Duchenne muscular dystrophy (DMD) is a degenerative muscle disorder characterized by a lack of dystrophin expression in the sarcolemma of muscle fibers. DMD patients acquire bone abnormalities including osteopenia, fragility fractures, and scoliosis indicating a deficiency in skeletal homeostasis. The dKO (dystrophin/Utrophin double knockout) is a more severe mouse model of DMD than the mdx mouse (dystrophin deficient), and display numerous clinically-relevant manifestations, including a spectrum of degenerative changes outside skeletal muscle including bone, articular cartilage, and intervertebral discs. To examine the influence of systemic factors on the bone abnormalities and healing in DMD, parabiotic pairing between dKO mice and mdx mice was established. Notably, heterochronic parabiosis with young mdx mice significantly increased bone mass and improved bone micro-structure in old dKO-hetero mice, which showed progressive bone deterioration. Furthermore, heterochronic parabiosis with WT C56/10J mice significantly improved tibia bone defect healing in dKO-homo mice. These results suggest that systemic blood-borne factor(s) and/or progenitors from WT and young mdx mice can influence the bone deficiencies in dKO mice. Understanding these circulating factors or progenitor cells that are responsible to alleviate the bone abnormalities in dKO mice after heterochronic parabiosis might be useful for the management of poor bone health in DMD. Duchenne muscular dystrophy (DMD) is the most common of the muscular dystrophies and is caused by mutations in the dystrophin gene ,2. ProgrThe mdx mouse (dystrophin−/−), a common DMD murine model ,8, has aIn this study, we investigated whether the bone abnormalities observed in dKO mice can be rescued through parabiotic pairing which will rejuvenate the dKO microenvironment by creating a shared circulation between dKO and young WT and mdx animals. Parabiosis is a process in which two mice are surgically connected to establish a shared blood circulation thus allowing for the exchange between the two animals of circulating blood, immune factors, circulating stem cells, hormones, growth factors and a myriad of other circulating molecules ,19,20. TTwo heterochronic pairs and two young mdx isochronic pairs died at two weeks after surgery due to “parabiotic intoxication”  which inTo confirm the formation of a shared blood circulation between the parabiotic animals, one pair of control parabionts of old/old dKO-hetero mice were tested at two weeks after parabiosis. Cross-circulation (chimerism) in paired animals was confirmed by tracking the flow of EBD from mouse “a” to mouse “b” following intravenous (tail vein) injection A. After We have reported the reduction of bone tissue, in both the lumber vertebra and the epiphysis of the proximal tibia, in dKO mice when compared with age-matched mdx and WT controls . In thisWe have reported that in addition to progressive bone loss, dKO-homo mice also demonstrated inferior bone healing capability . To testTo decipher the role of parabiosis in bone healing, bone marrow mesenchymal stem cells and hematopoietic stem cells were isolated and quantified from the contralateral tibia of dKO mice. Hematopoietic stem cell quantity was significantly increased in the dKO-homo mice after pairing with WT mice A. There Most of the work toward developing a treatment for DMD over the last 20 years has focused on skeletal muscle repair, with little effort being directed toward understanding the other musculoskeletal abnormalities observed in DMD patients. Despite the assumption that bone abnormalities are a secondary consequence to the muscle pathology, it is important to note that most DMD patients become wheelchair-bound after they experience a bone fracture, which demonstrates the importance of increasing our understanding of these bone abnormalities in order to improve the quality of life of DMD patients. Parabiosis is a process in which two mice are surgically connected to establish a shared blood circulation thus allowing for the exchange between the two animals of circulating blood, immune factors, circulating stem cells, hormones, growth factors and a myriad of other circulating molecules. Parabiosis has been used to evaluate disease progression in knockout mice by conjoining a healthy WT mouse to a diseased mouse model ,19,20. THeterochronic parabiosis studies were performed based on dydy dystrophic mouse models at early 60s with controversial results. Hall et al.  reportedIn the current study, parabiosis were used to answer the questions that if blood-borne factors may also be related to the development/progression of the bone deficiencies observed in dystrophic mice. Surprisingly, we observed significantly improved bone quality in old dKO-hetero mice that paired with young mdx mice and improved bone healing property in dKO-homo mice that paired with WT mice, which strongly suggests that blood borne factors are responsible, or at least play a role, for the bone abnormalities in DMD. Moreover, we also observed deleterious bone changes in young mdx mice that were paired with old dKO-hetero mice. These observations further indicate that it is indeed the circulating factors in the older dKO animals that are causing the deleterious bone changes. The next logical question to ask is which circulating factors are responsible to alleviate the bone abnormalities in old-dKO mice after heterochronic parabiosis procedure. To address that, the generalized “omics” approaches to reveal such factor(s) or cell(s) is needed based on this heterochronic parabiosis model. Although out of the scope of the current study, several studies have already shad lights on this topic. One study found that IL-6, a pro-inflammatory cytokine, significantly increased in the circulation of mdx mice and in DMD patients . BlockagIn addition to the circulating factors, it is possible that circulating cells from young mdx mice and/or WT mice might also contribute to the improved bone quality and bone healing in paired dKO mice. However, under current experimental settings, we cannot investigate the contribution of circulating cells due to lack of tracing markers. Further investigations by pairing dKO mice with reporter mouse that has specific genetic markers, will help to address those questions. Those investigations should be aimed to answer the questions that: (1) if there are circulating cells actually participate in the bone turnover and/or new bone formation in bone defect repair; (2) What is the characteristics of those circulating cells? Are those cells circulating progenitor cells that could actually integrate into the newly formed bone tissues or circulating immune cells that transitionally facilitate bone turn-over and bone repair processes? (3) If there are circulating cells that not directly participate the bone turn-over and bone repair processes but integrated into other organs which indirectly affect bone turn-over and bone repair via modifying the systemic milieu. In terms of circulating osteogenic progenitor cells, based on the published data ,36, evenAnother explanation of the improvements seen in parabiosed old dKO-hetero and dKO-homo mice might due to the improved nutrition provided by the relatively healthier or normal partner. At the moment, we could not exclude this possibility due to lack of a reliable system to monitor and control the food consumption of the two paired animals. However, this explanation seems improbable with the observation that the stronger parabionts always dominant the movement in the cage and the weaker part of the parabionts may not take as much food as it normally does, which suggests that the weak dystrophic mice paired with WT mice may theoretically take less food than their isochronic controls. Moreover, no significant body weight changes were found among groups, which also suggest that the nutritional explanation is less likely.There are some limitations of the current study. First, functional assays, such as bone mechanical tests both on cortical and cancellous bones, will give us more information regarding the changes of bone mechanical properties in addition to the 3D morphometric changes after parabiosis. Second, the overall health and/or skeletal muscle functionalities may also contribute to the general improvement of the bone parameters that we observed. More detailed systemic parameters such as body weight, food consumption, motor function and specific muscle contractility before and after parabiosis will help to refine the interpretation of the data. Third, one may argue that the beneficial effects seen in older dKO mice is just a reflection of the younger mice on older mice and it is not specific to dystrophic mice. A series rigorous age-matched pairing maybe helpful to rule out this possibility. Even that, it is also of great interests to identify those factors that represents the differences between young and old. Fourth, although data presented in the current study strongly suggested that the blood-borne factors or progenitors may play an important role in causing the low bone quality and defective bone healing of DMD, the exact mechanism(s) is still unclear. Further investigations based on this model are needed to elucidate: (1) whether absence or accumulation of circulating soluble mediator(s) in dystrophy negatively affects bone homeostasis; (2) whether defective circulating cellular mediator(s), such as inflammatory cells and/or progenitor cells may play a role in regulating bone turnover and bone healing process; or (3) a combination of both. Finally, high-throughput -omics based analytic assays on blood, skeletal muscle and bone tissues should be performed to identify possible factor(s) that contribute to the bone deterioration response induced by the lack of dystrophin.Nevertheless, the current study demonstrated that the parabiosis model provides a useful, and potentially powerful, model for investigating further the treatment options targeting blood-borne factors in managing musculoskeletal deficiencies in DMD. The findings of a robust effect on bone quality and bone healing in dystrophic mice after parabiosis raises the possibility that strategies could be devised to experimentally or therapeutically manipulate circulating factors in a manner that regulating bone homeostasis and even promotes regeneration.The University of Pittsburgh’s Institutional Animal Care and Use Committee  approved all experiments. Wild-type (C57BL/10J) mice and GFP transgenic mice (C57BL/6-Tg [CAG-EGFP] 10sb/J) were obtained from the Jackson Laboratory . Three different animal models of DMD were utilized in this study: mdx (dystrophin−/−) mice have mild skeletal muscle defects and potent regenerative capacity, whereas dKO-homo  mice display severe musculoskeletal changes but a very limited lifespan , while dKO-hetero  mice display a similar phenotype as dKO-homo with a longer life expectancy. All dystrophic animals were derived from our in-house colony originally from Dr. Davies . The metTo test if systemic circulating factors accumulated in DMD play a role in the progressive bone loss, three types of parabiosis pairs were performed, designed as follows: (1) heterochronic parabionts of old dKO-hetero (12–14 months) and young mdx (3–5 months) mice (10 pairs); (2) isochronic control parabionts of young/young mdx (10 pairs); as well as (3) isochronic control parabionts of old/old dKO-hetero mice (7 pairs). Three months after pairing, both animals were sacrificed, and bone tissues were collected for radiographical and histological analyses.To test if parabiosis can affect the bone healing, six pairs of heterochronic parabionts of four weeks old dKO-homo mice with same aged C57BL/10J WT mice were performed. The bone defects on proximal tibia were created on both mice two weeks after parabiosis. The bone healing process was monitored weekly by in vivo micro-CT scanning for three weeks. The animals were sacrificed at nine weeks of age and bone tissues were collected for further histological analysis. Two groups of isochronic parabionts, WT/WT and dKO-homo/dKO-homo, four pairs each, served as controls.2 L/min). Following the required surgical preparations and shaving of the skin along the flanks of mice to be joined, matching skin incisions from the olecranon to the knee were made along the opposing flanks of each mouse. The subcutaneous tissue was dissected to create a 0.5 cm free skin flap. The limbs of the two animals were joined laterally by silk sutures, followed by skin closure using continuous nylon sutures. Parabiotic pairs were housed separately and fed with mushed standard rodent chow with adequate water. Diet and water were offered ad libitum in a low-profile dish on the cage floor to make sure both parabionts can equally get nutrition. To confirm the formation of a shared blood circulation between the parabiotic animals (stable chimerization), at 14 days post-surgery, Evans blue dye (EBD), which binds to serum albumin were injected into the tail vein of one paired mouse and the successful transfer of the EBD into the other paired mouse were assayed in the circulation and tissues. The EBD transfer to the paired animal was visualized by the EBD dye on the skin and internal organs and the florescence signal from the damaged dystrophic skeletal muscle (Gastrocnemius-GC) [Parabiosis surgery was performed as described previously ,40 with mius-GC) . FurtherTwo weeks after parabiosis, mice were sedated as above, and unicortical, spherical defects  were created using a 0.9 mm burr  on the proximal tibia of the lateral knees of the paired mice, as described previously . Bone inLumbar spine, femur, and tibia were collected at sacrifice and fixed in 4% paraformaldehyde for 24 h. Micro-CT scans were performed to evaluate bone volume (BV) and architectures of lumbar vertebrae, mid-shaft of femur, metaphyseal part of proximal tibia using Viva-CT 40  with settings: energy 70 kV, intensity 114 µA, integration time 300 ms, and isotropic voxel size of 10.5 µm as descripted previously ,33. Regi5 nucleated BM cells per animal were seeded in triplicate into a 6 well-plate with 1.1 mL of MethoCult M3534 methylcellulose medium  or with Complete MesenCult medium . MethoCult M3534 and complete MesenCult media have been formulated to support the optimal growth of granulocyte/macrophage precursor cells and mesenchymal stem cells respectively. After seven to ten days in culture, the cell colonies in MethoCult medium (CFU-GM) were imaged using stereo microscope , and colonies (>30 cells) were counted. The cell colonies in MesenCult medium (CFU-F) were stained with Giemsa stain  and counted. Colony recognition and enumeration were performed according to StemCell Technologies guidelines.To evaluate progenitor cells, both mesenchymal stem cells (MSCs) and the hematopoietic stem cells (HSCs) from bone marrow (BM) were quantified by the colony-forming unit-fibroblast (CFU-F) and CFU-granulocytic-monocyte (CFU-GM) assays respectively. Briefly, single-cell suspension of BM cells from femur and tibia was collected. 10p < 0.05 was considered significant. Data are expressed as Mean ± SEM. GraphPad Prism 7 software was used for statistical analysis. Comparison between two groups was assessed by non-parametric Mann-Whitney U-test. Comparison among ≥3 groups was assessed by one-way ANOVA with Bonferroni Post Hoc test. In conclusion, heterochronic parabiosis with young mdx mice significantly increased bone mass and improved bone micro-structure in old dKO-hetero mice, which showed progressive bone deterioration. Furthermore, heterochronic parabiosis with WT C56/10J mice significantly improved tibia bone defect healing in dKO-homo mice. These results suggest that systemic blood-borne factor(s) and/or progenitors from WT and young mdx mice can influence the bone deficiencies in dKO mice. These results also suggest that understanding these circulating factors or progenitor cells that are responsible to alleviate the bone abnormalities in dKO mice after heterochronic parabiosis, might be useful for the management of poor bone health in DMD.


Is there an association between the neonatal care setting—a family integrated care (FICare) model in single family rooms with complete couplet-care for the mother-newborn dyad vs standard neonatal care in open bay units—and mental health and participation outcomes among mothers of preterm newborns?In this cohort study of 296 mothers of preterm infants, mothers reported experiencing less stress and participated more when they and their infants received care in wards using a FICare model. Participation in infant care mediated the beneficial association of the FICare model and mothers’ depressive symptomatology, self-efficacy, and mother-newborn bonding.These findings suggest that intervention strategies aimed at reducing mother-newborn separation and intensifying active maternal participation are warranted. This cohort study of Dutch mothers with infants born preterm compares stress and participation levels of mothers treated in family integrated care units with mothers receiving standard neonatal care. Active participation in care by parents and zero separation between parents and their newborns is highly recommended during infant hospitalization in the neonatal intensive care unit (NICU).To study the association of a family integrated care (FICare) model with maternal mental health at hospital discharge of their preterm newborn compared with standard neonatal care (SNC).This prospective, multicenter cohort study included mothers with infants born preterm treated in level-2 neonatal units in the Netherlands (1 unit with single family rooms [the FICare model] and 2 control sites with standard care in open bay units) between May 2017 and January 2020 as part of the AMICA study . Participants included mothers of preterm newborns admitted to participating units. Data analysis was performed from January to April 2021.FICare model in single family rooms with complete couplet-care for the mother-newborn dyad during maternity and/or neonatal care.Maternal mental health, measured using the Parental Stress Scale: NICU (PSS-NICU). Secondary outcomes included survey scores on the Hospital Anxiety and Depression Scale, Postpartum Bonding Questionnaire, Perceived Maternal Parenting Self-efficacy Scale, and satisfaction with care (using EMPATHIC-N). Parent participation (using the CO-PARTNER tool) was assessed as a potential mediator of the association of the FICare model on outcomes with mediation analyses.A total of 296 mothers were included; 124 of 141 mothers (87.9%) in the FICare model and 115 of 155 (74.2%) mothers in SNC responded to questionnaires . Mothers in the FICare model had lower total PSS-NICU stress scores at discharge  than mothers in SNC, and specifically had lower scores for mother-newborn separation . Mothers in the FICare model were present more  and participated more in neonatal care . Active parent participation was a significant mediator of the association between the FICare model and less maternal depression and anxiety , higher maternal self-efficacy , and better mother-newborn bonding .The FICare model in our study was associated with less maternal stress at discharge; mothers were more present and participated more in the care for their newborn than in SNC, which was associated with improved maternal mental health outcomes. Future intervention strategies should aim at reducing mother-newborn separation and intensifying active parent participation in neonatal care.NL6175Netherlands Trial Register identifier  Secondary objectives were to determine if the FICare model was associated with improved outcomes in maternal depression, self-efficacy, mother-newborn bonding, and satisfaction with care. We studied active participation in neonatal care as a potential mediator in the pathway between the FICare model and maternal mental health outcomes.NL6175). Hospital architectural design limited randomization between hospitals, and randomization within hospitals was impossible given the risk of cross-contamination. Therefore, we included infants consecutively who were admitted to participating units. This study followed the Transparent Reporting of Evaluations With Nonrandomized Designs (TREND) reporting guideline and A Guideline for Reporting Analyses of Randomized Trials and Observational Studies-Short Form (AGReMA-SF) checklist for reporting mediation analyses.23 This study was approved by the medical ethical review committee of Medical Research Ethics Committees United Nieuwegein, the Netherlands; participating parents provided written informed consent.This study is part of the AMICA study , a prospective observational cohort study comparing an innovative neonatal care model (FICare model) with standard neonatal care in open bay units  in the Netherlands were eligible. All participating units had a comparable patient population. Preterm infants (defined as infants born before 37 weeks’ gestation) with a hospital stay longer than 7 days and their parents were included after the parents provided informed consent. For this study, we analyzed the mothers of the families. Exclusion criteria were severe psychosocial problems , parents not proficient in Dutch or English, infant congenital abnormalities likely to influence neurodevelopment, and if death of an infant occurred  providing feedings by nasogastric tube, bottle or breast, providing skin-to-skin care, weighing, and temperature regulation. Family-centered rounds were implemented that included parents on medical rounds, involving them in patient management, and enabling them to hear first-hand the developments in their infant’s condition. Parents could provide information on their infant’s general well-being, ask questions, and participate in shared decision making.27The exposure setting comprised several aspects, including implementation of FICare principlesSNC in open bay units (OBUs) was provided in 2 different level-2 neonatal units in Alkmaar and Amsterdam, The Netherlands. These units had an open configuration with newborns staying together in 1 unit (with a maximum of approximately 18 infants admitted simultaneously)  to “extremely stressful” (5),28 for a maximum score of 130, with higher scores indicating more stress. Secondary maternal mental health outcomes included: measurements at discharge of maternal depressive symptoms and anxiety using the Hospital Anxiety and Depression Scale ,29 parent self-efficacy with the Perceived Maternal Parenting Self-efficacy Scale ,30 impaired mother-newborn bonding using the Postpartum Bonding Questionnaire ,31 satisfaction with care and empowerment using EMPATHIC-N (EMpowerment of PArents in THe Intensive Care–Neonatology) .32 Mothers filled out how they participated and collaborated with health care staff in neonatal care using the CO-PARTNER tool .The predefined primary outcome for this study was maternal stress as measured by the Parental Stress Scale: NICU (PSS-NICU) questionnaireAlso, mothers filled out a general questionnaire with details on their education, current job, and the cultural background they identified most with (classified by the participant). To improve response rates, mothers were reminded up to 2 times   procedure with parcel mean summary scores to missing data at the item level.34 All variables used in the analyses were included in the imputation model, as well as auxiliary variables related to the probability of missing data or to the variables with missing data itself. Variables that were multicollinear with other included variables were excluded from the imputation model. For all data sets, we performed 10 imputations and 50 iterations to obtain imputed data sets. Convergence was checked graphically with convergence plots. All analyses were performed on the imputed data sets and results were pooled by using Rubin Rules.35Baseline characteristics between mothers with and without outcome variables at discharge were compared. We assumed that the data were missing-at-random. The proposed guidance as explained by Sterne et alWe performed multivariable linear and logistic regression in imputed data sets estimating crude and adjusted associations between the FICare model and maternal mental health outcomes. Logarithmic transformations were applied to normalize skewed distributions, or, if unsuccessful, dichotomization. Potential confounders and effect modifiers were identified from the literature and assessed using statistical analyses (eMethods in the c-path)36 also in the absence of a significant total association (c-path) as described before.37We hypothesized that the FICare model (exposure) transmits its association on maternal mental health outcomes (the outcome) at discharge  through active parent participation  . Mediatia-path). In the second regression model, the association of the mediator  on outcomes (b-path) and the direct effect of the FICare model on outcomes (c’-path) were estimated. We calculated the indirect effect (the amount of mediation) in the single mediator models as the product of the a and b coefficients. Crude and adjusted mediation analyses were performed. In the adjusted analyses, confounders were added to all models. We used bootstrap 95% CIs based on 1000 bootstrap resamples around the indirect effects.39In addition to the total association model, 2 linear regression models were fitted. Total parent participation was included in single mediator models as an individual potential mediator of different mental health outcomes in mothers . In the 40 including the mice package for multiple imputation,41 the VIM package for analyzing missing data patterns,42 and the boot package for the bootstrap 95% CIs.43 For all tests, P < .05 was considered statistically significant. Data analysis was performed from January to April 2021.We used R version 3.6.1 for statistical analyses ,From May 19, 2017, through January 8, 2020, we recruited 309 families (145 in FICare and 164 in SNC), encompassing 358 infants and their parents . During P < .001, Mann-Whitney U Test) .Baseline characteristics for mothers were similar between the exposure and control groups with the exception of infant gestational age, which was lower in the FICare model  . InfantsOverall, mothers in the FICare model had significantly lower total NICU stress scores , lower stress from infant behavior, sights, and sounds , and lower stress scores due to parental role alteration  at discharge compared with mothers in SNC . In the P < .001). In the FICare model, mothers required less information compared with mothers in SNC .Mothers in the FICare model were present more than mothers in SNC . One hundred five of 124 mothers (84.7%) were able to be present for at least 8 hours in the FICare model compared with 42 of 115 (36.5%) in SNC  . Mothersab path) of the FICare model on maternal mental health outcomes through active parent participation. We also estimated the direct effect of the FICare model on maternal mental health outcomes that was not explained by increased active parent participation (through the c’ path).With mediation analyses we estimated the indirect effect (the ab path), better mother-newborn bonding  (ab path) and higher maternal self-efficacy  (ab path), at discharge  was associated with lower depressive symptomatology , better mother-newborn bonding scores , and higher self-efficacy scores . No beneficial direct effects (c’ paths) were found of the FICare model on maternal depression and anxiety, mother-newborn bonding, and maternal self-efficacy.Increased active maternal participation was a significant mediator of the association between the FICare model and less maternal depression and anxiety  (ischarge . In otheab path) (c’ path) of the FICare model on maternal NICU stress remained large after adjustment for active parent participation . Parent satisfaction was not different between the FICare model and SNC, and increased active parent participation was not a mediator of the association between the FICare model and satisfaction with care .The FICare model was associated with less stress for mothers at discharge compared with mothers in SNC. Increased active parent participation in the FICare model was a potential mediator of this association but did not reach statistical significance  (ab path) . The dirThis study showed that mothers of preterm infants experienced less stress at discharge when admitted to a setting with FICare in SFRs compared with SNC. Mothers in the FICare model were able to be present more and participate more in neonatal care, which was associated with improved mental health outcomes including less depression, better mother-newborn bonding, and higher self-efficacy.13 admitted to SNC settings. Mother-newborn separation is one of the main challenges health care professionals currently encounter when caring for mothers and infants postnatally, especially when both need medical care. Additionally, during the COVID-pandemic it has become apparent that restrictive policies and mother-newborn separation are of great concern.44 Parents have reported that restrictions limit their ability to bond with their infant, to participate in care, and negatively impact breastfeeding as well.47In concordance with previous research, our results indicated an association between mother-newborn separation and high stress levels in mothers of preterm infantsc’ path) with the FICare model—independent of active maternal participation—was present. This could indicate that the architectural design with complete couplet-care for the mother-newborn dyad in SFRs was an important factor associated with less maternal stress at discharge, as has been shown before.10 The architectural design may have been less important for the other maternal mental health outcomes, since we found no direct beneficial association (c’ path) for these outcomes. However, increased active maternal participation was a significant mediator of the association between the FICare model and less maternal depression and anxiety, better mother-newborn bonding, and higher maternal self-efficacy. These findings suggest that for maternal depression, mother-newborn bonding, and maternal self-efficacy, specific attention should be pointed toward active maternal partnership and collaboration in neonatal care. Improving active maternal participation and collaboration in neonatal care is feasible independent of the architectural design, as the FICare methodology was initially developed in an OBU.50For NICU stress, a direct association (19) and parents  could persist. Future studies should also explore how hospitalization of a preterm infant affects fathers or partners, as they too can experience adverse outcomes.54 Additional research can also focus on an exact definition of zero separation in this context, as one can still feel emotionally connected without being physically present. For instance, research studies could qualitatively focus on the perception of emotional closeness and the pathways toward emotional closeness that might be facilitated in our FICare model from parents’ perspectives.55Future research should focus on both parental and neonatal outcomes after discharge, as effects of NICU hospitalization on infants As this was a nonrandomized study, there were several limitations that should be considered. We had different enrollment numbers between the FICare model and SNC settings. This was mainly due to nonconsent in SNC and not from missed participants (these numbers were similar between settings). Also, potential baseline differences were present, specifically for gestational age. However, despite this, mothers in the FICare model still reported less stress due to parental role alteration, and specifically less stress from being separated from their infant.Additionally, the potential causality that might be suggested with mediation analysis should also be considered. Mothers who are less depressed, better bonded, and/or highly self-efficient might also participate more in care, and health care professionals should consider this when implementing programs aimed at increasing parent participation.In this study, setting up level-2 neonatal units with a FICare model in single family rooms with complete couplet-care for the mother-newborn dyad was associated with reduced maternal stress at discharge compared with SNC in OBUs with separate maternity care. In the FICare model, mothers could participate and collaborate more in neonatal care, which is associated with ameliorated maternal mental health. For future ward reconfigurations, zero separation between mothers and their newborn should be strived for. However, independent of the architectural design of the neonatal unit, mothers should be allocated as active partners in neonatal care.


All perceptual and cognitive circuits in the human cerebral cortex are organized into layers. Specializations of a canonical laminar network of bottom-up, horizontal, and top-down pathways carry out multiple kinds of biological intelligence across different neocortical areas. This article describes what this canonical network is and notes that it can support processes as different as 3D vision and figure-ground perception; attentive category learning and decision-making; speech perception; and cognitive working memory (WM), planning, and prediction. These processes take place within and between multiple parallel cortical streams that obey computationally complementary laws. The interstream interactions that are needed to overcome these complementary deficiencies mix cell properties so thoroughly that some authors have noted the difficulty of determining what exactly constitutes a cortical stream and the differences between streams. The models summarized herein explain how these complementary properties arise, and how their interstream interactions overcome their computational deficiencies to support effective goal-oriented behaviors. Feedforward and Feedback Processing in the Cerebral Cortex: Connectivity and Function to which it contributes. This circuit design benefits from the fact that all perceptual and cognitive neocortical circuits are organized into layers, typically six of them the bottom-up adaptive filtering process whereby neocortex compresses, or categorizes, familiar information in order to recognize it;(2)the horizontal grouping process whereby neocortex binds distributed features into coherent object representations that are sensitive to the contrasts and spatial arrangement of their inducing features;(3)the top-down matching process whereby neocortex uses learned expectations to focus attention upon critical feature patterns that predict valued outcomes, while suppressing irrelevant features; and(4)the developmental and learning processes whereby neocortex shapes its circuits in response to environmental constraints, while dynamically stabilizing adaptations, such as those in items (1)–(3), to avoid catastrophic forgetting.When these feedforward and feedback processes are combined into a canonical laminar cortical circuit, they clarify how seemingly unrelated psychological properties are mechanistically related. For example, they clarify how the fact that neocortex can develop and learn in a self-stabilizing way implies why top-down attention requires matched bottom-up inputs or volitional signals to fully activate cortical cells, whereas horizontal groupings do not.This article provides an overview and unified analysis of laminar cortical models of multiple types of biological intelligence, including 3D vision and figure-ground perception; attentive category learning and decision-making; speech perception; and cognitive working memory (WM), planning, and prediction. These models are consistent with descriptions and statistical analyses of cortical connectivity patterns  are manifestly complementary.The perceptual groupings that form due to boundary completion are sensitive to statistically significant spatial distributions of contour, texture, shading and depth cues in scenes and images Julesz, . IllusorA surface representation is formed after the brain compensates for variable illumination conditions by discounting the illuminant, and then uses the surviving feature contours to fill-in surface qualities like brightness and color within completed boundary contours, or perceptual groupings. The percepts induced by the four images in bipole boundary completion due to the fact that boundaries form inwardly between pairs of colinear inducers . Cells in the off-surround cannot fire, even if they receive a bottom-up excitatory input, because they are balanced by the top-down off-surround (one against one).critical features that are predictively important for classifying the object or event that the prototype encodes. These LTM traces occur at the ends, or synapses (denoted by green hemi-disks) of the top-down excitatory pathways in The adaptive weights, or long-term memory (LTM) traces in an ART Matching Rule top-down circuit can learn an expectation, or prototype, of initial top-down expectation must be able to match any feature pattern. Thus, initial top-down expectation signals are uniformly large across all feature detectors. As category learning proceeds, the top-down expectation is pruned until it learns a prototype whose top-down signals are positive only at critical features.A much greater omission occurs in As illustrated by the data of Barzegaran and Plomp , one canIn fact, depending upon how one counts, which is limited by the resolution of particular experimental methods, there are more than two pairs of feedforward and feedback streams.computationally complementary laws to accommodate the orientationally-tuned specializations needed for form perception and the directionally-tuned specializations needed for motion perception Self-stabilizing in the sense that it can dynamically stabilize memories formed by development and learning processes. Said in another way, the network does not experience catastrophic forgetting A seamless fusion of automatic, preattentive, data-driven, bottom-up processing and attentive, task-selective, top-down processing within the on-center off-surround layer 6-to-4 circuit that combines these constraints and chooses the best possible consensus of them to control network decision-making. Finally the network can do this while maintaining:(3)Analog coherence, or a solution of the binding problem for perceptual grouping without a loss of analog sensitivity. Said in another way, the network solves the noise-saturation dilemma.Another way to say how bottom-up, horizontal, and top-down pathways cooperate during decision making is as follows: the bottom-up inputs instate distributed spatial patterns of feature categories in a scene. The horizontal interactions preattentively bind these feature categories into emergent object boundary representations. The top-down interactions read-out familiar object boundary representations and use the ART Matching Rule to select, gain amplify, and synchronize object boundaries that fall within their modulatory on-center, while suppressing object boundaries that fall within their off-surround. These interactions can hereby create novel preattentive object boundaries across a scene while they attentively select the familiar ones that are of current interest upon which to base decisions and predictions.This kind of canonical circuit, suitably specialized, occurs in essentially all laminar perceptual and cognitive neocortical areas, not only the visual ones. Raizada and Grossberg  summarizIn response to unambiguous inputs, a fast feedforward sweep of activation can progress throughout multiple cortical areas, as Thorpe et al.  previousSelf-normalizing ratio processing can be interpreted as a kind of “real-time probability theory” . When ARanalog coherence combines the stability of digital processing—because the feedback signals store the chosen representation and buffer it against noisy perturbations—and the sensitivity of analog processing—because the shunting on-center off-surround interactions solve the noise-saturation dilemma.Again using vision as an example: recurrent shunting on-center off-surround networks are capable of binding distributed features within coherent boundary representations without a loss of analog sensitivity. This property of via the ART Matching Rule mutually influence one another. The current discussion focuses primarily on the circuit per se. A more sustained analysis is needed to explain how ART uses the ART Matching Rule to dynamically stabilize category learning in all perceptual and cognitive neocortical areas  can cause catastrophic forgetting when the inputs are presented over and over in the order ABCAD ABCAD etc., rossberg  summarizrossberg  as well modulatory. In other words, bottom-up inputs must occur in order to fire cells that are modulated by attention. Illusory contours seem to violate this constraint because, by their very nature, they complete suprathreshold boundaries over positions that receive no bottom-up inputs , so that the target layer 3B cell can then fire. It is thus a truly binocular cell which fires selectively only when the depth of the object in the world creates a binocular disparity that can activate both the left and right eye inputs to the corresponding disparity-selective layer 3B cells.This  Poggio,  is realiAs noted in Section 4.3, a binocular cell in layer 3B is part of a feedback loop that sends top-down signals to monocular simple cells in layer 4, and whose effect on layer 4 monocular cells is modulatory. This modulatory effect can now be understood as a special case of the ART Matching Rule for dynamically stabilizing the learned binocular fusion at layer 3B cells of V1 that ensures their disparity selectivity.binocular disparity. Different binocular simple cells, and the binocular complex cells in layer 2/3A to which they project  could not be stored in working memory without risking the catastrophic forgetting of previously learned memories of shorter chunks . Language, motor, and spatial sequential skills would then be impossible. As a consequence of LTM Invariance, as new items are stored through time in working memory, smaller stored sequences of items can continue to activate their familiar list chunks until they are inhibited by longer, and more predictive, list chunks; e.g., until MY is supplanted by competition from MYSELF through time .relative activities, or ratios, between previously stored working memory activities as new list items are stored in the working memory through time. Newly arriving inputs may, however, alter the total activity of each active cell across the working memory. In this way, the previous learning of chunk MY is not undermined, but the current activity of the chunk MY can be inhibited by MYSELF.The LTM Invariance Principle is achieved by preserving the item-list resonance with a Masking Field (Grossberg, Masking Fields have been used to explain and simulate psychophysical data from many experiments about how speech sounds are temporarily stored in an Item-Order-Rank working memory before they are categorized through learning, and recognized during an The ability of an Item-Order-Rank working memory to store the same item at multiple positions derives from a topographic projection from the parietal cortex to the ventrolateral prefrontal cortex. This projection converts numerical representations in the parietal cortex Dehaene,  into theItem-Order-Rank working memories illustrate the general hypothesis that a relatively small number of processing stages are needed to realize many properties of biological intelligence. In particular, these working memories form part of the predictive Adaptive Resonance Theory, or pART, neural architecture which also includes other processes that are needed to explain how our brains make our minds (Grossberg, The canonical laminar cortical architecture that is illustrated in The current article has focused upon how feedforward and feedback cortical connections can support the simplest properties of 3D vision; namely, those that depend upon early stages of binocular fusion in cortical layer 3B and boundary grouping in layers 2/3A of V1 and V2. As the 3D LAMINART model illustrates , multiplsurface-shroud resonances between V4 and parietal cortex support conscious seeing, and feature-category resonances between V4 and inferotemporal cortex support conscious recognition (Grossberg, Interactions with other brain regions are needed to consciously see and recognize this surface percept, so that it can be used as a basis for goal-oriented actions. In particular, The current article has also restricted its attention to how feedforward and feedback cortical connections can support vision in response to a stationary scene. It does not incorporate models that clarify how the apparent stability of 3D vision and figure-ground separation are realized when our eyes scan a scene during the learning of view-, position-, and size-invariant recognition categories. How this happens is explained and simulated using the 3D ARTSCAN Search neural model, which has been incrementally developed in a series of articles (McLoughlin and Grossberg, As noted by Grossberg et al. , achieviThe author confirms being the sole contributor of this work and has approved it for publication.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Immunotherapeutic agents harness the patient’s immune system to fight cancer cells. Especially immune checkpoint inhibitors, a certain group of immunotherapeutic agents, have recently improved treatment options for many cancer types. Unfortunately, clinical trials testing of these agents in pancreatic cancer patients have not confirmed promising results from laboratory experiments. Several characteristics of pancreatic cancer biology, especially the profound tumour microenvironment that inhibits the successful identification and elimination of tumour cells by immune cells seems to be responsible for the lacking efficacy of immunotherapeutics in pancreatic cancer. We summarise recently published clinical trials investigating immunotherapeutic strategies in pancreatic cancer patients and available data on how these treatments influence pancreatic cancer biology. Moreover, we identify potential strategies to improve experimental and clinical studies in order to generate more conclusive data and improve patient outcomes in the future.To date, extensive efforts to harness immunotherapeutic strategies for the treatment of pancreatic ductal adenocarcinoma (PDAC) have yielded disappointing results in clinical trials. These strategies mainly focused on cancer vaccines and immune checkpoint inhibitors alone or in combination with chemotherapeutic or targeted agents. However, the growing preclinical and clinical data sets from these efforts have established valuable insights into the immunological characteristics of PDAC biology. Most notable are the immunosuppressive role of the tumour microenvironment (TME) and PDAC’s characteristically poor immunogenicity resulting from tumour intrinsic features. Moreover, PDAC tumour heterogeneity has been increasingly well characterized and may additionally limit a “one-fits-all” immunotherapeutic strategy. In this review, we first outline mechanisms of immunosuppression and immune evasion in PDAC. Secondly, we summarize recently published data on preclinical and clinical efforts to establish immunotherapeutic strategies for the treatment of PDAC including diverse combinatorial treatment approaches aiming at overcoming this resistance towards immunotherapeutic strategies. Particularly, these combinatorial treatment approaches seek to concomitantly increase PDAC antigenicity, boost PDAC directed T-cell responses, and impair the immunosuppressive character of the TME in order to allow immunotherapeutic agents to unleash their full potential. Eventually, the thorough understanding of the currently available data on immunotherapeutic treatment strategies of PDAC will enable researchers and clinicians to develop improved treatment regimens and to design innovative clinical trials to overcome the pronounced immunosuppression of PDAC. Many cancer patients have benefited from novel immunotherapeutic approaches, most notably immune checkpoint inhibitors. This class of drugs harnesses the immune system for the fight against aberrant cancer cells by inhibiting co-inhibitory signals (immune checkpoints) allowing the undamped response of cytotoxic CD8+ T lymphocytes (CTL) to the cancer cells. Unfortunately, patients diagnosed with pancreatic ductal adenocarcinoma (PDAC) have to date not benefited from these and other immunotherapeutic innovations, suggesting the existence of comprehensive immune escape and immunosuppression mechanisms.Regarding a projected increase of PDAC cases, PDAC is expected to be the second most common cause of cancer-related death by 2030 in the United States . Still, Immune surveillance essentially contributes to the elimination of altered  cells and thereby prevents cancer onset under physiological conditions . MoreoveAccordingly, despite the lack of clinical efficacy to date, immunotherapeutic approaches represent the largest group of therapies currently under investigation for the treatment of PDAC patients . ImmunotIn this review, we outline the diverse mechanisms by which PDACs suppress and escape the activity of immune cells and summarise recently published data from preclinical and clinical trials exploring the therapeutic potential of immunotherapeutic strategies for the treatment of PDAC. As available preclinical and clinical data is rapidly mounting, we mainly focus on current data from the last three years with a special emphasis on the role of immune checkpoint inhibitors. Additionally, we outline some translational data obtained from the evaluation of patients treated with these therapies to identify recurrent patterns of immunologic responses that may be employed for improved combinatorial regimens. Finally, we highlight unresolved preclinical and clinical research questions arising from the latest developments of the field with a focus on standards for the translational evaluation of patient material, biomarker development, and the role of tumour heterogeneity for tailored treatment strategies.Like other solid carcinomas, PDAC is characterised by high tumour heterogeneity, which refers to the different geno- and phenotypes of the PDAC cells, as well as to the tumour microenvironment (TME). The latter often comprises more than 80% of the total tumour mass and, in addition to a distinct desmoplastic response, a variety of stromal cells, such as carcinoma-associated fibroblasts (CAF), endothelial cells, and diverse immune cell populations are found . Both thImportantly, a high number of CTL infiltrating the tumour tissue positively correlates with improved survival of PDAC patients, while a high number of regulatory FOXP3+ T-cells (Treg), functionally suppressing the activity of effector T-cells, correlates with poor outcomes . AdditioIn order to successfully induce T-cell mediated elimination of cancer cells, several steps of the cancer-immunity cycle resulting in CTL activation are necessary. These have been comprehensively characterised by Chen et al. . Tumour-Even though PDAC tissues comprise T-cell populations to varying amounts, it is meanwhile well appreciated that PDAC evades T-cell mediated cytotoxicity and hampers the activation of adaptive immunity by different means. Thus, immune evasion starts right from the beginning of tumourigenesis as PDAC harbours a comparatively low tumour mutational burden compared to other cancers such as melanoma . This reThe existing data support the rationale to develop immunotherapeutic regimens that increase the number and activity of tumour infiltrating lymphocytes (TIL) to elicit a potent anti-tumour T-cell response in PDAC patients. For this purpose, different strategies have been developed and explored for their efficacy in preclinical and clinical studies .Applied as cancer therapy, cancer vaccines aim at the delivery of tumour-specific antigens to elicit a specific and strong T-cell mediated anti-tumour response whilst not inducing autoimmunity. The main obstacles towards an effective cancer vaccination strategy have been the choice of appropriate antigen(s), effective adjuvants, the mode of application , and comGVAX is a cancer vaccine consisting of irradiated granulocyte-macrophage colony-stimulating factor (GM-CSF) secreting allogeneic pancreatic tumour cells. The induction of anti-tumour immunity by vaccination with irradiated GM-CSF expressing tumour cells had been explored preclinically in animal models for other cancers including melanoma and prostate cancer ,27 and an = 213) did not confirm the initial positive results for a combination of GVAX and CRS-207 and this regimen did not outperform conventional chemotherapy [A second phase II clinical trial assessed GVAX in combination with low dose cyclophosphamide as a T-cell activity modulating agent alone or in combination with live attenuated mesothelin-expressing Listeria monocytogenes (CRS-207) for treatment of metastatic PDAC . GVAX alotherapy . Interesotherapy .n = 303) adding it to a neoadjuvant standard of care chemotherapy and chemoradiation for the treatment of borderline resectable or locally advanced unresectable PDAC [Another vaccine approach for PDAC treatment is Algenpantucel-L, also known as the HAPa cancer vaccine, consisting of two allogeneic pancreatic cancer cell lines expressing the murine aGT gene. Mechanistically, Algenpantucel-L aims at exploiting hyperacute rejection to murine proteins to induce a strong immune reaction towards PDAC peptides. This agent was clinically evaluated as part of a neoadjuvant treatment strategy in a phase III clinical trial  in combination with Gemcitabine as an adjuvant treatment for stage I or II KRAS mutant PDAC in a phase I/II single-arm clinical trial . The meaAlthough several additional cancer vaccine strategies have been explored in clinical trials, none of them has significantly improved the survival and prognosis of PDAC patients ,36,37,38Despite great hopes, checkpoint inhibitors targeting either PD-1, PD-L1 or cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) applied as monotherapies have not yielded clinical improvements for PDAC patients in contrast to therapeutic successes in other cancers ,40,41,42To overcome the clinical inefficacy of checkpoint inhibitors for the treatment of PDAC, multiple combinatorial approaches to harness T-cell mediated responses have been clinically investigated. These include combinations of two checkpoint inhibitors simultaneously targeting the PD-1, PD-L1, and CTLA-4 axes (or triple therapy with stereotactic radiation), combined with small molecule inhibitors  inhibitors), combined with an oncolytic virus, combined with chemokine antagonists, with CD40 agonists or with cancer vaccines. This overview underscores the diversity of therapeutic approaches being used in the clinical trial landscape to overcome immunosuppression in PDAC patients, which is even broader considering ongoing or abstracted clinical trials . These iTwo recently published clinical trials investigated checkpoint inhibition with Durvalumab (PD-L1 inhibitor) alone or in combination with Tremelimumab s(CTLA-4 inhibitor) for the treatment of recurrent or metastatic PDAC patients with  or withoTGF-β is a key player involved in creating and maintaining an immunosuppressive TME in PDAC, e.g., by inducing transdifferentiation and ECM production in CAF and inhibiting Granzyme B mediated T-cell cytotoxicity . Based oSimilarly, BTK signalling contributes to the generation of the immunosuppressive TME. Mechanistically, BTK signalling is involved in the polarisation of macrophages towards an M2-phenotype. Tumour-associated macrophages (TAM) often exhibit an immunosuppressive M2-subtype which is characterised by secretion of chemokines and cytokines dampening T-cell activity, facilitating polarisation of Treg, promoting angiogenesis, and inducing ECM production by CAF . In a moAnother approach to sensitise PDAC for checkpoint inhibition is the combination with oncolytic viruses. By inducing selective lysis of tumour cells, oncolytic viruses have the property to alter the characteristics of immunologically non-accessible tumours. Virus-induced cell lysis results in increased tumour antigen levels at the site of cell death and augments invasion and maturation of macrophages and dendritic cells that play a key role in recruiting and activating T-cells via chemokine secretion, mostly of INF-γ and TNF-α . Based on = 90) in a phase I clinical trial revealed promising results for the PDAC cohort  [n = 24). However, an effective reduction of tumour-infiltrating Tregs in post-treatment biopsies compared to baseline could be monitored [As outlined above, Tregs are highly abundant in the TME and peripheral blood of PDAC patients even at the early stages of the disease and are key players in modulating the immune response in PDAC . Mechanieatment) . In thisonitored . Overalln = 37) had received one or more lines of systemic treatment, cohort 2 (n = 22) one previous line with a Gemcitabine-based regimen. Both cohorts showed a DCR of 34.5% and 77%, respectively. In cohort 2 of less-heavily pre-treated patients, ORR was 32% and the mean duration of response was 7.8 months. Due to these data showing a promising modest improvement of outcomes, a clinical investigation is ongoing in a second clinical trial (NCT02907099). Translational findings from IHC profiling of paired pre- and post-treatment biopsies of 24 PDAC tissues showed increased infiltration of different T-cell populations, especially activated CD8+ T-cells and decreased numbers of granulocyte-like MDSCs.Another recently explored combinatorial partner for checkpoint inhibition is BL-8040, a CXC chemokine receptor 4 (CXCR4) antagonist. CXCR4 is abundantly expressed on leucocytes and signalling via its ligand CXCL12 is functionally involved in chemotaxis and bone marrow homing of leucocytes. Pharmacological blockade of CXCR4 or abrogation of its main source CAFs by introducing a diphtheria toxin receptor-expressing Fap transgene leading to eradication of Fap expressing CAFs upon diphtheria toxin administration in a mouse model of PDAC altered the composition of the TME and increased T-cell invasion into the tumour. Furthermore, the combination with PD-1 inhibition resulted in tumour shrinkage in this mouse model . AdditioAnother approach to improve CTL mediated anti-tumour responses in PDAC represents the combination of the cancer vaccine GVAX (see above) and immune checkpoint inhibition. To this end, the safety and efficacy of GVAX in combination with the CTLA-4 inhibitor Ipilimumab were evaluated as a maintenance therapy in PDAC patients with stable disease or ongoing response after 8–12 doses of FOLFIRINOX in comparison to FOLFIRINOX continuation in a phase II clinical trial . The meaFinally, another approach to potently activate immune responses against PDAC is combining immune checkpoint inhibition with CD40 agonists. CD40 is a receptor expressed primarily by DC, B-cells, and myeloid cells. Functionally, CD40 is activated by its ligand CD40L, which is mostly expressed on CD4+ helper T-cells and mediates the maturation of DC allowing them to activate CTL . CD40/CDn = 21) investigating the CD40 agonist Selicrelumab  in combination with Gemcitabine for the treatment of chemotherapy-naïve non-operable PDAC. Whilst tolerability was good, 4/21 patients experienced a partial response and 11/22 stable disease [Based on this rationale, multiple clinical trials have been conducted. First, Beatty et al. published a small phase I single-arm clinical trial ( disease . The resn = 30) [A recently published phase I clinical trial investigated the CD40 agonistic antibody Sotigalimab in combination with Gemcitabine/Nab-Paclitaxel with or without additional Nivolumab for the treatment of therapy-naïve metastatic PDAC (n = 30) . The mOSn = 30) . InteresPreclinical and clinical data on a plethora of immunotherapy concepts for PDAC treatment are rapidly mounting. The lack of clinical responses towards a multitude of immunotherapeutic approaches has again shed light on the specific characteristics of PDAC as well as the limitations and challenges of transferring innovative research findings into effective clinical treatment of PDAC patients.While translation from preclinical models to clinical application is generally difficult, the lack of novel therapeutics entering the standard of care for PDAC might specifically highlight the limitations of available preclinical models of PDAC. As PDAC is characterised by a high proportion of dense tumour stroma and resulting cell–matrix and cell–cell interactions, meaningful models should allow studying these interactions. Moreover, patient cohorts subjected to novel treatment strategies have often been treated with at least one prior line of therapy including repeated drug exposure. Most tumour models do not recapitulate this characteristic which may significantly shape the response towards experimental therapeutic agents. Innovative in vitro models, e.g., patient-derived organoid co-culture models and ex vivo culture of tissue slices may help to overcome some of the limitations of 2D cell cultures as they enable tumour cell/immune cell interactions in the presence of experimental drugs and have been demonstrated to recapitulate drug effects observed in patients ,84,85.Moreover, increasingly ambitious translational research programmes have been developed that accompany clinical trials and improve the understanding of PDAC immunology even if clinical endpoints are not met. Ideally, these programmes will result in “reverse translation” by experimentally establishing concepts to overcome resistance towards immunotherapeutic strategies. However, in terms of translational methodology, a lack of established standards for patient material sampling and methods to evaluate immune responses becomes apparent. While peripheral blood samples are often collected longitudinally and blood cell populations are assessed by flow cytometry-based assays, only a subset of clinical trial protocols includes the longitudinal collection of tumour material for histological evaluation and functional analysis of TIL. This is understandable as tumour access is anatomically difficult in PDAC patients and repeated sampling is associated with an imminent risk of complications. However, a comprehensive comparison of pre- and on- or post-treatment biopsies may substantially improve the understanding of biological processes in general and in individual patients. Moreover, additional factors such as the gut and intratumoural microbiomes determine responses to immunotherapy ,87 and hSummarising the available translational data sets from clinical trials presented in this review, a recurring pattern of immune response to diverse immunotherapeutic strategies becomes obvious. Multiple clinical trials testing the efficacy of immunotherapeutic strategies reported decreased Treg numbers or increased T-cell proliferation in peripheral blood or TME ,70 or evStrikingly, none of the above-mentioned trials selected or stratified patients based on molecular or immunological criteria. This is surprising, as intra- and inter-tumour heterogeneity are well-defined characteristics of PDAC  and haveIn summary, the extensive efforts to investigate immunotherapeutic strategies for the treatment of PDAC have resulted in important insights into PDAC immunology and the plethora of ongoing clinical trials will add to this repository . However


The current review outlines the pivotal role this novel molecular imaging technique has played in improving our understanding of native and bioprosthetic aortic valve disease, as well as providing insights into its feasibility as an important future research and clinical tool.Calcific aortic valve disease is the most common valvular disease and confers significant morbidity and mortality. There are currently no medical therapies that successfully halt or reverse the disease progression, making surgical replacement the only treatment currently available. The majority of patients will receive a bioprosthetic valve, which themselves are prone to degeneration and may also need replaced, adding to the already substantial healthcare burden of aortic stenosis. Echocardiography and computed tomography can identify late-stage manifestations of the disease process affecting native and bioprosthetic aortic valves but cannot detect or quantify early molecular changes. The online version of this article (10.1007/s12350-020-02411-x) contains supplementary material, which is available to authorized users. Aortic stenosis affects 1-2% of the general population >65 years old, conferring with it increased mortality.The aortic valve is located at the junction between the left ventricle and aorta and functions to prevent backflow of blood into the left ventricle during diastole while allowing unimpaired systolic ejection. Anatomically, the aortic valve consists of three leaflets that are anchored to a crown-like anulus. During diastole, the high aortic pressures force the leaflets to coapt, closing the valve and forming three blood-filled sinuses; two of which contain coronary arteries and facilitate blood flow. The aortic leaflets are highly specialized structures that must be compliant enough to open without resistance to blood flow, while being strong enough to withstand the repeated mechanical stresses applied throughout the cardiac cycle. In aortic stenosis, diseased valve leaflets become stiff and lose compliance, increasing the intraventricular pressure required to generate the same flow across the valve. The current model of native aortic valve pathology is thought to involve an initiation phase, where initial valvular injury leads to inflammation, immune activation and initial calcium deposition, followed by a propagation phase, involving a damaging cycle of increasing calcification activity.Due to a strong overlap between risk factors for developing atherosclerosis and aortic stenosis, the lipid-lowering effect of statins was initially investigated as a means of slowing aortic stenosis progression. Frustratingly, multiple randomized controlled trials did not demonstrate a reduction in aortic valve velocity or calcium score progression over medium-term follow-up.in vivo, researchers performed hybrid positron emission tomography combined with computed tomography (PET/CT), comparing 18F-fluorodeoxygloucose, (18F-FGD), a non-specific marker of inflammation, and 18F-sodium fluoride (18F-NaF), which preferentially binds to developing microcalcification, in a cohort of patients with varying degrees of aortic stenosis. The results demonstrated that calcification activity dominated over inflammation in aortic stenosis, particularly in the latter stages of moderate or severe stenosis  as well as clinical events .15To explore the relative contributions of inflammatory and calcific processes to aortic stenosis 18F-NaF is a potentially useful biomarker, recent advances in imaging protocols have improved image quality and reproducibility of measurements, thereby enhancing this technique. A detailed description of standardized analysis techniques have been previously outlined.18F-NaF uptake in the valve from adjacent structures such as the aorta and coronary arteries. Motion correction techniques have proved similarly important. Motion of the valve throughout the cardiac cycle produces a wider distribution of PET signal, therefore, ECG-gating that captured the valve in diastole  was employed to improve reproducibility.While the early studies described above demonstrate that 18F-NaF becomes a crucial tool for elucidating novel insights in to the pathology of aortic stenosis and bioprosthetic valve degeneration as well as an efficacy endpoint in randomized controlled trials of novel therapies.When the above advances are combined, scan–rescan measurement error falls from >60% to 10%.18F-sodium fluoride activity, the relationship between possible disease instigators and aortic valve disease can be investigated. Both lipoprotein(a) and oxidized phospholipids have been genetically linked to an increased incidence of aortic stenosis in the general population; however, whether these lipid markers are also associated with the propagation phase of aortic stenosis and faster disease progression remained uncertain.18F-NaF uptake, with this increased PET activity translating in to faster progression of the aortic valve CT calcium score, and more rapid progression of haemodynamic gradients on echocardiography.18F-NaF uptake as a primary outcome.22Using PET/CT 18F-NaF to measure disease activity and predict disease progression and clinical events in aortic stenosis, is there a role for this technique in clinical practice?18F-NaF PET/CT means that it would need to provide incremental information to established echocardiographic and CT approaches. However, the studies reported to date have suggested that CT calcium scoring provides similar prognostic and predictive information to PET.18F-NaF PET activity and CT calcium scores. While at present this argues for wider use of CT and against routine 18F-NaF PET imaging, it does provide important pathophysiological insights. In particular, it suggests that calcium in the valve leaflets encourages further calcification activity and faster disease progression, perhaps via the increase in associated mechanical stresses within the valve. The result is a vicious cycle of progressive calcification that drives the propagation phase of aortic stenosis and suggests that calcification should be the predominant target for future therapies. Two ongoing randomized controlled trials are investigating this hypothesis, assessing the effect of denosumab or alendronate  and vitamin K2  on aortic stenosis progression  developed new valve dysfunction, with two requiring urgent valve replacement and one death directly related to valve failure. Crucially, in multivariable linear regression analysis, baseline 18F-NaF was the only predictor of bioprosthetic valve dysfunction outperforming age, echocardiographic findings, CT findings, valve age and gender Electronic supplementary material 1 (PPTX 1851 kb)Below is the link to the electronic supplementary material.


Saccharomyces cerevisiae, and the fission yeast Schizosaccharomyces pombe, for deciphering the molecular mechanisms underlying chronological ageing. Automation accompanied with appropriate computational tools allowed manipulation of hundreds of thousands of colonies, generation, processing and analysis of genome-wide lifespan data. Together with barcoding and modern mutagenesis technologies, these approaches have allowed to take decisive steps towards a global, comprehensive view of cellular ageing.Cellular models such as yeasts are a driving force in biogerontology studies. Their simpler genome, short lifespans and vast genetic and genomics resources make them ideal to characterise pro-ageing and anti-ageing genes and signalling pathways. Over the last three decades, yeasts have contributed to the understanding of fundamental aspects of lifespan regulation including the roles of nutrient response, global protein translation rates and quality, DNA damage, oxidative stress, mitochondrial function and dysfunction as well as autophagy. In this short review, we focus on approaches used for competitive and non-competitive cell-based screens using the budding yeast  Two major cellular lifespan paradigms are currently utilised in the field of biogerontology: replicative (RLS) and chronological lifespan (CLS). RLS is the number of cell divisions that a mother cell can undergo until it reaches senescence and death . CLS is Saccharomyces cerevisiae) and the fission (Schizosaccharomyces pombe) yeasts have been pivotal in this aspect providing molecular insights and having huge conceptual contributions in the field. Characterising the contribution of individual mutants in ageing is a continuously active and informative activity in the field. On top of these studies, genome-wide screens have provided insights on the role of evolutionarily conserved processes and signalling pathways in ageing such as nutrient response [Organismal ageing depends on both types of cellular lifespan. Ageing hallmarks and age-related diseases can be related to depletion or pathological physiology of either dividing adult stem cell pools or differentiated postmitotic cell populations . In otheresponse , 18, proresponse , 20, mitresponse , 22 and response  opening response , glucoseresponse .A pivotal step for conducting genome-wide lifespan screens has been the availability of deletion libraries for both yeasts . ConductFollowing a multitude of environmental, nutritional and drug screens, yeast deletion libraries have been used for lifespan studies, with the budding yeast system pioneering within the field. A first step using mutant library resources has been the contribution of all non-essential genes in lifespan regulation. For his reason, a genome-wide study of genetic factors affecting chronological lifespan was devised in budding yeast  using smAcceleration and efficiency of relevant screens was achieved through automation and in combination with relevant pharmacological interventions targeting specific ageing pathways. Robotics have revolutionised cell-based screens addressing questions on toxicity, stressor and drug sensitivity or resistance as well as genome-wide genetic interactions in the presence and absence of drug treatments revealing insightful positive and negative gene relationships. Such approaches are conducted through spotting colonies on solid media . RelevanBeyond characterisation of short and long-lived mutants a quest in the field of biogerontology is to identify compounds that can increase cellular fitness and lifespan. Indeed, chemical genetic screens using yeasts have been very fruitful. A chemical genetic screen for compounds that extend the chronological lifespan of fission yeast led to the discovery of eight natural products with such properties . FurtherTwo models of chronological lifespan have been developed in fission yeast. During the glucose depletion (or stationary phase) model, cells are arrested in G2 or a mixture of G1 and G2 depending on the media used , and can survive in this state for several days , 10, 45.Caenorhabditis elegans mutants. Moreover, although a trend towards an overlap with genes enhancing replicative lifespan was observed, it was not significant. This study also showed through a screen for reduced acidification of the culture medium that 11 out of 76 deletions strains exhibiting reduced culture medium acidification were chronologically long-lived. Beyond indicating that acidification of culture media is an important determinant of survival during chronological lifespan, this work is important for the field as it has suggested that it is of benefit to assay the yeast CLS under multiple media conditions [Although the gene list of lifespan-controlling genes has been continuously growing due to multiple genome-wide genetic and chemical screens, the relationship of these genes with ageing in other model systems has not been fully addressed. Towards this issue, Burtner and colleagues  performenditions  to speciAn important aspect of genome-wide cell-based assays is the overlap of their results. In a study comparing three different ageing screens in budding yeast , among 3Efforts towards efficient determination of lifespan in a high-throughput manner without the need of CFU determination led to alternative approaches in budding yeast with the use of flow cytometry (and propidium iodide stain) . The metIn addition to the flow cytometry method as alternative to the CFU approach, fast colorimetric approaches suitable for high-throughput screens have been devised. Notably, one of these methods makes use of the reduction of the yellow tetrazolium compound MTT -2,5-diphenyltetrazolium bromide) to a purple-coloured formazan product with an absorption peak at 550 nm within live cells [Contrary to non-competitive screens where individual mutants are separately grown either in liquid media or on solid media through manual or robot-mediated pinning . Followide novo purine biosynthesis pathway, which ultimately produces IMP and AMP were among the long-lived ones [A competitive ageing assay was performed in budding yeast where samples from the ageing pool were collected at specific timepoints . Mutantsved ones . Validatved ones .An interesting screen pipeline was proposed in budding yeast that utilised fluorescently labelled deletion strains (e.g. with RFP) and wild-type/control (e.g. with CFP) , 61. Delclg1 [Beyond using existing and commercially available deletion collections, scientists have developed an insertion vector-based method of generating independent barcode-tagged fission yeast insertion mutant libraries. The approach, with the potential of generating viable mutations in both essential and non-essential genes  led to aclg1 . Clg1 prclg1 . This stet al. [ppk31  and SPBnsporter ). Furthensporter .While most ageing pool experiments involve glucose starvation similar experiments have been conducted using fission yeast and nitrogen starvation. Parallel mutant phenotyping of a prototroph version of the haploid deletion library  using BaThe clonogenic, revivability CLS assays are based on CFU determination and can be time-consuming requiring several parallel cultures, serial dilutions and multiple plating repeats followed by colony counting. Biogerontologists using cellular model systems are continuously trying to devise new reliable and user-friendly methodologies and computational tools that will allow faithful CLS determination in a medium to high-throughput manner in a plethora of conditions. Here we present a few of these tools developed for yeast lifespan analysis.Analyser   is a sofYeast Outgrowth Data Analyzer (YODA) , is an aPyphe is a package providing viability scores from phloxine B staining or colony growth curves . The proet al. [Throughout the manuscript we have referred to regrowth or outgrowth from ageing cultures and using growth parameters as a proxy for survival of the examined mutants . Traditiet al.  developeet al. . These pet al. .High-throughput screens have now revealed many genes and signalling pathways implicated in yeast chronological lifespan. In addition, mutant libraries and saturation mutagenesis in combination with chemical compound screens have uncovered life-prolonging chemicals and gene-drug genetic interactions in ageing.Many genes are having orthologues in animal models and their role in ageing is validated in further downstream analyses or separate studies. Pharmacological treatments in combination with genetic and genomic approaches such as global genetic interactions  canGenome-wide ageing screens using yeasts have identified anti-ageing and pro-ageing genetic factors and signalling pathways.Screens can be non-competitive where thousands of mutants are grown separately, or competitive where mutants are pooled, and their abundance is quantified using mutant-specific barcodes.Wet lab protocols as well as computational tools have been developed for faster lifespan assays, quantitation and appropriate statistics.


Nature, Choi et al.1 demonstrated that corticosterone, the mouse equivalent to human cortisol, controls the activity of hair follicle stem cell (HFCS) quiescence by regulating the gene expression of Gas6.In a recent paper published in 2 The hair follicle is a highly interactively regulated mini-organ that enables the study of stem cell functions. During hair formation, hair follicles undergo orchestrated and dynamic cycles of growth (anagen), regression (catagen) and quiescence (telogen). The niche of HFSCs is known as the bulge region where quiescent HFSCs are localized. HFSCs are transiently activated, and proliferate to form the hair germ. Matrix cells of the hair bulb proliferate and differentiate, elongating the hair shaft in the growth (anagen) phase. In the regression (catagen), the hair bulb and the lower part of the hair follicle regress leading to the telogen (resting) phase where bulge cells remain inactivated, and HFCSs are quiescent.3 In the telogen phase, under normal conditions, HFSC quiescence is regulated by cell intrinsic as well as extrinsic signals from the bulge niche environment. This is precisely orchestrated by Wnt and bone morphogenetic protein (BMP) signalling. In early telogen, HFCSs are silenced through BMPs, including BMP6 and fibroblast growth factor 18, secreted from Keratin 6-positive cells of the inner bulge, BMP4 from dermal papilla cells that mantain a low Wnt low niche environment and BMP2 and BMP4 from surrounding fibroblasts and adipocytes.3 Transcriptional factors such as Nfatc1 or Foxp1 are important intrinsic regulators of HFSC quiesence that control BMP signalling.2 TREM2-positive macrophages in surrounding tissues secrete oncostatin M4 which activates JAK-STAT5 signalling to mantain HFSC quiescence. During the early telogen phase, the HFSC niche environment is BMP-high and Wnt-low to mantain HFSC in quiescence. Dermal papilla (DP) cells play a critical regulatory role as they secrete various mediators in a paracrine manner either to keep HFSC inactivity or to enter the early anagen phase by secreting BMP inhibitors and transforming growth factor-beta-2, Fibroblast growth factor 7 and 10 activate Wnt signalling leading to the early anagen phase again  remained proliferative, their hair shafts and hair follicles became longer, and their hair follicles entered anagen phases more synchronized with longer cycles compared with the results obtained in mice with intact glands. To study the direct effect of corticosterone on hair follicle growth, mice were fed oral corticosterone. Hair follicles prolonged their telogen phase in contrast to that observed in mice without adrenal glands. During ageing, mice showed increased levels of corticosterone and long telogen phases could be reversed by re-entry in the anagen phase by adrenalectomy, even in old mice. Anagen entry remained intact throughout life. In a mouse model of growth receptor gene deletion, they revealed that the dermal papilla fibroblasts surrounding the hair follicle play a crucial role in connecting the corticosterone effects with the prolonged resting phases. By comparing the different mouse models, DP cells revealed a candidate gene, Growth Arrest Specific Protein 6 (Gas6), as a regulator of hair growth that is stress sensitive. In DP cells, Gas6 encodes a gamma-carboxy glutamic acid-containing secreted protein that predominantly binds to AXL, a member of the TAM -family of receptor tyrosine kinases and can be suppressed by corticosterone. Interestingly, recombinant Gas6 was also able to activate hair growth when supplemented in vitro with isolated HFSCs and in vivo via adenovirus-associated injection.Chronic stress can result in continuous hair loss. This leads to telogen effluvium where hair follicles enter a resting phase followed by considerable hair loss. Even worse, it can also result in an autoimmune condition called alopecia areata where hairs are lost due to an attack of the immune system,High levels of BMPs secreted from fibroblasts, and dermal adipocytes can promote HFSC quiescence in early (refractory) telogen. Since the effects of corticosterone remain constant, Choi and coworkers observed that Gas6 expression was opposite to BMP signalling. Gas6 expression in the dermal papilla was low in early telogen and high in late telegon. BMP suppressed the expression of Gas6, and when Noggin, a secreted BMP inhibitor, was overexpressed, increased levels of Gas6 were observed. Corticosterone (a systemic hormone) and BMP  represent two upstream signals that suppress Gas6 levels in DP. Restoration of Gas6 expression via adenovirus-associated injection was sufficient to promote HFSC activation even in a high-corticosterone environment, suggests that corticosterone regulates HFSC activity by inhibiting Gas6 expression in dermal papilla cells.This work is of interest because the orchestrated signalling process of mammalian HFSCs during maintenance and repair, already known to be quite complex, has been a primary research focus. Nevertheless, the influence of external stimuli such as corticoids , on the regulation of HFSCs, has not yet been well characterized. Chronic stress, as well as long-term corticoid therapy, has many undesirable side effects. It seems to directly impact the regulation of hair follicle stem cells via non-autonomous, paracrine effects keeping those cells in both, a prolonged quiescent state and prolonged telogen phase. This results in a noticeable stop of hair growth and potential subsequent telogen hair loss, which is a further stressor for patients.


Postmenopausal breast cancer is the most common obesity-related cancer death among women in the U.S. Insulin resistance, which worsens in the setting of obesity, is associated with higher breast cancer incidence and mortality. Maladaptive eating patterns driving insulin resistance represent a key modifiable risk factor for breast cancer. Emerging evidence suggests that time-restricted feeding paradigms (TRF) improve cancer-related metabolic risk factors; however, more flexible approaches could be more feasible and effective. In this exploratory, secondary analysis, we identified participants following a low-glucose eating pattern (LGEP), defined as consuming energy when glucose levels are at or below average fasting levels, as an alternative to TRF. Results show that following an LGEP regimen for at least 40% of reported eating events improves insulin resistance (HOMA-IR) and other cancer-related serum biomarkers. The magnitude of serum biomarkers changes observed here has previously been shown to favorably modulate benign breast tissue in women with overweight and obesity who are at risk for postmenopausal breast cancer. By comparison, the observed effects of LGEP were similar to results from previously published TRF studies in similar populations. These preliminary findings support further testing of LGEP as an alternative to TRF and a postmenopausal breast cancer prevention strategy. However, results should be interpreted with caution, given the exploratory nature of analyses. High obesity rates among women in the United States and worldwide are leading to a continued rise in obesity-related cancers, most notably postmenopausal breast cancer , which iIn a seminal Phase II feasibility study of a 6-month intensive lifestyle intervention conducted in postmenopausal overweight and obese women at increased risk for breast cancer, Fabian et al. demonstrated that weight losses of at least 10% effectively reduced serum biomarkers, including insulin resistance (HOMA-IR), at a magnitude that favorably modulated benign breast tissue biomarkers ,13. WhilIntermittent fasting paradigms have become increasingly popular among researchers and health-conscious individuals. These eating paradigms aim to align meal-timing with circadian rhythms. Restricting the consumption of energy intake to a daily timespan of 4–10 h  enhances synchronization between the central circadian clock (synchronized by light) and peripheral circadian clocks (entrained by nutrient intake) ,20. DesyEating when pre-prandial  glucose levels are low (“low-glucose eating pattern”) is an evidence-based strategy to improve maladaptive eating patterns. Research shows that eating without physiological hunger is a modifiable health risk behavior associated with excessive weight gain and increased metabolic risk ,32. Cons2) [The modification of glucose eating patterns by GGE is feasible ,36 and h2) . Insulin2) ,41. GGE 2) , suggestThe key aspect of the GGE intervention is eating when glucose is low, defined as under one’s personalized glucose threshold. The goal of the current study is to explore the impact of low- vs. high-glucose eating patterns on changes in body weight and the selected serum biomarkers of breast cancer risk after 16 weeks and compare these results with those reported in recent TRF studies in similar populations of older women and with the intensive lifestyle intervention conducted by Fabian et al. in postmenopausal overweight and obese women at increased risk for breast cancer. The findings of the current study are intended to support further testing of GGE to promote a low-glucose eating pattern as a strategy to reduce breast cancer risk in postmenopausal women. Therefore, this exploratory, secondary analysis aims to examine the potential effect of a low-glucose eating pattern on postmenopausal breast cancer risk.Project Take Charge  was a 16N = 50) were predominantly White, non-Hispanic older women who were well-educated; lived in the Houston, Texas, metropolitan area; and had a BMI > 27 kg/m2. This study was approved by the Institutional Review Board and registered at clinicaltrials.gov (NCT03546972). Women provided informed consent prior to initiating the study.As part of a randomized feasibility study, GGE was added to a 16-week version of the DPP intervention that targeted women at risk of postmenopausal breast cancer  . ParticiThe Project Take Charge protocols were fully described elsewhere . BrieflyN = 19 women.The women enrolled in Project Take Charge additionally provided blinded CGM data using Dexcom G5  at week 0 (baseline), week 8, and week 16 (post-intervention) for up to 10 days at a time. From the collected CGM data, the mean amplitude of glycemic excursions (MAGE) was calculated using EasyGV  as a meaWomen were categorized into eating patterns based on week 16 dietary and CGM data. Those who consumed at least 40% of their recorded meals when their pre-prandial glucose levels were below their personalized threshold will be referred to as following a “low-glucose eating pattern (LGEP)”; whereas those who ate less than 40% of their meals when pre-prandial glucose was below their threshold will be referred to as following a “high-glucose eating pattern (HGEP)”. The threshold of 40% eating events was chosen to define the groups post hoc to maximize between-group differences in reductions of HOMA-IR at 16-weeks. LGEP and HGEP were quantified at all three time points using blinded CGM was calculated as the percentage of reported eating occasions where a participant’s glucose was equal to or less than their computed, personalized threshold .p-values or other tests of significance were not reported due to the secondary and exploratory nature of this analysis. TRF studies were identified using MedLine. Only those articles with predominantly older women at or near an age consistent with the onset of menopause (>45 years) were considered.Outcomes between the original intervention groups were similar. Specifically, the intervention groups (DPP-only vs. DPP + GGE) had comparable changes from baseline to 16 weeks in weight (−5.0 kg vs. −4.9 kg) and HOMA-IR (−0.3 vs. −0.4). Therefore, for this analysis, the data from the DPP-only and DPP + GGE groups were combined. The 16-week changes in weight and serum biomarkers were compared between women in the LGEP and HGEP groups using SPSS version 28. The means, standard deviations, medians, and ranges are reported here and compared to findings from Fabian et al.  and publN = 19) were predominantly older, White, non-Hispanic, college-educated women, with a BMI in the obese range at baseline. Out of the 19 women with CGM and dietary data, eight (42%) were identified as following a low-glucose eating pattern at post-intervention (week 16) and categorized in the LGEP group. Women in the LGEP were comparable demographically to those in the HGEP group with modestly greater BMI (ater BMI .At baseline (prior to starting the intervention), nearly 70% of reported eating events occurred when glucose was above fasting levels (“HGEP”). By week 8 in the LGEP group, the majority of reported eating events occurred when glucose levels were below the personalized thresholds (approximately 60%). This change in the LGEP group was maintained at week 16. In the HGEP, fewer reported eating events occurred when glucose levels were below personalized thresholds from baseline to mid-intervention to post-intervention .Women in the LGEP group experienced notable improvements in adiponectin, HOMA-IR, fasting insulin, and glycemic variability  . These cWhen results of this study are compared to those from published TRF interventions in similar populations, it suggests that LGEP induces nominally larger average weight loss and reductions in HOMA-IR and fasting insulin that is not explained by changes in energy intake . The comThis study supports the potential efficacy of a low-glucose eating pattern (LGEP) to improve metabolic and cancer risk biomarkers, including insulin resistance, in older women. Importantly, these data support a viable alternative to TRF for improving health outcomes. Furthermore, the positive metabolic effects of an LGEP might be achieved without eating all meals under the personalized glucose threshold, further supporting the flexibility of LGEP and the robust effects of LGEP in relation to metabolic health. Specifically, following LGEP at ≥40% of eating events is associated with significant improvements in weight and serum markers of cancer risk over time. These findings are similar to previously reported findings of the GGE intervention, where modest protocol adherence was associated with clinically relevant weight loss and improvements in eating behavior . HoweverComparing our results to those from TRF studies suggests that LGEP could be as effective or more effective at reducing the risk of postmenopausal breast cancer. We hypothesize that GGE could effectively teach women to follow LGEP to achieve these outcomes. The results shown here suggest it is worthwhile to conduct a clinical trial aimed at comparing the effects of these interventions on biomarkers of postmenopausal breast cancer. Key features of such a trial should include adherence for a range of population groups and durability of effects after the intervention has ceased. A previous pilot study showed that GGE is acceptable from a patient perspective and outlined adherence barriers and enablers . FurtherOf note, this and the related Project Take Charge studies to exemplify the benefit of using biological feedback (here glucose levels) to motivate and support effective behavior change . While systematic reviews demonstrated the utility of glucose monitoring in obesity research , limitedStrengths of this study include objective quantification of LGEP through passive glucose monitoring and the range of biomarkers tested. However, this analysis is limited by our small, homogeneous sample, which limits the generalizability of our findings. Our findings are most appropriate for hypothesis driving rather than hypothesis testing, and results should be interpreted with caution given the exploratory nature of analyses. Furthermore, the TRF studies were of shorter duration than the current study, which could have implications on comparing the magnitudes of observed effects. It is also unclear why women in Project Take Charge, who were randomized to the DPP-only intervention, changed to their LGEP without additionally receiving the GGE intervention. Future DPP intervention research could test the robustness of these findings. While other clinical trials have tested GGE as a standalone intervention ,36, follThis exploratory analysis of the impact of LGEP on weight and metabolic markers offers direction for the next steps in testing GGE as an intervention to prevent postmenopausal breast cancer. The adherence goal of 40% offers a feasible target for future GGE interventions and potential for health benefits, most critically a reduction in risk of postmenopausal breast cancer.