Despite its reputation as a radioresistant tumour, there is evidence to support a role for radiotherapy in patients with melanoma and we summarise current clinical practice. Melanoma is a highly immunogenic tumour and in this era of immunotherapy, there is renewed interest in the potential of irradiation, not only as an adjuvant and palliative treatment, but also as an immune stimulant. It has long been known that radiation causes not only DNA strand breaks, apoptosis, and necrosis, but also immunogenic modulation and cell death through the induction of dendritic cells, cell adhesion molecules, death receptors, and tumour-associated antigens, effectively transforming the tumour into an individualised vaccine. This immune response can be enhanced by the application of clinical hyperthermia as evidenced by randomised trial data in patients with melanoma. The large fraction sizes used in cranial radiosurgery and stereotactic body radiotherapy are more immunogenic than conventional fractionation, which provides additional radiobiological justification for these techniques in this disease entity. Given the immune priming effect of radiotherapy, there is a strong but complex biological rationale and an increasing body of evidence for synergy in combination with immune checkpoint inhibitors, which are now first-line therapy in patients with recurrent or metastatic melanoma. There is great potential to increase local control and abscopal effects by combining radiotherapy with both immunotherapy and hyperthermia, and a combination of all three modalities is suggested as the next important trial in this refractory disease. When larger daily doses are administered, this is termed hypofractionation. Early clinical series reported up to double the complete response rate when more than 4 Gy per fraction was delivered  and formon size) , the weaA particular advantage of radiation over surgery is in the primary therapy of choroidal melanoma, where irradiation with protons can avoid enucleation and achieve local control rates exceeding 95% and a 5-year survival similar to surgery , 8. RadiPostoperative irradiation of the primary site is not standard practice as increasing awareness of melanoma has led to the diagnosis of earlier stage tumours and wide local excision can achieve 95% local control rates. Adjuvant irradiation should be considered in cases of invasive melanoma with positive histological margins despite optimal surgery or desmoplastic histology with margins <1cm where reresection is not feasible and/or with extensive neurotropism . A singlMucosal melanoma carries a worse prognosis than the cutaneous form and the benefit of adjuvant radiotherapy has been controversial. A meta-analysis of over 1593 patients with head and neck mucosal melanoma from 12 studies reported a highly significant hazard ratio of 0.51  in favour of PORT, again with no survival advantage . The widRadiotherapy in patients with melanoma is most frequently delivered in the palliative setting, particularly for nodal, satellite, and in-transit metastases that are unresectable or have progressed despite systemic therapy, and for the management of brain metastases. Historically, whole brain radiotherapy (WBRT) was standard practice, but the QUARTZ trial has shown that patients with poor performance status and short life expectancy do not benefit more from WBRT than from steroids alone . Due to The potential neurocognitive deficits , fatigue, and alopecia along with the lack of survival benefit associated with WBRT are the compelling rationale for radiosurgery for brain metastases. Stereotactic radiosurgery (SRS) is the terminology for the very precise delivery of high dose irradiation to small volumes in one to six sessions (fractions) with a steep dose fall-off outside the irradiated volume. SRS is now well established as a nonsurgical treatment alternative for brain metastases when histological confirmation is not required, there is no mass effect, or the lesion is not surgically accessible. A series with over 300 melanoma brain metastases reported one year local control rates of 91% following radiosurgery, not inferior to other histologies . HypofraThere is a growing momentum behind stereotactic body/ablative radiotherapy (SBRT or SABR) for the control of oligometastases (up to five metastases at less than three metastatic sites). Local ablative irradiation is an attractive noninvasive and relatively nontoxic option for the management of oligorecurrence , or oligoprogression in the case of a mixed response to defer the next line of systemic therapy. Typical SBRT fraction sizes range from 9 to 20 Gy and retrospective series including melanoma patients have shown the utility of this therapeutic modality . A phaseRadiation induces not only single- and double-strand DNA strand breaks resulting in apoptosis and necrosis, but also immunogenic modulation. Immunogenic cell death leads to the release of danger-associated molecular patterns (DAMPs) including calreticulin and ATP. Such DAMPs both recruit and activate dendritic cells to take up and to cross-present tumour antigens to naïve T cells thus initiating antitumour immune responses . Further In vitro evidence suggests a greater immunogenic potential of melanoma cells following irradiation combined with hyperthermia, culminating in increased apoptotic and necrotic melanoma cells as compared with irradiation alone [Radiotherapy can achieve good palliation of metastases; however, the overall response rate can be significantly enhanced by combination with hyperthermia. The superior local control rates achieved at two years with combined radiation and hyperthermia (46%) as compared with radiation alone 28%) in patients with recurrent or metastatic disease in the randomised trial ESHO 3-85 are proof of concept for melanoma . Heating8% in paton alone . Severalon alone –32.Since 2011, the systemic treatment options for melanoma have expanded considerably from chemotherapy with limited efficacy, to tyrosine kinase inhibitors  for patients with a B-RAF v600 mutation, augmented by a MEK inhibitor (trametinib) , and T-cIpilimumab was the first FDA-approved CTLA-4 inhibitor. Its negative regulation of T-cell activation results in an enhanced cytotoxic antitumour T-cell response. Following complete resection of cutaneous melanoma, a randomised phase III trial showed a statistically significant increase in recurrence free survival: 46.5% with adjuvant ipilimumab over 34.8% with placebo, but drug-related adverse events led to discontinuation of treatment in 52% of patients and death in 1% of patients . The PD-A different approach to enhancing the immune detection and destruction of cancer cells is through vaccination . SeveralCurrently, the main radiation oncology research focus in patients with melanoma is the development of safe and efficacious combined modality treatments. There have been some very serious toxicities including death following radiosensitisation by concomitant B-RAF TKi with SBRT , 47 and The published studies of ipilimumab combined with irradiation have been recently summarised ; howeverImmune therapies have very long half-lives and it is inevitable that the immune response will have been initiated and still be active at the time of radiosurgery if immunotherapy has already begun. A randomised trial without immunotherapy in the control arm is now unlikely in recurrent or metastatic melanoma; thus, patient data registries such as the ongoing German radiation oncology association (DEGRO) ‘TOaSTT' registry, established to collect toxicity data from patients treated with targeted therapies within 30 days before or after cranial or extracranial radiosurgery, will be valuable to ascertain the toxicities seen in practice.10 is more immunogenic than conventional fractionation and that fraction sizes above 12 Gy are necessary to induce immune effects [ In vivo studies suggest a threshold dose of 12-18 Gy in a single fraction, after which larger fraction sizes are less immunogenic than smaller doses per fraction [As well as selecting the best systemic agent(s) and optimising timing of delivery , the optimal radiation fractionation must also be established. It has been shown that BED 100 Gy effects –66. In vfraction . Ipilimufraction . Interesfraction .As described above, control rates of 60-90% at one year have been reported with SRS for melanoma brain metastases. There is some potential to increase local control and a definite need to reduce distant brain relapse, hence the numerous current studies of combined modality therapies in melanoma patients with brain metastases. Combinations with B-RAF TKIs and the checkpoint inhibitors are predominantly being explored. In a retrospective study, median survival was greater in patients who received a B-RAF TKI after, rather than during or before, SRS for brain metastases but the incidence of intracranial haemorrhage was higher (10.4%) in the B-RAF TKI group as compared with 3% in the those who did not receive the targeted therapy . In a reAlso of interest in the therapy of melanoma brain metastases is temozolomide, an established radiosensitiser in primary brain tumours through its inhibition of MGMT, a DNA repair protein. In addition, temozolomide is metabolised to dacarbazine, one of the chemotherapeutics used in melanoma, and demonstrates selective depletion of regulatory T cells in vitro, thus offering, in theory, a triple-pronged attack. Results to date are modest however . Many syThe radioresistant reputation of melanoma and nihilistic perception of the prognosis of patients with metastatic disease prior to the availability of tyrosine kinase inhibitors and immune therapies are probable reasons behind the lack of recent clinical trials of radiation with hyperthermia in this disease entity. Ongoing preclinical research aims to deepen our mechanistic understanding . Recent Whilst historically the role of radiation in the management of melanoma has been questioned due to perceived radioresistance, data and clinical experience support radiotherapy for cranial and extracranial palliative indications, the adjuvant irradiation of primary cutaneous and mucosal tumours at high risk of local recurrence, and the adjuvant therapy of patients at high risk of nodal recurrence. Radiotherapy can be more effective when delivered as radiosurgery or when combined with hyperthermia and the combination of radiotherapy with immunotherapy may increase the real but elusive abscopal effects. As radiation and hyperthermia are well tolerated, and radiation and immunotherapy generally do not show additive toxicity, a future trial evaluating radiation combined with both hyperthermia and checkpoint inhibition could be important for patients with relapsed melanoma.


Exposure to potent synthetic opioids such as illicitly manufactured fentanyl (IMF) has fueled the escalating overdose crisis in the USA, particularly in the east coast. Drug checking services, which allow people who use drugs (PWUD) to learn about the contents of their drugs, remain limited and even criminalized in many states. Further, there is a persistent belief that PWUD are not willing or able to change their behaviors despite being aware of their potential exposure to fentanyl through drug use.N = 334) were recruited in Boston (n = 80), Providence (n = 79), and in Baltimore (n = 175). At the time of the survey, no legal drug checking services were available in these cities.We conducted a multi-site cross-sectional study among PWUD to assess what behaviors, if any, were employed in the case of suspected fentanyl exposure, and the correlates of engaging in harm reduction behaviors (HRB). PWUD (n = 196), 39% reported employing HRB including using less of the drug (12%) or abstaining altogether (10%), using more slowly (5%), and doing a tester shot (5%). In adjusted logistic regression models, the odds (aOR) of practicing HRB after suspecting fentanyl exposure were increased among PWUD who were non-White  and older . Daily injection , using drugs in public , using drugs alone , and experiencing multiple recent overdoses  were associated with decreased odds of practicing HRB.The majority of PWUD (84%) expressed concern about fentanyl. Among those who suspected fentanyl exposure prior to using their drugs (These data illustrate that PWUD employ a number of practices to reduce overdose risk in a context of unknown drug purity and content. Results may also guide efforts to identify early adopters of drug checking services and engage them in peer-outreach to target the most socially and structurally vulnerable PWUD, who are not reporting behavior change, with harm reduction messaging. Drug overdoses have been increasing at unprecedented rates in recent years and are now a leading cause of death and driver of reduced life expectancy in the USA . The emeGiven the unknown purity and content of illicit street drugs and the evidence that many people who use drugs (PWUD) are uncertain of their exposure to fentanyl before use , 11, manA critique of employing drug-checking approaches is the belief that PWUD do not care about their own health and are unable to make rational decisions altering their behavior even with the necessary information to do so. This notion has been refuted, most extensively in literature detailing behavior change and HIV risk among PWUD  and moren = 80) and Providence (n = 79) were recruited through syringe service programs (SSP) and harm reduction services. In Baltimore, Baltimore City Police Department, drug arrest data from 2016 was used to identify regions for street-based targeted sampling [n = 175). PWUD were eligible for inclusion if they were at least 18 years of age, spoke English, and reported the use of non-medical heroin, fentanyl, cocaine, methamphetamine, or opioid pills in the past 30 days.The Fentanyl Overdose Reduction Checking Analysis Study (FORECAST) is a multi-site study measuring the validity and feasibility of drug-checking tools. The comprehensive methodology for the FORECAST study are described elsewhere , 33, 34;sampling  to recruAt the time of the study, FTS were not being widely promoted or distributed in Boston, Providence, or Baltimore. All study participants provided informed oral consent and were compensated $25 USD upon completion. The study was approved by the Johns Hopkins Bloomberg School of Public Health and the Rhode Island Hospital Institutional Review Boards.Sociodemographic data , knowledge and experience of fentanyl, and history of drug use, overdose, criminal justice interaction, and use of health and harm reduction services were ascertained. Age was analyzed as a dichotomous variable  and as a continuous variable  in regression analyses. In regression, race was also considered a binary term (White/non-White) due to small sample sizes of Hispanic and mixed- or other racial groups. Polydrug use was analyzed as a binary variable denoting the use of the median number of 4 or more substances in the past 6 months. The usual setting of drug use was also considered as a binary variable, with public use inclusive of public  and semi-public spaces , compared with private use in a home setting.N = 334) were first asked whether they had ever used a drug they thought had fentanyl in it (yes/no); those who had (n = 256) were then asked what they did in response. Fifty-seven participants suspected contamination after using; of the remaining 199, 196 answered questions regarding behavior change and were included in this analysis. Survey response options for behavior change were adapted from prior studies in Rhode Island [use as originally intended (nothing different); use less than originally intended; go slower; do a tester; sniff/toot/smoke instead of shooting (not inject); use with someone else around; share/take turns when I use; use the drug somewhere safe (like where someone could find me); throw them out; give them away; sell them; get naloxone/Narcan; tell the dealer/supplier; get money back from dealer/supplier; beat up dealer/supplier; tell other people who use the same dealer/supplier; stop going to that dealer/supplier; other (specify). Categories were collapsed and a dummy variable was created classifying those responding “use as originally intended” as “no behavior change” and those reporting a change to their method of use or acquisition to minimize exposure risk  as practicing HRB. We additionally included behaviors that amounted to total, primary prevention  as HRB in this setting, given that they were adopted specifically in response to suspected adulteration. Responses among participants who selected “other” were reviewed and coded as HRB , no behavior change , or excluded .To define and measure the primary outcome, practicing HRB, participants  in the sample were reported and correlates were modeled using multivariable logistic regression with clustered variance estimation to adjust for differences by site. We did not analyze the likelihood of behavior change according to SSP attendance in subsequent regression models due to potential bias, as the recruitment of participants occurred through SSP in two of our sites . If the distribution of HRB differed by any other correlate at a level of p < 0.1 using either χ2 or bivariate regression (with clustered variances), they were considered for inclusion in the final adjusted model; only the subset of items informed by existing literature and with demonstrated statistical association with HRB were included to achieve the greatest parsimony given a small sample size. We excluded “using drugs alone” from regression models due to confounding with one of the behaviors defining the outcome (“use with others”). We conducted a sensitivity analysis with additional adjustment for fentanyl preference (not shown); however, given the lack of significant influence on the outcome and the absence of other qualitative or substantial quantitative changes to model output, we omitted this variable from the final model. Analyses were conducted using Stata S/E 14.2 .We compared the frequency of reported HRB by city and across sociodemographic, drug use history, criminal justice, and health-related correlates of interest using Pearson’s p = 0.007) as did Black, Hispanic, and other non-Whites compared to White participants .Participants were predominantly male (59%) and White (47%), with age ranging from 20 to 65 years (mean 42 years). The majority of the population was homeless (69%) and lacked stable employment (88%). Sociodemographic characteristics of the sample are shown in Table p = 0.003). Significantly fewer participants who used drugs in public or semi-public spaces reported HRB (30%) compared with those using in private settings .The most prevalent reported drugs used were heroin (93%), cocaine (87%), crack (73%), alcohol (63%), and benzodiazepines (53%), and 53% of the sample reported the use of more than four substances in the past 6 months. Employing HRB was less common among people who inject drugs (PWID), daily (29%), or at all in the past 3 months (44%), relative to those who did not inject . A suggestive dose-response pattern was detected with reducing the prevalence of behavior change correlating with greater number of overdose events within the past year (p = 0.074).The majority (71%) of the sample had experienced overdose, and 48% had done so within the past year. The reported prevalence of HRB was lower among individuals who had a history of overdose than among those who had not . When treatment was examined by category, we observed significantly higher reported HRB among PWUD with recent counseling, support group attendance, or mental health services, relative to those without .Practicing HRB was higher among participants receiving drug treatment in the past 6 months relative to those who did not, though this was marginally non-significant . Participants expressing a preference for fentanyl (37%) had a significantly lower frequency of reporting HRB compared to those who did not .The vast majority of PWUD expressed concern about fentanyl (81%), and this was higher among those who practiced HRB (94%) relative to those who did not . Participants who were not White had twice the odds of reporting HRB during last suspected fentanyl exposure . Drug use characteristics that were significantly associated with a decreased likelihood of practicing HRB in response to fentanyl exposure were daily injection  and using in public .Adjusted correlates of reporting behavior change are shown in Table 3This study characterized the adoption of a range of harm reduction behaviors among PWUD after suspecting fentanyl exposure in three cities with extremely high rates of fentanyl-related morbidity and mortality and limited exposure to drug checking services. Eighty-four percent of PWUD were concerned about fentanyl in their drugs, and 39% reported practicing HRB, such as doing a tester shot or using drugs more slowly or not at all when they perceived their drugs to be adulterated. These data illustrate that PWUD are concerned about their risk of exposure and employ harm reduction approaches in a context of unknown drug purity and content. Our findings can inform messaging around safe drug use in the context of fentanyl-adulterated markets with and without drug checking.We observed a number of important factors associated with HRB. Non-White participants had twice the odds of practicing HRB in response to suspected fentanyl exposure. This is consistent with some prior work illustrating higher adoption of protective behaviors like using a new needle  , 38 amonunder review). However, it is important to note that even among the minority of individuals who prefer fentanyl in our study, some (18%) still also reported HRB, highlighting that PWUD may make decisions based on their perceived risks, risk setting, or scenario, rather than their preferences. Substance use and misuse literature highlight lack of perceived risk due to factors like impulsivity, lack of appreciation of consequence, and susceptibility to peer norms as a driver of adopting high-risk practices in young adulthood [Younger individuals less frequently practiced harm reduction around suspected exposure. This is consistent with literature detailing changes in opioid initiation and use by birth cohort and may be explained by differences in drug content and availability . For exadulthood . Data hadulthood . This madulthood .We detected several markers of extreme vulnerability to overdose that were also associated with lower employment of HRB, including frequent injection drug use and use in public or semi-public spaces. This finding is consistent with the theory that forms of social and structural disadvantage interact to produce a risk environment that can limit the capacity of PWUD to reduce drug-related harm , 45. ThiThe proportion of PWUD practicing HRB in these three cities is nearly identical to that reported in a recent study assessing responsive behavior change after distribution of FTS in North Carolina . After aThis study has important programmatic implications for health and outreach services. As drug checking programs are increasingly decriminalized in many contexts, these data can help to guide strategies to target PWUD as such programs are enacted. Individuals who are more concerned about fentanyl and already practicing harm reduction may be rapid adopters of expanded services and may be engaged in peer outreach and training efforts to increase knowledge, use FTS and other tools, and behavior change among fellow PWUD. This will enable PWUD who are already practicing HRB do so more in a more informed manner and can empower and engage them in peer-led overdose prevention programs. Given that the majority of PWUD reported are also selling drugs in the past 3 months, strategies to engage dealers in drug checking and other harm reduction practices may also be appropriate  in this Our findings should be interpreted in light of study limitations. We rely on self-report to capture whether and how PWUD enact behavior change and as such data are subject to biases due to either poor recall or systematically inflated responses based on the desirability of responses. However, we note that the use of anonymous, computer-assisted surveys likely reduces the impact of the latter , 60. TheFinally, this work raises a number of important questions for future research. As previously noted in the literature, harm reducing behaviors are not simply determined by access to knowledge of contaminants, but multiple interacting and often upstream contingencies , 44. ThoThis study demonstrates that in three hotspots of the fentanyl epidemic, there is a will and a way to practice harm reduction when faced with fentanyl exposure and risk of death. These data complement the existing body of evidence refuting the notion that PWUD are indifferent to the value of their lives and unable to enact rational decision-making and provide channels for future research to assess how to increase HRB among PWUD at high risk of overdose. Providing PWUD with tools to protect themselves is an ethical imperative in the era of widespread adulteration of a range of substances and rampant overdose fatalities. Access to harm reduction education and services such as drug-checking should be broadly expanded in these settings and beyond.


Skin architecture and function depend on diverse populations of epidermal cells and dermal fibroblasts. Reciprocal communication between the epidermis and dermis plays a key role in skin development, homeostasis and repair. While several stem cell populations have been identified in the epidermis with distinct locations and functions, it is now recognised that there is additional heterogeneity within the mesenchymal cells of the dermis. Here, we discuss recent insights into how these distinct cell populations are maintained and coordinated during development, homeostasis, and wound healing. We highlight the importance of the local environment, or niche, in cellular plasticity. We also discuss new mechanisms that have been identified as influencing wound repair and cancer progression. Different epidermal and dermal cell populations not only contribute to tissue homeostasis but also to wound repair and underlie tumour heterogeneity and progression.Skin cellular behaviour and fate are regulated by interactions at the level of single cells, termed microniches, as well as on higher scales.Individual niche components and transcription factors can be identified that significantly affect stem cell maintenance and behaviour.Cell population-specific mechanisms regulate cellular plasticity and behaviour during wound repair and tumour development.Epidermal stem cell plasticity is achieved by a loss of lineage specificity during wound healing, and may become permanent in skin cancer. The skin consists of two layers, the upper epidermis and the lower dermis, which are separated by a basement membrane, and harbours specialised structures such as hair follicles (HFs) and sweat glands . The epidermal papilla (DP) , which is connected close to the HF bulge and is responsible for piloerection  composition (reviewed in (DP) see  at the bIn this review we highlight recent advances in dissecting different cell subpopulations in the epidermis and dermis. We discuss how epidermal and dermal cells interact with each other during homeostasis, wound healing, and cancer.+ and CD34+/Krt15+) bulge to the upper HF (Gli1+ and Lgr6+) and junctional zone (Lrig1+) (interfollicular epidermis (IFE), which is located between HFs and comprises the largest pool of keratinocytes in the skin Box 1In the IFE and HF maintenance of distinct SC populations relies on a tight interplay of intrinsic factors and extrinsic factors that define the cellular microenvironment or niche . The comKey intrinsic factors include SC-specific regulation of metabolism, gene expression, and chromatin arrangement. For example, HFSCs produce more lactate than other epidermal cells, suggesting that differences in cellular glycolytic metabolism influence SC activity Within the niche interactions between neighbouring cells are important regulators of cell behaviour. Upon epithelial SC ablation neighbouring cells are able to replace niche SC even if they are committed progenitors Extrinsic niche factors can act on a single cell level and are either produced by the SCs themselves Physical parameters such as tension, pressure, or temperature contribute to the cellular microenvironment, are sensed by distinct cell populations, and influence cellular fate. For example, in epidermal SCs a mechanosensory complex consisting of emergin, nonmyosin-IIA, and actin affects gene transcription and lineage commitment It remains to be determined whether force-induced changes in chromatin structure are transient or if SCs functionally adapt and memorise the stress response. It was recently shown that epithelial SCs exhibit a memory of previous inflammation upon tissue damage by maintaining chromosomal accessibility at key stress response genes, promoting faster transcription Epidermal β-catenin activation not only results in cell-intrinsic effects but also alters the behaviour of neighbouring cells It is conceivable that many more specialized SC subpopulations, maintained and defined by a complex array of niche factors, will be discovered; indeed that all epidermal cells in contact with ECM have SC properties.Alt-text: Box 1Multiple epithelial stem cell (SC) populations have been shown to contribute to skin homeostasis. In mice a highly diverse pool of SCs has been identified in the HF, ranging from the lower HF (Lgr5(Lrig1+) A. In undHF placodes, which are characterised by expression of adult SC markers such as Sox9 niches such as the HF bulge, during hair bud development, SC specification is achieved by asymmetric cell division, differential levels of Wnt signalling, and the response to sonic hedgehog (SHH) of basal and suprabasal cells isthmus and sebaceous gland  cell division of a homogeneous population of keratinocytes, referred as the neutral drift modelparakeratotic scale IFE that is not associated with HFs, and the other to the orthokeratotic interscale IFE located close to the HFs, each being generated and maintained by a different pool of basal cells + cells contribute to the interscale but not to the scale IFE Beside epithelial cells (keratinocytes and Merkel cells), the adult IFE harbours melanocytes and immune cells (Langerhans cells and γδT-cells). Until recently the organisation of epithelial SCs within adult mouse IFE was a matter of some debate. Initial Lineage tracing experiments have revealed that while cell clones associated with HF show a rapid increase in size during the HF growth phase, distant clones cycle more slowly, yet can be mobilised upon tissue injury cellular states.Single cell transcriptomic analysis of mouse dorsal epidermis and cultured human epidermis has identified at least two distinct IFE SC transcriptional signatures, even though there appears to be a single terminal differentiation programme To gain further insights into the proliferative dynamics of epidermal cells with age, in recent yearsclonal analysis has been applied to human epidermis by making use of sunlight induced mutations in cancer-associated genes, such as p53, as markers pericyte, and immune and endothelial cell populations that dynamically change with age and influence the properties and cellular behaviour of the overlying epidermis cell lineages that differentiate into distinct subpopulations and contribute to the dermal layers Beside its role as an ECM-rich scaffold, the dermis harbours highly diverse fibroblast, During development, gene expression in dermal fibroblasts is highly dynamic and there is a swift change in dermal fibroblast behaviour on the tissue scale during dermal maturation In vitro studies suggest that there is not a common nonproliferative state in fibroblasts but that it is rather an accumulation of different states that are actively maintained While the nature of the switch between fibroblast proliferation and ECM production is unknown, gene expression analysis of neonatal (proliferative) and aged (nondividing) dermal fibroblasts suggests that it is controlled by epigenetic changes at the chromatin level Reciprocal signalling between epidermis and dermis plays a key role in skin development, homeostasis, wound repair, and cancer. A prime example of tight temporal and spatial regulation involves the DP fibroblasts at the base of the HF and the overlying HF bulb  inhibitors creates distinct microniches along the epithelial–mesenchymal interface in the DP The HF bulge SC niche harbours a distinct combination of ECM components. Bulge cells have been shown to create microniches for APM cells by depositing specific ECM components including nephronectin in the basement membrane In contrast to the HF, in the IFE the spatial and temporal coordination of epidermal–dermal interactions has not been defined at single cell resolution. However, in tail skin, leucine-rich repeats and immunoglobulin-like domains protein 1 (Lrig1) expression is selectively upregulated in dermal fibroblasts that underlie the interscale epidermal compartment, pointing to different dermal niches in the interscale and scale One of the key pathways of epidermal–dermal communication is via canonical Wnt signalling. Activation of Wnt/β-catenin in basal keratinocytes of adult mouse epidermis induces a rapid increase in fibroblast proliferation and stimulates ECM remodelling + DP fibroblasts maintain their identity in cell culture It seems likely that, similar to the epidermis, dermal subpopulations are maintained by a complex combination of extrinsic and intrinsic factors . On the panniculus carnosus muscle, a structure that is absent in human skin , while Lrig1+ cells develop trichoadenomas (a rare benign follicular tumour with cornifying cysts) and the Lgr6+ population gives rise to dermatofibromas within the IFE + cells in the IFE and HF infundibulum can initiate basal cell carcinoma (BCC) formation Similar to wound healing, different epidermal and dermal populations contribute to cancer in different ways . This isin vivo live imaging approach, it was observed that healthy epithelial cells routinely recognise, surround, and eliminate mutant cells to restore tissue homeostasis, revealing an innate cellular ability to prevent over proliferation and tumour initiation During tumour initiation, mutant epidermal cells have the ability to engage nontransformed  cells via paracrine signalling, such as Wnt ligand secretion, to induce aberrant growth of the whole tissue tumour stroma, comprising all nontransformed tissue components associated with a tumour, can have both tumour-promoting and -inhibitory effects. Besides endothelial and immune cells a major component of the microenvironment are cancer-associated fibroblasts (CAFs), which play an important role in the evolution of solid tumours. Similar to myofibroblasts, CAFs seem to originate from different mesenchymal populations, ranging from normal fibroblasts and MSCs to transdifferentiated epithelial and endothelial cells. In contrast to normal fibroblasts, CAFs either reside within the tumour margin or infiltrate the tumour mass and show increased proliferation, migration, ECM deposition, and secretion of growth factors and other ECM modulators .In recent years our appreciation of epidermal and dermal cell heterogeneity has grown, together with a realisation that cells are capable of exhibiting plasticity and changing fate through dedifferentiation and Outstanding QuestionsHow is the behaviour of cell populations in the epidermis and dermis coordinated during wound repair and tumour development on the tissue scale?What defines and distinguishes cellular state and cellular type in the skin?What are cell type specific and universal mechanisms for establishing and maintaining cellular identity and plasticity in the skin?What are the key changes that create a pro-oncogenic environment?How do distinct epidermal and dermal cell populations contribute to tumour heterogeneity and progression?What are the dynamics and organisation of microniches in the dermis and epidermis?As more single cell gene expression profiles become publically available and tools to make those datasets readily accessible to researchers with a biological background improve


In developed countries heart failure represents a heavy epidemiological burden, with a prevalence of 1–2% of the adult population, rising to ≥ 10% among people with > 70 years of age . In patiRecommendations for the Evaluation of Left Ventricular Diastolic Function by Echocardiography are in fact explicit in warning that none of the echocardiographic indices should be used in isolation, as some measurements may fall in the normal range despite the presence of diastolic dysfunction due to the several hemodynamic factors that may affect each signal [HFpEF demands a multi-parametric echocardiographic assessment. In this retrospective analysis of data from a previous observational study on stable hypertensive patients, Del Rios et al. considered a single echocardiographic variable in order to assess concordance of EPs and cardiologists in the diagnosis of diastolic dysfunction: medial/lateral averaged mitral annular tissue doppler (TDI) protodiastolic velocity. Other authors have recently investigated the possibility of diagnosing HFpEF by EPs receiving a dedicated training in cardiac bedside ultrasound , or suggh signal .tau although it is not entirely governed by relaxation [TDI acquisition can be technically challenging. Althought E/E’ is key to assess diastolic dysfunction with good reproducibility and reliability, a series of technical issues can affect the accurate measurement of mitral annular TDI. Protodiastolic mitral annular velocity (TDI E’), the cornerstone of diastolic dysfunction diagnosis, reflects mitral annulus motion that precedes filling; it correlates well with invasive measures of the time constant of myocardial relaxation laxation . There alaxation , 14. AllTDI interpretation conceals several pitfalls that should not be neglected. There are a variety of pathological conditions where TDI E’ could be normal in presence of altered cardiac dynamics: constrictive physiology , patientLimited bedside ultrasound diagnostic tests are accurate in specific patient populations, and only when interpreted in tight conjunction with clinical data. One of the basic assumptions of Focused Cardiac UltraSound (FOCUS)  is that the limited ultrasound exam is sufficiently informative only when it is part of a clinical-ultrasound integrated approach , 19, notCore of HFpEF diagnosis is the demonstration of elevated left atrial pressures. Patients may exhibit diastolic dysfunction  unrelated to their respiratory symptoms, i.e. without having high left atrial pressures and congestive HF. Rather than diastolic dysfunction per se, what matters in the approach to patients with suspected acute heart failure is the finding of positive echocardiographic indices of increased left atrial pressures . These sPreliminary data on the feasibility of a proposed simplified approach to diagnose diastolic dysfunction, “more suitable for the use by emergency physicians (EPs) with limited experience in echocardiography”, have been recently reported on the Critical Ultrasound Journal . AlthougThe application of echocardiography in the assessment of diastolic function and cardiovascular pathophysiology requires advanced competences and skills (compared to FoCUS) to both acquire high-quality pictures and signals and to interpret them in the context of the acute cardiac care setting. A recent meta-analysis showed how despite the application of the guidelines for the diagnosis of diastolic dysfunction  this diaThe inherent limitations of FoCUS in the diagnosis of congestive heart failure in the ER can though be partly circumvented by means of clinically integrated multi-organ ultrasound , 26, 27.One of the pivotal rules in medicine is that the difficult path to diagnosis requires a process of integrative, systematic, acquisition of signs, symptoms and data. This is even more true when a complex operator-dependent tool is applied in critical scenarios. Echocardiography is extremely powerful in the study of cardiovascular pathophysiology, and any message supporting the concept of simplifying a complex diagnosis by studying a single echocardiographic parameter risks to be definitively simplistic and misleading, rather than just hazardous. A comprehensive approach which integrates clinical, biochemical, FoCUS, lung ultrasound, and echocardiographic findings is advised to better approach the suspected diagnosis of diastolic dysfunction causing congestive heart failure.


Wolbachia may be selected to interfere with caste determination, whilst others have discounted this possibility on theoretical grounds. We argue that there are in fact three distinct evolutionary scenarios in which maternally transmitted symbionts might be selected to influence the process of caste determination in a social hymenopteran host. Each of these scenarios generate testable predictions which we outline here. Given the increasing recognition of the complexity and multi-faceted nature of caste determination in social insects, we argue that maternally transmitted symbionts should also be considered as possible factors influencing the development of social hymenopterans.The separation of individuals into reproductive and worker castes is the defining feature of insect societies. However, caste determination is itself a complex phenomenon, dependent on interacting genetic and environmental factors. It has been suggested by some authors that widespread maternally transmitted symbionts such as  The defining feature of eusociality is reproductive division of labor  to influence caste determination in the social hymenoptera. Heritable symbionts are extremely widespread in insects and vary enormously in terms of their effects on host organisms  that the fidelity of transmission of the symbiont from parent to offspring is perfect, i.e., infected queens produce only infected offspring; (2) that only a single maternal lineage is present within a colony; (3) that any symbiont present has no interest in distorting the sex ratio of its host. In reality, each of these assumptions is violated in nature and each creates a different arena for conflict over caste determination between MTSs and social hymenopteran hosts. Here, we discuss each of these scenarios in turn and describe how they might be experimentally investigated.In many instances, the fidelity of transmission of MTSs to the offspring of their host is impressively high, and essentially all the offspring of an infected female are themselves infected  two independent evolutionary origins, rather than a single origin followed by horizontal gene transfer between taxa (Penz et al. It could be argued that MTSs have only evolved a limited number of manipulative phenotypes, i.e., cytoplasmic incompatibility, male-killing, feminization and parthenogenesis induction, during the course of tens of millions of years of evolutionary history in association with an enormous number of arthropod hosts (Engelstädter and Hurst Caste determination in social hymenopterans is increasingly recognized as a complex phenomenon, with multiple interacting causes. We suggest that heritable symbionts should also be considered as additional factors that may influence caste determination, because there are at least three distinct evolutionary scenarios that could select for caste-biasing genes in MTSs. Such symbionts are extremely widespread, easy to detect using a range of standard laboratory techniques, and all three evolutionary scenarios outlined here generate testable predictions concerning caste determination in social hymenopterans. Maternally transmitted symbionts are already renowned for their ability to influence fundamental aspects of metabolism, immunity, behavior and reproduction in their hosts and we suspect that caste determination will be no exception to this.


Polyploidy does not usually occur in germinal cells of mammals and other highervertebrates. We describe a unique example of mosaic autotetraploidy in themeiosis of a human male. Although the original observations were made in thelate 1960s, we did not publish them at that time, because we expected to detectfurther examples that could be described together. However, this did not occurand we have now decided to make the observations available to demonstrate thatpolyploidy in mammalian male meiosis can arise at a higher frequency thanexpected by random polyploidization of individual meiotic cells, by either DNAduplication or cell fusion prior to synapsis. This is the first description of apopulation of primary spermatocytes exhibiting multivalent formation atleptotene /diakinesis in human spermatogenesis, with ring, chain, frying pan andother types of quadrivalents, typical of autotetraploidy. As many of thepolyploid configurations showed apoptotic breakdown, it is likely that diploidand/or aneuploid spermatozoa would have rarely or never resulted from thismosaic autotetraploid meiosis. There is only one published report describing pairing and meiotic crossing over byelectron microscopic analysis of the synaptonemal complex of a single tetraploid primaryspermatocyte during spermatogenesis in the mouse . Later, We wish to bring to your attention a unique example of mosaic autotetraploidy in humanmale meiosis based on observations made in the late 1960s at the MRC Population GeneticsUnit in Oxford. We did not publish the results at the time in anticipation that othercases would be discovered that could be published together; regrettably, this did notoccur. Now, ~ 50 years later, given the continuing uniqueness of the observations, wewish to make them available for the historical record whilst we still can, using theoriginal chromosome photographs made at that time. These demonstrate that true polyploidmeiosis, presumably resulting from polyploidy arising in spermatogonia or spermatocytesprior to synapsis (chromosome pairing), was detected in a mosaic, single humantesticular biopsy, at an estimated frequency of ~3%.The images we present in Chrysanthemum leucanthemum (Ox-eyed daisy) shows thetypical multivalent chromosome structures of autotetraploidy  and lower vertebrate species , but is either extremely rare or non-existent in mammals. A single claim ofputative allo-tetraploidy in the red vizcacha rat , it is likely thatthe majority of multivalent bearing spermatocytes would have failed to complete meiosisand produced viable diploid sperm. Regrettably, we did not examine the DNA content ofthe patient’s sperm at the time, although the method was available to us, so were unableto conclude whether the observed mosaic tetraploid meiosis increased the proportion ofeither diploidy and/or aneuploidy in his spermatozoa.Diploid sperm are found in ~ 0.1% (range 0.05% - 0.47%) of sperm in normal fertile males observed in many of the polyploid cells is one of the mainbarriers shown to prevent progression of abnormal progenitor cells throughspermatogenesis


Bacillus subtilis has the capacity to choose between two mutually exclusive lifestyles: biofilm formation and flagellum-mediated swimming motility. Interestingly, this choice is made at the individual cell level, with bacterial cells in a population expressing genes required for biofilm formation or genes required for swimming motility but not both. Bacillus subtilis has the capacity to choose between two mutually exclusive lifestyles: biofilm formation and flagellum-mediated swimming motility. Interestingly, this choice is made at the individual cell level, with bacterial cells in a population expressing genes required for biofilm formation or genes required for swimming motility but not both. A bistable switch controls the biofilm-versus-swimming decision, resulting in an evolutionarily favorable strategy known as “bet hedging” that ensures that subpopulations of bacteria continue to grow as conditions change and/or become unfavorable. In a recent issue of mBio, J. Kampf and colleagues  reported the use of a combination of genetics and microfluidics to reveal that the interplay that occurs between the SinR and YmdB proteins underlies the B. subtilis choice between biofilm formation and swimming motility. Their report suggests that B. subtilis experiences selective pressure to form biofilms while maintaining reserve cell subpopulations with the capacity to swim away. Bacillus subtilis form biofilms, but this behavior is bistable in that individual bacteria within the biofilm can express genes for motility or genes for biofilm formation but generally not both at the same time. These bistable B. subtilis cells are programmed to form a biofilm or to swim and thereby typify a behavior known as “bet hedging”  and autolysin proteins  gene to hag, the flagellin gene, and of the cyan fluorescent protein (CFP) gene to tapA, a gene involved in biofilm formation, they were able to image expression patterns over time in individual cells in both ΔymdB and wild-type strains. Subpopulations of cells became readily detectable over time, with wild-type bacteria giving rise to cells expressing neither motility nor biofilm genes, cells expressing just motility genes, cells expressing just biofilm genes, and even a few cells expressing both motility and biofilm genes. The results of those experiments provide a glimpse into the dynamic variation within the population and the interconversion of different cell types over time.Kampf and colleagues first verified that the loss of YmdB from ymdB mutant populations did not give rise to many cells expressing biofilm-associated genes. The majority of cells expressed hag-CFP or expressed neither reporter gene, although cells expressing tap-YFP did infrequently become visible over time. Kampf et al. characterized independently isolated mutants based on biofilm restoration to the ΔymdB mutant to determine how suppressor mutations compensated for the loss of YmdB and restored biofilm formation, with the ultimate goal of gaining insight into the direct targets of YmdB that influence the activity of the SinR regulator. Among 14 mutants containing suppressor mutations in ymdB deletion strains, 12 contained point mutations within the sinR gene and 2 contained deletions of sinR and expressed biofilm genes and motility genes simultaneously. Thus, rather than finding indications of YmdB substrates that might lead to the identification of, for example, second messenger nucleotides as targets for YmdB phosphodiesterase activity, all suppressor mutations affected the expression or activity of SinR. This finding reveals that the homeostasis of SinR is the major function of the YmdB phosphodiesterase.As anticipated, ΔWhile the precise role of YmdB has yet to be determined, the work by Kampf et al. clearly establishes the close ties between YmdB function and SinR activity in regulating the bistable switch between biofilm formation and motility. Biochemical analyses of five SinR suppressor mutants with single amino acid substitutions shed additional light on the functional regions of SinR and its multimeric interactions.sinR transcripts. This may explain why suppressor mutations that affect SinR activity, rather than phosphodiesterase target molecules, were identified in this work.The phosphodiesterase activity of YmdB is important for biofilm production, and yet previous work by those authors excluded the involvement of YmdB in the hydrolysis of second messenger nucleotides . It is pymdB mutants appear to readily acquire suppressor mutations that restore biofilm formation suggests that there is selective pressure with respect to this bacterial behavior. Whatever the advantages of biofilm formation, the ability of individuals to switch and thus maintain a subpopulation of swimmers through SinR regulation provides the bet hedging that guarantees bacterial survival via the swim to freedom should conditions suddenly prove less than favorable for the more recalcitrant biofilm members.Finally, the fact that


Implementation of new programs within healthcare systems can be extraordinarily complex. Individuals within the same healthcare organization often have different perspectives on how implementation of a new program unfolds over time, and it is not always clear in the midst of implementation what issues are most important or how to address them. An implementation support team within the Veterans Health Administration (VHA) sought to develop an efficient method for eliciting an ongoing, detailed and nuanced account of implementation progress from multiple viewpoints that could support and inform active implementation of two new VHA programs.The new Prospectively-Reported Implementation Update and Score (“PRIUS”) provided a quick, structured, prospective and open-ended method for individuals to report on implementation progress. PRIUS updates were submitted approximately twice a month. Responding to the prompt “What are some things that happened over the past two weeks that seem relevant from your perspective to the implementation of this project?”, individuals scored each update with a number ranging from + 3 to − 3.In 2016–17, individuals submitted over 600 PRIUS updates across the two QI projects. PRIUS-based findings included that staff from different services reported fundamentally different perspectives on program implementation. Rapid analysis and reporting of the PRIUS data led directly to changes in implementation.The PRIUS provided an efficient, structured method for developing a granular and context-sensitive account of implementation progress. The approach appears to be highly adaptable to a wide range of settings and interventions. Implementation of new programs within healthcare systems can be extraordinarily complex.  This occIf there was a systematic and efficient way to collect and analyze data about how the implementation process unfolds over time, such a method could enable implementation leaders, organizational stakeholders and researchers to monitor implementation progress more closely, make revisions and course corrections as needed, assess the impact of any changes, and understand how local context influences the implementation process. In the midst of active implementation it is not always clear what issues are most important or how to address them, and a structured approach that drew independently upon the viewpoints of multiple individuals and organized their responses for rapid evaluation could identify opportunities for improving implementation on a continuous basis. Collecting data prospectively and longitudinally could also help maintain accuracy in the reporting of implementation-related information, as the ultimate success or failure of program implementation can introduce hindsight bias and retrospective evaluation can introduce recall bias. Several approaches already in use in mixed methods research and health services research precede and inform any new method concerned with the systematic capture and rapid analysis of implementation-related developments. For example, Rapid Evaluation and Assessment Methods (REAM) have long offered a way for researchers in diverse settings to collect and analyze data on an accelerated timetable while retaining rigor. –8 MatrixA major limitation across these methods in terms of their practical application is that they tend to place substantial demands on researchers and evaluation teams in terms of labor, time and resources, and thus have largely been applied retrospectively to gain insights into past episodes of implementation that have already happened. A need remains for a quick, structured, open-ended and longitudinal method that can capture and report on implementation progress that can be applied prospectively in ways that inform and influence implementation as it unfolds.To address this gap, a team led by health services researchers who provide implementation support for ongoing projects in the national Veterans Health Administration (VHA) system in the United States sought to develop a new, efficient method for eliciting an ongoing, detailed and nuanced account of implementation progress from multiple viewpoints. This implementation team hypothesized that this new approach, called the Prospectively-Reported Implementation Update and Score (PRIUS), could generate findings and insights in a timeframe and format that supported and informed active implementation of two new VHA programs.In 2016 and 2017, the Precision Monitoring (PRIS-M) QUERI  based at the Roudebush VA Medical Center in Indianapolis, Indiana was charged with supporting and studying two different quality improvement (QI) projects in VHA. The 7-person implementation team included three doctoral-level implementation scientists who collectively had been working in implementation science for over 30 years in VHA; a masters-level research nurse; a masters-level program manager; a project manager; and a research assistant. The implementation team was led by the senior implementation scientist and held regular weekly meetings, and was a formal part of the local QUERI program in Indianapolis; the QUERI program’s mission is to ensure that research gets used effectively to improve care for veterans. This research received institutional review board (IRB) approval.The first QI project was a new “TeleSleep” program implemented in 2015–16 at the Richard L. Roudebush VA Medical Center in Indianapolis, Indiana that brought the staff of the local Sleep Medicine and Telehealth services together for the first time. The prospective one-year QI project targeted Veterans newly diagnosed with Obstructive Sleep Apnea (OSA), compared a TeleSleep protocol to usual care, and operated from October 2015 through September 2016. In the TeleSleep protocol, Roudebush VA Sleep Service staff provided Positive Airway Pressure (PAP) set up , mask fitting, and education, and the Roudebush VA Telemedicine service followed patients using a TeleSleep protocol designed to adhere to Telehealth requirements and provide state-of-the-art sleep medicine care. The second QI project was a Tele-robotics Stroke Rehabilitation QI program designed to be implemented at four pilot sites around the United States. Based at the Atlanta VA Medical Center, the Tele-Stroke Robotic Rehabilitation program provides rural Veterans who have had a stroke with an innovative, in-home solution for physical rehabilitation that improves access to care by mitigating transportation barriers for Veterans who live in rural areas at a distance from Veterans Health Administration medical centers. In FY17, the PRIS-M implementation support team assisted the Atlanta-based team during its program expansion to incorporate additional Veterans Health Administration facilities, helped to develop and refine implementation strategies, attended in-person program kick-off meetings in Atlanta, Georgia and Birmingham, Alabama and conducted baseline interviews and surveys with participating Veterans Health Administration clinicians and Veterans. As Veterans’ access to the program has continued to expand through implementation at additional Veterans Health Administration facilities, the PRIS-M implementation support team partnered with the Atlanta-based team and key stakeholders from the national Veterans Health Administration Office of Rural Health to formalize procedures, policies, practices and approaches to ensure successful implementation and larger-scale deployment across diverse Veterans Health Administration medical centers at a national level.The core implementation strategies used in the implementation of both programs were facilitation and education. The TeleSleep program had both an executive facilitator and a coordinator facilitator, who worked in parallel to address and overcome facility-level barriers and promote implementation. The executive facilitator secured resources; conferenced with national VHA telehealth leadership; met with third-party vendors; communicated regularly with with service chiefs and facility leadership; and provided feedback to front-line clinical staff. The coordinator facilitator coordinated planning and development of the innovation; developed and coordinated the data systems including patient tracking system and CPRS templated notes; trained telehealth RNs in the TeleSleep protocols; and spanned the boundaries of traditional facility-level services  to facilitate implementation. The executive and coordinator facilitators met together in person weekly to plan, strategize, evaluate and reflect. In the Tele-robotics program, on-site kickoffs took place where members of the national program team travelled to the participating facilities to spend a half-day training local staff on how to use the new equipment, recruit new patients with an eye to both inclusion and exclusion criteria, conduct baseline assessments, and follow new protocols.The PRIS-M implementation support team sought to gain new insights into the implementation process for both projects and generate actionable findings that could provide specific guidance to implementation leaders regarding implementation progress and the potential need for mid-course adjustments. With these aims in mind, the team developed the new PRIUS approach and applied it to both projects.The PRIS-M implementation support team developed a new practical tool called the Prospectively-Reported Implementation Update and Score (PRIUS) to provide a quick, structured and open-ended method for individuals to report on implementation progress. Taking between five and fifteen minutes to complete, PRIUS updates were collected approximately twice a month. PRIUS updates could come from either participants  or from observers . The two-week interval was explicitly chosen as a way to collect data with sufficient regularity without imposing undue burden on individuals reporting PRIUS updates. The format of the PRIUS was heavily influenced by the prior experience of the implementation team members from earlier projects with assigning ratings to CFIR constructs, using matrix displays, and applying rapid evaluation and assessment methods.In the Tele-Sleep project, severe time constraints on staff participating in the program underscored the importance of finding a quick, structured, straightforward and open-ended method for participants to report on implementation progress. PRIUS was conducted as a brief verbal check-in, and began in January 2016 after the program went “live” and began to enroll patients. PRIUS sessions took place via a face-to-face or phone conversation between participants and implementation support team members , depending on the preference of the participant. Participants responded verbally while notetakers prompted participants as needed and recorded responses. Participants did not need to provide anything in writing, as the notetakers wrote down all relevant data following the standardized process described below. Before conducting their first PRIUS session, members of the implementation team attended a one-hour training session in January 2016 on how to administer the PRIUS. One month later, the team met again in person to compare PRIUS entries and calibrate the scoring process so that team members shared a common understanding of valence and magnitude.In the Tele-robotics project, individual implementation support team members independently filed their own PRIUS updates. The analytic target of the Tele-robotics project was how national implementation was progressing overall, as opposed to implementation at one specific site (as with TeleSleep).PRIUS updates were entered into a four-column Microsoft Excel document Table , with eaIn the first column, individuals responded to the PRIUS prompt: “What are some things that happened over the past two weeks that seem relevant from your perspective to the implementation of this project”? This structured, open-ended question was selected because it bounded responses in three specific ways: it provided a specific reporting timeframe; it engaged others by explicitly valuing their individual perspectives on implementation; and it focused attention on only the most notable  implementation developments.In the second column, individuals scored the perceived impact of each development on the implementation of the QI project. The scoring process prompted individuals to engage in a brief reflecting and evaluating activity for each item. Individuals scored each development by selecting a number ranging from + 3 to − 3 . Positive scores indicated a positive influence on the implementation process; negative scores indicated a negative influence; and zero indicated no discernible influence one way or the other. In terms of magnitude, 3 indicated a strong influence, 2 a moderate influence and 1 a weak influence. For example, a PRIUS update with a “+ 2” score would indicated that that the development was perceived to have a moderate positive impact on implementation of the QI project. The rationale behind the scoring process was to draw upon the experience and perspectives of individuals at the time they reported on specific implementation developments to sort each development into discrete categories of perceived impact. These numerical scores provided an additional factor with seven different possible values linked directly to the qualitative reports of implementation developments. This quantitative information provided another source of data for the implementation support team to use when sorting, analyzing, and reporting on implementation progress.In the third column, individuals provided a brief rationale for each score. For example, a individual might explain why they scored a particular development as a “-1.”The fourth column provided an optional space for additional comments as desired related to a particular development. This might include observed nonverbal cues like facial expressions, body language, and tone of voice; environmental factors ; and additional relevant details. This column was included for the notetaker to capture any important contextual details related to the reporting of specific implementation developments.After each PRIUS session was completed, the new entries were added to a unified Microsoft Excel spreadsheet in a secure online location. This master spreadsheet featured additional columns to capture date and respondent for each entry. The growing body of PRIUS entries was then reviewed, sorted, and searched by other project team members on a shared drive, where the unified spreadsheet was available for rapid feedback, formative evaluation, and making connections between local context, implementation processes and implementation outcomes. PRIUS reviews took place approximately every two months.In both the TeleSleep and Tele-robotics projects, implementation support team members met regularly to review the aggregated PRIUS updates to compare different perspectives on implementation-related developments, identify emergent trends, and make recommendations to implementation leaders to support ongoing implementation progress. When discrepancies in scores were observed across respondents for similar items, implementation team members flagged those implementation-related developments for further scrutiny and discussion in order to assess if the underlying source of the differences was a relatively minor semantic issue or if it reflected a deeper polarization of perspectives. If the latter, implementation team members brought these discrepancies to the attention of implementation leaders for further discussion and possible corrective action. Longitudinal analyses were conducted in two ways: by comparing PRIUS updates scored with similar values at two different timepoints ; and by comparing how scores changed over time for the same kind of entry .Figure In November 2016, several months after PRIUS data collection and analysis had ended for the TeleSleep program, implementation support team members independently wrote analytic memos about their experiences using the PRIUS, including perceived strengths and limitations of the method, and then convened a face-to-face meeting to discuss their findings.Twelve different staff members involved in the TeleSleep project participated in PRIUS sessions over a 6-month period in 2016, resulting in a total of over 190 different PRIUS updates. The Tele-robotics program generated 498 PRIUS entries related to program implementation over a 10-month period in 2017. The average PRIUS session had three or four items (or “rows” in the PRIUS template). New PRIUS updates were discussed regularly during implementation support team meetings, with PRIUS-based findings and insights shared with the lead investigators of the QI projects.Table Longitudinal analyses of PRIUS entries primarily identified major program-wide changes over time rather than individual-level changes. For instance, TeleSleep participants initially reported professional development and training activities as a strong positive (+ 3) in February 2016 but then reported the perceived need for additional training to meet the PAP needs of patients as a moderate negative (− 2) by April 2016. Strong positive (+ 3) influences reported in Tele-robotics corresponded initially most often to items that had been planned and successfully executed by the program  but six months later mostly related to enterprise-level administrative actions .Rapid analysis and reporting of the PRIUS data led directly to changes in implementation. In the TeleSleep project, for example, findings from the onset of data collection in February 2016 revealed that staff from different clinical areas reported fundamentally different perspectives on project implementation. The implementation support team shared this result with the TeleSleep implementation leader in a formally scheduled meeting to review the PRIUS data in February 2016. Reflection on these findings led to the realization that while managers and service chiefs related to sleep medicine and telehealth had regularly attended TeleSleep meetings, several key frontline staff could not make those meetings because of conflicting clinical duties. Key frontline staff from sleep medicine and telehealth, furthermore, did not know each other personally, and did not know what happened when a patient transferred from one service to the other when enrolled in the TeleSleep program.In March 2016 patient enrollment in TeleSleep nearly ground to a halt, with only 5 patients enrolled when the original target had been 30. As a direct result of the earlier PRIUS discussion, the implementation leader together with the implementation support team decided to host a voluntary, catered “appreciation” lunch for all interested TeleSleep staff in early April 2016. As part of this lunch, the implementation leader would thank staff personally for their participation in the program and share TeleSleep data demonstrating how effective the program had been to date in terms of improving patient outcomes. The appreciation lunch was well-attended and positively received, with frontline staff from the two services engaging in personal conversations for the first time. Program recruitment by clinical staff improved so dramatically after this lunch that within a month, enrollment had to be temporarily suspended until the TeleSleep staff could catch up with the new patients. PRIUS updates collected after the lunch clearly identified this event as a turning point in program implementation, with several individuals independently reporting that it was not until this event that they understood how the TeleSleep program worked overall, the positive impact they were having on patients through the program, and the perspectives of other staff involved in the program from different service areas. As originally intended, the PRIUS enabled the implementation leader of TeleSleep to monitor implementation progress more closely, to make a needed course correction, and assess the impact of that intervention.Several months after PRIUS data collection and analysis had ended for the TeleSleep program, implementation team members wrote analytic memos and convened an in-person meeting and group discussion in November 2016 to reflect on their experiences on using the PRIUS. The main strengths identified were that the structured but open-ended PRIUS approach helped implementation team members establish rapport quickly with participants, and that participants seemed eager to share their perspectives on implementation. In fact, there were no refusals; rather, participants were universally willing to talk with the implementation team. Several participants reported that they actively looked forward to new PRIUS sessions so they could share the latest updates from their vantage point. One nurse in the TeleSleep program even contacted the implementation team to request the initiation of PRIUS sessions when they had not yet been scheduled with that individual. Participants were also not shy about reporting developments they viewed as negative ; the PRIUS appeared to create a safe space for venting negative concerns in a constructive way in an organizational context where individuals might otherwise not feel comfortable divulging critical remarks or observations. The biggest reported limitation was that numerical scoring of PRIUS items on a + 3 to − 3 scale occasionally felt forced for participants; this awkwardness was sometimes alleviated by first introducing the + 3 to − 3 scale to participants but then using terms  rather than numbers when scoring items.The PRIUS mapped directly onto several implementation strategies listed in the Expert Recommendations for Implementing Change project, including audit and feedback; facilitation; purposely reexamining the implementation; tailoring strategies; and using implementation advisors . FurtherNo special software is required to collect, organize or analyze PRIUS entries. By generating succinct descriptions of implementation-related developments along with their corresponding scores, the PRIUS offers implementation leaders, stakeholders, and researchers a pragmatic approach for monitoring how implementation unfolds in specific healthcare settings and for making mid-course adjustments as needed.This study had several limitations. It involved only two implementation projects in Veterans Health Administration, and may not generalize to use in other projects or in other healthcare systems. The amount of time required to capture PRIUS updates was not formally tracked and cost data were not formally collected. The PRIUS method has not yet been formally validated against patient outcomes or other measures of implementation success. The PRIUS may be more feasible when applied to pilot projects when the total number of participants and stakeholders remain relatively small. We recommend future evaluations of the method include assessments of its validity and limitations, as well as its cost and time requirements.PRIUS provided an efficient, structured tool to elicit and capture perspectives of diverse participants on local implementation-related developments over time. The method turned out to be straightforward, quick and interactive, capturing valuable implementation-related data that might otherwise prove evanescent. The PRIUS allowed for the development of an ongoing, detailed and nuanced account of implementation progress that could support and inform active implementation of two diverse programs in healthcare settings.The Veterans Health Administration PRIS-M QUERI in Indianapolis has since applied PRIUS to additional projects and found the tool to be highly adaptable to a wide range of settings. As a prospective, longitudinal and standardized approach to capturing and analyzing implementation-related developments over time, PRIUS offers a new way to develop a granular, context-sensitive account of implementation progress. Ongoing collection, review and analysis of PRIUS entries can result in new insights and actionable recommendations that support the dynamic and complex process of implementation within healthcare settings.


Epithelial-to-mesenchymal transition (EMT) programs contribute to the acquisition of invasive properties that are essential for metastasis. It is well established that EMT programs alter cell state and promote invasive behavior. This review discusses how rather than following one specific program, EMT states are diverse in their regulation and invasive properties. Analysis across a spectrum of models using a combination of approaches has revealed how unique features of distinct EMT programs dictate whether tumor cells invade as single cells or collectively as cohesive groups of cells. It has also been shown that the mode of collective invasion is determined by the nature of the EMT, with cells in a trailblazer-type EMT state being capable of initiating collective invasion, whereas cells that have undergone an opportunist-type EMT are dependent on extrinsic factors to invade. In addition to altering cell intrinsic properties, EMT programs can influence invasion through non-cell autonomous mechanisms. Analysis of tumor subpopulations has demonstrated how EMT-induced cells can drive the invasion of sibling epithelial populations through paracrine signaling and remodeling of the microenvironment. Importantly, the variation in invasive properties controlled by EMT programs influences the kinetics and location of metastasis. The acquisition of invasive ability  is an esThe acquisition of invasive traits by tumor cells mirrors the phenotypic changes of epithelial-to-mesenchymal transitions (EMTs) that take place during embryogenesis and wound healing . The EMTEpithelial tissue is comprised of adherent polarized sheets of cells that, depending on tissue type, are sculpted into ducts and lobules . Tumors EMT programs are normally initiated by ligands that bind to transmembrane receptors capable of activating intracellular signaling pathways. Examples include members of the TGFβ family, growth factors that bind to receptor tyrosine kinases (RTKs), and WNT ligands . These sThe most established mechanism by which EMT programs promote cell migration is the suppression of the cell–cell adhesion protein E-cadherin. Reduced E-cadherin expression correlates with poor patient outcome ,22,23,24The loss of epithelial features alone is not sufficient to promote migration and invasion. EMT-TFs also induce mesenchymal genes that promote alterations in cell morphology, enhance migratory properties, and influence the ability to remodel the ECM . EMTs alThere is heterogeneity in the composition and functions of EMT programs. The elements of epithelial suppression and mesenchymal induction just described are not a part of a single EMT program through which cells progress over time. Thus, it should not be assumed that a feature of one EMT program is a trait of all EMT programs. There are a range of unique EMT states, reflecting distinct activating signals and intrinsic cell-lineage features, that determine the extent of epithelial gene suppression and mesenchymal gene induction that occurs as part of an EMT program. This heterogeneity in EMT programs contributes to the significant phenotypic variability observed in the modes of tumor cells invasion  Figure . Tumor cell invasion is frequently conceptualized as a process undertaken by solitary cells that detach from a multicellular tumor mass and migrate into the ECM. There are distinct modes of single-cell invasion . Cells cTumor cells frequently engage in a process called collective invasion, in which cells migrate through the ECM in groups of cells that retain cellular cohesion . During The trailblazer EMT program is distinguished by the induction of genes that are specifically required to form cellular protrusions that provide traction and reorganize collagen into parallel fibrils ,72. ThesOpportunistic EMT states can be induced by hybrid programs that confer mesenchymal features while allowing cells to retain epithelial character. Hybrid states are a general property of carcinomas  and collThe precise nature of hybrid EMT programs that confer an opportunist phenotype have begun to become unraveled. ΔNp63 is necessary for opportunistic invasion in multiple breast cancer models ,84 and cIn addition to the underlying transcription regulation conferring an opportunistic hybrid state, properties of hybrid EMT cells that directly promote collective invasion have begun to be investigated. In pancreatic cancer models, a hybrid EMT state correlates with an increased storage of E-cadherin in recycling endosomes, potentially due to the increased expression of Rab11 . This meEMT induction occurs in a fraction of cells in primary tumors ,63,88,89Trailblazer cells can promote sibling opportunist-cell invasion through a paracrine signaling independent mechanism. In this mode of interaction, paths in the ECM created by trailblazer cells promote the collective invasion of motile opportunist sibling tumor cells or normal mammary epithelial cells that lack the intrinsic capacity to initiate invasion . InteresGenes that are specifically required for trailblazer cell invasion that were described earlier, including, DOCK10, integrin α11, DAB2, and PDGFRA, are expressed at least four-fold higher in trailblazer cells, relative to opportunist siblings ,85. In lAnalysis of mouse models of breast and pancreatic cancer suggest that an EMT is induced in a subpopulation of cells prior to detectable primary tumor formation ,95,96,97It is well-established that EMT programs are activated in invasive primary tumor cells ,63. In pThe nature of the EMT program influences the ability of tumor cells to engage in colonizing metastatic growth. Stable and complete induction of EMTs promotes dissemination to new tissues ,103. HowExtensive investigation using an array of tumor models supported by patient tumor analysis has demonstrated that EMT programs contribute to tumor invasion and metastasis. The basic features of EMT programs that control invasion have been established. More recently, the diversity of EMT programs and the phenotypes they induce during tumorigenesis have been recognized. Building upon these discoveries, there are a number of challenges that must be addressed to understand the regulation and function of EMTs with the goal of improving cancer patient diagnosis and treatment. It is critical to determine at which point EMTs are induced during tumor progression to define precisely how EMTs influence metastasis. The contributions of EMTs towards metastasis has largely relied on models in which tumor cells have progressed to a near fully mesenchymal state. However, hybrid states are frequently detected in primary tumors and may be the predominant type of EMT . Yet the


Chemical communication plays many key roles in mammalian reproduction, although attention has focused particularly on male scent signalling. Here, we review evidence that female chemical signals also play important roles in sexual attraction, in mediating reproductive competition and cooperation between females, and in maternal care, all central to female reproductive success. Female odours function not only to advertise sexual receptivity and location, they can also have important physiological priming effects on male development and sperm production. However, the extent to which female scents are used to assess the quality of females as potential mates has received little attention. Female investment in scent signalling is strongly influenced by the social structure and breeding system of the species. Although investment is typically male-biased, high competition between females can lead to a reversed pattern of female- biased investment. As among males, scent marking and counter-marking are often used to advertise territory defence and high social rank. Female odours have been implicated in the reproductive suppression of young or subordinate females across a range of social systems, with females of lower competitive ability potentially benefiting by delaying reproduction until conditions are more favourable. Further, the ability to recognise individuals, group members and kin through scent underpins group cohesion and cooperation in many social species, as well as playing an important role in mother-offspring recognition. However, despite the diversity of female scent signals, chemical communication in female mammals remains relatively understudied and poorly understood. We highlight several key areas of future research that are worthy of further investigation. A defining feature of all mammalian taxa is high female investment in reproduction through the process of lactation  the urinary compound (Z)-7-dodencen-1-yl acetate elicits male flehmen responses, erections and premating behaviour -7-dodencen-1-yl acetate peaks just prior to oestrus, suggesting it functions to advertise the approach of female receptivity  that stimulates copulatory behaviour in males  are significantly less attracted to female odours immediately prior to parturition, although pre-parturition odours are still more attractive than male odours  found that although males showed plasticity in sperm production, this was caused by competitive cues from other males, rather than by female cues relating to mating opportunities : males housed alone or with a female show elevated testosterone following exposure to a novel female odour but males housed in family groups do not  are more attracted to scents from dominant females, but only if scents are from familiar females   token urination is performed almost exclusively by females and patterns of marking do not vary across seasons, suggesting it may function to maintain spatiotemporal separation between females  immediately visit and scent mark their shared border, suggesting that they are capable of recognising neighbouring individuals (Sliwa and Richardson Myodes glareolus) display higher rates of infanticide towards the offspring of unfamiliar females under semi-natural conditions  exposed to repeated simulation of intrusions by neighbours increase their rates of counter marking, suggesting that animals adapt their behaviour to neighbours depending upon the perceived level of threat  emit more worry calls and perform more inspection bouts in response to translocated scent marks from neighbouring groups compared to the scent marks of strangers (Müller and Manser Mus spicilegus) tend to be more aggressive towards their immediate neighbours, particularly when pregnant  but female-biased in female dominated species  deposit more scent marks than both dominant and juvenile females and concentrate most scent marking at territory borders  only display higher rates of scent marking than subordinates during intergroup encounters  or prairie voles (Microtus ochrogaster) suppress reproduction in the presence of their mother. In some species, puberty delay may not be inhibited by female signals, but females may require the presence of an unfamiliar male to stimulate reproduction  to an unfamiliar male accelerates development even in the presence of their reproductively active mother  females continue to exhibit reproductive suppression 30 days after being removed from the dominant female or her cues  anal gland secretions from high ranking females are preferentially overmarked by subordinates  found that despite being able to discriminate between the odours of own and alien young, females retrieved both equally . Here, females need to discriminate own from alien young to ensure that the benefits of parental care and lactation are received by own offspring. By contrast, in many altricial species, offspring are confined to a single den or nest until weaning (Gubernick and Klopfer Octon degus) used a radionuclide to track milk transfer from mother to young in communal nests (Jesseau et al. Recognition of young is expected to be particularly important for animals that raise their young in a communal nest. However, in many communally nesting species females nurse all young in the nest indiscriminately (degus: Ebensperger et al. The ability to recognise own mothers by offspring may also be important in mammals as unfamiliar conspecifics often behave aggressively towards alien young. Although recognition of mothers has been demonstrated in some species, few studies have directly investigated the role of olfaction in recognition (Lickliter and Heron Olfactory cues are also important in mother-offspring recognition in humans. Mothers can identify their own infant through odour alone (Kaitz et al. Evidence from several mammalian species shows that females release highly specific chemical cues to guide their young to the nipples and elicit suckling behaviour (Arteaga et al. Similar nipple searching behaviour is seen in other altricial species, although the mechanisms have not been investigated as thoroughly as in rabbits (Schaal and Al Aïn Odours are also important in initiating suckling behaviour in humans. Odour emitted from the breast of lactating females are attractive to newborns (Makin and Porter This review highlights the diversity of scent signals produced by female mammals, and the wide range of functions that these fulfil, including competitive signalling, sexual advertisement and facilitation of maternal behaviours. However, female chemical communication still remains poorly understood in the majority of mammals. We suggest several key areas for future study. First, although female scents can communicate a wealth of information about an individual, such as species, sex, reproductive state, health, dominance status and genotype (Brown and Macdonald