Electrospinning has been used for decades to generate nano-fibres via an electrically charged jet of polymer solution. This process is established on a spinning technique, using electrostatic forces to produce fine fibres from polymer solutions. Amongst, the electrospinning of available biopolymers , chitosan (CH) has shown a favourable outcome for tissue regeneration applications. The aim of the current review is to assess the current literature about electrospinning chitosan and its composite formulations for creating fibres in combination with other natural polymers to be employed in tissue engineering. In addition, various polymers blended with chitosan for electrospinning have been discussed in terms of their potential biomedical applications. The review shows that evidence exists in support of the favourable properties and biocompatibility of chitosan electrospun composite biomaterials for a range of applications. However, further research and in vivo studies are required to translate these materials from the laboratory to clinical applications. Electrospinning (ES) is a process that utilises an electric field to control the deposition of polymer fibres onto target substrates. Originally, the process was devised for the textile industry . It is nES has been adapted to obtain natural and synthetic polymer fibrous mats that mimic the extra cellular matrix (ECM). Amongst available biopolymers, CH and its naturally derived composites have been widely adapted for TE applications. Its versatility with respect to variations in the molecular weights and degree of deacetylation in order to target a clinical condition in the field of regenerative medicine is adapted very efficiently. For ease of understanding, there is a brief description of the ES process, various parameters affecting the fabrication and properties of electrospun nano-fibres. The aim of the current review is to assess the current literature about the electrospinning of chitosan and its composites with other natural polymers to be utilized in TE and regenerative applications.The process of ES is based on obtaining fine fibres from polymer solutions via electrostatic forces. The electrospun fibres have small diameters and significantly larger surface area compared to conventional fibres. The essential components of ES are: 1-high voltage power supply, 2-spinneret and a grounded collecting plate such as a metallic screen, and 3-plate or rotating mandrel .A direct current (DC) voltage is used to generate a potential difference between two terminals within the range of 1–30 kVs that injects a charge of a certain polarity into the polymer solution to accelerate a jet towards a collector of opposite polarity . A syrinIn brief, a polymer solution is held by its surface tension at the capillary tip that is subjected to the potential difference created between the spinneret and anode surface (collector). Due to this electric field, a charge is triggered on the liquid surface, changing the pendant drop to a falling jet. When this field reaches a critical value, the repulsive electric forces overcome the surface tension forces. Finally, an unstable charged jet of polymer solution is extruded from the tip of the Taylor cone . Consequently, an unstable and a rapid whipping jet ensues between the capillary tip and collector. The resultant evaporation of the solvent leaves the polymer nano-fibres behind on the collector ,19.The characteristics of electrospun fibres are governed by various factors related to the ES dope such as concentration, molecular weight, surface tension, viscosity and conductivity/surface charge density of the polymer solution. In addition, equipment factors such as voltage, feed or flow rate of polymer loaded in the syringe, type of collector and the distance between the collector and needle tip can affect the morphology of electrospun fibres . For insIn terms of collectors, several materials can be used to collect the electrospun nano-fibres ,26. AlumSolution parameters such as concentration and viscosity influence the electrospun fibre morphology. For instance, lower concentrations result in finer fibres and increased number of beads, while concentrated solutions result in thicker bead free fibres or a reduced number of beads . As the Solution viscosity is another vital parameter that affects the electrospun fibres in terms of size and morphology. ES of uniform bead-free nano-fibres requires the polymer solution to have an optimal viscosity. Very thick fibres can result if the viscosity is high enough to hinder the ejection of the polymer solution. At a viscosity lower than optimal, the continuous fibre formation is less likely and results in droplets or excessive bead formation. The viscous polymers result in extending the stress relaxation times and limiting the breakage of polymer jets. Consequently, fibres acquired at relatively higher viscosity showed more uniform fibres. Similarly, low surface tension of the solvent facilitates production of nano-fibres with less or no beads . PolymerThe molecular weight (Mw) affects the solution properties such as dielectric constant, conductivity, electrical, viscosity, surface tension, and rheological properties ,16. The Nanoparticles of various natural and synthetically derived materials have a significant impact on the process of electrospinning . AdditioA wide variety of natural and synthetic polymers and their composite blends have been used for ES. Examples of electrospun synthetic polymers include poly glycolic acid (PGA), poly lactide co-glycolide (PLGA), polycaprolactone (PCL), polyurethane (PU), poly lactic acid (PLA), polystyrene (PS) and poly vinyl alcohol (PVA). Some of polymers available naturally for ES are, silk, cellulose, collagen, gelatine, hyaluronic acid and CH. Natural and synthetic polymers are also used in combination to manipulate the materials properties  depending on the specific application. Other properties such as cellular affinity, morphological, structural, pore size and degradation can also be altered by copolymers .d glucan, (N-acetyl d glucosamine and 2 amino 2 deoxy-β-d glucans. The structure of the biopolymer is shown in Chitosan (CH) is a polysaccharide biomaterial composed of (1–4) acetamido 2 deoxy-β-The deacetylation of CH is performed either by chemical hydrolysis under harsh alkaline conditions or enzymatic hydrolysis . The alkFabrication of electrospun CH micro or nano-fibres is not an easy task. Common solvents used for dissolving CH are triflouroacetic acid (TFA) or composite solutions of diluted acetic acid copolymerized with polyethylene oxide (PEO) . Differed-glucosamine structures, high crystallinity and ability to form hydrogen bonding is responsible for the poor solubility in commonly available organic solvents  have been electrospun using aqueous solutions with suspended SF nanoparticles ..141].Bioactive ceramics such as HAp can also be incorporated in CH solutions prior to ES to produce bioactive scaffolds capable of accelerating osteoblast proliferation and bone formation  on electrospun PCL/CH fibrous scaffolds and enhancing the bone regeneration in vivo . More reEscherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus [Although CH is primarily used as quaternized form to deliver drugs to the implantation sites, the use of fibrous CH scaffolds as delivery media for various drugs has also been reported ,161,162.s aureus  indicatis aureus .2) particles exhibits higher healing properties in rats [S. aureus and E. coli, which are both organisms implicated in wound infections [S. aureus and P. aeruginosa [Considering the excellent porosity and drug-carrying ability of CH fibres, another major application is production of wound dressings . CH can  in rats . Similarfections . More refections . Moreoveruginosa .Garcinia mangostana extract in form of mucoadhesive mats which can be used by patients who may be unable to administer conventional oral hygiene measures to prevent dental caries [Recently, anti-carious mats constructed from electrospun CH fibres containing antimicrobial agents have been studied for anti-cariogenic potential. CH/thiolated CH mats blended with PVA can be used to deliver anti-caries agents such as l caries ,172.The diversity of electrospun CH fibres have led to their use as templates for hepatocyte, chondrogenic and myogenic differentiation. Feng et al. reported CH nano-fibre mesh liver TE applications and tested the biocompatibility using hepatocytes . In anotThe present review shows that there is a wealth of scientific evidence available in support of the favourable properties and biocompatibility of chitosan electrospun composite biomaterials for a range of TE and regenerative medicine applications. However, further research including in vivo studies are required to translate these materials from laboratory to clinical applications. Although investigators have been able to alter the instrumentation and solution parameters to mimic natural tissue structure and morphology, the continual process of reporting various possibilities needs further characterisation and clinical trials before their applications for treating medical diseases with predictability. Using electrospinning and augmenting this technique with additives, manufacturers can have further control of the final template. Moreover, clinicians and bioengineers, whilst working together, can solve unexplored regenerative therapies by harnessing the fibre diameter, size, morphology and orientations according to the desired clinical applications. Mimicking structural and functional aspects of natural tissues will have a significant impact on the future of electrospinning of these materials.


Glioblastoma is the most deadly brain tumor type and is characterized by a severe and high rate of angiogenesis, remaining an incurable disease in the majority of cases. Mechanistic understanding of glioblastoma initiation and progression is complicated by the complexity of genetic and/or environmental initiating events and lack of clarity regarding the cell or tissue of origin. To determine these mechanisms, mouse models that recapitulate the molecular and histological characteristics of glioblastoma are required. Unlike in other malignancies, viral-mediated mouse models of glioblastoma rather than chemically induced mouse models have been developed because of its sensitivity to viruses. Based on recent molecular analyses reported for human glioblastoma, this review critically evaluates genetically engineered, xenograft, allograft, viral-mediated, and chemically induced mouse models of glioblastoma. Further, we focus on the clinical value of these models by examining their contributions to studies of glioblastoma prevention, tumorigenesis, and chemoresistance. Glioblastoma is the most common and deadly primary brain tumor , and theOver the past few years, genomic and proteomic characterization along with robust animal models of glioblastoma have provided invaluable data. In addition, pre-clinical models and a better understanding of the core pathways disrupted in glioblastoma are providing renewed optimism for novel strategies targeting these devastating tumors. Here we discuss the current advances in rodent models, particularly mouse models, of glioblastoma and other gliomas, and how these developments have influenced strategies for therapeutic intervention.The 2016 World Health Organization Classification of Tumors of the Central Nervous System uses molecular parameters in addition to histology to define many tumor entities . In thisGlioblastomas are divided into three categories in the 2016 CNS WHO classification according to a key genetic prognostic marker, isocitrate dehydrogenase (IDH): glioblastoma, IDH-wildtype; glioblastoma, IDH-mutant; glioblastoma, NOS. IDH-wildtype (about 90% of cases) corresponds most frequently with clinically defined primary or de novo glioblastoma and predominates in patients over 55 years of age . IDH-mutPathological characteristics of glioblastoma is typically a highly cellular glioma, usually comprised of poorly differentiated, sometimes pleomorphic tumor cells with nuclear atypia and vigorous mitotic activity. Conspicuous microvascular proliferation and/or necrosis is an essential diagnosis characterization.Molecular characteristics typical of IDH-wild type glioblastoma include TERT promorter mutations (present in ~ 80% of cases), homozygous deletion of CDKN2A/CDKN2B (~ 60%), loss of chromosomes 10p ~ 50%) and 10q (~ 70%), EGFR alterations , PTEN mutations(25–30%), and PI3K mutations (~ 25%) , 5.0% and 10The use of mice to create suitable models for the study of specific tumors or to investigate the role of candidate genes has obvious advantages. Firstly, manipulation of the mouse genome to create specific genetic changes by microinjection of DNA into fertilized eggs or by homologous recombination in embryonic stem cells is relatively easy compared with other mammalian species such as rats. Another advantage is the availability of inbred strains of mice that are genetically identical, obtained by breeding sibling mice over 20 generations. Since these animals present the same genetic background, they can be compared for their response to a treatment or a genetic modification between different laboratories. The laboratory mouse shares extensive molecular and physiological similarities to humans and is a powerful tool for studying cancer.Transgenic mouse models offer an opportunity to develop and utilize an easily replenished, reproducible, spontaneously manipulated, and more accurate pre-clinical model of human cancers, which we can use to enhance our molecular knowledge and to test promising therapies. Therefore, mouse models that recapitulate human glioblastoma may be an invaluable tool. However, these conventional genetic approaches, such as transgenics and knockouts, are limited by the time and costs associated with extensive intercrossing of mouse lines. Several new viral vector-mediated genetic approaches offer the ability to directly modify the genome of somatic cells in mouse tissues and these have recently been applied to the rapid generation of complex mouse tumor models that harbor multiple genetic changes. On the other hand, xenograft and allograft models can be used to measure therapeutic responses to drugs more rapidly than genetically engineered or viral vector-mediated models. Chemical carcinogen-induced models are usually generated in rats, and only a small number of instances of chemical carcinogen-induced models are currently known.PTEN, TP53, CDKN2A, RB) or the activation of oncogenic pathways  [The molecular progression of gliomas, like many tumors, involves the accumulation of genetic and epigenetic alterations that result in the loss of tumor suppressor gene function  –8.GFAP gene regulatory elements in astrocytes.There are several examples of aberrant expression of relevant downstream signaling pathways in mouse glioma modeling. These include astrocytomas of varying grades resulting from glial fibrillary acidic protein (GFAP)-regulated expression of v-src . WeissenGFAP promoter [Ding et al. , 11 utilpromoter , 11.EGFR, CDK4, MDM2; decreased expression of CDKN2A, TP53, PTEN) are present [When the glioblastoma-like tumors are examined in these mice, additional genetic alterations such as those found in human glioblastomas  [Overexpression of relevant oncogenic receptors or downstream signaling pathways has also been employed in the development of mouse glioma models. These have been either alone or in combination with mice harboring specific knockouts of relevant cell cycle regulatory proteins. For example, S100 glial precursor promoter-regulated v-ERBB (an activated member of the EGFR family) transgenic mice develop oligodendrogliomas, which are potentiated in terms of shorter latency and increased malignancy when initiated in mice deficient for both p16 and p19 (ll mice) .(T2); Idh1fl(R132H)/+ developed small nodules (up to 1 mm diameter) originating from the subventricular zone at 2–6 weeks after tamoxifen injection.Briefly, Bardella et al.  have estThe nodules expressed proliferation markers, such as Ki67, and retained BrdU label, suggesting that they exhibited a variety of proliferative behaviors. Further, many nodule cells also expressed the astrocytic and NSC marker GFAP and in some lesions, a few cells expressed the neuroblast marker Doublecortin (Dcx).Table In recent years, viral vectors have been extensively used for the generation of mouse models of interest in the study of brain tumors. Several routes for viral vector delivery to the brain are available: intracerebral stereotaxic injection, intrathecal and intraventricular injection, and intravascular infusion with or without modification of the blood–brain barrier. The choice of route for viral vector administration needs to be carefully considered since it affects neuronal cell transduction efficiency and spatial distribution, as well as the level of transgene expression in the infected cells . IntraceVirally transduced expression of relevant gain-of-function alterations, in combination with transgenic mouse technology, allows one to model such somatic alterations at a later stage in life, though it does not lead to germline colonies. Although the link between a viral etiology and human gliomas is weak, retroviruses that have been engineered to express relevant gain-of-function genes have been used to create glioma models in mice and other mammals , 18. ThiPDGFB) injected into normal mice have yielded astrocytic tumors, with varying glioma types generated when injected in Cdkn2a-null mice. One of the best examples of coupling retroviruses to express somatically defined gain-of-function genes in varying cell lineages and genetic backgrounds to model gliomas is the RCAS-tva system [PDGFB, EGFRvIII, activated p21–RAS, activated AKT), the lineage of the cell expressing the tva receptor  and underlying genetic cell cycle alterations in the mice . For example, retrovirally transduced expression of v-sis or PDGFB in GFAP-tva mice resulted in oligodendrogliomas or mixed oligoastrocytomas in 40% of the mice, with 60% of NES-tva mice developing similar gliomas [Cdkn2a-null mice, the gliomas formed with a shorter latency and were of higher grade [EGFRvIII mutant into mice harboring activated RAS led to efficient formation of glioblastoma [Retroviruses carrying v-sis  infect mouse cells expressing Tv-a (the receptor for RCAS viruses) [Lentiviruses expressing oncogenes such as ckground , 23. Curand TP53 . Misuracand TP53  have estviruses) .For many years, immunodeficient rodents have been an important tool in modeling human glioblastoma. Propagation and testing of glioblastoma in such animals is most commonly accomplished in the subcutaneous flank location (heterotopic), although recent years have seen increased use of orthotopic  xenograft models. For both heterotopic and orthotopic studies, xenograft and allograft tumors are usually established from permanent human glioblastoma cell lines.Invasive orthotopic xenografts have also been established from surgical specimens that were first maintained as tissue spheroids in short-term culture , 28. FinGlioblastomas that have been continuously propagated as flank tumors recapitulate this very important and characteristic feature of human glioblastoma following intracranial transfer. Different from the heterotopic transplantation, the direct orthotopic transplantation denies the influences of in vitro culture, provides a proper microenvironment, and preserves the integrity of tumor-initiating cells , 32.Many human and mouse cell lines have been used in xenograft and allograft models Table . TateishA library of orthotopic glioblastoma xenograft models using surgical samples of glioblastoma patients has been established. These patient-derived glioblastoma xenograft (PDX) tumors recapitulated histopathological properties and maintained genomic characteristics of parental glioblastoma in situ , 43. SoePatient-derived stem cells are used to identify cell functions that are altered by disease, such as Alzheimer’s and Parkinson’s disease and thereby provide a target for drug discovery. Patient-derived glioblastoma stem cells have been generated from xenograft tumors of the glioblastoma surgically resected , 46. PatRecent progress and expansion in next-generation sequencing (NGS) technologies enable to characterize the cancer genome in a time frame that is corresponding to treatment decisions, providing the chance to potentially increase the therapeutic effect by targeting the genomic alterations driving tumor behavior . To chalThere are important caveats to this approach that still need to be addressed in xenograft models. Firstly, the mice do not have an intact immune system. Inflammatory cells may be a critical component to the biology of the tumor and its response to certain drugs, particularly immunotherapy. Secondly, the surrounding stroma and microenvironment is of mouse origin, not human, and may interfere with drug response.Previously, only a chemical carcinogen-induced mouse model, the GL261 model, have been derived from an intracranially-induced methylcholanthrene tumor in C57BL/6 mice . RecentlIn 1971, H. Fraser has described the first incidence of a spontaneous glioma within the SMA-560 mouse strain. Initially, these tumors, which resembled human anaplastic astrocytomas, were restricted to in vivo studies only, as tumorigenicity was lost with repeated in vitro passaging of tumor explant cultures .Glioblastoma is one of the most problematic cancers to treat. Despite advances in molecular profiling of the disease, information is still lacking, particularly regarding treatment.As genome-wide sequencing efforts continue in humans, mouse glioma models that better recapitulate the complex genomic landscape of human glioma will be generated. These models will provide increasingly powerful tools for the validation of hypotheses engendered by human genomic data, such as confirming the driver mutations that are causal to oncogenesis, as well as for preclinical testing of personalized therapy.


Fibrosis contributes to the development of many diseases and many target molecules are involved in fibrosis. Currently, the majority of fibrosis treatment strategies are limited to specific diseases or organs. However, accumulating evidence demonstrates great similarities among fibroproliferative diseases, and more and more drugs are proved to be effective anti-fibrotic therapies across different diseases and organs. Here we comprehensively review the current knowledge on the pathological mechanisms of fibrosis, and divide factors mediating fibrosis progression into extracellular and intracellular groups. Furthermore, we systematically summarize both single and multiple component drugs that target fibrosis. Future directions of fibrosis drug discovery are also proposed. Fibrosis, characterized by excess accumulation of extracellular matrix (ECM), is a common pathological process in many chronic diseases or injuries. Many irritations trigger the pro-fibrotic responses, including persistent infections, radiation, chemical agents, genetic disorders, and autoimmune diseases. The development of fibrosis is accompanied by the loss of a fraction of resident cells and their replacement by ECM, which would finally lead to tissue remodeling and organ failure. Fibrosis contributes to high morbidity and mortality in many diseases such as dilated cardiomyopathy and idiopathic pulmonary fibrosis (IPF)  and connective tissue growth factor signaling is required in kidney, liver, and lung fibrosis  owing to the imbalance of natriuretic peptide system pathway and renin angiotensin aldosterone system/TGF-β1 pathway in aging  could degrade ECM to prevent its excessive accumulation.Growth factors contain a huge family of proteins that stimulate cell growth and proliferation. They are secreted by fibroblasts, immune cells, and epithelial/endothelial cells, and are able to orchestrate cellular responses. While epithelium/endothelium are damaged, cells in these tissues massively upregulate the production of growth factors to promote the proliferation of immune cells and fibroblasts. Among growth factors, TGF-β is the “master” modulator in fibrogensis  work as the inhibitors of MMPs. The balance of MMPs and TIMPs modulates the process throughout fibrosis development, including the formation of multiple cell injuries, the activation of latent cytokines and myofibroblasts and mainly, the maintenance of the homeostasis of ECM  signaling pathway mediates EMT during the fetal development, and responds to injury through the repression of epithelial marker epithelial-cadherin by Intracellular factors, mainly multiple kinases, propagate the signaling received by cells through phosphorylation and other pathways. A common consequence of the signaling is that, transcription activators or inhibitors translocate into nucleus to regulate fibrosis-related gene expression and cell responses. In certain inflammatory pathways, intracellular factors also modulate the expression of extracellular factors, such as growth factors and cytokines, and secrete them out of cells to amplify inflammatory responses. Besides, epigenetic factors are emerging as a new way to affect fibrosis-related gene expression.col1a2, col3a1, and lox  pathway. An element of MAPK pathway, mitogen-activated protein kinase-activated protein kinase-2 mediates myofibroblasts differentiation and regulates the gene expression of several matrix proteins such as Nuclear receptors are receptors located in cytoplasm and nucleus that could receive signals from intracellular ligands and bind to DNA to regulate gene expression. For example, peroxisome proliferator activated receptor γ(PPAR-γ), may directly regulate type I collagen gene  differ among different fibrosis stages in NAFLD , galectin-3 and lysyl oxidase homolog 2 (LOXL2). ROS can be generated through tissue injuries, cell damages and NADPH oxidase activities. Extracellular ROS targets latency-associated peptides and then activates TGF-β signaling while intracellular oxidative stress induces p53-dependent apoptosis in lung fibrosis via the caspases-9/3 activation in mitochondria  and multi component (Table 4) drugs with their verified and potential targets in fibrosis.Motivated by huge clinical burdens, continuous intense researches on drug targeting fibrosis have been conducted, many of which have led to clinical trials. Due to the strong associations between inflammation and fibrosis, more efforts have been devoted to anti-inflammation drugs in the past few years , and angiotensin all have approved antagonist drugs. An antagonist for tyrosine kinase receptors of PDGF, Imatinib, has showed protective effect by reducing differentiation of resting fibroblasts in SSc mice model  therapies, usually appeared as herbal formula, have been studied for thousands of years as multi-component drugs (Wang et al., With multiple targets being discovered, it becomes apparent that more common targets will be shared across many fibrotic diseases with common mechanisms, though some proteins will not express in special conditions. Meanwhile, there will be more chance for drug repositioning, which indicate common drugs will be shared across different targets andfibroproliferative diseases.Fibrosis is a common pathological process in many diseases, causing a great clinical burden in recent years. The development of state-of-the-art technologies facilitate discovery of fibrosis therapies. Multi-omics analysis provides a more convenient and systematic way for researching on disease mechanisms (Fernandes and Husi, RZ and LZ conceived and designed the project. Each author has contributed significantly to the submitted work. XL drafted the manuscript. LZ, BW, MY, and RZ revised the manuscript. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer XT and handling Editor declared their shared affiliation.


Non-communicable diseases are the leading global causeof death and disproportionately afflict those living in low-income andlower-middle-income countries (LLMICs). The association between socioeconomicstatus and non-communicable disease behavioural risk factors is well establishedin high-income countries, but it is not clear how behavioural risk factors aredistributed within LLMICs. We aimed to systematically review evidence on theassociation between socioeconomic status and harmful use of alcohol, tobaccouse, unhealthy diets, and physical inactivity within LLMICs.We searched 13 electronic databases, including Embaseand MEDLINE, grey literature, and reference lists for primary research publishedbetween Jan 1, 1990, and June 30, 2015. We included studies from LLMICspresenting data on multiple measures of socioeconomic status and tobacco use,alcohol use, diet, and physical activity. No age or language restrictions wereapplied. We excluded studies that did not allow comparison between more or lessadvantaged groups. We used a piloted version of the Cochrane Effective Practiceand Organisation of Care Group data collection checklist to extract relevantdata at the household and individual level from the included full text studiesincluding study type, methods, outcomes, and results. Due to high heterogeneity,we used a narrative approach for data synthesis. We used descriptive statisticsto assess whether the prevalence of each risk factor varied significantlybetween members of different socioeconomic groups. The study protocol isregistered with PROSPERO, number CRD42015026604.After reviewing 4242 records, 75 studies met ourinclusion criteria, representing 2 135 314 individuals older than 10 years from39 LLMICs. Low socioeconomic groups were found to have a significantly higherprevalence of tobacco and alcohol use than did high socioeconomic groups. Thesegroups also consumed less fruit, vegetables, fish, and fibre than those of highsocioeconomic status. High socioeconomic groups were found to be less physicallyactive and consume more fats, salt, and processed food than individuals of lowsocioeconomic status. While the included studies presented clear patterns fortobacco use and physical activity, heterogeneity between dietary outcomemeasures and a paucity of evidence around harmful alcohol use limit thecertainty of these findings.Despite significant heterogeneity in exposure andoutcome measures, clear evidence shows that the burden of behavioural riskfactors is affected by socioeconomic position within LLMICs. Governments seekingto meet Sustainable Development Goal (SDG) 3.4—reducing prematurenon-communicable disease mortality by a third by 2030—should leverage theirdevelopment budgets to address the poverty-health nexus in these settings. Ourfindings also have significance for health workers serving these populations andpolicy makers tasked with preventing and controlling the rise ofnon-communicable diseases.WHO. Non-communicable diseases account for 70% of globaldeaths,Development agencies—mainly working with the poorest members oflow-income and lower-middle-income countries (LLMICs)—might be more likely torealign their activities to address non-communicable disease prevention if therewas clear evidence that non-communicable diseases and their risk factors affectthese populations.Evidence before thisstudyWe searched PubMed and Google scholar onJuly 28, 2015, with no language restrictions. Our searchterms were a list of World Bank-defined low-income andlower-middle income countries (LLMICs); MeSH and free-textterms for tobacco use, alcohol use, diet, and physicalinactivity; and socioeconomic status. Studies publishedbefore 1990 were excluded. There was a moderate risk of biasamong the included studies.From this search, we identified a 2005non-systematic review of surveys from 11 low-income andmiddle-income country (LMIC) WHO subregions and ameta-analysis of studies examining tobacco use and income.These studies report higher prevalence of tobacco use  and lower alcohol use in thepoorest strata of LMICs compared with more affluent groups.The most comprehensive analysis to date comes from ananalysis of LMIC World Health Survey data from 2002–04.Self-reports from 232 056 participants from 48 countries—ofwhich 25 were upper-middle-income and 23 were low-income orlower-middle income (LLMICs)—suggested that those with moreeducation and assets were more likely to be physicallyinactive and consume insufficient fruit and vegetables andless likely to smoke daily. The socioeconomic patterning ofheavy episodic drinking was mixed and inequalities were morepronounced in the least developed countries. The findingsfrom that study are now 10 years old, and largely drawn fromupper-middle or high-income countries.Added value of thisstudyTo our knowledge, this study is the firstsystematic review to examine the distribution of the mainnon-communicable behavioural risk factors across differentsocioeconomic groups within LLMICs and the first study toreport on physical activity and socioeconomic status indeveloping countries. This work supports ongoing efforts tolink non-communicable disease prevention with the globaldevelopment agenda and provides evidence for developmentagencies on how to engage with non-communicable diseases.Our study shows that lower socioeconomic groups are morelikely to drink alcohol, use tobacco, and consumeinsufficient fruit and vegetables than more advantagedgroups. Higher socioeconomic groups were found to be moreinactive and might consume more fats, salt, and processedfood. Our findings substantially augment the scant evidencefrom previous LLMIC-based reviews on individual riskfactors. With the use of broader measures of socioeconomicstatus, we found significant differences between castes,classes, sexes, and occupational groups with the widestdifferences observed across different educationalstrata.Implications of all the availableevidenceCombined with previous work, theassociation between non-communicable disease risk factorsand socioeconomic status seems to be dependent on setting,population, and exposure definitions. Tobacco use seems tobe almost universally more prevalent in low socioeconomicgroups than in high socioeconomic groups, whereas alcoholand diet require further investigation. Our findings haveimportance for the development community that have a part toplay in ensuring that their projects do not promoteenvironments that promote non-communicable diseases inlow-income settings. This study shows that there is a clearsocioeconomic gradient of non-communicable disease riskbehaviours within most LLMICs. Education was stronglycorrelated with healthier behaviour in most settings andmight be an important tool in controlling the epidemic.Other interventions should be focused on social groups thatare most at risk.Only a few studies on the intranational distribution ofbehavioural risk factors have been published: a 2005 non-systematic review ofsurveys from 11 low-income and-middle-income country (LMIC) WHOsubregionsNon-communicable diseases are the leading cause of death andindividuals living in LLMICs are 1·5 times more likely to die prematurely fromthese conditions than those living in high-income countries.We aimed to systematically review current evidence on theassociation between socioeconomic status and harmful use of alcohol, tobaccouse, unhealthy diets, and physical inactivity within LLMICs.We did a systematic review following a registered protocoland PRISMA guidelinesWe used English search terms  but did To accommodate differing views, capture all relevantstudies, and broaden the systematic review, we included household orindividual-level data measures of income, wealth, assets, socioeconomicstatus, education, caste, and occupation . Weexcluded studies that did not allow comparison between more or lessadvantaged groups. Authors were contacted for additional data wheresocioeconomic status and behavioural risk factors were measured but reportedindependently. We used the same dates and strategy for all searches,tailored to specific databases by LA and an experienced medical librarian(NR).With the use of a piloted form , LA and LA used a piloted version of the Cochrane EffectivePractice and Organisation of Care Group data collectionchecklistWe assessed data quality using a modified Newcastle-Ottawascale,The main outcome was differences in prevalence or relativerisk of non-communicable disease behavioural risk factors between differentsocioeconomic groups. We also planned to examine how age, sex, urban orrural location, and study quality affected findings. We assessed variabilitywithin studies in our quality scoring. This included considering theuniformity of training for those conducting the study and the instrumentsused to gather data. Significant heterogeneity between studies, particularlyin the exposure and outcome measures precluded quantitative synthesis andmeta-analysis. We used a narrative approach, grouping studies by outcomemeasure and WHO region. We analysed differences between outcomes, geographicregions, age groups, and sex. We also present sensitivity analyses for eachrisk factor having removed all medium and low-quality studies. The protocolof this study is registered with PROSPERO, number CRD42015026604. We usedExcel to generate simple descriptive statistics.An employee of the funder (BM) contributed to the studydesign and review of draft manuscripts. The funder did not have anyinvolvement in data collection, data analysis, or data interpretation. Thecorresponding author had full access to all the data in the study and hadfinal responsibility for the decision to submit for publication.Our literature search returned 4242 records and 106 additionalrecords were retrieved from other sources . Over10Five articles presented data for all risk factors, 41 articlesreported on a single risk factor, and the remaining 29 articles reported datafor two or three risk factors. Ten different socioeconomic indicators were used.One article presented Global Adult Tobacco Survey data fromtwo LLMICs,Over half of the included studies had been published since2010 and seven were published before 2000.40Overall, 13 studies were of low quality, 33 were moderate, and29 were of high quality, leading to a low risk of bias across studies. The mostcommon study weaknesses were loss to follow-up and failure to control forconfounding factors. Studies in African populations were more likely to benon-peer reviewed, to be of a lower quality, and have smaller sample sizes.Studies reporting data on Indian populations and tobacco use tended to be largerand of a higher quality than those for other populations and risk factors. Moststudies were funded by governments, public health agencies, developmentagencies, and non-governmental or non-profit organisations. A summary of allhigh quality studies is presented in the 29 studies from 15 countries reported measures of physicalactivity.There was a paucity of studies reporting adjusted results thatwere statistically significant; however, most studies found that individualswith a high socioeconomic status were less active than groups with a lowersocioeconomic status, irrespective of outcome and exposure measures. This trendwas consistent across studies from southeast Asia, the western Pacific, Africa,and the eastern Mediterranean. The notable exceptions were found in populationsfrom urban areas: residents of Aleppo,Most participants were aged 15–65 years old. Studies thatexcluded people aged over 60 years, or those younger than 30 years, still foundthat higher socioeconomic groups were the least active.24 studies from ten countries reported measures of alcoholuse.One study found that alcohol users were more likely toexperience impoverishment than non-users but this association was notstatistically significant.Analysing the findings by region, alcohol use—while notnecessarily at harmful levels—was most prevalent in low-income and less-educatedgroups across Indiavs 5·1%); however, no measures of significancewere presented.Six of the eight high-quality studies assessed alcohol userather than harmful use of alcohol. With the exception of a 2012 survey ofyounger Indian adults,26 papers from 11 countries reported on eight differentaspects of diet.78Studies from Indonesia, Syria, Nepal, Benin, Eritrea, andNigeria all found lower fruit and vegetable intake in less affluent and lesseducated groups.When we examined the effect of age on these dietary findings,we found that studies examining older populations came to the same conclusionsas other studies examining the same dietary component.50 studies reported data for tobacco use in 39 countries, almostThe popularity of chewing versus smoking tobacco varied bysetting, but women were more likely to chew tobacco than smoke.98One moderate quality survey of 233 young Keralan men found asignificantly higher prevalence of tobacco use in middle-class students,adjusting for age and other confounders.99The finding that low socioeconomic groups were more likely touse tobacco than high socioeconomic groups was the same in every geographicalregion. Studies that examined older populations came to the sameconclusions.Differences between educational groups were larger thandifferences between castes and income or wealth strata.Overall, low socioeconomic groups in most of the LLMICs inwhich evidence was available were more likely to use tobacco and alcohol, and toconsume less fruit, vegetables, fish, and fibre, and more meat than highsocioeconomic groups. High socioeconomic status groups tended to have higherlevels of physical inactivity and consume more fats, salt, and processed foodsthan low socioeconomic groups . While tThis systematic review identifies broad trends in globalbehavioural risk factors for non-communicable diseases, finding that lowsocioeconomic groups in many countries are more likely to drink alcohol, usetobacco, and consume insufficient fruit and vegetables than are highsocioeconomic groups. High socioeconomic groups tend to be more inactive andconsume more fats, salt, and processed food.This systematic review is the first to examine thesocioeconomic distribution of all four major behavioural risk factors withinLLMICs. Our findings substantially augment the scant evidence from previousLLMIC-based reviews on individual risk factors; Ciapponi andcolleaguesOur tobacco findings mirror the well established inequalitiesfrom high-income countries, where low-income groups are the most likely tosmoke, start smoking earlier, consume more tobacco, quit less successfully,experience more adverse health effects, and die at a younger age than affluentgroups.Our dietary findings complement studies from high-incomecountries that have consistently found a positive association betweensocioeconomic status and consumption of fruit, vegetables, fibre, andfish.Our finding that rural high socioeconomic status groups tendto be the most physically inactive departs from the experience of high-incomecountries.This systematic review was done in line with PRISMA andCochrane guidance, following a registered protocol and assessing risk of biasusing well established criteria. The bidirectional association betweensocioeconomic status and health is widely averred but infrequently assessedwithin LLMICs.Our method was designed to capture all studies onsocioeconomic status and non-communicable disease behavioural risk factors. As aresult, our findings are extremely heterogeneous and require carefulinterpretation. We treated the highest and lowest groupings of each exposure as if they were interchangeable eventhough each study tended to use a unique definition, cutoff, and studypopulation. This allowed us to identify broad trends for future research toexamine in detail; however, it meant that our findings should not be seen asdefinitive. The large amount of data also prevented us from presenting deepanalysis of each risk factor in this systematic review; however, ourcomprehensive data extraction and presentation of all original data and subgroupdescriptors in the Use of the 2013 World Bank classification of income excludedcountries that have only recently been reclassified as upper-middle-income;however, our focus on countries that are currently low-income enhances theusefulness of this systematic review as development agencies are moving awayfrom upper-middle-income settings.In view of the broad scope of this systematic review, the factthat over half of the countries classed as low-income or lower-middle-incomewere not represented in our search results is striking. Almost half of theincluded studies relate to India, and the evidence from the Americas, theeastern Mediterranean, and Europe is relatively scant. The fact that so manyLLMICs were not represented is a major finding, but also a weakness in itself;the excellent evidence from India is not generalisable to all low socioeconomicgroups in LLMICs and research is needed to explore whether the patterns weidentify hold true in countries where surveillance is non-existent.Of the 47 publicly available LLMIC-based WHO STEPSsurveys,Our findings provide an overview of the current evidence,underlining intranational trends and data gaps. Policy makers and nationaldevelopment agencies working in the countries where 82% of premature deathsoccur should review the evidence relevant to their setting and consider whethertheir current non-communicable disease prevention strategies are appropriate.Where low socioeconomic status correlates with non-communicable disease riskfactors, governments can use development funds to simultaneously improveliteracy, living standards, and income alongside health. The definitions used toidentify behavioural risk factors are inconsistent, and data are not availablefor most LLMICs. Rectification of these issues, with surveillance reporting riskfactors stratified by socioeconomic status, is an obvious research priority.Nevertheless, this should not delay action in the countries where dataexist.


Objectives: According to the CanMEDS’ Scholar competency, physicians are expected to facilitate the learning of colleagues, patients and other health professionals. However, most medical students are not provided with formal opportunities to gain teaching experience with objective feedback.Methods: To address this, the University’s Medical Education Interest Group (MEIG) created a pilot teaching program in January 2015 entitled ‘MedTalks’. Four 3-hour sessions were held at the University Faculty of Medicine, where first and second year medical students taught clinically oriented topics to undergraduate university students. Each extracurricular session included three 30-minute content lectures, and a 90-minute small group session on physical examination skills. Each medical student-teacher received formal feedback from undergraduate students and from faculty educators regarding teaching style, communication abilities, and professionalism. In addition, medical student-teachers self-evaluated their own teaching experience.Results: Over 50 medical students from the University participated as medical student-teachers. Based on quantitative and qualitative evaluation surveys, 100% of medical students agreed that MedTalks was a useful way to develop teaching skills and 92% gained a greater confidence in individual teaching capabilities, based largely on the opportunity to gain experience (with feedback) in teaching roles.Conclusions: A program designed to give medical students multi-source teaching experience  and feedback on their teaching  can improve medical student confidence and enthusiasm towards teaching. Future studies will clarify if medical student self-perceived enhancements in teaching ability can be corroborated by independent  observations of future teaching activity. In 2005, the Royal College of Physicians and Surgeons of Canada established seven formal roles to guide the practice of all Canadian physicians. In the role of Scholar, physicians are expected to act as teachers and facilitate the education of their patients, students, fellow healthcare professionals, and the public . Also, sLike any other skill, developing teaching acumen requires instruction, practice, and feedback . SeveralTo address this need, the University Medical Education Interest Group (MEIG) created a pilot teaching program in January 2015 entitled ‘MedTalks’. This novel educational initiative aimed to provide undergraduate medical students with the knowledge and skills to excel in teaching roles during their clinical careers and to provide teaching opportunities with formal observation-based feedback.The purpose of this report is to characterize the development of the local innovative MedTalks program, and evaluate the program via participant feedback, identifying both strengths and areas requiring improvement. The dissemination of the lessons learned from this study will help further the development of practical teaching programs at the undergraduate medical student level, complete with multi-level feedback.MedTalks was held at the University of Ottawa Faculty of Medicine between January 2015 and April 2015, and included four 3-hour evening sessions. Each session included three 30-minute lectures and a 90-minute small group session. These teaching sessions were centered on the anatomy, physiology, pathophysiology, and relevant clinical exam skills pertaining to the four session topics: the cardiovascular system, the respiratory system, the musculoskeletal system, and patient history.Medical student-teachers, individually or in pairs, delivered a 25-minute lecture, followed by 5 minutes of questioning from learners and open discussion. Lecture materials included: anatomy and physiology teaching slides related to muscles, heart, and lungs; electrocardiogram interpretations; and chest x-ray interpretations. All teaching material was prepared by medical student-teachers using standardized guidelines from the local medical program’s Physician Development Course to ensure quality and consistency of teaching materials.Medical student-teachers demonstrated clinical exam skills to undergraduate student learners in a small group teaching context. Clinical exam skills taught included: auscultation with a stethoscope, use of a sphygmomanometer, palpation, and percussion. Clinical exam skill teaching was based on the local Physician Skills Development course. Students were also brought to the medical gross anatomy lab at the University Faculty of Medicine. Under the supervision of anatomy teaching Faculty, medical student-teachers led student-learners through relevant objectives using prosected cadaveric specimens; these demonstrations were based on the medical curriculum that has been described previously .Two evaluation forms were created to evaluate and provide feedback regarding medical student teaching immediately after each session. These evaluation forms were based on the official undergraduate medical program’s evaluation forms, and included both five-point Likert scale items and gave the option for open-ended qualitative feedback. The first evaluation form  was used by undergraduate learners and faculty evaluators to evaluate the teaching activities, including clarity of lecture material, engagement of audience, and overall effectiveness. The second form  was provided to the medical student-teachers to reflect upon their teaching experience, and comment on any impact this program had on their willingness to participate in future formal  teaching activities.While the feedback student-teachers received from Faculty on their lectures were based on Faculty notes from an entire observed lecture, the feedback that student-teachers received from Faculty on small group teaching was limited to short ~5–10 minute observation windows (from the entire 90 minutes session) due to lack of Faculty participants  and the consequent need for Faculty to circulate room to room to provide some feedback to all students.Undergraduate learners and medical student-teachers also completed program evaluation surveys regarding the MedTalks program that included both quantitative  and qualitative (open-ended commentary) aspects .One hundred and fifteen undergraduate students from the University of Ottawa signed up for MedTalks, and 53 medical students from the University of Ottawa participated in MedTalks as medical student-teachers. Eleven faculty members, each with teaching experience in the local medical curriculum, volunteered their time to observe medical student-teachers during their teaching activities and provide written feedback via provided evaluation forms. The written feedback was collected by MedTalks organizers and provided to the medical student-teachers immediately after their teaching activities were completed.Undergraduate student learner feedback was very positive, with 97% of responding participants either agreeing or strongly agreeing that MedTalks was a useful and interesting program. The organization of MedTalks and professionalism demonstrated by medical-student teachers were also highly regarded. The major theme in the open-ended commentary regarding suggestions to improve the initiative was a greater focus on hands-on learning through the small group sessions and less time dedicated to didactic lectures. A related sub-theme that emerged was greater exposure to the anatomy lab.MedTalks was very well received by medical student-teachers as 53 (100%) of medical student-teachers either agreed or strongly agreed that MedTalks was a useful and interesting way to practice their teaching skills, and would be motivated to teach in the future, in other domains as well as at future MedTalks events. While 92% of the student-teachers found that this experience resulted in a greater confidence with individual teaching capabilities and felt comfortable leading their teaching sessions, preparation and training in teaching was identified as an area of weakness of the program by 2 (4%) of the medical student-teachers.Teaching is a crucial but widely overlooked skill in the everyday role of the clinician. The Royal College of Physicians and Surgeons in Canada has reaffirmed teaching competency as a necessary skill through the formally defined CanMEDS Roles since 2005, which highlights ‘Clinical Educator’ as a key competency to be achieved by all medical professionals, including trainees. However, despite these formal expectations of clinicians to develop as clinician-educators, few programs are characterized in the literature to be dedicated to teaching skill enhancement at the undergraduate medical education level, and most identified programs lack objective assessment of trainees . ApproxiIn its inaugural year of 2015, MedTalks received an overwhelmingly positive response from over 50 medical students interested in teaching, and over 100 undergraduates keen to learn from medical students. MedTalks was regarded as informative and well-organized by both participants and medical student-teachers. In addition, all medical student-teachers agreed that MedTalks was beneficial in providing an avenue to practice teaching and more similar opportunities were needed. Although the amount of prior teaching experiences the cohort had was not noted, most student-teachers found this experience resulted in a greater confidence with individual teaching capabilities. Moreover, all participating student-teachers noted that this program enhanced enthusiasm for future teaching opportunities.There were several improvements identified that could be implemented in future iterations of MedTalks, informed by the feedback to the pilot program. Although medical student-teachers were provided with the hands-on experience, the knowledge and theory behind effective teaching was lacking. MacDougall and Drummond state that although physicians are experts at what they teach, they lack expertise on how to teach effectively and therefore rely on personal experience rather than educational skill . WorkshoFeedback is essential in facilitating improvements in an educator’s teaching skills and performance . UnfortuIn a national survey of student-as-teacher programs in U.S. medical schools, Soriano et al noted the lack of long-term outcome evaluations such as assessment of student-teachers’ teaching competence in residency . AssessiMedical students have little opportunity to hone their teaching skills and receive constructive feedback on their teaching abilities, and yet are expected to teach proficiently as early as their first year of residency. The MedTalks initiative addresses these gaps by providing medical students with teaching opportunities, in practical medical education settings, complete with multi-source feedback. These early positive educational experiences may also prove to build enthusiasm and drive for future roles as medical educators.Click here for additional data file.


Even with an effective vaccine, an estimated 240 million people are chronically infected with hepatitis B virus (HBV) worldwide. Current antiviral therapies, including interferon and nucleot(s)ide analogues, rarely cure chronic hepatitis B. Animal models are very crucial for understanding the pathogenesis of chronic hepatitis B and developing new therapeutic drugs or strategies. HBV can only infect humans and chimpanzees, with the use of chimpanzees in HBV research strongly restricted. Thus, most advances in HBV research have been gained using mouse models with HBV replication or infection or models with HBV-related hepadnaviral infection. This review summarizes the animal models currently available for the study of HBV infection. One-third of the world’s population have experienced hepatitis B virus (HBV) infection, with 240 million people chronically infected. Although hepatitis B vaccination programs have greatly reduced HBV infection rates in adolescents in China, there are still 93 million chronic HBV carriers . ExistinTupaia belangeri), which also exhibits a close phylogenetic relationship with primates  recently identified using this system (Marmota monax) can led to persistent infection and hepatocellular carcinoma (HCC)  to be highly susceptible to WHV infection , and woodchuck hepatitis virus (WHV). Chimpanzees are the only non-human primate fully susceptible to HBV infection, though their use in HBV research is strongly restricted . Severalprimates . In addis system . Animalss system . Furtherma (HCC) . Recentlnfection . WoodchuThe chimpanzee is the only immunocompetent animal fully susceptible to HBV infection. After inoculation with serum from HBV patients, chimpanzees can develop acute and chronic HBV infection, accompanied by similar liver inflammation and cellular immune responses as patients with HBV infection , which c+ T cell response against HBV was determined to be a key factor in viral clearance through cytopathic and non-cytopathic pathways, with CD4+ T cell priming crucial in the early phase of HBV infection  belongs to Tupaiidae, Scandentia, Mammalia, and displays a close phylogenetic relationship with primates. Recently, the whole genome sequence of the Chinese tree shrew implied that the nervous, immune, and metabolic systems were similar to those of humans  mice with the urokinase-type plasminogen activator (uPA) transgene. The expression of the uPA gene can induce necrosis of hepatocytes, leading to subacute liver failure in young mice, and making it possible to transplant human hepatocytes into mouse livers. Transplantation of human hepatocytes into uPA-SCID mice results in a liver-humanized model with high human hepatocyte reconstitution rate and supportive of HBV and HCV infection  Table 1Table 1.-/-/Rag2-/-/Il2rg-/-Fah) mice, in which liver failure can be induced at will  epitope has been found to induce prolonged suppression of HBV in human hepatocytes transplanted into FRG mice . FurtherBecause chimeric animals are genetically immunodeficient, they are unsuitable for the study of adaptive immunity or immunotherapy strategies. Researchers have used a variety of strategies to develop chimeric mice dually reconstituted with both immune cells and hepatocytes of human origin, including AFC8  and A2/NAvihepadnavirus. Similar to HBV infection in humans, age and inoculum dose can influence the outcome of DHBV infection. For example, 7-day-old ducklings can exhibit persistent infection when inoculated with high-titer DHBV inoculum , a relative of the American woodchuck, to be highly susceptible to WHV, suggesting potential as a new animal model for HBV infection , HBV replication lasts for only three months because transduction of ad-HBV often results in a strong immune response against the adenovirus itself. This can be overcome by the injection of low doses of Ad-HBV  Table 1Table 1.in vivo, and evaluate antiviral compounds. Using this model, we discovered that Poly (I: C) cleared HBV through IFN-dependent pathways (Injecting a large amount of liquid containing naked DNA into mice by the tail vein over a short period of time terms as hydrodynamic injection, which can effectively transfer exogenous genes into liver cells. Hydrodynamic injection was found to successfully deliver replication-competent HBV genomes into the livers of immunocompetent mice, with viremia peaking after 6 d and rapidly declining thereafter . It shoupathways . Recentlpathways .HBV cccDNA plays a major role in HBV persistence and is responsible for the relapse of viral activity after antiviral treatment with polymerase inhibitors in chronically infected patients, while is not built in not only HBV transgenic mice but also HBV transfected mice. To overcome this large obstacle and closely mimic chronic HBV infection in humans, in vivo system requires considerable improvement. For the duck and woodchuck models, the differences between HBV and other hepadnaviruses should be considered. In addition, except for mice, the above models are not inbred and detection reagents are not readily available. Thus, all current animal models have specific limitations. Therefore, researchers need to carefully interpret their results from animal studies, and validation of their findings in multiple systems should be encouraged.The above animal models with HBV replication or hepadnavirus infection provide more options for HBV research. The identification of NTCP as an HBV receptor is a milestone in the field of HBV research. The NTCP-transfected hepatoma cell line is the first human cell line supporting HBV infection, while NTCP transgenic mice support HBV infection was ongoing. Transfected, transduced, or transgenic mice with HBV genomes can only support HBV replication, with the HBV life cycle incomplete due to the lack of viral entry, cccDNA formation, and viral spread. As human liver cells supporting HBV infection are transplanted into immunodeficient mice, these mice exhibit obvious immunodeficiency and their maintenance systems are very complex. The chimpanzee is an optimal model for HBV infection, but is strongly restricted and not easily available for HBV research. Tree shrews can be infected with HBV, but the


Research over the past decade has clearly shown that long non-coding RNAs (lncRNAs) are functional. Many lncRNAs can be related to immunity and the host response to viral infection, but their specific functions remain largely elusive. The vast majority of lncRNAs are annotated with extremely limited knowledge and tend to be expressed at low levels, making ad hoc experimentation difficult. Changes to lncRNA expression during infection can be systematically profiled using deep sequencing; however, this often produces an intractable number of candidate lncRNAs, leaving no clear path forward. For these reasons, it is especially important to prioritize lncRNAs into high-confidence “hits” by utilizing multiple methodologies. Large scale perturbation studies may be used to screen lncRNAs involved in phenotypes of interest, such as resistance to viral infection. Single cell transcriptome sequencing quantifies cell-type specific lncRNAs that are less abundant in a mixture. When coupled with iterative experimental validations, new computational strategies for efficiently integrating orthogonal high-throughput data will likely be the driver for elucidating the functional role of lncRNAs during viral infection. This review highlights new high-throughput technologies and discusses the potential for integrative computational analysis to streamline the identification of infection-related lncRNAs and unveil novel targets for antiviral therapeutics. Viral infections remain a major world-wide concern. Even though we have all but eradicated some once deadly viruses, many still elude effective treatment. HIV-1 is becoming a chronic disease since current anti-retroviral therapy can suppress the infection but cannot clear the virus. Influenza is still a major global concern that evolves rapidly and kills thousands of individuals every year. As shown by recent examples, like Zika and Middle East Respiratory Syndrome Coronavirus (MERS-CoV), the threat of emerging viral infections is constant. A better understanding of virus and host interactions is needed to accurately define viral pathogenesis and to rapidly develop new therapies.Long non-coding RNAs (lncRNAs), a new class of transcripts, have recently garnered interest in the field of infection, as studies of the host response to viral infection typically focus on protein-coding genes. LncRNAs are defined as RNAs greater than 200 nucleotides with insignificant coding potential, i.e., noncoding, but they can have very diverse regulatory functions ranging from active transcriptional regulation to epigenetics. Compared to the small number of proteins that are druggable, a large number of lncRNAs offer many new potential targets as they may be easily and accurately targeted using sequence specific oligonucleotides, and they are more cell type- and tissue- specific than coding genes in general. We and others have documented the association of lncRNAs in viral infections and innate immunity ,2,3,4,5.First, we summarize our current understanding of the diverse functions of lncRNAs and their potential relevance to viral infection . The anaLncRNAs are generated using the same machinery and components as mRNA (and other ncRNA), may possess 5′ caps, and may also be polyadenylated at their 3′ ends. Their main distinction from mRNA is the lack of a significant open reading frame. Unlike coding genes, lncRNAs tend to be poorly conserved at the primary sequence level ,10. LncRMuch of the initial interest in lncRNAs stems from the discovery of Xist and its involvement in X chromosome inactivation (XCI), a process that has been detailed in a number of extensive reviews over the years ,15,16,17LncRNAs have been established as significant players in epigenetic regulation. Several studies have characterized associations between lncRNAs and DNA methyltransferases (DNMTs): DNMT1 ,32, DNMTSeveral host- and virally-encoded lncRNAs have also been shown to function epigenetically. For example, PAN RNA  has beenThe role of lncRNAs in epigenetic regulation has also been described through the concept of extra-coding RNA (ecRNA). The term was first introduced by Di Ruscio et al.  upon theBiological scaffolding is a function typically reserved for proteins; however, it has recently been identified as a function of lncRNAs. In plants, RNA polymerase V-dependent lncRNAs are known to recruit AGO4 in the canonical RNA-directed DNA methylation (RdDM) pathway . The funLncRNAs can also act as sponges for miRNA and prevent the miRNA-mediated degradation of target transcripts. The mode of this function has been described through the competing endogenous RNA (ceRNA) hypothesis and has been extensively reviewed ,49. LncRThe notion that lncRNA can affect alternative splicing stems from the fact that these RNAs are often found in complex loci that also contain protein-coding genes. A recent study has shown that the highly conserved human h5S-OT lncRNA expressed within the 5S rDNA locus can impact alternative splicing by interacting with U2AF65, a core splicing factor that binds to intron-exon junctions . An antiIn the previous section, we discussed some of the characteristics and functions of lncRNAs. Due to their unique nature, methodologies developed for the interrogation of coding genes may not be applicable or may require modification to adequately investigate lncRNAs. This section will discuss selected methods for the discovery, prioritization, and validation of lncRNAs. We begin with a discussion of the modifications to RNA-seq that allow for the detection of lncRNAs , which rDuring viral infection there are a multitude of changes throughout the transcriptome, which includes lncRNAs. Therefore, a natural choice for discovering specific lncRNAs important for infections is to systematically profile lncRNA changes in response to infection. While multiple methods for the detection of lncRNAs are currently available, as highlighted in , transcrStandard RNA-seq focuses on mRNAs (mRNA-seq) by enriching transcripts with poly(A) tails using approaches like poly(dT) coated magnetic beads. This would be less desirable for lncRNA analysis as many lncRNAs lack poly(A) tails. Alternatively, the expression of both poly(A+) (mostly mature transcripts of coding genes) and poly(A-) (mostly non-coding RNAs) can be captured by the sequencing of total RNAs , as shown by our studies ,3 and otLncRNAs are frequently expressed at very low levels. Total RNA-seq or the sequencing of mRNA depleted RNAs may still be insufficient for the detection of lowly expressed lncRNAs ,64. InstLike any other study, the described RNA-seq analysis requires necessary computational infrastructure and bioinformatics expertise for data storage, processing, and statistical analysis. Ideally, this is planned out before the start of the study, and may be accomplished in-house, in-collaboration, or through core services. Particularly, the annotation of lncRNA genes and its completeness should be examined carefully. For viral infections in well-studied systems like humans or mice, there are large numbers of annotated lncRNAs. For example, there are 14,720 human lncRNA genes and 8980 mouse lncRNA genes in the current Ensembl annotation (release 90.38). In these cases, lncRNA expression analysis can be carried out simultaneously just as all other coding genes. However, after quality control, it would be very beneficial to align cleaned sequencing reads to viral reference sequences to verify the identity of infected samples and the percentage of total reads mapped to viruses.For other organisms with draft genome assemblies, it is likely that the annotation of lncRNA genes is largely incomplete. Therefore, it would be important to annotate lncRNA transcripts ab initio, using the collected RNA-seq data in combination with other available sequencing data. For example, we were able to expand the ferret genome annotation with about 40,000 intergenic loci, which were enriched with polyadenylated and non-polyadenylated intergenic ncRNAs . SimilarTranscripts assembled from RNA-seq data consist of known as well as novel transcripts. Tools like Cuffcompare, part of the Cufflinks package , may be While it is not covered here, we want to emphasize that as with any other experiments, proper experimental design should be carefully conducted before the start of infections and RNA-seq analysis.An exciting new development of RNA-seq is the analysis of large numbers of individual cell transcriptomes . TranscrA typical RNA-seq analysis of virus infected samples may identify hundreds if not thousands of lncRNAs that respond to infection. One of the more challenging analytical steps is the prioritization of these identified lncRNAs, i.e., the identification of a small subset of lncRNAs that may play more important roles in the phenotype of interest. We see from The prediction of individual lncRNA functions is often based on “guilt-by-association” analysis, wherein tightly co-expressed lncRNA and coding transcripts are assumed to share similar functions ,87,88,89There are multiple approaches for inferring putative lncRNA functions from co-expressed coding genes. As shown in ,63, for Alternatively, co-expressed lncRNAs and coding genes can be determined by clustering methods. Genome-wide clustering of similar gene expression profiles extrapolates lncRNA function based on known gene functions in the same cluster, where transcripts in the same cluster are considered to be co-regulated . The comAnother strategy for inferring putative functions of lncRNAs is to examine protein-coding genes located near lncRNAs of interest, as lncRNAs may have cis-regulatory effects on flanking genes ,95. For When portions of lncRNAs overlap a coding gene, it is expected that this region be conserved and under a strong selective pressure. A recent study found that the evolutionary age, overlapping configuration, and local genomic environment of an lncRNA-coding gene pair influence the expression correlation of the pair . While tWhen assessing the function of a gene, including lncRNAs, it is imperative that it is done in the context of evolution. Homology searches leverage annotation from closely related organisms in order to infer the function of novel lncRNAs. The existence of homology often indicates that there is some selective pressure for the lncRNA in question, thereby implying functional significance. However, lncRNAs pose computational challenges when it comes to identifying and ascertaining cross-species homology. LncRNAs have been previously categorized into subclasses, each with functions that reflect their evolutionary conservation . For exaIt is well-understood that lncRNAs often possess very little conservation at the primary sequence level ,10. For Positional information can aid sequence homology searches, whereby the homology of lncRNAs is inferred by their relative positioning near orthologous genes. This syntenic conservation can help overcome the shortcomings of lncRNA sequence homology searches . A recenThe lack of sequence conservation among most lncRNAs has also prompted researchers to look at higher-order structures to infer functional significance. It is commonly understood that canonical RNAs, such as rRNA and tRNA, can form complex secondary and tertiary structures. There is now mounting evidence that lncRNAs, unlike shorter ncRNAs, assume higher-order structures with functional significance. For example, MALAT1 is known to have a highly conserved 3′ cloverleaf structure, one of several structures described by Nitsche et al. . The mosMany of these approaches fall short of identifying conserved lncRNA structures, as their focus lies primarily at the sequence level. To overcome the low sequence identity observed among many ncRNAs, a recent approach has made use of CMfinder in a computational pipeline that makes structural alignments to successfully capture conserved RNA structures (CRSs) . The stuConsidering the large number of lncRNAs that are lacking functional annotation, large scale perturbation will be another attractive strategy for probing their roles in viral infection. As shown by earlier studies ,110,111,As summarized in , the CRIAlternatively, Cas9 is mutated to an endonuclease-deficient form (dCas9) and fused to a repressive Krüppel associated box (KRAB) motif preventing cytotoxicity due to DNA double-strand breaks  and inhiGilbert et al.  establisOften, lncRNA genes are not detectably expressed, or they may be repressed by a disease state. This limited expression of lncRNAs makes detecting meaningful changes by inhibitory assays difficult, if not impossible. Several CRISPR-Cas9 methods have been developed to induce gene expression ,124,125,An exciting development for these screening methods is to inhibit or activate two or more target genes within the same single cells ,128, as Another key consideration for designing large scale screens is the choice of readout for approximating the phenotypes of interest. Using single cell sequencing (scRNA-seq) as a readout represents a potential paradigm shift ,130,131.Several groups in the past year have made modifications to the dCas9/gRNA system, circumventing this shortcoming of pooled screens ,130,131.Overall, these large scale perturbation approaches are unbiased and especially suitable for identifying associations between diseases and genes with no known function such as many lncRNAs. The CRISPR-dCas9 system is scalable and provides sufficient throughput for handling the large number of lncRNAs that require functional interrogation. While these newer methodologies like gain-of-function, multiplexed library screens, and scRNA-seq have yet to be implemented in the interrogation of lncRNA functions in the context of viral infection, the utilization of this technology will ultimately hasten the discovery of infection-related lncRNAs.Cross-examining the results generated by different methods described above will likely produce a shorter list of lncRNAs of higher interest. The next challenge is to experimentally validate their associated phenotype, e.g., pathway modulation or resistance to viral infection. Validation is especially important when considering the results of in silico analyses; however, confirmation studies should also be performed with regard to large-scale library screening due to the size and complexity of those assays. Though many existing techniques for studying RNA are easily transferable for investigating lncRNAs, there are some specific considerations given the uniqueness of lncRNAs.One consideration is the subcellular localization of specific lncRNAs. A nuclear lncRNA may function through interactions with DNA methyltransferases, histone modifying enzymes, or other epigenetic machinery ,32,33,34This subcellular localization information will also inform the choice of experimental techniques. As described, inhibition studies are useful for studying candidate lncRNAs. RNAi, in the form of siRNA, degrades target RNA via DICER and has been used in many instances to inhibit the expression of lncRNAs . AnotherThe complexity of genomic regions of interest and the ability to target specific lncRNAs in a given region are also important considerations for validation studies. CRISPR inhibition or activation will potentially influence the expression of overlapping or neighboring coding genes, making it difficult to single-out the effects of a specific lncRNA. Goyal et al.  illustraSince lncRNA tends to have a lower expression abundance, overexpression might be better suited for lowly expressed lncRNAs and to gain information complimentary to inhibition studies. CRISPRa or ectopic plasmid expression ,140 are The low expression of lncRNAs is in part due to their cell-type specific expression. Low expression levels can be particularly pronounced when an lncRNA is identified from a heterogeneous cell population, such as blood or tissues. In these cases, it is important to determine the cell types of origin for these lncRNAs. Elucidation of cell type may be done by combining cell staining and FISH techniques coupled with flow cytometry or microscopy. Knowledge of the cell type may also assist in hypothesizing the lncRNA function  and desiOur understanding of the biological relevance of lncRNAs has greatly increased over the past decade. Unfortunately, many of these genes still lack sufficient functional annotation, and their functional role in host-pathogen interactions remains largely unknown. In order to efficiently elucidate the role of these lncRNAs during viral infection, multiple high-throughput methodologies coupled with computational strategies are being utilized to process large quantities of poorly characterized lncRNAs.RNA-seq has emerged as the most widely used technology for this purpose, which usually results in a large number of lncRNAs significantly associated with viral infection. In order to narrow down these lists of lncRNAs, complementary computational strategies like co-expression network analysis and evolutionary analysis may be leveraged to aid the annotation and prioritization of lncRNA genes. Alternatively, large-scale perturbation screens fueled by rapidly advancing CRISPR-Cas9 techniques are providing novel tools for investigating lncRNA functions in specific areas including viral infection and quickly expanding our knowledge of lncRNA functions. These additional layers of information will reduce the long list of potential interesting lncRNAs to a short list of high-confidence ‘hits’. Ultimately, verifying the mechanism and function of candidate lncRNAs identified by high-throughput strategies requires orthogonal experimental confirmations.In summary, the large number of less studied lncRNAs represent a great opportunity for uncovering novel insights into virus-host interactions and potentially new targets for intervention. To fully realize this potential, different high-throughput technologies can be leveraged. While efforts are underway to enable searching putative lncRNAs across large multimodal datasets , there i


Coordination between ROS regulatory systems and other pathways needs to be flexibly modulated to finely tune the mechanisms underlying responses of different types of tissues to heat stress, biotic stresses and their combinations during different growth stages. Especially, modulation of the delicate balance between ROS-scavenging and producing systems in reproductive tissues could be essential, because ROS-dependent PCD is required for the proper fertilization, despite the necessity of ROS scavenging to prevent the damage on cells under heat stress and biotic stresses. In this review, we will update the recent findings associated with coordination between multiple pathways under heat stress, pathogen attack and their combinations. In addition, possible integrations between different signals function in different tissues via ROS-dependent long-distance signals will be proposed.Regulatory systems of reactive oxygen species (ROS) are known to be integrated with other pathways involving Ca Plants are exposed to multiple abiotic and biotic stresses that may simultaneously occur in the natural environments. Although necessity of the researches deciphering the molecular mechanisms underlying response of plants to stress combinations has been proposed in many reviews, such studies are still scarce . In addi2+ signaling, kinase cascades, and hormone signaling to tailor the specific mechanisms to adapt to different types of biotic and abiotic stresses , which metabolizes the nitric oxide (NO) adduct S-nitrosoglutathione, were more sensitive to heat stress compared with WT plants . For example, appropriate timing of tapetal degeneration involving PCD regulated by RbohE-mediated proper temporal ROS regulation was shown to be essential in pollen maturation and fertility . In addition, ROS and NO also play an important role in pollen-pistil interactions, as well as pathogen defense . Prior to and during arrival of pollens to pistils, high level of NO and H2O2 in pollens and papilla cells, respectively, might be required for the recognition of pollens by papilla cells. Following the arrival of pollen grains on a tip of stigma, NO derived from pollens might function to decrease the level of H2O2 accumulation in papilla cells . During this process, NO-ROS redox signal might navigate pollen tube growth to the proper direction . In Arabidopsis, each different RBOH protein might function in different tissues and play key roles in the regulation of different biological processes . ROS-dependent signal might be integrated with heat-specific long-distance signal that might enhance ABA synthesis, but decrease the intermediate of sugar metabolism in leaves not directly exposed to heat stress when part of a plant was subjected to heat stress (2+ signaling that might integrate with ABA- and SnRK-dependent pathways (2+ signaling, metabolite sensing and hormone balance.It could be also interesting to study integration of long-distance signaling induced by heat stress or pathogens with carbon transport between sink and source tissues, because of the certain level of overlaps in the mechanisms between these processes (t stress . Ca2+ sipathways  was alsopathways . These fPhytophthora infestans (Defense responses might be modulated depending on temperature. In many cases, abiotic stresses negatively impact on disease resistance of plants . For exanfestans . The heanfestans . Neverthnfestans .Botrytis cinerea accompanied by lower accumulation of ROS. In this case, cross talk between heat response and defense response might be strictly regulated by fine-tuned mechanisms to balance between ROS scavenging to prevent oxidative damage caused by heat stress and ROS production necessary for the defense responses. This finding therefore suggests that mitochondrial ROS regulatory systems might play key roles in the ROS-dependent cross talks between heat response and pathogen defense. It should be also interesting how UCP-dependent mechanisms might modulate pathogen defense and PCD under heat stress in pollens that contain many mitochondria (Comprehensive expression analysis of HSPs in wheat revealed that different types of HSPs can be enhanced in response to different types of pathogens . NO and chondria .A recent study demonstrated that Xa7-mediated pathogen resistance in rice might function better under high temperature . This Xa2+ signaling and hormone signaling should be finely tuned in plants under heat stress, pathogen attack and their combination. Such coordination between multiple pathways might be modulated depending on the growth stages and type of tissues. Although appropriate ROS scavenging is essential to prevent damage on cells under heat stress (Based on the previous findings, we propose that coordination between ROS regulatory systems and other pathways such as Cat stress , ROS-dept stress . In thist stress .2+ signaling are shown to be modulated through the entire plants over the long distance. Furthermore, different RBOH proteins were shown to be specifically expressed in different tissues. Therefore, it should be necessary to address how heat acclimation and defense mechanisms in different tissues are linked via long-distance signaling under heat stress, pathogen infection and their combinations.ABA signaling and carbohydrate distributions that might be integrated with ROS regulatory systems and CaAll authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The inability to maintain the immaturity of stem cell populations in vitro restricts the long-term expansion of various types of human epithelial stem cells. However, recent technical advances in epithelial stem cell culture have led to the development of novel in vitro strategies for regenerating epithelial tissues and for closely mimicking human diseases such as cancer and inflammation. Specifically, improvements in culture conditions provided by small molecules in combination with three-dimensional (3D) culture approaches have facilitated the establishment of in vitro systems that recapitulate biological properties in epithelial organs, and these systems may be used to model disease. In this review article, we describe the biological significance of technical improvements in the development of these methods, focusing on human epithelial cells, including stratified and columnar epithelial cells. We also discuss the potential and future perspectives of this technology, which is only beginning to be explored. The isolation and long-term expansion of primary cells, particularly stem/progenitor populations, are fundamental and important basic techniques in various biological fields, including developmental biology and stem cell biology, and medical science. Cells in stratified and columnar epithelial tissues are highly regenerative and disproportionately accountable for many human cancers; however, cloning adult stem cells is limited by difficulties in maintaining these cells in an immature state. In recent years, technical innovations have resulted in rapid and dramatic progress in stem cell biology, such as the use of small molecules and growth factors to mimic tissue niche environments and facilitating “Organoid culture” .In 1975, Rheinwald and Green established the first successful example of human adult stem cell culture using human keratinocytes . SpecifiIn this review article, we provide an overview of recent research progress and accumulating evidence of a cell culture system that has led to technical breakthroughs in epithelial cell technologies. Novel culture strategies for both stratified epithelial cells and columnar epithelial cells have enabled human epithelial development to be recapitulated and can be used to generate a human disease model in vitro. We also discuss the potential and possible applications of normal epithelial cell culture technologies for regenerative medicine and highlight a cancer cell culture system that reproduces individual patient phenotypes.In stratified epithelial tissues, including glandular and pseudostratified epithelium, p63+ cells, which are localized on the basement membrane, can self-renew to maintain stem/progenitor populations and give rise to progeny that form functional tissues , 5. As mSchlegel and colleagues reported that a Rho-associated protein kinase (ROCK) inhibitor in combination with 3T3 feeder cells significantly increased the proliferative capacity of epithelial stem cells, including human keratinocytes, prostate cells, and mammary gland cells, and they termed this phenomenon “conditional reprogramming” , 18. TheCollectively, these technical advances, in combination with small molecules and feeder cells, can be used to continuously and efficiently expand stratified epithelial stem/progenitor populations in vitro. Another breakthrough in stratified epithelial culture, organoid culture, has been utilized to expand both basal and luminal human prostate progenitors. These human luminal progenitors were multipotent and formed prostate gland-like structures in vitro . HoweverAlthough intestinal stem cells possess the remarkable capacity to proliferate at a high turnover rate to maintain intestinal epithelia, and hepatocytes are highly regenerative in response to damage, the ability to clone stem cell populations from columnar epithelial cells is severely limited, presumably due to a lack of tissue niche signals in vitro. Over the past decade, Clevers and colleagues discovered LGR5 (leucine-rich repeat-containing G-protein coupled receptor 5), an intestinal stem cell marker, in a sophisticated mouse model (Lgr5-EGFP-ires-CreERT2 mice crossed with the Cre-activated Rosa26 LacZ reporter) and established a mouse intestinal organoid culture method that consists of villus-like structures and crypt-like zones with multiple intestinal cell types . In combOrganoid culture employs a Matrigel-based 3D culture platform and can be extensively used to stably culture diverse types of adult epithelial cells, including stratified epithelial cells, with stem/progenitor cell populations . HoweverThe same strategy was also applied to clone human gastric stem cells obtained from endoscopic biopsy. Specifically, clonogenic gastric cells were stably expanded on a 3T3 feeder layer in combination with growth factors and small molecules and differentiated into gastric epithelial lineages typically found in the stomach, such as pepsinogen-expressing chief cells . In addiSince the first cancer cell line, the HeLa cell line, was established from a cervical cancer patient in 1951 , cancer Culture methods for adult stem cells, such as organoid and feeder systems, are also applicable to different approaches that use patient-derived cancer cells. Specifically, Clevers and colleagues reported that organoid culture can be used to model pancreas , prostatModeling human disease is hindered by the limited accessibility of human diseased tissues. Nevertheless, advances in adult stem cell cultivation have allowed us to reproduce disease phenotypes in vitro by expanding stem cells and deriving mature cell types from small human biopsy samples. Because 3D culture methods, such as ALI and organoid culture, provide structures that consist of multiple cell types and resemble the epithelium architecture observed in vivo, they should be suitable for studying inflammatory diseases, including infectious and hereditary diseases. Specifically, reproducing the disease phenotype is simple when the pathogen (or main cause) and targeted cell type are known.Pseudomembranous colitis (PMC) is caused by a disproportionally increased population of Clostridium difficile (C. difficile) after antibiotics treatment. C. difficile is a Gram-positive, spore-forming bacterium, and produces the high-molecular-weight toxins TcdA and TcdB, which induce fluid secretion, inflammation, and colonic tissue damage. Colonic epithelial cells differentiated from clonogenic colonic stem cells in ALI culture were challenged with these toxins, which caused devastating epithelial damage in a time- and dose-dependent manner. This result indicated that the 3D culture model may be used to represent C. difficile pathology . SimilarAdult stem cells have also been used to model hereditary disease. Beekman and colleagues reported an intestinal organoid culture derived from cystic fibrosis (CF) patients. CF is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), which is normally expressed in the epithelial cells of many organs, such as lung and digestive tissues. Although normal intestinal organoid cultures exhibited robust swelling in response to Forskolin, the swelling response was not observed in CF organoid cultures . MoreoveDespite promising strategies that use human embryonic stem (ES) cells and induced pluripotent stem (iPS) cells for applications in regenerative medicine, few clinical trials of these strategies are ongoing, which is in part due to difficulties in lineage specification and the possibility of tumorigenesis. Because adult stem cells are essentially committed to specific tissue types, producing intended cell types is relatively easy, and the potential risk for tumorigenesis is low. Thus, therapeutic approaches are aiming to use adult stem cells as the cell source for transplantation. Although Green and colleagues established the human keratinocyte culture method in 1975 and the cultured cells were transplantable into patients with burns or chemical injuries, the long-term cultivation of other types of adult stem cells was subject to significant technical barriers. As described above, recent technical advances overcame this limitation for diverse types of epithelial cells. Hence, the ability to rapidly and efficiently expand stem cell populations is valuable for their use in regenerative medicine.For example, mouse Lgr5+ colonic stem cells have been expanded in organoid culture and transplanted into the damaged mouse colon, and engrafted cells that were able to self-renew and differentiated were detected even after 25 weeks . In a diBecause of the high turnover rates of many epithelial cells, transplanting stem cell populations is essential for long-term tissue maintenance. Theoretically, a single stem cell can reconstitute whole tissues, and several research groups empirically demonstrated this notion , 53. DesIn recent years, remarkable progress has been made in the development of in vitro culture system for epithelial stem cells. The realization of the long-term culture of epithelial stem cells allows us not only to reproduce physiological events in vitro but also enables the development of therapeutic platforms based on cell transplantation. An increasing number of studies of epithelial stem cells clearly indicated that understanding the basic biology of these cells will be closely linked with clinical studies of human disease pathology, such as cancer and inflammation. The interactions of biological networks during tissue development and disease progression are complex at the cellular and molecular levels. Building an in vitro epithelial structure model can simplify this complexity and provide comprehensive views of epithelial physiology and pathophysiology. Moreover, in vitro epithelial models can easily be combined with genomic and epigenetic approaches and single-cell analyses. In addition, genome editing, e.g., the CRISPR-Cas9 system, can also be readily incorporated into the model. One drawback of in vitro epithelial structure models derived from the stem cells is that epithelial structures lack stromal populations. Although a simplified system provides direct insight into epithelial physiology in most cases, the interaction between different cell types is important for reproducing a genuine phenotype because all tissues consist of multiple cell types, such as epithelial cells, endothelial cells, mesothelial cells, fibroblasts, and hematopoietic cells. One possible solution to this problem is a self-organizing method, in which several cell types are mixed in vitro and spontaneously form actual organ-like structures. Although improvements are required to recapitulate the in vivo behavior of human organs, the ability to expand epithelial stem cells and generate a 3D structure model holds great promise for both basic and clinical research.