Tert/Terc) are associated with increased risk of congenital as well as age-related diseases , dyskeratosis congenita, emphysema, nonspecific interstitial pneumonia, etc.). Thus, telomere length and maintenance can serve as an important prognostic factor as well as a potential target for new strategies of treatment for interstitial lung diseases (ILDs) and associated pulmonary pathologies.Telomeres are distal chromosome regions associated with specific protein complexes that protect the chromosome against degradation and aberrations. Telomere maintenance capacity is an essential indication of healthy cell populations, and telomere damage is observed in processes such as malignant transformation, apoptosis, or cell senescence. At a cellular level, telomere damage may result from genotoxic stress, decreased activity of telomerase enzyme complex, dysfunction of shelterin proteins, or changes in expression of telomere-associated RNA such as TERRA. Clinical evidence suggests that mutation of telomerase genes ( Telomeres are nucleoprotein complexes that assemble at the end of chromosomes. They comprise tandem repeats of a short DNA sequence, such as TTAGGG for humans and other vertebrates . TelomerInterstitial lung disease (ILD) is a diverse group of different pathological conditions that mainly affect the pulmonary parenchyma . IdiopatMost cases of IPF are sporadic; the occurrence of pulmonary fibrosis in multiple members of the same family, commonly referred to as familial pulmonary fibrosis (FPF), accounts for 2–20% of the overall cases of IPF, suggesting gene-environment interaction . The diaSmoking and aging are well-established risk factors for IPF. Various factors may be involved in the pathogenesis of ILD, such as chromosomal damage , DNA repWhile the precise origin of IPF remains elusive, the disease probably results from repetitive alveolar injury coupled with dysfunctional alveolar wound healing mechanisms . The disThere is no cure for the disease, and referral to lung transplantation is recommended. Two FDA-approved anti-fibrotic drugs (Pirfenidone and Nintedanib) are known to hinder the disease progression by reducing the annual decline in forced vital capacity (FVC). Especially in early treatment ,15.The holoenzyme of telomerase consists of a catalytic subunit (telomerase reverse transcriptase (Tert)), regulatory subunits and an RNA cofactor (telomerase RNA component (Terc)) . Telomer−/− nor Terc−/− telomerase mutations are associated with the development of spontaneous fibrosis [−/−) show a compromised alveolar epithelial type 2 integrity after being inbred for only four to six generations, but do not develop spontaneous fibrosis [The role of telomerase activity and the significance of telomere length to the ILD susceptibility in experimental models remains a contested subject. Degryse et al. found that neither Tertfibrosis . Lee et fibrosis .This could be explained by the fact that telomerase deficiency in itself does not immediately lead to a decrease in the length of telomeres . Instead−/−), combined with inbred generations (four to six), increases the susceptibility of mice to stress-induced pulmonary fibrosis. Povedano et al. showed that the doses of bleomycin required to initiate lung fibrosis were significantly lower than in wild-type animals [−/− mice following their exposure to smoke [Telomere shortening due to telomerase deficiency   and RNA Shelterin is a multi-protein complex consisting of telomeric repeat binding factors 1 and 2 (TRF1 and 2), TRF1-interacting protein 2 (Tin2), tripeptidyl peptidase 1 (TPP1), repressor-activator protein 1 (Rap1) and protection of telomeres protein 1 (POT1) . ShelterCST complex, which in humans consists of CTC1, STN1, and TEN1 proteins, is localized at the single-strand 3′ overhang and is involved in telomere end regulation . It has Fl/Fl mice, Alder et al. determined that telomere dysfunction leads to alveolar stem cell failure via the P53-mediated pathway [Shoeb et al. demonstrated that pulmonary fibrosis  is associated with disruption of TIN2-TPP1-POT1 shelterin complex leading to telomere damage . In expe pathway .lncRNAs are RNA transcripts that do not serve as protein templates but are instead involved in epigenetic regulation together with other non-coding RNA subtypes, such as siRNAs . Cao et One lncRNA is telomeric repeat-containing RNA (TERRA), which is of high importance to telomere stability . TERRA iThe population of cells involved should be regarded as a critical factor in research on telomerase function in pulmonary fibrosis. For instance, Liu et al. showed that in conditional-knockout murine models, mesenchymal-specific TERT inactivation leads to attenuation of bleomycin-induced pulmonary fibrosis .Trf1 in lung fibroblasts caused pulmonary edema, but not fibrosis, unlike deletion of Trf1 in alveolar epithelial type 2 cells, which, with an increase in age, led to lung remodeling and spontaneous fibrosis [−/− mice with shelterin Trf1 deletion, in alveolar epithelial type 2 cells, develop pulmonary fibrosis due to severe telomere protein cap dysfunction [Naikawadi et al. demonstrated that deletion of telomere cap shelterin gene fibrosis . Povedanfunction .Tert mRNA expression and activity decreased in murine type 2 lung epithelial cells under sufficient bleomycin exposure, compared with untreated cells, which led to increased apoptosis [Of note, these findings also indicate that telomere damage in alveolar epithelial type 2 cells is sufficient to cause spontaneous lung fibrosis in the elderly, and may be a major molecular defect causing IPF in humans . In our poptosis .Concomitantly, Arish et al.  and PoveThe first clinical association between telomerase and lung disease was found in dyskeratosis congenita .The characteristic features of dyskeratosis congenita are nail dystrophy, abnormal skin pigmentation and mucosal leukoplakia in the lungs. Bone marrow failure and pulmonary fibrosis are the primary causes of death. There are multiple patterns of inheritance, and the autosomal dominant form has a later age of onset. The X-linked form is due to mutations in the gene-encoding dyskerin, which is involved in processing the template RNA component of telomerase (Terc). Individuals with X-linked dyskeratosis congenita have low levels of Terc and, therefore, less telomerase activity .Tert, Terc, RTEL1, PARN or DKC1 are found to be associated with a higher risk of pulmonary fibrosis  [RTEL1 mutation has been evidenced in 5%–9% of cases of familial pulmonary fibrosis; patients with ILD and RTEL1 mutations present with various pulmonary and extra-pulmonary phenotypes, including sarcoidosis and RA-ILD. Hematological features are rare [Mutations in telomerase complex genes such as fibrosis . Indeed,is (FPF) . These gis (FPF) . RTEL1 mare rare . HeterozIt is believed that the loss of function of the telomerase complex may influence the turnover and healing of alveolar epithelial cells after a damaging stimulus, thus triggering IPF. Moreover, mutations in Tert/Terc are also associated with extra-pulmonary abnormalities, including premature hair greying, bone marrow failure and liver cirrhosis. The phenotype may be heterogeneous, even in patients with the same mutation .hTert or hTR. They did not identify any symptoms of dyskeratosis congenita in these patients [Tert was found in a subject with the sporadic form of the disease who had no family history of IPF [It is important to emphasize that IPF is an age-related disease—it is rare to find IPF in young patients under 50 years old. Therefore, the connection to the telomerase gene is inferred. IPF is a prototype of telomere-mediated disease in slow turnover tissue such as the lungs, liver and pancreas. In these slow turnover tissues, telomere dysfunction disturbs organ homeostasis because of cumulative hits in long-lived cells and eventually culminates in what appears as an irreversible adult-onset disease . Armaniopatients . Anotherpatients  followedShort telomeres are a risk factor for IPF . CronikhTelomeres were found to be significantly shortened in all forms of ILDs, including NSIP, cryptogenic organizing pneumonia (COP), hypersensitivity pneumonitis (HP), and others. However, it has been shown that telomeres in IPF were significantly shorter than in other forms of ILDs .Tert mutation can develop not only ILD but also emphysema.Smoking is a significant risk factor for developing IPF. One study showed that both current and former smokers had shorter telomeres than did age-matched nonsmokers . In addiIn two independent cohorts, Stanley et al. found 3 of 292 severe COPD cases carried deleterious mutations in Tert (1%). This prevalence is comparable to the frequency of alpha-1 antitrypsin deficiency (the most known mutation in young emphysema patients) documented in this population. The Tert mutations compromised telomerase catalytic activity, and mutation carriers had short telomeres. Telomerase mutation carriers with emphysema were predominantly female and had an increased incidence of pneumothorax. In families, emphysema showed an autosomal dominant inheritance pattern, along with pulmonary fibrosis and other telomere syndrome features, but it manifested only in smokers . This obMutations in telomerase genes and short telomeres influence the course of the disease and tend to have a worse prognosis . One obsToday, immunosuppressive medication and prednisone are contraindicated in IPF patients. The PANTHER trial demonstrated increased risks of death and hospitalization in patients with idiopathic pulmonary fibrosis who were treated with a combination of prednisone, azathioprine and N acetyl cysteine as compared with a placebo . Newton Shortened telomeres in patients with IPF who carry a Tert mutation may respond less to treatment with pirfenidone. Therefore, the role of antifibrotic therapy in these patients is still unclear, and it should be managed with caution once there is a risk of liver injury associated .Recently, in a prospective study, danazol, a synthetic androgen, was shown to increase telomere length, and seemed to stabilize DLCO among seven enrolled patients with pulmonary function tests available before danazol administration in telomerase mutation carriers. Pulmonary fibrosis scores based on CT were stable during the two years of treatment in all patients except one, who died from acute exacerbation of pulmonary failure .Tert, RTEL1 or PARN, whereas 231 (88.2%) did not. It was shown that IPF patients with a mutation in telomerase-associated genes had a significantly higher risk of death and chronic lung allograft dysfunction (CLAD) than patients without these variants. There was no difference in acute rejection or grade 3 primary graft dysfunction between the two groups [As mentioned before, lung transplantation is the only definitive treatment for patients with IPF. A few small cohort studies suggest that patients with a mutation in the telomerase gene have more complications and a worse outcome after lung transplantation ,67,68. To groups .p < 0.01) including relapsing-viremia episodes, end-organ disease and CMV resistance to therapy, as well as shorter time to viremia versus age-matched non-IPF control subjects (p < 0.001) [A recent study in lung-transplanted patients showed a high prevalence of relapsing CMV viremia in IPF in comparison with non-IPF patients. IPF-transplanted patients who had short telomeres had the highest risk of CMV complications (< 0.001) .Significant advancements made in the field of understanding the involvement of the telomeres in the pathogenesis of ILD leave open many possibilities for diagnostic and therapeutic attention . This isSignificant advances have been made in the field of telomere research and specifically on the role of telomere maintenance in ILDs. The regulation of telomerase activity in lung tissue cells under various conditions  has been elucidated in numerous studies ,40,41,74Nevertheless, many details remain unclear in the complex field of the role telomeres play in the pathogenesis of ILDs. For instance, although some factors such as GRN510  or TERRATo what extent the disruptions in function of CST complex observed under other pathological conditions  may applDKC1 (dyskerin gene) at the X chromosome raises the possibility of the impact of its mutations on the phenotype, including hereditary pathologies such as familial interstitial pneumonia. Nevertheless, a study by Kropski et al. [Cajal bodies are known to process telomerase RNA  and promi et al.  remains ATM (ataxia-telangiectasia mutated) protein is a dsDNA break response factor that activates a multitude of signal cascades and is known to participate in telomere maintenance via telomerase activation . AlthougTelomere maintenance and telomeric DNA linearization are intricately tied to cell cycle regulation and cell cycle arrest , which iIdiopathic pulmonary fibrosis is one of the most common manifestations of a telomere-related disease (ILDs). Genetic mutations related to telomere maintenance are one of many genetic defects found in patients with familial IPF. Mutations in telomerase genes and shortened telomeres influence the course of the disease and tend to have a worse prognosis and more extrapulmonary manifestations.Taken together, it is important to identify IPF patients early, to introduce antifibrotic drugs and refer them to a lung transplant center. However, and unfortunately, there are neither good and available screening tools nor efficient treatments for these patients in clinical practice.The evaluation of telomere length is not included in the diagnostic algorithm of IPF or other fibrotic diseases due to the limited evidence about the utility of this test. Furthermore, telomere length testing is only available through a minority of expert centers for interstitial lung disease. However, identification the clinical signs that increase the probability of telomere shortening is possible and could help in diagnostic and treatment decisions. Many questions remain open regarding screening and treatment, such as: Who should be screened, and what tests should be performed in suspected genetically determined IPF ? What is the best treatment for IPF patients? Could Nintedanib be more efficient that pirfenidone, giving its anti-cancer role? Should danazol be added to anti-fibrotic therapy or as supplementary therapy after lung transplantation in these patients?In summary, further research is needed in order to better understand how to manage IPF patients before and after lung transplantation, and to try to tailor personalized medicine.


Determining the effects of antimicrobial therapies on airway microbiology at a population-level is essential. Such analysis allows, for example, surveillance of antibiotic-induced changes in pathogen prevalence, the emergence and spread of antibiotic resistance, and the transmission of multi-resistant organisms. However, current analytical strategies for understanding these processes are limited. Culture- and PCR-based assays for specific microbes require the a priori selection of targets, while antibiotic sensitivity testing typically provides no insight into either the molecular basis of resistance, or the carriage of resistance determinants by the wider commensal microbiota. Shotgun metagenomic sequencing provides an alternative approach that allows the microbial composition of clinical samples to be described in detail, including the prevalence of resistance genes and virulence traits. While highly informative, the application of metagenomics to large patient cohorts can be prohibitively expensive. Using sputum samples from a randomised placebo-controlled trial of erythromycin in adults with bronchiectasis, we describe a novel, cost-effective strategy for screening patient cohorts for changes in resistance gene prevalence. By combining metagenomic screening of pooled DNA extracts with validatory quantitative PCR-based analysis of candidate markers in individual samples, we identify population-level changes in the relative abundance of specific macrolide resistance genes. This approach has the potential to provide an important adjunct to current analytical strategies, particularly within the context of antimicrobial clinical trials.The online version of this article (10.1186/s40248-018-0140-9) contains supplementary material, which is available to authorized users. As in all clinical disciplines, the management of patients with chronic respiratory diseases is subject to a process of ongoing refinement, including through the development of novel antimicrobial drugs and treatment strategies. Understanding the impact of antimicrobial treatments for individual recipients allows the personalisation of clinical management. However, determining the effects of treatments at a population level is also crucial, providing a means to predict shifts in the prevalence of respiratory pathogens, or the emergence of antimicrobial resistance, within large patient groups.Stenotrophomonas maltophilia as an airway pathogen [Staphylococcus aureus (MRSA) isolates increasingly show resistance to newer therapies, including linezolid [The impact that evolving treatment strategies can have on airway microbiology can be seen, for example, in changes in the cystic fibrosis (CF) airway microbiota during recent decades. Within this context, the use of anti-Pseudomonal treatments, including parenteral therapies and fluoroquinolones, have been implicated in the emergence of pathogen , 2. Likepathogen , 4. The pathogen . For exainezolid , 7, ceftinezolid  and tigeinezolid , presumainezolid .Despite the importance of understanding the impact of antimicrobial exposure on the airway microbiome in those with respiratory disease, characterising this process remains challenging. Assessments of antibiotic-associated changes in microbiology are typically limited to a small group of predefined pathogens or resistance genes. The standard analytical approaches employed in clinical antibiotic trials fail to assess major aspects of antibiotic resistance, including shifts in the composition of the wider airway microbiota, and the carriage of transmissible resistance determinants by populations of commensal microbes. The absence of suitable strategies to determine antibiotic impact has resulted in significant gaps in our understanding of how widely employed therapies affect the complex microbiota of the respiratory tract.Shotgun metagenomic sequencing is an emerging technology that allows highly detailed characterisation of airway microbiota through the analysis of total microbial DNA from clinical samples, including determination of the prevalence of virulence factors and resistance determinants . While mWe describe a novel, cost-effective, strategy to inform the use of assays for specific resistance genes or microbial taxa, based on deep metagenomic screening of pooled study cohort DNA. We illustrate the application of this approach through the analysis of samples from a previously reported randomised controlled trial of long-term low dose macrolide therapy in adults with bronchiectasis.n = 32, and n = 31, respectively). Patient baseline characteristics are described in Additional file The BLESS randomised placebo-controlled trial assessed the effect of 12 months of low dose erythromycin therapy  on exacerbation rates in adults with non-CF bronchiectasis [Sputum DNA extracts were pooled according to time-point and subject to microbial DNA enrichment (NEBNext® Microbiome DNA Enrichment Kit). DNA fragmentation was performed with TruSeq Nano DNA Library Prep Kit (Illumina), prior to 150 bp paired-end metagenomic shotgun sequencing using the Illumina HiSeq system at the South Australian Health and Medical Research Institute, Adelaide. Reads have been uploaded to the Sequence Read Archive (SRA) under BioProject ID: 397083.Sequences were quality filtered using Trimmomatic v0.32  and humaermA [, ermB [ermC [, 16S [smpB [hmrM were designed within this study .Specific resistance genes that were identified as associated with erythromycin treatment through metagenomics were subsequently quantified in DNA extracts from individual sputum samples by qPCR. Previously published assays were used for ermA , ermB [2 [, ermB , ermC [2mB [ermC , 16S [236S [smpB  genes. PA schematic of the pooled-template metagenomic sequencing strategy, and subsequent qPCR-based validation, is shown in Fig. hmrM, appeared to increase in response to erythromycin therapy. This gene is chromosomally-encoded by Haemophilus influenzae however, and subsequent qPCR analysis revealed hmrM levels to be correlated with H. influenzae levels . This phenomenon could explain apparent changes in the group-level abundance of other chromosomally-encoded resistance genes, such as an observed decrease in the relative abundance of patA, a chromosomally-encoded fluoroquinolone resistance gene carried by Streptococcus pneumoniae [aph(3′)-IIb, a chromosomally-encoded aminoglycoside resistance gene carried by Pseudomonas aeruginosa [A substantial proportion of the genes identified through resistome analysis were chromosomally-encoded, non-transmissible, resistance determinants. Changes in the level of carriage of these genes during the trial therefore reflected shifts in the relative abundance of the species in whose genomes they are encoded, rather than resistance gene acquisition or loss. For example, the multidrug efflux pump gene, eumoniae , and aphruginosa .Fig. 3CoermB, in subjects who received erythromycin (p = 0.007), but not in those who received placebo , we calculate the cost of our analysis to be approximately 15% of that required to analyse all of the samples individually . However, despite this substantial reduction in expense, it is important to be aware of some of the limitations that are inherent in this approach. For example, variations in bacterial load between samples from different patients mean that pooling DNA based on total concentration could result in the contribution of individual samples to meta-microbiome characteristics being unequal. In addition, the non-normal distribution of microbiome traits within a population could lead to the identification of traits as potential inter-group discriminators based on their particularly high abundance in a small number of individuals .A limitation of all metagenomic sequencing is the challenge to differentiate between changes in the carriage of resistance determinants due to direct selective pressure versus changes in resistance gene carriage, because of shifts in the relative abundance of the bacterial populations that encode them. Due to such limitations, the approach that we describe should be used as an additional means to identify markers for further analysis, rather than as a means to characterise antibiotic associated effects on airway microbiology in itself.As an illustration of the potential of the pooled-template metagenomic analysis, we examined shifts in the airway resistome. This application targeted the global health concern of monitoring of antibiotic resistance. Patients with chronic lung diseases have an increased exposure to antibiotics, with the emergence of resistance correlating closely with consumption . The resThe costs of metagenomic sequencing, and the associated costs of high performance computing required for bioinformatic analysis, are likely to continue to fall. However, by providing a low-cost means to perform unbiased surveys of large patient cohorts, strategies such as the one described here represent a useful means of identifying potentially important discriminatory microbiome features for follow-up analysis.Additional file 1:hmrM primers and patient characteristics. (DOCX 18 kb)


Nematostella vectensis challenges our long-standing conceptions on the evolution of cnidarian and bilaterian germ layers and guts.Digestive systems and extracellular digestion are key animal features, but their emergence during early animal evolution is currently poorly understood. As the last common ancestor of non-bilaterian animal groups  dates back to the beginning of animal life, their study and comparison provides important insights into the early evolution of digestive systems and functions. Here, I have compiled an overview of the development and cell biology of digestive tissues in non-bilaterian animals. I will highlight the fundamental differences between extracellular and intracellular digestive processes, and how these are distributed among animals. Cnidarians , the phylogenetic outgroup of bilaterians , occupy a key position to reconstruct the evolution of bilaterian gut evolution. A major focus will therefore lie on the development and cell biology of digestive tissues in cnidarians, especially sea anemones, and how they compare to bilaterian gut tissues. In that context, I will also review how a recent study on the gastrula fate map of the sea anemone  N-acetylglucosamine polymer). Lipids are important for energy storage and production, and membrane formation. Triacylglycerides are the most common animal storage lipid, consisting of three fatty acids linked to a glycerol molecule by ester bonds. Proteins are polymers of amino acids, and crucial for almost all enzymatic activity , lipids and proteins  Fig.  Alberts. TransmeFood particles or larger molecules  cannot be taken up directly through the membrane but necessitate vesicle-mediated uptake. Depending on particle or molecule size, these uptake processes are subdivided into two broad classes, together called endocytosis: phagocytosis and pinocytosis. Phagocytosis leads to the engulfment and uptake of large particles . In bilaterians, it is best known for its role during innate immunity Bayne . ParticlThe subclasses of pinocytosis most relevant for nutritive purposes are micropinocytosis  and macropinocytosis (independent of specific receptor-ligand interactions) . All major bilaterian groups, with a few exceptions (e.g. platyhelmintes or xenacoelomorphs), possess a through-gut with an anterior mouth and a posterior anus  and its associated glands , lophotrochozoans  and ecdysozoans  use intracellular digestion by phago- or pinocytosis, often in combination with extracellular digestion Fig. . CurrentAmphimedon queenslandica, Ephydatia fluviatis, Tethya wilhema, Spongilla sp.), calcareous sponges (e.g. Sycon sp.), glass sponges  and homoscleromorph sponges (e.g. Oscarella sp.). They are mostly marine filter feeders, but some molecular model species are freshwater sponges . The cell biology and physiology of feeding has been mainly investigated in demosponges, comprising the majority of all sponge species, and calcareous sponges (Leys and Hill Sponges (Porifera) are subdivided into four large groups: demosponges  shows however a highly specialised mode of potential extracellular digestion. Their members feed by digesting copepods, and lack a canal system and choanocyte chambers Vacelet . They enpiwi, Wnt signalling genes, Sox genes, NK genes) has so far not revealed any similarities with bilaterian gut or enterocyte cell types  are predatory, gelatinous animals with superficial similarities to cnidarian jellyfish. Recent molecular phylogenies consistently group ctenophores as unrelated to cnidarians with a branching near the basis of the animal tree, but their definite position remains highly debated Fig.  Dunn et. They coMmemiopsis leidyi, for example genes belonging to the TGFβ or Wnt signalling pathway, or a number of genes from the Sox, Tbx or Homeobox transcription factor families  (Bumann and Puls Pleurobrachia at acidic (ph 5.75) and neutral pH (pH 7.5)  in ctenophores makes a comparison of these regions on a molecular level currently difficult  and secretes digestive enzymes from the lower epithelium into a ‘digestive bag’ . Notably, gland cells located at the rim, which have traditionally been connected to secretion of digestive enzymes, are proposed to have neurosecretory functions with roles in behavioural responses to food presence  of lipophilic, gland and ventral epithelia cells compare to bilaterian and cnidarian digestive cell types. Their study will have a large potential to shed light on the evolution and homology of digestive cells between placozoans, bilaterians and cnidarians.Ciliated ventral epithelial cells (‘cylinder cells’) are the second major cell type in the lower epithelium. The vesicular uptake of ferritin, the presence of coated vesicles and a large number of microvilli suggest that nutrient uptake occurs by receptor-mediated endocytosis in these cells , scyphozoans , cubozoans , staurozoans  and hydrozoans . While anthozoans have only a sessile polyp stage, all other cnidarian groups have an additional pelagic medusa (jellyfish) life-stage. Coloniality is common among anthozoan, scyphozoan and hydrozoan polyps.Cnidarians are the phylogenetic sister group to bilaterians and thus occupy an important position for the purposes of reconstructing the digestive system and cell types of the last common ancestor of cnidarians and bilaterians. Cnidarians are further subdivided into five large phylogenetic groups Fig. : anthozoAll cnidarians are composed of two epithelial sheets: the outer epidermis and inner gastrodermis with an intermediate, jelly-like extracellular matrix (mesoglea) Fig. . Polyps Digestion in cnidarians is generally a two-step process: first, prey is broken up in the gastrovascular cavity by digestive enzymes secreted from exocrine gland cells (often termed ‘zymogen’ cells in the literature); then, food particles and nutrients are distributed throughout the GVC, and phago- or pinocytosed into gastrodermal cells where digestion is finalised by lysosomal enzyme activities  medusae  and digestive enzymes (chymotrypsin-like genes), suggesting a dual role of this tissue in extracellular digestion and venom release   Fig. . Phagocyme) Fig.  (Bouillome) Fig. . In colome) Fig. . The cniTripedalia cystophora. Similar to most hydrozoans, potential zymogen cells in Tripedalia are found predominantly in the oral gastrodermis   Fig. . Phagocye’) Fig.  (Chapmane’) Fig. .Anthozoan and scyphozoan polyps, in contrast to hydro- and cubopolyps, show a variable number of gastrodermal folds (termed ‘septa’ or ‘mesenteries’) that subdivide the GVC into inter-connected compartments Fig. . ScyphozAurelia polyps, but their presence elsewhere in the gastroderm remains unclear , the combination of exocrine and insulinergic cells is confined to tissues expressing the foxA transcription factor, which is a conserved marker of bilaterian endoderm, midgut or vertebrate foregut  implies that this region has a generally increased phagocytic activity of food particles. As juveniles have no obvious gonad, and as it is not clear if this median region will contribute to the adult gonad epithelium, I propose to re-name this juvenile region ‘trophic tract’ . These similarities are further corroborated on a molecular level: Nematostella orthologs of genes with conserved expression in bilaterian midgut tissues  and vertebrate pancreas development  consistently co-localise to the larval pharyngeal ectoderm of Nematostella , have almost exclusively been found expressed in different endodermally derived parts of the Nematostella gastrodermis  and physiological functions (digestive enzyme secretion and amino acid uptake) of The pharyngeal ectoderm, developing near the blastopore margin, is homologous to bilaterian endodermThe sea anemone endoderm is not homologous to both bilaterian endoderm and mesoderm, as previously suggested  Formation of the primary, apical-blastoporal body axis with a functional specialisation of the substrate-facing side in phagocytosis. This would resemble the situation also in Due to the consistent development of cnidarian-type endoderm into epitheliomuscular cells and gonad tissue, the primary reason(s) for its evolution were:Extracellular digestion has evolved after the appearance of the phagocytic cavity. The specification of exocrine cells within the ectoderm near the blastopore margin suggests an ancestral digestive or protective (defence against pathogens) function of this region, as partly proposed previously in the ‘mucociliary sole’ concept (Arendt et al. The gastrodermis of sea anemones, scyphozoans and ctenophores is derived from endoderm, and secondarily internalised pharyngeal ectoderm. As it would make most sense to secrete digestive enzymes into a digestive cavity, it sounds counter-intuitive that exocrine cells are specified within the outer ectoderm in these animals. In ‘design terms’, a direct specification and differentiation of exocrine cells in the inner endodermal germ layer would have been much more straightforward. The peculiar development of cnidarian-type endoderm and exocrine cells might therefore reflect their evolutionary history, and it can be speculated that:Altogether, recent data is in line with Haeckel’s assumption that the sea anemone endoderm reflects an ancient absorptive cavity. Future studies will potentially resolve if the cell types deriving from cnidarian endoderm derivatives are similar in their gene expression profiles and functional capacities to the cell types found in the ctenophore gastrovascular system, the endocytic lower epithelium of placozoans, or the phagocytic choanocytes or archaeocytes. The core of the gastraea theory, consisting of the idea that the bilaterian gut evolved from the endoderm of a cnidarian-like ancestor, is however not supported by recent data. I have put forward an alternative theory of germ layer and gut homology between cnidarians and bilaterians that can be put to the test in future studies.


Systematic evolution of ligands by exponential enrichment (SELEX) is an established procedure for developing short single-stranded nucleic acid ligands called aptamers against a target of choice. This approach has also been used for developing aptamers specific to whole cells named Cell-SELEX. Aptamers selected by Cell-SELEX have the potential to act as cell specific therapeutics, cell specific markers or cell specific drug delivery and imaging agents. However, aptamer development is a laborious and time-consuming process which is often challenging due to the requirement of frequent optimization of various steps involved in Cell-SELEX procedures. This review provides an insight into various procedures for selection, aptamer enrichment, regeneration and aptamer-binding analysis, in addition to a very recent update on all aptamers selected by Cell-SELEX procedures. Aptamers are short single-stranded DNA or RNA oligonucleotides that bind to specific targets through three-dimensional structural conformation and with a collection of other forces such as hydrogen bonding, stacking of aromatic rings and Van der Waals interactions ,2. This 15 unique sequences that undergo a series of selection cycles against a specific target of choice [Aptamers selected by conventional SELEX involve the use of a large oligonucleotide library of approximately 10f choice . Severalf choice . Cell-SEA general SELEX process involves repetitive series of selection cycles starting with the incubation of the target cell with a random sequence single strand oligonucleotides library . Each ofConsistent cell culture maintenance is critical for Cell-SELEX success, such as consistent confluency across all selection cycles, passage number and growth conditions . ProlongAfter each selection cycle, aptamer sequences within the oligonucleotide pool are enriched due to the continuous amplification of high affinity aptamers. However, the amplification of selected aptamers requires optimization to enhance yield without amplifying non-specific sequences due to mispriming. The number of amplification cycles can be optimized by analyzing the PCR products at different amplification cycles by gel electrophoresis. Excessive cycles would result in the amplification of non-specific products, whilst fewer cycles may result in insufficient yields . The numAsymmetric PCR is a simple method for ssDNA preparation in which PCR amplification is carried out with different ratios of primers to preferentially amplify the aptamer sequences without its complementary strand ,20,21. IA highly effective method includes the denaturation of the double stranded DNA with urea-containing denaturing polyacrylamide gel electrophoresis (PAGE) . The ureMagnetic beads exhibit magnetic properties and congregate when exposed to a magnetic field, which allows for the rapid separation of molecules from a mixture. The amplified aptamer library (dsDNA) are immobilized on streptavidin coated magnetic beads, generally through biotin-streptavidin interaction, allowing for the separation of the DNA strands by increasing the temperature or alkaline treatment . Once imLambda exonuclease digests phosphorylated DNA strand from 5′ to 3′ end, leaving the non-phosphorylated ssDNA strand intact . Lambda The ssDNA product from lambda exonuclease digestion requires purification from traces of digested oligonucleotides and the lambda exonuclease enzyme prior to Cell-SELEX progression. Purification can be achieved with phenol chloroform isoamyl alcohol removal followed by ethanol precipitation. However, this process may risk considerable loss of ssDNA yield . Alternad) of the aptamer. Determination of Kd also requires a blank treatment  to subtract the background noise from the other treatments. If using fluorescence labels, it is recommended to use amber tubes or microplates designed for fluorescence-based assays to minimize photo bleaching. Finally, it is highly recommended to use positive controls, such as aptamers or antibodies that are known to bind to their targets to confirm the efficacy of the binding assay being used.Binding assays serve as the first step of confirming the binding efficiency of the newly selected aptamers. It is recommended to perform the binding assays at various stages of the aptamer development process to confirm target-binding enrichment. There are already a number of established binding assays for confirming Cell-specific aptamer binding, such as flow cytometry, fluorescence or confocal microscopy, and enzyme-linked assay. While these methods are different in principle, they share similar steps in binding affinity and specificity determination. Firstly, initial binding affinity tests should be carried out in a similar environment used in the aptamer development process, such as temperature, buffer composition and incubation time. Secondly, it is recommended to block the cells with non-specific oligonucleotide sequences  to block non-specific aptamer binding, which is critical in determining binding specificity. Thirdly, binding affinity can be determined by incubating the target cells with increasing concentrations of the aptamer , which can be plotted on a non-linear regression curve to determine the dissociation constant  [Similar to traditional enzyme-linked immunosorbent assays (ELISA) used for specific substance detection, aptamers can be incorporated into enzyme-linked assays to determine their binding affinities, and was first reported in 1996 by Drolet  as a higs (TESA) , and hums (TESA) , or tumos (TESA)  were conCampylobacter jejuni [Although not semi-quantitative, confocal and fluorescence microscopy are among the most popular tools to screen aptamer binding to cells, and can also visually demonstrate the specificity of aptamers towards their targeted cells. While fluorescence and confocal microscopy are similar in principle in the excitation and emission of fluorescence signals, confocal microscopy offers the ability for depth field control, reduction of background and the ability to take serial optical sections. Indeed, aptamer binding affinity and specificity were confirmed with fluorescence and confocal microscopy on human cells, including SK-BR3 expressing human epidermal growth factor receptor-2 (Her-2) , HEK celr jejuni , trypanor jejuni  and salmr jejuni . Both li32P-labelled aptamers specific to IVB pili encoded by Salmonella enterica serovar Typhi pil operon and required filtration of the binding reaction through a nitrocellulose filter, followed by scintillation counting of the nitrocellulose filter [Scintillation counting offers high sensitivity detection of radiolabeled samples, making it a powerful tool for the detection of aptamer binding reactions to their target cells. For accurate measurements of aptamer binding affinities, separation of aptamer-bound targets from non-binding aptamers is essential and can be achieved by filtration or centrifugation. Aptamer binding assays using scintillation counting was applied to e filter . Similare filter , MCF-7 [e filter  and diffe filter  were conTraditional SELEX develops aptamers targeting homogeneous molecules, which offers a higher rate of success in selecting high affinity aptamers. However, in biological systems, the use of recombinant proteins as targets away from their native environment risks altering their structural conformation and stability. Native structural conformation is an essential component for aptamer selection intended for therapeutic purposes, and therefore the use of Cell-SELEX is generally favoured as it retains the aptamer selection targets in their natural environment. Although the aptamer targets in Cell-SELEX uses cells in a heterogeneous solution, such as growth media with serum, it remains a highly achievable process as evident by the many Cell-SELEX experiments performed in last few years that resulted in Cell-specific aptamers with high affinity and specificity .Not all cell-specific aptamers are developed by Cell-SELEX, but can also be developed by traditional SELEX against specific intra- or extra-cellular markers. Aptamers generated against cell markers with traditional SELEX vastly outnumbers aptamers generated by Cell-SELEX. Only a few cell-specific aptamers are presented in this section as more detailed reviews are available ,72. DeteThe development of cell-specific aptamers carries significant diagnostic and therapeutic potential. Their small size allows for efficient tissue penetration, ease of chemical modification permits longer systemic circulation and high binding affinities allows for rapid molecular detection. However, Cell-SELEX often carries more complications than traditional SELEX that uses pure forms of targets. Unlike the use of purified targets, it is more difficult to direct aptamer binding towards specific parts of the target cell, which may result in the development of non-specific aptamers. The definitive strategy to alleviate the issue of enriching non-specific binding aptamers is the use of a different, but similar, cell line as the negative control selection, one that shares the majority of the target cells’ markers but not the actual aptamer target molecule. In tumor Cell-SELEX, neighboring healthy cells that surround the targeted tumor cells can also serve in negative control selection. Optimized methods for aptamer library regeneration and binding assays play a significant part for the success of Cell-SELEX. Indeed, efficient regeneration and purification of RNA or ssDNA pool is a critical step for aptamer selection as oligonucleotide fragments and dsDNA can nonspecifically bind to cell surfaces and lead to enrichment. Moreover, enrichment of non-specific binding oligonucleotides can potentially give false positive results in non-optimized binding assays. By efficiently regenerating the enriched aptamer pool, non-specific binding can be eliminated from the selection process, and high affinity aptamers are preferentially selected. The DNA strand separation methods described in this review are efficient in regenerating the ssDNA pool for DNA aptamer development, and it is highly recommended to confirm the ssDNA product with gel electrophoresis to confirm the purity and yield. Furthermore, it is critical to use a binding assay method that is compatible with the cell target, such as flow cytometry that scans individual cells in a single-file stream, fluorescence and confocal microscopy for visual aptamer binding confirmation, and scintillation counting for high sensitivity assays. In addition, enzyme-linked assays and qPCR assays allow for the detection of internalized aptamers by analyzing cell lysates or excreted products. Cell-SELEX is a powerful tool, and its successful implementation leads to the development of high affinity aptamers with diagnostic and therapeutic potential, as well as the identification of new cellular markers that advances cell biology research.


West Nile virus (WNV) is an arthropod-transmitted flavivirus that causes West Nile fever and may infrequently cause neuroinvasive disease in humans. We present 2 cases of confirmed WNV infection, 1 of severe encephalitis and 1 of mild febrile illness, in a couple returning to the United Kingdom from South Africa. Approximately 80% of human infections are subclinical, although symptomatic cases can vary from influenza-like symptoms to neurotropic manifestations , and urine samples to the Public Health England Rare and Imported Pathogens Laboratory (RIPL) for testing  Figure. Empirical antimicrobial drug treatment was stopped, and the patient’s Glasgow Coma Score improved to 14, with residual mild cognitive impairment, ongoing balance disorder, and improving myalgia. Inflammatory markers and all other blood parameters normalized before discharge 5 weeks later. At follow-up 12 weeks after presentation, the patient’s cognition had improved, but she still required use of a cane for persistent balance disturbance.One day after the index patient’s admission, her 72-year old husband was admitted with an influenza-like illness. He had stage 3 chronic kidney disease but no other concurrent conditions. Urine and serum samples were obtained, and test results confirmed that he, too, had had a recent WNV infection. WNV IgM and IgG were positive in serum 5 days after symptom onset; WNV RT-PCR on urine was negative. He had no neurologic component to his illness and made a rapid clinical recovery.. WNV is sporadically reported from South Africa (WNV was initially isolated from a febrile patient in Uganda in 1937 (. Detection of WNV IgM in CSF is used to confirm neuroinvasive disease. WNV IgG appears shortly after IgM but remains positive in the long term and is therefore not a useful marker of recent infection unless seroconversion is demonstrated.Clinicians should bear in mind the value of urine sampling to detect WNV RNA by RT-PCR in addition to serologic testing. WNV is excreted in high concentrations in urine and may be detected for several weeks postinfection, whereas detectable virus in serum and CSF is transient and generally not present by the time of symptom onset (Plasmodium falciparum malaria. In addition, an outbreak of arboviral infections, particularly Sindbis virus, had been reported in the Johannesburg area coinciding with the travel dates of the patients we report (.Clinicians should be alert to the possibility of arthropod-borne infections in travelers returning from South Africa. These include WNV, African tick-bite fever, chikungunya, and Figure giving timeline of West Nile virus exposure risk, testing, and progress of illness in woman who returned to the United Kingdom from South Africa.


One of the main characteristics of patients with HFpEF is the intoleranceto exercise at different degrees and through diverse mechanisms.4-6Exercises are among the main therapeutic strategies for the treatment of heartfailure with reduced ejection fraction (HFrEF) and HFpEF, being important agents indecreasing the morbidity and mortality of these patients.7 The physical training programs offered to patientswith HF in cardiac rehabilitation services involve primarily aerobic exercisessupplemented by resistant exercises, stretching and, in some cases, respiratoryexercises.1Among the benefits of aerobic training in patients with HFpEF, we can highlight theimprovement in endothelial function and arterial stiffness, contributing to theimprovement of cardiovascular dynamics and symptoms.8 Kiviniemi et al. have recentlyreported that HIIT is superior to traditional continuous aerobic training inimproving cardiac autonomic function and suggested that the effect verified onpost-HIIT autonomic function was related to improved baroreflex modulation and vagalcontrol.9Aerobic exercises can be continuous, of moderate intensity or intercalating high andlow-intensity efforts. High-intensity interval training (HIIT) is currently one ofthe most effective methods for improving cardiorespiratory and metabolic function.HIIT involves repeated activities, from short to long ones, of high-intensityexercises combined with periods of active or passive recovery.10 Studies have also suggested that HIIT induces increasedbaroreflex sensitivity and reduces arterial stiffness.11There are several potential adaptations that explain the positive changes induced byHIIT on the autonomic cardiac function. One of the potential mechanisms related toHIIT-induced improvement in cardiac vagal tone may be angiotensin II, which inhibitscardiac vagal activity. Sedentary or physically inactive individuals have higherplasma renin activity when compared to those who are physically active. Exercisecauses angiotensin II suppression, which can, to some extent, mediate theimprovement in cardiac vagal tone.12 studied post-training changesin blood pressure (BP) and endothelial function in 16 patients with HFpEF. Asmain results, it was possible to demonstrate a significant increase in thebrachial artery diameter with a corresponding reduction in systolic BP. Thesefindings indicate the potential benefit of this type of training for patientswith HFpEF, with an improvement in blood pressure levels and, possibly, abeneficial effect on ventricular function.In this interesting study, designed for the assessment of the acute effects of asingle session of high-intensity interval training, Lima et al.13 The BP reduction after the exercise sessions tends tolast for hours, acting as powerful adjuvants to the vasodilation effects ofantihypertensive drugs, which are commonly used in HFpEF. BP control is amongthe main goals for symptom improvement in HFpEF, and exercises are crucial toattain this goal and improve diastolic function.14Although the authors did not find any significant changes in the flow-mediateddilation index, questions have been raised about the real importance andinterpretation of this measurement.This experiment used a single training group, without a control group for betterdefinition of the effects and lower chance of bias when assessing the results.Although the number of patients was small, the positive results encourage betterdesigned future researches, with a larger number of individuals to define the roleof this training modality in HFpEF. These patients have an expressive limitation toexercises and strategies that improve BP and diastolic function show great potentialfor benefits in functional class improvement and, likely, in morbimortalityreduction.


Recent advances in cancer cell metabolism provide unprecedented opportunities for a new understanding of heart metabolism and may offer new approaches for the treatment of heart failure. Key questions driving the cancer field to understand how tumor cells reprogram metabolism and to benefit tumorigenesis are also applicable to the heart. Recent experimental and conceptual advances in cancer cell metabolism provide the cardiovascular field with the unique opportunity to target metabolism. This review compares cancer cell metabolism and cardiac metabolism with an emphasis on strategies of cellular adaptation, and how to exploit metabolic changes for therapeutic benefit. The intermediary metabolism of substrates defines every living cell, including heart and cancer cells. Metabolism in mammalian cells has four specific functions: (a) to provide chemical energy from nutrients, (b) to convert exogenous nutrients into macromolecules or building blocks of cell components, (c) to assemble building blocks into proteins, nucleic acid, lipids, and other cell components, and (d) to synthesize or degrade biomolecules required in specialized functions of cells . AlthougCardiac metabolism is a dynamic process that adapts to stress by altering its activity to maintain cardiac contraction, thereby ensuring cell survival in the near term. Some metabolic responses shift cardiac metabolism toward an energetically unfavorable state, and turn an initial adaptation into maladaptation, which leads to further disease progression –9. PreciMetabolic adaptation in the heart supports contractile function by maintaining ATP provision. While cancer cells optimize metabolic flux to maximize cell proliferation and growth at pathological levels, the heart tries to optimize the use of energy-providing substrates to ensure cardiac contraction and cell survival. Both cancer cells and the heart show a high metabolic turnover to rapidly adapt themselves to changes in the chemical composition of their exogenous nutrients. The question arises: how do cancer cells successfully improve their cellular fitness by coopting metabolic machinery, while the heart fails to do so under long-term stress? Is it time to seriously rethink cardiac metabolism? To address this question, we will discuss modulation of energy substrate metabolism, biosynthesis of macromolecules, and redox balance in both cancer and the heart below. Regardless of whether specific metabolic activities provide benefits or liabilities to cancer cells or the heart, the rationale is that these activities may be exploited as therapeutic targets. For example, pharmacologic inhibition of fatty acid oxidation by agonists of the peroxisome proliferator-activated receptor (PPAR) ligand-activated nuclear hormone receptors decreases myocardial fatty acid oxidation; which, in turn, promotes increased glucose uptake and oxidation, and improves contractile function , 14. Theex vivo and in silico studies  leads to activation of glycolysis even in the presence of oxygen . Most of studies , 23, 27  studies , 30, 31. studies . This ch studies , 33. The2, and to provide ATP through β-oxidation. De novo fatty acid synthesis includes several key regulatory enzymes: ATP citrate lyase (ACL) which generates acetyl-CoA from citrate; acetyl-CoA carboxylase which catalyzes the irreversible carboxylation of acetyl-CoA from malonyl-CoA, and fatty acid synthase (FASN) which catalyzes the sequential addition of carbon-units to assemble long-chain fatty acids. In most tissues, including the heart, FASN expression and de novo fatty acid synthesis is relatively low, indicating that most cells preferentially take up exogenous or dietary lipids from the blood to provide energy and macromolecule biosynthesis. However, proliferating cells, like several human cancers, have been shown to up-regulate FASN expression (de novo synthesis. Increased fatty acid oxidation may be driven by increased activation of AMP-activated protein kinase (AMPK) by reduced [ATP]:[AMP] ratio in RAS-mutant cells. Several studies in cancer cell lines and other mammalian tissues showed that ATP and AMP availability regulate the activation of AMPK and the mechanistic target of rapamycin (mTOR), which, in turn, regulate fatty acid metabolism on the molecular level . The noe et al. , 121. Foe et al. –124. In e et al. , 87, 125e et al. . Recent e et al. . DepletiDespite striking similarities in the metabolism of the failing heart and cancer cells, there are fundamental differences that need to be considered for the development of pharmacologic treatments. Cancer treatments targeting metabolism have the goal to limit or prevent tumor growth and induce cell death. Interventions targeting cardiac metabolism during heart failure aim to reverse structural remodeling and improve cardiac function. Are there therapies that may target metabolism to reverse heart failure? Are there strategies that both protect the heart and target the cancer? Common pharmacologic strategies for both heart failure and cancer are summarized in Table Glucose metabolism is an attractive target for the treatment of cancer and heart failure, because many solid tumors, as well as the failing heart, upregulate glucose utilization , 168. GlAnother potential therapeutic target is Hexokinase II  and CPT1 have been either approved  or tested in clinical trials for both heart failure and cancer (Table de novo lipid synthesis by inhibiting FASN or ATP citrate lyase (ACL). FASN is the rate limiting enzyme for de novo lipogenesis, while ACL catalyzes the conversion of glucose-derived citrate to acetyl-CoA and regulates cytosolic acetyl-CoA levels. Similarly, pharmacologic inhibition of GRK2 partially inactivates PPARγ and inhibits FASN through mitogen-activated protein kinases (MAPK)  . Ongoings (MAPK) –177. By s (MAPK) , 178. InA further potential treatment strategy is targeting nucleic acid synthesis and amino acid metabolism. Among the various pharmacologic agents targeting nucleic acid synthesis that are available for cancer therapy, almost all have been reported to have cardiovascular side effects. Glutaminase inhibitors offer a potential way to inhibit mitochondrial amino acid metabolism Figure , to induWe presented several common metabolic strategies that both cancer cells and cardiomyocytes employ to optimize nutrient flux and cell growth. Metabolic reprogramming is a hallmark of both heart failure and malignant cells, which provides them with the ability to survive and sustain stress. Recent progress in molecular techniques  and metabolic flux analysis using stable isotope labeling improved our understanding of mechanisms, biological consequences, and vulnerabilities associated with metabolic reprogramming in heart disease and cancer. Somatic mutations in metabolic reprogramming predominately stems from redirections of metabolic intermediates and increased ATP demand in the context of decreased cardiac contractility. Intermediary metabolites serve as signals that activate signaling pathways, modulate posttranslational modifications of proteins and alter gene expression. Examination of these relationships has inspired pharmacologic strategies that aim to either correct or enhance metabolic vulnerabilities in cancers and the failing heart. In cancer, potential pharmacologic targets manifest in pathways that regulate energy homeostasis and macromolecule biosynthesis. In the heart, similar strategies are often accompanied by severe side effects and increased cell death. Developing rational therapeutic strategies for both cancer and cardiovascular diseases will be aided by integrating findings on a systems level from pre-clinical and clinical studies. Little is known about the metabolic interaction between tumors and the heart. However, recent studies show that oncometabolic dysregulation can promote cardiac dysfunction . DespiteAK drafted and wrote the manuscript. WS edited the manuscript, critically evaluated and searched the literature. HT provided guidance and edited 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. The reviewer XP and handling Editor declared their shared affiliation.


Inspired by work done in genetics and neuroscience, new modeling frameworks of cardiac electrophysiology have been recently developed that take advantage of modern computational capabilities and approaches, and account for the variance in the biological data they are intended to illuminate. In this review, we outline the recent advances in statistical and computational techniques that take into account physiological variability, and move beyond the traditional cardiac model-building scheme that involves averaging over samples from many individuals in the construction of a highly tuned composite model. We discuss how these advanced methods have harnessed the power of big (simulated) data to study the mechanisms of cardiac arrhythmias, with a special emphasis on atrial fibrillation, and improve the assessment of proarrhythmic risk and drug response. The challenges of using in silico approaches with variability are also addressed and future directions are proposed.In cardiac electrophysiology, there exist many sources of inter- and intra-personal variability. These include variability in conditions and environment, and genotypic and molecular diversity, including differences in expression and behavior of ion channels and transporters, which lead to phenotypic diversity . These variabilities play an important role in progression of heart disease and arrhythmia syndromes and outcomes of therapeutic interventions. Yet, the traditional  Beginning with the seminal paper by Hodgkin and Huxley, in silico approaches have led to important insights into arrhythmia risks, mechanisms of arrhythmogenesis, and variable response to drugs, they should be considered when determining the regulatory requirements for the proarrhythmia assessment and drug efficacy and safety evaluation of candidate compounds.In this review, we discuss the importance of accounting for variability when building models of cardiac electrophysiology in both physiological and diseased conditions, and describe new tools being developed to address the limitations of traditional modeling approaches. In particular, we focus on two computational approaches that have emerged as leading methodologies for studying variability in cardiac electrophysiology, which we will refer to as (1) population-based modeling and (2) sample-specific modeling. Both methodologies have provided valuable insights in the fields of neuroscience, cardiology, and pharmacology. Here we review how they have advanced our understanding of the basic mechanisms of cardiac arrhythmias, and particularly atrial fibrillation (AF). As these Sources of variability in cardiac electrophysiology encompass multiple spatial and temporal scales, and include inter-species  or chronic AF  channel are generally responsible for drug-induced long QT syndrome (diLQTS), but in a population this adverse response is highly variable, from minimum changes in the electrocardiogram (ECG) to induction of lethal ventricular arrhythmias  population-based and 2) sample-specific modeling  , or revealing association of certain parameter ranges or properties with specific physiological behaviors . Many relevant examples have recently been reviewed , are sensitive to changes in model parameters, . Because many parameters are often varied to generate the populations of models, multivariable linear regression  or the Na+/K+ pump current was more likely to produce FRD  and sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) in CaT amplitude. They found that in rat epicardial cells Ito plays a more important role than SERCA in regulating CaT amplitude, and this was analogous to human atrial cells, where both Ito and ultra-rapid delayed-rectifier K+ current (IKur) had greater impacts on CaT amplitude than did SERCA  current has little influence on APD in control cells but more markedly impacts AF cells; the analysis also revealed that IK1 upregulation plays a dominant role in APD shortening in AF, and that the L-type Ca2+ current (ICaL) significantly contributes to rate-dependent APD changes in both control and AF myocytes  and employing sensitivity analysis using multivariable regression has led to many important insights in cardiac electrophysiology , and demonstrated the accuracy of logistic regression using receiver operator characteristic curves . Applying logistic regression in studies of physiology is well-described by Lee et al. who examined CaThe main limitation of regression (both linear and logistic) analysis is that it only highlights how inputs are correlated to outputs, so the conclusions drawn from the analysis can be misleading if only a few outputs are considered. For example, it has been shown that completely different parameter combinations could produce essentially identical AP shapes but substantially different CaT amplitudes Figure  Sarkar . However+/K+ pump is a key determinant of susceptibility to repolarization abnormalities in human ventricular cardiomyocytes by applying arrhythmia-provoking conditions to a population of experimentally-calibrated cardiac cells  can help identify underlying determinants of different phenotypes, behaviors, and pathological conditions  and hERG block and found that the selective INaL blocker GS-458967 could suppress proarrhythmic markers after hERG block in ventricular myocytes , dofetilide, sotalol, quinidine, and verapamil, to quantitatively compare changes in AP biomarkers, and demonstrated good agreement with experiments for the selective IKr blockers  but not for the non-selective IKr inhibitors (quinidine and verapamil). Paci et al. utilized populations of in silico iPSC-CMs to evaluate antiarrhythmic effects of mexiletine and ranolazine to treat iPSC-CM long QT syndrome type 3 (LQT3) mutants and demonstrated that mexilitine stops spontaneous APs in more LQT3 models than ranolazine due to its stronger effects on the fast Na+ current (INa) . Mayourian et al. employed a comprehensive integrated approach to study the mechanisms of cardiac contractility and arrhythmogenicity using experimentally-calibrated human mesenchymal stem cells (hMSCs) , especially in eliminating false-positive outcomes generated by the baseline model parameters.Sample-specific models can be constructed by fitting the parameters of a baseline model so that the model outputs match the corresponding physiological behaviors seen in a single patient or myocyte Figure . Cardiac2+ handling  given their disproportionately large contribution at voltage steps of −120, +20, +40, and −30 mV, respectively  analyzing variability in cardiac electrophysiology, (2) assessing proarrhythmic risk, (3) determining the underlying factors contributing to variable drug response, and (4) identifying phenotype-specific (and in the future patient-specific) antiarrhythmic targets. Table  analyzinWe reviewed the most common methods used to account for variability in cardiac electrophysiology, which largely fall into the two categories of (1) population-based modeling and (2) sample-specific modeling. These methods complement each other well, as population-based methods can help characterize behavior in a particular patient group , and sample-specific modeling shows significant promise to develop personalized medical approaches for individual patients. Both methods have led to many important insights into the mechanisms of arrhythmogenesis and antiarrhythmic strategies. However, there are several important limitations to consider, which suggest potential future developments in modeling of cardiac electrophysiology.in silico population-based studies, differences in the nature of experimental and simulation studies may need to be considered. For example, some population-based techniques generate model population sizes (often sample sizes in the 1000s) that are much greater than could be achieved by experiments (often sample sizes of 3–12) or the traditional cardiac modeling approach alone. Therefore, given similar effects, results produced in the population-based simulations have greater statistical power to detect differences. Furthermore, because even very small effects can reach statistical significance with large samples, physiological significance should be assessed  with which a quantity, such as an electrophysiological output, can be given a value  variability  to maintain normal Ca2+ handling  is assessed sometimes  initiative, which aspires to develop better methods to predict TdP. Beyond exclusively using steady-state hERG block as the main predictor of arrhythmia and not at all using QT interval prolongation, the CiPA initiative attempts to gain a more comprehensive understanding of proarrhythmic risk by combining (1) mechanistically-based in vitro assays, (2) in silico reconstructions of cardiac electrophysiology, and (3) confirmation using human iPSC-CMs (Colatsky et al., The Computational approaches that have been developed over the past decade to account for variability in cardiac electrophysiology have led to important insights into mechanisms of arrhythmogenesis, etiology of disease, and variable response to drugs. The approaches outlined in this review are used in basic research studies, i.e., quite separately from actual clinical workflows, where decisions are made sometimes for a particular patient within minutes. Advanced computing facilities now allow near real-time simulations of anatomically realistic, biophysically detailed models of human cardiac electrophysiology (Niederer et al., HN, SM, and EG reviewed the literature 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.


Over the last decade it has become evident that under normal conditions connexin hemichannels are either not expressed  or are expressed in very low numbers with low open probability in various mammalian tissues ). However, both the number and activity of these non-selective channels are drastically increased in different cell types under pathological conditions. Since the inflammatory response is a consequence and/or the cause of most diseases, connexin hemichannels have been implicated as a common factor in numerous chronic pathological indications . However, further work is needed to fully understand hemichannel regulatory mechanisms and their involvement in the outcome of the different disease conditions.Currently, the molecular target of different clinically available anti-inflammatory agents block one (or at most two) intracellular inflammatory signaling pathways, leaving several others active, and which persist despite chronic treatment, and alter the phenotype of inflamed cells. The phenotypic changes explain why cells stop performing their normal functions and subsequently lead to organ dysfunction. Consequently, the chronic use of many available anti-inflammatory compounds can result in kidney, liver, or heart dysfunction, amongst other complications. Connexin hemichannel regulation offers an alternative approach. Connexin hemichannels have been shown to be permeable to calcium ions and ATP which activate intracellular inflammatory signaling pathways. Therefore, selective inhibitors of connexin hemichannels are expected to be useful for the long-term treatment of inflammatory disease. These agents may provide an entire new generation of anti-inflammatory pharmaceuticals. Indeed, it is important to emphasize the evidence for connexin hemichannels as new molecular targets in order to prevent the activation of most intracellular inflammatory pathways.International Journal of Molecular Sciences focuses on the involvement of connexin hemichannels in the inflammatory response, emphasising chronic pathological conditions. It brings together substantial evidence for connexin hemichannel-mediated tissue dysfunction associated with a diverse range of chronic disease indications, thus supporting the translation of hemichannel regulation into clinical application.This issue of the For instance, Valdebenito and co-workers  have pro2+ and reactive oxygen species (ROS) on cell damage, Pecoraro et al. [2+ homeostasis, but also to enhance the amount of connexin43 expression in addition to affecting its localization. Keeping in mind that intracellular Ca2+ and ROS activate connexin43 hemichannels, which are permeable to Ca2+ and ROS, it is tempting to propose that connexin43 hemichannel blockers might reduce the cardiotoxicity of doxorubicin. Notably, the same group [The role of connexin hemichannels in inflammatory responses within peripheral tissues such as retina, heart, liver, and kidney are also described. Mugisho and collaborators  have anao et al.  have focme group  demonstrClearly, more studies are required to fully understand how a reduction in gap junctional communication but, conversely, a gain in plasma membrane hemichannel activity, as well as possible hemichannel activity in mitochondria and channel-independent functions of connexins, may impact chronic disease. This special edition marks a significant step in the right direction.


Colloid Polym. Sci.173, 117–122 (doi:10.1007/bf01502416)) treats suspensions of non-motile particles as a continuum with an evolving preferred direction; this model describes fibrous materials as diverse as extracellular matrix, textile tufts and plant cell walls. Director-dependent effects are incorporated through a modified stress tensor with four viscosity-like parameters. By making fundamental connections with recent models for active suspensions, we propose a modification to Ericksen's model, mainly the inclusion of self-motility; this can be considered the simplest description of an oriented suspension including transversely isotropic effects. Motivated by the fact that transversely isotropic fluids exhibit modified flow stability, we conduct a linear stability analysis of two distinct cases, aligned and isotropic suspensions of elongated active particles. Novel aspects include the anisotropic rheology and translational diffusion. In general, anisotropic effects increase the instability of small perturbations, while translational diffusion stabilizes a range of wave-directions and, in some cases, a finite range of wavenumbers, thus emphasizing that both anisotropy and translational diffusion can have important effects in these systems.Suspensions of self-motile, elongated particles are a topic of significant current interest, exemplifying a form of ‘active matter’. Examples include self-propelling bacteria, algae and sperm, and artificial swimmers. Ericksen's model of a transversely isotropic fluid (Ericksen 1960  Once the wavenumber is large enough perturbations are dampened. The inclusion of the extra stress due to interactions between elongated particles and the surrounding fluid simply decreases the growth rate for corresponding wavenumbers as the volume fraction of particles is increased. This leads to a smaller range of wavenumbers when the perturbation is unstable when compared with results for an isotropic stress tensor.Considering an initially randomly oriented (isotropic) suspension at rest, the base state corresponds to a constant particle distribution function and zero fluid velocity, giving isotropic integrals for the first, second and fourth moments of the distribution function, which may be evaluated analytically. By following a similar method to Saintillan & Shelley ,32, we fActive suspensions and transversely isotropic fluids are both biologically relevant and physically fascinating states of matter. Linking these two fields of research enables knowledge transfer, enabling extension of the transversely isotropic model and identifying important components of the active suspension model. Transversely isotropic effects were found to produce some changes to linear stability analysis of nearly aligned and isotropic active suspensions in the absence of background flow. We argue that these aspects should be included in future modelling studies.


Circular RNAs are a new class of endogenous non-coding RNA that can function as crucial regulators of diverse cellular processes. The diverse types of circular RNAs with varying cytogenetics in cancer have also been reported.Circular RNAs can act as a microRNA sponge or through other mechanisms to regulate gene expression as either tumor inhibitors or accelerators, suggesting that circular RNAs can serve as newly developed biomarkers with clinic implications. Here, we summerized recent advances on circular RNAs in cancer and described a circular RNA network associated with tumorigenesis. The clinical implications of circular RNAs in cancer were also discussed in this paper.Growing evidence has revealed the crucial regulatory roles of circular RNAs in cancer and the elucidation of functional mechanisms involving circular RNAs would be helpful to construct a circRNA-miRNA-mRNA regulatory network. Moreover, circular RNAs can be easily detected due to their relative stability, widespread expression, and abundance in exosomes, blood and saliva; thus, circular RNAs have potential as new and ideal clinical biomarkers in cancer. More than 75% of non-coding RNAs have been found in transcription of the human genome . CirculaSome circRNAs discovered in human tissues have been related to diverse cellular processes, including senescence, growth and apoptosis, etc. , 13. MorThe regulation mechanisms of circRNAs have been revealed by increasing studies. The most notable of these mechanisms is that circRNAs can work as microRNA (miRNA) sponges. CircRNAs can block the binding of miRNAs with the 3’ UTR of a specific gene by directly binding to miRNAs, thus indirectly regulating the gene expression , 18. For6-methyladenosine (m6A) in human cells [Translation of ncRNAs is poorly noted due to the classic ORFs longer than 100 codon are lacking. With more research on small open reading frames (sORFs), the proteins or peptides with biological functions that are translated by ncRNAs have received more attention . CircRNAan cells . Recent an cells . There han cells  Extracellular vesicles released by cells can be divided into three categories according to origin and size, including microvesicles, apoptotic bodies, and exosomes . Many biRecently, a number of studies have found that more exosomes are released from cancer cells than from normal cells. It was reported that circRNAs in gastric cancer (GC) can be transferred from GC cells to normal cells via exosomes, indicating that exo-circRNAs are important in the peritoneal metastasis of GC . MoreoveAs microarray chip and next-generation sequencing technologies have been developed, many circRNAs were examined or identified in cancer samples. The expression profiles of circRNAs during the early stages of pancreatic ductal adenocarcinoma (PDAC) have been demonstrated, which revealed that deregulated circRNAs may participate in the progression of PDAC and potentially serve as a novel therapeutic biomarker , 38. In In gliomas, RNA-Seq data showed the existence of over 476 deregulated circRNAs . A recenInterestingly, by combining microarray circRNA expression profiles with bioinformatics target prediction and sequence analysis, many deregulated circRNAs with miRNA response elements (MREs) have been identified in basal cell carcinoma (BBC) and cutaneous squamous cell carcinoma (CSCC) , 46. MorAlthough the overall mechanisms of circRNAs in cancers have not been entirely elucidated, crucial regulatory roles of circRNAs in cancer have been revealed. Recent studies on circRNAs mainly focused on the roles as miRNA sponges, interactions with binding proteins and translation into proteins or peptides , 48. An CircRNAs regulate apoptosis in cancer. It has been shown that circ-Foxo3 can promote MDM2-induced degradation of p53 by binding to MDM2 and p53; however, circ-Foxo3 contributes more to repression of MDM2-induced Foxo3 ubiquitination by binding to Foxo3 and thus increasing the expression of the downstream gene PUMA to induce apoptosis in breast carcinoma , 51. In CircRNAs regulate the cell cycle in cancer. It has been demonstrated that miR-7 can inhibit cancer progression by suppressing CCNE1 and PIK3CD in hepatocellular carcinoma (HCC) , 54. A rCircRNAs regulate cancer proliferation. The overall expression of circ-CDR1 can also increase EGFR expression and lead to cell proliferation by sponging miR-7 in HCC . AdditioCircRNAs regulate invasion and metastasis in cancer. Upregulated androgen receptor (AR) expression can accelerate the development of clear cell renal cell carcinoma (CCRCC) by inhibiting miR-145 . RecentlAlthough the rough roles of several circRNAs in some cancers have been confirmed, the functions and regulation pathways of most circRNAs in cancer remain to be revealed.Differential expression profiling analysis and functional studies of circRNAs in tumors are important for the further understanding of circRNAs and cancer. Meanwhile, similar to microRNAs and lncRNAs, circRNAs also show potential as new independent diagnostic and prognostic biomarkers, which provides new approaches to improve clinical diagnosis and treatment. Here, we summarize currently known cancer-associated circRNAs related to clinical implications in Table Colorectal cancer has become the fourth most deadly cancer in the world, and its occurrence is related to changes in individual genetics . CircRNAHCC is responsible for nearly 90% of primary malignancies of the liver, and patients with advanced stage disease always have poor prognoses , 72. CirAlthough many efforts have been made to improve the diagnosis and therapy of GC, five-year OS rates in gastric cancer patients are still less than 30% . New bioIn breast cancer, downregulated circ-Foxo3 can enhance cell survival and decrease cell apoptosis . In bladThe emergence of chemoradiation resistance can lead to poor prognosis or recurrence –91. At pMany studies indicated that circRNAs, similar to miRNAs and lncRNAs, may have significant regulatory effects on pathophysiologic processes, including tumorigenesis. The connections of circRNAs with cancer has become a hot research field. CircRNAs can be easily detected due to their relative stability, widespread expression, and abundant presence in exosomes, blood and saliva, indicating that circRNAs might be novel and ideal diagnostic and prognostic biomarkers in cancer. In this paper, we drew conclusions about recent advances on circRNAs in cancer and presented a circRNA-mediated network involved in cell cycle control, apoptosis, proliferation, invasion and metastasis in cancer.At present, some circRNA expression profiles in several cancers have been identified; however, there are still many questions that need to be addressed. Further investigation is needed regarding the various gene regulatory mechanisms of circRNAs other than miRNA sponges. The important relationship between the exo-circRNAs and tumor metastasis and the development of novel and valid ways to predict target genes of circRNAs using bioinformatics, among other issues, need to be addressed. This will provide new insights into circRNAs to construct circRNA-miRNA-mRNA regulation networks, reveal cancer pathogenesis mechanisms and seek novel potential diagnosis biomarker or therapeutic targets for future cancer management.


Background: The psycho-physiological changes in brain-body interaction observed in most of meditative and relaxing practices rely on voluntary slowing down of breath frequency. However, the identification of mechanisms linking breath control to its psychophysiological effects is still under debate. This systematic review is aimed at unveiling psychophysiological mechanisms underlying slow breathing techniques (<10 breaths/minute) and their effects on healthy subjects.Methods: A systematic search of MEDLINE and SCOPUS databases, using keywords related to both breathing techniques and to their psychophysiological outcomes, focusing on cardio-respiratory and central nervous system, has been conducted. From a pool of 2,461 abstracts only 15 articles met eligibility criteria and were included in the review. The present systematic review follows the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines.Results: The main effects of slow breathing techniques cover autonomic and central nervous systems activities as well as the psychological status. Slow breathing techniques promote autonomic changes increasing Heart Rate Variability and Respiratory Sinus Arrhythmia paralleled by Central Nervous System (CNS) activity modifications. EEG studies show an increase in alpha and a decrease in theta power. Anatomically, the only available fMRI study highlights increased activity in cortical  and subcortical  structures. Psychological/behavioral outputs related to the abovementioned changes are increased comfort, relaxation, pleasantness, vigor and alertness, and reduced symptoms of arousal, anxiety, depression, anger, and confusion.Conclusions: Slow breathing techniques act enhancing autonomic, cerebral and psychological flexibility in a scenario of mutual interactions: we found evidence of links between parasympathetic activity (increased HRV and LF power), CNS activities  related to emotional control and psychological well-being in healthy subjects. Our hypothesis considers two different mechanisms for explaining psychophysiological changes induced by voluntary control of slow breathing: one is related to a voluntary regulation of internal bodily states (enteroception), the other is associated to the role of mechanoceptors within the nasal vault in translating slow breathing in a modulation of olfactory bulb activity, which in turn tunes the activity of the entire cortical mantle. Breathing is intimately linked with mental functions. In the millenary eastern tradition, the act of breathing is an essential aspect of most meditative practices, and it is considered a crucial factor for reaching the meditative state of consciousness, or “Samadhi” . The breath is called “Prana,” which means both “breath” and “energy” . “Prana-Yama”  is a set of breathing techniques that aims at directly and consciously regulating one or more parameters of respiration . Pranayama is primarily related to yoga practice, but it is also part of several meditative practices . Nevertheless, western science has paid little attention to the investigation of the effects of pure breathing control on neural correlates of consciousness, and on specific mental functions.Returning on meditative practices, the main issue in unveiling the basic mechanisms underlying their effects is to disentangle those related to breathing control from those associated with non-respiratory cognitive components such as focused attention and mental imagery.To our best knowledge, only ten dedicated reviews tackle the effects of Pranayama, without succeeding in the identification of a common psychophysiological model . These breathing techniques are often referred to as paced breathing  by systematically reviewing the scientific literature. Only studies involving healthy humans, avoiding thus possible confounding effects due to pathological conditions, and dealing with the voluntary modulation of breathing (Pranayama and paced breathing) were included. It is in fact crucial to distinguish between slow breathing techniques, and other techniques that simply direct attention to the act of breathing  or slow down breath as a consequence of other attentional practices . Studies based on self-reports instruments alone were not included, as their reliability is severely weakened by the absence of objective measures, a major and common problem when dealing with contemplative sciences  and functional Magnetic Resonance Imaging (fMRI), and autonomic activity, studied by Heart Rate Variability (HRV), Respiratory Sinus Arrhythmia (RSA), and Cardio-Respiratory Synchronization.To develop an effective search strategy, we adopted the Population, Intervention, Comparison, Outcomes and Study Design (PICOS) worksheet (see Methods and Table https://www.crd.york.ac.uk/prospero/).This systematic review followed the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines - Any technique of breath control that directly slows the breath down to 10 breaths per minute was used- A comparison technique  or control groups  was included- A physiological outcome was measured, related to cardio-respiratory system or central nervous system , together with a psychological/behavioral outcome (assessed with a psychometric quantitative approach).We considered eligible for the inclusion all studies assessing physiological parameters during slow breathing techniques (state effect), immediately after (state effect), and after long-term interventions (trait effect).Studies identified from the literature search were excluded if:- Young (<18 years) and/or old (>65 years) subjects were recruited- The population comprised any chronic or acute pathology- Breathing was paced at a frequency higher than 10 b/min- Techniques do not comprise an active and direct modulation of breathing, investigating instead “passive” breathing techniques - The intervention was not limited to breathing exercises, but included also other techniques as meditation, visualization, or required specific yoga postures - The protocol used active emotional induction - Measured a physiological parameter of no interest, or measured only a physiological or a psychological/behavioral parameter alone- They were case reports- The applied methodologies and/or techniques were not well-described or replicable- Not published in a peer-reviewed journal- Not available in full-text and/or in English language.The research of the studies, according to databases, terms and quantity of returned studies, is presented in Table Two independent reviewers (AZ. and AP) checked an early pool of 2,461 abstracts collected from the search engines' outputs. Titles and abstracts were screened, and 2,303 studies were removed either because they were duplicated or of no interest for the systematic review. The remaining 158 full-text papers were checked for the eligibility criteria. At the end of the analysis, 15 articles meeting the eligibility criteria were retained and included in the review. Seven studies , all these studies were excluded from the review, as they did not allow the unambiguous identification of the specific psychophysiological effects of breath modulation. A large number of studies were excluded as they focused on techniques not aimed at a conscious regulation of breathing, requiring, on the contrary, the meditator not to attempt any control on his/her own breathing rhythm, but rather to observe it in a non-judgmental way. Finally, several other studies lacked a rigorous description of the experimental set-up and of the applied methodologies, impeding thus the study replicability, and were consequently excluded from the review , using two different tools. Disagreements between the reviewers were resolved by discussion with a third reviewer (AG). As regards within-subject designs, the Single-Case Reporting Guideline In Behavioural Interventions (SCRIBE) Statement  seem to interact with the cardio-respiratory system by increasing HRV and RSA, suggesting thus a strong involvement of the parasympathetic nervous system  and psychological/behavioral outcomes of decreased anxiety Specify the mean breathing frequency and, if present, any significant breathing frequency variationsSpecify whether during respiration the air passes through the mouth or through the nostrils , or through both mouth and nostrilsSpecify the presence and the duration of inspiration (if any) and expiration pauses (if any)Specify the Inspiration/Expiration ratioSpecify whether the breath is thoracic or abdominalSpecify (if applicable) what type of metronome is usedSpecify (if applicable) the air pressure during the inspiratory phases.We found evidence of increased psychophysiological flexibility linking parasympathetic activity, CNS activities related to emotional control and psychological well-being in healthy subjects during slow breathing techniques. In particular, we found reliable associations between increase of HRV power and of LF power, increase of EEG alpha and decrease of EEG theta power, induced by slow breathing techniques at 6 b/min, and positive psychological/behavioral effects. This evidence is unfortunately weakened by the lack of clear methodological descriptions that often characterizes slow breathing techniques literature. Further studies are thus needed to unambiguously assess these links. Only few authors have attempted to systematically describe the psychophysiological effects of slow breathing techniques, and a fewer number have attempted to relate them to meditation practice. Breath seems to be confined to an “ancillary” role when compared to other important mechanisms such as cognitive or affective ones.Finally, more research is needed to disentangle the pure contribution of breathing in a variety of meditation techniques. As stated by Nash and Newberg , differeAG, EG, and AZ conceived the idea. AZ and AP conducted the literature search and analysis. AZ, AP, EG, and AG wrote the paper. ML, DM, and BN contributed to the writing. All authors reviewed 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.