Glioblastoma, a grade IV astrocytoma, is regarded as the most aggressive primary brain tumour with an overall median survival of 16.0 months following the standard treatment regimen of surgical resection, followed by radiotherapy and chemotherapy with temozolomide. Despite such intensive treatment, the tumour almost invariably recurs. This poor prognosis has most commonly been attributed to the initiation, propagation, and differentiation of cancer stem cells. Despite the unprecedented advances in biomedical research over the last decade, the current in vitro models are limited at preserving the inter- and intra-tumoural heterogeneity of primary tumours. The ability to understand and manipulate complex cancers such as glioblastoma requires disease models to be clinically and translationally relevant and encompass the cellular heterogeneity of such cancers. Therefore, brain cancer research models need to aim to recapitulate glioblastoma stem cell function, whilst remaining amenable for analysis. Fortunately, the recent development of 3D cultures has overcome some of these challenges, and cerebral organoids are emerging as cutting-edge tools in glioblastoma research. The opportunity to generate cerebral organoids via induced pluripotent stem cells, and to perform co-cultures with patient-derived cancer stem cells (GLICO model), has enabled the analysis of cancer development in a context that better mimics brain tissue architecture. In this article, we review the recent literature on the use of patient-derived glioblastoma organoid models and their applicability for drug screening, as well as provide a potential workflow for screening using the GLICO model. The proposed workflow is practical for use in most laboratories with accessible materials and equipment, a good first pass, and no animal work required. This workflow is also amenable for analysis, with separate measures of invasion, growth, and viability. Glioblastoma is the most common and most aggressive primary brain tumour, characterised by high recurrence rates and exceptionally poor prognosis . StandarFor glioblastoma to establish beyond the primary site within the brain, this subpopulation of cells must be able to self-renew, generate differentiated tumour cells, and spawn a heterogeneous tumour . GlioblaProof-of-principle genetic studies have shown that blocking the self-renewal of GSCs leads to prolonged survival in GSC patient-derived mouse efficacy studies . HoweverRecent studies have enabled drug screening using patient-derived glioblastoma 3D spheroid cultures ,17. UsinA major challenge in glioblastoma research is to develop disease models that mirror the cellular complexity, aggressive nature and treatment resistance observed in vivo. Previous research models for drug testing and development have relied on an oversimplified approach which has resulted in many of the drugs investigated in traditional preclinical models  yielding suboptimal results in clinical trials, culminating in an FDA approval rate as low as 3% for new oncologic therapies .Established commercial cell lines grown in vitro in 2D adherent culture, offer only modest real-world disease relevance, due to the lack of spatial organisation, cellular heterogeneity, and interaction with non-cancer support cells . TypicalAlternatively, transplantation of patient-derived tumour cells into the brains of immune-compromised mice (xenograft models) provides an in vivo-like model system to investigate the dynamic interplay between GSCs, which drive tumourigenesis, and the non-cancer microenvironment . Unlike Recent advances in cell culture technology have led to the development of organoid models that mimic in vivo organ development, increasing our ability to study cellular diversity and complex tissue structures . The estThe cancer stem cell (CSC) concept of cancer, first proposed more than four decades ago, states that tumour growth is comparable to healthy tissue generation, i.e., it is fuelled by a small population of dedicated stem cells . DecadesIn 2003, Singh et al. were the first to isolate CSCs from human brain tumours . These cThrough histological classification, glioblastoma has been traditionally classified as an astrocytoma, although the precise cell type from which the tumour originates is still a controversial issue. Some experts argue that the glioblastoma origin is a subpopulation of neural stem cells, while others propose that it is derived from differentiated astrocytes  43]. It. It43]. GSCs are thought to be the initiators of tumour recurrence and the major contributor to the aggressive nature of glioblastoma . They haGiven their vital role in glioblastoma, it seems logical to specifically target GSCs to achieve a durable treatment for glioblastoma. However, therapies aimed at intrinsic mechanisms of GSC proliferation have so far offered only limited success, partly due to the lack of suitable experimental model systems that recapitulate the complex invasive behaviour of GSCs in the human brain. The ability to understand and manipulate complex cancers such as glioblastoma–requires clinically relevant models which encompass the complexity of these tumours and can be used in drug development and testing . TherefoFortunately, the use of patient-derived tumour cells for the development of tumour spheroids and human cerebral organoids to both characterise and model glioblastoma has gone some way to fulfilling this need. Human cerebral organoids are powerful in vitro systems that recapitulate many aspects of human brain development and function .The GSC subpopulation has often been associated with invasion as well as radiotherapy and chemotherapy resistance . The intGSCs isolated from tumour biopsies have been shown to recapitulate the heterogeneity of the patient’s tumour when differentiated in culture or upon xenotransplantation into immune-deficient mice ,55. WhenPatient-derived GSCs can be grown and sustained under specific culture conditions in vitro; with media supplemented with growth factors such as epidermal growth factor and fibroblast growth factor-2 . These cFormation of human cerebral organoids involves stem cells, either iPSCs or AdSCs, which are sequentially exposed to specific and appropriate exogenous signals to stimulate the developmental process. These conditions allow stem cells to differentiate and aggregate to first form an organ bud/embryoid bodies, and over longer culture, form a cerebral organoid. Mature cerebral organoids can contain differentiated astrocytes, mature neuronal cell types, and even surprisingly microglia-like cells. Ramani et al., surprisingly observed microglial cells and astrocytes in their organoids . Even thDue to their potential utility for drug discovery and development, there are many organoid-glioblastoma models that have been developed recently. Hubert et al.  culturedAlong a similar vein are Bioprinted glioblastoma organoids, which are generated through patient-derived dissociated glioblastoma cells combined with a decellularised porcine brain ‘bioink’ composed of extracellular matrix proteins. On top of this, a layer of human umbilical vein endothelial cells is printed in the same bioink . This moGenetically modified cerebral organoids such as neoplastic cerebral organoids (neoCOR) have mutations introduced to induce the expression of oncogenes to cause tumourigenesis within developing iPSC-derived organoids ,65. NeoCA novel approach to overcome this disadvantage has been pioneered by the laboratories of Howard Fine and Amanda Linkous. This approach involves co-culture of patient-derived GSCs in the form of tumour spheroids, with iPSC-derived human cerebral organoids . The autThe behaviour of cancer cells in this system not only mimic the original tumour, but also maintain key genetic aberrations of the patient’s original tumour . EGFR amUnlike animal brains, human cerebral organoids provide a more species-specific microenvironment which is essential for GSCs to display their inherent characteristics . These m®), or a standard confocal microscope and imaging analysis software (such as TASI) [When glioblastoma spheroids are cultured in basement membrane extracts such as Matrigel or Cultrex, tumour cell invasion can occur and is able to be measured . Spheroias TASI) . The sigas TASI) . Additioas TASI) . This isMoreover, all of these techniques are starting to become more accessible, in high-throughput formats, using specific 96- and 384-well plates  and the Taking advantage of the GLICO model for higher-scale compound screening has not yet been achieved , likely Utilising a genetically engineered luciferase-based assay to measure proliferation, Linkous et al.  were ablThe ability to understand and manipulate cancers such as glioblastoma requires the disease models to be clinically and translationally relevant, replicating the cellular heterogeneity of such cancers. Therefore, brain cancer research models need to aim to recapitulate GSC function, whilst remaining amenable for analysis. Our proposed workflow of preliminary drug screening using glioblastoma spheroids as part of the GLICO model utilises patient-derived glioblastoma cells and cerebral organoids, providing the unique capability to investigate tumour–brain interactions. Despite their usefulness, the versatility and reliability of brain organoids in modelling glioblastoma remains to be standardised, with cost and time to culture being some of the greatest limitations. The GLICO model suffers from similar weaknesses as other organoid models, with the absence of vasculature and immune cells, which are necessary for accurate recapitulation of the tumour microenvironment. However, recent research is making significant steps to ameliorate these limitations. Our proposed model for smaller-scale drug screening is particularly practical for use in most laboratories, or for those wanting to move into 3D cell culture/organoid research; with accessible materials, and accessible equipment, good for the first pass, and no animal work required.


Tau is an important protein for maintaining the shape and normal function of nerve cells. There are many diseases that are identified by changes in this protein, yet examination of tau in these diseases shows robust differences in tau modifications. A question arises from these observations on whether tau can be used as a therapeutic target. In this review, we aimed to provide a general overview of tau-physiology and pathophysiology in neurodegenerative diseases and describe the current approaches for diagnosis and experimental/clinical trials. This review is intended to enhance the understanding of graduate students specializing in neurobiology.Tau, a member of the microtubule-associated proteins, is a known component of the neuronal cytoskeleton; however, in the brain tissue, it is involved in other vital functions beyond maintaining the cellular architecture. The pathologic tau forms aggregates inside the neurons and ultimately forms the neurofibrillary tangles. Intracellular and extracellular accumulation of different tau isoforms, including dimers, oligomers, paired helical filaments and tangles, lead to a highly heterogenous group of diseases named “Tauopathies”. About twenty-six different types of tauopathy diseases have been identified that have different clinical phenotypes or pathophysiological characteristics. Although all these diseases are identified by tau aggregation, they are distinguishable based on the specific tau isoforms, the affected cell types and the brain regions. The neuropathological and phenotypical heterogeneity of these diseases impose significant challenges for discovering new diagnostic and therapeutic strategies. Here, we review the recent literature on tau protein and the pathophysiological mechanisms of tauopathies. This article mainly focuses on physiologic and pathologic tau and aims to summarize the upstream and downstream events and discuss the current diagnostic approaches and therapeutic strategies. Tauopathies are a group of progressive neurodegenerative diseases characterized by tau protein inclusions in the human brain. Clinically, there are twenty-six different types of tauopathies that are recognized by complicated neurodegenerative symptoms, including dementia. A series of cognitive/behavioral and memory deficits manifest in individuals affected by tauopathies . From a According to the World Health Organization, nearly 50 million people are currently suffering from dementia, and each year, around 10 million new cases are diagnosed with dementia. It is predicted that by 2050, the number of patients will approximately reach 140 million worldwide. Due to the debilitating nature of dementia, these patients require constant care, which significantly impacts their careers, their families and society. In 2017, WHO proposed a “Global action plan on the public health response to dementia 2017–2025” in which dementia has been identified as a public health priority .,47.46,47Oxidation is another form of tau PTMs that has been identified as the inducer of tau fibrilization under oxidative stress conditions. This can affect five tyrosine residues in the 2N4R tau isoform, at positions 18, 29, 197, 310 and 394 . Under oPost-translational modification of tau, particularly tau phosphorylation and acetylation, can trigger a tau condensation mechanism known as liquid-liquid phase separation (LLPS) in neurons. This mechanism is reported in both physiological and pathological conditions and is regulated by electrostatic interactions between oppositely charged regions of tau isoforms or between tau proteins and RNA. LLPS leads to the formation of liquid droplets containing high levels of concentrated tau, which can electrostatically interact with other tau isoforms or RNA molecules. Although the mechanistic details of LLPS are poorly understood in neurons, it has been shown to accelerate neuronal tau fibrilization and facilitate neurotoxicity in tauopathies ,52. There are approximately 57 neurodegenerative-related mutations on the MAPT gene, which were mainly discovered in pedigrees with familial frontotemporal lobar degeneration (FTLD). Predominantly, inherited mutations in the MAPT gene (FTDP-17) are associated with neuronal and glial tau inclusions in patients with FTLD . The speThe second identified cause of pathogenic tau is chronic traumatic encephalopathy, which is also listed as a type of progressive neurodegenerative tauopathy. Mechanistically, brain injury can induce tau cleavage, its aberrant phosphorylation and aggregate formation. These processes can adversely affect axonal microtubule organization, resulting in tau-microtubule dissociation and subsequent tau accumulation in axons. The key link between brain injury and tau hyperphosphorylation is attributed to the reduction in alkaline phosphatase levels, which is mainly responsible for tau dephosphorylation. All these pathophysiological conditions can ultimately trigger chronic inflammation, apoptosis and neuronal degeneration in brain tissue . AccumulIn addition to the aforementioned stimulators, pathogenic tau can also be induced in people with metabolic syndrome. Examination of in vitro and in vivo reports as well as results from post-mortem human patients depict a strong relationship between tauopathy and neurons containing increased levels of phosphorylated insulin receptor substrate-1. This results in impaired insulin signaling, which can drive pathogenic tau aggregation through reducing phosphoinositide 3-kinase–Akt activity and activating GSK3β. This in turn induces tau hyperphosphorylation and its dissociation from microtubules  Figure .Additionally, pathogenic tau formation is shown to be triggered through neuron-to-neuron transmission in a prion-like manner. Various mechanisms are identified as the stimulators of this type of pathogenic tau aggregation. For instance, the pathogenic tau spreads trans-synaptically from one cell to another or through “exosomes” which are present in CSF and plasma ,60. ThesThe mechanistic target of rapamycin (mTOR) has been identified as another potential driver of pathogenic tau formation. This serin/threonine protein kinase plays a crucial role in neuronal growth and maintenance. In neurons, mTOR regulates prominent cellular functions including autophagy, signal transduction, metabolism and cytoskeletal dynamics . HyperacThe association of pathogenic tau with neuronal toxicity and disruption of neuronal networks, as well as glial damage, has been well documented in the literature. However, the exact mechanisms and physiological events that link pathogenic tau formation to neurotoxicity and cell death remain unknown. As AD is well-characterized microscopically by the combined presence of intracellular NFT aggregates and extracellular Aβ plaques, inevitably, the tauopathy-related studies are vastly focused on AD models. The impact of tau pathology on the cellular cytoskeleton and its negative effect on intracellular traffic of synapse machinery were discussed previously. We know that hyperphosphorylated tau can stabilize dendritic microtubules and induce synaptic dysfunction . These pImpaired axonal transport is another identified pathologic event that is attributed to tau toxicity. “Long-range transport” in neurons is mediated by kinesin motors, which can be inhibited by tau protein. Mechanistically, tau can limit cargo travel distance by reducing kinesin velocity . Under pDrosophila model, in which downregulation of thioredoxin-1 or superoxide dismutase was associated with increased tau hyperphosphorylation -fThe complexity and multifactorial nature of tauopathies is the major obstacle to better understanding the pathology and designing new therapeutic interventions for slowing the disease’s progress. The current clinical tools cannot be used for the detection of early-stage diseases, and the diagnosis can only occur in the late stages, when the currently prescribed drugs can only treat the symptoms. There is an urgent need for developing early diagnosis tools and developing disease-modifying drugs and, accordingly, many preclinical and clinical trials are currently in progress. Since AD is the most common form of tauopathy, the efforts to discover novel therapeutic interventions have also been mostly focused on this disease. However, considering the complexity of tau isoforms and inclusion bodies, it is not clear whether an effective drug for AD-associated tauopathies can also cure other forms of tauopathies. Whether treatments targeted for tauopathies may need to be personalized remains to be investigated. The timing of the application of any potential treatments represents the next challenge in human diseases. Notably, the pathologic events in tauopathies, including tau hyperphosphorylation and protein aggregation, occur a long time before the possibility of a clinical diagnosis, when experimental treatments are proven to be ineffective. These limitations urge the need for the discovery of novel biomarkers to allow diagnosis at earlier stages of the diseases. Two categories of tau biomarkers have been identified in CSF and blood. In CSF, increased levels of phosphorylated tau at residue 217 (p-tau217) and p-tau235 as well as p-tau231 are shown to be correlated with the early stages of AD , while pNumerous studies have demonstrated evidence of considerable achievement in designing therapeutic interventions for tauopathies. Proper identification of druggable pathophysiological mechanisms in tauopathies is fundamental to this achievement. Some of the potential mechanisms that can be targeted for this purpose include: (1) tau PTMs, particularly tau hyperphosphorylation, by phosphatase modifiers and kinase inhibitors, (2) targeting cytoskeleton/nuclear disruption and genomic architecture dysfunction and (3) reducing tau expression levels with small interfering RNA (siRNA) and antisense oligonucleotides (ASOs). Additional approaches include reducing the levels of plasma tau, inhibiting the aggregation of different forms of pathogenic tau such as PHFs, NFTs and small oligomeric tau aggregates, as well as enhancing tau clearance by active and passive tau immunization  Figure .Passive immunotherapy has been the focus of tau clinical trials, with the aim of slowing the neurodegenerative process through preventing the spread of pathogenic tau and removing its extracellular aggregations. The main barrier to finding an effective treatment for clinical purposes is finding a suitable epitope on tau protein that is commonly present in all patients despite their genetic background or the specific type of tauopathy disease. Active tau immunotherapy has also been explored. AADvac1 is a therapeutic tau vaccine candidate that has shown successful reduction of neurofilament light chain (NfL) in plasma and CSF in AD patients by recognizing a 12-amino acid sequence (KDNIKHVPGGGS) in tau MTBR. Reported results from its phase I clinical trial have raised hope for tauopathy treatment. According to these results, AADvac1 leads to reduced cognitive decline and lower brain atrophy in AD patients in mild to moderate stages ,92,93. ODrosophila and mouse models of tauopathy have revealed a new aspect of tauopathy that is characterized by nuclear envelope invagination and changes in gene expression. Interestingly, nuclear envelope invagination has also been confirmed in human post-mortem brain tissue, which results in the re-emergence of endogenous ancient retroviruses. Calorie restriction in Drosophila effectively attenuated the endogenous retroviral expression [Drosophila [Dementia is an umbrella term for many neurodegenerative diseases. As we age longer, the rate of dementia is exponentially increasing, imposing a significant emotional cost on the patients’ families and a financial burden on human societies. Tauopathies are a major contributor to dementia, and despite extensive research, the exact underlying neurodegenerative mechanisms remain to be elucidated. Tauopathy diseases present extensive neuropathological and phenotypical differences, which impose considerable challenges in developing diagnostic and therapeutic strategies. Despite significant transformative achievements in the field of tauopathy, several fundamental questions remain to be addressed. Novel drugs are in clinical trial phases with some promising outcomes; however, proving their safety and efficacy will require large human studies that may take several years to complete. Preclinical studies in pression . The proosophila  and in tosophila  as well osophila . While t


This specificity makes dsRNA a biosafe candidate for integration into the global conservation initiative. Our interdisciplinary perspective conforms to the UN’s declaration, and is facilitated by the Earth BioGenome Project, an effort valuable to RNAi development given its utility in providing whole-genome sequences, allowing identification of genetic targets in crop pests, and potentially relevant sequences in non-target organisms. Interdisciplinary studies bringing together biocontrol-enhancing techniques and RNAi are needed, and should be examined for various crop‒pest systems to address this global problem.Habitat loss and fragmentation, and the effects of pesticides, contribute to biodiversity losses and unsustainable food production. Given the United Nation’s (UN’s) declaration of this decade as the UN Decade on Ecosystem Restoration, we advocate combining conservation biocontrol-enhancing practices with the use of RNA interference (RNAi) pesticide technology, the latter demonstrating remarkable target-specificity  Crops dominate about 11% of Earth’s land, and these areas are associated with rapid losses of species , a trendAnother global initiative, the Earth BioGenome Project, aims to whole-genome sequence all extant eukaryotic species on Earth over the next decade, and provide open access to these genomes . Given tAgroecological communities are susceptible to changes in and around crops, likely due to multiple interacting stressors , includiOur aim here is to promote new ways to approach integrated pest management (IPM) research, a complex field that aims to identify holistic strategies that combine biological, physical and cultural tactics for optimizing the control of all classes of pests , to achieve sustainable production of crops in a way that minimizes health- and environmental hazards and potentially reduces costs, through a decision-based process . IPM parin planta  or exogenous  delivery of dsRNA molecules to host-plant tissues (Cautious use of pesticides is a pillar of IPM . However tissues .Bt) cotton and corn , GreenLight Biosciences (United States), RNAissance (United States) and Syngenta (Switzerland). h aphids . The useh aphids . On the via inadvertent coprophagy. This potentially useful application of RNAi in crop protection requires further investigation into its utility for various crop‒pest systems.Of notable utility to increasing cost-effectiveness of dsRNA production is the use of cell-free systems, as well as the use of dsRNase-deficient bacteria strains engineered to produce dsRNA-expressing genes. The latter can be cultured on growth media to produce large volumes of dsRNA-expressing bacteria, which can then be applied to plants as heat-killed bacteria, or as lysate after using a lysis buffer, or even as live cells. via the release of type I interferons   . This caApis mellifera (via other consequent food web associations). The hopeful outcome of acquiring whole-sequence information on a great diversity of species will be our ability to safeguard an unprecedented diversity of species in conservation initiatives; this high-resolution evidence of biosafety may prove to be reliably generated using bioinformatics tools . Choosing appropriate species for RNAi risk assessment depends on the crop species of interest and the trophic interactions existing in and around the focal agroecosystem.RNAi’s role in protecting biodiversity lies in the environmental safety associated with both transgenic and spray approaches. While risk assessment of dsRNA is still under conceptual development , resultsellifera , represein vivo metabolism of dsRNA. For reliable confirmation of RNAi risk assessment results, ring testing of potential- or final market products should be conducted when possible, due to potentially high variability between research laboratories, as well as between non-target species populations.The scarcity of high quality open access sequences currently impedes our ability to safeguard non-target organisms with certainty. However, it is critical to exploit currently available transcriptomes, representing unique taxonomic lineages, as templates for designing species-specific dsRNAs; and subsequently examine RNAi susceptibility across these taxa. In both insects and fungi, dsRNA uptake and RNAi efficiency can vary between- and within taxonomic groups , and botThe environmental fate of dsRNA presents an additional advantage to potentially exposed non-target organisms, in that dsRNA is a natural molecule that is rapidly degraded in soils, waterbodies and plants . The eliThe UN Environment Programme (UNEP) and the Food and Agriculture Organization (FAO) of the UN have acknowledged the urgency to restore damaged ecosystems; and that the required restoration measures are fundamental to sustainability goals including security of food and water resources, biodiversity conservation, poverty eradication and combating climate change. UNEP and FAO recognize the role of numerous restoration initiatives already led by citizens, farmers, businesses and governments. They also recognize that rural- and especially indigenous communities have been models for ecosystem preservation, and that building on their knowledge is vital for success of this decade’s global initiative. However, new technologies will play a pivotal role in achieving sustainability goals, especially when considering the current momentum in both technological development and human population growth. It is our view that combining conservation biocontrol-enhancing techniques with the use of both transgene- and spray-based RNAi technologies  represenOf notable importance for biocontrol of many crop pests is the continuous presence and services provided by specialist natural enemies such as parasitoids. It is conceivable that large-scale use and high efficacy of an applied pesticide  can reduce pest (host) populations to levels insufficient for sustaining local and/or regional parasitoid populations. This can be combated by preserving and/or restoring refuge habitats for pest species and their co-evolved parasitoids, both in and around the focal agroecosystem, a tactic that would help to suppress development of resistance, be applicable to all crop‒pest systems, and conform to the UN’s ecosystem restoration initiative.Given dsRNA’s sequence-specificity and the potential this holds for managing pathogens and animal pests without directly affecting non-target organisms, examining the benefits  of combining conservation biocontrol practices with RNAi technology represents promising and unexplored territory. Greenhouse studies could be the first step to study the combined, potentially synergistic efficacy of these- or similar IPM approaches; augmented biocontrol is already a common technique for regulating pests in greenhouses, and could act as a useful model for conservation biocontrol. Being limited to closed systems, greenhouse studies are well suited for disentangling complex interactions between techniques while avoiding potential confounds. Ultimately, greenhouse studies would guide subsequent investigations into the use of such strategies in practical field scenarios, and provide insight for the betterment of greenhouse- and vertical farm crop protection. Studies examining the benefits of combining conservation biocontrol practices with RNAi technology should be conducted with regard to various crop‒pest systems, in order to maximize scientific value in a global context.IPM has been the focus of much research for decades, yet there remains a global insufficiency of strategies mobilized for ensuring sustainability in both crop productivity and agrobiodiversity conservation. For example, regulators such as the EU define IPM policies, yet do not adequately support IPM practice . This isRNAi technology is not a stand-alone solution to achieving global food security and agrobiodiversity conservation. However, adopting sufficient conservation biocontrol measures, and combining these techniques with RNAi technology, may significantly increase the success of sustainable crop protection, given the low environmental risk of RNAi products compared to conventional pesticides. Non-RNAi plant biotechnologies with narrow-spectrum activity  represent additional techniques that may have roles in facilitating sustainable IPM alongside conservation biocontrol measures. These could also be examined with regard to compatibility with biocontrol-enhancing measures. The current push for reducing humanity’s ecological footprint through the UN’s global initiative aimed at restoring damaged ecosystems, together with the recent advancements in RNAi research and utility, calls for innovative solutions relating to the interdisciplinary perspective introduced here.


Excessive weight gain during pregnancy is associated with complications for both the mother and her infant including gestational diabetes, hypertensive disorders, operative delivery, and long-term obesity. A healthy diet during pregnancy promotes healthy gestational weight gain and determines fetal epigenetic programming in infants that impacts risk for future chronic disease.This project will examine the impact of grocery delivery during pregnancy on the weight, diet, and health outcomes of young pregnant women and their infants.A three-arm randomized controlled trial design will be performed. A total of 855 young pregnant women, aged 14-24 years, from across the state of Michigan will be enrolled and randomized equally into the three study arms. Participants in arm one (control) will receive usual care from the Special Supplemental Nutrition Program for Women, Infants, and Children (WIC); arm two will receive WIC plus biweekly grocery delivery; and arm three will receive WIC plus biweekly grocery and unsweetened beverage delivery. Weight will be assessed weekly during pregnancy, and total pregnancy weight gain will be categorized as above, below, or within guidelines. Additionally, dietary intake will be assessed at three time points , and pregnancy outcomes will be extracted from medical records. The appropriateness of pregnancy weight gain, diet quality, and occurrence of poor outcomes will be compared between groups using standard practices for multinomial regression and confounder adjustment.This study was funded in April 2021, data collection started in December 2021, and data collection is expected to be concluded in 2026.This study will test whether grocery delivery of healthy foods improves weight, diet, and pregnancy outcomes of young moms with low income. The findings will inform policies and practices that promote a healthy diet during pregnancy, which has multigenerational impacts on health.ClinicalTrials.gov NCT05000645; https://clinicaltrials.gov/ct2/show/NCT05000645DERR1-10.2196/40568 The United States has one of the highest adolescent pregnancy rates among high-income countries with almost 800,000 15- to 24-year-old women giving birth in 2021 . Among yA healthy diet during pregnancy promotes healthy weight gain among mothers  and impaGrocery delivery is a well-established and inexpensive service that removes logistical barriers to obtaining healthy foods and beverages. There are numerous grocery delivery services  that help make obtaining fresh food and groceries more convenient for those who cannot or do not shop in person. However, grocery delivery is predominantly used by affluent middle-aged women ,31, and To address this critical problem, we developed Special Delivery, a program that uses grocery delivery to overcome these logistical barriers to a healthy diet during pregnancy among pregnant young women with low income to prevent excessive gestational weight gain.The aim of this randomized controlled trial is to assess whether grocery delivery of healthy foods to the homes of young women during pregnancy will promote healthy pregnancy weight gain. The study will also assess secondary outcomes of diet quality, pregnancy complications, and delivery outcomes. Our long-term goal is to identify effective interventions to support a healthy diet during pregnancy and promote healthy weight gain among pregnant young women with low income.The primary hypotheses to be tested are that the home delivery of healthy foods will promote healthy weight gain during pregnancy and that the addition of unsweetened beverage delivery will promote more healthy weight gain. The findings will inform policies and practices that promote a healthy diet during pregnancy, which may have multigenerational impacts.This study is a three-arm randomized controlled trial with an additive parallel design. Enrollment in the study is rolling and starts as early as possible in the pregnancy (by 21 weeks’ gestation). The intervention period begins at enrollment and continues to the end of pregnancy/birth . Data coSpecifically, we will be testing the following hypotheses:Mothers who receive free grocery delivery of WIC-approved healthy foods will have healthier weight gain during pregnancy, higher-quality diets, and fewer perinatal complications than peers with usual WIC benefitsMothers who receive free grocery delivery of WIC-approved healthy foods plus unsweetened beverages will have healthier weight gain during pregnancy, higher-quality diets, and fewer perinatal complications than peers with usual WIC benefits and home delivery of WIC-approved foods onlyThe rationale for the study is that grocery delivery will address barriers to accessing healthy foods and will therefore facilitate a healthier diet and prevent excessive weight gain during pregnancy.This study was approved by the University of Michigan Institutional Review Board (HUM00190614).The study will include 855 young moms who are pregnant with their first child. Those with previous births are excluded to limit the impact of variable past pregnancy experiences and habits on the intervention. Participants will be included from across the state of Michigan based on the criteria below.Inclusion criteriaYouth (ages 14-24 years)Enrolled in WICGestational age ≤20 weeksSMS text message capabilityHealthy singleton pregnancyNulliparousConsume SSBsLiving within delivery zone of a grocery delivery serviceExclusion criteriaNon-English speakingParticipants who live at the same addressHigh-risk pregnancy requiring specialized careParticipants in this study will be referred from WIC offices, online study recruitment sites, or other clinical and community organizations that serve young pregnant women across the state of Michigan. Interested women complete an online screening and enrollment survey or call the study line for assistance in completing the survey. Consent is obtained verbally or in completion of the online enrollment form and a copy of the consent form is emailed to participants.Based on nationally representative studies of weight gain in pregnancy , we antiUpon completion of baseline screening and assessments, 855 pregnant young women will be randomly assigned to either the control group or one of the two experimental groups. We will use county-level block randomization to assign participants in a 1:1:1 ratio to the three study arms. The treatment assignments will be created and stored in the Consulting for Statistics, Computing and Analytics Research randomization tool managed by the University of Michigan. No treatment assignments will be made until after collection of baseline data . At this time, the participant will be notified of their arm assignment and additional details required for grocery delivery  will be collected from participants in arms 2 and 3. All personnel who assess study outcomes are masked to group assignment. Participants and staff implementing the grocery delivery are not masked to group assignment.All groups will receive usual WIC nutritional assessment and counseling benefits, including monthly nutritional counseling sessions based on a state-approved curriculum with trained nutritionists and peer counselors ,35. ThesThe control group (arm 1) will receive usual WIC counseling, as described above, and food benefits loaded onto their Electronic Benefits Transfer (EBT) card to use in person at approved grocery stores. At completion of study participation, arm 1 participants will receive a 1-year prepaid subscription to a grocery delivery service of their choosing.Participants in arm 2 (WIC counseling + food delivery) will choose WIC-approved foods from an online survey, which will be purchased and delivered to their home biweekly by the study team via a grocery delivery service at no cost to the participant. Fruit and vegetable options will be offered as well as whole grains  and proteins  as listed in the WIC-approved foods booklet  36]. Th. Th36]. VegetablesBaby carrots, broccoli, bell peppers, cauliflower, radishes, snap peas, green peas, avocados, tomatoes, cucumbers, celery, mushrooms, spinach, corn.FruitsApples, pears, oranges, clementines, grapes, bananas, nectarines, peaches, strawberries, blueberries, raspberries, blackberries, watermelon, pineapple.Proteins and grainsYogurt, cheese, milk, whole grain bread, whole grain buns, whole grain tortillas, brown rice, whole grain cereal, beans, lentils, peanut butter, eggs.Unsweetened beveragesSeltzer water, unsweetened tea, Spindrift, sparkling tea, bottled waterParticipants in arm 3 (WIC counseling + food plus unsweetened beverage delivery), the home delivery of healthy foods, as described above, will be supplemented with the inclusion of participant-selected unsweetened beverages, in quantities comparable to their current SSB intake (based on self-reported consumption). Unsweetened beverages are intended to replace current SSB consumption. Because SSBs contribute to a significant number of excess calories among youth, this group includes unsweetened beverage delivery to further promote healthy weight gain. The unsweetened beverage choices will include bottled water (flavored or unflavored), unsweetened seltzer water (flavored or unflavored), and unsweetened teas. Artificially sweetened beverages and SSBs will be excluded.Our study is designed to be low burden, so participants only have to maintain minimal communication via text, phone call, or email to remain in the study. In addition, participants are incentivized with Amazon gift cards for various components of the study. Participants in all arms receive US $20 for completing the enrollment process, baseline survey, first diet recall, and medical record request form. Participants in all arms will receive US $20 for each additional diet survey completed on their own throughout the course of the study . For each week participants use their study provided scale, they will receive US $1, and an additional US $10 is given for the end-of-study interview. Women in the intervention arms receive biweekly grocery deliveries during their pregnancy, while women in the control arm receive free access to 1 year of a grocery delivery service at the end of their pregnancy. The resulting maximum incentive for participation is US $160 for participants who enroll at 10 weeks’ gestation, complete all study questionnaires, and remain active for the remaining 7 months of their pregnancy.At baseline, interested and eligible participants will complete the consent process to participate and respond to baseline measures, described in detail below. In addition to the baseline surveys, participants will be asked to share weight, physical activity, and diet recall data at specified times throughout the study period. The complete list of outcomes, covariates, and timeline of assessments is shown in All sociodemographic data will be collected upon enrollment online or by phone with the study team. These include date of birth , self-reported race and ethnicity, highest level of education, free/reduced lunch status while in school  , full adPrepregnancy weight and height will be obtained by self-report and verified by medical record review after birth to determine prepregnancy BMI and identify target weight gain zones. Weekly weight measurements during pregnancy will be measured via a BodyTrace scale and recorded automatically using cellular connectivity (no Wi-Fi or cellular plan required) . The scaDietary intake will be measured with paired assessments using the Automated Self-Administered 24-hour (ASA24) dietary recall survey -48. ThisThe American College of Obstetricians and Gynecologists recommends an exercise program of moderate intensity exercise for at least 20 to 30 minutes per day on most or all days of the week for pregnant women . To measParticipants will complete the validated Pulvers Body Image  scale atParticipants will be prompted to complete questions adapted from the Eating Disorder Examination Questionnaire Short and Youth Eating Disorder Examination-Questionnaire at baseline and end of study to identify perceptions of loss of control of eating, binge eating, weight/shape overvaluation, and body dissatisfaction -61. AddiSMS text messages will be sent to participants in all arms using Textizen.com, a GovDelivery platform. Participants will receive 2 to 3 SMS text messages per week reminding them to confirm their delivery, place their order, step on their scale, and respond to physical activity questions. These SMS text messages will also prompt participants to share any updates they may have regarding their pregnancy, allowing participants an opportunity to report on infant birth, miscarriage, or any other outcomes. In addition, participants will be prompted via SMS text message to complete their diet recall surveys throughout their pregnancy.Medical records will be requested from participants’ prenatal and delivery care sites within a few weeks of delivery using the release forms completed at enrollment. Data collected will include all health care visits during pregnancy and up to 12 weeks postpartum, including prenatal visits (diagnoses and complications), nutrition visits (number and content of visits), emergency department visits, inpatient stays, WIC counseling visits (number and type: nutrition vs breastfeeding), quantity of WIC foods redeemed, laboratory results , vital signs , medications prescribed, complications during prenatal care , infant birth weight, and gestational age at delivery.A phone interview will be performed after each woman gives birth to collect qualitative insights into participants’ experience with grocery/beverage delivery; the impact of delivery on their diet; overall WIC experiences; and end-of-pregnancy body preference, self-reported weight gain, physical activity, any prenatal or childbirth complications, plans for breastfeeding, and food insecurity.To understand the effect of grocery delivery on pregnancy weight gain, diet quality, and health outcomes, weight will be measured weekly, dietary quality will be assessed twice per trimester, and perinatal outcomes will be assessed at the end of the pregnancy. All analyses will follow the intention-to-treat principle. Prior to analysis, assessment of missing data will be completed and appropriately imputed using multiple imputation methods .Our primary outcome is appropriate weight gain in pregnancy, which is based on prepregnancy BMI and includes weekly weight gain and total weight gain recommendations. Total weight gain at birth will be categorized as above, below, or within the Institute of Medicine guidelines ,33. MultThe trajectory of weight gain behavior, using each week’s categorized weight gain (within guidelines vs not), will additionally be assessed as the outcome in a clustered logistic regression model fit using a generalized linear mixed model framework. The study arm is the primary between-participant factor, while time (week) is the main within-participant covariate. The interaction term between time and arm is also a factor of interest, as it will allow us to assess changes in weight gain behaviors over time between study arms. A random participant-level intercept will account for within-participant clustering, and a random slope for time will be investigated to account for additional between-participant variability. Additional covariates and effect modifiers included in these regression models will be chosen using the same methods as before.Our secondary outcome is diet quality as measured by the HEI score based on two ASA24 measurements at baseline and in the second and third trimesters ,66. A coPrenatal complications, delivery complications, and birth weight  are also secondary outcomes that will be assessed. Comparison of the proportion of participants with complications between study arms will be analyzed using a logistic regression model in a manner analogous to the primary outcome of appropriate weight gain in pregnancy.The end-of-study interview transcripts will be analyzed using standard qualitative techniques -69. Two Finally, we will conduct a cost analysis focused on costs from the payer perspective during the pregnancy period. All costs will be adjusted to current dollars and will represent average cost per participant. Our analysis will estimate the costs of delivering the intervention, including cost of grocery delivery service, increased costs for WIC due to increased redemption of food benefits , print aThis study was funded in April 2021, data collection started in December 2021, and data collection is expected to be concluded in 2026. No results are available as of manuscript preparation.This protocol describes a randomized controlled trial to evaluate whether grocery delivery of healthy WIC-approved foods supports healthy pregnancy weight gain for young pregnant women with low income. We will additionally evaluate if the grocery delivery impacts diet quality, pregnancy complications, and delivery outcomes. We anticipate that the young pregnant women in the two intervention groups will have better rates of appropriate pregnancy weight gain, improved diet quality, and lower rates of pregnancy and delivery complications.Results from this study can be used to inform United States Department of Agriculture policies on online use of WIC benefits as well as to provide evidence about the cost-effectiveness of national implementation of grocery delivery for WIC beneficiaries . These oThere are several key strengths to this protocol. This work uses a low-cost well-established service to enhance an existing government program which enables the results to inform policies to improve the implementation of WIC services and other programs for vulnerable populations. COVID-19 has also led to a surge in online grocery ordering, which enhances the generalizability of this study . AdditioLimitations of this study are that it does not perfectly emulate the real-life application of integrating grocery delivery into WIC services, as study personnel are ordering and managing the delivery process, which reduces the generalizability of the results to program-based implementation. Specifically, participants request the groceries from the study team who then place the orders and manage the deliveries. Additionally, participants are able to access and use their WIC food benefits in addition to groceries provided, and it does not directly replace this benefit.Ultimately, this study will provide rigorous evidence regarding the impact of grocery delivery of healthy foods and unsweetened beverages among pregnant young women with low income and their infants. The findings from this study will provide invaluable information about the potential benefits of incorporating grocery delivery in WIC and about the costs to implement, sustain, and expand such a program.


This perspective article provides an overview of the impact of mindfulness meditation (MM) on social and moral behavior. In mindfulness research, prosocial behavior has been operationalized as helping behavior, altruistic redistribution of funds, reparative behavior, or monetary donation. Studies concerning moral behavior are still scarce. Despite inconsistent evidence, several studies found a beneficial effect of mindfulness on prosocial outcomes . However, since the employed tasks were reward-based, participants’ decisions also directly affected their own payoff by reducing it. Crucially, MM also affects self-control circuitry and reduces reward-seeking behaviors and reward salience by making rewards less tempting. We have discussed evidence suggesting how challenging it may be to dissociate the specific weight of enhanced other-oriented motivation from one of the decreased monetary reward salience in explaining meditators’ behavior. Future higher-quality studies are needed to address this open issue. The scientific interest in mindfulness meditation (MM) has significantly increased in the last two decades due to its well-documented beneficial effects on physical  and psycNeuroimaging evidence shows that MM might induce neuroplastic changes in the structure and the function of brain regions involved in attentional processes, memory, emotion regulation, self-awareness, exteroceptive and interoceptive body awareness, and self-control . A summaAlthough MM has classically investigated the individual domain, recent investigations have tried to understand whether the beneficial effects of mindfulness practice can extend individual boundaries, reaching the domain of social and moral traits and behaviors.Morality refers to a system of norms, values, and customs adopted by a specific cultural group to guide socio-moral behavior . Moral bN = 554), the authors found positive correlations among DM, moral sensitivity, moral identity, and prosocial tendencies. In a randomized controlled experiment (N = 99), they investigated the effect of an 11-week mindfulness intervention on moral identity and prosocial behavior in participants who underwent the training (N = 49) and in the wait-list control group (N = 50). The results showed that mindfulness practice improved self-compassion, also affecting prosocial behavior in high moral identity participants (N = 171). They found that DM predicted moral responsibility but not moral judgment. These studies suffer from methodological limitations, such as the employment of self-reports or hypothetical scenarios instead of the measurement of actual behavior in a morally relevant context.Recently, it was proposed that mindful states may impact the ability to detect morally relevant information and, consequently, moral behavior . Nonetheicipants . Small aProsocial behavior can be defined as any action that benefits one or more people other than the actor  and is cProsociality can be assessed by means of different measures , such asMeta-analytical evidence on the link between mindfulness and prosocial behavior reported mixed findings . LubertoIn mindfulness research, prosocial behavior has often been operationalized as helping behavior , altruisHere, we highlighted a potential problem in the interpretation of the effects of mindfulness on socio-moral behavior, namely, the employment of behavioral tasks that involve some kind of incentive or reward for the participants see . A summaPrevious evidence highlighted that CM practice may directly affect prosocial behavior but see . In addiA similar behavioral pattern emerged in studies investigating altruistic redistribution of money. Other studies investigated the impact of mindfulness on donation and charitable behavior. Recently, To summarize, some of the above-reviewed studies showed a beneficial effect of MM, CM, and LKM trainings on several prosocial outcomes (despite the studies investigating donation behavior reporting the most inconsistent findings). Since the employed tasks were reward based, this translates into a higher propensity to spend or give away money for the sake of other individuals after various types of training.As briefly mentioned in the introduction, MM also impacts an individual’s cognitive functioning  from decreased individual reward salience . In other words, does mindfulness induce people to give money to others for their sake or make money less tempting, making it easier to give it away?It is worth noting that not all studies investigating the effect of mindfulness on prosocial behavior adopted reward-based tasks. Prosocial behavior relies on several mental processes, such as socio-cognitive and socio-affective ones . A recenTo prevent interpretation biases, future studies should opt for experimental designs that parallelly manipulate both other-oriented motivation and monetary reward salience. They should also investigate the dynamic development of both processes, since they may both positively affect the socio-moral stance but with different timings or trajectories. Finally, future research should investigate whether the prosocial effects of distinct mindfulness training activities may derive from different psychological mechanisms: an increase in other-oriented motivation for LKM/CM and an enhancement in self-control for MM practice.The original contributions presented in this study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.All authors listed have made a substantial, direct, and intellectual contribution to the work, and approved it for publication.


Investigation of this aspect may not only alter the approaches of BC diagnosis and prognosis, but may also open a new avenue in using ncRNA-based therapeutics for BC treatment by targeting different metabolic pathways.Cancer cells reprogram their metabolisms to achieve high energetic requirements and produce precursors that facilitate uncontrolled cell proliferation. Metabolic reprograming involves not only the dysregulation in glucose-metabolizing regulatory enzymes, but also the enzymes engaging in the lipid and amino acid metabolisms. Nevertheless, the underlying regulatory mechanisms of reprograming are not fully understood. Non-coding RNAs (ncRNAs) as functional RNA molecules cannot translate into proteins, but they do play a regulatory role in gene expression. Moreover, ncRNAs have been demonstrated to be implicated in the metabolic modulations in breast cancer (BC) by regulating the metabolic-related enzymes. Here, we will focus on the regulatory involvement of  In. InncRNAer cells . On the er cells . A studylncRNA RPPH1 expression in breast cancer cells. They observed obvious changes in breast tumor size in vivo after transfecting shRNA–RPPH1. In other words, silencing lncRNA RPPH1 downregulates PKM2 gene expression by an indirect attachment with miR-122  technology can be a helpful approach in lncRNA-based therapies against cancers like breast cancer. In this strategy, usually exogenous or mimic double-stranded RNAs, such as short interfering RNAs (siRNAs) and short hairpin RNAs (shRNAs), are employed for special gene knockdown ,164. Thi miR-122 , which ic target . In anotactivity . As disccRNA H19 , lncRNA NA NEAT1  and lncRcRNA 030  can exerCircRNAs are involved in triggering and developing different tumors, including those of BC. It is reported that one of the primary activities of circRNAs is their function, as miRNA sponges and acts as an endogenous modulator of miRNA activity [circRAD18 facilitates cell proliferation and glycolysis through the upregulation of HK2 by sponging miR-613, indicating that circRAD18 could be valuable a therapeutic target for BC treatment [circYY1 sponges miR-769-3p, reducing cell proliferation, migration and the invasion of reprogramed breast cancer cells through inhibiting LDHA2-reduction mediated glycolysis [activity . Xing etreatment . In anotycolysis . All of ncRNAs and metabolic pathways, including glycolysis. There is a slight doubt that ncRNA has appeared as a versatile mediator in regulating the various aspects of BC metabolism. Several different members of ncRNAs, including lncRNAs, miRNAs and circRNAs, were found to play regulatory roles in BC metabolism, particularly in the glycolysis pathway, and in the metabolism of glucose, lipids and glutamine. Metabolic pathway regulation of BC by ncRNAs can enhance or suppress the tumorigenic process through the regulation of rate-limiting enzymes that mediate the glycolysis, lipids and glutamine metabolisms. Clarifying the special role of ncRNAs in BC with metabolism changes, may open a window for ncRNA-based regulators that are personalized and accuracy against BC. Interestingly, several miRNAs indicate promising outcomes in periclinal examination. However, the clinical application of miRNA-based therapies is limited by their unwanted and unessential interactions with targets that are not involved in the BC process. Unlike miRNAs, the relation of lncRNAs and circRNAs in BC, especially the mechanisms of breast tumor, to glucose, lipid and glutamine metabolism remains at an early stage and requires a plethora of further studies to establish their importance in treating BC. Since lncRNAs and circRNA can exert their regulatory effect to modulate the miRNAs involved in metabolic enzyme expressions, research to attain lncRNAs or circRNA-based therapies are very convincing. Overall, more studies into the mechanisms and actions of ncRNAs in BC could not only permit the finding of additional prognostic indicators, but could also be useful for the exploration of novel therapeutic methods.Metabolism reprograming is the hallmark characteristic of BC cells. In reprogramed BC cells, anaerobic glycolysis is mostly active to provide macromolecules that are essential for the rapid assembly of new cancer cells. In addition, lipid and glutamine metabolisms are changed to meet their needs. All these changes occur through a dysregulating expression of key enzymes that control metabolic pathways. Therefore, applying a strategy that uses the characteristics of tumor metabolism for reprogramed BC regulation and treatment might be promising. Although some metabolic modulating drugs have been used in clinical trials, the consequent results were not completely satisfactory. Researchers are now investigating to find certain therapeutic approaches that target the critical enzymes involved in different types of metabolic pathways. Therefore, it is crucial to study the relevance between


Astrocytes are known to actively participate in synaptic communication by forming structures called tripartite synapses. These synapses consist of presynaptic axon terminals, postsynaptic dendritic spines, and astrocytic processes where astrocytes release and receive transmitters. Although the transcription factor cyclic AMP response element (CRE)-binding protein (CREB) has been actively studied as an important factor for mediating synaptic activity-induced responses in neurons, its role in astrocytes is relatively unknown. Synaptic signals are known to activate various downstream pathways in astrocytes, which can activate the CREB transcription factor. Therefore, there is a need to summarize studies on astrocytic intracellular pathways that are induced by synaptic communication resulting in activation of the CREB pathway. In this review, we discuss the various neurotransmitter receptors and intracellular pathways that can induce CREB activation and CREB-induced gene regulation in astrocytes. Astrocytes are glial cells of the brain that perform diverse functions, including supportive functions such as brain barrier transport, energy molecule transport, ion concentration control, and neurotransmitter recycling. In the last two decades, the role of astrocytes, in addition to their supportive functions, has been largely studied. Molecular and behavioral studies have shown that astrocytes are major participants in information processing and learning in the brain. The astrocytic process establishes a structure morphologically and functionally similar to neuronal synapses, called tripartite synapses, which bidirectionally affect synapses using release factors and contact-dependent factors. Through tripartite synapses, astrocytes respond to neuronal activity and various neurotransmitters. A single astrocyte can occupy up to 100,000 synapses, suggesting that astrocytes may be the center for converging various neuronal inputs and modulating synapses . Many stvia the cAMP/PKA pathway; however, other factors such as phosphatidylinositol 3-kinase (PI3K)/Akt can also phosphorylate CREB . In Aplysia, serotonin (5-HT)-induced long-term facilitation (LTF) of synapses activates the cAMP second messenger pathway and phosphorylates CREB . Subsequ2+/calmodulin (CaM) kinases are also responsible for CREB activation  to phosphatidylinositol 3,4,5-trisphosphate (PIP3), leading to Akt activation  pathway in astrocytes in a calcium-dependent manner , which hGene expression by direct manipulation of the CREB transcription factor will further explain the importance of astrocytic participation in synaptic activity and cognitive function. Various transcriptional changes were induced by CREB activation in astrocytes . BDNF isAlthough CREB may or may not be activated by calcium signaling itself, CREB activity may modulate astrocytic calcium signaling. With regard to astrocytic calcium signaling, a study by The CREB transcription factor is involved in various neurodegenerative disorders, including Alzheimer’s disease. Reactive astrocytes have recently gained attention as a cause of neurodegenerative diseases, and CREB expression in reactive astrocytes has been reported to be neuroprotective . High GFWe summarize various types of receptors and pathways involved in synaptic communication that may activate CREB in astrocytes and briefly discusses the consequences of CREB activation. Adenosine and adrenergic receptors are GPCRs mainly studied for neuron-astrocyte communication and subsequent CREB activation, which is mediated by the cAMP-PKA pathway. Although other types of receptors, such as RTKs and Notch receptors, have been documented, these studies have scarcely focused on the CREB pathway. Different receptors are involved in separate downstream pathways; however, it is yet to be determined whether they participate in CREB activation. Finally, this review summarizes CREB-mediated gene expression in astrocytes. It is to be noted that only the genes that are known to be important for astrocytic and synaptic functions were selected for discussion, and there are many more genes regulated by CREB activation involving complex mechanisms, which should be examined thoroughly. In summary, the evidence presented in this mini-review suggests the importance of CREB activation in astrocytic communication with neuronal synapses and the possible pathways that mediate such effects.JK wrote the initial draft in consultation with B-KK. JK and B-KK reviewed and edited the manuscript. Both authors contributed to the writing of this review manuscript.


One of the main characteristics of cancer cells is their incredible plasticity, which is possibly the origin of tumor resistance to diverse therapeutics. During cancer treatment and in particular with targeted therapies, tumor cell evolution can be divided into three phases. First, the tumor shrinking phase characterized by the dramatic tumor mass decrease. Second, the residual disease phase during which persistent cancer cells are mostly “dormant,” and tumor mass remains undetectable at the radiological level. Third, the tumor escaping phase in which the reprogrammed genetic features of residual cancer cells cause an explosive tumor progression. One of the major questions regarding this stepwise evolution process is whether the surviving malignant cells are reprogrammed and/or selected during drug treatment, thus serving as a reservoir for the subsequent resistance and eventual tumor relapse. With the great advances in tumor resistance research during the last decade, non-genetic adaptive resistance mechanisms emerge as a quintessential component of cancer evolution. Multiple adaptive traits involved in drug resistance have been revealed, such as the capacity for tumor cells to alter their epigenetic states, rewire the intracellular signaling network, or co-opt extrinsic cell-to-cell symbiosis. In this Research Topic collection, we present the state-of-art in non-genetic adaptive drug resistance in cancer and cover topics that include tumor heterogeneity, accessible chromatin rearrangement in resistant cancer cells, mechanisms of drug efflux, gene rearrangement participating in cancer resistance, and important mechanisms that regulate genomic repeat elements in cancer.Rabé et al., explored the heterogeneity of a glioblastoma cell line under temozolomide treatment. Using an elegant multicolor barcoding strategy, the authors demonstrated that the resistance to temozolomide does not result from clonal selection, but rather that dynamical persistence is induced by the temozolomide treatment per se. They identified a four-gene signature encoding for proteins with extracellular activities that could be implicated in remodeling the microenvironment, thus inducing the survival and the regrowth of resistant cells.Wang et al. explored doxorubicin dependent non-genetic resistance in breast cancer. They showed that chromatin accessibility in resistant cancer cells dictates a specific gene expression landscape, in which putative therapeutic targets could be further investigated to improve therapeutic strategies.At the epigenetic level, Nicholson et al. identified PKCϵ as an important player of cancer cell aggressiveness and resistance in Acute Myeloid Leukemia (AML). PKCϵ was involved in the upregulation of the efflux pump P-GP, leading to a poor clinical prognosis. In consequence, their work highlights the importance of the drug efflux pump in cancer cell adaptation to treatment.Feng et al. identified a novel serine/threonine kinase 3 (STK3)-ALK rearrangement in a patient with advanced non-small cell lung cancer (NSCLC). Interestingly, this patient was resistant to the ALK inhibitor crizotinib but responsive to alectinib, a different drug that also inhibits ALK. This work underscores the potential benefit of large-scale, unbiased approaches  in dissecting cancer plasticity and proposing alternative strategies to overcome drug resistance.In a case report, Kermi et al. offered a systematic overview of the role of genomic repeat elements including retrotransposons in cancer aggressiveness and resistance to therapies. Their work opens a new route toward the exploitation of transposable elements that could be the “Achilles’ heel” of tumor cells, especially in the context of immunotherapy treatments. In fact, recent studies have shown that dysregulation of transposable elements could potentiate cancer cell immunogenicity by increasing neoantigen presentations.Taken together, this Research Topic collection shows the multilevel complexity of non-genetic adaptive resistance in cancer treatment and offers enthusiastic prospects in this domain. With the advent of novel single cell analysis technologies and lineage tracing, the underlying biological mechanisms of non-genetic tumor resistance will be further explored in a higher resolution. Future research will be further conducted in topics like drug resistance-associated immunosurveillance, adaptive genetic instability, cell invasiveness, and multicellular symbiosis.We would like to thank all the authors, co-authors, and reviewers who accepted our invitation and contributed to this Research Topic, and we sincerely hope that the data and information conveyed will be beneficial to the cancer research community.


Iron is necessary for essential processes in every cell of the body, but the erythropoietic compartment is a privileged iron consumer. In fact, as a necessary component of hemoglobin and myoglobin, iron assures oxygen distribution; therefore, a considerable amount of iron is required daily for hemoglobin synthesis and erythroid cell proliferation. Therefore, a tight link exists between iron metabolism and erythropoiesis. The liver-derived hormone hepcidin, which controls iron homeostasis via its interaction with the iron exporter ferroportin, coordinates erythropoietic activity and iron homeostasis. When erythropoiesis is enhanced, iron availability to the erythron is mainly ensured by inhibiting hepcidin expression, thereby increasing ferroportin-mediated iron export from both duodenal absorptive cells and reticuloendothelial cells that process old and/or damaged red blood cells. Erythroferrone, a factor produced and secreted by erythroid precursors in response to erythropoietin, has been identified and characterized as a suppressor of hepcidin synthesis to allow iron mobilization and facilitate erythropoiesis. However, Fe2+ can promote Fenton-type reactions generating highly reactive oxygen species (ROS) that cause lipid peroxidation and DNA damage, eventually leading to cell death by ferroptosis . Im. Im49]. Decreased oxygen availability caused by hypoxia or compromised oxygen transport/delivery induces HIF-1 dependent erythropoietin (EPO) synthesis in the kidney. Increased circulating EPO levels stimulate erythroid activity in the bone marrow, which is accompanied by higher expression of erythroferrone (ERFE) by erythroid precursors. By interfering with the BMP signaling pathway, ERFE downregulates hepcidin production in the liver. In turn, the lower hepcidin levels permit ferroportin (FPN)-dependent iron release into plasma out of the iron recycling (spleen macrophages) and iron absorption  compartments. The higher iron availability in the bloodstream satisfies the enhanced needs for red blood cell production.The role of hepcidin as a key regulator appears well established, but where does the iron needed to sustain a massive increase in erythropoietic drive come from?Optimization of iron transport mechanisms, including increased intestinal absorption and fast and efficient recycling of iron derived by processed RBCs, can provide iron to the erythroid bone marrow. Indeed, it has been shown that in healthy volunteers exposed to hypoxemic conditions (rapid ascent to 4559 m) hepcidin is repressed, and duodenal iron transport in biopsy specimens is rapidly upregulated . These cIt is less well established whether and how iron metabolism is regulated in residents at high altitude who are chronically exposed to low oxygen . More thRegarding the molecular mechanisms underlying hepcidin repression under conditions of enhanced erythropoietic activity, though a study proposed HIF1α-dependent repression of hepcidin transcription , subsequInitial studies showing (1) strong downregulation of hepcidin in mice injected with EPO ,52,73, , a member of the transforming growth factor-β superfamily, and twisted gastrulation factor 1 (TWSG1) have been suggested but not confirmed, and the hepcidin suppressing capacity of PDGF-BB  should bThe hormone erythroferrone (ERFE) appears to be the major suppressor of hepcidin expression in response to higher erythropoietic activity and EPO, i.e., the erythroid regulator hypothesized by Finch  16]. It. It16]. The role of ERFE in basal erythropoiesis was indicated by studies in ERFE knockout mice, which did not show abnormalities in iron and hematological parameters at baseline. However, they were not able to promptly suppress hepcidin in response to erythropoietic stress . On the ERFE plays a relevant role also in pathological settings involving altered erythropoiesis; in particular ERFE induction was found in various types of mouse anemia , and ERFERFE may also be important in AI. Studies in mice lacking ERFE showed that AI caused by injection of inactivated bacteria was more severe than in control animals and was associated with higher hepcidin levels and iron sequestration, thus suggesting that ERFE antagonizes AI . HoweverThe absence of an apparent phenotype in ERFE knockout mice under physiological conditions suggests the possible existence of other unidentified regulators linking the erythroid compartment with hepcidin expression in the liver. For example, it has been demonstrated that erythroid TfR1 is an ERFE-independent inhibitor of hepcidin in response to expanded erythropoiesis .In recent years, several key aspects of the regulatory system by which the erythroid compartment, which consumes the largest share of circulating iron, controls iron trafficking to obtain iron for Hb synthesis have been unraveled. ERFE produced by erythroid precursor cells is currently the best-characterized hepcidin inhibitor, and mechanistic insights into its interaction with the hepatic BMP/SMAD pathway have been provided. Given the role of dysregulated ERFE production in hematologic disorders, manipulation of ERFE levels may represent an innovative therapeutic approach for diseases such as ineffective erythropoiesis with iron overload or AI. The characterization of the mechanism based on the ERFE-hepcidin–FPN axis by which systemic iron homeostasis is modified has seen considerable advances. However, other mechanisms based on local iron supply for the development of erythroid precursors in the BM may play a relevant role in this context.In the BM, erythroblastic island macrophages (EIMs) are surrounded by erythroid progenitors, adhere to erythroblasts, favor proliferation and differentiation and phagocytose extruded nuclei, thus providing an environmental niche for RBC production . Since EThe situation might be even more complex; in fact, a recent study has shown that iron availability in the BM is relevant for the development of other hematopoietic precursors, as it controls hematopoietic stem cells (HSCs) self-renewal vs. differentiation decisions . Under sIn conclusion, present and future advances in our knowledge of the interaction between iron metabolism and erythropoiesis are expected to translate into better therapies for patients with disordered erythropoiesis.


Microorganisms naturally present at environmental contaminated sites are capable of biodegrading, biotransforming, or removing contaminants in soil and groundwater through bioremediation processes. Cleanup strategies and goals for site remediation can be effectively achieved by bioremediation leveraging the capabilities of microorganisms to biotransform contaminants into lesser or non-toxic end products; however, reproducible success can be limited by inadequate design or performance monitoring. A group of biological analyses collectively termed molecular biological tools (MBTs) can be used to assess the contaminant-degrading capabilities and activities of microorganisms present in the environment and appropriately implement bioremediation approaches. While successful bioremediation has been demonstrated through previously described lab-scale studies and field-scale implementation for a variety of environmental contaminants, design and performance monitoring of bioremediation has often been limited to inferring biodegradation potential, occurrence, and pathways based on site geochemistry or lab-scale studies. Potential field-scale application of MBTs presents the opportunity to more precisely design and monitor site-specific bioremediation approaches. To promote standardization and successful implementation of bioremediation, a framework for field-scale application of MBTs within a multiple lines of evidence (MLOE) approach is presented. The framework consists of three stages: (i) “Assessment” to evaluate naturally occurring biogeochemical conditions and screen for potential applicability of bioremediation, (ii) “Design” to define a site-specific bioremediation approach and inform amendment selection, and (iii) “Performance Monitoring” to generate data to measure or infer bioremediation progress following implementation. This framework is introduced to synthesize the complexities of environmental microbiology and guide field-scale application of MBTs to assess bioremediation potential and inform site decision-making. There is growing interest in applying natural approaches to contaminated site management, such as bioremediation which may have lower energy or resource consumption as compared to mechanical or chemical remediation approaches . BioremeDuring the past 20 years, molecular biological tools (MBTs) have been increasingly utilized due to scientific advancements and decreased analytical costs, to directly assess microbiological processes at environmental sites . MBTs, sWhile prior publications have focused on the advantages of employing MBTs in concert with contaminant chemistry and geochemistry evaluations to reduce site uncertainties and better characterize subsurface microbiology , it has In this perspective, a framework is introduced to synthesize the complexities of environmental microbiology and guide field-scale application of MBTs to assess bioremediation potential and inform site decision-making. This framework consists of following a staged process and a multiple lines of evidence (MLOE) approach to meet site-specific objectives associated with contaminant biodegradation or biotransformation using field-scale data. Example application of this framework can be found in another article in this research topic issue .2) (3–), oxidized manganese (Mn3+/4+), oxidized iron (Fe3+), sulfate (SO42–), and carbon dioxide (CO2). Since utilizing different electron acceptors yield varying energies , microorganisms prefer to utilize the electron acceptor with the highest energy gain first resulting in a preferential use sequence of O2 > NO3– > Mn3+/4+ > Fe3+ > SO42– > CO2  . The ele2– > CO2 . A schemAt the time of writing this perspective, commercially available and scalable MBTs which can be more readily applied at field-scale to support site management decision-making within a MLOE approach are nucleic acid-based analyses that include qPCR, reverse transcriptase qPCR (RT-qPCR), and next generation sequencing (NGS) of the 16S rRNA gene.Dehalogenimonas spp.  which reduces benzoyl-coenzyme A  , key funnas spp. . The preRT-qPCR is a variation of qPCR, which can be used to quantify gene expression  indicative of biological activity. The occurrence of mRNA expressed from genes linked to biodegradation indicates that the associated contaminant-degrading microorganisms are metabolically active and carrying out biodegradation processes . While pNGS of the 16S rRNA gene can be used to identify microorganisms in a sample and assess the composition of the microbial community. The 16S rRNA gene is evaluated to identify and assess prokaryotes (bacteria and archaea) at contaminated sites. NGS can most relevantly be used when gene targets linked to contaminant biodegradation  are not known, and to further screen trends in community composition to identify genera or species with potential contaminant-degrading capabilities . These dIn addition to these nucleic acid-based MBTs, there are other approaches that can be leveraged to measure or infer contaminant biodegradation, such as proteomics, metabolomics, and isotope-based methods. While proteomics and metabolomics measuring enzymes or intermediates are less often currently used at contaminated sites, expanded use of these MBTs in the future is anticipated. Further, isotope-based methods, such as compound specific isotope analysis (CSIA), are currently considerably used at contaminated sites and can be considered where applicable .The framework structure  proposedAt the onset of applying this framework, historical site data should be reviewed to understand the nature, extent, trends of contamination and associated site risk-drivers . HydrogeThe objective of the assessment stage is to identify potential biodegradation or biotransformation processes to inform the suitability of bioremediation. The assessment stage is where field-scale MBT data may be first generated to develop or refine the site biogeochemical CSM. While the assessment stage within this framework is not to be confused with project site characterization and is applicable across the project lifecycle where bioremediation may be considered , the generation of MBT data should be done intentionally to support answering site-specific questions or achieving site-specific objectives .A sampling plan should be developed and implemented in alignment with SSBOs, CSM, and regulatory requirements. Samples should be collected at target locations across the area(s) of interest related to contaminant(s) concentration and geochemical gradients and include unimpacted background location(s) . The goaSubsequently generated contaminant, geochemical, and MBT data can be interpreted through spatial and/or temporal comparative analysis to support determination of bioremediation potential. MBT data interpretation should be aligned with previously developed MLOE approaches and/or MNA guidance, and should consist of assessing contaminant trends for direct or indirect evidence of biodegradation, geochemical conditions to identify biodegradation mechanisms and potential electron acceptor/donor limitations of biological activity, presence and abundance of contaminant-degrading microorganisms or genes, and the inferred extent of intrinsic biodegradation . This apIf the results of Stage 1 suggest SSBOs have not been met nor determined infeasible to meet, the framework proceeds into the design stage (Stage 2). When a natural bioremediation strategy is posited to accomplish site remediation objectives, the generated contaminant, geochemical, and MBT datasets from Stage 1 should provide sufficient, conclusive evidence to support occurrence of intrinsic bioremediation across the area of interest. In addition to demonstrating natural bioremediation, the Stage 1 MLOE datasets should guide the monitoring program design including selection of sample locations  and sampling parameters to assess occurrence of natural bioremediation as a mechanism of natural attenuation.Dehalococcoides spp. and stimulated complete reductive dechlorination of tetrachloroethene (PCE), trichloroethene (TCE) and daughter products cis-1,2-dichloroethene and vinyl chloride to non-toxic end product ethene , lactate, formate], electron acceptor , nutrients , pH, temperature}, and/or microbial cultures . Additionally, Stage 1 data should be used to support the estimation of amendment dosing, location and frequency based on observed biogeochemical processes and stoichiometry. Field-scale pilot testing is recommended to be performed to refine amendment dosing and frequency, and field-scale feasibility and effectiveness prior to full-scale implementation. Geochemical parameters are utilized to monitor the biogeochemical effects of the approach while qPCR or other MBTs monitor the effects on the microbial population in response to biostimulation or bioaugmentation. This approach was employed in a pilot and full-scale remedial application to demonstrate organic carbon amendment addition promoted growth of t ethene .Performance monitoring (Stage 3) follows design and initial implementation of the natural or enhanced bioremediation strategy. While contaminant concentrations ultimately must show progress toward the remedial goals, sampling and analysis of select geochemical parameters and MBTs during remedy performance monitoring can improve understanding of remedy progress. MBT application may consist of qPCR analyses to assess whether contaminant-degrading microorganisms are sufficiently abundant to sustain desired biodegradation. Temporal and spatial trends for qPCR results can be particularly helpful to demonstrate positive, negligible, or negative effects on microbial processes to refine the remedial strategy across the treatment area, and in some cases serve as a leading indicator of remedy performance. Contaminant concentrations can, in some cases, be a lagging indicator of enhanced bioremediation due to various fate and transport mechanisms which may act as confounding factors impeding the ability to distinguish occurrence or enhancement of contaminant biodegradation. For example, as the rates of contaminant biodegradation are increased due to successful enhanced bioremediation, the rate of dissolution of residual non-aqueous phase liquids (NAPL) may also increase to maintain multi-phase equilibrium , 2009. AbssA gene (involved in the first step of anaerobic degradation of toluene and xylene; Data generated in Stage 3 should be continually reviewed to assess remedy performance monitoring. Data interpretation should be done as described in section “Stage 1: Assessment,” consisting of comparative analysis across contaminant and geochemical concentration gradients, within a MLOE approach, and compared to SSBOs, CSM, and regulatory requirements. In a study where sulfate was introduced to the subsurface, an integrated performance monitoring program of contaminants, geochemical parameters, and MBTs demonstrated that sulfate injection increased the rates of biodegradation of BTEX . As sulfAt the conclusion of the framework, whether having progressed through all three stages or solely Stage 1, a biogeochemical CSM should have been developed or refined to support subsequent site decision-making and strategy. Microbiology plays an important role in contaminant fate and attenuation, but is often times unmeasured, inferred, or not considered at contaminated sites. Applying this framework will aid in improving knowledge of microbial processes and broader contaminant fate at contaminated sites. Additionally, data-driven decisions based on this framework should improve the effectiveness of natural or enhanced bioremediation to meet site goals and achieve or progress toward site closure, as well as avoid the application of bioremediation where it may be infeasible.Bioremediation methods have been demonstrated to be favorable strategies for contaminated site management to naturally biodegrade, biotransform or remove contaminants in soil and groundwater. As discussed in this perspective, the increasing application of MBTs to directly measure microbiological processes at environmental sites has the potential to reduce uncertainty related to site-specific biogeochemical processes. However, field-scale MBT application, implementation strategies, and interpretation can be inconsistent across varying sectors, institutions, or individuals potentially posing barriers to increased uptake and acceptance. For this reason, the field-focused framework described herein was developed to synthesize complexities of environmental microbiology and assess bioremediation potential at the field-scale during various stages of planning and execution.Application of this framework should be considered for use at contaminated sites implementing bioremediation; further description of application can be found in another article in this research topic issue . It shouThe original contributions presented in this study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.TK and AM contributed to the conception and design of this perspective. SS and YW refined and sharpened conceptual layout and messaging of content. TK, SS, and AM wrote sections of the manuscript. All authors contributed to the manuscript review, read, and approved the submitted manuscript.