African swine fever virus (ASFV) represents a severe threat to global agriculture with the world's domestic pig population reduced by a quarter following recent outbreaks in Europe and Asia. Like other nucleocytoplasmic large DNA viruses, ASFV encodes a transcription apparatus including a eukaryote-like RNA polymerase along with a combination of virus-specific, and host-related transcription factors homologous to the TATA-binding protein (TBP) and TFIIB. Despite its high impact, the molecular basis and temporal regulation of ASFV transcription is not well understood. Our lab recently applied deep sequencing approaches to characterise the viral transcriptome and gene expression during early and late ASFV infection. We have characterised the viral promoter elements and termination signatures, by mapping the RNA-5′ and RNA-3′ termini at single nucleotide resolution. In this review, we discuss the emerging field of ASFV transcripts, transcription, and transcriptomics. Ornithodoros (soft) ticks can act as a biological vector between warthogs and/or pigs, though direct pig-to-pig infection is the most frequent cause of disease propagation in an agricultural context . Tr. Tr12]. in vitro transcription assays, to biochemically characterise ASFV transcription. Allowing also, for testing of chemical compound libraries for ASFV-RNAP inhibitory activity, and therefore antiviral drugs.Going forward the important task at hand is to study and characterise the molecular mechanisms underlying ASFV transcription. This includes (i) carrying out further transcriptome analyses using combinations of chemical inhibitors in order to unequivocally determine all ASFV gene expression stages. Thus, extending our understanding of how viral transcription initiation factors and cognate promoters co-operate to enable gene expression during early and late infection, as well as investigate whether host factors participate in ASFV transcription. Likewise (ii), an essential step for understanding these mechanisms requires determining the whole-genome distribution of ASFV-RNAP and initiation factors, to study transcription complexes throughout infection. Lastly (iii), it is additionally important to produce recombinant RNAPs and transcription factors for structural characterisation, and www.oie.int).Importance for the field: Outbreaks of African Swine Fever are spreading rapidly throughout Eastern Europe, Africa and Asia, making the threat of ASFV of critical socioeconomic importance for agriculture and pig farming industries worldwide. With disease control measures (such as culling) being the only available option, since there is no approved vaccine for this disease with a mortality rate up to 100% (World Organisation for Animal Health, Current thinking: Transcriptomics have provided essential insight into understanding mechanisms underlying ASFV gene expression and its interplay with that of the host. Specialised next generation sequencing techniques have also been used to map the 5′ and 3′ ends of ASFV transcripts genome-wide, which has enabled identification of promoter and transcription termination motifs, respectively, expanding our understanding of how ASFV modulates its gene expression.Future directions: Moving forward, work should focus on further transcriptomics to discern other viral gene expression stages and their respective promoters, complemented by whole-genome distribution studies of ASFV-RNAP and transcription factors. Additionally, production of these proteins recombinantly, will allow for their structural characterisation and functional assessment with biochemical assays, thus enabling screening for compounds showing viral transcription inhibition and therefore could act as anti-viral drugs.


Sabre-toothed carnivores are among the most famed vertebrate fossils in the world. The sabre-tooth ecomorph has been converged upon repeatedly by distantly related species throughout mammalian evolution. Lautenschlager et al. employ a range of biomechanical analyses to investigate the functional diversity of sabre-toothed skulls. Across 66 species, broad functional diversity is recovered with implications for prey specialization and niche partitioning, despite being morphologically convergent. Smilodon fatalis is immortalised today in pop culture, childrens’ toys and the hearts of fossil lovers everywhere. However, it was not the only sabre-toothed vertebrate to have ever lived. As is often the case with body forms in biology, the sabre-toothed skull has evolved multiple times in independent species, a process known as evolutionary convergence. Convergent evolution results in animals achieving similar shapes or functions, often with convergent animals occupying similar niches. In the case of the sabre-toothed skull, this can be simplified to oversized upper canine teeth and enhanced jaw gape, and this ecomorph is currently absent in modern fauna. The functional convergence of sabre-toothed carnivores has not been assessed in mechanical detail, until now.Few fossil species can compete with the allure and grandeur of dinosaurs, but a strong contender is the sabre-toothed cat. 1 conducted a detailed biomechanical analysis of the skulls of 66 sabre-, scimitar- and dirk-toothed fossil vertebrates, spanning 265 million years of evolutionary time from the Permian to the Pleistocene. Using finite-element models, biomechanical simulations, geometric morphometrics and phylogenetic comparative methods, the function and evolution of the sabre-toothed ecomorph was investigated.Lautenschalger et al.Gape angle, mandibular bending strength and bite efficiency were found to vary considerably across taxa and convey different information about hunting style and skull function. For example, within gorgonopsians, the authors suggest a specialisation to smaller prey is indicated by an effective gape angle below 60°. Across all species, bending strength of the mandible does not correlate with jaw gape, but interestingly the rate of jaw bending evolution varies a lot across groups. A significant correlation between canine length and jaw length indicates that maximum canine penetration is more important in the hunting styles of sabre-toothed animals than prey size.These results, along with others reported in the paper, suggest that there was a wide variety of functional convergence in sabre-toothed vertebrate ecomorphology not apparent with surface level observations of these skulls. The morphological convergence in these species does not correlate with the breadth of functional diversity reported by the authors in this exciting new study.


FN1 gene. Cellular FN isoforms differ in their solubility, receptor binding ability and spatiotemporal expression, and functions that have yet to be fully defined. FN induction at tumor sites constitutes an important step in the acquisition of biological capabilities required for several cancer hallmarks such as sustaining proliferative signaling, promoting angiogenesis, facilitating invasion and metastasis, modulating growth suppressor activity and regulating anti-tumoral immunity. In this review, we will first provide an overview of ECM reprogramming through tumor-stroma crosstalk, then focus on the role of cellular FN in tumor progression with respect to these hallmarks. Last, we will discuss the impact of dysregulated ECM on clinical efficacy of classical (radio-/chemo-) therapies and emerging treatments that target immune checkpoints and explore how our expanding knowledge of the tumor ECM and the central role of FN can be leveraged for therapeutic benefit.Normal tissue homeostasis and architecture restrain tumor growth. Thus, for a tumor to develop and spread, malignant cells must overcome growth-repressive inputs from surrounding tissue and escape immune surveillance mechanisms that curb cancer progression. This is achieved by promoting the conversion of a physiological microenvironment to a pro-tumoral state and it requires a constant dialog between malignant cells and ostensibly normal cells of adjacent tissue. Pro-tumoral reprogramming of the stroma is accompanied by an upregulation of certain extracellular matrix (ECM) proteins and their cognate receptors. Fibronectin (FN) is one such component of the tumor matrisome. This large multidomain glycoprotein dimer expressed over a wide range of human cancers is assembled by cell-driven forces into a fibrillar array that provides an obligate scaffold for the deposition of other matrix proteins and binding sites for functionalization by soluble factors in the tumor microenvironment. Encoded by a single gene, FN regulates the proliferation, motile behavior and fate of multiple cell types, largely through mechanisms that involve integrin-mediated signaling. These processes are coordinated by distinct isoforms of FN, collectively known as cellular FN (as opposed to circulating plasma FN) that arise through alternative splicing of the  Historically, studies addressing the genesis and progression of cancer have focused on the genotype of tumor cells. In the case of carcinomas, nascently transformed epithelial cells progress to an invasive phenotype by the accumulation of mutations. However, this is only part of the story, as schematized in The ECM proteins induced in tumor tissue are often development- and disease-specific isoforms generated by alternative splicing events. Such is the case with fibronectin (FN), as described below. Before focusing on FN and its multi-faceted role in the tumor setting, we will briefly discuss important notions and emerging themes regarding the production, organization and remodeling of ECM in tumor tissue. Numerous outstanding reviews are cited in this section to provide a more comprehensive picture of these themes.Gene expression screens have revealed that many genes encoding ECM components are dysregulated during tumor progression , 4. As tIn vitro and in vivo studies have established that matrix proteins expressed by malignant cells also become directly incorporated in the matrix. Sets of tumor cell-derived ECM proteins were elegantly identified using xenograft models in which human tumor cells were grafted in murine hosts  and fibroblast growth factor 2 (bFGF/FGF2) play critical roles in myofibroblast activation and fibrosis –42. In cReactive oxygen species (ROS) generated by cancer cells were shown to promote the fibroblast-to-myofibroblast transition by a mechanism involving TGF-β, PDGF, and CXCL12 signaling . CertainIn addition to the stimulation of ECM protein expression, tumor-fibroblast crosstalk profoundly impacts matrix assembly, cross-linking and remodeling. The assembly of matrix macromolecules into a 3D structure is a dynamic process largely carried out in the tumor stroma by CAFs. Fibrillar collagens are the major components of the tumor ECM and collagen architecture is severely altered in tumor tissue see , 50]. In. In50)].FN is a major core component of the tumor matrisome. Initially discovered as a “contaminant” in one of the steps of fibrinogen isolation more than 70 years ago , it is n1−12), two type II repeats (FNII1−2), and 15  type III repeats . pFN isin vitro and in vivo studies regarding EDB and EDA functions can be found in Muro et al. , as FN was named at the time, was significantly reduced in quantity upon malignant transformation of chicken and human fibroblasts infected with Rous Sarcoma Virus (RSV) , 140. HoIn vitro studies have underlined the role of FN in promoting cancer cell growth, survival, and invasion in glioma  are less clear. Post-natal deletion of endothelial cFN in a spontaneous RIPTag-driven model of carcinogenesis fails to inhibit tumor angiogenesis , 160, suin vitro. However, it is important to consider that beyond its role as ligand for signaling receptors on endothelial cells, FN in perivascular matrices constitutes an obligate scaffold for organization of the vessel-associated ECM and a repository for pro-angiogenic factors [reviewed in  has refined the molecular signatures of tumor cells and different stromal cell populations thus providing a closer look at FN expression and function in different cell types, and across cancer types. Moreover, former studies on tumors were largely centered on malignant cells. However, in most carcinomas FN is produced and assembled by stromal cells . Indeed,FN is a mesenchymal marker par excellence. In mesenchymal-like tumors, such as glioblastomas, FN expression and assembly by tumor cells has been shown to facilitate intercellular cohesion and collective invasion through a basement membrane-like ECM . FN knocFn1 gene in the liver, von Au and colleagues showed that a decrease in pFN reduces tumor angiogenesis, tumor growth and bone metastasis through an apparent feed forward upregulation of its own production and by modulating the response to VEGF  expression, and TGF-β, suggesting a protective role of FN-EDA against vascular oxidative stress .The striking implication of tumor ECM components and their cellular receptors in tumor progression and response to conventional and emerging therapies has led to the quest for novel TME-directed therapeutic strategies. Different approaches have been employed to target FN, or its receptors . The firin vivo  . αv-basein vitro and in vivo model of human non-small lung cancer, cetuximab promoted FN expression via p38-MAPK-ATF2 signaling (Integrin-mediated cell-ECM and in particular cell-FN interactions confer resistance to chemotherapy as well as to ionizing radiation –241. In ignaling . Cell adignaling . More reignaling . These rignaling .Our understanding of stromal reprogramming in the TME has advanced at a rapid pace in recent years, driven by technological advances and the integration of massive amounts of information from different fields, spanning multiple scales. As illustrated above, cFN is recurrently a central component of the tumor stroma that contributes to several cancer hallmarks and enabling characteristics. Further understanding of cFN production, assembly and remodeling in primary tumor beds, draining lymph nodes and at metastatic sites is needed to grasp the full complexity of tumor progression and metastatic spread. This knowledge should provide valuable insights into cell-ECM interactions and the physical and functional interplay between different cellular components of the TME. Elucidating the role of cFN variants in modulation of immune cell trafficking, phenotype and functional maturation is of particular importance for improving the current armatorium of immunomodulatory agents and for providing therapeutic alternatives that target the stroma.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


An ever-growing body of evidence has linked the gut microbiome with both the effectiveness and the toxicity of cancer therapies. Radiotherapy is an effective way to treat tumors, although large variations exist among patients in tumor radio-responsiveness and in the incidence and severity of radiotherapy-induced side effects. Relatively little is known about whether and how the microbiome regulates the response to radiotherapy. Gut microbiota may be an important player in modulating “hot” versus “cold” tumor microenvironment, ultimately affecting treatment efficacy. The interaction of the gut microbiome and radiotherapy is a bidirectional function, in that radiotherapy can disrupt the microbiome and those disruptions can influence the effectiveness of the anticancer treatments. Limited data have shown that interactions between the radiation and the microbiome can have positive effects on oncotherapy. On the other hand, exposure to ionizing radiation leads to changes in the gut microbiome that contribute to radiation enteropathy. The gut microbiome can influence radiation-induced gastrointestinal mucositis through two mechanisms including translocation and dysbiosis. We propose that the gut microbiome can be modified to maximize the response to treatment and minimize adverse effects through the use of personalized probiotics, prebiotics, or fecal microbial transplantation. 16S rRNA sequencing is the most commonly used approach to investigate distribution and diversity of gut microbiome between individuals though it only identifies bacteria level other than strain level. The functional gut microbiome can be studied using methods involving metagenomics, metatranscriptomics, metaproteomics, as well as metabolomics. Multiple ‘-omic’ approaches can be applied simultaneously to the same sample to obtain integrated results. That said, challenges and remaining unknowns in the future that persist at this time include the mechanisms by which the gut microbiome affects radiosensitivity, interactions between the gut microbiome and combination treatments, the role of the gut microbiome with regard to predictive and prognostic biomarkers, the need for multi “-omic” approach for in-depth exploration of functional changes and their effects on host-microbiome interactions, and interactions between gut microbiome, microbial metabolites and immune microenvironment. Radiotherapy is a core modality used for the treatment of solid tumors ; more thRadiotherapy is also associated with toxic side effects that negatively affect patients’ quality of life. Acute toxicities that may affect the patient’s ability to complete a treatment course include mucositis, dermatitis, cystitis, and bone marrow suppression. Chronic toxicities include fibrosis, vascular damage, or atrophy of the affected tissue or organ . HoweverThe gut microbiome can influence both the effectiveness of cancer treatment –13 and tGut microbiota may be an important player in modulating “hot” versus “cold” tumor microenvironment, ultimately affecting treatment efficacy , 23. TheAlistipes and decreases in Mucispirillum genera [Firmicutes and a 3% increase in Fusobacterium phyla [Duodenibacillus massiliensis was linked with the improved CR rate. Generally, the most significant changes in the gut microbiome associated with cytotoxic chemotherapy or radiotherapy are increases in Bacteroides and Enterobacteriaceae and decreases in Bifidobacterium, Faecalibacterium prausnitzii, and Clostridium cluster XIVa [Pseudoxanthomonas-Streptomyces-Saccharopolyspora-Bacillus clausii) highly predictive of long-term survivorship in both discovery and validation cohorts. Through human-into-mice fecal microbiota transplantation (FMT) experiments from short-term survival, long-term survival, or control donors, the tumor microbiome was differentially modulated, and tumor growth as well as tumor immune infiltration were affected. Logically, then, one could hypothesize that the gut microbiome also influences the immunostimulatory effects of radiotherapy  and those disruptions can influence the effectiveness of the anticancer treatments Table . Kim et m genera . A clinium phyla . A studyum phyla  analyzinter XIVa . Gut micter XIVa . A recenter XIVa  showed Hapy Fig. .Table 1SIndeed, one group, seeking to explore whether the gut microbiota could modulate antitumor immune response after radiation to non-gut organs, used mouse models of B16-OVA melanoma and TC-1 lung/cervical cancer and found that the antibiotic vancomycin (which acts on gut bacteria) potentiated the radiation-induced antitumor immune response and inhibited tumor growth. This synergy depended on cross-presentation of tumor-specific antigens to cytolytic CD8 + T cells and on interferon-γ . This grEvidence from both mouse models  and clinFusobacterium nucleatum has been shown to have a role in chemoresistance to colorectal cancer by activating autophagy [Another hypothesis involves the link between radioresistance and autophagy regulation . Digomanutophagy . However+ cytotoxic T cells and CD4+ T helper cells. Taken together, the complex inflammatory reactions launched by the immune system to an irradiated tumor and the surrounding stroma are neither wholly immuno-stimulatory nor immuno-suppressive.Inflammation may also have a role in the sensitivity or resistance of tumors to radiation. A component of the tumor microenvironment, cancer-associated fibroblasts, are involved not only in tumor initiation, progression, metastasis, and angiogenesis but also in immune modulation, including inflammation ; radiatiEnterococcus faecalis, Clostridium perfringens, Bacteroides thetaiotaomicron, and Escherichia coli were found to regulate FIAF production in colorectal cancer cell lines [Streptococcus, Lactobacillus, and Bifidobacterium spp, which led the authors to suggest that administering these probiotics may affect FIAF production and thus perhaps influence the course of colorectal cancer.Other insights about the microbial regulation of intestinal radiosensitivity come from studies of germ-free mice treated with whole-body gamma irradiation. One such study implicated fasting-induced adipose factor (FIAF), also known as angiopoietin-like 4 (ANGPTL4), a microbiota-regulated, epithelial-derived, secreted protein, in radioresistance, and suggested that FIAF may be useful as a gut radioprotector . In anotll lines . Transcrll lines , 45. IndGastrointestinal mucositis is a particularly debilitating side effect of radiotherapy that can lead to significant declines in quality of life as well as treatment delays or dose reductions, which in turn can compromise treatment outcomes . RadiothThe pathobiology of gastrointestinal mucositis has been described elsewhere , 48, butBacteroides, Dialister, Veillonella, and unclassified bacterial species and reduced Clostridium XI and XVIII, Faecalibacterium, Oscillibacter, Parabacteroides, and Prevotella [Clostridium difficile infection, which is associated with high mortality rates [Table evotella , 17. Somty rates . Researcty rates .Table 2SThe influence of the gut microbiome on the pathogenesis of radiation-induced gastrointestinal mucositis  is mediaThe gut microbiome interacts with toll-like receptors (TLR) expressed on epithelial and immune cells to maintain intestinal homeostasis. Depletion of the gut microbiome in mice by using broad-spectrum antibiotics has been associated with increased susceptibility to methotrexate-induced gastrointestinal injury, which is suppressed by the administration of TLR2 ligands . ConversStudies have begun to explore whether modifying the gut microbiome can maximize the response to treatment and minimize adverse effects . Agents Lactobacillus, Bifidobacteria, Saccharomyces boulardii, and Bacillus coagulans are the most common microbiome components used as probiotics [Probiotics are defined as live microorganisms that have a beneficial role in cancer prevention and treatment by reducing the translocation of harmful bacteria, promoting intestinal immune barrier function and antipathogenic activity , 62. Curobiotics , 62. Synobiotics .Propionibacterium freudenreichii and TNF-related apoptosis-inducing ligand (TRAIL) increased proapoptotic gene expression and decreased antiapoptotic gene expression in those cells, suggesting that P. freudenreichii may be useful as an adjuvant for TRAIL-based colorectal cancer therapy. Probiotics have been shown to decrease the incidence and development of carcinogen-induced colorectal cancer in experimental models [Lactobacillus casei has also been found to prevent atypia in colorectal tumors [Bifidobacterium lactis and resistant starch produced unique changes in the fecal microflora in another study of patients with colorectal cancer, it did not significantly alter any other fecal, serum, or epithelial biomarkers [A recent preclinical study of colorectal cancer cells  revealedl models –66. In al models . Among pl models , 69. Lacl tumors . Howeverl tumors , 72. Moromarkers . The automarkers .Indeed, studies of the effect of probiotics on radiation-induced gastrointestinal symptoms are difficult to evaluate, as they vary in the type of cancer patients recruited, the radiotherapy modalities used, the presence or absence of concomitant chemotherapy, end-point assessment, and the types of bacteria used as probiotics. A meta-analysis of six randomized controlled trials investigating probiotics and post-radiotherapy diarrhea suggested that oral probiotics could have beneficial effects in terms of reducing the incidence of diarrhea . AlthougIn 2016, the International Scientific Association for Probiotics and Prebiotics updated the definition of a prebiotic as “a substrate that is selectively utilized by host microorganisms conferring a health benefit.” This definition expanded the concept of prebiotics to include non-carbohydrate substances and applications to body sites other than the gastrointestinal tract , therebyF. nucleatum-positive colorectal cancer, but not F. nucleatum-negative colorectal cancer, suggesting that any association of diet with colorectal cancer risk significantly differed according to tissue F. nucleatum status [Evidence from cell culture and animal models suggests that the consumption of prebiotics can inhibit colorectal carcinogenesis –79. In hm status . In contm status .Notably, not all clinical studies of prebiotic or synbiotic therapies for colorectal cancer have shown conclusive results. Potential reasons for this include (a) differences in the pathogenesis of inflammation, genetic mutations, and epigenetic modifications in patients with colorectal cancer may result in prebiotics or synbiotics having multiple functions; (b) some specific species in the gut microbiome (pathogenic or not) could reduce or suppress the regulatory functions of prebiotics or synbiotics, or even “hijack” these agents to facilitate colorectal cancer progression under certain conditions; and (c) although prebiotics and synbiotics have important roles in modulating immune development and function and in maintaining balance in the gut microbiome, some cases of severe gut microbiome dysbiosis may not be controllable with prebiotics. Addressing these and other potential explanations may allow the use of oral prebiotics or synbiotics to prevent or control colorectal cancer in the future.Fusobacterium load, cancer cell proliferation, and overall tumor growth, which collectively suggested that antimicrobial interventions may be useful for patients with Fusobacterium-associated colorectal cancer [Antibiotics are well known to affect the composition of the gut microbiome, but how these effects interact with the development and progression of colorectal cancer is less clear. In one study of heme-induced carcinogenesis in rats, antibiotics were found to suppress the microbiome by reducing crypt height and proliferation, thereby implicating the microbiome in heme-induced promotion of colorectal cancer . Antibiol cancer . WhetherOther agents, including celecoxib, berberine, isoliquiritigenin, and curcumin, have also been found to decrease the incidence of colorectal tumorigenesis by modulating the gut microbiome –94. ReceAnother potential means of manipulating the gut microbiota has been the use of FMT, in which a fecal suspension is transferred from healthy donors into the gastrointestinal tract of other individuals, with goal of curing specific conditions or diseases by reconstructing the normal function and immune system of the gut microbiome. FMT transplants can consist of fresh stools or frozen fecal capsules, or extracts of bacterial flora from normal fecal flora. Although direct evidence is lacking at present to support the use of FMT for treating colorectal cancer, fecal microbiomes isolated from patients with colorectal cancer have been shown to promote intestinal carcinogenesis in germ-free mice and in mice given a carcinogen , 98. ThiNotably, the effects of FMT on the recipient immune system are complex and unpredictable, and the risk that FMT may lead to dissemination of unknown pathogens cannot be eliminated . NumerouOther novel approaches could include bioengineering the gut microbiome –106, theThe majority of published studies used 16S rRNA sequencing to investigate and compare the taxonomic distribution and diversity of gut microbiome between individuals. However, 16S rRNA only identify bacteria level other than strain level. The functional gut microbiome can be studied using methods involving metagenomics, metatranscriptomics, metaproteomics, as well as metabolomics. The above methods may provide significant functional information for network analyses, and identification of proteins and metabolites produced by gut microbiome.Sequencing the collection of genomes present in an ecosystem is known as metagenomics. Shotgun metagenomics provides an enormous amount of valuable functional information down to the strain level and for all types of microorganisms, therefore is now widely applied , 113. HoConsidering each ‘-omic’ technology provides its own unique perspective of the microbiome and its communication with the host, multiple ‘-omic’ approaches can be applied simultaneously to the same sample to obtain integrated results .The inter-individual variations seen in the response to radiation and in the severity of radiation-related toxic effects remain major challenges in the use of radiotherapy for cancer treatment. Considerable research effort has been devoted to identifying factors that could explain this variation, with particular interest expressed recently in how the gut microbiome influences radiation response and toxic effects. However, many unknowns still remain in attempts to clarify the complex, bidirectional relationship between the gut microbiome and radiation effects.First, the role of the gut microbiome in radiosensitivity is a new concept that has generated a lot of interest, but few original studies have yielded convincing results. The mechanisms underlying how the gut microbiome influences radiosensitivity are still obscure, and much more research is needed to clarify the links between the gut microbiome and variations in radiotherapy response.Second, radiotherapy is increasingly being combined molecular targeted therapy or immunotherapy in the treatment of solid tumors. The mechanisms underlying the synergistic effects of such combinations are a “hot topic” in research, and further information on how the gut microbiome participates in these effects is urgently needed to enhance radiation-based combined treatments for cancer. Because patients participating in clinical trials are already closely monitored, it will be important to include comprehensive microbiome assessments in this monitoring to fully understand the baseline microbiome in cancer patients and to study the effects of various therapies on specific bacterial families and their contribution to therapeutic outcomes.Third, aspects of the microbiome could be used to predict cancer risk, recurrence, response to therapies, and survival—in other words, aspects of the gut microbiome could be useful as predictive and prognostic biomarkers. Future research to investigate the influence of the gut microbiome on the incidence and severity of radiotherapy-induced mucositis is warranted, with a view toward modulating the microbiome composition to improve cancer therapy outcomes.Fourth, findings from most analyses of the gut microbiome undertaken to date have relied on next-generation sequencing. However, the presence of a gene or its transcript does not necessarily indicate protein expression; therefore, direct measurements of expressed proteins via meta-proteomics will be useful for providing precise functional information on the microbiome. Indeed, thorough examinations of the gut microbiome should include metaproteomic analysis, which can reveal both human and microbial functional changes indicative of the host-microbiome interactions. More recently, “microscomics” approach was conducted in human stool samples by transmission electron microscopy, which may further decline the inconsistencies observed with metagenomics and culturomics . This isFinally, previous studies showed that microbial metabolites produced locally can enter the bloodstream and act systemically Fig.  117, 11. Crossta


Metastases cause high mortality in several cancers and immunotherapies are expected to be effective in the prevention and treatment of metastatic disease. However, only a minority of patients benefit from immunotherapies. This creates a need for novel therapies that are efficacious regardless of the cancer types and metastatic environments they are growing in. Preclinical immuno-oncology models for studying metastases have long been limited to syngeneic or carcinogenesis-inducible models that have murine cancer and immune cells. However, the translational power of these models has been questioned. Interactions between tumor and immune cells are often species-specific and regulated by different cytokines in mice and humans. For increased translational power, mice engrafted with functional parts of human immune system have been developed. These humanized mice are utilized to advance understanding the role of immune cells in the metastatic process, but increasingly also to study the efficacy and safety of novel immunotherapies. From these aspects, this review will discuss the role of immune cells in the metastatic process and the utility of humanized mouse models in immuno-oncology research for metastatic cancers, covering several models from the perspective of efficacy and safety of immunotherapies. Primary, localized cancer can be sufficiently treated, resulting in survival rates above 90% for most cancers. The majority of cancer-related deaths are caused by metastases that remain mostly incurable ,2,3. To The metastasis process is influenced by many cells in the local tumor microenvironment (TME) including different immune cells. Blomberg and colleagues have reviewed the parts of the metastatic processes that can be regulated by immune cells . These iIn a tumor promoting inflammation, immune cells create an immunosuppressive microenvironment that enhances tumor growth and progression . A tumorImmune cells can modulate extracellular matrix (ECM) composition and blood and lymphoid vessel formation, and increase motility of cancer cells, leading to increased intravasation and possibility for formation of metastases. ECM can be modulated by TAMs ,19 and TA crucial factor for tumor progression in tissue microenvironment is hypoxia that increases the formation of metastases, and distinct metastatic sites are differentially hypoxic. For example, one of the most hypoxic environments is bone marrow, where hypoxia affects not only tumor cells but also bone marrow and immune cells ,30. HypoAfter being disengaged from the tumor, circulating tumors cells (CTCs) can be killed by different immune cells, which can be prevented by forming multi-cellular aggregated with different immune cells and fibroblasts . CTCs foTumor cells prepare the new metastatic microenvironment already before their arrival, creating so-called pre-metastatic niche. Three major factors crucial for the formation of the pre-metastatic niche are primary tumor -derived components, tumor-mobilized bone marrow-derived cells and local stromal microenvironment of the host . The preAs described above, the formation of metastases is greatly influenced by many immune cells. For this reason, it is rational to think that the use of immunotherapies could be beneficial for the prevention of formation of immunosuppressive microenvironment that would later lead to tumor cell escape for the primary location, and to disable metastases from forming and growing. Immunotherapies are currently studied in different metastatic cancers as mono- and combination therapies . TargetiThe expanding research done in immuno-oncology has created a need for more translational models that would allow the study of interactions between human tumor and human immune cells and importantly, to predictably assess efficacy and safety of immunotherapies . Most prPreclinical models used in immunotherapy assessments have long been limited to using either syngeneic models with injected mouse cancer cells or inducible carcinogenesis models. Human and mouse proteins vary in structure and function, and although cross-reactive antibodies or small molecule inhibitors are developed, intracellular signaling ,58 and pHumanized mice are commercially available, but many laboratories also produce their own mice for research needs. Some experimental protocols are also published for creating these mice ,65,66. BMouse models of reconstituted human immune system (HIS) are often referred as HIS-models that can be created by injecting intravenously either peripheral blood mononuclear cells (PBMCs) or hematopoietic stem cells (HSCs) into recipient hosts at young age. The choice of cells used for engraftment affects the immune cell population in the models. T cells are most abundant in both models, but HSC engrafted mice also have other leukocytes such as B cells and NK cells. Furthermore, depending on the mouse strain used for engraftment, other immune cells may also differentiate, which is discussed later in this section. PBMCs are typically isolated from adult donors and HSCs typically from cord blood, but also human liver and thymus can be used as a source for HSCs ,68. CD34PBMC engraftment allows studies to commence fast because immune cells mature within one to two weeks. However, study length is often limited because of the early onset of graft-versus-host disease (GVHD), limiting the study length to 3–4 weeks . Though The recipient host for human immune cells needs to be immunodeficient. The used mouse strains are most commonly super immunodeficient hosts such as NOG, NSG or BRG mice ,79,80. TThe first used host for the injection of CD34+ cells was the NOD.Scid mouse that showed good engraftment of human immune cells in peripheral blood, spleen and bone marrow . In thesIn these above-mentioned HSC engrafted models, mainly T and B cell mediated effects can be studied due to low number of myeloid-derived cells. However, the myeloid-derived cells have an important role in TME and in metastasis, and the novel humanized mouse models need to also support the development of these cells. One of the first models developed was the transgenic human interleukin 6 (IL-6) NOG mouse . When thHumanized mouse models are relatively new and as they are more used in research, we will gain better understanding of their translational power and limitations in oncology research. It is important to understand the limitations of the models as they affect study planning and interpretation of the results. Major limitations include donor-caused and donor-independent differences in the engraftment and differentiation of human immune cells, and in a larger content, the variability between different mouse strains used as hosts for human immune cells. Limitations related to immune responses observed in these mice may include the lack of certain human cytokines that cause immune activation, and incomplete HLA-matching that causes a non-specific immune reaction especially in tumors with low antigen presentation. Finally, GVHD is a major limitation especially in PBMC models, which limits the timelines for performing a study, as PBMC engrafted mice develop GVHD within 3–4 weeks. On the other hand, HSC engrafted mice are already over 16 weeks old when entering the study and their long-term survival should be noted.When deciding a model for immuno-oncology research, the main criteria should be the human cells of interest and how they are presented in the humanized mouse models. In immuno-oncology studies, follow-up of tumor size should not be the only main outcome, and attention should be paid to the expression of immune markers in TME and immune cell infiltration. In the next section we will focus on the use of humanized models in metastasis research.Humanized mouse models have not yet been widely utilized in metastasis studies. Development of metastases in humanized mice has mostly been monitored only from subcutaneous and orthotopic cancer cell injection models. Generally, it is thought that tumor growth, especially in subcutaneous models, does not vary if the models are performed in immunodeficient or humanized models . Still, Gammelgaard and colleagues studied the growth and metastasis of human melanoma A375 cell line and two human triple-negative breast cancer (TNBC) cell lines, MDA-MB-231 and MDA-MB-468 in CD34+ HSC transplanted BRGS mice . In theiShafiee and colleagues developed an advanced bone metastasis model with both human bone  and human CD34+ human immune cells . When miWagner and colleagues also studied bone and used SAOS-2 human osteosarcoma cells in orthotopic humanized tissue engineered bone constructs (ohTEBC) placed in the femur of mice engrafted with human CD34+ cells . OsteasaThe above cited studies show that immune cells have a role in the formation and growth of metastases in preclinical models. The essential question that arises is: are different immunotherapies able to prevent or treat metastasis in these models? This has been evaluated, for example, in the models for TNBC, melanoma, bladder, adrenocortical carcinoma, osteosarcoma and bone metastasis .TNBC is unresponsive to hormonal therapies and often highly metastatic, which increases the need for new therapeutic strategies, including immune targeted therapies. Rosato and colleagues studied TNBC using the patient-derived xenograft (PDX) tumor line MC1 in CD34+ HSC-engrafted NSG mice in comparison to non-humanized NSG mice . They obMetastatic melanoma has a poor prognosis and the development of novel therapies is an unmet clinical need . Kuryk aIn order to improve models where immunotherapies could be tested against bladder cancer and metastasis, Blinova and colleagues established subcutaneous PDX models of basal, luminal and p53 subtypes of primary and relapsed non-muscle invasive bladder cancer (NMIBC) in human lymphocyte transplanted mice . All modAdrenocortical carcinoma (ACC) is an aggressive malignancy with very limited treatment options that eventually develops into metastatic disease in most patients. There is rationale to assess efficacy of immunotherapies in ACC , althougImmunotherapies have often been studied in context to solid tumors, and there are less studies relating to musculoskeletal tumors such as osteosarcoma. Zheng and colleagues studied the effects of an anti-PD-1 antibody (nivolumab) on osteosarcoma growth and metastases using human KHOS cells injected subcutaneously in a PBMC humanized mouse model . NivolumIn context to bone, novel bone metastasis models have been established in humanized mice. While observing anti-tumor efficacy of pembrolizumab in a primary orthotopic TNBC model with MDA-MB-231(SA)-luc cells, no efficacy was observed against the tumor or tumor-induced bone changes in an intratibial model that mimics growth of bone metastases . In a prTaken together, the data available indicates that metastasis can be modelled in humanized mice and the metastatic prolife and immunological contexture of human tumors is preserved when the tumors are grown in humanized mice. Furthermore, both in preclinical models and patients, metastases can respond to immunotherapies differently than primary tumors. Also, metastases growing in different locations such as lung and liver may have differences in their response to immunotherapies. One promising example of translational power of humanized mouse models in metastasis research has been shown in ACC where preclinical findings of anti-PD-1 efficacy in humanized mice correlates with clinical responses. A more detailed analysis of metastases can help to guide immunotherapy development towards more effective novel compounds that could be beneficial especially for metastatic patients.Immunotherapies induce immune-related adverse events (irAEs) that can occur in patients early or late after treatment . As manyWeaver and colleagues studied nivolumab in a BLT humanized mouse model in NOG and transgenic hGM-CSF and IL-3 NOG (NOG-EXL) mice . Though The life-threatening complication cytokine release syndrome (CRS) is a potential adverse effect of immunotherapies  and testThe anti-CTLA-4 antibody ipilimumab that has been approved for the treatment of metastatic melanoma and advanced renal cell carcinoma is associated with severe irAEs, including rash and colitis . Du and IL-2 based therapies have been accepted for the treatment of malignant melanoma and renal cell carcinoma. They expand T cells and are associated with dose- and dosing schedule-dependent severe toxicities. Li and colleagues developed a HIS model that recapitulated clinical symptoms of IL-2 immunotherapy, including vascular leak syndrome and cytokine storm . The advOur own research also revealed unexpected adverse effects in a female CD34+ humanized mouse model . HumanizTaken together, clinically observed irAEs can be predicted especially in BLT humanized mouse models. This is probably caused by more complete antigen presentation and enhanced immune activation in BLT models compared to HSC models as discussed earlier. Furthermore, these models provide an improved translational tool for studying adverse effects because they have the biological and functional human immune system. Species-related differences in predicting adverse effects has been shown for example in the TGN1412 analogue studies, where CRS was observed in humanized mice but not in other model systems such as non-human primates. As immunotherapies induce major irAEs in patients, these novel models should be increasingly used in early phases of preclinical development in order to develop safer products and predict the type and onset of irAEs in clinical trials.Immunotherapies are an emerging treatment option for metastasizing cancers that are currently undergoing clinical investigations in many cancer indications. As discussed in this review, immune responses to metastases may vary depending on the tumor type and stage, taking into account the local tissue microenvironment, which makes it more challenging to interpret not only preclinical but also clinical findings. Lack of understanding interactions between immune system and metastasis in preclinical models highlights the need for further basic and translational research in this field. The data cited in this review shows the potential of humanized mouse models in metastasis research. However, new emerging data is still needed to show the true translational power of these models when preclinical findings are validated in clinical studies.Novel preclinical metastasis models in humanized mice give us the possibility to understand the role of immune regulation in tumor growth and metastasis. Still, limitations of these models should be noted, and new preclinical metastasis models need to be established. Development of models with multiple humanized components and focusing especially on the metastatic tumor microenvironment will be of great value. These novel metastasis models will hopefully provide new opportunities for preclinical drug development both in terms of efficacy and safety assessment of immunotherapies.


Despite great advancements in early detection and therapeutic strategies, the 5-year survival rate for patients with metastatic prostate cancer remains low . Targeting prostate cancer-associated genes has emerged as a promising treatment for this devastating disease. This review summarizes recent findings in silencing genes that are involved in prostate cancer pathogenesis. Moreover, novel nanotechnology-based platforms for effective delivery of therapeutic RNAs to prostate cancer cells have been discussed. Information provided in this review will benefit both researchers and clinicians to design and develop novel therapeutic approaches for patients suffering from prostate cancer.Short interfering RNAs (siRNAs) have provided novel insights into the field of cancer treatment in light of their ability to specifically target and silence cancer-associated genes. In recent years, numerous studies focus on determining genes that actively participate in tumor formation, invasion, and metastasis in order to establish new targets for cancer treatment. In spite of great advances in designing various siRNAs with diverse targets, efficient delivery of siRNAs to cancer cells is still the main challenge in siRNA-mediated cancer treatment. Recent advancements in the field of nanotechnology and nanomedicine hold great promise to meet this challenge. This review focuses on recent findings in cancer-associated genes and the application of siRNAs to successfully silence them in prostate cancer, as well as recent progress for effectual delivery of siRNAs to cancer cells. Prostate cancer (PCa) is the most commonplace malignancy in men along with the second most frequent cause of cancer-related death in males in the Western population, just behind lung cancer . In 2020Recent advancements in genetic research have provided numerous innovative tools for treatment of various abnormalities, from inflammation to diverse types of cancers. In line with this, RNA interference (RNAi) has attracted tremendous attention as a novel therapeutic tool in the clinical setting among the research community merely after it was discovered in the 1990s . Short iBRCA1 (a human tumor suppressor gene), consequently triggering significant tumor growth and poor survival rate. Using siRNAs to target EF2K significantly declines cell proliferation, migration, and invasion of the cancer cells. Additionally, delivery of EF2K-specific siRNAs into BRCA1-mutated breast cancer in an orthotopic xenograft model by silica-coated cobalt-ferrite nanoparticles considerably diminishes tumor growth and metastasis [There is growing evidence proving that siRNAs can be considered as promising therapeutics for many human pathologies, namely cancer, cardiovascular diseases, and other abnormalities of genetic etiology ,19. Canctastasis . Furthertastasis . Consisttastasis . These dNumerous proteins are upregulated in cancers and have crucial roles in cancer development. The precursor of nerve growth factor is one of the proteins that was recently shown to be overexpressed in pancreatic cancer tissues and cell lines. It was reported that knockdown of this specific growth factor by siRNA diminishes cell proliferation, migration, and invasion, and promotes anoikis of pancreatic cancer cells . SimilarsiRNAs in combination with other therapeutic agents including chemotherapeutic drugs have exhibited synergetic effects in the treatment of various cancers. It has been shown that gene silencing of Notch-1, Wnt/β-catenin, and STAT3 alone or in combination significantly enhances the chemosensitivity of both doxorubicin-sensitive and -resistant MCF-7 breast cancer cells to doxorubicin, indicating the potential role of siRNA in the enhancement of the anticancer efficacy of conventional therapies . FurtherBRCA1/2 mutant tumors [BRCA1/2 mutation. PARP1 silencing reduces cell migration and invasion in vitro and inhibits tumor growth in a PC3 xenograft model. Notably, knockdown of PARP1 increases the induction of apoptosis in PCa treated with docetaxel, implying that PARP1-siRNA may be a potential therapeutic agent against PCa by application either alone or in combination with other therapeutic tools [As alluded to above, RNAi offers a novel and beneficial strategy in the field of cancer therapy. Despite the late discovery, siRNAs have been developed to silence countless genes responsible for diverse PCa hallmarks, including angiogenesis, invasion, and metastasis. In line with this, knockdown of a dual specificity protein kinase TTK, which is involved in chromosome segregation during mitosis, by designed siRNA, has been reported to be able to reduce proliferation, invasion, and migration of PC3 and DU145 PCa cells and initiate cell death . Once apt tumors . Lai et t tumors  reportedic tools . BMI1) and a transcriptional factor (COMMD3) has been recently identified in PCa, which triggers disease recurrence and poor survival [COMMD3:BMI1 fusion gene with siRNA leads to reduced c-MYC expression and decreased tumor cell proliferation both in vitro and in metastatic tumors in a xenograft mouse model of PCa [TMPRSS2:ERG gene fusion is the most common genomic alteration identified in PCa, leading to overexpression of the transcription factor ERG [Fusion genes are a class of oncogenes that have been found in many cancer types including PCa and caused by genomic rearrangements . Gene fusurvival . It was l of PCa . Moreovector ERG . It was ctor ERG . Several cell membrane proteins that are overexpressed in PCa have been shown to play decisive roles in cell development and tumor progression. For example, transmembrane channel-like 5 (TMC5) is upregulated in PCa cells, and knockdown of TMC5 using siRNA leads to inhibition of PCa cell proliferation through cell cycle arrest at the G1 phase . MoreoveHypoxia is one of the key characteristics of tumors and correlates with poor prognosis of cancer patients. Dai et al.  demonstrThere are several proteins in cells that are overexpressed during prostate malignancy and play crucial roles in both tumor formation and invasion. For instance, the association between overexpression of a small ribosomal protein subunit 7 (RPS7) with tumor growth has been highlighted in various studies ,49. It wAnother imperative factor in tumor progression is a proteoglycan protein named endothelial cell-specific molecule-1 (ESM-1). It is overexpressed in various malignancies and mediated by inflammatory cytokines and proangiogenic growth factors . InteresIn summary, there are myriad genes that are overexpressed during prostate malignancy and participate actively in cell proliferation, invasiveness, and tumor progression. Targeting these upregulated genes offers a promising strategy for preventing tumor progression and increasing survival among patients suffering from PCa. As validated in various studies, designing and developing siRNAs that can effectively silence genes involved in tumor pathogenesis has shown encouraging outcomes in PCa treatment. Hence, they might be applied as alternative tools to conventional treatments or be used in combination with other treatments to improve their efficacy. Drug resistance in tumor cells remains the central cause of treatment failure, leading to tumor recurrence and metastasis . Drug reIn recent years, cancer immunotherapy has gained vast attention in light of its capabilities of augmenting the host immune response towards cancer cells . siRNA hDespite the fact that siRNA therapy is a promising strategy for cancer treatment, there are some concerns regarding their applications in clinical therapy. Naked siRNAs have very short half-lives in vivo and undergo rapid clearance . MoreoveOwing to their unique and easy modification properties, dendrimers have emerged as a promising system for gene/drug delivery in cancer therapy ,82. For Both the EGFR signaling pathway and survivin are involved in cancer cell proliferation, tumor vascularization, and metastasis . Lui et Since its discovery, siRNA holds great promise to treat a number of diseases, including cancer, owing to its specificity to target and silence genes that are correlated with the pathogenesis of cancer, from cell proliferation and invasion to immunosuppression . Recentl


The observation that cancer acquires significant changes in its metabolism dates back nearly a century, to Otto Warburg noting that cancer cells preferentially utilize glycolysis even when there are no hypoxic conditions in the growth media. Altered energetics are thus considered a hallmark of cancer. However, it has become clear that altered metabolism is not limited to cellular energetic pathways. Alterations in amino acid synthesis and catabolism, lipid biogenesis, and other pathways such as polyamine processing are commonly seen in cancer. Additionally, alterations in metabolism do not only have profound effects for cancer cells but also affect their surrounding microenvironment. With new cancer therapeutics targeting the immune microenvironment, these effects may have implications on cancer growth and response to therapy. These interactions are profound in lung cancer, further demonstrating the manifold interactions between developing tumors and the inflammatory microenvironment. Here, we discuss how dysregulation of metabolism in cancer alters its microenvironment and how this newfound knowledge can be exploited for anticancer treatment. As first reported by Warburg in 1923, cancer cells prefer glycolytic fermentation even in the presence of oxygen –8. GivenThe Warburg effect is a common finding in cancer, and increased glucose utilization has been exploited to aid in detection, for instance, using fludeoxyglucose as a tracer in positron emission tomography (PET) scans . ResearcWhile the Warburg effect is a common observation in cancer cells, and glycolytic inhibitors are currently being studied as potential therapeutics, it is clear that there is significant metabolic plasticity and heterogeneity both within cancers and between cancer cells and their microenvironment. Many cancers continue to generate energy using standard oxidative phosphorylation in the mitochondria, even while utilizing the glycolytic Warburg pathway . AdditioCancer cell energetics thus serve as a broader paradigm for oncometabolomics in a number of ways. Dysregulation is often a consequence of oncogenic mutation, but also may be a consequence of the evolutionary processes by which cancer cells overcome nutrient limitation and immune surveillance. Metabolic dysregulation within the cancer cells leads to profound changes both within cells and the microenvironment. While dysregulation of a metabolic pathway may not have a solitary cause, the same changes are observed frequently and thus serve as an enticing target for therapy. However, linkage between pathways, redundancy, heterogeneity, and plasticity may mean that metabolic inhibition may form only a piece in a larger puzzle. A similar example can be seen in folate and nucleobase synthesis pathways, which both support oligonucleotide synthesis and function as antioxidants to mitigate the effect of ROS. These changes may arise through oncogenic mutations such as those which activate the mammalian target of rapamycin (mTOR) pathway but also may represent the surviving cancer cells that can best support their unchecked cell division requirements and oxidative burden. These pathways are tied closely both to energetics and amino acid metabolism, and depletion of precursor molecules such as serine from the microenvironment has direct effects on T-cell function. Finally, this pathway is a classic example of how cancer metabolism can be targeted as antimetabolites targeting this pathway such as aminopterin, methotrexate, and fluorouracil were among the first chemotherapeutic agents to come into wide usage. However, these agents are commonly used as one part of a combination chemotherapy regimen, for instance, the combination of fluorouracil with irinotecan as current first-line therapy in metastatic colorectal cancer.Examples of these highly interregulated pathways, with effects in the tumor cell and the microenvironment, are emerging throughout oncometabolomics. In lung cancer, there is a close relationship between developing cancers and the immune microenvironment, with both pro- and anti-tumor interactions changing dynamically through carcinogenesis –20. HereDepletion of the essential amino acid tryptophan and accumulation of its catabolites including kynurenine (Kyn) lead to a microenvironment that is suppressive of an immune response and thus leading to promotion of tumor growth phenanthridin-4(1H)-one) (glutaminase inhibitor 968), Bis-2-ethyl sulfide (BPTES), and telaglenastat (CB-839). Glutaminase inhibitor 968 is an allosteric inhibitor of KGA and GAC , whereasde novo nucleotide synthesis and increasing reliance on generation of endogenous glutamine via glutamine synthetase (GS). Blocking of GS reduced FOXP3+ Treg cell proliferation and maintenance under glutamine restriction .Collectively, these studies suggest that targeting glutamine metabolism in tumors may attenuate glutamine depletion in the local tumor microenvironment and enable a more potent antitumor immune response. To this end, previous studies in a CT26 colon cancer syngeneic mouse model demonstrated that the combination CB-839 plus anti-PD-1/anti-PD-L1 yielded superior anticancer efficacy compared to either treatment alone . More rein vitro were first reported over 70 years ago (Arginine has multiple metabolic fates depending on cell type, developmental stage, and state of health or disease. Normally, a semi-essential amino acid in humans and most other mammals, endogenous arginine synthesis proceeds primarily through the “intestinal–renal axis”: citrulline precursor derived from glutamine, glutamate, or proline in the intestine is converted to arginine in the proximal tubule cells of the kidney . Elevateears ago . Many caears ago . Deprivaears ago –77. Whilears ago .de novo sensitivity to platinum chemotherapy (In contrast to arginine auxotrophs, many epithelial tumor types overexpress ASS1, particularly those that are characterized by moderate to high otherapy . These iotherapy , 81. In otherapy . Downstrotherapy .Metabolic alterations in cancer are not only a consequence of oncogenic mutation or of evolution through nutrient limitations. Rather, there is a complex interplay of metabolic precursors and by-products between a cancer cell and its surrounding environment. Recent advances highlight that these metabolic shifts modulate not only tumor growth but the host immune and microenvironmental response. Given the profound effect of these pathways in lung cancer, these pathways hold tremendous potential for biomarker development and intervention.EO, JF, and JV prepared and reviewed the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Most persons with fetal alcohol spectrum disorders (FASDs) remain undiagnosed or are diagnosed in later life. To address the need for earlier diagnosis, we previously assessed miRNAs in the blood plasma of pregnant women who were classified as unexposed to alcohol (UE), heavily exposed with affected infants (HEa), or heavily exposed with apparently unaffected infants (HEua). We reported that maternal miRNAs predicted FASD-related growth and psychomotor deficits in infants. Here, we assessed whether fetal sex influenced alterations in maternal circulating miRNAs following prenatal alcohol exposure (PAE). To overcome the loss of statistical power due to disaggregating maternal samples by fetal sex, we adapted a strategy of iterative bootstrap resampling with replacement to assess the stability of statistical parameter estimates. Bootstrap estimates of parametric and effect size tests identified male and female fetal sex-associated maternal miRNA responses to PAE that were not observed in the aggregated sample. Additionally, we observed, in HEa mothers of female, but not male fetuses, a network of co-secreted miRNAs whose expression was linked to miRNAs encoded on the X-chromosome. Interestingly, the number of significant miRNA correlations for the HEua group mothers with female fetuses was intermediate between HEa and UE mothers at mid-pregnancy, but more similar to UE mothers by the end of pregnancy. Collectively, these data show that fetal sex predicts maternal circulating miRNA adaptations, a critical consideration when adopting maternal miRNAs as diagnostic biomarkers. Moreover, a maternal co-secretion network, predominantly in pregnancies with female fetuses, emerged as an index of risk for adverse birth outcomes due to PAE. Prenatal alcohol exposure (PAE) is common. For instance, in a recent state-wide study in Texas, we found that 8.4% of assessed newborn infants were positive for a blood alcohol metabolite, indicative of at least 3rd trimester PAE  and unplIn attempting to address the critical need for better diagnostic tools, we and others previously reported that maternal plasma miRNAs in both sheep  and humaSex differences in miRNA secretion emerge as early as the blastocyst stage of embryogenesis and influence maternal endometrial function , and sexCollectively, the above studies support the hypothesis that fetal sex is one determinant of the maternal response to alcohol exposure. Moreover, if maternal plasma miRNAs are to be used as predictive biomarkers for the effects of PAE, the association between fetal sex and alterations in maternal miRNAs needs to be assessed. However, small sample sizes in typical FASD studies in human populations are a significant barrier since the disaggregation of data by fetal sex results in decreased sample size and, therefore, decreased statistical power. To overcome this limitation, we employed a statistical strategy of iterative bootstrap resampling with replacement  to simulAll study protocols were approved by the Institutional Review Boards at the University of California San Diego and Texas A&M University and for data collection at Lviv National Medical University in Ukraine. The study sample was drawn from a larger prospective cohort recruited at perinatal clinics in two regions of Ukraine, in the Rivne and Khmelnytsky provinces, by OMNI-Net teams. Recruitment methods and cohort characteristics have been described elsewhere , 17, 20.Plasma samples were processed and assessed for 752 miRNAs using the Exiqon/Qiagen Human panel I+II, V5, miRCURY LNA miRNA miRNome PCR Panel, as described previously, including the assessment of absorbance at 414 nm and mRNA expression of Slc4a1/Band3 to detect hemolysis .https://github.com/nihalasalem/Ukraine_Analysis). For each of the 153 miRNAs, maternal samples for each exposure group  were resampled with replacement for 2000 iterations, consistent with published recommendations .


In addition, the use of cardiac decellularized extracellular matrix has been recently suggested as promising biomaterial to develop novel therapeutic strategies for myocardial repair, as either source of molecular cues for regeneration, biological scaffold for cardiac tissue engineering or biomaterial platform for the functional release of factors. In this review, we will specifically address the translational relevance of these two approaches with ad hoc interest in their feasibility to rejuvenate endogenous mechanisms of cardiac repair up to functional regeneration.True cardiac regeneration of the injured heart has been broadly described in lower vertebrates by active replacement of lost cardiomyocytes to functionally and structurally restore the myocardial tissue. On the contrary, following severe injury  the adult mammalian heart is endowed with an impaired reparative response by means of meager wound healing program and detrimental remodeling, which can lead over time to cardiomyopathy and heart failure. Lately, a growing body of basic, translational and clinical studies have supported the therapeutic use of stem cells to provide myocardial regeneration, with the working hypothesis that stem cells delivered to the cardiac tissue could result into new cardiovascular cells to replenish the lost ones. Nevertheless, multiple independent evidences have demonstrated that injected stem cells are more likely to modulate the cardiac tissue via beneficial paracrine effects, which can enhance cardiac repair and reinstate the embryonic program and cell cycle activity of endogenous cardiac stromal cells and resident cardiomyocytes. Therefore, increasing interest has been addressed to the therapeutic profiling of the stem cell-derived  Cardiovascular disorders significantly affect life expectancy; according to the World Health Organization (WHO), by 2030 about 23 million people annually will be severely affected by heart failure . Cardiacde novo generation of cardiomyocytes can actually occur, although via very low self-renewal rate (ca. 0.5–1% per year), which is not therapeutically relevant per se . All such stem-like cell populations can be isolated from discarded tissue obtained during heart surgery or endocardial biopsy according to different protocols, as extensively reviewed in Evidences that the mammalian heart possesses some restorative potential have been supported by the identification of resident stromal mesenchymal cells, defined as cardiac progenitor cells (CPC). CPC were originally reported in 2003 when describing Linke cells . Since ti) preservation of viable myocardium during injury or disease and (ii) replacement of cardiomyocytes to restore structural and functional integrity of the damaged heart activating endogenous CPC  the stimulation with stem/progenitor cell-derivatives and (b) the innovative use of cardiac decellularized extracellular matrix dECM, . In thisData from clinical and preclinical studies on stem cell-based therapy for cardiac disease have shown that cells transplanted into compromised myocardium are very unlikely to contribute to novel cardiovascular cells by means of differentiation . Multipladvanced therapy medicinal product (ATMP) that could be manufactured via scale-up in vitro culture systems. Therefore it could offer the big advantage of being promptly accessible to cardiovascular patients, whenever needed; moreover, this strategy may overcome limits and concerns related to canonical cell therapy, including, for instance, donor cell engraftment, immune-compatibility and cost-effective, time-consuming procedures to provide an high amount of cells to be injected  or clinical waste material at birth ; specifically, they are endowed with remarkable proliferative potential along with ease of isolation, while avoiding ethical issues, as extensively reviewed in in vitro amplification challenging  as suitable source. MSC with relevant paracrine potential for cardiovascular disease have been described as isolated from either bone marrow or adipose tissue, which represent easily accessible cell options. Likewise, fetal and perinatal MSC obtained from extra-embryonic annexes such as placenta tissue , umbilicllenging .Within the stem cell paracrine scenario, increasing attention has turned toward cell-secreted EVs, as functional biological conveyors of modulatory influence. EVs are phospholipid micro- and nano-vesicles that act as key mediators of inter-cellular communication affecting cellular functions. EVs are very heterogeneous and can be further sub-classified based on their size: from nano-scaled exosomes (ranging from 35 to 150-200nm) to medium-sized micro-vesicles (from 200 to 500nm) up to apoptotic bodies (>500 nm) . EVs carin vivo infiltration along with quenching of severe inflammation following skeletal muscle tissue damage  signaling , produced a pro-angiogenic secretome driving tubulogenesis in HUVEC cells  within the injured myocardium. Systemic delivery of putative therapeutic paracrine factor(s), as the less invasive and more clinically compliant option, may be significantly restrained by the meager homing of the treatment to the cardiac tissue, as likely rapidly sequestered by off-target organs/tissues; thus, optimization of local administration would offer significant improvements.In order to implement heart regenerative strategies, devices made from ECM and other biomaterials were recently developed as systems capable of delivery therapeutic factors. These ECM-derived scaffolds, gels, and protein suspensions, are able to both convey cells and factors, and to enhance survival and regeneration of the heart. Importantly, significant therapeutic benefits were obtained when these ECM-derived scaffolds were used stand-alone in post ischemic models . These sInitially, tissue ECM was considered a biologically inert space, able to provide only a physical support to the attached cells. However, it is now clear that ECM is a dynamic and complex network of fibrous and adhesive proteins, serving as reservoir of different bioactive peptides and growth factors. Cell behavior is influenced by biochemical and biomechanical signaling present in the tissue microenvironment, including ECM, which is a dealer of cellular processes such as proliferation, differentiation, migration, and survival . In ordeWithin the cardiovascular scenario, a variety of naturally derived dECM sources have been investigated (with or without cells/factors) as cardiac patch or injected directly into the myocardium as hydrogel . Since iin vivo implantation could allow improvement in the mechanisms of cardiac repair and regeneration. From one hand, dECM could provide the infarcted myocardium with incisive mechanical compensation. In fact, tissue mechanical properties are mainly determined by ECM. From the other hand, the structural, ultrastructural and mechanical characteristics of dECM could serve as physical messenger for the delivered cells or infiltrated host cells to augment cardiovascular differentiation and tissue regeneration. Together with the mechanical properties, preservation of biochemical cues within dECM could be desirable for cell attachment, proliferation and stem cell differentiation, both in vivo and in vitro, as demonstrated in many studies  are easy to obtain, but may carry residual immunogenicity and may be contaminated with biological agents. On the other hand, human allogeneic tissues would be the ideal material to obtain dECM suitable for implantation or generation of hydrogel. Together with cadaver organ donation, human tissue Biobanks are important sources to augment organ availability , especiain vitro . More rein vitro .When considering possible clinical translation of the most recent advances in cardiovascular research, there are relevant open questions that still need to be addressed. These include the optimal time point of intervention following injury/during disease, and the most suitable strategy to pursue in terms of enhancement of cardiac repair over stimulation of endogenous regeneration, according to the cardiovascular patient’s specific needs . In this scenario, innovative cardiac regenerative strategies, based on either paracrine or cell-therapy approaches, may be challenged by poor cardiac tropism of cells/factors injected systemically and may require local or intra-myocardial administration to enhance their effects. Novel bio-scaffold/hydrogel formulations can increase and enhance retention and release; yet they may depend on topical delivery, and thus being indicated as concomitant strategy during heart surgery or percutaneous coronary intervention/angioplasty. While not ideal for any kind of cardiovascular patient, they can still be therapeutically relevant for acute treatments, such as prompt intervention on patients experiencing MI. The functional combination of prompt EV modulatory effects with the tunable biological properties of dECM hydrogels may represent an ideal delivery platform to enhance intrinsic mechanisms of cardiac restoration. Another relevant cardiovascular scenario is then represented by congenital heart defects, which may affect up to ca. 35.000 newborns annually, significantly impacting on patient morbidity and mortality . PalliatEM: manuscript writing, analytical discussion of decellularized extracellular matrix biology and applications, and figure design. PG: manuscript writing, critical discussion of stem cell paracrine biology. RQ: manuscript writing, contribution to critical discussion on cardiac repair. MP and SB: conception and design, manuscript writing and supervision. All authors reviewed the manuscript and approved it.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.


Mounting evidence indicates that the presence of cardiovascular disease (CVD) and risk factors elevates the incidence of cognitive impairment (CI) and dementia. CVD and associated decline in cardiovascular function can impair cerebral blood flow (CBF) regulation, leading to the disruption of oxygen and nutrient supply in the brain where limited intracellular energy storage capacity critically depends on CBF to sustain proper neuronal functioning. During hypertension and acute as well as chronic CVD, cerebral hypoperfusion and impaired cerebrovascular function are often associated with neurodegeneration and can lead to CI and dementia. Currently, all forms of neurodegeneration associated to CVD lack effective treatments, which highlights the need to better understand specific mechanisms linking cerebrovascular dysfunction and CBF deficits to neurodegeneration. In this review, we discuss vascular targets that have already shown attenuation of neurodegeneration or CI associated to hypertension, heart failure (HF) and stroke by improving cerebrovascular function or CBF deficits. The cerebral circulation plays a critical role in matching nutrient and oxygen supply to neuronal activity and thus, is intimately linked to proper brain function . Howeverhypertension and heart failure (HF) (stroke), but also for reducing the risk of developing CVD-associated neurodegeneration that may lead to the development of CI and dementia , the most prevalent disease burden worldwide , structuure (HF) . Thus, cdementia . Althougdementia , it is sdementia . This arChronic hypertension is the most prevalent cardiovascular disorder and the leading cause of cardiovascular and cerebrovascular morbidity and mortality worldwide . It is oAngiotensin II (AngII), the primary effector hormone of the renin angiotensin system (RAS), is considered the main contributor to impairments of neurovascular coupling independent of blood pressure . It is sRecent evidence has emerged that statins, cholesterol-lowering drugs, may be beneficial in hypertension, specifically for the reversal of cognitive decline . HyperteHeart failure-associated morbidity has grown as the frequently accompanying cognitive decline majorly affects disease outcome, accelerating disease progression by reducing the ability to execute self-care activities and treatment compliance . EpidemiIn healthy patients, cerebral auto-regulatory mechanisms can compensate for fluctuations in cardiac output and blood pressure by lowering cerebrovascular resistance . During Another target of pre-clinical investigation in attempt to link HF and neurodegeneration is inflammation associated to CBF deficits. In a mouse model of slowly developing HF, vascular CI was observed during the early stages of HF and interestingly, preceded the development of left ventricular dysfunction . PreciseStroke is associated with the highest incidence of severe disability, of which neurological deficits are reported in 50% of all patients 6 months after a stroke event . IschemiClinicalTrials.gov Identifier: NCT03686163). Although effects on CBF are warranted for most of these studies, angiogenesis-boosting therapy to re-establish proper perfusion evolved as promising therapeutic target in acute stroke therapy. Besides testing the classical growth factor-mediated therapy approaches in rodent models, AT1R blockers, which are widely used anti-hypertensive medications, were re-discovered to show pro-angiogenic effects after stroke and were reported to increase CBF in the ipsilateral hemisphere  represents one of the most researched targets to improve CBF after stroke despite the controversy regarding its effects. Studies using VEGF treatment post-stroke revealed a clear importance of administration route and timing. VEGF injected systematically in the acute post-stroke phase induces BBB leakage and hemorrhagic transformation  accompanmisphere . Similarmisphere . Nonethemisphere , 2005, wmisphere .Similar to cerebrovascular ATR effects, modulation of S1P receptors has shown beneficial effects on cerebrovascular function and CBF in experimental stroke. The selective S1PR1 agonist SEW2871 revealed positive effects on CBF, potentially resulting from increased diameter of leptomeningeal collateral vessels and enhanced vasodilatation in leptomeningeal anastomoses associated with increased phosphorylation of endothelial NOS in the ipsilateral hemisphere during chronic cerebral hypoperfusion . BesidesIn a model of subarachnoid hemorrhage (SAH), a form of stroke that originates from a hemorrhage but often exacerbates through secondary ischemia , increasOverall, targeting the cerebral vasculature and improving vascular function and thus, normalizing CBF post-stroke evolved as promising path to improve post-stroke recovery. More studies are needed that evaluate CBF effects of different treatment strategies in addition to the commonly used assessment criteria for stroke outcome, such as infarct volume and neuroscore.We have come a long way to appreciate the association between CVD and cognitive function. Hence, efforts to preserve and restore cerebrovascular function and integrity drastically increased. The identification of important drivers of vasoregulation and thus, CBF is more and more valued to possess the capacity as therapeutic intervention early in disease processes to either prevent neurodegeneration or boost neuro-regeneration not only in CVD but also in classical neurodegenerative diseases. However, our mechanistic knowledge relating CVD-associated cerebrovascular dysfunction to neurodegeneration and potentially CI is still very fragmented, with only few targets with larger applicability emerging thus far. Particularly, the modulation of AT1R or S1P signaling has shown promising effects on cerebrovascular function and CBF and in turn, neuronal function in different pre-clinical disease models . Other eLV, HM, and AM wrote the first draft of the manuscript. ND-D and FU wrote sections of the manuscript. All authors contributed to the manuscript revision, read, and approved the submitted version.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.


Sleep-wake disorders are highly prevalent disorders, which can lead to negative effects on cognitive, emotional and interpersonal functioning, and can cause maladaptive metabolic changes. Recent studies support the notion that metabolic processes correlate with sleep. The study of metabolite biomarkers (metabolomics) in a large-scale manner offers unique opportunities to provide insights into the pathology of diseases by revealing alterations in metabolic pathways. This review aims to summarize the status of metabolomic analyses-based knowledge on sleep disorders and to present knowledge in understanding the metabolic role of sleep in psychiatric disorders. Overall, findings suggest that sleep-wake disorders lead to pronounced alterations in specific metabolic pathways, which might contribute to the association of sleep disorders with other psychiatric disorders and medical conditions. These alterations are mainly related to changes in the metabolism of branched-chain amino acids, as well as glucose and lipid metabolism. In insomnia, alterations in branched-chain amino acid and glucose metabolism were shown among studies. In obstructive sleep apnea, biomarkers related to lipid metabolism seem to be of special importance. Future studies are needed to examine severity, subtypes and treatment of sleep-wake disorders in the context of metabolite levels. Sleep is a pivotal operating state of the central nervous system, which controls organismal well-being and might be the most well-preserved activity throughout the evolutionary timescale ,2. SleepOverall, many individuals are affected by sleep disorders. Insomnia has a prevalence of 10% to 15% in the general population. Sleep apnea represents the second most prevalent sleep disorder with about 10%, followed by other sleep disorders, such as restless legs syndrome and circadian rhythm disorders . GrowingRecent studies support the notion that metabolic processes correlate with sleep . The gloThe following chapters summarize studies on the use of metabolomics approaches to reveal metabolic changes occurring due to alterations in sleep-wake activity. Therefore, a literature search was conducted using the Scopus, PubMed, and APA Psycinfo databases. Research articles in scientific journals on experiments using animal models or human subjects were considered. The search was conducted in July 2020, with no limitations on the publication date. Articles were identified by searching for titles using the following search terms: “metabolom*” AND “sleep* OR insomnia* OR apnea”. Despite the strategic literature search strategy, the current study does not follow PRISMA  guidelines for systematic reviews .Insomnia is regarded as a disorder in itself that needs independent clinical attention . TherefoStudies suggest that poor or decreased sleep causes metabolic changes in peripheral metabolism . In indiOverall, most studies conducted thus far imposed short-term sleep deprivation and/or sleep restriction protocols in normal subjects; however, these are not reflective of long-term sleep habits . A recent study aimed to fill this gap by investigating real world sleep habits in insomnia in humans. More specifically, metabolomics was applied on fasting plasma samples from 277 individuals over the course of a year where participants kept to their normal sleep habits. Here, 64 metabolites were found to be associated with sleep timing, with representation from all classes detected . ElevateDespite a lack of  chronic insomnia models that might enable the identification of insomnia biomarkers, some biomarkers can be suggested. Among those, alterations in BCAA and glucose metabolism show similarities among studies ,31,32.Taken together, these changes suggest that bedtime metabolic activity is shifted towards catabolism in patients with insomnia .Within the category of breathing-related sleep disorders, most research has been conducted on obstructive sleep apnea (OSA), which refers to a disturbance in breathing during sleep. The diagnosis is based on the number of episodes of upper airway collapse while sleeping, measured via polysomnography through overnight monitoring. Nocturnal breathing disturbances can cause snoring, gasping and breathing pauses during sleep, but also daytime impairments, such as sleepiness, fatigue or unrefreshing sleep despite sufficient opportunities to sleep . Sleep aMetabolomic approaches have been applied in the study of OSA, whereby they can be categorized into two types of studies, including (1) metabolomic studies of exhaled breath ,39,40, aExhaled breath is well suited for metabolomics analyses as its condensate can be easily obtained by cooling exhaled air from spontaneous tidal breathing and thus represents a non-invasive sampling method . AlthougThe general notion is that metabolic biomarkers obtained from blood and/or urine samples are potentially useful in understanding OSA mechanisms and OSA management. A systematic review of individual metabolites detected by chromatography and/or mass spectrometry supports this conclusion . SeveralAlthough continuous positive airwave pressure (CPAP) is the treatment of choice for patients with OSA , there iSleep deprivation (no sleep opportunity), sleep restriction  and circadian clock disruption are associated with negative effects on mental health as well as metabolic disorders ,64. TablDifferences in the plasma  and urinIn men, sleep deprivation caused a change of about half of the identified urinary metabolites. More specifically, it caused an increase of eight urinary metabolites , while another eight metabolites  decreased . FurtherBesides metabolomics studies conducted on plasma and urine samples, animal models have been used to obtain direct information about brain changes due to sleep deprivation. A recent study conducted in mice investigated the metabolome of the prefrontal cortex during sleep, sleep deprivation and spontaneous wakefulness . OverallMetabolomics approaches have also been applied in a study investigating treatment responses. In a study conducted in rats submitted to sleep deprivation, the potential of extracts from Panax ginseng to restore serum and brain metabolic profiles was investigated . Panax gOverall, sleep restriction better reflects the real life situation than total sleep deprivation. Several studies have investigated changes in the metabolic profile of individuals experiencing insufficient sleep .Reducing sleep has been shown to be associated with pronounced shifts in the lipid metabolome. Biomarkers of insufficient sleep were investigated in plasma samples of 16 normal-weight participants . The indRodent studies provide further insights into the effect of sleep restriction on energy metabolism and on the clinical complications of sleep restriction, including obesity, diabetes and neurological disorders. A study conducted in rats revealed altered hepatic profiles indicative of changes in hepatic energy metabolism, as well as alterations in nicotinamide adenine dinucleotide salvage pathways and transmethylation pathways. Findings suggest that sleep restriction reduced activity of the tricarboxylic acid cycle, which went along with a concomitant increase in lipogenesis .Sleep fragmentation refers to brief arousals occurring during sleep. Arousals occur frequently in patients with sleep disorders, such as OSA, but are also often induced by external factors, such as noise, bright light or high temperature during sleep . Sleep fAnimal models are used to study the pathophysiological mechanism of sleep fragmentation as summarized in Previous studies revealed that the hippocampus is especially vulnerable to sleep fragmentation, showing a reduction in hippocampal volume, learning and cognition deficits and reduced neurogenesis . To reveThe role of the gut microbiome for mental and physical health disorders received increasing research interest in recent years ,85. A stThis review on the application of metabolomics in the study of sleep disorders clearly suggests alterations in specific metabolic pathways which likely contribute to the association of sleep-wake disorders with other psychiatric disorders and medical conditions. Although differences in study populations and technologies used for the profiling of metabolites complicate comparison of these studies, a common pattern seems to arise in terms of changes in BCAA metabolic pathways. Furthermore, glucose and lipid metabolism, as well as antioxidative status, were found to be affected by changes in physiological sleep-wake cycles in several studies.Overall, the observed results suggest that especially the BCAA metabolism is associated with the sleep-wake regulation. The three BCAAs L-leucine, L-valine, and L-isoleucine must be acquired through diet, as they cannot be synthesized de novo . In seveIn general, BCAAs are transported through the blood–brain barrier via the L-type or large amino acid transporter 1 (LAT1) to serve as a major nitrogen source for the brain . As LAT1While on the one hand, elevation of BCAAs in healthy individuals can negatively affect the sleep/wake rhythmicity and metabolic health, on the other hand, supplementation of BCAAs has been suggested as a viable therapy for treating sleep/wake disturbances in individuals suffering from traumatic brain injury . TraumatDespite the overwhelming consensus that metabolomics bears potential for biomarkers in the management of sleep-wake disorders, there are still limitations in the existing metabolomics work. A major limitation is that existing studies mainly focused on relatively small sample sizes under limited clinical conditions . AlthougFinally, it can be concluded that metabolomics provides important insights into the metabolic pathways linked to sleep disorders; however, the usability in clinical practice is limited to date as no references for normal ranges of metabolites exist.