Quercetin is a flavonoid present in fruits, vegetables and plants with antioxidant, anti-inflammatory and anticancer properties. Its beneficial activities have been demonstrated in different human pathologies, including hepatoprotective effects against liver disorders. High mortality and late diagnosis of the primary liver tumor hepatocarcinoma (HCC) makes this cancer an interesting target for the study of quercetin effects. Our aim was to systematically review antitumor activities of quercetin in HCC preclinical studies employing single, encapsulated, combined or derived quercetin forms. Literature search was conducted in PubMed, Scopus and Web of Science (WOS), and 39 studies were finally included. We found that 17 articles evaluated quercetin effects alone, six used encapsulated strategy, 10 combined this flavonoid, two decided to co-encapsulate it and only four studied effects of quercetin derivatives, highlighting that only nine included in vivo models. Results evidence the quercetin antiproliferative and proapoptotic properties against HCC either alone and with the mentioned strategies; nevertheless, few investigations assessed specific activities on different processes related with cancer progression. Overall, further studies including animal models are needed to deeper investigate the precise mechanisms of action of quercetin as antitumor agent, as well as the potential of novel strategies aimed to improve quercetin effects in HCC. Quercetin  is one of the main components of the polyphenol family of flavonoids  and it iLiver injury is largely caused by obesity or metabolic syndrome, in addition to high alcohol consumption ,7. HepatHCC is the most common primary liver cancer and the sixth tumor with higher incidence, ranking as the fourth deadliest neoplasm worldwide . Liver dHigh mortality and treatment efficacy limitations of HCC makes it an interesting target for the study of potential antitumor effects of the natural flavonoid quercetin. Nonetheless, there are not specific review articles which comprise results from researches that evaluate properties of this flavonoid in HCC models. This systematic review is the first that summarizes quercetin antitumor activity against such liver tumor, providing clearer perspective and reliable evidences on potential use of quercetin in HCC therapy. The aim of this article was to systematically review all evidences available from in vitro and in vivo studies in which quercetin effects against HCC were analyzed, including its use as single agent as well as encapsulated, combined and derived forms of the flavonoid.This systematic review was done based on the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) .Following criteria were used for inclusion of articles that met all of them: (i) studies that employed quercetin as single or combined agent, free or encapsulated, or quercetin-derived forms; (ii) studies that used in vitro or in vivo models of HCC; (iii) studies that reported effects directly generated by quercetin treatment.The following criteria were used for the exclusion of articles that met any of them: (i) conference or congress communications; (ii) review articles; (iii) articles in other language than English; (iv) full articles not available; (v) studies that evaluated plant extracts effects; and (vi) studies published earlier than the last 10 years.The article search was conducted using the electronic databases PubMed, Scopus and Web of Science (WOS) on September 2019. MeSH terms used were: “quercetin AND hepatocarcinoma”, with the “[All fields]” tag in PubMed, and the fields “” in Scopus and “[Topic]” in WOS search. After removing duplicates, title and abstract screening of all obtained articles was done against previously established study inclusion criteria. Selected articles were subjected to a full-text analysis excluding those that met any of the exclusion criteria. Remaining articles were considered relevant studies and were included in this systematic review.Data collection from each study was extracted using a standardized form and following variables were used: first author name, year of publication, quercetin administration strategy, experimental model (in vitro or in vivo), cell line or in vivo HCC induction method, general effects , molecular alterations . Articles employing different samples did not allow quantitative estimates of quercetin effects in experimental HCC models. Hence, performing a meta-analysis was discarded. Data extracted from included articles were summarized and comprised in a table.The study selection was performed as it is described in The main characteristics of the articles included in this review are summarized in Quercetin antitumor effects have been described in different cancer types, including HCC . In 25 oOtherwise, though cell cycle regulation is a common mechanism found to be altered in HCC cells, only eight publications have evaluated the effects of quercetin in this process, showing contradictory results ,51,52,53Apoptosis has been clearly established as one of the mechanisms of quercetin-induced cell death in HCC ,51,52,53Angiogenesis and metastasis are tightly associated with HCC progression and represent very common targeting processes in cancer treatment ; howeverIn addition to the described properties, quercetin has been shown to exert antioxidant activity in the human HepG2 cell line ,31,57. N1 (AFB1) [Besides, beneficial effects of quercetin against HCC were evaluated focusing on the sensitization of chemoresistant liver cancer cells. It has been suggested that the Frizzled homolog protein 7 (FZD7)/β-catenin pathway participates in resistance mechanisms mediated by the family of ATP-binding cassette (ABC) transporters and quercetin was able to suppress it . Contrar1 (AFB1)  and etha1 (AFB1) , or DNA 1 (AFB1) .By last, despite the numerous studies conducted with quercetin, only three publications have included animal models to complete in vitro results ,23,24. GAltogether, the wide variety of antitumor effects of quercetin along with its demonstrated efficacy against HCC cells, set this flavonoid as a promising therapeutic agent in the treatment of HCC.Drug delivery systems have emerged as a novel mechanism of targeting cancer progression, enhancing drug efficacy through its encapsulation . In thisIn vitro models of HCC have also evaluated lipid-based formulations as quercetin-encapsulation strategies ,44. SoliEven though few studies analyzed quercetin nanoencapsulation as a drug delivery system in liver cancer cells, it has arisen as a novel therapeutic approach that could improve quercetin properties against HCC progression by specifically targeting tumor and increasing drug cellular uptake.Although a great number of antitumor properties of quercetin have been established in HCC treatment—mainly in cell line but also in animal models—some researchers have focused on searching for synergistic combinations with this flavonoid with the aim of improving its effectiveness against liver cancer ,52,53,54Increment of quercetin apoptosis induction was also observed after its co-administration with the following compounds, ZD55-TRAIL , two malRegulation of oxidative stress and ROS production by quercetin was also evaluated after the addition of 3′5′-dimaleamylbenzoic acid and 3′5′-dimaleimylbenzoic acid . This stEnhancement of antiproliferative effects of quercetin in animal models was evaluated employing nude mice subcutaneously injected with HuH7 ,47 or HeIt should be mentioned that two independent studies put together both quercetin combination and encapsulation strategies and evaluated its cytotoxic actions both in vitro and in vivo models ,56. LactGlobally, co-treatment strategy of quercetin with different compounds may enhance its effectiveness by mainly raising its antiproliferative and proapoptotic effects and leading to improve quercetin single-therapy properties against HCC.O-glucoside (Q3G) were reported in an in vitro study with HepG2 cell line, along with S and G2/M phase arrest of cell cycle and morphologic alterations [Quercetin glycosides are one of the main naturally occurring forms of quercetin, which makes them interesting compounds for cancer treatment . This leerations . This querations . Later, erations .O-methyl quercetin (3′MQ) and quercetin-3-O-glucuronide (Q3GA) [On the other hand, 3,4-dihydroxyphenylacetic acid (DOPAC), a catabolite of some quercetin glycosides produced by colonic microflora, has been shown to augment the expression of different aldehyde dehydrogenases  as well as ALDH activity in the Hepa1c1c7 mouse hepatoma cell line . Besidese (Q3GA) . Resultse (Q3GA) . These pe (Q3GA) .Bioactive compounds derived from quercetin have been shown to abrogate cancer progression in liver cancer cells; nonetheless, a greater number of studies would be needed to search for more quercetin derivatives and study underlying mechanisms of its antitumor action against HCC cells.This systematic review aimed to summarize scientific evidences concerning activities of the natural flavonoid quercetin as HCC treatment. Studies evaluating beneficial properties of quercetin encapsulation and/or combination, as well as of quercetin derivatives, were included considering its relevance in the purpose of this review. Out of the 39 included articles, 17 investigated mechanisms of signaling pathways and cellular processes alteration by quercetin alone in HCC models, including seven researches where its effectiveness as single agent besides its combination was analyzed. Although quercetin has been shown to act as a potent antitumor drug in liver cancer cell lines, only three out of the 17 publications included in vivo experiments to demonstrate its positive effects against HCC tumors. In order to accomplish greater efficiency, six studies designed quercetin-loaded nanoparticles, 10 combined this flavonoid with different compounds and two joined both strategies evaluating effects of quercetin co-encapsulation. In these cases, animal models where employed in six articles, corresponding one to flavonoid nanoencapsulation, three to its combination and the remaining two to quercetin co-encapsulation with other drugs.Results presented in this article collect a great variety of antitumor actions of either quercetin or the mentioned strategies of combination, encapsulation and derived compounds. Cell growth inhibition, in addition to apoptosis stimulation, were the main processes described as quercetin mechanisms of action against HCC, properties that have also been observed in different tumors, such as non-small cell lung cancer  and breaBlockade of cell cycle progression seems to be part of the mechanisms responsible for the efficacy of quercetin, as it was reported in HCC by different researches, and not only with quercetin ,51,52,53Beyond the above-mentioned tumor processes, several studies showed a great number of mechanisms modulated by quercetin. These include autophagy ,24, oxidImprovement of quercetin efficacy has become one of the purposes of some researchers, focusing on the design of nanocarriers which increase delivery efficiency and cellular uptake of this flavonoid. In the present work, six studies evaluated different nanoparticles and demonstrated higher cytotoxic effects of quercetin in both in vitro and in vivo HCC models ,42,43,44Natural presence of quercetin in many occurring forms in plant-derived beverages, vegetables and fruits, and its antitumor effects convert quercetin-derived molecules into interesting drugs for cancer treatment. Regardless of this, quercetin derivatives were evaluated in only four of the total articles included in this systematic review, demonstrating an HCC growth inhibitory effectiveness comparable to that of quercetin ,58,59,60The limitations present in this systematic review are mainly due to the wide and heterogeneous set of articles included. Several studies reported contradictory results of oxidative stress regulation properties of quercetin as well as its specific role on cell cycle arrest, which evidences the need for homogeneity and a good design in the performance of experiments. Despite angiogenesis and metastasis are well-recognized characteristics of HCC, only two articles studied the quercetin effects in them, employing in one of them co-encapsulation strategy with sorafenib. Moreover, chemoresistance is often developed in patients with HCC; however, quercetin benefits on sensitizing tumor resistant cells was solely reported in one research. Although several publications showed a great variety of cellular processes and signaling pathways that were modulated by this flavonoid, each activity was demonstrated in one or at most two studies and in vivo model was not employed to validate the in vitro results. One of the main limitations found was the low number of in vivo experiments that were carried out, with only two researches evaluating other characteristics than tumor weight and volume decrease after quercetin treatment. Quercetin combination is a conventional strategy to improve drug efficacy; nevertheless, most articles were limited to assess only its antiproliferative properties without analyzing specific mechanisms. Lastly, few studies used quercetin derivatives to prove its positive effects in HCC.In conclusion, results presented in this systematic review suggest a clear antiproliferative and proapoptotic effect of quercetin in HCC, and likely a modulating role on tumor cell cycle progression which needs to be investigated further. This flavonoid seems to have antitumoral efficacy through the alteration of a great variety of cellular processes and signaling pathways, though more studies are required to further elucidate its mechanisms of action against HCC. Arising strategies of combination and drug-delivery systems may improve such cancer inhibition properties and, along with emergent use of quercetin derivatives with anticancer efficacy, broaden the therapeutic options for HCC patients.


Drosophila melanogaster, has been of paramount importance in deciphering the significance of these alterations. In fact, many of the ASD associated genes, such as FMR1, Neurexin, Neuroligins and SHANK encode for proteins that have conserved functions in neurons and during synapse development, both in humans and in the fruit fly. Drosophila is a prominent model in neuroscience due to the conserved genetic networks that control neurodevelopmental processes and to the ease of manipulating its genetics. In the present review we will describe recent advances in the field of ASD with a particular focus on the characterization of genes where the use of Drosophila has been fundamental to better understand their function.Autism Spectrum Disorder (ASD) is a complex group of multi-factorial developmental disorders that leads to communication and behavioral defects. Genetic alterations have been identified in around 20% of ASD patients and the use of genetic models, such as  Autism Spectrum Disorder (ASD) is a complex developmental neurological disease characterized by persistent deficits in social behaviors , presence of repetitive and restrictive comportments and is often associated with motor deficits and sleep abnormalities, among others. Among individuals suffering from ASD, there is a high frequency of intellectual disability and mental retardation, although the described frequency is variable due to the difficulty in assessing cognitive performance in certain groups of ASD patients . Autism The identification of ASD susceptibility genes involved in various aspects of synapse biology, lead to the hypothesis that aberrant synaptogenesis/synaptic function might be a central process in ASD . MultiplDrosophila is an excellent model to study ASD to understand the consequences of genetic alterations found in ASD patients and to identify the molecular mechanisms underlying the role of ASD related genes in synaptic function and plasticity , or less frequently, by point mutations in FMR1  is a neuro-developmental disease that leads to intellectual disability and is the most common form of autism of monogenic origin . FXS is  in FMR1 , that le in FMR1 . FMR1 enon mRNAs .Drosophila harbors only one FMRP ortholog, dfmr1, that shares high homology with its mammalian counterpart . Mutations in dfmr1 affect synaptic transmission at histaminergic photoreceptor synapses  and glutamatergic NMJ synapses   .dfmr1 controls brain development and neural circuit assembly  and lateral neurons (LNvs), and neurite-branching abnormalities in DC neurons. Interestingly, loss and gain of function of dfmr1 lead to similar phenotypic defects, indicating that the levels of dfmr1 are critical for brain development , a higher hierarchy circuit involved in olfactory learning and memory  and projection neuron (PN). In fact, the dendritic arborizations of these neurons are enlarged in dfmr1 null animals. MVP2 and PN neurons respond in the opposite way to this activity by remodeling their dendritic arbor and dfmr1 is required for this function  ( (dVMAT) .dfmr1 mutants show also impairment in odor-induced attraction and aversion, due to reduced lateral interactions across the olfactory glomeruli and impairment of the lateral inhibition in the antennal lobe caused by weaker inhibition from GABAergic interneurons (rneurons . These rdysfunctions in sensory processing (SPD), meaning they respond to a certain behavioral stimulus differently than individuals in the average population (Drosophila stress odorant (dSO) , and the cAMP-dependent PKA activator and the cGMP dependent phosphodiesterase inhibitor 8-CPT (8-(4-Chlorophenylthio)adenosine 3′,5′-cyclic monophosphate), suggesting a potential use of these drugs In ASD treatments  . dfmr1 neatments .pathogenic mutations have been identified in FXS patients. The most studied is an isoleucine to asparagine substitution (I304N) within the second K-homologous (KH) domain of the human FMRP, which is associated with very severe FXS  found in a patient with FXS. This mutation generates a novel peptide sequence with a premature stop codon, resulting in the truncation of the FMRP protein at the C-terminus and loss of the arginine-glycine-rich motif (RGG box), which is one of the FMRP RNA-binding domains. Overexpression of a mutant form of C+NLSΔdfmr1 , which closely mimics the human variant, in LNvs, results in axons that fail to extend medially, leads to aberrant bifurcations of axonal bundle and to the formation of axonal “tangles.”Up to now only few potentially vere FXS . However animals . More retrans-synaptic binding partners involved in synaptogenesis  and Neuroligins (Nlgs) are adhesion molecules that function as ogenesis . Severalpatients .Drosophila, loss of dNlgs (Drosophila harbors 4 dNlgs) and dNrx results in developmental defects at the NMJ such as an altered number of boutons, aberrant presynaptic/postsynaptic structure, and impaired synaptic transmission , one of the key players in assembly . In partassembly . dNrx isassembly .Drosophila strongly support that dNlgs and dNrx have a primary role in synapse formation/maintenance and outline how these signaling pathways might be further assessed as pharmacological targets.In mammals, Neurexins and Neuroligins are also central for the establishment of functional synaptic networks . The finSHANK3 mutations are one of the most prevalent monogenic causes of ASD, accounting for at least 0.69% of all cases, and patients harboring SHANK3 truncating mutations display autism combined with moderate to severe intellectual disabilities. Moreover, 22q13.3 deletion syndrome, also known as Phelan–McDermid syndrome, which is characterized by ASD or ASD-traits, is caused by deletions and mutations that lead to the loss of a functional copy of SHANK3  is composed of three members: SHANK1, 2 and 3. These proteins are scaffolding proteins present at the post-synaptic density in glutamatergic synapses. SHANK3 deletions, duplications, and mutations have been frequently reported in patients with ASD . SHANK3 f SHANK3 . Recent f either . This stDrosophila harbors only one ortholog of the SHANK family called Prosap/Shank  pathway at the post-synaptic terminal. Shank affects the internalization of the Frizzled-2 (Fz2) receptor, most likely by organizing molecules associated with its internalization and trafficking to the nucleus  and Choline acetyltransferase (ChAT). These abnormalities result in significant impairment of the olfactory learning in Shank mutants.ap/Shank . Harris  nucleus . A more A genome-wide association study (GWAS) of copy-number variation (CNVs) in patients with autism that lead to defective gene family interaction networks (GFINs)  identifimGluR theory” states that loss of FMRP in FXS results in increased glutamatergic signaling via mGluR5, leading to uncontrolled increases in local mRNA translation , during development and adulthood, restores the naive courtship levels of the dfmr1 mutants. Similar treatments also rescue dfmr1 defects in immediate recall-memory and the lack of short-term memory. Moreover, the treatment with mGlur antagonists greatly reduces axon growth defects (β lobe overgrowth) observed in the MB of dfmr1 mutants. Interestingly, the free running rest-activity rhythm defects of dfmr1 mutant flies are not rescued by these treatments, suggesting that not all the phenotypes observed in dfmr1 null flies are due to upregulation of mGluR signaling  network has been widely associated with ASD, where mutations in genes of the DA signaling, such as the Dopamine transporter (DAT), Synataxin 1 (STX1), the DA-receptors, and enzymes involved in DA metabolism, have been associated with autism. Work from several groups suggested that dopamine imbalances in specific circuits of the brain could lead to ASD related behavior . MoreoveDAT gene (hDAT-T356M) was identified. This mutation results in reduced ability to accumulate intracellular DA, due to an increased dopamine efflux  and in STX1A (STX1A-R26Q) genes. The analysis of these mutations showed defects in the reverse transport of DA that leads to behavioral abnormalities  feeding stimulates Drosophila locomotion but only in the presence of a fully functional DA network. Moreover, expression of a dominant negative form of CK2, mimicking the STX1A-R26Q variant, in DA neurons renders flies insensitive to AMPH. On the other hand, flies harboring the hDAT-R51W mutation increased their locomotion upon AMPH significantly less than wt hDAT expressing flies, confirming the reduced ability of AMPH to cause DA efflux in hDAT R/W mutants . The fruit fly allows for the analysis of the effects of multiple genetic modifications in different subsets of cells, allowing for the discrimination of the contributions of combinations of genetic alterations co-occurring in ASD patients.in vivo model with a robust and comparable neuronal development, will be essential to gain insight into the pathogenesis of these disorders.Therefore, the combination of genomic analysis of ASD patients together with the use of an easy to manipulate Both authors listed contributed to writing and reviewing the 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.


Endogenous methylarginines were proposed as cardiovascular risk factors more than two decades ago, however, so far, this knowledge has not led to the development of novel therapeutic approaches. The initial studies were primarily focused on the endogenous inhibitors of nitric oxide synthases asymmetric dimethylarginine (ADMA) and monomethylarginine (MMA) and the main enzyme regulating their clearance dimethylarginine dimethylaminohydrolase 1 (DDAH1). To date, all the screens for DDAH1 activators performed with the purified recombinant DDAH1 enzyme have not yielded any promising hits, which is probably the main reason why interest towards this research field has started to fade. The relative contribution of the second DDAH isoenzyme DDAH2 towards ADMA and MMA clearance is still a matter of controversy. ADMA, MMA and symmetric dimethylarginine (SDMA) are also metabolized by alanine: glyoxylate aminotransferase 2 (AGXT2), however, in addition to methylarginines, this enzyme also has several cardiovascular protective substrates, so the net effect of possible therapeutic targeting of AGXT2 is currently unclear. Recent studies on regulation and functions of the enzymes metabolizing methylarginines have given a second life to this research direction. Our review discusses the latest discoveries and controversies in the field and proposes novel directions for targeting methylarginines in clinical settings. The biological and clinical significance of endogenous methylarginines as cardiovascular markers was demonstrated in multiple epidemiological studies. At the same time, experimental studies demonstrated that at least in some pathological conditions, endogenous methylarginines can directly contribute to development of cardiovascular, metabolic and renal damage. Despite these promising findings the interest towards methylarginines eventually plateaued, presumably because of the lack of therapies that selectively lower their concentrations. However, the recent discovery of new functions of the enzymes that metabolize endogenous methylarginines has led to a renewed impetus in this field. The aim of this review is to discuss these discoveries and their therapeutic potential, as well as to address the key controversies and unanswered questions.Despite significant advances in prevention and therapy, cardiovascular disease (CVD) remains the leading cause of morbidity and mortality worldwide, thus warranting the search for novel markers for early identification of individuals at high risk for cardiovascular complications and new therapeutic approaches to further decrease cardiovascular morbidity and mortality. Endogenous methylated analogues of the essential amino acid l-arginine: (1) NG-monomethyl-l-arginine (NMMA), (2) symmetric NG,N’G-dimethyl-l-arginine (SDMA), and (3) asymmetric NG,NG-dimethyl-l-arginine (ADMA) (50) of about 2–3 μM [There are three different types of endogenous methylated analogues of e (ADMA)  1]. Two. Twol-ar (NMMA),  symmetriMA), and  asymmetrt 2–3 μM ,5, so moThe association between elevated ADMA levels and endothelial dysfunction was first demonstrated by Vallance and colleagues in 1992 . SubsequIn addition to being elevated in cardiovascular, renal and metabolic diseases, ADMA has been shown to be a strong predictor of cardiovascular complications and increased mortality in patients at risk. The Ludwigshafen Risk and Cardiovascular Health Study, a large prospective study with a median follow-up of 5.5 years, documented that plasma ADMA was independently associated with all-cause and cardiovascular mortality in patients with stable and unstable ischemic heart disease . SimilarIn their original work, Vallance and coauthors showed that the acute infusion of ADMA in healthy volunteers leads to endothelial dysfunction in the brachial artery . Kielstel-arginine . Th. Th35]. In order to determine the physiological and pathophysiological roles of DDAH isoforms, several groups generated genetically modified models with either upregulation or downregulation of DDAH expression. Dayoub and colleagues showed that transgenic overexpression of DDAH1 in mice decreases ADMA concentrations in plasma and tissue and increases NO production . DDAH1 tMost of the existing evidence suggests that therapeutic strategies aimed at upregulation of DDAH activity and subsequent ADMA lowering might be beneficial to patients with CVD. As a result, several groups performed experimental screens for modulators of DDAH activity to identify DDAH-activators ,60,61. TDDAH1 but not of DDAH2 gene in cultured human umbilical vein endothelial cells led to increased accumulation of ADMA in the cell culture medium [Ddah1, but not of Ddah2 gene led to increase in plasma ADMA concentrations in rats [Ddah2 gene [Ddah2 gene in mice did not lead to changes in plasma ADMA concentrations, but nevertheless resulted in decreased ADMA concentration in urine, myocardium and kidneys as well as in impairment of endothelial function in aortic rings [Ddah2 knockout mice also had decreased expression of Ddah1 gene in the kidneys as compared to their wild type littermates, which could have affected ADMA metabolism independently of DDAH2 [There is still a debate in the field about the relative contribution of DDAH2 towards the total DDAH activity. In contrast to the initial observation that DDAH2 is active towards ADMA in vitro, Pope and colleagues could not detect any significant increase in total DDAH activity in the lysate of endothelial cells with massive overexpression of DDAH2 even though those cells were producing higher amounts of NO . Knockdoe medium . siRNA-m in rats . On the ah2 gene . DDAH2 tah2 gene . Knockouic rings . Howeverof DDAH2 . With thBoth DDAH isoforms have been suggested to possess some nonenzymatic effects  from ADMA, symmetric α-keto-dimethylguanidinovaleric acid  from SDMA  and α-keAGXT2 is a pyridoxal phosphate-dependent mitochondrial enzyme that is expressed in the liver and in the epithelial cells of Henle’s loop of the kidney ,75. It wAGXT2 can also utilize D-beta-aminoisobutyric acid (BAIBA), endogenous nonproteinogenic amino acid, as an alternative amino donor . There aAGXT2 is also involved in the metabolism of some other bioactive substances. One of them is β-alanine, a nonproteinogenic amino acid which is a rate-limiting precursor of carnosine, a compound needed for high-intensity performance . InteresIt still remains to be established whether AGXT2 can play a direct role in the pathogenesis of diseases associated with decreased homoarginine concentrations.Considerable clinical and experimental evidence suggest that reduction in NO availability might facilitate the development of chronic kidney disease (CKD) and kidney impairment ,91,92. IOur discovery of the role of AGXT2 in homoarginine metabolism further increases the complexity of the potential cardiovascular effects of AGXT2. On one hand upregulation of AGXT2 would be expected to have a beneficial influence on the cardiovascular system via lowering systemic concentrations of ADMA and SDMA. On the other hand, upregulation of AGXT2 may also result in some negative cardiovascular effects via lowering systemic concentrations of homoarginine and BAIB, with the latter being suggested to play a role in browning of adipose tissue, which is also thought to be beneficial for the cardiovascular system, and in mediating beneficial effects of exercise on endothelial function . AnotherThe work presented in this review has significantly advanced our understanding of the multiple cardiovascular and metabolic roles of endogenous methylarginines and the enzymes involved in their metabolism. Furthermore, it has allowed identification of the novel therapeutic targets and created the basis for future translation of basic research findings into the clinical practice. The most promising research directions in this field include the further search for small molecules upregulating DDAH activity and the identification of the binding partners and regulatory posttranslational modifications of DDAHs, which could also be potentially targeted therapeutically. A better understanding of the regulation of AGXT2 activity with a specific focus on the role of the enzyme in liver and kidney disease and on the potential therapeutic approaches modulating the relative substrate specificity of AGXT2 is also of a significant importance in parallel to further exploration of the biological effects of its individual substrates.


Moreover, the functional characteristics of different synthetic promoters, their modes of regulating the expression of candidate genes in response to different conditions, and the resulting plant trait improvements reported in the past decade are discussed.Advances in plant biotechnology provide various means to improve crop productivity and greatly contributing to sustainable agriculture. A significant advance in plant biotechnology has been the availability of novel synthetic promoters for precise spatial and temporal control of transgene expression. In this article, we review the development of various synthetic promotors and the rise of their use over the last several decades for regulating the transcription of various transgenes. Similarly, we provided a brief description of the structure and scope of synthetic promoters and the engineering of their  The application of plant biotechnology techniques has revolutionized the field of plant science and greatly contributed to the progress of crop improvement and modern agriculture . The demcis-acting elements started with analysis of promoters from native plant genes and Agrobacterium which express in plant cells. Interestingly, like their counterparts in animals, plant gene promoters contain TATA box, initiator element as well as well-defined transcription initiation site about 20 to 30 bp downstream to TATA element. Whereas, in the upstream region, several gene promoters were found to contain positive or negative regulatory elements, some of which were characterized as enhancers or silencers (CaMV promoter or its –90 bp derivative) is fused to a heterologous promoter sequence at its 5´ end and to a reporter gene  at its 3´ end. Such constructs are introduced in plant cells by Agrobacterium, electroporation, or biolistics and evaluated either for transient expression of the reporter gene or for its expression in stable transgenic system. The pattern of expression conferred by heterologous promoter sequences has been helpful in defining characteristics of developmental as well as inducible regulatory promoters . The core-promoter region  usually contains a TATA-box necessary for recruiting RNA polymerase II and the assembly of general transcription factors to form the preinitiation complex. Thus, the multiplication of the elements, their reorganization, and ligation with native and synthetic cis-sequences are used to obtain synthetic promoters. The development and use of such a novel promoter can successfully regulate transgenes in trait- and environment-specific manners  that have been commonly exploited in plant biotechnology. In contrast, synthetic promoters are typically smaller in size but generate strong constitutive or inducible expression. The primary elements  of different promoters can be linked together from diverse origins to form a synthetic promoter for the spatial and temporal control of a transgene in a genetically engineered plant , switchgrass Ubiquitin 2 (PvUbi2), and CaMV 35S minimal promoters were used as positive controls  in P. pastoris also suggested that the engineering of novel cis-regulatory elements is required for synthetic motif library screening, an experimental line of tracking and bioinformatics tools, because these approaches greatly affect the strength of transcription and its regulation. It was also demonstrated that there are multiple Cis-regulatory elements in the promoter region of Catharanthus roseus CrWRKY1 that regulate its relevant expression. In another study they revealed that the promoter is active in both natural/native and heterologous systems, Cis elements can also be utilized for the isolation of regulatory factors  of lantCARE , TRANSFAlantCARE , and PLAlantCARE  are help factors .Cis-regulatory elements. A comprehensive bioinformatics analysis of the soybean genome was conducted for de novo discovery of soybean cyst nematode (SCN)-inducible motifs  platform, could become a powerful approach for the evaluation and discovery of novel cis-sequences in response to plant-pathogen interactions or any other specific stimuli  and used for the expression of transgenes in plants (PtMCP) isolated from Populus tomentosa; PvUbi1 and PvUbi2 isolated from switchgrass; and CsCYP, C2 (CsGAPC2), and CsEF1 isolated from citrus  of Arabidopsis failed to enhance protein expression in microsporangia or seed coats of Arabidopsis, and the modified actin promoter of rice did not cause xylem expression in rice plants  was incorporated into the enhancer region (−90 to −941) of CaMV 35S, yielded interesting results. The Mac promoter overexpressed GUS compared to double CaMV 35S promoters in the leaves and roots (CaMV 35S and Actin-1 from rice) with a compact chimeric Bt gene and cryIA(b) of Bacillus thuringiensis were joined (in vivo gene expression and regulation properties of a G-box motif with different flanking sequences were investigated (GUS gene expression driven by 11 different G-box tetramers fused to a promoter (CaMV ± 90/35S) was studied in transgenic tobacco plants  in an Agrobacterium-based leaf-infiltration transient assay in Nicotiana tabacum (Vigna radiate aminocyclopropane-1-carboxylate synthase (VR-ACS1) gene coupled with a CaMV 35S promoter and β-glucuronidase (GUS) reporter gene  for high level expression of transgenes in different plants. Ten recombinant promoters with different upstream activation sequences were generated by incorporating the Mirabilis mosaic virus sub-genomic transcript  and TATA-containing core domains of the Figwort mosaic virus sub-genomic transcript promoter . The successful regulation of recombinant promoters for GUS and GFP was confirmed in tobacco protoplasts. The engineered promoters revealed stronger activity compared to the CaMV 35S constitutive promoter (promoter .cis elements are shuffled can be advantageous in developing transgenic plants that express the trait of interest. MUSASMSCP) by incorporating the upstream activation sequence of Mirabilis mosaic virus (MMV) full-length transcript (−279 to −38) to the 5´end of the MMV sub-genomic transcript (−306 to −125) promoter fragment. Subsequently, the transient activity of the MUASMSCP promoter was evaluated in Petunia hybrida and tobacco protoplasts and showed constitutive expression of the transgenes at levels comparable to that of the CaMV 35S promoter  were developed, and each chimeric/hybrid promoter drove the constitutive expression of multiple reporter genes in different plant species, including tobacco, spinach, Arabidopsis, petunia, and tomato. In addition, promoter activity was induced by salicylic acid and abscisic acid  transporter protein that confers broad-spectrum resistance against fungal pathogens in wheat plants at adult stages. Ta-Lr34res that was expressed under the control of the pathogen-inducible Hv-Ger4c promoter in transgenic barley conferred enhanced resistance against powdery mildew and leaf rust, even at the seedling stage. These results suggested that Ta-Lr34res driven by the pathogen-inducible Hv-Ger4c promoter is agronomically useful in barley, and that the negative effects of the endogenous promoter can be eliminated without compromising disease resistance . A promoter consisting of tandem copies of the ethanol receptor inverted repeat binding site was used in combination with a minimal promoter sequence to yield an ethanol-inducible promoter. This promoter conferred enhanced sensitivity and significantly higher expression levels in a modified alc gene expression system  was evaluated in Arabidopsis  reporter gene, and GUS expression was analyzed in A. thaliana. GUS activity under stress was significantly higher in transgenic plants with the synthetic promoter constructs than in plants with the control construct where GUS expression was driven by the responsive to dehydration 29A (rd29A) promoter. In particular, EKCM showed a 1.29-fold increase in GUS activity in different tissues of transgenic A. thaliana under dehydration conditions (CARK1) and regulatory components of ABA receptor 11 (RCAR11) with synthetic promoters resulted in drought stress tolerance. It was also reported that exogenous ABA or co-transformation with the effector dehydration-responsive element binding protein 2A (DREB2A) induced the activity of the synthetic promoters (Dp.RCAR11-Ap.CARK1 and ANDp.CARK1) can be used to generate drought stress resistance in different plants.nditions . Synthetnditions . The comromoters . They cocis-acting elements, F and E17, in dimers and in combination, and placed them upstream of the minimal CaMV 35S promoter. The resulting constructs, pGEE, pGFF, and pGFFEE, were analyzed after transformation into canola (Brassica napus) plants. GUS histochemical analyses revealed that the synthetic promoters responded to phytohormone treatments and fungal elicitors  gene, in Brassica juncea phloem exudates. A number of different seed-specific promoters have also been identified for the purposes of enhancing the nutritive value of rice, corn, and legumes. Promoters active in seeds, such as the glutelin promoter in rice, the zein promoter in maize, and the arcelin (arc5-I) promoter in the bean Phaseolus vulgaris, were able to transform and greatly improve the nutritive value of seeds with heterologous proteins (Prrn and psbA have been used to transform tobacco chloroplasts for the production of insulin (CTB‐Pins) and other protective antigens  deaminase under the control of the root locus o plants . Koramutproteins . Recentlantigens . One arevaccines .hex-3 and hex-1 elements of the wheat histone gene promoter construct synthetic promoters 4H3-46 and 4H1-46. Only a tetramer of hex-3 in transgenic tobacco was shown to overexpress the target gene upon exposure to saline (NaCl) and elevated concentrations of ABA  that specifically regulates the activity of acid vacuolar invertase in potato tubers at low temperature, but has no effect in other tissues or in tubers under normal conditions. Transgenic potato lines were constructed with a vector containing 955 bp of antisense StvacINV1 sequence under the control of the pCL promoter to confirm the function of the pCL promoter in cold-induced sweetening (CIS) in potato. These results demonstrated the utility of using the pCL promoter to prevent CIS in potato tubers  in a quest for a better-quality flower-specific promoter. The study combined CaMV 35S or the OCS enhancer with a CHSA core promoter fragment, from either petunia or lily, along with an omega (Ω) element and evaluated the levels and tissue specificity of GUS expression in transgenic Torenia fournieri. Among these chimeric promoters, pOCS-PCHS-Ω showed high expression levels in colored corollas  was constructed and evaluated in transgenic tobacco plants  with high bidirectional expression efficiencies specifically in green tissues, which can be widely applied to agriculture biotechnology. Furthermore, bidirectional green tissue‐specific promoters have important application prospects in genetic engineering and crop genetic improvement. However, there is no report on the application of them, mainly due to undiscovered natural bidirectional green tissue‐specific promoters and the lack of a comprehensive approach for the synthesis of these promoters  gene  or enzymes for genome modification  will obviously facilitate metabolic engineering in plant synthetic biology. The activation domains of synthetic ZF-TFs have been used for targeted activation of genes in II) gene , and thethaliana . These sA. thaliana after salt and cold treatment when TALE DNA-binding domains were fused to the EAR-associated repression domain (SRDX) for both endogenous and transgenic RD29A promoter expression  and systems  have been designed; their application in crop plants will definitely enhance our understanding for increasing the availability of desired products. The combined use of synthetic promoters and synthetic transcription tools have the capability to modernize plant genetic modification and genetic technology. Similarly, the use of tools, such as CRISPR-Cas, RNAi, and other plant genomic techniques, will soon revolutionize the field of plant biotechnology, enabling precise and targeted genome editing and the development of novel plant species with desired traits.In this review, we surveyed the structure, synthesis, and expression of synthetic promoters reported in the preceding decade. Parameters such as specificities and strength were chosen to categorize the reviewed promoters, which included synthetic constitutive promoters, synthetic inducible promoters, and synthetic tissue-specific promoters 3. Such SA and WK have made substantial, direct, and intellectual contribution to conceptualize the review outline and wrote the 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.


This necessitates the exploration of alternative EC sources. Although there has been a recent surge in the use of pluripotent stem cell derived endothelial cells (PSC-ECs) for various cardiovascular clinical applications, current differentiation protocols yield a heterogeneous EC population, where their specification into arterial or venous subtypes is undefined. Since arterial and venous ECs are phenotypically and functionally different, inappropriate matching of exogenous ECs to host sites can potentially affect clinical efficacy, as exemplified by venous graft mismatch when placed into an arterial environment. Therefore, there is a need to design and employ environmental cues that can effectively modulate PSC-ECs into a more homogeneous arterial or venous phenotype for better adaptation to the host environment, which will in turn contribute to better application efficacy. In this review, we will first give an overview of the developmental and functional differences between arterial and venous ECs. This provides the foundation for our subsequent discussion on the different bioengineering strategies that have been investigated to varying extent in providing biochemical and biophysical environmental cues to mature PSC-ECs into arterial or venous subtypes. The ability to efficiently leverage on a combination of biochemical and biophysical environmental cues to modulate intrinsic arterio-venous specification programs in ECs will greatly facilitate future translational applications of PSC-ECs. Since the development and maintenance of arterial and venous ECs in vivo occur in disparate physio-chemical microenvironments, it is conceivable that the application of these environmental factors in customized combinations or magnitudes can be used to selectively mature PSC-ECs into an arterial or venous subtype.Endothelial cells (ECs) are required for a multitude of cardiovascular clinical applications, such as revascularization of ischemic tissues or endothelialization of tissue engineered grafts. Patient derived primary ECs are limited in number, have donor variabilities and their  Endothelial cells (ECs) can either be derived as primary ECs from donors or differentiated from stem cells for various clinical applications. Primary ECs are limited by donor availability and are subjected to donor-dependent variabilities  are proposed as engineered alternatives to vein grafts to replace occluded peripheral and coronary vessels  on collagen coated nanofibrils  regulate the secretion of vasodilating and constricting factors, such as Nitric oxide (NO) and endothelin, respectively, to actively modify the vessel volume in response to hemodynamic pressure by acting on the smooth muscle cells in the tunica media. In contrast, veins experience passive blood flow with lower flow rates (1–5 dyne/cm2)  , which czona occludens), adherence junction (zona adhaerens), and gap junctions consisting of connexin proteins  is a key transcription factor, which determines venous specification of ECs, and inhibits the expression of the arterial marker, Notch1 , which in turn regulates the expression of arterial-promoting VEGF and NICD (Notch intracellular domain)  reminiscent of immature ECs present in embryonic vasculatures, and (2) phenotypically and functionally heterogenous  on the expression of arterial and venous markers in human ESC-derived ECs. They reported a significant upregulation of the arterial marker EphrinB2 and downregulation of the venous marker EphB4 as compared to static control in the presence of 10 dyne/cm2 shear stress. The authors postulated that shear stress activates Notch signaling to increase EphrinB2 expression, although the mechano-sensing mechanism is still unknown. Another study by Sivarapatna et al. (2 and 5 dyne/cm2) for arterial and venous enrichment, respectively. The iPSC-ECs showed an upregulation of arterial markers, EphrinB2 and Notch1, and the venous marker, EphB4, when subjected to both 5 and 10 dyne/cm2 of shear stress. However, the venous transcription factor, COUP-TFII, did not demonstrate any significant change in the presence of both shear stress magnitudes. Taken together, it appears that shear stress is positively correlated to an arterial phenotype; although it is still unclear how shear stress will affect venous specification or whether there exists a threshold shear stress magnitude that can toggle between arterial and venous maturation.Fluid-induced shear stress is an important biophysical factor for regulating EC homeostasis. Disturbances in healthy flow promote vascular complications, including atherosclerosis, tissue ischemia and myocardial infarction  on human ESC derived ECs to investigate their dose-time response to shear stress. The study discovered a threshold shear stress magnitude of ~4 dyne/cm2 where an enhancement of Notch1 and EphrinB2 arterial markers could be observed , which further supports arterial ECs differentiation. Lanner et al. (2) treatment followed by normoxia increased the pan-EC as well as arterial EC marker expressions in comparison to conditions with consistent normoxia (21% O2). This shows that hypoxia is an important regulator of EC differentiation in early embryonic phase.Oxygen concentration is another factor that differentiates the arterial and venous endothelium microenvironment. Although the oxygen tension in a healthy mature artery is much higher than a mature vein, vasculogenesis and arterial EC specification appear to be supported by a low oxygen environment. Vascularization in both mature ischemic and early embryonic tissues has been shown to be promoted by oxygen deficiency. Indeed, multiple studies have shown an enhancement in EC differentiation from human or mouse PSC as well as mesenchymal stem cells under hypoxic conditions  Chip platform (Moe et al., tunica intima is also different. Therefore, ECs in arteries and veins also experience different tissue stiffness. An artery consists of more smooth muscle cells due to the need of it to dilate in response to blood pressure leading to higher stiffness of the arterial wall. Culture substrates with tunable stiffness thus, can be used to evaluate whether substrate stiffness can affect arterial-venous specification. For example, Xue et al. (Besides the basement membrane, the wall composition of artery and vein as determined by the amount of smooth muscle cells present in the e et al.  demonstrArterial and venous ECs are required for multiple clinical and research applications. Deriving them using PSC-ECs will provide a cell source that is not limited by cell number and accessibility. An important hurdle to overcome to realize the practical translation of these cells is to mature them into specific subtypes with functional performance comparable to mature ECs. The strategies discussed here for maturing PSC-ECs into arterial or venous subtypes are predominantly based on a body of work on how environmental factors regulate vascular development. These include both biochemical and biophysical cues , of whicSA, EY, and Y-CT contributed to the idea conception and the study design. SA did the literature review. SA, Y-CT, and EY wrote the 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.


Foot-and-mouth disease virus (FMDV) is an important livestock pathogen that is often described as the greatest constraint to global trade in animal products. The present study utilized a standardized pig-to-cow contact exposure model to demonstrate that FMDV infection of cattle initiates in the nasopharyngeal mucosa following natural virus exposure. Furthermore, this work confirmed the role of the bovine nasopharyngeal mucosa as the site of persistent FMDV infection in vaccinated and nonvaccinated cattle. The critical output of this study validates previous studies that have used simulated natural inoculation models to characterize FMDV pathogenesis in cattle and emphasizes the importance of continued research of the unique virus-host interactions that occur within the bovine nasopharynx. Specifically, vaccines and biotherapeutic countermeasures designed to prevent nasopharyngeal infection of vaccinated animals could contribute to substantially improved control of FMDV. The pathogenesis of foot-and-mouth disease virus (FMDV) in cattle was investigated through early and late stages of infection by use of an optimized experimental model for controlled contact exposure. Time-limited exposure of cattle to FMDV-infected pigs led to primary FMDV infection of the nasopharyngeal mucosa in both vaccinated and nonvaccinated cattle. In nonvaccinated cattle, the infection generalized rapidly to cause clinical disease, without apparent virus amplification in the lungs prior to establishment of viremia. Vaccinated cattle were protected against clinical disease and viremia; however, all vaccinated cattle were subclinically infected, and persistent infection occurred at similarly high prevalences in both animal cohorts. Infection dynamics in cattle were consistent and synchronous and comparable to those of simulated natural and needle inoculation systems. However, the current experimental model utilizes a natural route of virus exposure and is therefore superior for investigations of disease pathogenesis and host response. Deep sequencing of viruses obtained during early infection of pigs and cattle indicated that virus populations sampled from sites of primary infection were markedly more diverse than viruses from vesicular lesions of cattle, suggesting the occurrence of substantial bottlenecks associated with vesicle formation. These data expand previous knowledge of FMDV pathogenesis in cattle and provide novel insights for validation of inoculation models of bovine FMD studies.IMPORTANCE Foot-and-mouth disease virus (FMDV) is an important livestock pathogen that is often described as the greatest constraint to global trade in animal products. The present study utilized a standardized pig-to-cow contact exposure model to demonstrate that FMDV infection of cattle initiates in the nasopharyngeal mucosa following natural virus exposure. Furthermore, this work confirmed the role of the bovine nasopharyngeal mucosa as the site of persistent FMDV infection in vaccinated and nonvaccinated cattle. The critical output of this study validates previous studies that have used simulated natural inoculation models to characterize FMDV pathogenesis in cattle and emphasizes the importance of continued research of the unique virus-host interactions that occur within the bovine nasopharynx. Specifically, vaccines and biotherapeutic countermeasures designed to prevent nasopharyngeal infection of vaccinated animals could contribute to substantially improved control of FMDV. Aphthovirus, family Picornaviridae), is a contagious livestock disease of global concern (Foot-and-mouth disease (FMD), caused by FMD virus  associated with the appearance of characteristic vesicular lesions in the oral cavity and in the interdigital clefts and coronary bands of the feet , 4. MortFMDV is infectious in low doses and can be transmitted by both direct and indirect mechanisms, including movement of infected animals as well as mechanical transfer by contaminated vehicles and fomites. Cattle are specifically sensitive to FMDV infection via the respiratory route , 5. EarlHistorically, challenge of cattle during experimental FMDV studies has been done through intraepithelial injection on the dorsal surface of the tongue . This inSeveral investigations have used contact exposure to experimentally infect cattle with FMDV , 20; howThis study reports the outcome of FMDV infection in cattle after time-limited, direct contact exposure to FMDV-infected pigs. Six vaccinated and six nonvaccinated cattle were monitored and sampled through 35 days after contact exposure for the objective of investigating FMDV infection dynamics, as well as the occurrence and characteristics of FMDV persistence. Additionally, 12 nonvaccinated cattle were euthanized for tissue harvest during early infection (6 to 72 h postexposure [hpe]) for temporo-anatomic mapping of FMDV in bovine tissues during the acute phase of infection .The pigs that were used as virus donors during contact exposure were infected through heel bulb inoculation. The dynamics of infection were highly synchronous across all 12 pigs. All pigs were viremic, as determined by detection of FMDV RNA in serum, and were shedding detectable quantities of FMDV RNA in oropharyngeal and nasal secretions at 24 h postinoculation (hpi) . The firA total of 18 nonvaccinated cattle were included in the investigations . Twelve Six vaccinated cattle were included in the investigation for monitoring of FMDV infection dynamics and persistence following natural virus exposure. None of these cattle developed any clinical signs of FMD, and there was no detection of FMDV RNA in serum. However, all six cattle were subclinically infected, based upon detection of FMDV in secretions and/or tissues. Declining levels of FMDV RNA were detected in oral and nasal secretions in all animals from the end of contact exposure at 1 dpe until approximately 4 to 6 dpe  and C. STwelve nonvaccinated cattle were euthanized during the early phase of infection (6 to 72 hpe) for determination of the temporo-anatomic progression of infection following natural virus exposure. The most consistent detection of FMDV in tissues prior to the onset of viremia occurred in the nasopharyngeal tissues. Specifically, infectious FMDV was isolated from the dorsal soft palate and/or the dorsal nasopharynx of all four cattle that were euthanized at 6 to 12 hpe . AdditioAll four cattle that were euthanized at 24 hpe were viremic, but had not yet developed any clinical signs of FMD. In these animals, moderate to large quantities of FMDV RNA were consistently detected in the nasopharyngeal tissues, concurrent with isolation of virus. Smaller quantities of FMDV RNA were detected in the tongue and oropharyngeal epithelium and oropharyngeal tonsils . InfectiThe four cattle that were euthanized at 48 and 72 hpe were viremic and had characteristic vesicular lesion in the oral cavity and/or on one or two feet. During this more advanced stage of infection, FMDV was isolated from all sampled tissues , interprAll 6 nonvaccinated cattle and 5 out of 6 vaccinated cattle that were monitored through to the persistent phase of infection were FMDV carriers. Infectious virus and FMDV RNA were consistently detected in nasopharyngeal tissues  from the persistently infected carriers, regardless of vaccination status . One nas+ cells (presumptive antigen-presenting cells) was seen in close proximity to FMDV-infected areas. At 48 to 72 hpe, substantial FMDV replication was detected in epithelial crypts of the palatine tonsils . Similarly, viral RNA quantities in air samples declined in all rooms during the first 24 h after removal of the infected pigs from the rooms (2 dpe) . From 2 Samples obtained from cattle and pigs in room A were subjected to FMDV deep sequence analysis by next-generation sequencing (NGS). This experimental cohort included 3 pigs that were all euthanized at 72 h postinoculation (hpi), as well as 6 cattle, of which 2 were euthanized for tissue collection at each of 6, 24, and 72 hpe  .p-distance = 0.002; 13 nucleotides [not shown]). However, the frequency distribution of subconsensus viral variants provided insights into FMDV pathogenesis events.It was not possible to identify definitive transmission chains based on FMDV consensus sequence phylogenetic analysis due to the high similarity between consensus sequences (maximum 147 (end of the G-H loop) and asparagine to aspartate at 3A136—were detected at the consensus level in at least one sample from all 3 donor pigs and were also present at high frequencies in all sampled cattle. Of the remaining synonymous changes, three  occurred in at least one sample from every animal in the study.The original inoculum used to infect the pigs contained a highly diverse viral population. Consequently, during early infection, viral populations in pig-derived samples had abundant synonymous and nonsynonymous single nucleotide polymorphisms (SNPs) compared to the inoculum consensus sequence . A singlVesicular lesions obtained from cattle during the clinical phase of disease (72 hpe) contained viral populations that were substantially less diverse  than samples obtained from nasopharyngeal tissues at corresponding time points. Pulmonary tissue samples contained virus populations of mixed characteristics: these were either highly diverse, and thus, similar to nasopharyngeal tissues , or cloMultiple previous works have investigated FMDV pathogenesis in cattle. However, differences in study design, specifically with regard to methods used for virus exposure, have led to various findings. The objective of the current investigation was to utilize a natural but controlled system of direct contact exposure to achieve a detailed understanding of the progression of FMDV infection in cattle, from primary infection through systemic generalization to establishment of persistent infection.The experimental model included inoculation of donor pigs with a cattle-derived FMDV strain. Infected pigs were subsequently moved into clean rooms housing naive or vaccinated recipient cattle, at a time point that corresponded to the early clinical phase of FMD in the pigs. Initiating the contact exposure at a time at which the pigs were known to be shedding large quantities of FMDV  and limiThe onset and progression of FMD in exposed cattle were rapid and consistent. The first detection of viremia in nonvaccinated cattle occurred as early as 18 h after the start of contact exposure. Similarly, the majority of nonvaccinated cattle were viremic at 24 hpe, and all had vesicular lesions in the mouth or on at least one foot at 48 hpe. This very rapid disease progression is similar to that which occurs after conventional tongue inoculation of cattle with virulent FMDV strains , 22. AddSimilar to previous investigations, vaccination with an adenovirus-vectored FMD A vaccine 14 days prior to virus exposure conferred complete protection against clinical FMD and viremia , 12. AllAnalysis of tissue samples from early and persistent stages of disease by immunomicroscopy confirmed localization of both primary and persistent FMDV infection to specialized regions of epithelium of the nasopharyngeal mucosa overlying the mucosa-associated lymphoid tissue (MALT). This finding confirms that the simulated natural, aerosol , 23, andDespite the identical microanatomic locations of virus associated with primary versus persistent FMDV infection, there were notable differences in histopathological characteristics. Consistent with previous investigations , 10, priViral RNA quantities detected in air samples in room C, with 6 nonvaccinated cattle, increased from 2 to 3 dpe, after removal of the pigs, suggesting that the infected cattle contributed to airborne virus during the clinical phase of disease. Contrastingly, the quantities of viral RNA detected in room D, with 6 vaccinated cattle, decreased continuously after removal of the pigs from the room. Despite this, infectious FMDV was isolated as late as 7 dpe in filters from both rooms. The combination of sequence similarity between pig samples and air samples and previous observations confirming that in a similar setting, vaccinated and protected cattle did not shed detectable virus into the air (unpublished data), suggests that the airborne virus in the room of the vaccinated cattle may to a large extent have been derived from the pigs.Analysis of the frequency of FMDV genome substitutions below and above the consensus level provided interesting insights into the complexities of FMDV pathogenesis during early infection. This part of the investigation included only a limited subset of study animals and was limited to unpassaged sample material to avoid introduction of artifacts occurring during viral amplification in cell culture. Overall, there was a consistent trend that viruses obtained from vesicular lesions of cattle were highly homogenous, approaching clonality, whereas the viruses obtained from nasal swabs, air, and most tissues had highly heterogenous virus populations (swarms). This finding is consistent with previous reports that concluded that FMDV samples from vesicular epithelium were less diverse than esophagopharyngeal scrapings, suggesting within-host bottleneck events in association with formation of FMDV vesicles , 28. HerThe virus samples obtained from the inoculated pigs were highly diverse, reflecting the high diversity found in the original virus inoculum. In contrast to cattle-derived samples, a similar level of diversity was seen in samples from the site of primary virus replication (tonsil swabs), as in vesicular epithelium (coronary band lesions) in the pigs. This suggests a lesser extent of bottleneck events within the pigs compared to the more extensive effects that occurred as part of the process of transmission, adaptation, and pathogenesis within a new host species. Additionally, the species-specific virus populations may represent differential species-specific viral fitness defined by host selective pressures.The different levels of within-animal virus diversity across the different cattle may be suggestive of different mechanisms of virus transmission or the occurrence of bottlenecks associated with transmission from one host to another. For example, the striking homogeneity of virus samples obtained from animal 16-28 suggests that infection in this animal was likely founded by a narrow population. In contrast to this, the greater diversity of viruses in animals 16-27 and 16-29 suggests that these animals were likely exposed to different or more diverse founding virus populations. The highly synchronous infection dynamics across the cattle suggests that all animals received a comparable challenge dose. However, the differences in the diversity of within-host virus populations suggest that there may still have been qualitative differences in how these animals acquired the primary infection.The primary output of this investigation was confirmation of the importance of the bovine nasopharyngeal mucosa as the site of both primary and persistent FMDV infection after natural contact exposure. This confirms the output of many previous studies that reached similar conclusions using simulations of natural infection. Additionally, natural virus exposure did not cause substantial FMDV replication in the lower respiratory tract prior to establishment of viremia. The standardized experimental system of time-limited exposure to FMDV-infected pigs provided a stringent and consistent virus challenge across different cohorts of animals, providing a useful tool for FMDV pathogenesis studies. The output of the FMDV sequence analyses demonstrated that FMDV quasispecies compositions differ substantially between primary and secondary replication sites. The current findings provide novel insights on the within- and between-host evolution of FMDV, which are being explored in greater detail in ongoing investigations within our laboratory.24 Cruzeiro that has been characterized in detail in previous pathogenesis studies  biocontainment facilities at the Plum Island Animal Disease Center, New York. All procedures were performed in compliance with an experimental protocol approved by the Institutional Animal Care and Use Committee (protocol 231.03-14-R). Cattle were Holstein heifers weighing approximately 150 to 200 kg when delivered. Pigs were castrated Yorkshire males weighing approximately 25 to 30 kg. Pigs and cattle were housed in separate isolation rooms before virus challenge.5 BTID50 (50% infectious doses titrated in bovine tongue epithelium), diluted in minimal essential medium (MEM) to a total volume of 0.4 ml. At 48 h postinoculation (hpi) of the pigs, the pigs were moved into different isolation rooms that housed the cattle. After 24 h of cohabitation, the pigs were removed from the rooms and euthanized. During each contact exposure trial, 3 pigs were housed together with 6 cattle in a room with an available floor area of 27 m2, a total air volume of approximately 135 m3, and 28 complete air exchanges per hour. The study included replicate challenge trials performed in four separate rooms. Rooms A and B housed nonvaccinated cattle that were included in investigations of FMDV pathogenesis during early stages of infection. These animals were euthanized at predetermined time points between 6 and 72 h postexposure (hpe). Cattle in rooms C and D were kept for 35 days for the objective of investigating FMDV persistence. Among these animals, there were 6 vaccinated and 6 nonvaccinated individuals. During contact exposure of cattle in rooms C and D, 3 vaccinated and 3 naive cattle were housed together with 3 donor pigs to ensure uniform exposure conditions for all cattle. Vaccinated and nonvaccinated cattle were subsequently separated and moved into different rooms after 24 h of cohabitation with the infected pigs: nonvaccinated in room C and vaccinated in room D.The study included a total of 24 cattle and 12 pigs. Pigs were inoculated through intraepithelial injection in the heel bulb of one foot as previously described . Each piFMDV infection dynamics and virus shedding in pigs were monitored through collection of blood, nasal, and oropharyngeal (OP) swabs at 24-h intervals from 0 to 72 hpi. Swabs from pigs were immersed in 2 ml of MEM with 25 mM HEPES. The progression of clinical FMD in pigs was recorded using a cumulative scoring system as previously described , 33. SamFor the objective of evaluating tissue distribution of FMDV during early infection, two nonvaccinated cattle were euthanized for tissue harvest at 6, 12, 48, and 72 hpe, and four animals were euthanized at 24 hpe. For evaluation of tissue distribution during the persistent phase of infection, six vaccinated and six nonvaccinated cattle were euthanized at 35 dpe. A standardized necropsy procedure with collection of 18 to 22 distinct tissue samples  and 2 waAir sampling was performed using a model 1000 air pump developed by the Program Executive Office for Chemical Biological Defense (PEO-CBD), fitted with an original DFU filter assembly holding two separate Lockheed Martin polyester filter discs  as previously described . The airTC) values were converted to RNA copies per milliliter or milligram using an equation derived from analysis of serial 10-fold dilutions of in vitro-synthesized FMDV RNA of known concentration. The equations of the curve of RNA copy numbers versus TC values were further adjusted for average mass of tissue samples and specific dilutions used during processing of samples. The qRT-PCR results reported in 10 GCN/µl for all animals sampled at each time point. Results for analysis of air filters are reported as TC values.Two aliquots of each tissue sample collected at necropsy were thawed and individually macerated in tissue culture medium, using a TissueLyser bead beater  and stainless steel beads . Air filters were cut into quarters and disrupted using a similar approach as for tissue samples, but with washed glass beads . The air filter “homogenate” was subsequently centrifuged to extract the fluid that had been absorbed by the filter. Total RNA was extracted from tissue macerates, air filter supernatants, serum, swabs, and OPF samples using Ambion’s MagMax-96 viral RNA isolation kit  on a King Fisher-96 magnetic particle processor . Extracted RNA was analyzed using quantitative real-time reverse transcription PCR (qRT-PCR), targeting the 3D region of the FMDV genome  with forAliquots of macerated tissue samples, air filter supernatants, and TTE-treated probang samples were cleared from debris and potential bacterial contamination by centrifugation through Spin-X filter columns . Samples were subsequently analyzed for infectious FMDV through virus isolation (VI) on LFBK-αvβ6 cells , 42, folAfter screening of tissue samples for FMDV RNA and infectious virus by qRT-PCR and VI, respectively, detection of antigen in cryosections by immunohistochemistry (IHC) and multichannel immunofluorescence (MIF) was performed as previously described , 24. Sli24-specific primers with the SuperScriptIII kit per the manufacturer’s instructions (Thermo Fisher Scientific). Amplicons were quantified using the Qubit 2.0 fluorometer, and NGS libraries were constructed using the Nextera XT DNA library kit  and run on a NextSeq 500. Reads were quality trimmed and mapped to the inoculum consensus sequence using CLC Genomics Workbench v9.5 (Qiagen). Full ORF consensus sequences were subjected to a minimum coverage of 10 reads with quality scoring/vote used for ambiguous base calls. Consensus sequences were aligned using MUSCLE . This experimental cohort included 3 pigs that were all euthanized at 72 hpi, as well as 6 cattle, of which 2 were euthanized for tissue collection at each of 6, 24, and 72 hpe . g MUSCLE , implemeg MUSCLE , and a pMH746921, and animal and air samples, MH559773 to MH559806. NGS data for animal and air samples are available at the Sequence Read Archive with sample identifiers SAMN09580658 to SAMN09580694.FMDV consensus sequence data are available at GenBank with the following sample identification numbers: inoculum,


The model predicts that competition between transient PLK4 activity maxima for PLK4-STIL complexes destabilizes the PLK4 ring and produces instead a single PLK4 spot. Weakening of competition by overexpression of PLK4 and STIL causes progressive addition of supernumerary procentrioles, as observed experimentally.Centrioles, the cores of centrosomes and cilia, duplicate every cell cycle to ensure their faithful inheritance. How only a single procentriole is produced on each mother centriole remains enigmatic. We propose the first mechanistic biophysical model for procentriole initiation which posits that interactions between kinase PLK4 and its activator-substrate STIL are central for procentriole initiation. The model recapitulates the transition from a uniform “ring” of PLK4 surrounding the mother centriole to a single PLK4 “spot” that initiates procentriole assembly. This symmetry breaking requires autocatalytic activation of PLK4 and enhanced centriolar anchoring of PLK4 by phosphorylated STIL. We find that   •Kinase PLK4 and scaffold STIL form the core of the centriole initiation network•Dual PLK4-activity-based positive feedback breaks symmetry of the PLK4 localization•Competition between nascent PLK4 activity maxima determines the single procentriole•In situ degradation of PLK4 cannot break the symmetry in the absence of competition Biological Sciences; Developmental Biology; In Silico Biology Symmetry breaking, an overarching principle of modern physics, explains the emergence of new order in initially disordered systems and has long been suggested to drive self-organization of biological systems, albeit very few specific examples have been elucidated . Thus bith century  via the well-characterized interactions with the scaffolds CEP192/Spd2 and CEP152/Asl . Rapid rartwheel . Phosphoartwheel , may be artwheel .trans, but still within the same PLK4-STIL complex  of PLK4 in the losing contender compartment. Namely, all PLK4 molecules residing within this compartment were virtually labeled at the time point indicated by vertical line in Drosophila has revealed intermediate states with multiple PLK4 maxima, which could correspond to the model contender sites  cannot be realized. Remarkably, a very similar quantitative trend had been observed in experiments in which the number of centrioles had been carefully assayed in response to progressive increase in PLK4 abundance  and formation of the unique cluster of PLK4-STIL complexes (“spot”) that initiates biogenesis of the procentriole. Positive feedback that drives symmetry breaking consists of two converging arms . In the de novo centriole formation. An important consequence of the activity-dependent retention is that out of two unequal PLK4-STIL clusters, the bigger one will grow faster. This property undermines neutral coexistence of multiple PLK4-STIL clusters. Because the clusters can exchange their material via the common cytoplasm, they, in fact, engage in an antagonistic winner-takes-all competition, which, under physiologically normal conditions, results in the emergence of a single procentriole. We, therefore, predict that improvement in the spatial and temporal resolution of live-cell imaging of centriole duplication will reveal the emergence of multiple competing PLK4 maxima and their subsequent resolution toward a single procentriole. We hypothesize that the principles highlighted by our model are instrumental for self-organization of unique cellular structures, regardless of the details of the molecular mechanisms. Although our model is formulated for mammalian cells, the principles of PLK4 symmetry breaking outlined above are likely to be conserved also in other organisms. Further concerted experimental and theoretical efforts will be required to increase the biological realism and predictive power of the model by the refinement of molecular mechanisms and incorporation of additional molecular players, such as CEP85, which was recently implicated in PLK4 activation (An important feature of our model is that the two feedback arms synergize . Indeed,tivation .in vivo using the current experimental methods. Although we have made a conscious effort to incorporate published measurements of protein abundances and reaction rates, the numerical values of molecular concentrations and reaction rates on the axes of our figures should be considered only as indicative and not as exact predictions of the model.Our model, like other efforts to model intracellular dynamics, is limited by the fact that most protein concentrations and reaction rate constants are unknown and, frequently, cannot be reliably measured Although our model is consistent with the majority of published experimental results, other models may also potentially explain the known experimental observations. When this paper was in the final stages of review, a study posted on bioRxiv  proposedAll methods can be found in the accompanying


Human adult stem cell research is a highly prolific area in modern tissue engineering as these cells have significant potential to provide future cellular therapies for the world’s increasingly aged population. Cellular therapies require a smart biomaterial to deliver and localise the cell population; protecting and guiding the stem cells toward predetermined lineage-specific pathways. The cells, in turn, can provide protection to biomaterials and increase its longevity. The right combination of stem cells and biomaterials can significantly increase the therapeutic efficacy. Adult stem cells are utilised to target many changes that negatively impact tissue functions with age. Understanding the underlying mechanisms that lead to changes brought about by the ageing process is imperative as ageing leads to many detrimental effects on stem cell activation, maintenance and differentiation. The circadian clock is an evolutionarily conserved timing mechanism that coordinates physiology, metabolism and behavior with the 24 h solar day to provide temporal tissue homeostasis with the external environment. Circadian rhythms deteriorate with age at both the behavioural and molecular levels, leading to age-associated changes in downstream rhythmic tissue physiology in humans and rodent models. In this review, we highlight recent advances in our knowledge of the role of circadian clocks in adult stem cell maintenance, driven by both cell-autonomous and tissue-specific factors, and the mechanisms by which they co-opt various cellular signaling pathways to impose temporal control on stem cell function. Future research investigating pharmacological and lifestyle interventions by which circadian rhythms within adult stem niches can be manipulated will provide avenues for temporally guided cellular therapies and smart biomaterials to ameliorate age-related tissue deterioration and reduce the burden of chronic disease. Pioneering experiments in stem cell research are often credited to Canadian scientists Till and McCulloch in 1961–1963, who were the first to carry out clonal colony formation assays on hematopoietic stem cells (HSCs) in murine bone marrow, indicating their multipotency , which can be more appropriately referred to as adult progenitor cells, are being used to treat an ever-increasing array of human conditions, varying from heart disease to leukaemia. But in order for ASCs to become a therapeutic reality, these tissue regenerative processes must fully be understood, and the environmental factors that regulate this regeneration process fully uncovered, which is vital for the repair and replenishment of most tissues in the body.Adult stem cell (ASC) classification has become highly complex since the original terms ‘haematopoietic’ and ‘mesenchymal’ covered the main types of these pluripotent cells. Currently, they tend to be classified based on their tissue of origin and differentiation potential, and one should perhaps increasingly replace the use of the term adult ‘stem cells’ in favour of adult ‘progenitor cells’. Adult progenitor cells, being somatic stem cells, can be derived from all parts of the body, where they are found in tissue-specific stem cell niches. Progenitor cells have been derived and utilised from cord and peripheral blood, blood vessels and bone marrow , the brain , skin , skeletal muscle (muscle satellite cells/myogenic stem cells), teeth , heart (cardiac stem cells), gut , liver (hepatic stem cells), ovarian epithelium (ovarian stem cells), breast (mammary stem cells) and testis (testicular stem cells).ASCs are multipotent and are, by that definition, limited to giving rise to different cell types from their tissue of origin. They divide either symmetrically to produce two identical cells which self-renew, proliferate and expand in number, or asymmetrically to produce one identical stem cell and one committed daughter cell which maintain progeny population Fig. . The forWhen comparing embryonic stem cells to ASCs, embryonic stem cells (ESCs) are pluripotent, meaning that they are capable of differentiating into any one of the three germ layers; endoderm, mesoderm or ectoderm. Since their discovery, there has been a great interest in their use in regenerative medicine and tissue engineering, due to their pluripotent differentiation capabilities. However, the pluripotency of ESCs makes it difficult to direct their differentiation in a reliable, long term and reproducible manner. Furthermore, many in vivo studies have shown embryonic stem cells, following implantation, can spontaneously differentiate and form a type of tumour called a teratoma  and Cryptochrome (Cry1 and Cry2) as well as numerous clock-controlled genes (CCGs). PER and CRY proteins then heterodimerise and translocate to the nucleus. They form a negative arm of the feedback loop, and repress target gene expression, including their own transcription, by inhibiting the CLOCK:BMAL complex  and Ror . These bind to retinoic acid-related orphan receptor response elements (ROREs), which are present in many clock-controlled gene promoters as well as the core clock gene Bmal. REV-ERB represses transcription of Bmal, whereas ROR activates it  in the anterior hypothalamus of the brain, coordinates a number of independent central nervous system (CNS) and peripheral tissue oscillators, acting as a pacemaker to regulate a coherent rhythm at the level of the whole organism . Morphologically, old MSCs appear larger and more flattened when they reach approximately 40 population doublings and stop proliferating , Rev-erbα and Rev-erbβ , both of which are regulated by the core molecular clock through rhythmic noradrenaline secretion. This implies that the clock-driven release of HSCs is stimulated by the CNS during the animal’s resting phase and can promote the tissue regeneration and regulate the function of a hematopoietic stem cell niche.There has been a substantial amount of evidence that suggests that not only do ASCs express the functional core circadian machinery, but that circadian clocks have important role in a variety of tissue homeostatic functions thus influencing the activation of stem and progenitor cells  and, in hematopoietic cells, wherein it stimulates DNA damage responses and p53-dependent apoptosis. Therefore, Per2 has been identified as a negative regulator of lymphoid-biased HSCs, immune function and lymphopoiesis. Another core clock gene that has been implied in cancer stem cells is Per3. Per3 has been shown to have an important role in colorectal cancer, being downregulated in colorectal cancer stem cells  leads to disruption in the establishment of the molecular clock oscillations in differentiating mouse ESCs. It has been reported that, when there is a failure of clock oscillation development, an increase in Kpna2 (Importin-α2) and altered subcellular PERIOD protein localization can be observed; therefore differentiation-coupled transcription of specific genes may regulate circadian clock development in mouse ESCs  show clear circadian rhythms in murine adipose tissue , which has been shown to be a key regulator of brown adipose tissue development  in the subgranular zone (SGZ) of the adult hippocampus also express components of the molecular clock and proliferate in a rhythmic manner  differentiating to mature neurons in the dentate gyrus. It has been postulated that mPer2 provides a functional link by influencing the early cellular events that lead to post-mitotic granule cell production underlying adult hippocampal neurogenesis . Overexpression of Bmal1 in C2C12 myoblasts conversely led to accelerated myogenesis and attenuation of Wnt signalling, indicating that Bmal1 is required for myoblast differentiation . Interestingly, the negative proliferation effects of Per2 deficiency can be rescued by activating PI3K/Akt/FoxO signalling. Indeed, PER2 and CXCR4 directly interact in EPCs, showing that PER2 is essential in maintaining early EPC function  function during angiogenesis. PER2 is abundantly expressed in early EPCs, whilst EPCs from Pln, Kcne4, Tspo, Cav1 and Rgs2  are critical in determining how the intestine regenerates in order to replace dying cells. The PER transcription factor has been discovered to be essential in intestinal regeneration, and numerous gene transcripts that are regulated by the circadian clock have been uncovered within intestinal stem cells, including those involved in stress response and regeneration pathways  that reside within the bulge   in several gene promoters encoding enzymes involved in glutathione synthesis and utilisation. The lack of NRF2-mediated antioxidant defence provides a potential mechanism for the premature onset of age-related pathologies seen in the absence of Bmal1 in mice (Pekovic-Vaughan et al. Furthermore, circadian clock is implicated in several cellular protective mechanisms that are important for stem cell homeostasis such as antioxidant stress responses (Kondratov et al. In regenerative medicine, there is a great need to generate mature functional tissues in vitro, especially given our increasingly ageing society. Therefore, it is essential to discover effective means to direct differentiation of stem cells into tissue-specific cells in a controlled manner, which requires appropriate biochemical and biophysical signals within cellular microenvironments. Biomaterials provide a 3D environment equipped with biological, mechanical and chemical cues, which can stimulate cells to proliferate, differentiate, secrete extracellular matrix and form functional tissues (Hwang et al. Npas2 and Bmal1 were upregulated around the implant and diminished by vitamin D deficiency, whereas the Per2 expression pattern showed the opposite trend. It was thus concluded that the circadian rhythms, along with the extracellular matrix, may be involved in osseo-integration establishment under vitamin D regulation.The circadian rhythms have recently been implied in dental and orthopedic implant integration. In order to investigate causes of failure in osseo-integration, an implant failure model under vitamin D deficiency was utilised to identify crucial gene networks that underpin this process (Mengatto et al. PER1 expression and upregulated NPAS2. When further investigated using Npas2 KO mice, the titanium biomaterial-induced suppression of Per1 was not rescued and the expression of other clock genes Per2, Per3, Bmal1 and Clock was not affected, suggesting that the altered expression of Per1 was independent of Npas2. The authors concluded that titanium-based biomaterials can influence BM-MSC circadian rhythms, and altered BM-MSC rhythms may be important factor in determining the rate of titanium-based biomaterial integration into bone. This exciting area of research examining how materials influence the stem cell clock warrants further research, and the authors of this review are optimistic that it will be a very prosperous area of research to come.In a recent follow up study by Hassan et al. , the cirThe circadian clock has recently been implicated in regenerative medicine as it has been shown to have a profound effect on the wound healing response in mice and humans (Hoyle et al. Zeitgebers. The most common of these cues is light which, in mammals, entrains rhythms in the SCN through retino-hypothalamic tract. The SCN relays this temporal information to the rest of the brain and peripheral tissue clocks via diffusible signals and neuroendocrine factors (Silver et al. Zeitgeber, as was initially shown by subjecting cultured fibroblasts to rhythmic temperature oscillations (Brown et al. The circadian clock has a period of approximately, but not exactly, 24 h. Therefore, it must be reset daily by external cues, known as One emerging entrainment mechanism of circadian clocks is mechanical stimulation. Indeed, in the body, different cells in various anatomical locations are subjected to varying amounts of mechanical strain. Published data has demonstrated that a uniaxial strain between 5 and 15% with a frequency of 1 Hz has notable effects on MSCs both on proliferation and collagen synthesis (Sun et al. Drosophila melanogaster. It has been shown that rhythmic mechanical stimulation of the chordotonal organs can synchronize the Drosophila circadian clock (Simoni et al. Period gene led to impaired ability of mechanical synchronization, highlighting the importance of a functional clock system for mechanical entrainment. Research from our own group has recently demonstrated that cyclical mechanical stretch can be used to synchronise circadian rhythms of human ASCs (Rogers et al. Bmal1 influences adipogenesis through β-catenin pathway. Whether BMAL1 has a role in regulating mechano-transduction pathways to influence adipogenesis is currently unknown and warrants further investigation. A summary of the circadian synchronising mechanisms that have been investigated in ASCs is presented in Table Recent research has also demonstrated that mechanical vibrations have the capability of resetting the clock in A recent study has demonstrated the importance of cell–matrix interactions for stem cell clocks and discovered that the mammary epithelial clock is regulated by the mechanical stiffness of the cellular microenvironment (Yang et al. The field of circadian stem cell biology is a dynamic area, which has provided us critical insights into the temporal control of stem cell function and maintenance. Whilst these are exciting developments, there is much fundamental research that is still needed as our understanding in this crucial area continues. Key aspects to build on over the next few years include the bi-directional effects of the biomaterials and biomechanical factors on stem cell clocks. Future research assessing the role of epigenetic factors on stem cell clocks, which are well documented to influence both circadian rhythms and ASC function, will be of great importance to the application of circadian biology to stem cell research. In summary, the invaluable knowledge pertaining to temporal regulation of stem cell physiology and metabolism by tissue-specific circadian clocks will provide novel insights into the dynamic processes that change during stem cell ageing, and allow appropriate optimisation of smart biomaterials and mechanical cues essential for steering stem cells into particular cell fates. Such critical findings are essential for future design of novel cellular therapies to ameliorate and/or slow down a number of age-related diseases and begin to tackle their prevention.