Atrial fibrillation (AF) with multiple complications, high morbidity and mortality, and low cure rates, has become a global public health problem. Although significant progress has been made in the treatment methods represented by anti-AF drugs and radiofrequency ablation, the therapeutic effect is not as good as expected. The reason is mainly because of our lack of understanding of AF mechanisms. This field has benefited from mechanistic and (or) statistical methodologies. Recent renewed interest in digital twin techniques by synergizing between mechanistic and statistical models has opened new frontiers in AF analysis. In the review, we briefly present findings that gave rise to the AF pathophysiology and current therapeutic modalities. We then summarize the achievements of digital twin technologies in three aspects: understanding AF mechanisms, screening anti-AF drugs and optimizing ablation strategies. Finally, we discuss the challenges that hinder the clinical application of the digital twin heart. With the rapid progress in data reuse and sharing, we expect their application to realize the transition from AF description to response prediction. The most common sustained arrhythmia atrial fibrillation (AF) not only has high morbidity and mortality, but also is very difficult to prevent, diagnose and treat, bringing a huge economic burden to individuals, countries and society . AF is oThe 2020 European Society of Cardiology guidelines endorse the Atrial Fibrillation Better Care (ABC) pathway as a structured approach for AF management, addressing three principal elements: “A” - avoid stroke , “B” - patient-focused better symptom management, and “C” - cardiovascular and comorbidity risk factor reduction and management . The mobMany dynamic predisposing factors, including modifiable and non-modifiable risk factors, contribute to the onset and progression of AF. The identified non-modifiable risk factors include age, sex, ethnicity and genetics, while modifiable factors consist of smoking, alcohol consumption, hypertension, lipid profile, diabetes, vascular disease, coronary artery disease, heart failure, obesity, physical inactivity, chronic kidney disease, obstructive sleep apnoea, chronic obstructive pulmonary disease, valve disease and inflammatory diseases . These rPotential AF patients are usually diagnosed with long-term electrocardiogram (ECG) monitoring to determine the temporal patterns . In addiRate and rhythm control strategies are two cornerstones of symptomatic AF management. For preventing mortality and morbidity from cardiovascular causes, the effectiveness of the two strategies is comparable . Due to Digital twin technologies are expected to overcome existing difficulties. The digital twin was firstly presented by Michael Grieves in 2003 and was initially described as a virtual representation of a physical product . Its defAlthough personalized atrial computer models from either imaging data or electroanatomical maps have been developed, their standardization has just begun. Lately, Razeghi et al. published the CemrgApp platform for image processing to provide MRI segmentation, including fibrotic tissue distribution derived from late gadolinium enhancement (LGE) intensity in a semi-automatic and userfriendly way . In addiRecently, several hybrid studies utilizing both mechanistic and statistical approaches investigated AF mechanisms. An example of the use of digital twin techniques is investigations of atrial electrophysiological variability . AlthougAt the cellular level, several studies concentrated on identifying potential determinants of inter-subject variability in calcium transient , action ICaL block as a rhythm control strategy depends on the availability of Na+ and Ca2+ currents  were investigated by altering the conductance or the gating kinetics. Scholz et al. introduced a mathematical description of IKur blockade into models of normal and remodeled atrial electrophysiology and found antiarrhythmic effects of IKur inhibitors are dependent on kinetic properties of blockade  by cardiac ablation emerged as a feasible strategy in AF ablation and has evolved from segmental ostial pulmonary vein ablation to the guide ablation with the 3D electroanatomical mapping, to wide-area circumferential ablation with verification of conduction block. For long-term ablation success, PVI using point-by-point radiofrequency or with the cryoballoon has evolved substantially, with multiple energy sources and a variety of ablation tools being available to make it safe and effective. These emerging tools include numerous novel radiofrequency catheters  and alternative energy sources . Although PVI has been shown to have a high success rate in patients with paroxysmal AF in proximity to the PV regions, it is insufficient in the most patients with persistent AF outside the PV regions. Over the past 2 decades, numerous anatomical structures have been suggested as sites from which non-pulmonary vein triggers might occur, including the posterior wall of the left atrium, the left atrial appendage, the superior vena cava, the crista terminalis, the fossa ovalis, the coronary sinus, the ligament of Marshall and adjacent to the atrioventricular valve annuli. Unfortunately, strong evidence to support improved clinical outcomes for any adjunctive ablation strategies is lacking and identifying functional localized target sites for ablation remains challenging . This maOne of the applications of digital twin techniques is to link biomarkers to tissue properties. For example, Corrado et al. found combing CV and APD with the atrial surface area can improve the accuracy in identifying regions that tether re-entrant activation patterns using both biophysically detailed computational models of the atria and a support vector machine classifier . Godoy ein vivo dataset and found the digital twin techniques can overcome a single voltage cut-off value to identify fibrotic tissue from intracardiac signals . In the digital twin heart, on the one hand, potential pathological mechanisms are explored through personalized multi-scale modeling and simulation; on the other hand, AF phenotypes are identified through a data-driven statistical model. Mechanistic and statistical models complement each other’s strengths to facilitate AF mechanism understanding and therapeutic evaluation. As experimental methods and imaging techniques continue to advance, more abundant and high-quality data will facilitate the development of digital twin hearts. Standardization of data acquisition and improved attention to re-usability will accelerate the development of digital twin technologies , while tAlthough there is a palpable exuberance in AF research regarding the potential of digital twin techniques, limitations of the various approaches and challenges in ensuring their clinical application remain. Whether it is the development of digital twin hearts or their clinical applications, the main challenge is the limited availability of experimental data at present. In order to achieve tailored AF treatment, we need to develop a more detailed personalized mechanistic model, but the functional and structural data required to build personalized atria are lacking. Except for the electrophysiological function data of the right atrial appendage of AF patients, other microstructural data, especially from the healthy atrium, are currently very scarce. Although individual structural data represented by patient-specific anatomy and fibrosis distribution can be obtained with LGE-MRI, the limited spatial resolution makes modeling fiber orientations and atrial fibrosis patterns difficult. Even if patient-specific models of the heart can be personalized, we still need to address the issue of intra-individual heterogeneity, including variability in atrial structural and functional properties. Due to the lack of massive experimental and clinical data, these heterogeneous features and their effect on the overall behavior of AF are poorly understood. For statistical models, supervised algorithms require significant amounts of high-quality labeled data. Annotation of data with labels is labor-intensive and datasets with poor data seriously affect the performance of algorithms. Therefore, data with its many aspects presents challenges to the digital twin heart adoption in AF management.AF continues to pose a significant risk of death, in part due to knowledge gaps in the fundamental AF mechanisms and treatment strategies. These clinical challenges in understanding AF mechanisms, screening anti-AF drugs and optimizing AF ablation strategies might benefit from the digital twin techniques. Although limited by the availability of experimental data, the digital twin heart remains a promising path towards the vision of precision cardiology and its clinical applications are emerging.


Circular RNAs (circRNAs) are expressed and are regulated in many biological processes but little is known about their ability to directly control mRNA homeostasis. We show that circRNA zinc finger protein 609 (circZNF609) interacts with several mRNAs increasing the final protein levels, which in the case of the cytoskeleton-associated protein 5 (CKAP5) leads to a stabilized microtubule cytoskeleton and an enhanced tumor cell proliferation. Within this frame of research, growing interest is dedicated to circular RNAs (circRNAs), a well-established class of RNA molecules originating from a back-splicing event in which a downstream splice-donor site is joined to an upstream splice-acceptor site, yielding a covalently closed circular RNA. CircRNAs show several peculiar features, such as evolutionary conservation and tissue-specific expression, but above all, they have been found to be deregulated in many pathological conditions, including cancer. Several studies indicate that circRNAs elicit their function as miRNA sponges, but the cell-type-specific microRNA signature restrains this mechanism as a general mode of action. In our work3we explored the regulatory roles of the RNA-RNA interactions involving the circRNA zinc finger protein 609 (circZNF609), a circRNA previously reported to be regulated and to control human primary myoblast and embryonal rhabdomyosarcoma (ERMS) cell proliferation.5 CircZNF609 was also found to be upregulated in several types of human cancers, such as prostate cancer, breast cancer, gastric cancer, nasopharyngeal carcinoma, and hepatocellular carcinoma, where its depletion has been linked with reduced aggressiveness of tumor cells.The latest research showed the importance of RNA molecules as a key factor in the regulatory circuitries. To carry out these fine-tuning activities, RNA not only interacts with proteins and chromatin but often directly pairs with other RNA moleculesin vivo, we found that circZNF609 pairs directly with a few mRNAs, among which is the mRNA of the cytoskeleton-associated protein 5 (CKAP5). A more detailed molecular study points to the back-splicing junction of circZNF609 as the sequence responsible for this RNA-RNA pairing, underlining the specificity of this interaction in front of the linear ZNF609 mRNA counterpart. As a result, the CKAP5 mRNA is stabilized and its translation is enhanced. We demonstrated that this stabilization is produced by ELAV like RNA Binding Protein 1 , an RNA-binding protein (RBP) known to induce mRNA stabilization and translation and to control lncRNA metabolism.7 Specifically, circZNF609 contains several-binding sites for ELAVL1 in its sequence, and the interaction between the circRNA and CKAP5 mRNA will facilitate ELAVL1 loading from the circular onto the messenger RNA  ends, sustains their elongation and regulates their dynamicsCKAP5 mRNA interaction, through either small interfering RNAs (siRNAs) against circZNF609 or locked nucleic acid (LNA)-modified oligonucleotides against the pairing region, sensitizes rhabdomyosarcoma cells to microtubule-targeting chemotherapeutic agents. Moreover, we demonstrated that this regulatory mechanism is also conserved in other cancer cell models such as neuroblastoma and chronic myelogenous leukemia, setting such blockage as a potential therapeutic coadjuvant in cancer treatment.Microtubule dynamics is essential to all processes depending on the cytoskeleton, including cell migration and differentiation, the building-up of the mitotic scaffold and chromosome segregation. Several first-line chemotherapeutic agents, such as vincristine or Paclitaxel/Taxol, are designed to target either the microtubule assembly from tubulin or their dynamic activity; they are effective chemotherapeutic agents and are still in use to treat cancer of various origins, including rhabdomyosarcoma. We demonstrated that preventing the circZNF609/CKAP5 mRNA interaction, through either small interfering RNAs (siRNAs) against circZNF609 or locked nucleic acid (LNA)-modified oligonucleotides against the pairing region, sensitizes rhabdomyosarcoma cells to microtubule-targeting chemotherapeutic agents. Moreover, we demonstrated that this regulatory mechanism is also conserved in other cancer cell models such as neuroblastoma and chronic myelogenous leukemia, setting such blockage as a potential therapeutic coadjuvant in cancer treatment.Microtubule dynamics is essential to all processes depending on the cytoskeleton, including cell migration and differentiation, the building-up of the mitotic scaffold and chromosome segregation. Several first-line chemotherapeutic agents, such as vincristine or Paclitaxel/Taxol, are designed to target either the microtubule assembly from tubulin or their dynamic activity; they are effective chemotherapeutic agents and are still in use to treat cancer of various origins, including rhabdomyosarcoma. We demonstrated that preventing the circZNF609/CKAP5 mRNA interaction, through either small interfering RNAs (siRNAs) against circZNF609 or locked nucleic acid (LNA)-modified oligonucleotides against the pairing region, sensitizes rhabdomyosarcoma cells to microtubule-targeting chemotherapeutic agents. Moreover, we demonstrated that this regulatory mechanism is also conserved in other cancer cell models such as neuroblastoma and chronic myelogenous leukemia, setting such blockage as a potential therapeutic coadjuvant in cancer treatment.Microtubule dynamics is essential to all processes depending on the cytoskeleton, including cell migration and differentiation, the building-up of the mitotic scaffold and chromosome segregation. Several first-line chemotherapeutic agents, such as vincristine or Paclitaxel/Taxol, are designed to target either the microtubule assembly from tubulin or their dynamic activity; they are effective chemotherapeutic agents and are still in use to treat cancer of various origins, including rhabdomyosarcoma. We demonstrated that preventing the circZNF609/


Metacognitive therapy (MCT) is a novel and promising transdiagnostic psychotherapy intervention based on the Self-Regulatory Executive Function model of conceptualizing emotional disorders. It was developed by Adrian Wells in 2009. Its therapeutic response occurs by reducing dysfunctional metacognitive beliefs regarding worry and rumination, often seen in patients with psychiatric disorders. Since its inception, it has been increasingly applied to a wide spectrum of psychiatric illnesses, but mainly focusing on mood and anxiety disorders. To our knowledge, no study has detailed its existing therapeutic scope in psychiatry. In this comprehensive narrative review, we describe the various psychiatric illnesses in which MCT has been used, the advantages of MCT, and the limitations of the MCT research. In addition, we propose some solutions to systematically examine its place in psychiatry. We encountered its potential role in treating trauma and stress-related disorders, obsessive-compulsive spectrum disorders, personality disorders, psychotic disorders, substance use disorders, and sexual disorders. Metacognitive therapy (MCT)  is a relMethodologyWe performed an exhaustive search on PubMed and Google Scholar to find all relevant publications on MCT and various psychiatric disorders that were available till November 2021. We used the following all-embracing keywords for our search: “Metacognitive therapy,” “Metacognitive therapy AND psychiatric disorders,” “Metacognitive therapy AND mental illness,” “Metacognitive therapy AND psychiatric illness,” “Metacognitive therapy AND mental disorders.” Because MCT is well established for depressive and anxiety disorders in the literature, we excluded studies that involved the use of MCT in patients with any of the following: major depressive disorder (MDD), dysthymia, postpartum depression, generalized anxiety disorder (GAD), social anxiety disorder, panic disorder, and phobias as their primary psychiatric diagnosis or when the above-mentioned disorders occurred secondary to a medical/neurologic illness. For this review, we exclusively focused on studies that described the use of MCT based on the manual developed by Adrian Wells , metacogRole of the metacognitive therapy in various psychiatric disordersAdjustment Disorder Secondary to a Physical IllnessAdjustment disorder is common among patients with potentially life-threatening illnesses . AccordiProlonged Grief DisorderPGD is a new diagnostic entity with limited evidence regarding the effectiveness of pharmacological and non-pharmacological interventions ,13. It iPosttraumatic Stress DisorderMetacognitive processes such as persistent negative thinking and the use of avoidance coping strategies predict symptoms of PTSD among patients following a traumatic event . The metTo summarize, there is burgeoning evidence regarding the benefits of MCT in PTSD. However, the psychological mechanisms underpinning the improvement brought by MCT in these patients have not been investigated yet. More studies are needed with a focus on outcome measures that explore the change in metacognitive beliefs, thought control strategies, and coping styles commonly used by patients with PTSD. Future trials should implement larger sample sizes and longer follow-up periods to assess the long-term success of MCT in PTSD. The effectiveness of MCT compared to other psychotherapeutic approaches traditionally used in the management of PTSD remains to be established. Future studies should also focus on the application of MCT in more complex trauma and/or different types of traumas.Obsessive-Compulsive DisorderAccording to the metacognitive model of OCD, the three key factors that are important in the development of OCD include the importance that an individual assigns to commonly occurring obsessional intrusive experiences, the strategies of self-regulation that the individual uses to process these experiences, and the experiential perspective that they take in relation to these experiences ,23. The More recently, attempts have been made to compare the effectiveness of MCT for OCD against other psychological interventions. In a pilot RCT, the effectiveness of MCT was compared with ERP among patients with OCD . The stuOverall, these findings suggest that MCT is an efficacious treatment that can be offered over a brief duration to manage OC symptoms.Illness Anxiety DisorderIllness anxiety disorder is characterized by persistent and excessive preoccupation and fear associated with the belief that one has or could acquire a serious illness, as well as high levels of anxiety about health . It has Body Dysmorphic DisorderBDD is characterized by excessive and persistent preoccupation with perceived defects or flaws in one’s appearance . It is aEating DisordersThe current understanding of the possible role of MCT in eating disorders is very limited. Disordered eating may be associated with perseverative thinking, dysfunctional metacognitive beliefs, and attentional biases ,46. DespBipolar DisorderThe effectiveness of MCT in the management of bipolar disorder is yet to be established. One published case series examined its role in treating bipolar disorder type II . At treaPsychosisThe standard and most widely used treatment of psychosis is pharmacotherapy . HoweverOverall, it can be concluded that MCT is a feasible and acceptable treatment in managing psychotic illnesses. It has shown promising results in managing positive symptoms including delusions and general psychiatric symptoms of schizophrenia among patients with treatment-resistant psychosis in the short term. The feasibility of MCT has also been established in reducing psychotic-like experiences and improving overall functioning among individuals at high risk of developing psychosis. Future studies should focus on examining the effectiveness of MCT among patients with psychosis using larger samples and rigorous methodological design.Borderline Personality DisorderMCT has also been applied in the management of BPD. MCT for BPD has three main components . The firAlcohol Use DisorderThe S-REF model of psychopathology has also been applied in the conceptualization and treatment of alcohol use disorder ,62. AccoSexual DisordersThere is no MCT model described for sexual disorders. A novel study by Ramezani et al. 2017)  studied 7  studieRole of the metacognitive therapy in managing psychological symptomsWork-Related StressA recent feasibility study by de Dominicis et al. 2021) tested the impact of MCT on managing chronic work-related stress among four individuals . Only pa tested tWe propose that a metacognitive model for stress should be developed, and larger randomized controlled studies are required to fully determine the efficacy of MCT on work-related stress.Emotional Distress Due to CancerFisher et al. (2017)  studied Buoyed by the above study’s success, Fisher et al. (2019)  conducteDiscussionMCT has begun creating an impact on the treatment of a wide variety of psychiatric disorders worldwide. It is making ground in the treatment of major depression and anxiety disorders, two areas of research where most of its thrust had been directed till now -5,69-75.To summarize, MCT appears quite encouraging in its ability to deliver across a gamut of psychiatric disorders. Treatment using MCT has led to remission of various psychiatric symptoms including OCD, PTSD, illness anxiety disorder, BDD, adjustment disorder due to physical illness, prolonged grief disorder, binge eating, binge drinking, hypoactive sexual desire, depressive episodes in bipolar affective disorder, and positive symptoms and delusional beliefs among patients with psychosis. Gains obtained through treatment remain stable for relatively longer durations (up to one year). Moreover, MCT is highly efficacious in reducing emotional distress experienced during chronic life-threatening illnesses such as cancer, as well as in day-to-day work-related stress. In the future, we encourage researchers at different centers globally to conduct replication studies of MCT for the psychiatric disorders discussed in this review and to test its effectiveness in other psychiatric disorders not investigated so far. Researchers should intend to recruit larger patient samples, use rigorous methodological designs , and employ longer follow-up duration. This exercise may prove to be quite rewarding because our field is grappling with a crisis of unsatisfactory and inadequate treatments for most psychiatric disorders.


Necrotizing enterocolitis (NEC) is a gastrointestinal disease frequently prevalent in premature neonates. Despite advances in research, there is a lack of accurate, early diagnoses of NEC and the current therapeutic approaches remain exhausted and disappointing. In this review, we have taken a close look at the regenerative medical literature available in the context of NEC treatment. Stem cells from amniotic fluid (AFSC) administration may have the greatest protective and restorative effects on NEC. This review summarizes the potential protection and restoration AFSCs have on NEC-induced intestinal injury while comparing various components within AFSCs like conditioned medium (CM) and extracellular vesicles (EVs). In addition to therapeutic interventions that focus on targeting intestinal epithelial damage and regeneration, a novel discovery that AFSCs act in a Wnt-dependent manner provides insight into this mechanism of protection. Finally, we have highlighted the most important aspects that remain unknown that should be considered to guide future research on the translational application of AFSC-based therapy. We hope that this will be a beneficial frame of reference for the guidance of future studies and towards the clinical application of AFSC and/or its derivatives as a treatment against NEC. Necrotizing enterocolitis (NEC) is a gastrointestinal disease frequently prevalent in preterm neonates. Of those, 5%–12% of very-low birth weights are at an increased risk . With adIn recent years, investigation into preventative actions have shown to be promising in reducing the incidence, morbidity and mortality of the disease . Breast via cell surface markers and release of transcription factors grants self-renewal capabilities , can potentiate the treatment effects of experimental NEC. In this article, we will review the current evidence and discuss how promising regenerative medicine and stem cell therapy may be on the clinical translation of AFSC-based therapy against NEC. Moreover, we have highlighted aspects that remain unknown, which should be considered to guide future research of the translational application of AFSC-based therapy.via increased enterocyte apoptosis in NEC , which is principally composed of water and solutes . These ss in NEC . Interess in NEC . This stEvidence of the potential of stem cell therapy against intestinal diseases has been a growing area of research including NEC. Adult stem cells, such as the mesenchymal stem cells (MSCs), were of the first to demonstrate protection from experimental NEC in rats in 2011 .in vitro, to be cultured infinitely in an undifferentiated state, and to produce clonal lineages and teratomas in vivo  dependent mechanism. These unique therapeutic effects and promising potential launched the focus on AFSC-based therapy against NEC.Extraordinarily, AFSC-based therapy against NEC was first discovered by Zani et al. in 2013 . AFSCs iSeveral experimental animal NEC studies have been conducted to identify the efficacy of different types of stem cells, such as amniotic fluid-derived stem cells (AFSC), bone marrow-derived MSC (BM-MSC), or enteric neural stem cells (E-NSC). McCulloh et al. compared these stem cell types and found that, generally, all demonstrate a reduction in the incidence and severity of NEC , 30. In via stable intestinal proliferation, multipotency, and immunomodulatory abilities.Both AFSC and MSC have been investigated as potential treatment strategies in lowering NEC incidence and reducing the intestinal inflammation. However, AFSCs, not MSCs, administered prior to the onset of NEC have beneficial effects on prevention of intestinal epithelial injury , which ivia amniocentesis, the AFSCs are isolated in cultured medium then administered via an intraperitoneal injection. The AFSCs can integrate into the injured bowel acting on the damaged intestinal area due to its preferential attraction to areas of intestinal injury. There are various modes of action by AFSCs , governed by the ER stress central regulatory protein binding immunoglobulin protein (BiP). BiP controls three downstream ER transmembrane stress sensor proteins: inositol-requiring enzyme 1α (IRE1α), activating transcription factor 6 (ATF6) and pancreatic endoplasmic reticulum kinase (PERK). Furthermore, BiP activates these proteins with anti-apoptotic properties and promotes cell survival. However, prolonged ER stress activates the C/EBP homologous protein (CHOP), a part of both pro- and anti-apoptotic pathways which include encoding genes within the BCL2-family proteins. Since NEC is associated with increased intestinal epithelial cell and TJ apoptosis, inhibiting apoptotic signaling via CHOP activation may rescue barrier function  present on intestinal epithelial cells . TLR4 exThe rescue of intestinal stem cell (ISC) function and differentiation is paramount to intestinal epithelial rescue and impairment as a lack of cell proliferation and differentiation grants further destruction of epithelial integrity. Leucine-rich repeat-containing G-protein coupled receptor 5 (Lgr5) is a well-studied stem cell proliferation marker. During homeostasis, Lgr5+ ISCs located at the crypt base are responsible for mediating the constant renewal of the intestinal epithelium. These stem cells undergo self-renewal to maintain an undifferentiated ISC reservoir, and differentiation to give rise to major intestinal epithelial cell types, such as absorptive enterocytes, goblet cells, Paneth cells, tuft cells and enteroendocrine cells .via Wnt signaling can be attributed to canonical Wnt pathways as demonstrated by blocking Wnt signaling via inhibitors of Porcupine (Porcn) protein  improved NEC survival and reduced intestinal injury . Specifiin vivo models of NEC. Over 1500 proteins were found in the CM, mostly enriched by exosomes. In addition, several clusters included immunomodulation, cell cycle and stem cell regulation, which is in line with study results previously described. Interestingly, a large functional cluster of this secretome analysis was vesicle-mediated transport, which led to the interest in the function of these extracellular vesicles in the context of NEC.Moreover, the secretome of the hAFSC-CM was assessed to gain a further understanding of its composition to explain the beneficial effects within The paracrine communication of AFSCs, as widely discussed in previous research findings, has now been accredited to the function of EVs –58. EVs via administration of human AFSC-derived EVs (hAFSC-EVs)  . This stvia Wnt signaling in intestinal organoids . It. It44). Overall, AFSC-based therapy has proven to be a novel therapeutic option in the context of experimental NEC. The pluripotent nature of these cells allows for their vast differentiation capabilities. In addition to its direct benefits to patients, the significant reduction of ethical issues of this less invasive tool in comparison to embryonic stem cells and other stem cell origins increases its attractiveness in tissue engineering and treatment of suffering neonates. Though there is a significant amount of evidence proving AFSCs to be valuable in the treatment of NEC, there remains a lack of clinical trials as indicated by our recent search in ClinicalTrials.gov.Optimization of stem cell and stem cell derivative expansion is vital to clinical application. Moreover, various factors must be considered prior to the induction of experimental animal model techniques of AFSC administration, such as identifying: (1) the ideal gestational age to harvest AFSCs, (2) markers eliciting optimal cells, (3) an optimized standard for AFSC expansion (4) optimized dose concentrations of AFSC derivatives, (5) stem cell conditioning for enhanced applications, (6) proving that animal study results can be translated to a human setting without detrimental effects. In the following paragraphs, these factors are explained in more detail.The general quality of AFSC cultures is significantly correlated with gestational age of the fetus when AF is harvested. In studies with experimental spina bifida patients, MSCs and neural SCs show to have been affected by gestational age . Recent via analysis of CD90 and CD105. It is well studied that the consequences of NEC go on to affect neurodevelopment and brain morphology, therefore, these results support AFSC-mediated protection via the use of optimal cells  was studied in the clonal expansion of AFSC cultures, however, results yielded noncompliance with good manufacturing practice (GMP) (The culture medium in which cell expansion occurs has been optimized for ce (GMP) , 66. Thoce (GMP) . Lyset, ce (GMP) .In recent research, short-term small molecule treatments are a growing interest within the field of regenerative medicine. These molecules can improve stem cell characteristics to further reinforce their therapeutic potential and efficiency. A study assessed the effect of various concentrations and mixtures of small molecules known to participate in cell repair and regeneration on AFSCs, such as surface marker and pluripotency associated gene expression . HDAC inTo overcome these hurdles within this research field of AFSC-based therapy, key knowledge must further be elucidated prior to the implementation of randomized control trials in human neonates. Experimental NEC trials are currently completed in animal models, thus further investigation is required to investigate the effects of this therapy on human models, such as intestinal epithelial maturation, barrier function and innate immune responses. For example, working with human-derived organoids would be a great first step to preclinical human models .via preclinical trials. This review provides a beneficial frame of reference towards clinical application of AFSC and/or its derivatives as a novel treatment against NEC.To conclude, we have identified important bodies of knowledge that continue to lack within the scope of clinical translation of AFSC-based therapy. Future research must confirm whether this treatment strategy will benefit neonates suffering from NEC


The integrity of blood vessel structure and function ensures continuous and well-regulated blood supply well match to energy demand, plays a pivotal role in the transport of amino acids, peptides, ions, neurotransmitters, and other signaling molecules, as well as immune surveillance and hemostatic balance. Numerous studies have confirmed that vascular pathology may be a pathogenic factor in the occurrence and progression of dementia, diabetes and cardiovascular diseases . Clinicach as AD . The Resd-aspartate (NMDA) receptor antagonist due to a general lack of a reliable molecular biomarker or well defined pathological mechanism. A large number of clinical trials have shown that Traditional Chinese medicine (TCM) therapy can improve the prognosis of debilitating VaD. In this Research Topic, by constructing complex target gene (CTM) miRNA, Liu et al. discovered the potential molecular mechanism of total flavonoids from Dracocephalum moldavica L (TFDM) in treating VaD. They found that the neuroprotective effects of TFDM were mainly contributed by kaempferol, apigenin, luteolin, and quercetin. They could act on 43 targets that were mediated by 8 miRNAs. Among them, miR-3184-3p and miR-6875-3p were further validated in oxygen-glucose deprivation (OGD) cell model. This study explored the miRNA expression profile in the process of VaD angiogenesis, uncovered potential pharmacological mechanism of TFDM on VaD, and offered an effective approach to explore the therapeutic mechanisms of TCM. However, given that flavonoids here may be pleotropic compounds that can interfere with bioassays via different mechanisms, more in-depth work should be carried out to exclude this possibility  is necessary to support neuronal survival and function. The reduction of CBF may contribute to cognitive decline by initiating or amplifying the amyloid cascade. Pathological vascular injury or vascular aging can lead to chronic cerebral hypoperfusion (CCH), which causes various molecular cascades driving downstream structural and functional changes such as blood-brain barrier (BBB) damage, glial activation and neuron damage, which further aggravated cognitive impairment. The current treatment strategies for VaD are performed using acetylcholinesterase inhibitor or N-methyl-sibility .via thrombolysis or thrombectomy agents like alteplase and urokinase are first-line options that reduces disability by restoring blood flow. In addition, reparative therapy of cerebral tissue warrants development for the treatment of IS. However, the lack of treatment options after IS has spurred the search for new stroke therapy. Salvianolic acid for injection (SAFI) serve as a TCM preparation. Previous clinical evidence have suggested a promising prospect for IS since it has no obvious side effects on liver and kidney function. Li et al. utilized network pharmacology and computational prediction as well as experimental verification to systematically analyze the potential mechanism of SAFI on IS. They confirmed 38 common genes shared by SAFI targets and main compound targets as well as genes associated with IS. Among them, PTGS1 and PTGS2 activity were inhibited by SAFI in a dose-dependent manner. SAFI also inhibited LPS-induced prostaglandin E2 production by RAW264.7 macrophages and BV-2 microglia. All the above findings suggest a underlying molecular mechanism of action of SAFI in IS. In addition, Kong et al. used zebrafish to study the protective effect of isopropyl caffeic acid (KYZ) on ischemic diseases, and used network pharmacology and molecular docking to predict angiogenesis through a variety of signaling pathways and predict the target of KYZ toward vascular dysfunction related diseases.IS caused by occlusion of the cerebral artery, where interrupted blood flow to the brain leads to a thrombosis or embolism. Different types of cells undergo different morphological changes during the pathogenesis of IS. In the ischemic core, the cytoplasm and nucleolus of neurons swell and axons disappear. Glial cells transform into an “activated state”, accompanied by the production of pro-inflammatory cytokines and chemokines. Dysregulation of NVU cells plays a vital role in the pathophysiology of IS, which is characterized by disruption of the permeability of BBB and triggering downstream cascades. Reperfusion treatment for IS Liu et al. attempted to detect Treg-associated genes (TRGs) in PH patients using transcriptome and Tregs infiltrates data. A gene signature using 25 hub TRGs can better distinguish PH patients from healthy controls. They further verified these predicted genes in lung tissue of PH mouse model. Overall, this study facilitates understanding of the molecular mechanisms associated with Tregs in PH development.PH is a progressive, fatal vascular disease that is histologically defined by vascular remodeling and clinically characterized by proliferative and apoptosis-resistant cellular phenotypes and adverse pulmonary vascular remodeling, with pathological processes associated with inflammatory cell metabolism. Previous studies have shown that advanced vascular remodeling can be reversed by addressing specific immune processes, and the extent of pulmonary vascular remodeling is closely related to the severity of inflammatory cell infiltration. In physiological states, regulatory T cells (Tregs) take part in suppressing and halting immune responses to prevent autoimmune and tissue damage. Accumulating evidence shows that Tregs are the main cell type of tissue self-tolerance, limiting vascular endothelial damage caused by PH. In this Research Topic, However, several issues should be acknowledged regarding these studies. First, the directly binding targets by TCM or active components have still not validated for corresponding diseases in the current work. In addition, as an essential factor in various disease, endophenotype module for vascular dysfunction can be built to characterize disease pathogenesis for efficacious therapeutic development and biomarker discovery. Third, network-driven analysis tends to highlight highly promiscuous natural products like polyphenols. These compound are usually undruggable or difficult to be drugs. Finally, due to the increasing interdisciplinary technologies that may be used in this field, as well as the complexity of the pathogenesis of vascular dysfunction, more wider scope and in-depth work is still needed.In summary, the article Research Topic in the Research Topic “Data Mining and Network-driven Analysis: Drug Discovery and Mechanism Exploration by Targeting Vascular Dysfunction” explored the molecular mechanism of drug candidates toward vascular dysfunction related disease and elucidate underlying immune-related mechanisms against diseases due to vascular dysfunction by data mining and network pharmacology.


Even when sharing etiologic factors, the incidence of DM-1 is low in patients with schizophrenia. Both diseases can lead to cognitive impairment, but its difficult to define its origin. 33 years old male, with DM-1 and schizophrenia referred to Therapeutic Community for psychotic symptomatology control, cannabis consumption withdrawal, improvement of self-care and hipoglycemia control reachNowadays toxic abstinent and adequate consciousness of disorder. Remarkable persistence of hallucinations both auditive and visual, mostly shown as delirium, pharmacologic treatment-refractary. During last months, he shows excessive absent-mindedness, recent memory failure and verbal declarative memory and psychomotor slowdown Analysis: unbalance glycosylated hemoglobin. MR: cortical-subcortical atrophy, very shocking his age. Endocrinology follow up it was decided to stablish an insulin pump, so metrics were regulated.Neurological profile of the patient (deficit and slowdown attention capability) aggravation of symptoms according to glycaemia and disturbances in image test could lead to vascular origin. Attention deficit and excessive focus are symptoms of schizophrenia, but they are shown in the beginning, they tend to stabilize during years. Verbal declarative memory disruptions can be produced in both disordersBetter glycemic control and changed to Lurasidone 37mg and Cariprazine 3mg objecting higher reactivity and less absent-mindednesCognitive impairment in DM is frequent in adults with severe and long evolving hypoglycemic episodes Regardless of its origin, the cognitive impairment in schizophrenia leads to serious impact in functional and pragmatic areas Further investigation will allow us to quantify the magnitude of cognitive effect in metabolic control so according strategies could be developed


A growing number of variants associated with risk for neurodevelopmental disorders have been identified by genome-wide association and whole genome sequencing studies. As common risk variants often fall within large haplotype blocks covering long stretches of the noncoding genome, the causal variants within an associated locus are often unknown. Similarly, the effect of rare noncoding risk variants identified by whole genome sequencing on molecular traits is seldom known without functional assays. A massively parallel reporter assay (MPRA) is an assay that can functionally validate thousands of regulatory elements simultaneously using high-throughput sequencing and barcode technology. MPRA has been adapted to various experimental designs that measure gene regulatory effects of genetic variants within cis- and trans-regulatory elements as well as posttranscriptional processes. This review discusses different MPRA designs that have been or could be used in the future to experimentally validate genetic variants associated with neurodevelopmental disorders. Though MPRA has limitations such as it does not model genomic context, this assay can help narrow down the underlying genetic causes of neurodevelopmental disorders by screening thousands of sequences in one experiment. We conclude by describing future directions of this technique such as applications of MPRA for gene-by-environment interactions and pharmacogenetics. Genome-wide association studies (GWAS) of neurodevelopmental and psychiatric disorders have demonstrated that the majority of common variation associated with these disorders is found in noncoding regions of the genome –8. SimilIn addition to the lack of regulatory code, GWAS alone cannot pinpoint variants that are causing a disease because of linkage disequilibrium (LD), the nonrandom inheritance of nearby alleles on the genome. A genome-wide significant (GWS) locus typically contains tens to hundreds of single-nucleotide polymorphisms (SNPs) that are associated with a trait or disease. Only a subset of these SNPs are thought to be causal. While it is commonly thought that the index SNP, the SNP most significantly associated with the trait at a locus, is causal, growing evidence portrays a more complex picture . The leaSeveral experimental and computational designs have been used to predict the causal variant at a given locus. Fine-mapping tools computationally predict potential causal variants based on association statistics and LD patterns –23, but Functional validation assays fill the existing gap by experimentally demonstrating how genetic differences lead to phenotypic effects . Gene reMassively parallel reporter assays (MPRA) have advanced the throughput of luciferase assays by enabling the simultaneous functional validation of regulatory activity of thousands of variants on a massive scale Fig. B, often MPRA has incredible potential for studying the noncoding genetic variants associated with neurodevelopmental disorders. Whereas the majority of efforts have been made to characterize common variants identified by GWAS, wide application of WGS would further expand the utility of MPRA in characterizing various classes of variants located in the noncoding genome.In this review, we will discuss the broad application of MPRA to functionally validate variants within the various regulatory contexts that encompass transcriptional and posttranscriptional regulation. We will then add important considerations for conducting MPRA, including limitations of MPRA experiments. We conclude by providing future directions of MPRA.The canonical MPRA design includes a CRE, a generic promoter, a reporter gene, and a unique barcode assigned to each regulatory element Fig. B. GeneraMutations and variants in promoter regions can have a profound impact on gene expression. MPRA has been used to test the impact of variants in promoters on a massive scale. In comparison with the canonical MPRA design, promoter MPRA lacks a CRE and alters the DNA sequence of the promoter region. Patwardhan et al. utilized promoter MPRA with saturation mutagenesis to screen the activity of mutated promoter sequences with attached barcodes Fig. A 40]. B. B40]. BIn addition to introducing variation in the promoter sequences, a similar approach can be used to characterize promoter activity of any given sequence. Boer et al. developed a gigantic parallel reporter assay (GPRA) that measured the promoter activity of over 100 million randomly synthesized sequences . The comWhile many neurodevelopmental disorder-associated variants have been shown to be enriched in promoter regions , 47, 48,Enhancers are CREs that TFs bind to and activate gene expression . DiseaseDDHD2 . S. S42]. SSilencer MPRA has been applied to detect thousands of CREs acting as silencers, which were enriched for disease-associated SNPs , highligMPRA can be combined with methods that sequence populations of cells binned by fluorophore expression, called Sort-seq , to studIKBKAP . T. TSox2 vionation –92 and oionation , 93.Though MPRA is an incredibly useful tool to experimentally verify variant function, this assay is not without limitations. Enhancer, promoter, and silencer MPRA can effectively identify variants and elements with regulatory activity, but these assays cannot inform which gene(s) that the variants act on. Therefore, MPRA results need to be combined with other functional genomic data such as eQTLs and chromatin interaction profiles (Hi-C) to identify target genes . While fA complementary approach to address this gap is Perturb-seq, which employs a pooled library of gRNAs associated with a unique barcode that modulates expression of the gRNAs’ target genes. A Perturb-seq gRNA library could be designed for the MPRA-validated regulatory elements and introduced into a cell line expressing Cas9 protein. Introduced gRNAs can perturb the region of interest, and transcriptomic alterations can be profiled via scRNA-seq. The resulting data can explain the regulatory impact of perturbed elements within a given cell . MoreoveAnother disadvantage of MPRA is that it uses exogenous DNA constructs that do not model the endogenous regulatory environment at the variant location. Exogenous DNA constructs are either episomal or inserted into the DNA at random locations. An episomal construct is not subject to cis-regulatory effects such as chromatin accessibility and conformation. Instead, its activity is only modulated by trans-regulatory effects, like transcription factors. In contrast to episomal MPRA, MPRA delivered through a lentiviral vector (LentiMPRA) randomly integrates into the host genome, hence enabling functional characterization of regulatory elements and variants within the context of the host genome . HoweverThe effect of genomic context has been evaluated with a new technique called PatchMPRA . This teRegulatory elements are often only functional within a given tissue or cell type , 98–101.MPRA is most commonly performed, out of convenience, on cell types easily cultured and transfected in a lab (such as HEK293 cells ). BecausR = −0.326) between regulatory activity of CRE barcode expression in these two cell types [The cell type specificity of MPRA has been previously demonstrated with an MPRA on random CREs tested in two cell lines: U87 glioblastoma cells and induced pluripotent stem cell (iPSC)-derived human neural progenitor cells (hNPCs) . This stll types . The difll types . These rTo delineate variant effects on gene regulation in a cell type-specific manner in vivo, Cre recombinase-dependent MPRA packaged within an adeno-associated virus (AAV) has been developed . In a prJust as cell and tissue types are important to MPRA, the development stage is a critical factor to consider when performing MPRA. This is especially important in studying genetic etiology of neurodevelopmental and psychiatric disorders which, by definition, have neurodevelopmental origin , 107. AsOne critical, unanswered question in the field is the extent to which variant function is influenced by gene-environment interactions. External stimuli can alter a cellular pathway that has downstream effects on TF abundance and binding properties. Applying MPRA in this context can uncover a new class of variants that gain (or lose) regulatory effects upon exposure to external stimuli. Here, we propose a term “response MPRA” to describe MPRA performed in response to an external stimulus, such as exposure to hormones, drugs, or other biomolecules, as well as measuring gene regulatory effects in the context of a particular cell state Fig. .Fig. 5A trans retinoic acid (ATRA). ATRA, an acid derivative of retinol, activates the retinoic pathway, which has been implicated with the risk for MDD [In an example of a response MPRA, Mulvey et al.  interrog for MDD . ATRA ad for MDD . As retiAn additional application of the response MPRA follows the paradigm of measuring gene regulatory activity in the context of a particular biological process. In neurons, a classic example of this involves measuring CRE response upon stimulation with known modulators of neuronal activity. In an early adaptation of this approach, Nguyen et al. compared promoter and enhancer activities in neurons treated with potassium chloride with their unstimulated controls . While sGiven the limited therapeutic options available for neurodevelopmental and psychiatric disorders, response MPRA can provide a high-throughput framework to investigate the impact of a drug candidate on variant function, potentially leading to high-throughput testing for pharmacogenomics and personalized therapies based on genetic background. An example of such an approach could be testing ADHD-associated variants with methylphenidate (a common ADHD medication ) to deteFinally, response MPRA can provide a useful tool to study gene-environment interactions by interrogating the variant function upon exposure to environmental factors associated with disease risk. Maternal exposure during pregnancy to valproic acid (VPA), a commonly prescribed antiepileptic medication, has been associated with risk for ASD , as wellMany variants in noncoding regions have, and will continue to be, identified by large-scale studies such as GWAS, WGS, and QTL. Understanding the function of those variants, and which variants within a haplotype block are causal, is the next key step in moving from association to biological understanding. In this review, we outlined how MPRA can validate variant function in a wide range of regulatory elements such as enhancers, promoters, silencers, and TF binding sites. We also described how MPRA can be used to garner mechanistic understanding of posttranscriptional regulation such as splicing, RNA modification, RNA stability, translation, and RNA localization. MPRA results can change based on cell type, stimulus state, and developmental time period, so these parameters must be carefully considered when designing an MPRA experiment. MPRA has limitations in associating regulatory effects to a target gene and lacks epigenetic context. Complementary approaches that range from other functional genomic resources  to other screening platforms  will extend knowledge gained from MPRA to provide a greater understanding of the mechanisms by which genetic variants affect brain structure, function, and development.


Mechanisms devoted to the secretion of proteins via extracellular vesicles (EVs) have been found in mammals, yeasts, and plants. Since they transport a number of leader-less proteins to the plasma membrane or the extracellular space, EVs are considered part of Unconventional protein secretion (UPS) routes. UPS involving EVs are a relatively new field in plants. Aside from their role in plant physiology and immunity, plant extracts containing EVs have also been shown to be beneficial for human health. Therefore, exploring the use of plant EVs in biomedicine and their potential as drug delivery tools is an exciting avenue. Here we give a summary of the state of knowledge on plant EVs, their crosstalk with mammalian systems and potential research routes that could lead to practical applications in therapeutic drug delivery. Originally considered as cellular waste, their discovery has revolutionised our understanding of cell-cell communications and transfer of biological information from 1 cell to another. Since leaderless proteins loaded in these vesicles bypass the Golgi and are recruited in EVs from the cytosol, most EVs are considered part of the unconventional secretion pathway (UPS). Exosomes, a particular type of EV, are particularly interesting in this context for the following reasons: the mechanism of cargo loading an cells , exosomean cells . Consequan cells . Plants an cells . While kUnconventional protein secretion (UPS) involves a range of mechanisms that allow proteins to reach the extracellular medium, bypassing at least part of the conventional ER-Golgi-PM secretory pathway. While this conventional pathway usually involves the presence of signal peptides at the N-terminus of proteins, UPS leads to the secretion of leaderless soluble proteins in the extracellular medium or trafficking of membrane proteins via an alternative route than through the Golgi . These mhttps://clinicaltrials.gov/ct2/show/NCT01294072).To address some of these challenges in terms of cost-effective production and lack of ethical issues, plants might offer an alternative source of exosomes and EVs. As a result, a growing number of studies are looking into their potential health benefits. For example, the effect of plant extracellular vesicles loaded with curcumin are currently being tested in clinical trials (NCT01294072) to evaluate their impact on surgery of newly diagnosed colon cancer patients  or exosomes-like nanoparticles (ELNs) are terms used to refer to vesicles that have been isolated from total plant extracts and usually contain a mix of EVs and other cellular microvesicles . Since tWhile Exo70E2 protein has been identified as a marker of EXPO vesicles, it has been reported that exosomes specifically contain TET8, a tetraspanin protein . This asThe processes described to isolate plant EVs depend on the nature of the plant material. Apoplastic fluids are usually extracted from leaves, while blending/juicing is performed on fruits or roots. EVs can also be isolated from liquid plant exudates . AlthougArabidopsis thaliana, Nicotiana benthamiana) or seeds (sunflower)  . Additionflower) . Recentlnflower) .Enriched EV fractions have been obtained through blending plant matter such as ginger roots, herbs, wheat, and dandelion . JuicingFusarium oxysporum, Botrytis cinerea and Alternaria alternata) suggesting an antifungal activity in plants . Nevertheless, PDNVs contain a range of bioactive molecules such as proteins, lipids, or metabolites with therapeutic effects summarised in . Amongstal cells . Nanovesal cells . DespiteResearch on EVs (obtained from the apoplast of plants) as potential drug delivery systems is far more restricted than those on PDNVs. So far, to our knowledge, only one study has shown that purified apoplastic small EVs (sEVs) are efficiently taken up by human ovarian cancer cells OVAR5 . This paβ-glucocerebrosidase protein expressed in carrots has been approved as being safe and efficient and successfully used for 2 decades . Nevertheless, preliminary data seem very promising such as the efficient uptake of plant EVs by human cells, their expected low immunogenic character (associated with nutrition) and their positive effect on human health. As a consequence, using plant EVs as a drug delivery tool might represent a powerful future alternative to classical therapeutic systems.Extracellular vesicles (EVs) are associated with Unconventional protein secretion (UPS) routes. They are released in the extracellular space through mechanisms that are still poorly understood. The field of plant EVs is relatively new but is proving to have great prospects in biomedicine. The potential to produce pure plant EV subtypes such as exosomes through biopharming and be able to deliver therapeutic molecules is very appealing. Additional advantages include the engineering capability of


Nanodrug platforms with redox characteristics can not only reduce the adverse effects of oxidative stress on tissues by removing excess oxidants from the body but also have multienzyme-like activity, which can play a cytotoxic role in tumor tissues through the catalytic oxidation of their substrates to produce harmful reactive oxygen species such as hydroxyl radicals. In this review, various redox nanomaterials currently used in disease therapy are discussed, emphasizing the treatment methods and their applications in tumors and other human tissues. Finally, the limitations of the current clinical application of redox nanomaterials are considered.Redox imbalance can trigger cell dysfunction and damage and plays a vital role in the origin and progression of many diseases. Maintaining the balance between oxidants and antioxidants  The bal2O2), singlet oxygen (1O2), hydroxyl radicals (•OH), and superoxide anion radicals (O2•-). Among them, hydrogen peroxide is a stable non-radical ROS, which can be converted into other highly active ROS under catalytic conditions, such as 1O2, •OH, O2•-, or peroxynitrite (ONOO−)  . Further hypoxia . An incr hypoxia . The mai hypoxia . HoweverBecause of the remarkable breakthroughs in the field of nanotechnology, particularly nanochemistry and nanofabrication technologies, a variety of nanomaterials with unique redox-modulating properties have been prepared . NumerouCurrently, the use of redox nanomaterials in biomedicine has received increasing attention. While some studies have unilaterally reported the classification of redox nanomaterials or their applications in tumor therapy or other fields, few articles have integrated the existing data to provide a comprehensive summary. It may thus be difficult to fully understand the applicability of redox nanomaterials in the treatment of various diseases. In this review, we briefly summarized the redox nanomaterials that have been or are projected to be used across a wide range of biomedical applications, focusing on the different material types and their redox properties. We have also described the therapeutic pathways underlying the use of redox nanomaterials in tumor tissue and some of the research advances in other human tissue applications . FinallyIn recent years, efforts have focused on the exploitation of sophisticated and effective nanomaterials that can be transported toward specific tissues with accurate release using spatial and temporal controls . SustainRedox-responsive nanosystems based on inorganic nanocarriers are of great interest due to their unique physicochemical properties, such as robustness, cost-effectiveness, stability, and facile synthesis/modification . The pro2O2 and O2•-(in situ polymerized polyplatinum (IV)-coated gold nanorods which can amplify tumor accumulation using mild hyperthermia for enhanced thermo-chemotherapy  and its nanocomposites, a redox-active transition metal dioxide nanomaterial, have been shown to be advantageous in cancer therapy due to their unique structural and physicochemical properties  and reduced graphene oxide (rGO) are more widely studied and used in nanomedicine due to their better solubility and dispersibility. GO and rGO are often used as antibacterial/antimicrobial agents due to their peroxidase-like redox ability . The bas60) is commonly used as an antioxidant to protect neural tissues as it scavenges excess free radicals  have good biocompatibility , highly In addition to the inorganic redox nanomaterials mentioned above, selenium nanoparticles (SeNPs) also have a wide range of biomedical applications due to their redox-regulating properties. In the human body, selenium is present as selenocysteine in various antioxidant enzymes, such as glutathione peroxidase, thioredoxin reductase, and selenoprotein P. As the redox center for these enzymes, selenium is vital in maintaining cellular redox homeostasis . The conAlthough inorganic nanomaterials are easy to prepare and are highly modifiable , their cLiposomes are composed of phospholipid membranes that mimic natural membranes and have features such as easy development with controlled size distributions and no innate toxicity; they are often used to construct nanodrug carriers . LiposomPolymeric micelle nanoparticles are usually composed of amphiphilic copolymers, whose hydrophobic parts self-assemble in an aqueous solution into a core surrounded by the hydrophilic part as a shell, forming a typical core-shell structure that contributes to creating a drug loading carrier . Like liAmong the many organic nanocarriers, nanosized hydrogels offer a controlled-release pattern of drugs in targeted tissues and passive targeting . The fleIn contrast to the redox properties of most inorganic redox nanomaterials, organic redox nanomaterials are also often redox-responsive due to their attachments to various chemical bonds or groups with redox properties, such as dendrimers  and variComposite redox-responsive nanomaterials that integrate inorganic and organic redox nanomaterials combine their respective strengths and improve their physicochemical properties and often exhibit synergistic effects . Numeroum-TTFTB) with Dy3+ under solvothermal conditions, m-TTFTB) through post-synthetic redox-tuning and the incorporation of Ag nanoparticles in situ for promoting the tuned photothermal NIR response  are unique MOF nanomaterials formed by alternating ferric and ferrous irons coordinated with cyanides  consist of metal-containing nodes , comprising an attractive class of composite nanomaterials . A largeresponse . The rescyanides . PBNPs ecyanides . PBNPs wcyanides . The dis2O2) or reducing equivalents (such as glutathione and NAD+). This observation inspired the development of smart redox-responsive nanomedicines with enhanced ROS-generating/scavenging efficiencies to help maintain the redox balance in different tissues. The functional mechanisms and applications of redox nanomaterials in tumors and other human tissues are briefly discussed.The dynamic cellular redox balance is maintained by the balance between the production and elimination of ROS  Figure . Some diTumor tissues usually include the parenchyma and stroma. The tumor stroma has different morphological characteristics compared to the stroma in normal tissues, and is often referred to as a tumor microenvironment (TME). Due to the abnormal growth of tumor cells and their excessive demand for nutrients, most tumors show dysregulated angiogenesis with leakage of vascular structures, increased stiffness, interstitial pressure, immunosuppression, the overexpression of enzymes, acidity, hypoxia, and an overactivated redox environment . These aEvidence has suggested that when compared with normal cells, most cancer cells produce more ROS, and this increase in ROS levels may play an important role in the occurrence and development of cancer . To bala2O2 level in tumor microenvironment to catalyze it to produce oxygen and improve the tumor hypoxia environment.High ROS level is a significant histological feature in tumor microenvironment. We can not only use this property as a marker to distinguish tumor tissue from normal tissue, but also kill tumor cells by changing the level of ROS or inducing the production of highly toxic ROS . In addi−3 M KO2 solution) for 24 h. In the experimental group, the relative molecular weight of the polyMTO decreased sharply to 400 g mol-1 after being incubated with a KO2 solution for 6 h, suggesting that the degradation of polyMTO was based on the ROS-triggered cleavage of the thioketal linker. The in vitro and in vivo experiments showed that the polyMTO could respond to intracellular ROS in tumor tissues with a chain-breakage patterned release of intact anticancer drugs, thus generating significant inhibition of tumor cell growth.Acidic environments can cause the breakage of some acid-unstable bonds and lead to the disintegration of nanomaterials . Similar3O4 nanoparticles and indocyanine green (ICG) hybrid nanogels (FIGs-LC) to potentially fulfill ROS regulation in tumor tissues. The endogenous ROS content in the tumor tissue cannot generate sufficient free radicals to induce tumor cell apoptosis -ss-poly (ethylene glycol)-ss-poly (ε-caprolactone) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[folate (polyethylene glycol)-2000]), to combine PTT and PDT in tumor treatments  exhibited precise tumor targeting, penetration, and efficient and responsive release. An in vivo mouse tumor model showed that Ang-NCss(siRNA) had a significant inhibitory effect on U87MG glioblastoma while exhibiting good biocompatibility, indicating that it is a promising, safe, and efficient tool for GBM gene therapy.GSH controls the redox environment of the cell mainly through the formation and breakage of disulfide bonds and reactions with excess ROS  from a mAs the GSH is overexpressed in the TME, the disulfide bond has been widely used as the “golden standard” for the design of redox-responsive drug delivery systems . In addivia diselenide bonds, and then coupled with telmisartan (TM)-embedded gelatin nanoparticles. Under the mediated action of the matrix metalloproteinase-2, the gelatin nanoparticles of G (TM)PPSP were degraded in the TME, and TM was released. The remaining portion of the G (TM)PPSP then entered the tumor cells through endocytosis, which disintegrated and released PTX at high GSH levels to induce tumor cell apoptosis combined with the photothermal effects of platinum nanoparticles. via GSH cleavage. In addition, when compared to disulfides and other common adducts, the bond expanded the reduction timescale. Oscillatory rheology experiments have also demonstrated the excellent stability of the succinimide–thioether bond. The degradation rate of the succinimide-thioether containing hydrogels was 10 times slower than that of disulfide-crosslinked hydrogels, suggesting that the succinimide-thioether-containing adducts have good blood stability and longer administration times. Compared with the disulfide bond, the trisulfide bond contains an extra sulfur atom, which is the key linkage in biomolecules with functions ranging from subtle modulation of the conformation and assembly stability in proteins and peptides , a contrast agent, in polyethylene glycol (PEG)-modified dendritic mesoporous silica, for multimodal tumor imaging-guided synergistic therapy. In vitro and in vivo experiments showed that Mo5+ in DCDM can catalyze hydrogen peroxide to produce singlet oxygen according to the Russell mechanism. Subsequently, the generated Mo6+ can consume GSH, reduce the GSH level at the tumor site, and weaken the antioxidant capacity of the tumor, thus further accelerating the generation of singlet oxygen and enhancing the efficacy of CDT. Under multimodal in vivo imaging guidance, DCDM exhibits superior therapeutic outcomes at a cellular level and possesses remarkable anticancer abilities in U14 tumor-bearing mice.It is not uncommon for nanomaterials to regulate the tumor tissue redox balance through both the ROS and GSH response pathways. Furthermore, their therapeutic mechanisms involve both aspects, such as chemodynamic therapy, which is based on transition metal-based Fenton responses or Fenton-like responses. Many highly valent transition metal ions, such as Mnoxic ROS . Compareoxic ROS . Dong etoxic ROS , which eIn vitro experiments using 10 mM GSH showed that the PPA-CS NPs exhibited a distinct increase in size over time after incubation with the 100 μM H2O2 for 19 h. In addition, transmission electron microscopy and mass analysis both confirmed that PA-CS NPs could sequentially respond to high levels of ROS and GSH. Compared with the control group, the tumor tissue in the PTX@PPA-CSNPs group showed the largest necrotic area and the most significant anti-tumor effect.ROS/GSH double-responsive nanomaterials, which involve the synthesis of two stimulation-responsive nanomaterial therapeutic systems that are modified with responsive functional groups on their surface, have attracted a wide range of research interest. Although more obvious therapeutic effects may be achieved, the nano-therapeutic platform designed by combining ROS responsiveness and GSH responsiveness will undoubtedly have a more complicated structure, which means more complex synthesis process or higher production cost, and also means that this kind of nanomaterials will face more experimental investigation and difficulties in clinical practice. Therefore, how to balance high efficiency and low price is an inevitable consideration of redox nanomaterials in the field of tumor therapy and even other medical fields.2 to generate O2•-, then be further converted into ROS, such as H2O2, •OH, and ClO−via various catalytic reactions /Ce(IV) valency switch provides ROS scavenging abilities . This abin vivo experiments showed that RNPs maintained normal redox reactions in healthy cells; however, they could reduce oxidative stress significantly in liver tissue and promote improvement in liver fibrosis and inflammation upon delivery to the liver tissue.Previously, we mentioned that mitochondria are the main organelles that produce ROS and that large amounts of ROS can lead to mitochondrial dysfunction, disrupting cellular function. Noteworthy, mitochondria are also functional organelles for intracellular lipid metabolism, whose damage and reduction in the number can affect lipolysis, leading to lipid accumulation, insulin resistance, and eventually, obesity . NumerouAs well as treating obesity and related diseases, redox nanomaterials have also been shown to have promising antibacterial effects. Undoubtedly, diseases caused by various pathogenic bacteria have historically posed serious global challenges threatening public health . AntibioOverall, the balance between oxidants and antioxidants are essential for mechanisms related to biological growth, health, and aging, and they are vital for the physiological processes that allow cells to function properly. Whether it is a tumor or other tissues or organs in the body, an imbalance in the redox state within a cell or tissue can lead to various malfunctions in its internal metabolism. Due to the rapid development of nanotechnology, scholars have explored various redox nanomaterials with unique properties to influence redox homeostasis in different tissues . For exaIn this review, we have briefly summarized the various redox nanomaterials used in biomedical applications and grouped them into three categories according to their structural composition, namely: inorganic, organic, and composite. Furthermore, in the field of tumor therapy, where redox nanomaterials are most widely used, three types of redox nanomaterials were identified: ROS-responsive, GSH-responsive, and ROS/GSH dual-responsive. The applications of these materials are described according to their therapeutic principles. In addition, the application of redox nanomaterials in other human tissues in relation to brain diseases, cardiovascular diseases, anti-aging, antibacterial, and obesity, was briefly reported.in vitro and xenotransplantation-based animal experiments. However, these model experiments are different from actual tumors or other pathological changes, especially metastatic tumors, and it is difficult to accurately imitate the damage to cells or tissues caused by various oxidative stress conditions in the human body. The lack of demonstration in clinical trial results is the key factor limiting the clinical application of redox nanomaterials. In the following statements, we briefly analyzed why it is difficult to apply redox nanomaterials to clinical experiments and provided future perspectives on redox nanomaterials.Although redox nanomaterials are widely used for the prevention and treatment of various diseases in the preclinical stage due to their advantages, clinical applications are still rare. Most research on redox nanomaterials has focused on in vivo comprises the basis for biocompatibility evaluations. At the same time, in order to establish a mature evaluation index of biocompatibility, not only systemic toxicity should be tested, focusing on evaluating the retention of nanomaterials in important organs such as the liver and kidney, but also the corresponding human cell lines should also be established to evaluate biosafety in vitro in different disease tissues and promote the development of redox nanomaterials in clinical trials.1) Considering the complexity of the human internal environment, the current evaluation methods for the biocompatibility of different redox nanomaterials are insufficient. Understanding the specific toxicity mechanisms of redox nanomaterials and improving the detection methods of cytotoxicity 2) Although redox nanodrugs lasts longer than traditional drugs and the side effects is smaller, their overall therapeutic effects are currently too low to be extended to large-scale clinical applications. In addition, the design cost for the complex structures of the redox nanodrugs, especially the multifunctional nanoplatform therapy system, is too high to be suitable for a wide range of applications. When designing redox nanodrugs, materials that are easy to synthesize and have a low cost should be selected. Meanwhile, it is necessary to adopt simple and effective synthesis methods, and rationally construct composite redox nanomaterials to achieve higher therapeutic effect.3) Finally, the literature has widely shown that redox nanomaterials with stronger permeability and retention effects tend to have drug delivery efficiencies of ≤5%. Therefore, efforts to eliminate pre-leakage and improve the precise drug-controlled release ability of redox nanomaterials in time and space must be made.We believe that as nanotechnology and biomedicine are rapidly developing, redox-responsive nanomaterials will gradually overcome these problems and become more applicable in clinical medicine in the future.


In recent years, bone tissue engineering (BTE), as a multidisciplinary field, has shown considerable promise in replacing traditional treatment modalities . Since bone is such a complex and dynamic structure, the construction of bone tissue composite materials has become an attractive strategy to guide bone growth and regeneration. Chitosan and its derivatives have been promising vehicles for BTE owing to their unique physical and chemical properties. With intrinsic physicochemical characteristics and closeness to the extracellular matrix of bones, chitosan-based composite scaffolds have been proved to be a promising candidate for providing successful bone regeneration and defect repair capacity. Advances in chitosan-based scaffolds for BTE have produced efficient and efficacious bio-properties via material structural design and different modifications. Efforts have been put into the modification of chitosan to overcome its limitations, including insolubility in water, faster depolymerization in the body, and blood incompatibility. Herein, we discuss the various modification methods of chitosan that expand its fields of application, which would pave the way for future applied research in biomedical innovation and regenerative medicine. Human bone is a three-dimensional 3D) composite porous structure with several roles within the body consisting of 30% organic matter and 70% inorganic matter . It perfD composiRecently, advances in tissue engineering and technologies in this field could provide more efficient treatment ,10. BoneThe marine environment accounts for about half of the global biodiversity, and nearly 70% of the earth’s surface is covered by oceans . Among tTo date, there have been considerable efforts to explore modified CS-based materials for BTE. Despite the phenomenal success of implants of BTE particularly in the realms of dentistry and orthopaedics, there are still challenges to overcome. The failure of implants resulting from infection, prosthetic loosening, and non-union continue to be the most serious examples . As we kBone injury and chain reactions mediated by free radical species  generation are other critical factors. ROS affect the long-term stability of bone/implants and mediate the apoptosis of osteoblasts and osteocytes, leading to osteoclastogenesis and thereby favoring bone resorption . Post-imSeveral studies have been developed in order to combat the aforementioned complications. CS and its derivatives show an intensified anti-inflammatory cytokine induction . StudiesDysphania ambrosioides (A) extract on the bone repair process in vivo. Results showed that when CS-based spheres exhibited the ability to guide bone repair and osteoinduction they stimulated osteogenesis by recruiting osteoprogenitor cells . M. M200]. 2O2). Enzymatic cross-linking provides high reaction rates under physiological conditions. It is also a “green” approach with mild reactions and biocompatible catalysts for hydrogel synthesis [®”. The spatial control of rhBMP-2 bioactivity at the cellular level was confirmed by fluorescence expressed in osteoblast and pre-osteoblast cells. The retained rhBMP-2 in the HPGC + BMP implant could localize osteoprogenitor recruitment and osteogenesis while also minimizing rhBMP-2 diffusion loss at the implantation site , tyrosinase (Tyr), lysyl oxidase, phosphatases, and horseradish peroxidase (HRP), and hydrogen peroxide -based composite scaffolds have great potential in BTE. CMCS is an anionic CS derivative. Modification of CS by carboxymethylation of hydroxyl and amino groups affords better solubility of aqueous solution at different pH values, which achieves the processability in BTE. Chelation with more Ca2+ is enabled due to the introduction of carboxymethyl groups in CMCS . FurtherThe improvement in compressive strength makes it an attractive candidate for bone defect repair at the same time. Because of its porosity, gel-forming characteristics, simplicity of chemical manipulation, and high affinity for in vivo macromolecules, CMCS will be a viable contender as a supporting material for BTE .Bone substitutes exhibiting osteoconductivity and antimicrobial activity are increasingly needed to prevent and treat contaminated or infected bone defects. Hydroxypropyltrimethyl ammonium chloride chitosan (HACC) is a water-soluble CS derivative with a strong cationic nature. The addition of quaternary ammonium groups to the CS molecule preserves the characteristics of raw CS and significantly weakens hydrogen bonds, improving water solubility and antibacterial activity . ForemosStaphylococcus strains [Antibiotic-loaded polymethyl methacrylate (PMMA) has successfully treated and prevented osseous infections. It represents the current gold standard for local antibiotic delivery systems in orthopedic surgery . However strains . FurtherAnother CS derivative is SCS. It is the general term for sulfonated and sulfated CS derivatives. SCS is the production of sulfonation reaction, the incorporation of sulfonate groups onto CS . Due to The most attractive characteristic of SCS is that it has been employed for its blood anticoagulant properties ascribable to their chemical structure similar to that of heparin ,229. SCSGlycol chitosan (GCS) is a water-soluble derivative of CS wherein the C6 hydroxyl groups are functionalized with glycol groups . It is iGCS is also an excellent drug carrier for delivery systems owing to its physicochemical features, biocompatibility, biodegradability, and mucoadhesiveness . In the Guanidine is an essential class of organic compounds. It is present in many medicinally important compounds that show biological activity and therapeutic functions, such as broad-spectrum antimicrobials and antidiabetic drugs. Polymers functionalized with amine groups can be easily converted to guanidinium groups. Attachment of the guanidine group onto CS can introduce a positive charge onto the polymer backbone, which would result in better aqueous solubility at neutral pH and shows potentially good antimicrobial activity . Surface3O4 nanoparticles and CS [There are also some other structure-modified strategies of CS. It has been modified with 5-fluorouracil to serve as an in vitro anti-tumor drug delivery system . It has s and CS . These Cs and CS .During the last decades, CS has been widely modified by graft copolymerization with a multitude of polymers to improve some CS weaknesses to broaden its application in BTE , such asFurthermore, developing hybrid biomaterials by including peptide sequences into biopolymers is an attractive alternative to confer functional cell–biomaterial interaction . RGD pepOverall, pure CS could be structure-modified through amino and hydroxyl groups such as acylation, carboxylation, etherification, graft copolymerization, and ring opening of epoxides to produce a series of CS derivatives . These CThe past few decades have seen revolutionary advancements for designing dynamic living constructs and replacing malfunctioned bone tissues in BTE applications. Intelligent hydrogels that respond rapidly to the exterior stimulus changes have attracted considerable attention from many scientists . These “The present contribution focuses on synthesizing and characterizing a pH- and thermo- responsive system based on CS modified with isopropyl side chains. CS-based injectable hydrogels present considerable potential for bone remodeling with sensitivity to the pH value. The use of poly  (PAA) to modify CS is crucial for forming pH-sensitive hydrogels. These hydrogels are designed to target and control the release of drugs for treating bone diseases or infection ,293, whiTemperature is another critical factor that influences the formation of intelligent CS hydrogels. The excellent moldable ability of thermo-responsive CS injectable hydrogels proved to be a new class of bone substitute materials that respond to temperature. This hydrogel exerts no potential adverse effects on the surrounding cells/tissues . ReportsComparing intelligent CS-based hydrogels to other presently available conventional materials shows great advantages, as they have the ability of in-situ osteoblastic differentiation induction and interesting biological functions. The incorporation of growth factors and other bioactive molecules as well as nano-particles as smart modifications can enhance cell differentiation, proliferation, and attachment and therefore may improve new bone formation . Even thBTE is regarded as the best alternative approach to conventional bone grafting techniques. A scaffold is an essential subunit that provides mechanical strength, a site for cell attachment, proliferation, and differentiation. Polymer selection and scaffold fabrication techniques are the key factors. The scaffold can be fabricated using various techniques based on the implantation site, the nature of polymers, and their characteristics.CS is a readily available polymer with good biodegradability and biocompatibility. It has non-toxic, mucoadhesive, hemostatic, and antimicrobial properties employed in a broad range of biomedical and biopharmaceutical research. CS has displayed significant osteoconductivity but minimal osteoinductive properties . The maiTo solve the limitations above, several modifications at C2 or C5 positions of CS by introducing various reactive groups or by copolymerization with other polymers of interest or grafting with biological and synthetic macromolecules are explored nowadays, which render appropriate bone regenerative properties. Modified CS offers unique bio functions, such as water solubility, antibacterial properties, and pH and thermo-sensitivity. The inclusion of bioceramic materials in modified CS may facilitate the spreading of bone marrow stromal cells and significantly enhance the mechanical property ,301,302.Although investigators have fabricated a variety of biomaterials that mimic the morphology and functions of natural bone tissue and have verified the effectiveness in promoting bone regeneration at the cellular and animal levels, there are still many obstacles and challenges in the BTE application of modified CS ,305. FutThis review summarized modified CSs for their application in BTE. Modified-CS-based scaffolds/hydrogels displayed superior physical, chemical, mechanical, and biological properties, unlike their counterparts, serving as excellent vehicles for accelerating bone regeneration. Future studies should focus on optimizing novel modifying methods and evaluating their performance parameters during modifications of bone scaffolds.This review showed that the empirical refinement of CS has candidly thrown open new avenues for the treatment of bone defects. Thus, an effort was made here to provide insights into the past and current trends in using modified CS polymers. Integration and processing of the different properties offered by various modified CS composites would be further beneficial for treating bone and bone-related defects.


Here, we review the current approaches to engineering spatial precision for in vivo bacterial diagnostics and therapeutics using synthetic circuits, and the challenges and opportunities this technology presents.The mammalian gut and its microbiome form a temporally dynamic and spatially heterogeneous environment. The inaccessibility of the gut and the spatially restricted nature of many gut diseases translate into difficulties in diagnosis and therapy for which novel tools are needed. Engineered bacterial whole-cell biosensors and therapeutics have shown early promise at addressing these challenges. Natural and engineered sensing systems can be repurposed in synthetic genetic circuits to detect spatially specific biomarkers during health and disease. Heat, light, and magnetic signals can also activate gene circuit function with externally directed spatial precision. The resulting engineered bacteria can report on conditions  The gastrointestinal tract is a spatially heterogeneous environment, with variable oxygen, pH, nutrients, host immune and antimicrobial factors, and microbiota composition . DiseaseThe role of the gut microbiota in therapy is increasingly apparent: microbes modify and sequester orally delivered drugs, impacting efficacy on a patient-to-patient basis ; many drA promising strategy to achieve these goals is using the bacteria residing in the gut for clinical applications. Bacteria have been used as probiotics to improve human health for over a century  and, morin situ biosensors for diagnosis, recording, and therapeutic delivery  .• Stimulus Counters “count” small numbers of independent induction events using recombinases, riboregulated transcriptional cascades, or genetic transcriptional switches .• Biocontainment Systems prevent engineered bacteria from surviving outside a specified environment. Examples include engineered physical containment , auxotro• Logic Gates and Complex Signal Processing can be used to compute multiple signals, for example using recombinases or toehold switches , enablinThis field is poised for rapid growth, with an expanding toolbox of synthetic biology tools available to engineer an ever-increasing range of bacteria . Basic sdelivery .• BinaryFor further detail we direct readers to recent reviews giving an overview of engineered bacterial biotherapeutics .Herein, we focus on synthetic gene circuits that enable spatial precision within the mammalian gut . These cBacteria have a naturally diverse catalogue of transcriptional, translational, or post-translational sensing mechanisms. These include, one-component systems (OCS), two-component systems (TCS), and extracytoplasmic function (ECF) sigma factors. Spatially precise and disease-specific gene regulators are used naturally by bacteria to control their function in the gut, making them an exciting option to control spatially precise functions of engineered bacteria. Synthetic signalling systems are also being developed to further expand this toolbox .Biosensing of infection and disease is a key goal for diagnostics but can also be used to activate synthetic circuits specifically at disease sites to create localised therapeutics. Many drugs, in particular broad-spectrum antibiotics, have off-target effects on the native microbiota, which can compromise colonisation resistance and promote opportunistic pathogen growth among other negative consequences . The devEscherichia coli was engineered to detect N-acyl homoserine lactone (AHL) from the pathogen Pseudomonas aeruginosa and respond by lysing to release a toxin and a biofilm destabilising enzyme  to detect cell-to-cell proximity and density  over difg enzyme . This synfection . While tnfection  or some QS can also be used for density-dependent delivery mechanism, which are being actively developed in the context of tumours , and couBacillus subtilis-based system that specifically detects multiplexed environmental DNA sequences, including human pathogens within a community. An alternative system using Acinetobacter baylyi detected tumour-associated host DNA mutations within the mouse gut (DNA-responsive biosensors could offer a more specific signal of proximity to pathogens and other sites of disease. This may be particularly useful in situations where QS circuits suffer from crosstalk between strains. Two recent pre-prints have co-opted the natural competence of some bacterial strains to develop DNA-responsive biosensors . Cheng eouse gut . It remaLive diagnostics have also been engineered to sense and respond to molecular disease biomarkers such as nitric oxide, nitrite and nitrate, thiosulfate, tetrathionate, and haeme . While tE. coli strains detect regional gut niches through hormones, oxygen, biotin, acetate, and bicarbonate to avoid activating metabolically burdensome virulence processes in unfavourable locations (Bacterial gene expression changes based on molecular cues that exist in natural gradients within the gut . These cocations . Clostriocations . SimilarE. coli throughout the mouse gut (E. coli strains were engineered to preferentially grow in specific regions of the mouse gut based on either hypoxic or low pH (Whilst promoters have been identified that exhibit consistent expression in ouse gut , the catr low pH . The firHelicobacter species bind specific glycans in the small intestine and stomach epithelium (Fusobacterium nucleatum bind a host factor overexpressed in colonic adenomas (Streptococcus gallolyticus expressed in E. coli to target cancerous colorectal epithelial cells (E. coli allowing specific adherence and colonisation of solid tumours (Localised activity can also be achieved by spatially restricting bacterial growth, which is possible through natural tropisms of a chosen chassis strain, engineered tropism through expression of targeted binding proteins, and through non-genetic mechanisms such as the adherence of antibodies to the bacteria’s surface  or usingithelium , and Fusadenomas , both caal cells , synthet tumours , or reco tumours  and in t tumours .Targeted binding can also simultaneously activate a desired gene of interest. E. coli chemotactic protein CheZ under the control of hydrogen peroxide, nitric oxide, or quorum molecule sensitive promoters in a CheZ KO strain allowed “pseudotaxis” towards the respective signals (Another method for spatial targeting involves placing key motility genes under the control of a sensor module to direct the biotherapeutic to a target site. Placing the  signals . AlthougA powerful complementary approach to control engineered bacteria is using externally applied physical stimuli, such as light, temperature, or electromagnetic fields. As with the molecular sensing approaches described above, these tools aim to create minimally disruptive, non-invasive methods to activate diagnostics and therapeutics within specific regions of the gut. Unlike the molecular approaches, physical stimuli could be directed externally by a health care professional or an individual for on-demand activity.E. coli with a 115-fold dynamic range (Optogenetic systems use light-sensitive proteins or protein domains to regulate downstream cellular processes upon exposure to specific wavelengths of light . They aric range , and benTechnological advances in photonics for medical applications are accelerating the use of optogenetics towards clinical contexts. For example, the poor tissue penetration of visible/UV light can be overcome using upconversion nanoparticles, which convert an externally applied NIR stimulus into internal UV or visible light . This apTemperature-sensitive systems can also provide non-invasive spatially precise circuit induction. Recently, focussed ultrasound was used to control release of immune checkpoint inhibitors for anti-cancer immunotherapy in a murine solid tumour model . The mecMagnetism is an alternative external physical inducer commonly used in the clinic in the form of MRI. Whilst magnetic-sensitive genetic circuits have been developed in eukaryotic cells , they haThe catalogue of spatial sensors that have been tested and validated in the mammalian gut is limited. New sensors can be developed through genome mining, direct targeted design, or directed evolution of existing sensors to improve or change their desired specificity or sensitivity . Bacteriin situ with high spatial precision techniques when possible, or integrated engineered memory systems when not (An additional fundamental challenge is that any biosensor will be inherently limited by the combination of the spatial precision of the signal, the temporal characteristics of the synthetic circuits, and movement of bacteria within the gut. Small molecules that readily diffuse from their target site may activate distant sensors in a less targeted manner. Studying engineered probiotic activity when not , will hein situ non-invasive diagnosis, to complex signal processing power, and targeting for reduced off-target effects, will offer worthy rewards for overcoming these in a range of clinical scenarios. Advances in spatial and functional precision will need to go hand-in-hand with advances in biosafety, biocontainment and patient acceptance that are already being tackled more broadly for the use of engineered bacteria as live biotherapeutic products (Engineered biotherapeutics are a promising technological opportunity for spatially precise investigations of the gut, and for diagnosis and treatment of gut diseases. While the field is still in its infancy, particularly with respect to clinical delivery, spatially precise engineered microbes are already primed to serve as exciting scientific investigative tools for non-invasive sampling of the complex gut microenvironment in animal models . There aproducts . The nexproducts . Technol