There is an increasing frequency of reports regarding the persistence of the Ebola virus (EBOV) in Ebola virus disease (EVD) survivors. During the 2014–2016 West African EVD epidemic, sporadic transmission events resulted in the initiation of new chains of human-to-human transmission. Multiple reports strongly suggest that these re-emergences were linked to persistent EBOV infections and included sexual transmission from EVD survivors. Asymptomatic infection and long-term viral persistence in EVD survivors could result in incidental introductions of the Ebola virus in new geographic regions and raise important national and local public health concerns. Alarmingly, although the persistence of filoviruses and their potential for sexual transmission have been documented since the emergence of such viruses in 1967, there is limited knowledge regarding the events that result in filovirus transmission to, and persistence within, the male reproductive tract. Asymptomatic infection and long-term viral persistence in male EVD survivors could lead to incidental transfer of EBOV to new geographic regions, thereby generating widespread outbreaks that constitute a significant threat to national and global public health. Here, we review filovirus testicular persistence and discuss the current state of knowledge regarding the rates of persistence in male survivors, and mechanisms underlying reproductive tract localization and sexual transmission. Filoviridae  includes three genera of single-stranded, negative-sense RNA viruses that are enveloped, filamentous, and non-segmented [Ebolavirus, one species of Marburgvirus, and one species of Cuevavirus [Ebolavirus include: Bundibugyo ebolavirus (BDBV), Zaire ebolavirus (EBOV), Reston ebolavirus (RESTV), Sudan ebolavirus (SUDV), Taï Forest ebolavirus (TAFV), and the recently discovered Bombali ebolavirus (BOMV) [Marburgvirus comprises a single species, Marburg marburgvirus, that encompasses the Marburg virus (MARV) and the Ravn virus (RAVV). The genus Cuevavirus contains one species, Lloviu cuevavirus (LLOV), which has yet to be isolated [The family egmented ,2. Thereevavirus ,4. Specis (BOMV) . The genisolated .Ebolavirus species to have emerged outside of Africa and has been associated with multiple epizootics in captive Philippine macaques [Marburg virus disease (MVD) was first recognized in 1967 in West Germany and Yugoslavia following a number of infections transmitted from African green monkeys imported from Uganda, and Ebola virus disease (EVD) was recognized in 1976 when EBOV and SUDV emerged in the Democratic Republic of the Congo (formerly Zaire) and South Sudan (formerly southern Sudan), respectively ,11,12. Mmacaques ,18.The number and magnitude of EVD and MVD outbreaks have increased over time, likely due in part to greater interaction between humans and intermediate or reservoir host populations . HistoriThe recent review by Baseler et al. summarizes the current state of knowledge regarding EVD pathophysiology in humans following observations from the West African EVD epidemic, and additional reviews are available elsewhere ,31,32,33In the past, the spontaneous emergence and inaccessible geographic locations of filovirus outbreaks hindered direct studies of viral pathogenesis in humans ,48. CultImmune responses consist of innate and adaptive cells that detect pathogens, recruit additional immune cells to the site of infection, and initiate antiviral activity . MVD andTo minimize damage to host cells, pro-inflammatory immune responses are followed by a release of anti-inflammatory factors. Tissues at greater risk of damage have modified local immune responses that heavily suppress pro-inflammatory responses ,79. ThesPathogen persistence is the sum result of positive and negative immune regulation resulting in an immune-tolerant environment that can mean a lack of visible symptoms . Thus, iThere is a paucity of information regarding the persistence of viral pathogens in immune-privileged sites. Infection of immune-privileged sites is likely a targeted strategy of persistence, in which acute infections shift to a persistent nature in response to an increasing host immune response . Viral mAlthough filoviruses emerged as high-consequence human pathogens over 40 years ago, there is growing concern regarding viral testicular persistence as a novel transmission mechanism. First reported in 1968 following the MVD outbreak, filovirus testicular persistence has grown from an anomaly to a major public health concern based on observations from the West African EVD epidemic ,101. RecResearch on EBOV persistence has been ongoing to varying degrees since the 1995 EVD outbreak in Kikwit, Democratic Republic of Congo ,103. AltTo date, there have been sporadic studies examining filovirus testicular persistence in vivo. These investigations date back to the initial MVD outbreak, when guinea pigs were used to detect the virus in patient samples and for subsequent viral propagation and isolation . ExaminaThe use of animal models to examine EBOV persistence has increased following the West African EVD epidemic. Bird et al. recently demonstrated that wild-type EBOV infection in humanized mice results in symptomatic disease with fatality rates similar to those seen in humans . In addiFilovirus reproductive tract persistence has been examined in non-human primates. A recent investigation found that asymptomatic testicular persistence occurred in 9.82% of non-fatal EBOV infections in rhesus macaques . The virThere are many post-exposure treatments under investigation for EVD. Recently, ZMapp, an antibody cocktail, and Favipiravir, a small molecule therapeutic, were deployed for patient treatment during the West African epidemic ,120,121.Remdesivir (GS-5734), an adenosine nucleoside analogue that has demonstrated efficacy in EBOV-infected non-human primates, has the potential to enter immune-privileged sites ,123. RemNumerous case reports have been published on filovirus infections in human patients since the emergence of filoviruses . These iPathological investigations of deceased patients were performed on five of the seven individuals that succumbed to infection during the 1967 MVD outbreak in Marburg, Germany . Eighty In early case reports, EBOV and MARV were identified in sperm cells or seminal fluid similar to that observed during the recent West African EVD epidemic  25,26,2,104,128.Collectively, decades of clinical data have demonstrated that filovirus family members likely have different pathological presentations associated with testicular persistence ,104. DesSeveral studies conducted during the West African EVD epidemic centering on testicular persistence and sexual transmissions with advanced methodologies have provided important insights into this biological phenomenon . One of the first reported cases of testicular persistence during the epidemic is thought to have resulted in persistence for 199 days post-EVD onset . During A US healthcare worker infected in Sierra Leone was transported to the National Institutes of Health Special Clinical Studies Unit in Bethesda, MD, following EVD onset and recovered with the aid of advanced supportive care . High viThere have been nine seminal/testicular persistence longitudinal cohort studies since 1995 that have followed a total of 158 persistent male survivors with seven studies conducted during the West African EVD epidemic ,131,133.One of the first longitudinal studies was conducted on a cohort of five EVD patients following their return to the U.S. for treatment during the West African EVD epidemic . PersistBy mid-late 2015, the seminal persistence of EBOV garnered increased attention in the scientific community as well as in mainstream media outlets due to the potential for sexual transmission . From JaAdditional longitudinal studies conducted towards the end of the West African EVD epidemic have demonstrated that persistence can extend to over a year in male EVD survivors. Soka et al. examined 38 men in Liberia from 2015–2016 with persistent EBOV genomic material in their semen . PersistA longitudinal cohort in Guinea from late-2015 to mid-2016 identified seminal persistence of EBOV genomic material in 15 males . PersistAlthough results have shown the potential for EBOV testicular persistence to extend over long periods of time (>16 months) in a small subset of males, it has yet to be determined if this includes infectious virus in addition to viral genome material ,132,133.Rates of EBOV testicular persistence within EVD survivors are available based on multiple longitudinal cohort studies. The comparison of these data will provide important information about both the rates of persistence during the recent epidemic as well as those that can be expected in future outbreaks.An analysis of persistence sought to determine the total cases of testicular persistence that resulted from the West African EVD epidemic and included data up to November 2015 from the World Health Organization (WHO) and from a cohort studied by Deen et al. ,135. TheIn the cohort examined by Deen et al., 100% of males had persistent testicular infections within 3 months of treatment center discharge, 62% of males remained positive from 4–6 months following discharge, 25% were positive 7–9 months after discharge, 15% were positive 10–12 months after discharge, 11% were positive 13–15 months after discharge, and 4% were positive for 16–18 months following discharge . A rate An investigation by Soka et al. in Liberia found that men over 40 encompassed 50% of the persistent testicular infections identified within the study but comprised only 23% of the cohort, suggesting that older men may be predisposed to increased susceptibility to persistent infections . Nine peThe studies performed to date suggest that up to 10% of male EVD survivors will have persistent testicular infections that extend beyond the nine-month abstinence window recommended by the WHO ,62. DataSince the first MVD outbreak in 1967, there have been nine filovirus sexual transmission events . AlthougThe first reported case of potential filovirus sexual transmission occurred during the MVD outbreak in 1967, where the wife of a male MVD patient fell ill two months following her husband’s recovery from the disease . Her husThe first confirmed case of filovirus sexual transmission occurred during the West African EVD epidemic in Liberia ,130. ThiAlthough each of the described sexual transmission events is unique in its own right, there are similar trends that are important to note. As described in three reports of sexual transmission, the infected female only displayed mild symptoms or was described as asymptomatic after sexual transmission ,139,140.Although EBOV transmission was largely controlled towards the end of the West African epidemic, sporadic transmission events continued to occur. Asymptomatic EBOV testicular persistence, and subsequent sexual transmission, contributed to these events. Longitudinal studies of EVD survivors have demonstrated great variability in the duration of viral persistence. Alarmingly, EBOV genomic material has also been detected in semen from EVD survivors in the absence of physiological symptoms of disease following multiple prior negative samples. The development of EBOV vaccine candidates and novel therapeutics has resulted in mass vaccination campaigns during recent outbreaks and could hold promise for combating EVD in regions where the virus is endemic. However, although 36 clinical trials have been completed, only a single study has demonstrated clinical efficacy ,146. Thu(i)What cells are targeted by filoviruses in the testis?(ii)What are the molecular mechanisms that contribute to filovirus testicular transmission?(iii)How do viral fitness and host response modulation contribute to these asymptomatic EBOV testicular infections?(iv)What are the kinetics of infectious virus and viral RNA shedding from sperm?(v)What is the rate of EBOV sexual transmission? Despite a growing number of reports describing the testicular persistence of EBOV, and additional filoviruses, in disease survivors, there is little information regarding the molecular mechanisms underlying these clinical sequelae. Currently, there is limited knowledge regarding the events that result in filovirus transmission to, and cell tropism within, the testis. Several critical questions remain, including: These questions are of critical importance given the increasing frequency of filovirus outbreaks as they will help inform public health agencies to allow them to develop effective strategies to prevent human-to-human transmission.


The lack of an appropriate intracellular delivery system for therapeutic nucleic acids (TNAs) is a major problem in molecular biology, biotechnology, and medicine. A relatively new class of highly symmetrical hyperbranched polymers, called dendrimers, shows promise for transporting small TNAs into both cells and target tissues. Dendrimers have intrinsic advantages for this purpose: their physico-chemical and biological properties can be controlled during synthesis, and they are able to transport large numbers of TNA molecules that can specifically suppress the expression of single or multiple targeted genes. Numerous chemical modifications of dendrimers extend the biocompatibility of synthetic materials and allow targeted vectors to be designed for particular therapeutic purposes. This review summarizes the latest experimental data and trends in the medical application of various types of dendrimers and dendrimer-based nanoconstructions as delivery systems for short small interfering RNAs (siRNAs) and microRNAs at the cell and organism levels. It provides an overview of the structural features of dendrimers, indicating their advantages over other types of TNA transporters. The targeted regulation of expression of therapeutically relevant genes using various types of nucleic acids (NAs) has been demonstrated in numerous in vitro and in vivo biological model systems ,2,3. GenRecent achievements in genetic research have provided many new tools for manipulating gene expression, each possessing its own individual advantage. For example, the natural mechanism of gene expression regulation by RNA interference (RNAi) , discoveRNA interference as a gene therapeutic tool provides the advantage of a much broader choice of target proteins than traditional treatment approaches . GeneralLarge numbers of therapeutics associated with siRNA and microRNA action are currently in the process of preclinical and clinical trials ,14,15,16Nowadays, all vectors for gene delivery can be divided into two large principal classes: viral and non-viral (synthetic). Recombinant retroviruses, adeno-associated viruses, and lentiviruses, etc. serve as viral vectors . These wTherefore, the development of new materials capable of binding, transporting, and releasing TNAs is high on the scientific agenda. The scope of the current review covers the design of synthetic delivery systems for small therapeutic RNAs based on dendrimer-related polymeric materials: dendrimers and/or dendrons , describing their efficiency in in vitro and in vivo models The description and comparison of dendrimer-based delivery vehicles with other non-viral vectors can be found in other reviews ,22,23. Dendrimers seem to be promising platforms for developing non-viral RNA delivery systems. They are highly symmetrical hyperbranched polymers with tree-like structures; their branches of monomeric units diverge in all directions from a central core . All denDendrimers are attractive carriers for small RNAs owing to a combination of properties that distinguish them from other polymer classes. Their main advantage is monodispersity  ,28, in cl-lysine (PLL) and phosphorus dendrimers—are mainly used for delivery of siRNAs and microRNAs  ,46 and al-lysine fragments linked by amide bonds. The lysine residues thus constitute the branches and act as branch points. PLL dendrimers are excellent examples of combining the properties of linear biocompatible polymers with the advantages of the dendritic effect. The main advantage of polylysine dendrimers is their easy biodegradation under the action of proteases and, in consequence, low cytotoxicity [PLL dendrimers are hyperbranched monodisperse analogues of linear oligopeptides widely used for transfection and for drug and gene delivery . They artoxicity ,55,56,57Phosphorus-containing dendrimers generally consist of a thiophosphate or cyclotriphosphazene core and extended branches containing stable aromatic thiophosphohydrazones ,59. SuchDendrimers bearing cationic groups inside and outside their structures can bind negatively charged nucleic acids effectively, mainly by electrostatic interactions, thus forming complexes called dendriplexes by analogy with polyplexes ,65,66. TDuring the past two decades tremendous progress has been made in the dendrimer-based delivery of genetic material and therapeutic molecules. Nevertheless, the toxicity of cationic polymeric macromolecules remains an acute problem. Cationic dendrimers, as a rule, have protonated amino groups on their periphery. Unbound charged groups interact with negatively charged cell membranes, mainly electrostatically. As a result, this interaction leads to membrane thinning, formation of pores and damage to membrane integrity, which in turn leads to necrotic cell death . Such noThe scope of this review is to summarize experimental data on the use of dendrimers for delivering short therapeutic RNAs in vitro and in vivo. Papers that are mostly concerned with the possibility of using dendrimers to deliver siRNAs that inhibit the expression of non-therapeutic targets, such as a green fluorescent protein or luciferase, are not reviewed here . DescripSmall interfering RNAs  are exogenous double-stranded RNAs of 21–23 base pairs in length. They can mimic the functions of natural regulators of gene expression (endogenous microRNAs) without undergoing shortening by the Dicer enzyme and can be directly embedded in the RNA-induced silencing complex (RISC) . Most ofDespite of some disadvantages, unmodified dendrimers are effective as carriers of RNA interference regulators. Cationic TEA-PAMAM G5 can deliver siRNA precursors Dicer-substrate siRNAs,(dsiRNA) for multitarget suppression of the human immunodeficiency virus (HIV). Zhou et al. achieved a more than a twofold decrease in the expression of p24  in human lymphocytes in vitro. In vivo studies on HIV-infected humanized mice models yielded even more impressive results: injections of TEA-PAMAM G5 complexes with a cocktail of 4 dsiRNA  into some mice led to undetectable viral particle levels accompanied by a decrease in the total CD4+ lymphocyte count . OtherwiThe overwhelming majority of studies on siRNA delivery involve modified PAMAM dendrimers. Owing to the commercial availability of these dendrimers, scientists have plenty of options to increase their biocompatibility, reduce their toxicity, and improve targeted delivery by numerous chemical modifications . A detaiPeng et al. conducted a series of studies to identify the most efficient PAMAM dendrimer modification as a delivery vehicle for siRNA siHsp27 ,83,84,85Small RNAs are able to bind to not only dendrimer surfaces but also to inside branch sites if internal amines became protonated, as shown by internally quaternized PAMAM dendrimers with acetylated surface aminogroups . Transfer of binding points from the surface to internal cavities allows the terminal cationic charge to be decreased by substitution with hydrophobic groups, and this is followed by a significant reduction of general side-effect toxicity . FollowiThe above-mentioned compositions combine the advantages of dendrimers  and CPP (targeted and efficient cell penetration). One of the key factors in dendrimer-based siRNA carrier design is the number of oligopeptides per dendrimer molecule. Since cell-penetrating peptides usually possess a positive charge, their combination in high concentrations with cationic PAMAM dendrimers results in an overcharge, which in turn can lead to increased toxic side-effects and reduced delivery efficiency both in vitro and in vivo .One widespread method for neutralizing dendrimer-associated toxicity is partial substitution of cationic groups with various di- or oligo-saccharides . In geneAnother way to improve dendrimer-based delivery is to make the dendrimer amphipathic by adding ‘lipid tail’-like alkyl chains. Spherical dendrimers carrying long linear hydrocarbon chains on the surface can form analogy of micelles, like lipids, called dendrisomes ,86. The N-methyl-d-aspartate. A similar construction based on the β-cyclodextrin core and PAMAM G3 dendrons showed accelerated wound healing in rats with diabetes: injected dendriplexes carrying metalloproteinase-9 siRNA suppressed the relevant protein expression in vivo [Replacement of the ethylenediamine dendrimer core with highly branched rigid structures  allows more biocompatible dendrimers to be created. The rigid polyphenylenevinylene core in combination with the flexible PAMAM branches results in a highly effective siRNA carrier for neurological treatment. It induces a high level (>90%) of siRNA penetration into neurons with subsequent protection of the cells from apoptosis  and auto in vivo . A brancAn interesting approach is to combine the advantages of dendrimers with the amphiphilic properties of lipids . The mosl-lysine [There have been significantly fewer studies recruiting other types of dendrimers for siRNAs delivery. They are represented by several groups of cationic dendrimers: poly-l-lysine ,98 and cl-lysine ,101,102,l-lysine ,49,100.Carbosilane dendrimers were designed and synthesized by F. Javier de la Mata and Rafael Gómez and their colleagues. NN16 dendrimers possess easily hydrolysable Si-O bonds in their internal architecture and diimine groups on the periphery and seem to have an optimal balance between efficiency and cytotoxicity among naked dendrimer carriers for delivering short-chain RNAs. The NN16 dendrimer of the second generation makes complexes with oligonucleotides up to 200 nm in size because it is significantly smaller than third- and fourth-generation PAMAM dendrimers ,106,107.MicroRNA (miRNA) and siRNA are both short non-coding nucleic acids that regulate the synthesis of proteins in numerous cell types via RNA interference mechanism .These moSimilar to siRNA, the synthetic mimics of microRNAs (miRs) or their precursors can be introduced into the cell with subsequent target gene silencing triggered by RNA interference.Otherwise “malignant” endogenous microRNAs in the cells can be arrested by synthetic oligonucleotides called microRNA antagonists (antimiRs). These oligonucleotides form strong duplexes with microRNAs and block their activity .MicroRNAs can control metabolic, pathological, and homeostatic processes in cells by both gene downregulation and upregulation ,113. ForHomo sapiens origin, “mir” refers to a precursor and “miR” to a mature microRNA, the number (8204) is a unique identifier (microRNAs with lower id numbers were discovered earlier), and the letter after the number indicates there are two or more mature microRNA with very similar sequences. There are more rules for microRNA nomenclature relating to other parameters, which are used in many databases [During the past 10 years there have been numerous studies on microRNA action. These studies have developed from recent discoveries of (1) dysregulation of microRNA expression profiles in pathological cells (creation of microRNAs and their target libraries); and (2) the capacity of microRNAs to affect cell phenotypes after altering the expression of several target genes simultaneously [atabases ,116. As the libraries grow, unique microRNA expression profiles could provide a basis for personalized therapy and a tool for effective monitoring . Some neAlthough much fundamental knowledge about microRNAs has been acquired, their therapeutic use has been severely limited by lack of an appropriate delivery system; microRNA mimics and siRNAs encounter several biological and physiological barriers once they are inside the organism. Most preclinical studies on dendrimer-based miR delivery  have beeThe mimic of miR-7 microRNA was studied as a human glioma growth inhibitor in vitro and in vivo; miR-7 regulates the expression of several target proteins simultaneously, including the tumor growth and apoptosis regulators EGFR, PI3K, and AKT-2, in human glioma U251 cells. PAMAM dendrimers with conjugated folic acid residues delivered miR-7 into the U251 cells efficiently in vitro with subsequent significant silencing of EGFR, PI3K, and AKT-2. The ability of the FA-PAMAM conjugate to overcome the blood–brain barrier, to transfect cells effectively, and to be selectively accumulated in cancer cells resulted in significant inhibition of tumor growth in vivo and prolongation of survival time in mice xenografted with gliomas .Zhou et al. synthesized 1500 different modular dendrimers consisting of a polyamide core, carbosilane branches with hydrolysable bonds, and various thiol-containing compounds on the surface . Such a The only study that did not concern cancer treatment was published by Gray et al. . They haThe second microRNA-based therapeutic strategy is intracellular introduction of antimiRs. This approach does not require an obligatory delivery system. MicroRNA antagonists are single-stranded oligonucleotides complementary to mature upregulated endogenous miR. Since their mechanism of action is not associated with recognition by intracellular proteins, various chemical modifications of oligonucleotides are widely used: 2’-O-methylation, locked nucleic acids (LNA), unlocked nucleic acids (UNAs), etc. Such modifications protect antimiRs from recognition by endogenous nucleases and binding to immune system proteins ,122, butConcerning the second strategy, only a few recent studies have been published dealing with delivery of the antimiR-21 microRNA antagonist in combination with anti-cancer chemotherapeutics. This is discussed below.The distinctive hyperbranched structure of dendrimers enables them to incorporate low molecular mass chemotherapeutics into their internal cavities and simultaneously bind functional nucleic acids to their surfaces . Thus, iCause–effect relationships between resistance to cancer chemotherapy and alterations in microRNA expression profiles have recently been established. In the “dendriplex + drugs” approach, low molecular mass drugs are delivered and released inside the cell simultaneously with siRNA/microRNA; this allows drug resistance to be blocked and at the same time enhances the effect of the corresponding chemotherapeutics. Moreover, the incorporated chemotherapeutics could have significantly increased solubility, enhanced intracellular penetration, and improved targeting, leading in consequence to a notable reduction of dosage, which in turn significantly reduces the severity of side effects.As for prostate cancer chemotherapy, resistance can be weakened by knocking down the MAPK protein (mitogen-activated protein kinase) in the MAPK/ERK signaling pathway, which has a significant role in prostate carcinogenesis. Thus, inhibition of p42-MAPK expression potentiates the antitumor effect of the anti-diabetic drug metformin . AlthougDrug resistance can be overcome by targeting the major vault protein (MVP) in breast cancer cells. MVP hampers the entry and persistence of doxorubicin (DOX) molecules in tumor cell nuclei where DOX acts, and thus causes DOX resistance. The combined delivery of MVP-siRNA and DOX by PAMAM G5 functionalized by hyaluronic acid resulted in a significant increase in cytotoxicity in vitro. The authors reported that delivery within dendriplexes enhanced tumor targeting, increased blood circulation time, and lowered in vivo toxicity in comparison to DOX alone. A synergetic action is also achieved through increased accumulation of DOX in cancer cells both in vitro and in vivo when it is delivered by the dendrimer via an enhanced permeability and retention (EPR) effect along with the receptor-specific interaction between the hyaluronic acid residues with CD44 receptors that are hyperexpressed on the tumor cells .In contrast to siRNA and microRNA, antimiRs are not involved in the cell machinery so intracellular enzymes do not recognize them. Thus, in vivo application of antimiRs does not require a delivery system, since their chemical modification enables them to circulate long in the body with no loss of functional activity. That is why few articles on dendrimer-based delivery of antimiRs can be found. One of the newest therapeutic concepts is the application of antimiRs to block the microRNAs responsible for chemotherapy resistance . A combOnly 20 years passed after the discovery of RNA interference as a gene silencing tool  before tAlong with viral, liposomal, and polycationic particles, dendrimers are now being actively investigated as carriers for small RNAs. A unique tree-like compact structure distinguishes dendrimers from other polymers. The combination of numerous active sites for binding with the presence of internal cavities, and flexibility in the synthesis and modification of dendrimers, led to the development of two distinctive therapeutic concepts: (1) chemical modifications of cationic dendrimers that allow their toxicity to be significantly reduced while maintaining delivery efficiency; and (2) the combined delivery of chemotherapeutic drugs inside the cavities and small RNAs at the periphery, which has shown particular effectiveness against chemoresistant cancer cells of various etiologies.The current review has highlighted preclinical studies on microRNA- and siRNA-containing constructs developed against cancers, HIV, neurodegenerative diseases, diabetes, and hepatitis. Thus, delivery systems based on dendrimers could potentially become a universal therapeutic approach. The key factor in the functional difference of such constructs is the small RNA sequence, which determines its specific intracellular target. The possibility of selecting any target protein for siRNA, along with microRNA targets, offers great potential for the development of personalized medicine using dendrimers.


Biomolecule labeling using chemical probes with specific biological activities has played important roles for the elucidation of complicated biological processes. Selective bioconjugation strategies are highly-demanded in the construction of various small-molecule probes to explore complex biological systems. Bioorthogonal reactions that undergo fast and selective ligation under bio-compatible conditions have found diverse applications in the development of new bioconjugation strategies. The development of new bioorthogonal reactions in the past decade has been summarized with comments on their potentials as bioconjugation method in the construction of various biological probes for investigating their target biomolecules. For the applications of bioorthogonal reactions in the site-selective biomolecule conjugation, examples have been presented on the bioconjugation of protein, glycan, nucleic acids and lipids. Biological processes in living systems are extremely complicated but also highly required to be unveiled for biological and biomedical research. To study the molecular details of biological processes, active biological probes toward these processes are needed, for which various bioconjugation strategies have been developed to construct these probes . MonocloBioorthogonal reactions are chemical reactions that can proceed in biological systems without interaction with the inside biomolecules or interference on the whole system ,3. The iIn this review paper, we will summarize the often used bioorthogonal reactions and their development, followed by introduction on their applications in bioconjugation of proteins, glycans, nucleic acids and lipids.Bioorthogonal chemistry has found impacted applications in the field of chemical biology. In the past decade, a limited number of reactions including Staudinger ligation, click reaction, tetrazine ligation and photo-click reaction have been well developed, which have also been widely utilized for labeling biomolecules in the context of living cells and whole organisms. In this section, we will summarize the development of these bioorthogonal reactions.in vivo bioconjugation.The Staudinger ligation was the first example of bioorthogonal ligation reaction. Its prototype reaction in which azides are transformed into primary amines upon reducing by phosphines was reported by Staudinger in 1919 and was first called the Staudinger reduction . BertozzUp to now, the most commonly used and famous bioorthogonal reaction is the “click” reaction, which is the cycloaddition reaction between azides and alkynes. The prototype reaction was first reported by Michael in 1890s  and thenTo avoid using the toxic copper catalyst, Bertozzi used strained cyclooctynes instead of linear alkynes . The alk−1·s−1. With the employment of tetrazine ligation, successful pretargeted live cell imaging was achieved ,31−1·s−1 glycans .Inspired by the photoactivated cycloaddition reaction between 2,5-diphenyltetrazole and methyl crotonate reported by Huisgen in 1967 , Lin expProteins are the most abundant biomolecules in living systems and participate in almost all the biological processes. So far, the introduction of bioorthogonal reporters into proteins, in combination with the bioorthogonal reactions, provides the most powerful tools for the exploration of protein expression, localization and function in their native environment. A variety of strategies have been developed to selectively incorporate the bioorthogonal chemical groups into the proteins of interest, including chemical or enzymatic modifications, residue-specific incorporation using the cell’s translational machinery and more precise strategy—site-specific incorporation by exogenously introduced aaRS-tRNA pair. The site-specific incorporation is more favorable as its less interference with the protein’s structure and function.in vitro through introduction of tetrazole group into proteins by chemical modification and fluorescent labeling of microtubules in living cells was achieved by labeling taxoid with tetrazole group and application of photo-click reaction thymidine or BrdU, which requires autoradiography, or DNA denaturation and antibody staining [Nucleic acids including DNA and RNA are essential units for cellular biological processes and studying nucleic acids in their native environment is thus of great importance in the field of chemical biology. Nucleotides and their analogs could be incorporated into the genomes of replicating cells by endogenous enzymes, which has made direct labeling of nucleotides the most commonly used method for nucleic acid detection . Metabolstaining ,59. Howein vivo based on the incorporation of 5-ethynyl-2-deoxyuridine (EdU) into cellular DNA during DNA replication, the ethynyl groups of EdU could be imaged by performing CuAAC with fluorescent azides  constitutes the sixth base in the mammalian genome and has potential applications as epigenetic marker. He developed an efficient and selective bioorthogonal labeling method to differentiate 5-hmC from 5-methylcytosine (5-mC) and determined the distribution of 5-hmC in mammalian genomes . β-GlucoLipids play critical biological roles in regulating many vital biological pathways and pathophysiological events, such as enforcing protein membrane binding and attaching lipids onto protein . In thesSaccharomyces cerevisiae [Alkyne labeled choline lipids were metabolically incorporated into phospholipids and the resulted lipids were selectively and sensitively labeled in cells through CuAAC . The biorevisiae . After previsiae . With thE. coli to identify bacterial lipoproteins that interacted with fatty acids, allowing sensitive imaging and large-scale discovery of unknown lipoproteins in gram-negative bacteria [Protein fatty-acylation is fundamental and critical in biological processes, which is also limited to study due to the lack of methods for protein lipidation. By combining Staudinger ligation, fatty-acylated proteins in mammalian cells were detected and characterized with azide modified fatty acids . And by bacteria .in vivo bioconjugations. Improvements in current bioorthogonal reactions are still needed to broaden their applications. In addition, chemical probes with multi-functionality are usually needed for detailed studies of complicated biological processes, in which orthogonal bioconjugation strategies are needed [Up to now, only a limited number of bioorthogonal reactions have been developed. Except the mostly used bioorthogonal reactions described above, other reactions such as Pd-mediated coupling reaction and cycloaddition reaction between quinone methide and vinyl thioether have also been recently developed for bioconjugation ,77,78,79e needed . Then, ne needed . With th


Microbial communities play essential and preponderant roles in all ecosystems. Understanding the rules that govern microbial community assembly will have a major impact on our ability to manage microbial ecosystems, positively impacting, for instance, human health and agriculture. Here, I present a phylogenetically constrained community assembly principle grounded on the well-supported facts that deterministic processes have a significant impact on microbial community assembly, that microbial communities show significant phylogenetic signal, and that microbial traits and ecological coherence are, to some extent, phylogenetically conserved. From these facts, I derive a few predictions which form the basis of the framework. Chief among them is the existence, within most microbial ecosystems, of phylogenetic core groups (PCGs), defined as discrete portions of the phylogeny of varying depth present in all instances of the given ecosystem, and related to specific niches whose occupancy requires a specific phylogenetically conserved set of traits. The predictions are supported by the recent literature, as well as by dedicated analyses. Integrating the effect of ecosystem patchiness, microbial social interactions, and scale sampling pitfalls takes us to a comprehensive community assembly model that recapitulates the characteristics most commonly observed in microbial communities. PCGs’ identification is relatively straightforward using high-throughput 16S amplicon sequencing, and subsequent bioinformatic analysis of their phylogeny, estimated core pan-genome, and intra-group co-occurrence should provide valuable information on their ecophysiology and niche characteristics. Such a priori information for a significant portion of the community could be used to prime complementing analyses, boosting their usefulness. Thus, the use of the proposed framework could represent a leap forward in our understanding of microbial community assembly and function. Microbes represent most of the Earth’s biodiversity and a large fraction of its biomass and play essential and preponderant roles in ecosystem processes . While iOur knowledge of microbial communities’ composition, distribution, and to a certain extent function has greatly increased over the last decade, chiefly thanks to the advent of high-throughput sequencing technologies. Significantly, large microbial community composition inventories have been generated from a myriad of microbial ecosystems . HoweverIn this regard, the past few years have seen the progressive recognition of the role that Vellend’s synthesis of community ecology  can playThe present work provides a conceptual framework for the phylogenetically constrained assembly of microbial communities. First, I discuss the observed microbial community and genomic characteristics on which the framework is grounded. Then, I enumerate a series of hypotheses and predictions that form the backbone of the framework and provide supporting evidence from the literature, as well as dedicated supporting results. I continue by considering the effect of ecosystem patchiness, social interactions, and sampling pitfalls on the model, and finally ponder on its limitations, implementation, and utility.Despite the pervasive horizontal gene transfer phenomenon among bacteria, tendency to gene loss, or convergent evolution, many traits are conserved across the microbial phylogeny . EvolutVibrionaceae of many ABC transporters and two-component systems likely related to niche space [Euryarchaeota and acetoclastic methanogenesis in Methanosarcinales [Cyanobacteria [The link between phylogenetic relatedness and ecological function or gene content similarity was further substantiated through the literature review of 990 microbial traits  or the che space , methanorcinales , oxygenibacteria , or the bacteria .Community assembly is often probed in terms of two opposing theories : neutralThe niche-versus-neutral dichotomy framework has been surpassed thanks to a series of reports indicating that both neutral and deterministic processes play a simultaneous role in shaping community composition , 41. MorWithin most microbial communities, bacteria tend to co-occur with phylogenetically related populations more often than expected by chance , 45, 46,Here, I synthesize the abovementioned facts that trait-based deterministic processes have a significant impact on microbial community assembly, that microbial communities most commonly show significant phylogenetic signal (clustering or overdispersion), and that microbial traits and ecological coherence are, to some extent, phylogenetically conserved, to produce a new community assembly principle.Selection acts on particular combinations of traits, where such combinations may or may not show phylogenetic conservation. The only plausible explanation for the pervasive existence of phylogenetic signal in most microbial communities is the existence, within such communities, of prevalent combinations of abiotic and/or biotic factors selecting for particular combinations of traits showing phylogenetic conservation. Hence, I conceptualize selection as divisible into two niche categories: (i) niches whose occupancy requires a specific phylogenetically conserved set of traits (from now on “phylo-niches”) and (ii) niches whose occupancy requires specific sets of traits not showing strong phylogenetic conservation (from now on “non-phylo-niches”). It becomes necessary at this point to clarify that the notion “niche occupancy” is used in the present framework to signify the ability to persist in the ecosystem by surpassing a particular combination of abiotic and/or biotic filters.From here, I derive the following predictions that form the basis of the framework. First, any microbial ecosystem can present phylo-niches and non-phylo-niches. Second, for each phylo-niche, there must be a discrete portion of the phylogeny  whose members share a phylogenetically conserved set of traits allowing the occupancy of their respective phylo-niche. Third, for each non-phylo-niche, there must be a group of microbial populations sharing a set of traits not showing phylogenetic conservation allowing the occupancy of their respective non-phylo-niche.Thus, each instance of the same microbial ecosystem type should present populations from each PCG (occupying phylo-niches) and non-phylogenetic-core populations (occupying non-phylo-niches). Populations occupying each of both the phylo and non-phylo type niches should present a high degree of intra-group ecological coherence, due to their shared traits, and hence, their intra-group structure should be governed by neutral processes and likely show intra-group competition.The assembly principle presented is well in line with results from recent works studying community assembly through different approaches. Lu et al.  evaluateBurns and co-workers  showed tRussel et al.  analyzedLactobacillus species appear to compete for the same niche in the vaginal microbiome of reproductive-age women [Turning to a simpler host-associated microbial community, four preeminent ge women . These lge women  host-derhypothesized that taxonomic convergence might reflect selection by functions that are conserved at the family level. Such selection was obviously enforced not only by abiotic factors (the synthetic media and culturing conditions employed) but also by biotic interactions, since they also showed that individual ESVs from the different taxa analyzed were able to independently grow on the substrate. The parallelism between their findings and hypothesis, and the predictions described above are evident; the “attractors” indicate the existence of phylo-niches, which in turn prompt the appearance of populations from each PCG (the dominant families) in all instances of each microbial ecosystem type. The PCGs in their more exhaustive glucose experiments related to the Enterobacteriaceae and Pseudomonadaceae families. The authors postulated that the emergence of such families could be related to competitive advantages associated with the uptake capabilities of their phosphotransferase system and adenosine triphosphate-binding cassette transporters, respectively. This conclusion links to the above prediction that PCGs share a phylogenetically conserved set of traits allowing them to surpass a particular combination of filters.Recently, Goldford et al.  studied The assessment of compositional “cores” in microbial communities in terms of bacterial groups of varying phylogenetic depths has previously almost exclusively been attempted in terms of taxonomic assignments . HoweveCandidatus accumulibacter excluded each other within each patch (granule), but coexisted in the metacommunity (reactor) [Before we can propose a community assembly model that can aspire to recapitulate the observed patterns of diversity in complex microbial ecosystems, we need to address a few additional factors; patchiness in microbial ecosystems, microbial social interactions, and scale-related sampling pitfalls. As recently reviewed by Cordero and Datta , microbireactor) . Hence, Another important aspect is the possible existence of strongly interacting species (SIS)  within tEnterobacteriaceae and Pseudomonadaceae as PCGs when diverse natural communities were cultivated ex situ on glucose [Pseudomonadaceae-affiliated ESVs. This behavior represents the simplest scenario of intra-PCG ecology; intra-group competition and dominance of a single PCG population. However, within the Enterobacteriaceae, the replicates presented either a Klebsiella-affiliated ESV or a guild consisting of variable compositions of ESVs affiliated to the Enterobacter, Raoultella, and/or Citrobacter genera. In this case, we find again an agreement with the prediction of intra-PCG competition. However, in the case of the Enterobacteriaceae, the intra-group structure was partitioned among two alternative structural states: either an ESV affiliated to Klebsiella or a variable guild of ESVs affiliated to three different genera. While it is not possible without further analysis to anticipate which of the abovementioned mechanisms is responsible for the phenomenon, it serves to demonstrate how the stochastic moiety of community assembly can result in biotic interactions with an impact on local community structure. Moreover, while the assembly principle postulated states that only particular populations can occupy each specific niche, in reality, either a single population or a functionally cohesive guild of populations can occupy each particular niche, provided that the guild as a whole presents the set of traits  required by the niche.Returning to the abovementioned Goldford et al.’s study featuring the emergence of  glucose , the difFinally, we need to take into account that collected environmental samples comprise multiple different abiotic microenvironments and local communities , with thHaving conceptually split selection as acting on particular trait combinations showing either phylogenetic conservation (phylo-niches) or not (non-phylo-niches), and accounting for a second tier of effects related to patchiness, biotic interactions, and higher-scale sampling, I propose the following step-wise assembly model which aspires to recapitulate the observed patterns in microbial community composition. We start by considering a single patch  within the microbial ecosystem presenting particular abiotic conditions Fig. . The patMicrobial communities show remarkable functional stability despite large species turnover, a phenomenon here explained by intra-niche stochasticity among populations featuring functional coherence.Microbial communities are highly diverse, show large species richness, and feature the coexistence of theoretically competing populations. These patterns arise from the pervasive existence of different patch assemblages and patch types in environmental samples as discussed by Cordero and Datta  , drift, and dispersal may affect the ecosystem’s patch structure, as explained above. Speciation is not explicitly accounted for in the model, which pertains mainly to community assembly, yet new genetic types are still subject to the proposed assembly rules. Finally, higher-scale sampling and abiotic microheterogeneity dictate that most analyzed microbial samples should be understood as representing a regional community formed by multiple local communities and abiotic patch types.The immediate practical value of the proposed framework rests on the fact that high-throughput inexpensive phylogenetic profiling of microbial community samples can be accomplished using 16S marker gene amplicon sequencing. From that point, PCGs detection and thus phylo-niche description in terms of sets of traits being selected can be accomplished using available bioinformatic resources. In this regard, microbial ecosystems of interest could be analyzed using the following general pipeline Fig. : First, Acidobacteria are ubiquitous in soil environments. Recently, Eichorst et al. [Acidobacteria-affiliated group of populations prevalent in the ecosystem, which in turn would have provided a more relevant core pan-genome for analysis and description of their respective phylo-niche.Similar overall approaches have been carried out previously. For instance, members of the t et al.  comparedAdditionally, the results from both the PCG detection/phylo-niche description approach and the assembly rules proposed can be used to prime network analysis, metabolic modeling, and individual-based models. While I cannot advance a general pipeline as above, it seems reasonable to argue that a greatly increased knowledge of the ecology (intra-group structure) and ecophysiology (niche description) for a substantial fraction of the community  should improve our ability to understand the role of non-PCG populations in the ecosystem, for instance, by reducing the complexity of the system collapsing intra-PCG populations as ecologically equivalent entities. On the other hand, the assembly rules proposed could be used in community assembly simulation and individual-based models. Finally, it should be more commonly acknowledge that co-occurrence networks will, in most cases, bear mixed signals; co-occurrence may arise from positive biotic interactions or from the shared ability to thrive in a particular abiotic patch type.Ulva australis, as it was reported that U. australis samples presented very high phylogenetic variability [The usefulness of the proposed approach will be affected by the ability of the available sequencing depth to adequately sample the different patch types in the ecosystem. It will also vary from ecosystem to ecosystem, mainly governed by the proportion of phylo to non-phylo-niches. For instance, the framework may not be too effective in the study of the microbial community associated with the green macroalga iability . The oppiability . FurtherThe framework is grounded on the theoretical existence of similar phylo-niches in all(most) instances of the ecosystem under study. However, while such instances may present a similar abiotic environment, the activity of niche modifying populations may impact niche structure, as mentioned earlier. When the impact is sufficiently important, it translates into different microbial regimes for the same initial environment. Also, different microbial regimes for the same ecosystem may be due to hidden abiotic differences. For example, different microbial regimes have been reported in the rumen for the same abiotic environment , in the Notably, the 16S rRNA gene presents low resolution at the highest and lowest phylogenetic depths , and thuThe common patterns of microbial community organization observed in nature suggest the existence of fundamental community assembly rules. In this regard, the lack of an adequate microbial community assembly model is hindering our ability to understand and manage microbial ecosystems, hampering our capacity to improve a plethora of environmental, agricultural, and health-related practices. Here, I have presented a community assembly conceptual framework built on well-known microbial community and genomic characteristics, supported by the recent literature and dedicated results, and able to recapitulate the characteristics most commonly observed in microbial communities. By pinpointing the theoretical existence of phylo-niches, the framework has the potential to increase our understanding of microbial ecosystems; PCGs’ identification is inexpensive and relatively straightforward using 16S sequencing, and subsequent bioinformatic analysis of their phylogeny and estimated core pan-genomes should provide valuable information on their ecophysiology and niche characteristics. Providing such a priori information for a significant portion of the community should greatly enhance the capacity of complementing analyses to further explain microbial community assembly and function. Thus, the use of the proposed framework should represent a leap forward in our ability to understand, manage, or remediate microbial ecosystems.The prediction that microbial communities should present PCGs has so far been substantiated by the analysis of various datasets and ecosystems. Future work should study to which degree the different populations within each PCG present shared ecological functionality, and how such particular functionality relates to the overall ecosystem’s function, and the ability of the group to occupy a particular niche within the ecosystem. It should also be assessed if these intra-core group populations exhibit competitive interactions, as could be expected by their theorized ecological redundancy. Lastly, alternative community assembly models should adequately explain the observed existence of extensive phylogenetic signal in microbial communities, the presence of PCGs in analyzed microbial ecosystems, and the link between invasion success and the presence in the resident community of closely related populations.Additional file 1. Supplementary Materials to “A conceptual framework for the phylogenetically-constrained assembly of microbial communities.”


Metastasis remains the leading cause of cancer-related deaths worldwide, and our inability to identify the tumour cells that colonize distant sites hampers the development of effective anti-metastatic therapies. However, with recent research advances we are beginning to distinguish metastasis-initiating cells from their non-metastatic counterparts. Importantly, advances in genome sequencing indicate that the acquisition of metastatic competency does not involve the progressive accumulation of driver mutations; moreover, in the early stages of tumorigenesis, cancer cells harbour combinations of driver mutations that endow them with metastatic competency. Novel findings highlight that cells can disseminate to distant sites early during primary tumour growth, remaining dormant and untreatable for long periods before metastasizing. Thus, metastatic cells must require local and systemic influences to generate metastases. This hypothesis suggests that factors derived from our lifestyle, such as our diet, exert a strong influence on tumour progression, and that such factors could be modulated if understood. Here, we summarize the recent findings on how specific metabolic cues modulate the behaviour of metastatic cells and how they influence the genome and epigenome of metastatic cells. We also discuss how crosstalk between metabolism and the epigenome can be harnessed to develop new anti-metastatic therapies. Summary: In this Review the authors summarize recent findings on how specific metabolic cues modulate and influence the behaviour, genome and epigenome of metastatic cancer cells. They also discuss how the crosstalk between metabolism and the epigenome of metastatic cells can be harnessed to develop new anti-metastatic therapies. Metastasis is the leading cause of cancer-related deaths worldwide yet, at the cellular level, it is an inefficient process – only a small fraction of cells shed from a primary tumour into the bloodstream or lymphatic system will successfully complete all the sequential steps of the metastatic cascade . The mecAnaplerosis: the replenishment of TCA cycle intermediates that have been used for biosynthetic processes.Dormant/quiescent cancer cells: cells that remain in a state of dormancy or quiescence in response to intrinsic or extrinsic stimuli that arrest mitosis and cell growth.Driver mutation: mutational events that confer cancer cells with either growth or survival advantages and therefore favour (drive) tumour development.Enhancers: short DNA regions that regulate gene expression by recruiting transcription factors that establish and maintain cell-specific transcriptional signatures. They can be located far away from the genes they regulate.Epithelial-to-mesenchymal transition (EMT): a process by which epithelial cells gain mesenchymal cell properties, including migratory and invasive traits. EMT occurs during embryogenesis, wound healing and during the malignant transformation of cancer cells.Ketogenic diets: low-carbohydrate and high-fat diets that force the body to preferentially mobilize stored lipids for energy production, resulting in increased levels of ketone compounds.Metformin: a drug commonly used to treat type-2 diabetes that inhibits OXPHOS and consequently reduces ATP production in mitochondria, favouring cellular ATP production via glycolysis.Omental fat pad: an area of fat tissue in the abdominal cavity that surrounds the intestines.Orthotopic model: an animal model, most commonly mouse, in which human tumour cells are injected or implanted into the equivalent organ or tissue that the human cancer originated from.Oxidative phosphorylation (OXPHOS): this process is the most efficient way to produce ATP in eukaryotic cells via the electron flow that occurs in the mitochondrial inner membrane.Pioneer transcription factor: a transcription factor that can bind compacted chromatin to recruit chromatin remodelling proteins and other transcription factors; these transcription factors are important for determining cell fate.Preconditioned niche: an environment at a distance from the primary tumour that is modified and generates specific signals prior to the arrival of disseminated cancer cells; this environment can facilitate subsequent tumour cell infiltration and colonization.Tricarboxylic acid (TCA) cycle: this metabolic pathway, also known as the Krebs cycle, produces electron carriers for ATP generation through the electron transport chain, which takes place in mitochondria.Triple-negative breast cancer (TNBC): breast carcinoma histologically assessed as negative for estrogen receptors (ER−), progesterone receptors (PR−) and HER2 receptors (HER2−).Warburg effect: process by which tumour cells increase their glucose uptake and convert pyruvate to lactate through aerobic glycolysis under normal conditions of oxygen and glucose availability, yielding ATP, increased NADPH reductive power and metabolic intermediates, instead of coupling glycolysis to the TCA cycle and OXPHOS pathways.in vivo suggest that metastatic cells reach distant organs early during primary tumour growth, yet can remain dormant  in different types of tumours allows us to explore what distinguishes metastatic cells from their non-metastatic counterparts . One parIn this Review, we discuss recent insights into the metabolic plasticity of cancer cells and the way in which their metabolic processes can contribute to their metastatic transformation. We highlight the emerging role of lipid metabolism as an important source of cancer metabolic heterogeneity, provide an overview of the crosstalk that occurs between metabolic processes and the cancer cell epigenome, and examine how lifestyle influences, such as diet, might affect cancer progression. We also discuss the therapeutic potential of targeting metabolism during cancer progression, highlighting novel and experimental drugs currently under preclinical investigation.Cancer stem cells (CSCs) sustain the growth of the tumour mass and are responsible for therapy failure and patient relapse . The ide+ pancreatic CSCs display enhanced mitochondrial respiration and impaired glycolytic plasticity relative to their CD133− non-CSC counterparts, rendering these cells more vulnerable to metformin  cycle  and oxidetformin , which ietformin .de novo fatty acid (FA) synthesis and lipid desaturation, through the activity of stearoyl-coA desaturase 1 (SCD1), to promote tumour initiation in a nuclear factor kappaB (NFκB)-dependent manner  affects tumour initiation . Howeverin vivo experimental model of metastasis in mice, disseminated human colorectal cancer cells were found to secrete the protein creatine kinase brain-type (CKB) in the liver, where it catalyses the conversion of extracellular ATP and hepatic creatine into phosphocreatine, which is incorporated into metastatic cells via the creatine transporter, SLC6A8. Increased phosphocreatine availability in the extracellular space fuels the generation of ATP within metastatic cells, which is essential to meet the intense energetic requirements involved in colonizing the liver . The hypoxia-inducible transcription factor 1 alpha , is an important transducer of hypoxic signals and drives the expression of GLUTs and glycolytic enzymes, such as hexokinase 2 (HK2), phosphofructokinase 1 (PFK1) and lactate dehydrogenase A (LDHA), while inhibiting the expression of pyruvate dehydrogenase kinase 1 (PDK1). This response reduces the flux of pyruvate into the TCA cycle, thereby decreasing the rate of OXPHOS and oxygen consumption . In certic cells . In addiic cells . HoweverOne interesting aspect of tumour cells is their ability to shape the metabolism of their TME to ensure a plentiful supply of energy, through a process known as metabolic coupling. Metabolic coupling can occur either between tumour cells or between tumour and stromal cells. For instance, metastatic ovarian cancer cells induce the release of stored lipids from the omental fat pad  they colThese findings suggest new therapeutic avenues by which to prevent the successful colonization of distant sites by metastatic cells by inhibiting the metabolic crosstalk between cancer cells and their environment. For instance, mouse models of both melanoma (B16) and Lewis lung carcinoma (LLC) have been shown to possess a hyper-glycolytic intra-tumour endothelium that favours metastatic spreading . InteresWe are only just beginning to understand how different local metabolites, and the metabolic programmes they elicit, shape the ability of tumour cells to colonize different sites. In the next section, we discuss recent findings on how specific metabolic cues modulate the behaviour of tumour cells, with an emphasis on the role of FA and lipid metabolism in feeding metastatic progression.Recent data suggest that the metabolic profiles of metastatic cells differ as they colonize different organs. For instance, in a murine orthotopic model  of metasAnother study concluded that breast cancer cells with a broad metastatic potential  could engage both OXPHOS and glycolysis-dependent metabolic strategies, whereas liver metastatic cells relied on glucose uptake and glycolysis, while lung metastatic cells relied on glutamine uptake and OXPHOS to metastasize .Although these studies provide insights into the dynamic metabolic programmes of tumour cells during cancer progression, further studies will be necessary to mechanistically connect altered gene expression, and the associated bioenergetic behaviour of the cell, to each step of metastatic progression.in vitro three-dimensional (3D) models have revealed that breast cancer cells rely on the non-essential amino acid proline, through proline dehydrogenase enzyme (PRODH) activity, to form spheroids in vitro and lung metastases in vivo  in metastatic breast cancer cells, which in turn converts pyruvate to oxaloacetate, thereby enhancing anaplerosis  Christe. Additio in vivo . Convers in vivo . Cancer  gliomas .Thus, the availability of specific nutrients at different organs might impose unique metabolic states on metastatic cells. However, future studies are required to determine how specific metabolic routes sustain the high energy demand of metastatic cells while modulating the signalling pathways that allow metastatic growth.+ OSCC metastatic cells in mouse orthotopic models are exquisitely sensitive to circulating blood fat levels, and a high-fat diet or stimulation with palmitic acid strongly boosts these cells' metastatic potential  induces EMT in liver cancer cells, thereby increasing their migration and invasion in in vitro assays , and the FFAs it produces, are elevated in aggressive human ovarian cancer cell lines and in primary ovarian tumours . Intereser cells .Myc and tyrosine-protein kinase Src, two well-known signalling regulators of metastasis , has been extensively documented in a number of malignancies, including breast and hepatocellular carcinomas , HK2, PFK1 and LDHA genes. A second enzyme, ACSS2, which is involved in controlling the pool of acetyl-CoA from acetate, translocates to the nucleus, where it can affect the incorporation of acetyl-CoA into histones (in vivo models (Acetyl-CoA-derived acetyl moieties are transferred to lysine residues on histone tails by histone acetyltransferases (HATs). Histone acetylation leads to the opening up of compact chromatin and to increased transcription of the genes that are located within the acetylated chromatin region. Global transcriptional control through chromatin remodelling in the nucleus has been linked to the enzyme ATP citrate lyase (ACLY) . In the histones . Althoughistones . Acetyl-o models . ACSS2 eo models . ACSS2-do models . Additioo models .Enhancer regions  are a foin vitro studies in various cancer cell lines have shown that acetyl-CoA obtained by FA oxidation provides acetyl groups that strongly activate a transcriptional programme involved in FA-related processes , this supplementation fails to rescue the global loss of histone acetylation . Howeverrocesses .Taken together, these data suggest that multiple carbon sources control the pool of acetyl-CoA to regulate histone acetylation at specific loci in tumour cells. Therefore, acetyl-CoA might be a major force that establishes the transcriptional networks that shape the metabolic flexibility and heterogeneity that cancer cells exhibit as tumours progress.in vitro methionine restriction in HCT116 cells (Histone methylation can occur at the arginine (R) and lysine (K) residues of histone 3 (H3) and histone 4 (H4), and is associated with transcriptional activation or repression, depending on the residue targeted and on the number of methyl groups deposited in the N-terminal region of either histone. Histone methylation is tightly regulated by histone methyltransferases (HMTs) and histone demethylases. Similar to DNA methylation (discussed below), alterations in methionine metabolism and in the availability of S-adenosyl methionine (SAM) directly modulate histone methylation. For instance, the levels of histone H3K4me3, a histone mark typically associated with promoter activation, decrease upon 16 cells . Importa16 cells . Signifi16 cells .The methylation of CpG islands generally impedes promoter activation and consequent gene expression, although it can also correlate with enhanced transcription factor binding . DNA metDNA methylation is vitally important for establishing stable epigenetic states during mammalian development . HoweverKRAS in pancreatic cancer results in tumours with increased glycolytic flux and serine biosynthesis, in which DNA methylation is fuelled by the increased availability of SAM. This results in the over-activation of DNMTs that control the transcriptional silencing of specific retrotransposons by regulating their methylation status. Hypermethylation of these repetitive elements might promote oncogenic transformation and growth through transcriptional control of host genes  inhibitor, had anti-tumour effects  is enhanced in a number of primary tumours, and its inhibition by the drug JZL184 results in reduced pathogenicity in murine models of melanoma and ovarian cancer . InteresFinally, the intimate interplay between metabolism and epigenetic mechanisms in cancer opens the possibility of targeting both to obtain synergistic effects . The rec. Such studies should provide clues as to the requirements for treatment and might also identify a window of opportunity for treatment, even leading to disease prevention prior to clinical detection.Despite intense investigation, the identity of metastatic cells remains elusive, preventing the research community from developing effective anti-metastatic therapies. Several recent studies discussed in this Review indicate that metabolism plays a causal role in the different phenotypic states exhibited by cancer cells during cancer progression and metastasis. Given the plethora of metabolic mechanisms in each organ, it is not surprising that the CSCs that initiate and promote metastasis need to exhibit metabolic plasticity in order to hijack multiple metabolic pathways and to survive hostile microenvironments during the metastatic cascade. Future investigations are needed to characterize and identify the cellular metabolic requirements for initiating metastasis, and to profile the metabolome of pro-metastatic and anti-metastatic nichesThe idea that local and systemic factors might confer metastatic cells with enough plasticity to successfully colonize distant organs suggests that influences derived from lifestyles, such as our diet or other daily habits, exert a strong influence on tumour progression, and that such factors could be easily modulated if understood. Increasing data supports the importance of diet in the crosstalk between metabolism and epigenetic regulation , and thehttp://dmm.biologists.org/collection/cancermetabolism.This article is part of a special subject collection ‘Cancer Metabolism: models, mechanisms and targets’, which was launched in a dedicated issue guest edited by Almut Schulze and Mariia Yuneva. See related articles in this collection at


The cerebral microcirculation holds a critical position to match the high metabolic demand by neuronal activity. Functionally, microcirculation is virtually inseparable from other nervous system cells under both physiological and pathological conditions. For successful bench-to-bedside translation of neuroprotection research, the role of microcirculation in acute and chronic neurodegenerative disorders appears to be under-recognized, which may have contributed to clinical trial failures with some neuroprotectants. Increasing data over the last decade suggest that microcirculatory impairments such as endothelial or pericyte dysfunction, morphological irregularities in capillaries or frequent dynamic stalls in blood cell flux resulting in excessive heterogeneity in capillary transit may significantly compromise tissue oxygen availability. We now know that ischemia-induced persistent abnormalities in capillary flow negatively impact restoration of reperfusion after recanalization of occluded cerebral arteries. Similarly, microcirculatory impairments can accompany or even precede neural loss in animal models of several neurodegenerative disorders including Alzheimer's disease. Macrovessels are relatively easy to evaluate with radiological or experimental imaging methods but they cannot faithfully reflect the downstream microcirculatory disturbances, which may be quite heterogeneous across the tissue at microscopic scale and/or happen fast and transiently. The complexity and size of the elements of microcirculation, therefore, require utilization of cutting-edge imaging techniques with high spatiotemporal resolution as well as multidisciplinary team effort to disclose microvascular-neurodegenerative connection and to test treatment approaches to advance the field. Developments in two photon microscopy, ultrafast ultrasound, and optical coherence tomography provide valuable experimental tools to reveal those microscopic events with high resolution. Here, we review the up-to-date advances in understanding of the primary microcirculatory abnormalities that can result in neurodegenerative processes and the combined neurovascular protection approaches that can prevent acute as well as chronic neurodegeneration. Cerebral microcirculation is a fundamental element for proper cerebral functioning since it is the main transport and distribution system for oxygen and nutrients that fuel the high and continuously changing metabolic demand of the brain tissue. The anatomical and physiological features of small vessels, which mainly consist of vasculature <100 μm in diameter  or feed-back (the metabolic consequences of increased activity regulates blood flow) mechanisms  through a cranial window in rodents provides high resolution angiogram of the capillary network and, red blood cell (RBC) flux and speed estimation within individual capillaries  and temporal (in milliseconds) resolution. Most of the available information on capillary flow and oxygenation is based on magnetic resonance imaging (MRI) as well as direct illaries –14. Phosillaries , 15–22. illaries , 23–26. vasively .2 distribution is highly heterogeneous, some capillaries have very low oxygen, while not necessarily making the tissue around them critically hypoxic but particularly vulnerable to fluctuations in blood flow and/or increase in metabolic demand  tight junctions between endothelial cells form the critical layer of the blood brain barrier (BBB) that controls material passage into the parenchyma. In the healthy setting, endothelium prevents paracellular plasma leakage by tight junctions and there is a low level of transcytotic vesicle trafficking through endothelial cells , 46. OxyAstrocyte end-feet around microvessels are critical elements for neuron-microvessel communication. Nearby neuronal activity is sensed by astrocyte processes as extracellular adenosine triphosphate (ATP) and glutamate increase around synapses. Calcium signals triggered in astrocytes lead to cyclooxygenase activation and release of vasodilatory prostaglandins from end-feet surrounding the capillaries , 59. ThiThe mechanisms of neurovascular regulation should be recognized in the setting of an integrated vascular network, where different levels of microcirculation may work in concert in addition to a tight compartmentalization. For example, arterioles appear to favor the vasoactive signals coming from interneurons coding the activity in a larger cohort of neurons in contrast to capillary regulation responding to very focal demand translated by adjacent astrocytes , 78. Art2 extraction in poorly perfused tissue  may not always reflect accurate supply-demand match at microscopic level. In routine clinical practice of cerebrovascular medicine, main focus is on the evaluation of adequate arterial flow and luminal patency, as they can be easily imaged by widely available radiological techniques. In the setting of a CBF drop, particularly to ischemic levels (below ~20 ml/100 g/min in humans) , there id tissue , 82. Thed tissue , 85. MRId tissue . These td tissue , 82, 86.The capillary dysfunction can arise from almost any element within the neurovascular unit. A thin (0.5 μm) glycoprotein coating in the luminal side of the endothelium, called glycocalyx, facilitates passage of blood cells –89. GlycEndothelial cell dysfunction can significantly contribute to dysregulated capillary flow. This dysfunction includes a reduction in endothelium-derived nitric oxide availability. Moreover, gap-junction uncoupling between endothelial cells increases functional shunting by introducing irregularities to the microvascular cross-sectional profiles, caused by desynchronized regulation of smooth muscle or pericyte tone , 92. A dPericytes maintain the BBB, interact with immune cells  and, sinFinally, the physicochemical properties of plasma or blood cells themselves can introduce microcirculatory heterogeneity. As noted above, RBCs and WBCs have large diameters compared to capillaries therefore they need to squeeze through the narrow capillary lumen, significantly making passage challenging and vulnerable to disturbances . Even suThe tissue can tolerate a certain degree of capillary dysfunction by regulating upstream arteriolar flow (either increasing or decreasing) to optimize oxygen extraction , 118–120Small vessel disease is a clinical and imaging phenomenon caused by different etiologies and characterized by pathological changes in vasculature with a diameter <100 μm, including, arterioles, venules, and capillaries . It is aCurrently, the only approved treatment of an acute ischemic stroke is prompt recanalization within few hours either by mechanical thrombectomy or thrombolysis with intravenous or intra-arterial tissue plasminogen activator. At the present, this treatment approach only takes large vessel recanalization into account. However, tissue reperfusion is usually incomplete, hence oxygen extraction is not optimal even after complete recanalization . In the In the ischemic brain, activated neutrophils can also temporarily plug capillaries with their large size and stiff cytoplasm, and can disturb the RBC transit . As a clThe ischemia-induced microvascular dysfunction progresses in a graded fashion. With a temporary decrease in perfusion pressure in a normal and compliant microvasculature, the cells transiting the microvasculature slow down, since there is less driving force due to lower pressure gradient. Accordingly, MTT values increase. The CTH calculated based on the standard deviation of transit times in multiple pathways across the capillary bed proportionally increases as well. Thereby, the relative transit heterogeneity (RTH = CTH/MTT) remains unchanged and oxygen delivery to the tissue can still be sufficient to match the demand. However, with ongoing ischemia, structural irregularities, like constrictions, and glycocalyx damage in capillaries emerge, making cellular passage complicated . This tiChronic cerebral hypoperfusion, even if it doesn't cause full-blown ischemia, can have long-term effects on microcirculatory structure. Experimental OCT data in mice with carotid artery occlusion show a local micro-heterogeneity of capillary flow and oxygen supply even at non-ischemic global blood flow levels . The fun2 is lost and an inverse neurovascular coupling response, i.e., constriction instead of expected hyperemia with sensory stimulation is observed 24 h after experimental subarachnoid hemorrhage  can trigger arteriolar and microvascular vasospasms . Hemoglomorrhage . Whethermorrhage .Ischemic injury is a well-known histological hallmark of traumatic contusion injuries . A reduc2 extraction by slowing flux rates , a cause of early onset dementia and psychiatric disorders especially in boxers or American football players and also in military veterans exposed to blast injuries . AutopsyA reduction in CBF is observed early in Alzheimer's disease (AD) patients and this has been proposed as a predictor of progression to overt AD from mild cognitive impairment –198. ManElectron microscopy has identified capillary wall damage, basal membrane thickening and pericyte degeneration in autopsy brains from AD patients . In MRI in vivo two photon microscopy in transgenic mice with excessive amyloid deposition demonstrated that an increased number of cortical capillaries had stalled blood flow due to neutrophils adhered to vessel wall , resulting in accelerated cognitive decline , 209–211Accumulating evidence suggests that several other progressive neurodegenerative conditions may also involve primary microcirculatory pathologies, Like AD and CTE as discussed above, other tauopathies such as corticobasal degeneration (CBD), progressive supranuclear palsy (PSP) and amyotrophic lateral sclerosis (ALS) exhibit possible microvascular involvement .In Parkinson's disease, both experimental and clinical data suggest microvascular involvement. Capillary loss and abnormal fragmented capillaries or endothelial clusters can be detected in substantia nigra, cerebral cortex and brain stem in pathological brain samples . There aCBD and PSP are two atypical and rapidly progressive forms of parkinsonism. In postmortem brains of CBD and PSP patients, prominent accumulation of hyperphosphorylated tau in close proximity to vasculature, astrocytic proliferation, and hypertrophy as well as reduced capillary diameter have been detected , 226. Thin vivo two-photon microscopy, however, identified individual capillaries with pericyte contractions and accompanying stalls in blood flow in hippocampus of epileptic mice during seizures and those abnormal capillary vasodynamics created an environment with irregular capillary flow and possibly microscopic hypoxia, which was associated with neurodegeneration in close proximity of constricted capillaries , has been shown to improve microcirculation in a mouse model of cerebral ischemia, improving tissue injury and functional outcome . Since N+2-activated chloride channels can also potentiate pericyte depolarization and constriction and can be another target  increase capillary clogging, stalls and heterogeneity, blood viscosity, and endothelial adhesion could be one of the possible targets. Phosphodiesterase inhibitors like pentoxifylline increase RBC flexibility and decrease blood viscosity , 273 andDecreasing leukocyte count has been reported to provide a beneficial outcome in animal models of cerebral ischemia . A monocOther innovative approaches to modulate rheological parameters of blood cells are continuously being developed. Drag-reducing polymers improved flexibility of RBCs, decreasing cell stasis in capillaries, and plasma separation in capillary branching points, improving capillary flow heterogeneity in cerebral and myocardial ischemia models –282. UptOur review can help the recognition that many microvascular pathogenetic mechanisms are shared across a variety of acute and chronic neurological conditions and these mechanisms are not simply by-standers but are actually role-players, emerging in very early phases of neurodegenerative conditions. With advancement of optical microscopy, MRI-based imaging tools having higher spatiotemporal resolution as well as computer models, the physiology and vulnerabilities of the microcirculation will be better elucidated. Investigators in the field should not be surprised to see the introduction of previously unknown details, which will eventually lead to much better neuroprotectants for many neurological disorders within the next decades.ŞE drafted the manuscript and prepared the figures. TD drafted and critically revised 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.


Neurodegenerative diseases affect millions of people worldwide, yet there are currently no effective treatments. Because risk of neurodegenerative disease substantially increases with age, greater life expectancy with a concomitant aging population means more individuals will be affected in the coming decades. Thus, there is an urgent need for understanding the mechanisms driving neurodegenerative diseases in order to develop improved treatment strategies. Inflammation in the nervous system, termed “neuroinflammation,” has become increasingly recognized as being associated with neurodegenerative diseases. Early attention focused primarily on morphological changes in astrocytes and microglia; however, brain and CNS resident mast cells are now receiving attention as a result of being “first responders” to injury. Mast cells also exert profound effects on their microenvironment and neighboring cells including behavior and/or activation of astrocytes, microglia, and neurons, which, in turn, are implicated in neuroinflammation, neurogenesis and neurodegeneration. Mast cells also affect disruption/permeability of the blood brain barrier enabling toxin and immune cell entry exacerbating an inflammatory microenvironment. Here, we discuss the roles of mast cells in neuroinflammation and neurodegeneration with a focus on development and progression of four prominent neurodegenerative diseases: Alzheimer’s Disease, Parkinson’s Disease, Amyotrophic Lateral Sclerosis, and Huntington’s Disease. Selected processes most relevant to neurodegenerative diseases are described below.In the brain and CNS, microglial cells are the guardian immune surveillance effectors that constantly monitor the surrounding microenvironment for injury and pathogen entry, which elicit microglial activation encompassing the release of cytokines/chemokines, phagocytosis of cellular debris and antigen presentation to T cells . Cross-tWhile microglial cells provide immune surveillance to the brain and CNS, other immune cells have recently been recognized for their contributions to neuronal degenerative diseases. Notably, tissue-resident mast cells have garnered much attention as primary communicators and mediators between the peripheral immune system and the nervous system during inflammatory responses . Mast ceInteractions between mast and microglial cells involves complex cross-communication that can be both unidirectional and bidirectional. Activated microglia release IL-6 and chemokine (C-C motif) ligand 5 (CCL5), which affect surface expression levels of toll-like receptors, TLR2 and TLR4, on mast cells thus modulating the ability of mast cells to respond to endotoxins . ConversThe blood-brain barrier (BBB) plays a critical role in controlling the entry of molecules, pathogens, and toxins into the CNS. The primary barrier units of the BBB are tight junctions between endothelial cells (ECs) that limit paracellular transport. Tight junctions are composed of transmembrane proteins such as claudin and occludin . BBB-speAs a result of their ability to quickly release diverse immuno- and neuromodulators, the relatively small numbers of mast cells in the brain and CNS have a substantial influence on the behaviors of neurons and glial cells. Direct influences via release of TNF-α, histamine and proteases empower mast cells with the ability to potentiate neuroinflammation, neurogenesis, neurodegeneration and BBB disruption/permeability. Nevertheless, to date all evidence regarding the direct influence of mast cells on neurodegeneration derive from animal studies . It therAlzheimer’s Disease (AD) is the most common cause of dementia among the elderly population, with 5–7 million new diagnoses annually . PatientRecent studies increasingly implicate neuroinflammation in AD neurodegeneration . For exain vitro as well as in the mast cells of Panx1-/- mice  is characterized by progressive motor deficits including rigidity, bradykinesia and resting tremor, alongside non-motor deficits that may evolve later in the disease course . Recent in vitro. The authors further characterized this phenomenon by exposing mast cells to glial activating factors in co-culture with fetal mouse brain-derived astrocytes, neurons, and/or mixed glia/neurons. Release of specific inflammatory mediators and neurite outgrowth were both quantified. By comparing all possible combinations of cell types, the authors demonstrated that mast cells specifically release mediators including tryptase. Furthermore, mast cell-specific mediators were shown to trigger CCL2 and MMP-3 release by astrocytes and glia in this model system. Taken together, these results suggest mast cell interactions with neurons and glial cells may play a role in PD pathogenesis  involves progressive degeneration of both upper and lower motor neurons, often accompanied by cognitive and/or behavioral symptoms. ALS is the most common and aggressive form of motor neuron degeneration in adults, with a heterogenous but invariably progressive and fatal disease course . The undSOD1-transgenic mice  of ALS patients . High moin vitro  causes excitatory neurotoxicity in inhibitory medium spiny neurons (MSNs) in the striatum and cortex. Patients experience involuntary jerking movements as well as dystonia, rigidity, cognitive, and neuropsychiatric symptoms (Huntington’s Disease (HD) is an autosomal dominant disorder caused by expansion of a CAG triplet in the symptoms .Although the role of mast cells in HD pathogenesis has not yet been established, substantial evidence supports a strong role of neuroinflammation via interactions between neurons, microglia and astrocytes. Expression of mHTT in astrocytes downregulates production of neuronal growth factors, while mHTT expression in microglia promotes expression of proinflammatory cytokines  and toxic metabolites. These processes converge to cause neurodegeneration, exacerbated by mHTT expression in neurons that triggers cell-autonomous apoptosis and degeneration. Components of dead neurons may be detected and phagocytosed by microglia in a similar fashion to Aβ detection in AD, resulting in further production of proinflammatory mediators, astrocyte activation, and a feed-forward loop of neuronal damage .Plasma levels of IL-6 and IL-8 correlate with functional scores in HD patients . SeveralEmerging studies implicate chronic stress in a variety of neuroinflammatory processes  that mayin vitro model systems, as well as validation in human tissue specimens. Further studies are also needed to elucidate the multifaceted cross-talk between mast cells and microglia, astrocytes and neurons. The scale of this problem may require informatics-based approaches; for example large genomic datasets derived from experimental mouse models may enable in silico discovery of target genes and guide rational validation in appropriate model systems  of mast cells in neuroinflammation and neurodegenerative diseases, the possible neuroprotective roles of mast cells also warrant further investigation.With respect to AD, PD, ALS, and HD, discussed in the present review, mast cell perturbation of the BBB appears to share a commonality. Moreover, mast cells have been found to home to sites of amyloid deposition in AD; and, an inhibitor of mast cell function was shown to reduce cognitive decline in AD patients. Mast cell interactions with neurons and glial cells have also been implicated in PD pathogenesis. Emerging evidence suggests that mast cell autocrine signaling may contribute to ALS: The mast cell chemoattractant, IL-15, is elevated in the serum and cerebrospinal fluid of ALS patients; and, mast cells expressing IL-17 have been found in the spinal cord of ALS patients. Plasma levels of cytokines , known to affect mast cell activation, have been correlated with functional scores in HD patients suggesting the possible involvement of mast cells in the pathogenesis of HD. Future considerations include validation of animal and All authors participated in the conceptualization, literature review, drafting, and editing of 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.