocation of potential pathogens.13–15 Toll-like receptors maintain intestinal homeostasis via their interaction with commensal bacteria.16 SCFAs including butyrate have a role in providing an energy source for intestinal epithelial cells as well as influencing T helper cell responses.17 In the presence of commensal bacteria, dendritic cells selectively induce immunoglobulin A (IgA), which also has an important immune function in prevention against invasive disease.18Modulation of innate and adaptive immune cells to enhance mucosal immunity is also an important role of microbially-secreted metabolites and microbial-associated molecular patterns (MAMPs).19 Such disruption of the microbiota not only alters its composition, but additionally may reduce the protective functions that it provides, including colonization resistance. Microbiome disruption and loss of colonization resistance is recognized to increase the risk of pathogens causing invasive disease and aberrant immune responses.Perturbation of the intestinal microbiota can be driven by factors such as medications , diet, surgery, host immune status, and comorbid conditions.20 There are two particular attractions about FMT as an approach as a ‘microbiome therapeutic.’ Firstly, from a theoretical perspective, this is a ‘whole microbiome’ approach, attempting to rest and restore both the entire composition and functionality of an ecological community. Secondly, from a clinical perspective, there is already sound evidence from the scenario of recurrent C. difficile infection (rCDI) that this approach may be highly effective and overall safe. In this review, we will discuss the rationale and utility of FMT in a range of infectious diseases and potential further applications.From a clinical viewpoint, manipulation of the gut microbiome to counter this perturbation and restore premorbid microbiome functionality is a relatively novel approach to reinstate colonization resistance, and may be a strategy that could be exploited for the treatment of particular infectious diseases; such new approaches are of particular pertinence and interest in an era of rising antimicrobial resistance. Gut microbiome manipulation strategies that have been explored encompass several modalities, such as prebiotics, probiotics, phage therapy, dietary manipulation, and fecal  microbiota transplantation (FMT).21 see Supplementary Material) is the transfer of screened healthy donor stool to a recipient’s gastrointestinal tract via routes including nasogastric tube, enema, colonoscopy or capsules. The express aim of the procedure is manipulation of an affected intestinal microbiome to restore premorbid microbiome composition and function, as well aiding recovery of host-microbiome interactions.22 Importantly, good tolerability of the procedure has been seen in immunocompromised patients.23 FMT administration should strongly adhere to international guidelines to ensure donor blood and stool are screened for potentially transmissible pathogens.24–26 A fatality from an ESBL–producing Escherichia coli bacteremia transmitted from FMT donor stool in the United States has been previously reported after transmission to two patients that was not screened for ESBL producing organisms.27 Additionally, systemic infection from Shiga toxin–producing Escherichia coli (STEC) from a single donor to seven patients has also been reported.28 These recent complications have prompted the Food and Drug Administration to issue additional warnings regarding donor testing and quarantine.29 However, reassuringly, no significant delayed complications or adverse effects related to infections have been described in longitudinal studies looking at long term follow up of patients who have received FMT administration.30–32Fecal microbiota transplantation ’;C. difficile infection (CDI) is a healthcare associated cause of diarrhea, precipitated by the use of antibiotics, and rCDI carries a significantly higher mortality than a single occurrence.33 FMT has been seen to confer a high success rate in the treatment of rCDI.34 The success in treatment of rCDI has led to a greater understanding of the wide interplay between the intestinal microbiome and defense against invading pathobionts and the role that FMT can play to restore and protect against invasive infection.35 The exploration into the mechanisms that contribute to the success of FMT in this field has led to the potential role of FMT to be explored in the treatment or prevention of other diseases with a link to the intestinal microbiome, including other infections. There currently exists an urgent need to seek non-antimicrobial options to address infectious diseases due to the global epidemic of antimicrobial resistance. Although life-saving, antimicrobials impact on the function of the intestinal microbiome and the subsequent long-term health consequences of their use is also increasingly being recognized, therefore FMT as a modality to restore the body’s own protection against invasive infection is of great interest. 90 and are therefore not reviewed further here).Enterobacterales, CRE – carbapenem-resistant Enterobacterales, ESBL – extended-spectrum beta-lactamase, ESBL-E – extended-spectrum beta-lactamase Enterobacterales, FMT – Fecal  Microbiota Transplantation, HBeAg – hepatitis B virus e-antigen, HBsAg – hepatitis B surface antigen, HIV – human immunodeficiency virus, HSCT – hematopoietic stem-cell transplantation, MDR – multidrug-resistant, MDRO – multidrug-resistant organism, MRSA – methicillin-resistant Staphylococcus aureus, n/a – not available, NDM – New Delhi metallo-beta-lactamase 1, No. – number, rCDI – recurrent Clostridioides difficile infection, UTI – urinary tract infection, VRE – vancomycin-resistant Enterococci.Abbreviations: allo-HSCT – allogeneic hematopoietic stem-cell transplantation, COVID-19 – coronavirus disease 2019, CPE – carbapenemase-producing 91 A relatively consistent finding between both CDI and non-CDI FMT studies is that high donor microbiota diversity and/or enrichment in particular commensal bacteria appear to be associated with FMT success.92 Studies in which either commensal bacteria cultured from healthy stool donors94 or spores derived from alcohol-shocked donor stool95 have been given as alternatives to conventional FMT in rCDI patients support the concept that transfer of commensal bacteria from donor to recipient is a central component of the efficacy of FMT, at least in this setting. However, the further demonstration in a pilot study that sterile, filtered FMT may have comparable efficacy to conventional FMT in the treatment of rCDI96 suggests that soluble components within FMT – including metabolites, microbial proteins, and/or bacteriophages and other nonbacterial microbiome components – may also be key mediators to the efficacy of FMT.In the best-studied application of FMT to infectious disease – rCDI – it has been shown consistently that successful FMT is associated with the rapid and sustained restoration of a gut microbiome with high diversity and taxonomic profile similar to that of healthy donors.97 After FMT for rCDI, there is restoration of a range of SCFAs within the gut from very low levels up to levels similar to healthy donors;98 this includes the five carbon SCFA, valerate, which directly limits the growth of C. difficile.7 In addition, successful FMT is associated with the restoration of microbial bile salt hydrolases and an associated recovery of the premorbid gut bile acid milieu, removing bile acids which act as potential germination triggers (such as taurocholic acid) and restoring secondary bile acids which limit the growth of C. difficile.99 Such changes in gut microbial metabolites may act beneficially beyond just a direct effect upon a specific gut pathogen itself, and impact upon host responses more generally; for instance, FMT-related changes in gut bile acid profiles have also been associated with altered farnesoid X receptor-pathway signaling,100 and the secondary bile acids that are enriched post-FMT are associated with an impact upon regulatory T cells.101A summary of both established and proposed mechanisms of FMT is presented in 102–105 low gut levels of Candida albicans is associated with successful FMT in CDI patients106 but failure of FMT in UC.107 Given the established role of the virome in colonization resistance108 – including a role for bacteriophages in lysing infected cells and reducing bacterial fitness109 – there is a clear rationale as to why these changes in bacteriophage profiles may contribute to the efficacy of FMT.A number of studies have described FMT-related changes in gut bacteriophage or fungal profiles, or defined profiles that predict treatment success, although these specific profiles are heterogeneous between conditions. For instance, while low relative abundance of Caudovirales bacteriophages in the gut predicts response to FMT in both patients with CDI and those with ulcerative colitis (UC),110 and may even reverse a CDI-related immunosenescent phenotype through its impact upon T cell repertoires.111 Both mouse and early human studies have associated successful FMT with increased interleukin-10 production by innate and adaptive immune cells, reduced interleukin-17 production, and reduced ability of macrophages, monocytes and dendritic cells to present MHCII-dependent bacterial antigens to colonic T cells.113 FMT-related changes in the gut microbiome have also been associated with changes in circulating and intestinal tissue microRNAs114 and reduction in complexity of serum N-glycan profiles toward that found in healthy donors,115 providing a potential link between the gut microbiome and epigenetic changes that may affect several aspects of host physiology, immune and otherwise.FMT-related changes in host immune responses have also been increasingly well-defined; for instance, FMT for rCDI has been associated with restoration of gut bacteria-IgA interactions116 and commensal bacteria in FMT outcompete C. difficile for proline as an energy source in the gut of a mouse CDI model;117 however, comparable studies have not been published using human samples at present.There are grounds for expecting that FMT may also restore other aspects of colonization resistance, although there are limited data from human studies at present. For instance, FMT has been demonstrated to transfer bacteriocins in piglets,92 In addition to exploring microbiota ‘signatures’ of donors or recipients that may predict response to FMT, a further focus of research is investigation of microbiota functions that may also be predictive. Other potentially relevant factors relating to donors and recipients within this scenario may include genetics, immune status, and clinical factors (e.g. coexisting medications). Factors including modality used to prepare FMT, use of any gut preparation prior to the procedure and/or diet of donor and recipient may also be relevant to consider.92While stool derived from almost any donor who passes screening protocols appears to work effectively in FMT to treat recurrent CDI patients, experience of FMT in non-CDI conditions demonstrates much more heterogeneity in response overall.118 Selection pressure from antimicrobials increases the genetic size of the resistome.119 Multidrug-resistant organisms (MDROs) are defined as bacteria resistant to more than three classes of antibiotics.120 Resistant ESKAPE pathogens that all colonise the intestine  have been given priority status for new therapy development by the World Health Organization.121 The intestinal niche of MDROs has health implications to the host, as invasive infection can occur after translocation across the gut barrier or fecal contamination of other body sites. Infections from MDROs have poorer outcomes than infections sensitive to first line therapy, due to the poorer efficacy and worse toxicity of second line therapy, as well as the increased cost of these agents122 and this is of particular significance in immunocompromised patients where mortality with MDRO infections are significantly higher.123 Attempts to stop the spread of MDROs includes use of antimicrobial stewardship programs and hospital infection control procedures; however, more targeted therapies such as probiotics and selective digestive decontamination have had variable outcomes.124The intestinal microbiome is recognized to act as a reservoir for pathogens that carry antimicrobial resistant genes (ARGs), the collection of which is known as the resistome.Barnesiella was associated with intestinal clearance of vancomycin-resistant enterococci (VRE);125 in addition, a four-strained consortium of commensal bacteria containing Blautia producta reduced susceptibility to VRE in a rodent model, with this protection attributable to production of a lantibiotic.126 Studies of FMT in the treatment of rCDI in humans have demonstrated a reduction in diversity and number of ARGs post-FMT;127 more recently, a similar finding has also been described in patients being treated with FMT for liver cirrhosis.128Mouse models have provided proof of concept of the impact of FMT upon intestinal MDRO dynamics. For instance, administration to mice of FMT containing the commensal bacteria 130 One of the perceived benefits of using FMT for this purpose has been that patients pose a lower nosocomial risk to others in a healthcare setting. Results from studies looking at intestinal decolonization of MDROs following FMT have been highly variable, in part due to the heterogeneity of the study design, patient cohorts, and FMT administration protocols.131 Biliński and colleagues reported decolonization rates or 75% in 20 patients,41 and Saïdani and colleagues also reported similarly high decolonization rates of 80% in 10 patients at 14 days (with a decolonization rate of 10% in a comparator arm). However, conversely, Davido and coworkers reported decolonization rate in eight patients as low as 37.5% after three months;43 in comparison, spontaneous decolonization rates for intestinal MDROs are reported as high as 48.2% after 90 days.132 The only reported randomized control trial (RCT) to date in this area demonstrated a non-significant decrease in rates of ESBL-E and CPE carriage in FMT-treated patients compared to the control group; in part, this was attributed to the low number of patients recruited and early termination of the trial by participants due to diarrhea.52 Although the reduction in the burden of ARG carriage in the gut has been described, the role of FMT as an infection control or an intestinal decolonization measure is still uncertain.Collectively, this evidence has led to the exploration of the use of FMT as a tool to “decolonize” the intestine to eradicate carriage of MDROs, which has been reported in a range of case reports, case series, and a single randomised trial see .36–46,48133 Studies reporting outcomes on the impact of FMT upon rCDI have reported a decrease in bloodstream infections (BSIs) post-FMT.134 Additionally, in studies investigating the impact of FMT in MDRO-colonized patients, a reduction in both MDRO-related and all-cause BSIs post-FMT has also been observed.50 The impact of FMT on the reduction of MDRO infections is currently being studied in two clinical trials, one looking specifically at patients with renal transplants .In vulnerable populations with a disrupted gut microbiota , the risk of bloodstream infections (BSIs) has been noted to be increased.135 and the frequent need for broad-spectrum antibiotic therapy136 impacts the intestinal microbiome – lower intestinal microbial diversity is seen to correlate with worse survival post-HSCT.137 An associated increased risk of BSIs in HSCT patients with intestinal domination with Gram negative organisms is also seen, and increased mortality in those colonized with MDROs.138–141 In terms of infection outcomes, studies looking at the use of FMT in HSCT patients have noted a reduction in days of fever,142 and reduction in number of BSIs in HSCT post-FMT.50143 The causes for disruption within the intensive care setting include hypoxic injury, enteral feeding, use of medications , and intestinal dysmotility, collectively resulting in a reduction in diversity and beneficial functional output of commensal bacteria.144 These changes within the gut microbiome are associated with an increase in infectious complications and mortality in patients with severe systemic inflammatory response syndrome (SIRS).145 Mouse models have reported improved survival in septic mice following FMT with an improvement in the gut barrier function.147 To date, several case reports have noted a decrease in SIRS response including fever in patients in the intensive care setting following FMT;72–74 in addition, a case series of 18 ICU patients with antibiotic-associated diarrhea were treated with FMT, with full resolution of symptoms occurring in eight out of eighteen patients.71Prevention of invasive disease in the Intensive Care Unit (ICU) setting using FMT has also been explored. Critical illness is recognized to dramatically impact the ecology of the microbial communities within the gut.148), or cohorts recognized to be at increased risk of BSIs due to the risk of microbiome disruption related to preceding drug therapy (i.e. chemotherapy or prolonged antibiotics) or chronic disease.Future targets to use FMT as a safe and cost-effective method to prevent BSIs could be aimed patient cohorts who are recognized to be at particular risk from their colonizing MDRO (utilizing a scoring system such as the INCREMENT scoreIn hematopoietic stem cell transplant (HSCT) patients – where preceding chemotherapy149 and the risk of antimicrobial resistance increases with recurrent courses of anti-infectives.150 Increased abundance of uropathogenic organisms in the gut has been seen to be a direct risk factor for occurrence of UTIs with the same organism;151 therefore, re-establishment of the composition of the intestinal microbiome to restore colonization resistance and reduce the burden of invasive infection has been explored using FMT.An important subgroup explored in the prevention of invasive infection is that of rUTIs. Non-antimicrobial options to treat rUTIs have limited evidence,80 Three case reports also describe use of FMT specifically to attempt to treat rUTIs, where no further UTIs were noted in patients after 8–12 weeks.76 FMT has also been used specifically to attempt to prevent rUTIs in renal transplant patients. These patients are recognized to have a lower intestinal microbial diversity than healthy controls152 and rUTIs are recognized to impact on the kidney graft function in these patients;153 as such, the restoration of the intestinal microbiota with FMT and prevention of invasive disease in this cohort could be of prognostic value. Two case reports and a case series have all reported a reduction in the occurrence of UTIs in renal transplant patients post-FMT despite no change in the risk factors predisposing the patients to recurrent infection.75Patients who were treated with FMT for rCDI were also noted to have a reduction in their occurrence of rUTIs.Blautia obeum, which has been demonstrated to block infection from Vibrio cholera via hydrolysis of bile acids.154 Another commensal bacterium, Clostridium scindens, has been recognized to possess antimicrobial features against infection from Entamoeba histolytica and C. difficile via the biotransformation of primary to secondary bile acids.156 Mouse studies have shown that FMT reduced intestinal bacterial load of Campylobacter jejuni, a common cause of foodborne gastrointestinal infection, and additionally lowered cell damage caused by the bacteria, as well as susceptibility to the disease.158 In humans, successful eradication of chronic Salmonella infection after FMT has been reported twice in the literature, firstly in two patients with Salmonella infection alongside prolonged carbapenem usage81 and secondly as a modality to eradicate asymptomatic chronic Salmonella carriage in two patients where carriage had an impact on their occupation in the food industry.57 The exploitation of FMT to treat enteric infections in humans has not been fully explored to date; however, the restoration of microbiota-derived metabolites seen in preliminary studies of these infections have also been seen in the mechanisms explored as contributors to the success of FMT for rCDI.35 This modality therefore may have either a prophylactic role or use in chronic or relapsing infections where long term antibiotic use has a detrimental effect on the host.As seen in rCDI, the intestinal microbiota is the first line of defense against enteric infections, and a deeper understanding of the role of the gut microbiota has arisen from studying the relationship between these pathogens and commensal bacteria. Examples include the commensal 159 Infection with respiratory viruses such as respiratory syncytial virus and influenza have been noted in mouse models to result in transient changes in intestinal microbiota composition, with an increase in Bacteroidetes and a decrease in Firmicutes phyla abundance as well as increased levels of lipocalin-2 concentrations, suggesting inflammation, posing an increased potential risk of subsequent bacterial infection.160 A recent study indicated that patients with a higher load of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in the stool had lower levels of commensal bacteria that produce protective metabolites such as short-chain fatty acids and tryptophan and higher levels of pathobionts such as Collinsella aerofaciens and Morganella morganii.161 A report on patients treated with FMT for rCDI during the coronavirus disease 2019 (COVID-19) pandemic noted full resolution from COVID-19 in two patients with concurrent CDI and COVID-19 infection.83 A further case series of two patients treated with FMT for rCDI with coexisting SARS-CoV-2 infection suggested that FMT may have had a role in the shortened recovery time seen in these patients.82 Another trial looked at administering FMT to patients one month following hospital discharge post-COVID-19, and noted improvement in both gastrointestinal symptoms and in microbial diversity.84The microbial communities in the intestine and respiratory system have a shared mucosal immune system that may be referred to as the ‘gut-lung axis,’ suggesting that there may be a role for FMT as a preventative or supportive measure in respiratory disorders.163 Response to treatment for hepatitis B is measured serologically using hepatitis B e-antigen (HBeAg) and hepatitis B surface antigen (HBsAg), where seroconversion of HBsAg is the ideal endpoint. Changes in HBeAg post-FMT has been seen in two case series: one with five patients (where decline in HBeAg was seen without any cases of seroconversion) and another study of twelve patients .86 A further case series reported HBsAg decline following FMT in HBeAg negative patients with a shift in the microbiota composition.87 Similarly, in human immunodeficiency virus (HIV) infection, disturbance of the microbiome is recognized to be related to virus response; the depletion of CD4+ cells is seen first in gut-associated lymphoid tissue, and this impacts on gut barrier function and Th17 cells function.164–166 A study of FMT administered to six macaques infected with simian immunodeficiency virus showed some immune restoration with significant increases in the number of peripheral Th17 and Th22 cells and reduced CD4 + T cell activation in gastrointestinal tissues, indicating some potential to enhance the immune system via T cell integrity. In humans, an early study looking at the use of FMT for rCDI in a severely immunocompromised patient noted an improvement in CD4+ counts as well as full recovery from rCDI.167 Sustained increase in microbial diversity similar to donor stool for up to 24 weeks was seen in another study of FMT administered to six HIV-infected patients – however, no changes were seen in inflammatory markers.89 A more recent study administered weekly oral capsules of FMT for eight weeks to 14 HIV positive patients and followed them up for 48 weeks; researchers described a sustained increase in bacterial diversity and reduction in intestinal fatty acid binding protein, which is recognized as a marker of gut barrier dysfunction.88 The clinical impact of this improvement in diversity is yet to be fully established in HIV patients. In terms of viral enteric infections, a case series of non-rCDI indications for FMT reported on a case attempting to trial FMT on a patient with chronic norovirus infection, without any change in their symptoms following FMT.57A similar ‘gut-liver axis’ is also purported to exist. Hepatitis B-infected patients with chronic carriage or decompensated liver cirrhosis are recognized to have a lower diversity of commensal bacteria and lower levels of microbial metabolites than healthy controls or those with asymptomatic hepatitis B carriage, which suggests that the gut microbiota may be responsible for modulating the effects of the virus on the liver.168 Manipulation of the microbiota is an attractive target for infections due to the global effort to reduce the use of antibiotics and the worldwide antimicrobial resistance crisis; however, large scale RCTs are needed to confirm the true utility in each of these different conditions.As the understanding grows of the role of the intestinal microbiota in both the defense against infection but also as a potential reservoir of pathogens, so our knowledge also expands regarding postulated strategies for reversing a perturbed microbiome. The role for FMT in protecting against and treating infection appears to be most valuable in vulnerable cohorts and the scenario of chronic or relapsing infection; however, a limitation is that current evidence for a number of potential applications are at present derived from rodent studies only or small, early phase human studies. Although the administration of FMT is becoming more refined in terms of the availability and ease of capsulized administration, with further understanding of mechanisms of actions, refined bacterial consortiums that could potentially be personalized to target specific organisms would be of greater value, and would overcome the potential risks identified with FMT administration.Click here for additional data file.


Development involves a delicate patterning of cell division and differentiation in three dimensions. This is beautifully illustrated in leaves, where three axes are visible: proximodistal (from base to tip), mediolateral (from the midvein to the margin/edge of the blade), and adaxial–abaxial (from upper to lower surface of the blade). Years of work have identified genes that control patterning along these axes, but how their activity is organized in time and space to control leaf development is less well understood.The Plant Cell, In this issue of trifoliate (tf-2) mutant, which produces only three leaflets—far fewer than WT. This allowed the authors to disentangle differentiation patterns due to leaf age (shared between WT and mutant) from those due to morphogenetic potential (much reduced in tf-2). When they visualized cell divisions by staining developing leaves with 5-ethynyl-29-deoxy-uridine (EdU), they found that cell divisions are higher in the margin compared to the rachis, but tf-2 mutants have fewer cell divisions where additional leaflets develop in WT, suggesting that TF promotes cell division during leaflet formation.They compared the development of wild type (WT) leaves with those of the Light Harvesting Chlorophyll A-B binding (CAB) genes and confirmed that they are upregulated in the rachis as predicted.They next used RNAseq to analyze transcriptional patterning across the proximodistal and mediolateral axes in WT. Using laser capture microdissection they sampled six regions of the P4 leaf: top, mid-region, and base along the proximodistal axis, each of which was divided along the mediolateral axis into rachis vs. margin see . This retf-2 mutant to make leaflets, and tested this by visualizing regions of high auxin response using the DR5:Venus reporter as well as localization of the SlPIN1 auxin transporter. The expression of both was reduced and more diffuse in the tf-2 mutant, suggesting the lack of leaflets may be due to reduced auxin response.To characterize detailed expression differences between the rachis and margin, the authors used self-organizing maps analysis to generate clusters of genes that are coregulated differentially between rachis and margin. In the margin, this revealed many genes known to be involved in margin identity, as well as genes related to auxin transport, biosynthesis, and regulation. They hypothesized that loss of auxin response in the margin may underlie the reduced ability of the tf-2 and showed that the gene Blade on Petiole (SlBOP2) is significantly upregulated in the margins of tf-2. To test the functional role of SlBOP2, the authors analyzed Slbop2 mutants and showed that leaves produce ectopic meristems and more leaflets, as previously demonstrated (TF binding site in the SlBOP2 promoter, suggesting that TF may promote leaflet formation by regulating SlBOP2 expression.They compared expression differences between WT and nstrated . FinallyAs well as painting a detailed picture of specific genetic regulators, these findings show that many genes expressed in the leaf margin are related to regulators of the shoot apical meristem. More work is needed to understand exactly how these genes have evolved to control diverse developmental processes, but this study leaves no doubt that the margin plays a central role in leaf morphogenesis. F1 Crossref missing


Mitigating and adapting to climate change requires an understanding of the magnitude and nature by which climate change will influence the diversity of plants across the world’s ecosystems. Experiments can causally link precipitation change to plant diversity change, however, these experiments vary in their methods and in the diversity metrics reported, making synthesis elusive. Here, we explicitly account for a number of potentially confounding variables, including spatial grain, treatment magnitude and direction and background climatic conditions, to synthesize data across 72 precipitation manipulation experiments. We find that the effects of treatments with higher magnitude of precipitation manipulation on plant diversity are strongest at the smallest spatial scale, and in drier environments. Our synthesis emphasizes that quantifying differential responses of ecosystems requires explicit consideration of spatial grain and the magnitude of experimental manipulation. Given that diversity provides essential ecosystem services, especially in dry and semi-dry areas, our finding that these dry ecosystems are particular sensitive to projected changes in precipitation has important implications for their conservation and management. The responses of terrestrial ecosystems to changes in precipitation patterns are highly context-dependent. Here the authors perform a quantitative synthesis of field rainfall manipulation experiments, showing stronger effects of precipitation on plant diversity at small spatial scales and in arid biomes. These changes in precipitation will, in turn, influence the structure and functioning of the altered ecosystems and the biodiversity therein2. While model-based projections for how ecosystems will change in the face of changing precipitation regimes are valuable3, experimental manipulations of climate change represent the prima facie evidence for predicting the structure and function of future ecosystems. While there have been many dozens of precipitation experiments across the world, synthesis has been difficult10. For example, Yue and colleagues10 conducted a meta-analysis of precipitation addition experiments, and found no overall effect of the treatment on biodiversity. Furthermore, Komatsu and colleagues7 synthesized data from global change experiments, including precipitation change, and also found no overall effect of precipitation on species richness. However, standard meta-analyses on reported effect sizes cannot disentangle complex biodiversity responses, such as the effects of the treatments on underlying components of abundance, evenness and aggregation that ultimately results in scale-dependent emergent patterns of species richness13. Ideally, a synthesis would explicitly consider (i) the magnitude and direction of the precipitation manipulation8; (ii) multiple aspects of biodiversity across multiple spatial scales12 and, (iii) information on background climate conditions, such as ambient levels of precipitation, which might influence how plant communities respond to precipitation change15.Human-caused climate change has dramatically altered temperature and precipitation distribution across the planet. While there is certainly variation in temperature changes, precipitation changes are even more variable, with both positive and negative changes in different parts of the world8, and we expect that biodiversity responses will increase with the magnitude of the manipulation and depend on whether precipitation is experimentally augmented or decreased16. Experiments that reduce precipitation and those that increase precipitation differ methodologically  and biologically 17, and could therefore cause a non-linear relationship between the magnitude of precipitation manipulation (which ranges from highly negative to highly positive) and biodiversity. Biodiversity responses to environmental factors often vary across spatial grains of investigation11, due to changes in the abundance and composition of individuals within and across communities12, and it is reasonable to expect that precipitation will influence the abundance, dominance, and spatial turnover of species.Experiments vary widely in the magnitude and direction of precipitation manipulations21. However, productivity is often a poor predictor of species richness22, and thus changes in productivity in response to climatic factors doesn’t necessarily coincide with changes in biodiversity23, due to scale-dependent biodiversity responses25. Nevertheless, plants located in arid ecosystems with low annual precipitation may show more dramatic biodiversity responses, since these communities are already facing hard physiological limitations to water availability26.Plant productivity in dryer ecosystems tends to be more responsive to precipitation than less water-limited systems12. To accomplish this, we required studies that contained information on the sampling effort and relative abundances of plant species12. In all, we were able to acquire such detailed data from 72 experiments, embedded in 34 plant communities contained within 23 studies (see “Methods” section). These studies came primarily from North America and Europe, but also included other biogeographical regions . However, this effect was tempered at larger spatial grains . Most likely these climate-dependent effects of precipitation manipulation on species richness were primarily associated with stronger changes in the total cover of plants , at least on the local scale. When we used potential evapotranspiration (PET) instead of mean annual precipitation, results were largely similar, but PET significantly changes the slope of the evenness across the more common species at the local scale  was related with higher probability of the dominant species of being monocarpic, i.e., annual or biennial . This suggests the possibility that the stronger biodiversity responses of dryer communities might be rather driven by longer-term species losses due to mortality, and species gains due to immigration from the species pool.Mechanisms underlying these differential responses from drier to wetter environments may involve stronger responses of dryer communities with different dominant life history strategies (monocarpic vs. polycarpic), and associated differences in seed bank dynamics 27. For example, at drier sites with generally sparse vegetation, added precipitation can increase biodiversity by increasing the possibility of establishment on open patches14. At wetter sites with denser vegetation, biotic interactions , may be relatively more important and thus dilute responses of biodiversity to precipitation29. However, we have much higher certainty for how biodiversity changes in dry and warmer ecosystems compared to wetter and colder ecosystems, such as tropical/subtropical systems and tundra, due to low sample sizes in the latter systems  report R2 values within the same range as ours . We also suggest that there are many important additional factors that, if measured consistently and reported across studies, could be incorporated into future synthetic analyses.Unlike other recent meta-analyses31, which cover ~40% of the earth’s surface32, accommodate unique biological diversity33, and provide essential functions and services for at least one third of the human population worldwide34.Our results show environment-dependent effects of precipitation change on biodiversity, which may have important implications for defining management strategies for mitigating and adapting to global climate change. Furthermore, we show that these differential responses became evident only when spatial grain and magnitude of experimental manipulation were explicitly taken into account. While previous studies have suggested that temperature-limited ecosystems are particularly vulnerable to temperature increases with climate change, our synthesis shows that dryland ecosystems are particularly vulnerable to changes in precipitation patterns associated with climate change. This likely emerged because experimentally increased levels of precipitation typically increased the possibility of establishment of species from the species pool into open patches, whereas experimental reductions in precipitation in these already dry communities most likely led to higher mortality and thus a decline in species richness. This more pronounced effect of precipitation change on biodiversity in drylands is likely to represent a threat to these ecosystemsWe conducted a systematic literature research in the Web of Science and Google Scholar by using the search terms (precip* change OR precip* OR water OR irrigation OR drought OR climate change) AND (biodiversity OR rich* OR diversity OR abundance OR evenness) AND (plant community OR plant*AND terr* ecosystems) AND (field experiment OR climate change experiment OR climate manipulation OR simul* climate change). This literature search yielded around 1700 published studies from which we screened the title and abstracts. We also followed relevant references from the papers and from systematic reviews and meta-analysis, which yielded 20 additional studies  in order to have a consistent measure among studies. When community responses to climate manipulations were followed across multiple years and different seasons, we only used the endpoint of the time series at peak biomass for our analysis.We extracted information about the location of the experiment , the duration of the experiment (number of years) and the magnitude of experimental manipulation relative to the mean annual precipitation (%). We extracted climate variables from the Chelsa database36. Specifically, for each experiment, we measured the following:As a measure of how densely the plot was vegetated, we calculated the sum of the total abundances per sampling unit either from the absolute abundances (%) or from the total number of hits a plant touches the pin per plot .S)—at two scales. We calculated local  S for each replicate in each treatment as the average number of species of species in a single plot/replicate (range of plot size: 0.08–2.5 m2). We calculated rarefied regional  S as the combination of all species observed in a given treatment/site combination  rarefied to the minimum number of replicates (n = 3)37. Because S does not include measures of relative abundance, it provides comparatively more weight to rarer relative to more common species.We calculated the total number of species—species richness S and SPIE at the local (average of replicates) and site scales . This resulting ratio , captures differences in the spatial heterogeneity in species composition among treatments for all species  and for primarily the most common species .To elucidate treatment-level effects on patterns of community compositional differences among replicates within treatments , we calculated the ratio of both We investigated the effects of precipitation manipulation on multiple response variables and scales40, SPIE was calculated using the inverse of the Simpson index in the vegan package41. To calculate an effect size of the precipitation treatment, we used the log response ratio  – ln (control)) as effect size42. Separate LRRs were calculated for each of the biodiversity components described above. A negative effect size indicates a decrease in the response variable due to the precipitation treatment, while a positive effect size indicates an increase in that response variable due to the treatment.Rarefied richness was calculated using the iNext package43. Model selection was performed based on the Akaike information criterion for small sample sizes (AICc44). Mean effect sizes and 95% confidence intervals were bootstrapped using 1000 iterations and extracted from the simplest model including that factor45  affected the LRR of species richness and effective number of species (SPIE), and how this in turn depended on mean annual precipitation  of the experimental location. All co-variates were scaled to the mean and two standard deviations before running the models49.Main analysis (Supplementary Table −1), was used instead of MAP (Supplementary Table P on the response variables (Supplementary Table P and the direction of manipulation into the model to test for different slopes in these two (non-overlapping) groups of data (Supplementary Table SPIE) at the gamma scale, where the quadratic term performed better , or polycarpic , and tested if the probability of being monocarpic was related to MAP/PET using a mixed effects logistic regression with study as random term. Next, we tested whether systems dominated by monocarpic species differed in the effect sizes of precipitation manipulation on biodiversity response variables using mixed effects models with study as random term, life history as a fixed factor with two groups (monocarpic vs. polycarpic) and effect size of biodiversity response variables (log response ratio) at local and regional scales.2) as a separate random effect or weighting the analysis by the square root of the sample size qualitatively influences the results . Following an approach by Spake et al.13, we checked for study-level scale dependence that could obscure our synthesis by inspecting ubiquitous co-dependencies between the mean/variance in the effect size of our biodiversity response variables with sample size and plot size (Supplementary Fig. P, and duration (Supplementary Fig. Effects of plot size and sample size: We examined if including plot size (mFurther information on research design is available in the Supplementary InformationReporting Summary


Luminal lipid droplets that emerge into the nucleoplasm do so at regions of the inner nuclear membrane that become enriched in promyelocytic leukemia (PML) protein. The resulting nLDs that retain PML on their surface are termed lipid-associated PML structures (LAPS), and are distinct from canonical PML nuclear bodies (NB) as they lack key proteins and modifications associated with these NBs. PML is a key regulator of nuclear signaling events and PML NBs are sites of gene regulation and post-translational modification of transcription factors. Therefore, the subfraction of nLDs that form LAPS could regulate lipid stress responses through their recruitment and retention of the PML protein. Both nLDs and LAPS have lipid biosynthetic enzymes on their surface suggesting they are active sites for nuclear phospholipid and triacylglycerol synthesis as well as global lipid regulation. In this review we have summarized the current understanding of nLD and LAPS biogenesis in different cell types, their structure and composition relative to other PML-associated cellular structures, and their role in coordinating a nuclear response to cellular overload of fatty acids.The nucleus harbours numerous protein subdomains and condensates that regulate chromatin organization, gene expression and genomic stress. A novel nuclear subdomain that is formed following exposure of cells to excess fatty acids is the nuclear lipid droplet (nLD), which is composed of a neutral lipid core surrounded by a phospholipid monolayer and associated regulatory and lipid biosynthetic enzymes. While structurally resembling cytoplasmic LDs, nLDs are formed by distinct but poorly understood mechanisms that involve the emergence of lipid droplets from the lumen of the nucleoplasmic reticulum and  Nutrien1) . LDs the1) . The def1) , a commo1) , defecti1)  and lipi1) . When co1) .The lipid storage function of LDs relies on a complement of core proteins on their surface that regulate the storage and release of lipids in response to nutrient signaling. Proteomic analysis has revealed an expanded repertoire of associated proteins that suggest a wide-ranging role for LDs in cell physiology. These include the MAX dimerization (MLX) protein and related glucose-sensing transcription factors , histoneCaenorhabditis elegans  and potential functions in the nucleoplasm, with a focus on the association of nLDs with promyelocytic leukemia (PML) protein in lipid-associated PML structures (LAPS).Since cytoplasmic lipid droplets (cLD) are more abundant than their nuclear counterparts, nLDs visualized by wide-field microscopy could be cLDs trapped in invaginations of the nuclear envelope (NE). However, serial sections of human liver revealed that nLDs are not connected to the NE and associate with heterochromatin . nLDs viin situ biogenesis at the INM and nLD budding into the nucleoplasm and 2) ER luminal lipid droplets (eLD) that migrate into type-1 nucleoplasmic reticulum (NR) invaginations that rupture to release a nascent nLDs into the nucleoplasm  leoplasm .In situ nLD biogenesis is documented in Saccharomyces cerevisiae (de novo from glycerol-3-phosphate and fatty acids by the concerted activity of glycerol 3-phosphate acyltransferases (GPAT) and 1-acylglycerol 3-phosphate acyltransferases (AGPAT) to produce phosphatidic acid (PA), which is dephosphorylated by Pah1 (S. cerevisiae) and Lipins  to produce diacylglycerol (DAG). Finally, DAG acyltransferases (DGAT) 1 and DGAT2 produce TAG  targynthesis  and 3) cynthesis .in situ pathway. Similar to cLDs  mutant grown at the non-permissive temperature had a 6-fold increase in nLDs due to the shift toward TAG storage. Cds1 synthesizes CDP-DAG, the common substrate for synthesis of S. cerevisiae phospholipids, and is one of many lipogenic enzymes upregulated by the Ino2-Ino4 transcription factors  that is targeted to the ER and supports lipoprotein secretion . However, it remains to be determined if a paralogous PML-like protein, for example containing the highly conserved tripartite motif (TRIM) domain found in PML and many E3 ubiquitin ligases  in the ER lumen transfers TAG and phospholipids to newly synthesized ApoB to form a VLDL precursor  and choline phosphotransferase (CPT) in the ER and ER/Golgi, respectively . This ceDrosophila embryogenesis , which is required for Torsin A activity, displayed impaired VLDL secretion, hepatic steatosis and nLD accumulation  diseases are caused by the expansion of polyQ repeats that are deleterious to neurons . A commoDuring mitosis, PML proteins form a unique SUMO-negative structure known as mitotic accumulations of PML protein (MAPP)  Dellair. MAPPs aThe recently characterized LAPS represent a new PML subnuclear compartment that associates with nLDs. Most LAPS (∼75%) share similarities to the MAPPs in the sense that they lack SUMO, DAXX and SP100 . HoweverRNA-mediated silencing of PML-II in U2OS cells did not significantly affect total nLD abundance and size  indicatiHigh resolution imaging of immunostained LAPS in U2OS cell revealed that CCTα and PML occupy different regions of the LAPS and have minimal overlap. Clues as to how CCTα and PML form this association with LAPS comes from HGPS fibroblasts in which CCTα is associated with nuclear lamina threads like those observed for PML . These tPml in Pten-null tumours resulted in the hyperactivation of SREBP and ultimately, in a SREBP-dependent aggressive metastatic phenotype  store and supply lipid precursors for membrane biogenesis and signalling molecules in the nucleus, and 2) regulate the cellular response to fatty acid stress. Although two different pathways for nLD biogenesis have been identified, both involve maturation by the Studies of the nLD maturation process has also revealed an important role for PML, and particularly PML-II, in the formation of a discrete subset of nLDs called LAPS. LAPS represent a novel PML-containing subnuclear structure that adds to our understanding of how the PML protein responds to various cellular stresses and disease states by altering the structure, composition, and localization of PML NBs. In the case of LAPS, there is much work to be in done to fully elucidate what role(s) PML might play in LAPS biology beyond LAPS formation and the recruitment of CCTα and Lipin-1. The presence of these lipid enzymes and the concurrent loss of SUMO, SP100 and DAXX from LAPS indicates they are unique PML-containing structures specialized in regulating lipid metabolism. It will be important to identify the proteome of nLDs and the role that PML plays in the association of client proteins with LAPS. In the absence of SUMO-SIM interactions, which drive the majority of protein associations with PML NBs, one might expect a more limited and specific repertoire of LAPS-associated proteins that rely on conventional protein-protein or protein-lipid interactions to be recruited and retained at LAPS. In this situation it is possible that specific PML isoforms, through their unique C-terminal regions, could recruit LAPS-associated proteins in a more direct way. Alternatively, or in addition to, PML-containing portion of LAPS could retain the phase separation properties of PML NBs to recruit and retain proteins in liquid condensates. In addition, it is still unclear if LAPS are associated with chromatin regions or specific gene promoters in the same way as PML NBs, although we do know that nLDs can modify nuclear architecture and chromatin dynamics. Therefore, PML association with nLDs through LAPS formation might provide a mechanism for the cell to effect transcriptional changes as part of the lipid stress response in much the same way that PML NBs regulate gene expression through chromatin contacts as well as recruitment and modification of transcription factors and chromatin modifying enzymes. The lack of key PML NB components at LAPS would, however, indicate that the mechanism for gene regulation at LAPS involves a different complement of PML-associated proteins that do not interact with LAPS via SUMO-SIM interactions. Collectively, these findings usher in new paradigms for not only PML-based nuclear structures but also for nLD biology.


Sexual minority women are more likely to drink alcohol, engage in heavy drinking, and experience alcohol-related problems than heterosexual women. However, culturally tailored interventions for this population have been slow to emerge.This type 1 effectiveness-implementation trial examines the feasibility and efficacy of a gamified, culturally tailored, personalized normative feedback (PNF) alcohol intervention for sexual minority women who psychologically identify as lesbian, bisexual, or queer (LBQ).The core components of a PNF intervention were delivered within LezParlay, a fun, social media–inspired, digital competition designed to challenge negative stereotypes about LBQ women and increase visibility. The competition was advertised on the web through social media platforms and collaboration with LBQ community organizations. After 2 rounds of play by a large cohort of LBQ women, a subsample of 500 drinkers already taking part in the competition were invited to participate in the evaluation study. Study participants were randomized to receive 1 of 3 unique sequences of PNF  over 2 intervention rounds. Randomization was fully automated by the web app, and both researchers and participants were blinded.Analyses will evaluate whether PNF on alcohol use reduces participants’ drinking and negative consequences at 2 and 4 months postintervention; examine whether providing PNF on stigma-coping behaviors, in addition to alcohol use, further reduces alcohol use and consequences beyond PNF on alcohol alone; identify mediators and moderators of intervention efficacy; and examine broader LezParlay app engagement, acceptability, and perceived benefits.incognito intervention approach is uniquely oriented toward engaging and preventing alcohol-related risks among community populations of LBQ women who may view their heavy drinking as normative and not in need of change because of the visibility of alcohol use in sexual minority community spaces. Thus, this intervention strategy diverges from, and is intended to complement, more intensive programs being developed to meet the needs of LBQ women already motivated to reduce their consumption.This ClinicalTrials.gov NCT03884478; https://clinicaltrials.gov/ct2/show/NCT03884478DERR1-10.2196/24647 The Institute of Medicine first identified sexual minority women (SMW) as a medically underserved population that disproportionately engage in hazardous drinking and carry the burden of alcohol dependence two decades ago . At presRecent research suggests that personalized normative feedback (PNF) may represent a welcomed low-risk, high-reward strategy for reducing alcohol use among SMW, particularly those who psychologically identify as lesbian, bisexual, or queer (LBQ) -16. FounAlthough social norms are among the most predictive and commonly targeted antecedents to alcohol use in other heavy drinking populations -23 and aIn contrast, web-based PNF interventions have the potential to cost-effectively reach and motivate reductions in drinking among SMW not seeking treatment and those who do not yet view their drinking as excessive or a risk to their overall health. Similar to the heavy drinking populations of college students ,43, miliFew longitudinal studies have simultaneously examined the perceptions of sexual identity–specific substance use norms and concurrently experienced sexual minority stressors as predictors of substance use , and no true norm. This study directly examines whether interpersonal stigma exposure moderates the efficacy of PNF designed to correct misperceived norms for LBQ peers’ drinking and coping behaviors.However, 2 longstanding social psychological theories support the prediction that correcting misperceived sexual identity–specific drinking norms may be maximally effective among SMW who experience severe interpersonal sexual minority stressors such as violence and harassment because of the sexual minority status. Specifically, self-categorization theory -50 conteAdditional potential moderators of PNF intervention efficacy examined in this trial include preintervention alcohol consumption and negative consequences, sexual identity, age, race or ethnicity, and relationship status. Relative to lighter drinkers, heavier drinkers naturally have greater room for behavior change following PNF’s correction of drinking norms ,22. ThusAn extensive study of web-based PNF interventions among college students suggests that these interventions lead to reliable but relatively short-term and modest reductions in drinking . ResearcOne tool commonly used to increase the credibility and appeal of health promotion programs for minority populations is cultural tailoring, which refers to the development of interventions, messages, and materials to conform with specific cultural characteristics of the target group . RecommeOne round of LezParlay was played monthly over an 8-month period, with a variable cash prize awarded monthly to the top scoring player exhibiting the greatest accuracy in their perceptions of LBQ peers. During the first 3 weeks of each round, players were invited to size-up fellow LBQ players by browsing their social media–like profiles, submit guesses about negative stereotype-related behaviors and experiences of age- and sexual identity–matched LBQ peers , select an amount of points to wager on these guesses being true of other age- and sexual identity–matched players, and earn points for reporting on their own corresponding behaviors and experiences. In the last week of each month, all players received individualized, detailed results  for a subset of the round’s questions. Animated charts and text detailed the accuracy of the player’s perceptions, how their behaviors and experiences compared with LBQ peers, summarized the stereotypes challenged, and provided their perceptual accuracy–based rank and score. Importantly, all actual norms featured in the detailed results were organically derived from the round-specific reports of players’ behaviors and experiences. This registered clinical trial sought to evaluate whether LezParlay-delivered PNF on alcohol use reduces alcohol consumption and negative consequences relative to PNF on control topics (aim 1); examine whether providing PNF on coping behaviors, in addition to alcohol use, further reduces alcohol use and consequences beyond alcohol PNF alone (aim 2); identify mediators  and moderators  of intervention efficacy (aim 3); and examine broader LezParlay competition engagement, acceptability, and perceived benefits (aim 4). The following sections provide an overview of the trial design, LezParlay competition promotion efforts, randomized controlled trial (RCT) subsample recruitment, measures, and analysis plan.replay bonus, which invited players to boost their scores by guessing, betting, and reporting on alcohol use and control topics a second time. Following the competition, 4 months postintervention, evaluation study participants then completed a feedback survey assessing competition acceptability, perceived benefits, and feature requests for the next version of the competition. At the end of this survey, participants reported their alcohol use one final time.Following recent recommendations for testing the real-world feasibility and impact of normative feedback interventions ,103, LezLezParlay is a low-cost, device-responsive, HTML5 progressive web application integrated with Facebook Connect and Construct 3 game engine as well as a text message application programming interface, and email server. Although it provided a native app–like feel on Android and Apple smartphones, the app could be accessed on any internet-connected electronic device and did not require players to visit an app store or download any software. Instead, SMW simply accessed the LezParlay web app by URL  and wereThe competition was open to all LBQ women aged 21 years or above, regardless of birth sex. Players learned about LezParlay through 1 of 4 promotion strategies taking place over a 3-month period. First, before the launch of the first round, local SMW were invited to sign up through flyers and promotional items distributed at LBQ community events in Los Angeles . Next, as the first round began, marketing campaigns on the HER social app, the leading dating or social app for LBQ women, invited users in their 3 largest markets  to LezParlay via push notifications and in-app advertisements. An advertisement was also placed in the electronic newsletter of Autostraddle, the leading independently owned news website for queer women. During the first 3 rounds of the competition, targeted campaigns on Facebook and Instagram (Facebook Inc) also advertised LezParlay to LBQ women residing in the United States. All recruitment materials were linked to LezParlay’s informational landing page . This paAfter consenting to take part in the competition, users were prompted to link a valid mobile phone number to their account, and they could elect to log in with a unique email address and password combination or use their existing Facebook credentials. Next, users created their LezParlay public profile, which included a username of their choice and their sexual identity, age group, relationship status, and pronouns. Users also had the option of uploading a profile photo or Bitmoji to represent them; entering a brief textual self-description; and connecting their Facebook, Twitter, and/or Instagram accounts so that other players could learn about them. Following account creation, players were directed to a home screen that displayed a timer counting down to the close of the current round as well as buttons to play the current round, browse player profiles, submit and vote on the questions to be parlayed in future rounds, view round winners and leaderboards, edit public profile, and change account settings. The specifics of round play and the format of the detailed results  delivered at the end of each round are detailed in official testers.There was no upper limit on the number of SMW who could take part in LezParlay, and new players were accepted on a rolling basis throughout the competition. We aimed to recruit a minimum of 1200 LBQ women to sign up during the first 2 monthly rounds to ensure that meaningful and stable sexual identity– and age group–specific actual norms for drinking and coping behaviors could be delivered in intervention rounds 3 and 4. From this larger pool of players, 500 drinkers were recruited to participate in a LezParlay evaluation study (RCT) during the third month of play. Acting as baseline (T1) for the RCT, round 3 featured questions about alcohol use, stigma experiences, and a group of nonhealth-related control questions submitted by players. Upon submitting answers to alcohol-related questions in round 3, players were covertly screened for evaluation study eligibility based on their answers  as well as their geolocation and the number of previous rounds played. Those who played at least one previous round, were in the United States, and reported drinking alcohol on 3 or more days per week or having 3 or more drinks on their peak drinking occasion were invited to take part in the evaluation study at the end of the round. Interested potential participants advanced to an informed consent screen that explained that the goal of the study was to evaluate the impact and format of detailed results received in LezParlay and gather player feedback to inform the next version of the competition. The information further detailed that participation in the evaluation study simply involved playing and viewing detailed results in subsequent rounds and completing a brief feedback survey at the end of the competition. Participants could earn up to US $40 in electronic gift cards of their choice to play subsequent rounds and complete the feedback survey. Those who checked a box indicating that they understood what the study participation entailed and desired to participate were welcomed into the study as LezParlay fixed to open to their randomly assigned feedback topics regardless of the door they selected in rounds 3 and 4. Upon completing the feedback survey at the end of the competition, participants were debriefed regarding the study’s research questions and the fixed sequences of health or control feedback they were randomized to receive in rounds 3 and 4 of the competition.The web app’s Qualtrics integration ensured that Qualtrics Research Suite’s automated randomizer, commonly used in RCTs evaluating psychosocial interventions ,41,55, cAll players taking part in the third round of LezParlay estimated the drinking behaviors of the typical same-sexual identity player in their age group during the previous 2 months, reporting on their perceptions of the typical player’s (1) maximum number of drinks consumed on a single occasion, (2) average number of drinks consumed per occasion, and (3) average number of drinking days per week . PlayersPlayers taking part in round 4 of the competition were prompted to think about how other players deal with stress and sexual minority stigma and asked to estimate the percentage of time  a typical player in their sexual identity and age group tried to feel better during the past month by (1) drinking alcohol; (2) taking a drug; (3) meditating, using relaxation techniques, or exercising; and (4) talking to a close other or mental health professional. Players were then prompted to think about how they themselves dealt with stress and stigma and responded to parallel items. The actual norms variably delivered to evaluation study participants in PNF at the end of rounds 3 and 4 were derived by computing the actual average response of all players submitting responses in each sexual identity and age group.Participants randomized to receive PNF on control topics received detailed results for nonhealth-related topics in round 3  and round 4 . At sign up, all players reported their sexual identity, relationship status, and age group. Upon enrolling in the evaluation study, participants also reported their race, ethnicity, and actual age in years. The feedback survey at the end of the competition prompted the study participants to rereport their relationship status and sexual identity.As described previously, perceived drinking norms and alcohol-use behaviors were assessed organically in competition rounds 3  and 7  by items modeled after Baer’s quantity, frequency, and max measure  in combiInterpersonal stigma exposure was also assessed at baseline (round 3) and follow-up (as a replay bonus topic in round 7). Players guessed about the stigma experiences of other players and reported on their own stigma experiences over the previous 2 months. Stigma-related norms were not corrected in the competition, and these perceptions were only assessed to make sense for players to report on their own recent interpersonal stigma exposure  at the same time that they were reporting on their alcohol use and negative consequences in the game. Players’ recent exposure to severe interpersonal stigma was assessed by their responses to 2 items: (1) “During the past 2 months, how many times have you been physically harmed due to your sexual identity?” and (2) “During the past 2 months, how many times have you been verbally harassed or threatened (online or in person) due to your sexual identity?” Associations between pairs of stigma items at each timepoint are expected, and we anticipate combining responses to derive severe interpersonal stigma scores.Data from Google Analytics and the app’s back end will allow us to examine the total number of players who signed up to participate in the LezParlay competition in the absence of traditional study participation incentives; identify the promotional channels that brought them to the app; and detail the players’ demographic characteristics, states of residence, average number of log-ins, and number of rounds completed.Feedback surveys prompted study participants to rate numerous aspects of the competition  on Likert-type scales ranging from did not like at all (0) to liked very much (5).yes in response were asked to enter text describing the perceived benefits.A single yes or no item asked participants whether they felt that participating in the LezParlay competition was psychologically beneficial. Those selecting A final free-response item asked participants to share recommendations they had for improving the competition and describing the features they would like to see in the next version.This project was funded by the National Institute on Alcohol Abuse and Alcoholism in April 2018. The institutional review board approval was granted in August 2018, and the LezParlay app was developed per approved protocol between August 2018 and November 2018. The competition launched in January 2019, and the collection of all efficacy and feasibility data was completed in September 2019. A total of 2677 LBQ women participated in the LezParlay competition, with 500 LBQ drinkers recruited into the efficacy trial. Data cleaning and analysis were delayed by several months because of COVID-19–related delays and is underway as of January 2021. The results are expected to be published in summer 2021.An intent-to-treat approach will be used to examine LezParlay treatment effects at 2 and 4 months postintervention on 4 outcomes: composite alcohol use, estimated number of drinks per week, peak number of drinks on 1 occasion, and number of negative alcohol-related consequences. Preliminary analyses will examine potential biases related to attrition and missing data ,112, insAt 2 and 4 months after the delivery of treatment PNF, participants in both conditions receiving treatment PNF on alcohol use  are expected to report reduced drinks per week, peak drinks, and negative consequences relative to those in the control PNF condition. Furthermore, participants in the alcohol+coping condition are expected to exhibit larger reductions in their alcohol use and negative consequences at postintervention follow-ups than participants in the alcohol-only PNF condition. Multilevel models MLMs ,114) wit wit114])Tests of mediation will examine whether perceived drinking norms at the 2-month follow-up mediate relationships between condition and alcohol-use outcomes at the 4-month follow-up. PROCESS bootstrap tests ,116 willd=0.22 [d=0.46 [P<.05, and an effect size of Cohen d=0.30 indicate a sample size of 375 (125 participants in each condition) to be sufficient to detect small-to-medium effects using repeated measures MLMs  as well as tests of mediation and moderation. Thus, our sample size of 500 will allow us to detect modest effects with even 30% attrition.Informed by previous research examining the effects of web-based alcohol PNF on changes in normative perceptions and drinking in other populations , th [d=0.46 ), power Descriptive statistics will allow us to assess SMW’s level of interest in the LezParlay competition and engagement with the app , recruitment origins , acceptability , and perceived psychological benefits . Qualitative text entry responses to items assessing the perceived benefits of the LezParlay competition and improvements or features requested for the next version will also be coded by theme or category using a generic inductive qualitative coding approach .Joining the growing number of gamified health interventions being developed for sexual minority men and youth -126 and This hybrid trial also follows recent recommendations for improved design and evaluation of social norms–based health interventions , as bothThe examination of demographics in terms of both LezParlay engagement and moderators of intervention efficacy will identify the groups of SMW most engaged and impacted by this strategy. Furthermore, as previous work with SMW has not examined potential interactions between perceptions of sexual identity–specific drinking norms and experiences with violence or harassment because of sexual minority status, examining the potential interplay between these established predictors of drinking allows this project to make a significant contribution to the larger sexual minority health literature. Similarly, as normative feedback interventions have not been widely considered as potential strategies for reducing problematic substance use or other health-risk behaviors among members of stigmatized health disparity populations, determining whether minority identity–based violence and harassment makes PNF more or less efficacious is a critical research question that may also carry intervention development implications for these populations .minimally viable product taking the form of an extremely low-cost, progressive web app designed and coded by the first author, who is a member of the target population. Thus, representing the preliminary step in a larger program of LezParlay-gamified PNF intervention research, findings from this trial will not speak to the feasibility or efficacy of delivering the intervention through a more sophisticated and polished native smartphone app that would likely be more desirable and user-friendly and thus better equipped to attract and retain LBQ women. In addition, PNF is the only intervention component featured in the initial version of LezParlay and, furthermore, only static descriptive norms for drinking and coping are corrected. Findings from this study may suggest high feasibility for the gamified approach and stereotype challenge framing , but treatment PNF fails to meaningfully reduce drinkers’ consumption and negative alcohol-related consequences relative to control. In this event, the stereotype challenge concept and game mechanics may be retained; however, future versions of the LezParlay app might expand PNF to correct additional types of alcohol and coping norms  and/or deliver additional intervention components such as skills training around healthy coping strategies and/or local alcohol treatment information .Funded by the National Institutes of Health’s exploratory/developmental R21 grant mechanism, the goal of this initial trial is to evaluate the feasibility and efficacy associated with the LezParlay competition as a The intervention’s real-time, organic generation of actual norms from LezParlay players’ round-specific reports of their behaviors represents both a point of innovation and a potential limitation. Foremost, this approach diverges significantly from traditional PNF interventions that derive actual drinking norms from an existing data source or a separate norms documentation study conducted before the recruitment of the intervention sample -22. As PAnother limitation pertains to this study’s organic assessment of alcohol outcomes and some moderators within the rounds of the competition. Although this is a major strength, in that it eliminates the demand characteristics that often plague transparent alcohol intervention studies and substantially decreases trial costs, this also meant that the key constructs could only be assessed by a few items, and the language of the items could not be too formal or clinical in tone. Although Baer’s frequency, quantity, and max measure  fit wellA final limitation pertains to this study’s narrow focus on the direct effect of PNF treatment on alcohol-related outcomes. Norms for other health behaviors  were also corrected within the broader competition; however, the RCT was not designed to examine potential PNF-related changes in these behaviors. Similarly, players were expected to overestimate several undesirable stereotypical behaviors among same-sexual identity peers in nonintervention rounds of the competition . Although outside the scope of this initial trial, revealing and reinforcing true norms for these experiences and attitudes in LezParlay may carry psychological benefits for SMW partaking in the competition . Thus, assessing pre-post competition changes in these constructs and evaluating the extent to which challenging negative stereotypes through the larger LezParlay competition might buffer stigma-related processes and thereby reducing drinking and improving other health outcomes remain to be critical next steps in the larger program of research.incognito, gamified intervention approach is uniquely oriented toward engaging and preventing alcohol-related risks among LBQ community members who may view their heavy drinking as normative and not in need of change because of the visibility of alcohol consumption in LBQ community spaces. Thus, this intervention strategy diverges from and is intended to complement more intensive intervention programs being developed to meet the needs of SMW already motivated to reduce their consumption and those seeking culturally tailored treatment for alcohol-use disorder and comorbid mental health problems [This hybrid trial will examine the efficacy and feasibility of an innovative, culturally tailored, evidence-based alcohol intervention for LBQ moderate-to-heavy drinkers, thereby narrowing the disparity in alcohol intervention research and practice. This problems ,41.