Diversity within the fungal kingdom is evident from the wide range of morphologies fungi display as well as the various ecological roles and industrial purposes they serve. Technological advances, particularly in long-read sequencing, coupled with the increasing efficiency and decreasing costs across sequencing platforms have enabled robust characterization of fungal genomes. These sequencing efforts continue to reveal the rampant diversity in fungi at the genome level. Here, we discuss studies that have furthered our understanding of fungal genetic diversity and genomic evolution. These studies revealed the presence of both small-scale and large-scale genomic changes. In fungi, research has recently focused on many small-scale changes, such as how hypermutation and allelic transmission impact genome evolution as well as how and why a few specific genomic regions are more susceptible to rapid evolution than others. High-throughput sequencing of a diverse set of fungal genomes has also illuminated the frequency, mechanisms, and impacts of large-scale changes, which include chromosome structural variation and changes in chromosome number, such as aneuploidy, polyploidy, and the presence of supernumerary chromosomes. The studies discussed herein have provided great insight into how the architecture of the fungal genome varies within species and across the kingdom and how modern fungi may have evolved from the last common fungal ancestor and might also pave the way for understanding how genomic diversity has evolved in all domains of life. Fungi are currently organized into eight taxonomically distinct phyla2. The most studied and well-characterized fungi belong largely to the Ascomycota and Basidiomycota phyla, which collectively make up the subkingdom Dikarya. The six most basal phyla, also known as early-diverging fungi, are relatively understudied compared to Dikarya fungi and include the Blastocladiomycota, Chytridiomycota, Mucoromycota, Zoopagomycota, Cryptomycota, and Microsporidia phyla3.The fungal kingdom is estimated to consist of 2.2 to 3.8 million different species, making it the most diverse kingdom within the eukaryotic domain7. The phenotypic and ecological diversity across the fungal kingdom is reflected, in part, in the diversity observed within fungal genomes.The diversity within the fungal kingdom is evident from genetic, phenotypic, and ecological perspectives. Fungi display a range of morphologies from macroscopic, multicellular filamentous fungi to environmentally ubiquitous, single-celled yeasts and obligate intracellular pathogens associated with animal hosts. This diversity is further demonstrated by the variety of ecological roles fungi fulfill, the importance of fungi as model organisms in scientific research, and the diversity of applications of fungi in industrial settings. The diverse and important roles of fungi combined with technological advances in high-throughput sequencing, also known as next-generation sequencing, have motivated broad efforts to sequence thousands of fungal genomes11. Introgression, meiotic drive elements, and horizontal gene transfer (HGT) from other fungal species as well as cross-kingdom genetic transfer from prokaryotes represent additional examples of small-scale genomic evolution in fungi19. Compounding elements of small-scale genomic change can mediate genomic macroevolution, which refers to evolution at and above the species level.In this review, we focus on recent advances in understanding the evolution of fungal genomes. Both small- and large-scale variation among fungal genomes contributes to stable or transient genotypic and phenotypic variation as well as speciation. Small-scale genomic changes include changes at the nucleotide and gene level, and often influence genomic microevolution, or the changes of allelic frequencies within a species . Increas24 , which overlap with the open-reading frames of genes in an antisense orientation and regulate expression of their cognate transcripts. It has been shown that loss of RNAi in fungi is correlated with expansion and increased abundance of ASlncRNAs, suggesting that these elements play an important role in modulating gene expression and therefore in the stability and evolution of fungal genomes77. Additional deep RNA sequencing among numerous and diverse fungi complemented with molecular genetic analyses will provide further insight into the diversity of lncRNAs and their structure, function, evolution, and impact on overall genomic evolution.The breadth and depth of RNA sequencing in fungi has revealed that long non-coding RNAs (lncRNAs) are abundant and diverse within fungal genomes, are transcribed from intergenic and genic regions alike, and carry out a number of distinct roles62. Previous studies focused on centromere identification in relatively few ascomycete fungal species, includingS. cerevisiae,S. pombe, andCandida albicans79. These studies revealed that centromere length in fungi could range from a few hundred nucleotides to 300 kb in length and varied significantly among related species. These studies also established that centromeres are defined by the binding of a conserved histone H3 variant, CENP-A, along with many other conserved kinetochore proteins. Over the past several years, whole-genome sequencing with long-read Nanopore and PacBio sequencing platforms has led to the identification of centromeres in many other fungi and a better understanding of centromere structure and evolution. One such study revealed that centromeres across theSchizosaccharomyces group harbor a similar architecture but vary in their sequence, suggesting sequence is not critical for centromere function25. Centromeres in the plant-pathogenic ascomyceteMagnaporthe oryzae were found to consist of long AT-rich sequences, a feature conserved in related species26.Despite having a conserved role in proper chromosome segregation, centromeres are one of the most rapidly evolving sequences among eukaryotesCryptococcus pathogenic species complex revealed that centromeres inCryptococcus are long, rich with retrotransposons, and syntenic among different species81. However, centromere lengths vary significantly among three pathogenic species in this complex due to the truncation of retroelements as a result of RNAi loss in one of the species81. A more recent study in another basidiomycete group ofMalassezia species identified short centromeres (less than 1 kb), which were highly AT-rich and devoid of any repeat content82. Following the pattern inSchizosaccharomyces and theCryptococcus species complex, centromeres inMalassezia are also syntenic between closely related species.Characterization of centromeres in theMucor circinelloides, an emerging model organism83. It is important to note thatM. circinelloides lacks CENP-A but harbors other conserved kinetochore proteins. Identification of centromeres in this species revealed a mosaic nature of centromeres where kinetochore binding was restricted to a small region, flanked by multiple copies of a retrotransposon. Each of these studies emphasizes the fact that fungal centromeres are highly diverse with few conserved features and are rapidly evolving, even among closely related species. Interestingly, all centromeres identified thus far are in ORF-free regions that are mostly devoid of transcription, and many centromeres are AT-rich in nature. Combining these features together might help in predicting centromeres in fungal species where experimental tools have not been applied or are not yet available.The nature of centromeres in the Mucoromycota phyla of fungi have also been characterized usingCandida auris andAspergillus fumigatus as well as in the plant fungal pathogenPyrenophora teres f.teres65. Interestingly, in the emerging fungal pathogenC. auris, a particular genetic lineage that has not been involved in outbreaks exhibits high mutation rates and genomic instability, especially in subtelomeric regions. This subtelomeric variation is hypothesized to be attributable to a loss-of-function mutation inDCC1, a gene involved in regulating telomere length, genome instability, and the cohesion of sister chromatids in yeast84. Similar to subtelomeric regions, the regions harboring effector genes, also known as effector compartments, in fungal plant pathogens are also prone to frequent expansion and contraction events11. These regions are enriched in transposable elements and are regularly located near chromosome rearrangement breakpoints, which contributes to their largely lineage-specific identity11. Effector compartments make up the rapidly evolving portion of the “two-speed” genomes of many plant fungal pathogens. Rapid evolution in these loci is attributed to the expansion, contraction, and recombination mediated by repetitive transposons within the region as well as repeat-induced mutation (RIP) and epigenetic silencing10.Other genomic regions in which rapid evolution has been observed are the subtelomeres and the repeat-rich regions of plant pathogens that harbor effector genes mediating virulence. Subtelomeric loci are prone to rapid expansion and contraction and often encode virulence-associated genes as well as biosynthetic gene clusters for many secondary metabolites. The impact of rapid subtelomeric evolution on virulence has been highlighted in the human fungal pathogens91.The transmission of alleles between species can have a profound impact on genomic evolution. Instances of inter- and intra-kingdom HGT, genetic introgression following hybridization, and meiotic drive elements have all been shown to be strong forces of genomic evolution and have thus received increased attention over the last decade. Robust whole-genome sequencing of isolates within a species as well as the increasing diversity of fungal species sequenced have allowed for the identification of these allelic transmission events and mechanisms and also shed light on their origins92. Previously, prokaryotic species were thought to experience frequent HGT events, while relatively few HGT events were thought to have occurred in fungi. However, advances in whole-genome sequencing have, by increasing efficiency and reducing costs, expanded the number of fungal genome sequences available and allowed the identification of numerous HGT events across the fungal kingdom; many instances of HGT events in fungi have been extensively characterized and reviewed19. HGT events appear to be more frequent in organisms that have undergone significant niche adaptation such as commensal organisms like the saprophytic skin commensalMalassezia species, which have acquired a bacterial flavohemoglobin as well as many other bacterially derived genes93. Similarly, obligate intracellular pathogens belonging to the Microsporidia and Cryptomycota phyla have experienced significant HGT, in which up to 2% of genes are estimated to be derived from HGT events85. This work has helped to markedly clarify the number of HGT events in Microsporidia because only a small number of HGT events involving bacteria- and animal-derived genes had been previously documented, and these events were thought to be rare96. In addition to the relatively common HGT of bacterially derived genes in fungi, an exciting new study has identified instances of trans-kingdom genetic transfer between an arbuscular mycorrhizal fungal species and its symbiotic plant partner97. Another case of trans-kingdom HGT was shown in basal fungi (Chytrids) where a key cell-cycle regulator protein evolved from a viral protein86. This study provides key insights into the evolution of cell-cycle regulating machinery that evolved differently in fungi and animals. Further characterization of genomic diversity within and among species across all domains of life will help to identify additional instances of HGT.HGT refers to the lateral exchange and integration of genetic material between two species, unlike the vertical inheritance pattern between parent and offspringKluyveromyces lactis, which was demonstrated to be a result of introgression of a subtelomeric locus fromKluyveromyces marxianus, and 2) finding that a lichen-formingRhizoplaca species likely originated from an introgressive hybridization event13. In another intriguing study, introgression following hybridization was shown to be the primary force of evolution in the fungal pathogen responsible for Dutch elm disease, driving changes in host–pathogen interactions via introgression of virulence-associated genes and changes in sexual reproduction via introgression of mating-type loci14.Introgression, also known as introgressive hybridization, refers to the integration of genetic alleles from one genetic lineage into another following hybridization. Population-based studies in fungi have revealed the high frequency with which introgression can occur. Two recent examples include 1) the loss and regain of fermentation capacity byNeurospora many decades ago98. Subsequent studies have further characterized the meiotic drive elements inNeurospora, known as Spore Killers, and have revealed other Spore Killer meiotic drive elements inPodospora102. Over the past several years, elegant and thorough research inSchizosaccharomyces species has identified several novel meiotic drive elements104. Studies have also shown that in addition to the genomic loci that can act as meiotic drive elements, supernumerary chromosomes in the plant pathogenZ. tritici show evidence of meiotic drive91. The ability of genetic elements to bias their inheritance without conferring any adaptive benefits or potentially reducing fitness can greatly influence the genomic landscape and evolutionary trajectory of a species.Population genomics approaches have also revealed the prevalence of meiotic drive elements throughout the fungal kingdom. Meiotic drive elements are genetic elements that cause non-Mendelian patterns of inheritance by either biasing their inheritance in sexual progeny or by killing progeny that do not inherit the element, also known as killer meiotic drive elements. Fungal meiotic drive elements were first identified inInstances of large-scale genomic evolution include chromosomal structural rearrangements and numerical increases or decreases in chromosome copy number. Chromosomal structural changes are rearrangements of chromosome segments that result from recombination or DNA damage repair. Numerical alterations include variation in the chromosome number due to aneuploidy or polyploidy as well as changes due to accessory chromosomes. The past several years have seen a significant increase in studies characterizing such events.107. These rearrangements can occur within a single chromosome  as well as across different chromosomes . Many different mechanisms are known to mediate rearrangements, including meiotic recombination, double-stranded break (DSB) repair during mitosis, and transposable elements109.Structural variations or chromosome rearrangements occur when chromosome segments are shuffled across the genome. These structural variations include deletions, inversions, duplications, and translocations. Chromosome rearrangements are known to occur in all eukaryotic genomes112. Some of these rearrangements are also correlated with gene loss, the generation of genetic diversity, and changes in pathogenicity113. A study in the basalTaphrina genus showed that chromosome rearrangements can drive the evolution of effector superfamily genes and host adaptation as well as speciation114. Another study comparing the genomes of 71 ascomycete species suggests that the rate of chromosome rearrangements accelerated the process of speciation in these species115. A comparison of two different subphyla showed that the number of rearrangements is directly correlated with species richness in each respective subphylum. The same study also showed that whole-genome duplication and pathogenicity might elevate gene order divergence and genome rearrangements115.The prevalence and mechanisms of chromosome rearrangements in fungi have been known for a long time, but recent advances in long-read sequencing have highlighted the importance of these events in multiple cellular processes. Several studies comparing the genome evolution of closely related species revealed that chromosome translocations can drive the evolution of secondary metabolite-producing gene clusters, and many of these translocations coincide with transposable elements116. While centromere-mediated translocation has been proposed to occur based on genome comparisons, we showed its direct impact on the sexual cycle. Such centromere-facilitated chromosome rearrangements have been observed inMalassezia andCandida and are proposed to play a role in both chromosome and karyotypic evolution in these species118. Centromere-mediated chromosomal translocation has also been proposed as a driving force during the evolution of the mating-type locus inCryptococcus from an ancestral outcrossing tetrapolar system to a derived bipolar inbreeding state80.Using a direct experimental approach, we recently showed that centromere-mediated chromosome rearrangements, induced by targeting DSBs at centromeric retrotransposons with CRISPR, can alone drive reproductive isolation and thus play a role in speciationCombined, these studies show that structural variations play a critical role in the evolution of a diverse array of functions in fungal species, including metabolism, pathogenicity, host adaptation, and speciation. Their role in the evolution of secondary metabolite-producing clusters has been observed in multiple fungal species complexes, suggesting that this is a common mechanism via which metabolic potential can be altered or expanded. Additionally, the role of chromosome translocation in speciation has received significant support as a result of these studies. Future studies will reveal the unexplored significance of these chromosome structural changes in fungi. One key aspect to focus on will be the impact of these changes on pathogenicity and how they contribute towards the evolution of virulence in fungal pathogens.24. Indeed, analysis of the whole-genome sequence ofSaccharomyces revealed that one such whole-genome duplication event was responsible for the evolution of this genus and likely involved an interspecies hybridization event119. Despite the presence of polyploidy across the fungal kingdom, studies analyzing the polyploidy of fungal genomes have started to re-emerge23. Many clinically relevant fungi show variation in ploidy, mainly in response to stressful or new environmental conditions. Some fungi can also exist in multiple different ploidy states and exhibit ploidy transitions from one state to another. Studies inS. cerevisiae, as well asC. albicans, found that ploidy variation can drive rapid adaptation in response to environmental conditions and vice versa121.Whole-genome duplication or polyploidy refers to scenarios in which a cell or organism possesses three or more complete sets of chromosomes compared to the standard haploid karyotype. Polyploidy is found across the fungal kingdom, and one route to its generation involves whole-genome duplicationC. albicans122. During this process, tetraploidC. albicans cells undergo ploidy reduction to generate more stable diploid cells, which can be selectively advantageous123. This process is accompanied by concerted chromosome loss and generates aneuploidy124. Recent studies have provided intriguing insights into this process. TetraploidC. albicans cells were found to be metabolically hyperactive, generating reactive oxygen species and DNA DSBs, resulting in increased genomic instability125. Another study found that the genome reduction in this parasexual cycle involves recombination, similar to meiosis observed in other organisms, and thus has been termed para-meiosis126. A closely related species,Candida tropicalis, was found to exhibit a combination of same- and opposite-sex mating to generate polyploidy, which in turn increases genetic diversity127.A well-studied process of ploidy transition is the parasexual cycle inC. neoformans, has been shown to exist in haploid or hybrid diploid states.C. neoformans can also exist in a polyploid state, known as titan cells, produced in response to host conditions129. These titan cells produce haploid and aneuploid progeny and thereby enhance stress adaptation130. A concerted effort from three independent studies established that this phenotype can be induced by multiple environmental as well as genetic signals133. Similar toC. albicans, the ploidy reduction in these titan cells is mediated by the activation of meiotic genes134. While these studies explore the reduction of a polyploid genome to a more stable state, the exact role of polyploidy and the factors responsible for inducing polyploidy are not well understood. The presence of different levels of ploidy inS. cerevisiae natural isolates hints toward its role in adaptation to environmental conditions, but such natural resources have not been explored in other fungal species yet.Another human fungal pathogen,24. It has been proposed that aneuploidy may represent an intermediate, transient state that can promote evolutionary adaptation136. Experimental evolution of multipleS. cerevisiae strains showed that aneuploidy frequently occurs during the course of evolution137. Similar results were observed when 47 non-laboratory isolates ofS. cerevisiae were analyzed138. The geneSSD1 and its contributions to mitochondrial physiology were identified as the genetic basis of aneuploidy tolerance in these natural isolates139. A genome-wide genetic deletion screen inS. cerevisiae found that genes involved in a ubiquitin-mediated proteasomal degradation pathway also confer tolerance to aneuploidy141.Aneuploidy refers to the stage when a cell deviates from its balanced genome by gaining or losing chromosomes. Aneuploidy has been described in a large number of fungi but mainly with respect to stress conditions. While aneuploidy is commonly thought of as a deleterious state, and in humans is invariably associated with disease states or is lethal, many studies have found naturally occurring aneuploid isolates in different fungal speciesC. albicans andC. neoformans144. In both cases, drug resistance involved amplification of fluconazole target genes likeERG11 and genes responsible for the expression of drug efflux pumps, either by isochromosome formation inC. albicans or aneuploidy inC. neoformans. Newer studies have shown that aneuploidy can play roles in osmotic stress, flocculation, and ethanol survival as well as responses to starvation conditions inS. cerevisiae147. We have also shown that aneuploidy can be essential to overcome reproductive barriers between two strains harboring chromosomal translocations116. Overall, aneuploidy seems to be a conserved fungal response to stress or harsh environmental conditions. However, the advances discussed here reveal a role for aneuploidy in adaptation to growth conditions as well as pathogenicity in fungal species. With their well-characterized role in drug resistance, mechanisms leading to aneuploidy also present potent targets for drug development.Aneuploidy has been previously known to play a crucial role in drug resistance in pathogenic fungi like22. Accessory chromosomes are well studied in animals and plants but have also been observed in several plant fungal pathogens. Earlier reports suggested that these chromosomes are required for virulence and can be horizontally transmitted149. More recent studies have focused on the characterization of these chromosomes at the molecular level inZ. tritici andM. oryzae. InZ. tritici, B chromosomes were found to be epigenetically indistinguishable from the core set of chromosomes, whereas the B chromosome  inM. oryzae was found to be more enriched with transposons relative to the core chromosomes151. B chromosomes in both of these species were also found to harbor active centromeres150. However, the B chromosomes inZ. tritici are unstable and frequently lost during vegetative growth152.Accessory, supernumerary, or B chromosomes are all different names for extra chromosomes that are present in a cell beyond the reference karyotype. These extra chromosomes segregate in a non-Mendelian fashion, are dispensable, and do not undergo recombination or pairing with the main, essential  chromosomes153. The mini-chromosome inM. oryzae was proposed to be involved in the shuffling of AVR effector genes between the mini-chromosome and core chromosomes, thus aiding in host adaptation151. While the exact function of genes present on these accessory chromosomes is still unknown, they significantly influence the pathogenicity of the fungi in which they are present. For this purpose, an in-depth characterization of accessory chromosomes is essential and should include genetic as well as epigenetic approaches. The identification of B chromosomes in more species will advance the field and may reveal new insights into their roles in pathogenesis, adaptation, evolution, and possibly speciation.Interestingly, the stability of B chromosomes also depends on the host genotype and thus plays a vital role in host–pathogen interactionsA multitude of advances in high-throughput sequencing, also known as next-generation sequencing, and molecular biology methods have helped to reveal and characterize the forces driving evolution in fungal genomes. Efficiency and improved affordability of high-throughput whole-genome sequencing on Illumina sequencing platforms have promoted some of the largest advances in our understanding of fungal genome evolution by enabling the sequencing of numerous strains within a single species. The whole-genome data of various isolates within a species provides a pan-genome resource that spans the population variation within a species and also provides an opportunity to assess genomic microevolution and potentially macroevolution from a population genetics and genomics approach. Pan-genome sequencing has been especially helpful for identifying rates and dynamics of intraspecific genomic exchange, including the evolution and propagation of meiotic drive elements, as well as identifying inheritance of interspecific genetic material, including HGT events and instances of hybridization leading to introgression.S. pombe, in which PacBio and Nanopore sequencing platforms were able to accurately predict 16-fold more genomic structural variations than short-read technologies28. Additionally, long sequencing reads often span repetitive regions and transposons, allowing sufficient coverage of unique flanking regions, which is essential for assembling repeat-rich regions of genomes. This particular feature has allowed the identification of centromeres in many fungal species, especially in cases where centromeres harbor repetitive elements or transposons. Another significant feature of long-read sequencing methods is the ability to capture telomere repeat sequences, thus allowing telomere-to-telomere genome assembly. The identification of telomere repeat sequences at the ends of mini-chromosomes or B chromosomes provided support for their existence and helped differentiate these supernumerary elements from core chromosomes. Similarly, it was also helpful in the determination of chromosome number in several species. Another type of technique that has helped to achieve enhanced fungal genome assemblies is chromosome conformation capture, which includes methods such as Hi-C155. By defining the interactions between DNA sequences, Hi-C has aided in resolving genome assembly conflicts, thus improving assemblies157. It is important to note that these long-read sequencing techniques are highly error-prone and thus need to be corrected with less error-prone short-read Illumina sequencing prior to gene prediction or gene calling for annotation.The technological advancements of the long-read PacBio and Oxford Nanopore Technologies sequencing platforms have been critical in both further understanding macroevolution in fungi and significantly advancing the identification and characterization of chromosome structural variations. Using these technologies, many fungal genomes were sequenced and compared to develop synteny plots, and these synteny plots have allowed the detection of chromosome translocations. Some of these translocations were found to result in loss or gain of effector genes and also to have contributed to the evolution of secondary metabolite genes. A striking example of the advantages long-read sequencing technologies can have over short-read sequencing techniques was recently demonstrated inResearch over the last decade has provided unprecedented insight into the evolution of fungal genomes from both micro- and macro-evolutionary perspectives. The ease, efficiency, and affordability of sequencing combined with great community efforts have promoted research and given rise to findings that illuminate how small-scale genomic changes such as hypermutation, mobilization of various genetic elements, allelic transmission, and rapidly evolving regions can impact the evolution of fungi. Due to advances in long-range whole-genome sequencing technologies and the generation of telomere-to-telomere assemblies, researchers can now characterize the evolution of ploidy and chromosome structure, particularly in highly repetitive regions, with high accuracy and confidence. Advances in understanding fungal genome evolution at the nucleotide, gene, chromosomal, and nuclear levels are illustrated inMethods and techniques that attempt to further improve long-range sequencing and its accuracy as well as resolve artifacts and biases generated through library preparation, sequencing itself, and subsequent analyses will also improve our understanding of fungal genome evolution.Improvements are still especially needed in RNA sequencing and sequencing library preparation. Because of the high abundance of some RNA species, like ribosomal and translational RNAs, it remains difficult to detect rare RNA species, such as non-coding RNAs and alternatively spliced transcripts. Developments on this front will aid in efforts to further identify and characterize lncRNA transcriptomes in fungi and can help to determine how lncRNAs vary at the pan-genome level and influence genomic evolution.Continued progress with sequencing and other genomic technologies will provide more insight into the driving forces and consequences of genomic evolution. Additional sampling and sequencing of novel fungi and less well-characterized species will provide further insight into genomic diversity in the fungal kingdom. Owing to the variety of fungal niches and lifestyles, sufficiently culturing fungi for sequencing efforts can be tedious and often impossible based on current knowledge and techniques. Therefore, advances in single-cell sequencing methods could be highly advantageous to characterize the genomes of unculturable species or species that are difficult to isolate. This will also help the sequencing and study of fungi beyond Ascomycota and Basidiomycota, which have been the focus of the majority of research until now. Studies involving fungi from Zygomycota and Chytridiomycota are only beginning and are already providing exciting insights into the diversity and uniqueness of these fungal phyla.In addition to the analyses conducted on the nuclear genomes presented in many of the studies discussed here, it will be interesting to conduct population genomics studies on mitochondrial genomes in parallel to characterize the diversity, inheritance dynamics, and evolution of these important organelles. Research focused on identifying and characterizing fungal-associated viruses, or mycoviruses, is also garnering increased attention, and developments on this subject will be interesting, especially with regard to their propagation dynamics and impacts on host genome evolution. These potential future research avenues present great and exciting opportunities to further advance the field of fungal genetics.


Breast-cancer-acquired lymphedema is routinely diagnosed from the appearance of irreversible swelling that occurs as a result of lymphatic dysfunction. Yet in head and neck cancer survivors, lymphatic dysfunction may not always result in clinically overt swelling, but instead contribute to debilitating functional outcomes. In this review, we describe how cancer metastasis, lymph node dissection, and radiation therapy alter lymphatic function, as visualized by near-infrared fluorescence lymphatic imaging. Using custom gallium arsenide (GaAs)-intensified systems capable of detecting trace amounts of indocyanine green administered repeatedly as lymphatic contrast for longitudinal clinical imaging, we show that lymphatic dysfunction occurs with cancer progression and treatment and is an early, sub-clinical indicator of cancer-acquired lymphedema. We show that early treatment of lymphedema can restore lymphatic function in breast cancer and head and neck cancer patients and survivors. The compilation of these studies provides insights to the critical role that the lymphatics and the immune system play in the etiology of lymphedema and associated co-morbidities. The immune system is well known to play significant roles in cancer progression and treatment. Many cancers metastasize through the lymphatics to regional draining lymph nodes (LNs), where immune priming against cancer can be interrupted and/or immune tolerance to cancer can be established–both resulting in immunological hijacking that facilitates metastatic spread . RadiatiWithin the past decade, paradigm-changing discoveries have dramatically revised how we view the role of lymphatics in health and an array of chronic conditions, including cancer-acquired lymphedema (LE). In 2010, Levick and Michel showed that capillary filtrate carrying oxygen and nutrients to interstitial tissues is returned along with cellular waste products primarily through the lymphatics, not through the venules as predicted by Starling’s principle of 1896  or increased to 40 Gy in one fraction, persistent lymphatic dysfunction occurred, as visualized by NIRF-LI . Strikin × 5 Gy oIn an ongoing prospective and longitudinal NIRF-LI surveillance study of breast cancer patients receiving neoadjuvant chemotherapy, axillary lymph node dissection (ALND), followed by RT, we observed dermal backflow pre-ALND and post-ALND, both pre-RT and post-RT . In almost every case, dermal backflow preceded arm swelling. The variable appearance times for dermal backflow imply that any one (or a combination) of cancer treatment modalities may contribute to lymphatic dysfunction.In a longitudinal study, Rasmussen et al.  imaged hWhether aberrant lymphatic dysfunction after RT results from the impaired clearance of macrophages and dendritic cells activated by DAMPs, which in turn secrete pro-inflammatory cytokines that impair contractile activity, remains to be investigated. Nonetheless, these results suggest the confounding effect of RT on lymphatic function that may be one of the most significant drivers for lymphatic dysfunction and the onset of cancer-acquired LE.Clinical LE diagnosis can be difficult because classic physical signs  of LE are unreliable—sensitivity and specificity of such clinical signs in predicting lymphoscintigraphy-confirmed LE are 17% and 88%, respectively, with overall accuracy of 47% . ClinicaAs described above, we have identified the imaging phenotype of dermal backflow as one of the earliest signs of lymphatic dysfunction. In the absence of clinically overt swelling, dermal backflow may become an objective, prognostic criterion for development of clinical LE, its sequela, and comorbidities.For example, in study subjects at risk for unilateral BCRL but without any measurable arm volume change, patient-reported outcome of regional “heaviness” was accompanied by dermal backflow in that region a. In theIn a case of an HNC patient four weeks after RT, we imaged dermal backflow and poor drainage to cervical LN that, unlike in prior studies of untreated HNC patients, dissipated after two weeks of home treatment with an advanced pneumatic compression device (APCD) d. IndeedBoth of these examples in breast and HN cancers suggest that adding diagnostic NIRF-LI to the examination toolbox could prompt more timely initiation of therapeutic strategies at sub-clinical stages of lymphatic dysfunction, and perhaps minimize or even reverse LE development. “Cure” for cancer-acquired LE may require early intervention to reverse lymphatic dysfunction prior to the onset of swelling and accumulation of inflammatory waste products and immune cells. MLD and APCD therapy are generally contraindicated until patients are cancer-free, based on the entirely theoretical concern that they might facilitate cancer spread. The basis for this indication is that metastasis through the lymphatics may be augmented with MLD and APCD therapy. However, as described above, early restoration of drainage to existing or newly formed LNs may prevent LE, and both may potentially enhance the benefits of RT by ensuring anti-tumor immunity.It is also noteworthy that, in a prior review of NIRF-LI studies of unilateral BCRL patients, contralateral, undiagnosed arms exhibited increasing surface coverage of dermal backflow with stage and duration of diagnosed contralateral LE, even though cancer treatments were confined to the ipsilateral arm . Other sWhile prospective double-blind studies remain to be performed to show that remediation of dermal backflow as an early sign of lymphatic dysfunction can prevent clinical LE, strategic deployment of current treatments for clinical LE may, in the meantime, be enhanced with NIRF-LI diagnostics. Treatments include complete decongestive therapy (CDT), advanced pneumatic compression , and lymphatic surgeries. Because the intradermal depot of ICG provides a source of contrast for several hours, if not longer, pre- and post-treatment imaging can be performed to assess efficacy.NIRF-LI has shown that manual lymphatic drainage (MLD), as part of comprehensive CDT, can stimulate lymphatic contractile function by the lymphangions in the affected limbs of patients with diagnosed LE, but is more effective in contralateral “normal” limbs . One mayWhen conventional CDT fails, surgical options may improve quality of life for LE patients. Surgeries include the LYMPHA preventive technique, which is performed at the same time as ALND, and connects transected main lymphatic trunks to a lateral branch of the axillary vein distal to a competent valve . Two othOther common conditions result in NIRF-LI features of dermal backflow and reflux as seen in early stages of cancer-acquired LE, and in advanced stages, result in LE diagnoses.Chronic venous disease encompasses a wide range of lower extremity symptoms ranging from unsightly superficial telangiectases to chronic venous insufficiency that, if left untreated, can progress to venous ulceration. We have shown that patients with venous leg ulcers have profound lymphatic dysfunction , consistLymphatic dysfunction has also been linked to rheumatoid arthritis ,118,119,Staphylococcus aureus and Streptococcus groups A, G, and B have been blamed as the causative bacteria in most cellulitis cases [Cellulitis, a complication of LE, is an inflammatory event in the inner layers of skin, occurs in approximately 25–50% of LE patients, and in 2.5% of the general population ,129,130.is cases ,137.The established dogma for cellulitis states that LE patients experience cellulitis because of diminished infection clearance. Pathogenic infection is accompanied by the release of pro-inflammatory cytokines that allow further damage to lymphatic vessels and increased limb swelling, thus creating a vicious cycle. Protein-rich lymphatic fluid, which is a feature of LE, is believed to provide a fertile medium for bacterial growth . These eOf note, 30% of cellulitis is misdiagnosed , and NIRStagnation in lymph vessels most likely contributes significantly to cellulitis, because moving lymph with massage-mimicking advanced pneumatic compression devices in LE patients lowered rates of cellulitis episodes from 21.1% to 4.5% in one study  and fromThe lymphatic vasculature is an open, unidirectional system providing afferent and efferent paths for mounting and resolving immune responses and clearing inflammatory waste products. Cancer progression and treatment directly involve the lymphatics and therefore can be expected to acutely impact immune responses as well as lymphatic function. Using a technique which we developed nearly two decades ago, we show that the earliest phenotype of lymphatic dysfunction occurs in breast and head and neck cancer patients before and during cancer treatment, and in some cases, can be resolved with complementary medicine approaches. Still, in other cases without intervention, early dysfunction progresses and persists for months to years even without the accepted clinical symptoms of LE. Because changes in lymphatic function seen before and after cancer treatment are also observed with other chronic conditions, one might speculate that etiological aspects of LE may be shared in common with some of these chronic conditions. It remains to be demonstrated whether we can “cure” LE by interceding early, before the onset of irreversible clinical symptoms, as we attempt to intercede before venous ulcer formation in patients with venous disease or before irreversible bone loss in patients with rheumatoid arthritis.The lymphatic vasculature is an essential part of the immune system that is involved in chronic conditions (including cancer) that are associated with the process of aging. The lack of routine approaches to image the lymphatics has limited our understanding of its role in diseases, as revealed in NIRF-LI studies of lymphatic dysfunction of cancer progression and treatment. In the hemovascular system, vascular interventions were not enabled until the advent of angiography. In the case of the lymphatics, the predominantly clear lymph and transparent lymphatic vessels escape notice in clinical surgery practice or in preclinical studies, further contributing to the lack of understanding of the role and responses of the lymphatics to disease conditions and treatment. It is highly likely that the changes in lymphatics with disease progression and treatment seen in cancer-acquired lymphedema may be shared by other chronic diseases that involve the immune system. The ability to clinically evaluate lymphatic function in “point-of-care” studies could accelerate more effective strategies to treat these diseases. For example, we recently used NIRF-LI techniques to demonstrate the lymphatic delivery of a TNF inhibitor to restore contractile lymphatic function in an animal model of rheumatoid arthritis  and of a


Tumors of the central nervous system are the most frequent solid tumor type and the major cause for cancer-related mortality in children and adolescents. These tumors are biologically highly heterogeneous and comprise various different entities. Molecular diagnostics are already well-established for pediatric brain tumors and have facilitated a more accurate patient stratification. The availability of targeted, biomarker-driven therapies has increased the necessity of longitudinal monitoring of molecular alterations within tumors for precision medicine-guided therapy. Nevertheless, diagnosis is still primarily based on analyses of the primary tumor and follow-up is usually performed by imaging techniques which lack important information on tumor biology possibly changing the course of the disease. To overcome this shortage of longitudinal information, liquid biopsy has emerged as a promising diagnostic tool representing a less-invasive source of biomarkers for tumor monitoring and therapeutic decision making. Novel ultrasensitive methods for detection of allele variants, genetic alterations with low abundance, have been developed and are promising tools for establishing and integrating liquid biopsy techniques into clinical routine. Pediatric brain tumors harbor multiple molecular alterations with the potential to be used as liquid biomarkers. Consequently, studies have already investigated different types of biomarker in diverse entities of pediatric brain tumors. However, there are still certain pitfalls until liquid biomarkers can be unleashed and implemented into routine clinical care. Within this review, we summarize current knowledge on liquid biopsy markers and technologies in pediatric brain tumors, their advantages and drawbacks, as well as future potential biomarkers and perspectives with respect to clinical implementation in patient care. Tumors of the central nervous system (CNS) are the most frequent solid tumor type in children and adolescents and exhibit the highest mortality rate of pediatric cancer types . AlthougOver the last decade substantial progress has been made in the understanding of the molecular pathogenesis of pediatric brain tumors through multi-omics screening methods ,15,16,17Circulating tumor cells were described and documented in the blood of a patient with metastatic disease for the first time in 1869 in Australia ,40. MoreDNA fragments originating from cells outside of the blood circulation are known to be found in the bloodstream and other bio-fluids from patient and healthy humans. They are released from the cells into the fluids by several cellular processes, such as apoptosis or necrosis, respectively ,43. UsuaSeveral studies have demonstrated that multiple cells, including tumor cells and tumor stem cells, release extracellular vesicles and exosomes and internalize exosomes secreted from other cells ,48,49,50Micro-RNAs constitute an evolutionary highly conserved class of non-coding RNAs that play key roles in the regulation of gene expression. They are genome-encoded short, single-stranded approximately 22 nucleotide-long RNA molecules that silence or contribute to degradation of mRNA by binding to 3′-untranslated regions of mRNAs. Their complete function is not yet fully understood, but recent research has shown small, non-coding microRNAs to be master regulators of cellular processes, and expression patterns have revealed their potential as diagnostic, prognostic, and treatment response biomarkers . FurtherAs already mentioned, the extensive interest in detailed knowledge from the molecular biology of pediatric brain tumors and the trend to precision medicine has fueled development of novel molecular methods in pathology. Technological developments and advances of extremely sensitive and reliable methods have improved the feasibility of analyzing liquid biopsy samples from brain tumor patients. However, there are still certain challenges and pitfalls to avoid false positive or negative results. In this chapter, we will discuss the major pitfalls of liquid biopsy.The quality and handling of the liquid biopsy sample is an important point in the pre-analytical phase. The stability of liquid biopsy markers and structures are mainly affected by several steps during the pre-clinical workflow, such as what kind of blood collection tube is used followed by the storage temperature and time-to-processing as well as centrifugation speed . All theBeside the pre-analytical pitfalls in handling of body fluids, circulating tumor cells are represented in an extreme low concentration in liquid biopsy samples in early stage tumors . Especia2EDTA blood collection tubes with centrifuged plasma, shipped frozen on dry ice. Thereafter, they analyzed the Epidermal growth factor receptor (EGFR) mutation status and demonstrated the same high significance and concordance of the cell free DNA tubes shipped on room temperature as the current method (K2EDTA blood collection tube). In summary, the simple handling of the cell-free DNA collection tubes for the shipping and storage on room temperature for up to 5 days indicates a preferable method for future clinical settings in routine labs [KRAS in ctDNA isolated from patient serum samples.The major challenge of using cf/ctDNA for liquid biopsies is their extremely low abundance in serum. Depending on the tumor stage, the ctDNA ranges between 0.01% to more than 10% in total isolated cfDNA . To incrine labs . The isoine labs . Beside In general, exosomes are extreme stable in the bloodstream and would be the perfect liquid biopsy tool. Their content includes miRNAs, proteins and, recently, researchers discovered the presence of dsDNA from the parental tumor cell . CertainMicro-RNAs have been found to be stable within extracellular vesicles and exosomes isolated from CSF and plasma samples. Balzano and colleagues addressed the question of the stability of miRNAs in frozen vs. fresh plasma samples . There, In this chapter we discuss the latest findings of sensitive techniques for the detection of markers in liquid biopsies Next-generation sequencing (NGS)-based technologies provide high-throughput screening of the biopsy samples and improved accuracy with respect to mutation allele frequency (MAF), gene fusions or DNA amplification ,75. As pOver the past decade, substantial progress has been made in the development of more sensitive PCR-based methods. The new explored dPCR and ddPCR ,75 methoKRAS mutation and wild type and hybridized the sample. The clamp sequence for the mutation (target) of interest was excluded in the mixture. Thereafter, the heterogeneous solution including ctDNA and binding partners were applied to the PNA probe-modified microsensor. The target sequence lacking clamp partner hybridized to the PNA probes on the modified nanostructured microchip. Beside the specific PNA design, Das and colleagues coated the Au nanostructure chip with a thin layer of Pd to obtain nanostructured microelectrodes and reached a higher sensitivity of the binding and measured finely the differential pulse voltammetry (+target or −target). They tested this new assay design successfully in undiluted serum samples of lung and melanoma cancer patients [During the last few years, the electrochemical biosensor technology has made substantial progress in the sensitive detection of nucleic acids in liquid biopsy samples ,79. Untipatients .Recently, another breakthrough in the development of electrochemical microfluidic biosensors was achieved. Bruch et al. in cooperation with our group developed a microfluidic chip combined with the novel Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR/Cas13a) technology ,82. ThisHaving high potential for improved patient management, several studies have already investigated different methods for liquid biomarker detection in pediatric brain tumors. The following section and BRAF-fusions, mutations) which are promising candidates for liquid biopsy tumor detection. These alterations  may also be found in a subset of high-grade glioma. Indeed, first reports demonstrate that detection of BRAFV600E in liquid biopsies is feasible [BRAFV600E exist and have been shown to effectively inhibit tumor growth in these patients [H3F3A, HIST3B) which are frequently found in the midline and are thus not suitable for resection or re-biopsies. Consequently, the possibility of other means for monitoring tumor development appears important. H3K27M-mutations have been shown to be reliable detected in both blood and CSF [H3K27M-detection by ddPCR was about 90% in both CSF and plasma [ACVR1, IDH1 or TP53 can be reliably detected [NTRK1/2/3, ALK, ROS1) have been found in a subset of pediatric glioma and could also serve as biomarkers for the respective targeted therapeutics which are already approved. However, studies investigating this aspect in pediatric brain tumors are still lacking. Last, copy number aberrations (CNA) such as amplification of MYC/PDGFRA or deletion of CDKN2A/PTEN are frequently found in high-grade glioma and could also serve for disease monitoring. Regarding miRNAs, one study has already shown that miR-21, miR-15b, miR-23a, and miR-146b are elevated in the serum of pilocytic astrocytoma patients and that the level correlated with tumor size and therapy response [Low- and high-grade gliomas cover a wide clinical spectrum ranging from long-term treatment and follow-up to highly aggressive tumors with limited treatment options and short overall survival. Molecular analyses have revealed distinct genomic drivers and uncovered inter- as well as intratumoral heterogeneity within this entity. Importantly, the majority of low-grade gliomas is driven by alteration of the BRAF-pathway  may serve as biomarker [Similar to medulloblastoma, ependymoma subgroups identified by DNA methylation patterns are well-established biomarkers  and thusia ctDNA . With rey fluids . In someiomarker . The utiiomarker . Importaiomarker ,98 and cSMARCB1 and would be both accessible to liquid biopsy technologies. Moreover, they comprise diverse subtypes which can be determined by DNA methylation analysis [Molecular dissection across the landscape of pediatric brain tumors has uncovered numerous novel tumor entities with specific genomic alterations which could be used for liquid biopsy monitoring. Atypical teratoid rhaboid tumors (ATRT) are characterized by mutations or deletion within analysis . On a pranalysis . Embryonal tumors with multilayered rosettes (ETMR) is another rare pediatric brain tumor type which harbors either a characteristic focal amplification of the C19MC-cluster or mutations in DICER1 , both geBCOR (HGNET-BCOR), FOXR1 (HGNET-FOXR1) or MN1 (HGNET-MN1) are newly described subtypes all characterized by distinct molecular alterations [Next, CNS high-grade neuroepithelial tumor with genetic alterations in either erations . Apart fOver the past decade, interest in biological markers isolated from patient body fluids have emerged in the scientific world and have already revolutionized the diagnosis and tumor surveillance of certain entities. However, there are still multiple pitfalls to bypass, beginning with the pre-analytical workflow of the sample processing up to the sensitivity, stability and reliability of the potential biomarkers. The first studies have already shown that this approach is also feasible for pediatric brain tumors providing potential biomarkers and further development is currently ongoing. However, the costs of the equipment for liquid biopsy techniques are still expensive and the establishment of a routine liquid biopsy lab in each center currently appears uneconomical. Consequently, centralized liquid biopsy analysis would be a feasible strategy to ensure broad availability of these methods. In addition, certain techniques such as electrochemical biosensors also harbor the potential for decentralized diagnostics in the future. Taken together, liquid biopsy could offer a more precise and comprehensive diagnosis and monitoring of pediatric brain tumors in the future, in particular in guiding personalized anti-cancer therapies, but further efforts are required until liquid biopsies can be integrated into the clinical routine and patient care.


Gastrointestinal cancers are a significant cause of cancer mortality worldwide and have been strongly linked with chronic inflammation. Current therapies focus on epithelial/cancer cells; however, the importance of the tumor microenvironment in the development and treatment of the disease is also now well established. Cancer-associated fibroblasts (CAFs) are a major component of the tumor microenvironment, and are actively participating in tumor initiation, promotion and metastasis. They structurally and functionally affect cancer cell proliferation, tumor immunity, angiogenesis, extracellular matrix remodeling and metastasis through a variety of signaling pathways. CAFs originate predominantly from resident mesenchymal cells, which are activated and reprogrammed in response to cues from cancer cells. In recent years, chronic inflammation of the gastrointestinal tract has also proven an important driver of mesenchymal cell activation and subsequent CAF development, which in turn are capable of regulating the transition from acute to chronic inflammation and cancer. In this review, we will provide a concise overview of the mechanisms that drive fibroblast reprogramming in cancer and the recent advances on the downstream signaling pathways that regulate the functional properties of the activated mesenchyme. This new mechanistic insight could pave the way for new therapeutic strategies and better prognosis for cancer patients. CAFs are an essential component of the tumor microenvironment and accumulating evidence supports their substantial contribution to cancer development and progression . They orIn this review, we summarize current knowledge on how fibroblasts are converted to CAFs, particularly focusing on gastrointestinal cancers, including colorectal cancer (CRC), pancreatic ductal adenocarcinoma (PDAC), hepatocellular carcinoma (HCC), and gastric cancer (GC). We specifically analyze the three major types of CAF reprogramming, namely synthetic, epigenetic and metabolic, and focus on the signals and downstream molecular pathways that regulate this transition .A variety of signals, such as growth factors, cytokines, chemokines, Toll-like receptor (TLR) and Hedgehog ligands, mechanical forces and ECM components activate fibroblasts to produce effector molecules that are responsible for CAF functions . TGFβ anTGFβ is a dominant effector in all gastrointestinal cancers and mediates the conversion of fibroblasts to CAFs. Its importance is highlighted by the association of a CAF-specific TGFβ signature with poor prognosis, immune cell exclusion and resistance to immunotherapy in CRC . The ple+ fibroblasts, contributing thus to EGF-dependent serrated polyp formation in the mouse cecum /Smoothened (Smo) signaling pathway has also been shown to regulate CAF activation in gastrointestinal cancers, although it seems to exert opposite functions depending on the organ affected. Several studies have shown that genetic deletion or pharmacological inhibition of Hh signaling leads to depletion of the PDAC-associated stroma and enhanced drug delivery, xenograft growth inhibition, as well as reduction of HSC activation and concomitant liver fibrosis . In contBesides driving CAF activation, other pathways have been shown to suppress or reverse it and could be promising candidates for therapeutic interventions. A prominent example is the vitamin D receptor (VDR), which acts as a modulator of CAF reprogramming in gastrointestinal cancers . MechaniFinally, beyond soluble mediators, the biophysical properties of the TME have been also implicated in CAF activation and the induction of a synthetic phenotype. Mechanical stress induces collagen overexpression and crosslinking, fiber rearrangement, ECM deposition and degradation by fibroblasts, which can thus mediate the remodeling of the ECM and increase matrix stiffness . Matrix Epigenetic abnormalities, including changes in DNA methylation, abnormal patterns of histone modifications and post-transcriptional regulation through micro and long non-coding RNAs support genetic changes in cancer cells to drive tumor initiation and progression. Similar genetic mutations driving CAF differentiation are rare . HoweverHOXB6  and hypoxia, which through downstream activation of HIF1 and NFκB promote the metabolism of glucose to lactate and glutamate and mediate the loss of caveolin-1 (CAV1)  Martine. Inflamm+ T cells in an antigen-dependent manner, while they can be also converted to myofibroblasts (Differences in reported CAF functions, including tumor-promoting and restraining roles have long led to the hypothesis that distinct CAF subpopulations could exist within tumors . Represeroblasts . These sroblasts .+ and an ECM-expressing FAP+ population, although further functional characterisation of these is still missing (Single-cell RNA transcriptomic analyses have also been performed for colorectal and head and neck cancer. These revealed the presence of normal fibroblasts along with two CAF subsets, a myofibroblastic αSMA missing . NeverthIn this review, we have provided a concise overview of the molecular mechanisms underlying fibroblast reprogramming in gastrointestinal cancers. This is especially important as CAF phenotype reversal has been proposed as a potential therapeutic strategy in cancer . Both soM-TM, NC, and MS wrote sections of the manuscript and prepared the figure. VK critically revised the manuscript. All authors 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.


Cardiopulmonary arrest (CPA), the acute cessation of blood flow and ventilation, is fatal if left untreated. Cardiopulmonary resuscitation (CPR) is targeted at restoring oxygen delivery to tissues to mitigate ischemic injury and to provide energy substrate to the tissues in order to achieve return of spontaneous circulation (ROSC). In addition to basic life support (BLS), targeted at replacing the mechanical aspects of circulation and ventilation, adjunctive advanced life support (ALS) interventions, such as intravenous fluid therapy, can improve the likelihood of ROSC depending on the specific characteristics of the patient. In hypovolemic patients with CPA, intravenous fluid boluses to improve preload and cardiac output are likely beneficial, and the use of hypertonic saline may confer additional neuroprotective effects. However, in euvolemic patients, isotonic or hypertonic crystalloid boluses may be detrimental due to decreased tissue blood flow caused by compromised tissue perfusion pressures. Synthetic colloids have not been shown to be beneficial in patients in CPA, and given their documented potential for harm, they are not recommended. Patients with documented electrolyte abnormalities such as hypokalemia or hyperkalemia benefit from therapy targeted at those disturbances, and patients with CPA induced by lipid soluble toxins may benefit from intravenous lipid emulsion therapy. Patients with prolonged CPA that have developed significant acidemia may benefit from intravenous buffer therapy, but patients with acute CPA may be harmed by buffers. In general, ALS fluid therapies should be used only if specific indications are present in the individual patient. Untreated cardiopulmonary arrest (CPA), the acute cessation of blood flow and ventilation, is uniformly fatal, and the only known treatment to reverse it is cardiopulmonary resuscitation (CPR). CPR is delivered in two phases. The first and most important phase, basic life support (BLS), consists of chest compressions to re-establish blood flow and oxygen delivery to the tissues, and ventilation, to oxygenate the arterial blood and excrete carbon dioxide. The second phase of CPR, advanced life support (ALS), consists of adjunctive therapies that are targeted at improving the efficacy of the BLS interventions to maximize oxygen delivery to the tissues through drug therapy and defibrillation.Intravenous fluid bolus therapy has been commonly used to increase preload during CPR in an attempt to increase cardiac output. In addition, specific fluid therapies to address acid-base and electrolyte disturbances that occur commonly in patients with CPA have been proposed. Finally, the use of intravenous lipid emulsions for the treatment of CPA secondary to specific toxicities has been studied. In 2012, the Reassessment Campaign on Veterinary Resuscitation (RECOVER) Initiative published the first evidence-based veterinary CPR guidelines after an extensive review of the primary literature  is unlikely. The coronary arteries arise from the base of the aorta just as it leaves the left ventricle and supply oxygen to the myocardium. Large epicardial vessels branch into smaller intramuscular vessels and ultimately to the myocardium before entering the capillary beds where oxygen delivery occurs. Venous drainage from the capillary beds feed into myocardial and then epicardial coronary veins. The latter ultimately coalesce into the coronary sinus, which drains into the right atrium. During systole, the myocardium contracts and compresses the myocardial coronary arteries, dramatically increasing resistance. Therefore, in the spontaneously beating heart, the bulk of myocardial perfusion occurs during ventricular diastole when the myocardium relaxes and myocardial coronary arterial resistance decreases. The driving pressure for blood flow into the myocardium during diastole is the aortic diastolic pressure (ADP). However, this driving pressure is resisted by the pressure within the left ventricle at the end of diastole (LVEDP) since the myocardial coronary arteries serving the left ventricle are exposed to left ventricular pressures as they course into the endocardium. During CPR, left ventricular end-diastolic pressures  are very low, commonly less than the right atrial pressure (RAP), and coronary arterioles are maximally vasodilated . TherefoSimilarly, the pressure gradient leading to cerebral blood flow is cerebral perfusion pressure (CePP). The primary driving pressure for cerebral blood flow is mean arterial pressure (MAP), and the counteracting outflow pressure is either central venous pressure (CVP) or intracranial pressure (ICP), whichever is greater (CePP = MAP-CVP or MAP-ICP).The dependence of tissue blood flow on perfusion pressures has a substantial impact on fluid therapy during CPR. Intravenous fluid loading has the potential to increase tissue driving pressures by increasing ADP and MAP during CPR. However, such fluid loading may also increase the counteracting outflow pressures, both RADP and CVP, which will decrease CoPP and CePP, respectively, and worsen oxygen delivery to myocardial and cerebral tissue beds. Ultimately, intravenous fluid loading should only be administered during CPR if it is likely that an increase in driving pressure (ADP or MAP) rather than in outflow pressure (RADP and CVP) will occur. By definition, this is more likely in patients that are effectively hypovolemic, either due to intravascular volume losses or inappropriate vasodilation . However, patients that are already fully fluid loaded or euvolemic will not likely experience significant increases in driving pressure. Rather, a further rise in intravascular volume in these patients will predominantly increase the outflow pressure (RADP and CVP), which will decrease perfusion pressures and result in decreased blood flow to the tissues.These theoretical concerns have been documented in experimental studies. In a study of 18 euvolemic dogs with induced CPA, infusion of 11 ml/kg of either lactated Ringer's solution or whole blood resulted in a 34% increase in cardiac output, but a concurrent 26% decrease in left ventricular blood flow and a 35% decrease in cerebral blood flow. This was explained by a 22% reduction in CoPP that resulted from a disproportional increase in RADP compared to ADP . SimilarIn patients with acute CPA, assessment of intravascular volume status can be challenging. Typical physical examination parameters that may be helpful, such as capillary refill time, mucous membrane color, heart rate and pulse quality, cannot be assessed in patients with CPA. Therefore, review of pre-arrest physical exam findings, an understanding of the precipitating causes of the arrest , or evidence of acute blood loss  are required to make the best intravenous fluid therapy plan during CPR. The RECOVER ALS guidelines recommend a conservative approach to fluid administration during CPR, reserving fluid boluses only for patients with known or suspected hypovolemia or distributive shock .As described previously, for hypovolemic patients in CPA, fluid boluses are recommended because they are likely to increase tissue driving pressures more than outflow pressures and improve CoPP and CePP. Generally, isotonic crystalloid fluid boluses of 1/4–1/3 of the total blood volume over 15–20 min  are recommended to treat hypovolemia.There is experimental evidence that hypertonic saline (HS) may have beneficial effects beyond volume expansion during CPR. It is posited that HS may have neuroprotective effects by reducing cerebral edema, neuronal excitotoxicity, inflammation, and neuronal apoptosis. A recent experimental study in rats showed improved neurologic outcome post-CPR and reduced hippocampal apoptosis in rats receiving hypertonic saline during CPR compared to rats receiving isotonic crystalloids or synthetic colloids . ImportaThere is limited clinical data available to inform a decision about the use of HS during CPR. A prospective, randomized controlled clinical trial (RCT) of 200 human patients with out-of-hospital cardiac arrest (OHCA) in which 100 patients received 2 ml/kg of HHS and 100 patients received 2 ml/kg of HES over 10 min showed no difference in short or long-term outcomes between the groups . A retroTaken together, this evidence suggests that HS may have a benefit over isotonic crystalloid fluids during CPR in patients with hypovolemia or vasodilatory shock. Given the consistent finding that intravenous fluid boluses of any kind lead to decreases in CoPP and CePP, and in the absence of any long-term outcome studies demonstrating a survival or neurologic function benefit to the use of HS during CPR in euvolemic patients, it is difficult to recommend volume expansion of any kind in patients known or suspected to be euvolemic during CPR. Taken in context with recent evidence of harm associated with the administration of HES  and the Where CPA is caused or compounded by hypovolemia, fluid resuscitation is recommended as outlined above, and where hypovolemia is the consequence of severe hemorrhage, those fluids should include blood products . While tSuccessful resuscitation from traumatic CPA is resource intensive. In addition to massive transfusion therapy, effective management of severe traumatic hemorrhage requires a comprehensive strategy potentially including damage control surgery, minimization of crystalloid administration, treatment of hyperfibrinolysis , permissive hypotension and other interventions , 28. In Hypothermia protects all tissues, but in particular the brain, from the consequences of ischemia and reperfusion by a multitude of mechanisms . The posExperimental studies using intra-arrest cooling by ice-cold fluid bolus infusion are limited to swine , 38. In A single randomized controlled trial in humans including 245 subjects with OHCA of any rhythm studied the effect of intra-arrest cooling with intravenous cold saline compared to no such cooling . Up to 2Animal research suggests that early induction of hypothermia after ROSC is superior to delayed cooling . IntraveIn summary, available evidence from animal studies suggest that mild hypothermia itself, either applied intra-arrest or early after ROSC mitigates ischemia-reperfusion injury. However, the harm associated with administration of large intravenous boluses of cold isotonic crystalloids during or after CPR suggests that other avenues for cooling should be considered in most cases. As dogs and cats are generally of lower thermal mass than humans, hypothermia  often occurs spontaneously during treatment of CPA and rapid administration of cold fluids is likely not necessary.Under special circumstances, the intra-arrest administration of calcium, magnesium, potassium and dextrose can be considered, but evidence of broad benefit is lacking.Hyperkalemia can occur for a multitude of reasons and can lead to life-threatening cardiotoxicity and cardiopulmonary arrest once serum potassium concentrations are severely increased  . IntraveAs for calcium, there is no conclusive evidence for an overall benefit of routine administration of magnesium during cardiac arrest, despite several RCTs in people examining the utility of magnesium in OHCA and IHCA . NeverthExperimentally induced hypokalemia in dogs below 3.0 mEq/L was found to increase vulnerability to ventricular tachycardia and fibrillation through a variety of arrhythmia mechanisms –57. WhilThe use of intravenous lipid emulsions (ILE) has been extensively reported in veterinary medicine to treat a wide range of poisonings and has recently been reviewed . In the The general recommendation for the clinical use of ILE in dogs and cats is (a) to administer 20% formulations, as 10% solutions are less effective; (b) to use inline filters (1.2 micron) as lipid aggregates are common; (c) to administer the solution as a bolus (1.5 mL/kg IV over 1 min) followed by an infusion (0.25 mL/kg/min IV CRI) for 30–60 min; (d) to limit the daily dose to 10 mL/kg . To faciPoisoning with other drugs than local anesthetics may also warrant the administration of ILE during CPR. Treatment with ILE is recommended in humans, and in extension animals, with CCB overdose alongside calcium gluconate, ALS measures as per standard guidelines and possibly other measures such as insulin/dextrose , 47. TwoAdverse effects of ILE therapy, while infrequent, deserve mention . In a ra2 rose above 350 mm Hg   or THAM  were tested as alternatives to bicarbonate, but studies in dogs and swine did not show any benefit over NaBic  , 87, 89. in vivo . Non-CO2er NaBic , 87, 88.er NaBic . In facter NaBic , 93. MorIn people, 2 RCTs and one systematic review that incorporates the findings of observational studies with a total of more than 18,000 patients showed no overall benefit of intra-arrest buffer therapy, with some studies reporting worse outcomes with buffer therapy –96. One The last evidence evaluation by the International Liaison Committee on Resuscitation examining intra-arrest buffer therapy was conducted in 2010 and did not recommend routine administration of NaBic or any other buffer . HoweverDF and MB co-wrote and critically revised the manuscript. Both authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer SD declared a past co-authorship with one of the authors (DF) to the handling Editor.


Dear Editor,"Exercise is Medicine" (EiM) is a United States-based health initiative that was co-launched by the American College of Sports Medicine (ACSM) and the American Medical Association (AMA) in 2007. Public health and research institutions in several countries and regions have embraced this philosophy, and over 40 countries have embraced EiM as a health initiative. EiM focuses on making exercise in the promotion of health care . UndoubThe logic behind EiM recognizes that non-communicable diseases  are closely related to sedentary lifestyles . ExerciThe goal of all medical treatments is to cure abnormal physical and mental condition, and to restore physiological indicators normal or near-normal values. In contrast, exercise applies an optimal stimulus to the body to promote better health and well-being. Acute exercise causes physiological indicators  to gradually approach boundary values by swaying from average levels; chronic endurance exercise training induces a series of adaptive changes in the cardiovascular system, such as exercise-induced sinus bradycardia and exercise-induced cardiac hypertrophy. These are accepted as normal "exercise-induced adaptations" but are considered to be "abnormal phenomena" in clinical medicine. Moreover, long-term resistance exercise training increases muscle mass and dimensions, so that the larger body mass index (BMI) is not clinically classified as “obese”.EiM requires positive feedback of health-related physiological indicators  to maintain exercise compliance. However, changes in physiological indicators often require a period of adaptation and accumulation, and there are obvious individual differences due to some non-motor factors such as ethnicity, genetics, gender, and physical activity levels , which uses medicine as a logical starting point, actively promotes the health and social aspects of physical activity, and counteracts the health risks of a sedentary lifestyle. However, EiM rarely considers many important aspects of exercise science. We need to acknowledge that exercise and medicine are both important components of maintaining and promoting health, but the blanket statement that “exercise is medicine” maybe misleading as some people engage in unscientific and unsuitable physical exercise, and also causing sports and exercise scientists to deviate from exploring the various physiological components of exercise.The authors declare no conflict of interest.


To ward off against the catastrophic consequences of persistent DNA double-strand breaks (DSBs), eukaryotic cells have developed a set of complex signaling networks that detect these DNA lesions, orchestrate cell cycle checkpoints and ultimately lead to their repair. Collectively, these signaling networks comprise the DNA damage response (DDR). The current knowledge of the molecular determinants and mechanistic details of the DDR owes greatly to the continuous development of ground-breaking experimental tools that couple the controlled induction of DSBs at distinct genomic positions with assays and reporters to investigate DNA repair pathways, their impact on other DNA-templated processes and the specific contribution of the chromatin environment. In this review, we present these tools, discuss their pros and cons and illustrate their contribution to our current understanding of the DDR. DNA double-strand breaks (DSBs) are the most cytotoxic DNA lesions. Their detection, signaling, and repair require a comprehensive cellular response collectively known as the DNA damage response (DDR). The DDR requires the activation of the ATM kinase, a member of the phosphoinositide 3-kinase (PI3K)-related protein kinase family , which iDNA double-strand breaks repair can be achieved by different means that are commonly grouped in two broad categories depending on the use or not of a homologous DNA sequence as a template. Repair by non-homologous end joining (NHEJ) involves direct resealing of the two broken ends independently of sequence homology. Although being active throughout the cell cycle, NHEJ is relatively more important during G1 . A schemIn contrast to NHEJ, homologous recombination (HR) requires a homologous DNA sequence to serve as a template for DNA-synthesis-dependent repair and involves extensive DNA-end processing . As expein cis to the DSB and this break-induced chromatin landscape contributes to recruiting DNA repair factors, thanks to interactions between histone modifications and their readers . In addition, during DSB repair, the destabilization of nucleosomes further enhances accessibility and regulate the mobility of the broken DNA ends  causes the appearance of a plethora of different genomic lesions . They caOf interest, upon DSB induction following exposure to radiation, many DDR factors tend to accumulate temporarily at sites of DNA damage, forming the so-called Ionizing Radiation-Induced Foci (IRIF) . Importain situ ligation followed by proximity ligation assay (DI-PLA), allows detection and imaging of individual DSBs in cells , the binding, recruitment, retention or release of specific proteins can be studied using a chromatin fractionation approach . First uDNA resection can be specifically investigated with high resolution using single-molecule analysis of resection tracks (SMARTs). This is a modified DNA combing approach, in which resection of broken DNA ends leads to the exposure of otherwise inaccessible BrdU-epitopes previously incorporated in the DNA. When combined with an immunodetection protocol using fluorescence microscopy, SMARTs allows the direct visualization and quantification of individual tracks of resected DNA after IR .Finally, approaches such as pulsed-field gel electrophoresis (PGFE) or single-cell gel electrophoresis  can also be combined with IR exposure to directly investigate DSB repair . IR-indu(+) Radiation exposure provides an easy and robust way to analyze the recruitment of proteins to sites of DNA damage (provided that their level of binding is high enough) and to study the DNA repair kinetics using different approaches .(−) Radiation not only induces DSBs but also a plethora of other damages in the cell, and creates “dirty” ends, mostly in a random manner on the genome, hence likely biased toward the untranscribed genome in higher eukaryotes . Moreover, since radiation induces DSBs at unknown locations, and in a non-homogenous manner in the cell population, locus-specific analyses of DDR factor recruitment or chromatin modifications using chromatin immunoprecipitation (ChIP) studies, for instance, is not possible.∗ Additionally, each readout of these stochastic DSBs has its own pros and cons. For example, ssDNA observed by SMART, RPA or BrdU foci might reflect unwound DNA; the COMET assay also detects apoptotic cells, albeit the tail shape is different; chromatin accumulation of some factors might occur independently of DNA damage and in response to other signals. Thus, in all cases, appropriate controls must be used.In addition to IR-induced DNA damage, in which cells are exposed to an X-ray lamp or a Cesium irradiator, non-ionizing radiation can also be used to study DSBs. For instance, ultraviolet A (UVA) radiation can be used to create hundreds of DSBs along the path of a laser beam (line or spot) through laser scanning microscopy . UVA doeCombined with the expression of fluorescently labeled proteins, laser microirradiation has provided unprecedented temporal resolution of the sequence of events following DNA damage . This ap(+) Laser microirradiation represents the best technique today to temporally resolve the sequence of events at DSBs, allowing to observe very early (<10 s) and/or transient repair proteins recruitment and chromatin modifications.(−) Microirradiation induces a large number of localized, clustered DNA lesions (not only DSBs), that may also initiate specific responses. Moreover, it is neither amenable for molecular characterization of the repair outcome at the sequence level, nor for ChIP, which limits the spatial resolution that can be achieved.In contrast to radiation treatments, that require specialized and expensive equipment, chemical-induction of DSBs is cheap and easy to implement in any laboratory and can be coupled with almost any experimental protocol. Usually, cells are treated with a defined concentration of a chemical agent for a fixed amount of time. It is important to distinguish between acute (from minutes to a few hours) versus chronic (for days) treatments, as the responses will vary enormously. Many types of chemical agents can indirectly cause DNA breaks. For example, chemical inhibition of topoisomerases I and II causes SSB and DSB respectively . SSBs caOf importance, given their different modes of action, all the above-mentioned drugs will produce different types of DSBs: either located at different genomic regions and/or introduced during different cell-cycle stages. For instance, DSBs created by radiomimetic drugs show a bias toward specific sequences . MoreoveThe analysis of DSBs induced by chemical agents can be performed by the same approaches described for irradiation-induced breaks . In addi(+) Treatment with genotoxic compounds represents an easy to implement and robust way to analyze the recruitment of DSB repair factors and to study the repair kinetics using different approaches .(−) Drugs produce different types of DNA damage, at different genomic loci, and most show a preference for specific cell cycle stages, which should be carefully considered during data interpretation.Different labs have sought to develop tools for the site-specific induction of DNA breaks making use of restriction enzymes targeting integrated exogenous cleavage sites, otherwise absent from the genome. Such tools overcome the ambiguity of DNA lesions introduced by previous methods and allow the inspection of protein recruitment during the DDR to a site-specific DSB and the assessment of chromatin remodeling events with nucleosome resolution. Moreover, they can be combined with strategies to control the timing of DSB induction, for instance by controlling the nuclear translocation of the restriction enzyme, affording a valuable strategy to measure kinetic parameters of the DDR in live cells .SceI  (SceI enzyme fused to a glucocorticoid receptor (I-SceI-GR) that translocated to the nucleus upon triamcinolone acetonide (TA) addition, allowed the controlled induction of a DSB at the L-I-SceI-T array and the live-cell tracking of the broken DNA ends in real-time  . Expresseal-time .FokI nuclease domain fused to the mCherry-LacI creates DSBs at the lac operator array  generated at sites of DNA damage . To inve(+) I-SceI or FokI mediated DSB induction on transgenic loci are powerful systems to investigate the response to clean DSBs. These systems allow analyzing the repair event at a molecular resolution, the repair frequency (thanks to designed reporters cassettes) and the DNA damage repair/signaling in single cells (using imaging approaches). They can be combined with additional reporters to investigate with great detail the functional links between the DDR and transcriptional activity, chromatin modification and spatial organization, or DNA replication, for instance.(−) These systems rely on transfection, transcriptional regulation, or nuclear localization of the endonuclease. Consequently, they cannot provide the same temporal resolution achieved using microirradiation, where DSB induction is immediate and highly synchronized. Moreover, the transgenic nature of the analyzed loci calls for caution, especially when repeat-rich transgenes are used . Finally, the accurate repair of endonuclease-created breaks reconstitutes the target site, therefore being re-cleavable until the target site has been mutated. Hence, most of the outputs measured in these experimental contexts address mutagenic repair, leaving faithful repair out of reach.In order to bypass the need for introducing a transgene and to avoid potential, non-generalizable, side effects of transgenic loci on the repair process , efforts have been made recently to develop alternative systems where DSBs can be induced at endogenous, annotated loci on the genome . On one PpoI, which has a recognition sequence of 15bp, to form site-specific DSBs within endogenous target sites of the human genome  was fused to I-PpoI. The addition of 4-hydroxytamoxifen (4-OHT) promotes rapid nuclear localization of ER-I-PpoI and the subsequent time-dependent cleavage of the endogenous sites. Using this system, the Kastan lab disclosed the distribution of Nbs1 and ATM, and histones  system, where multiple annotated DSBs can be induced after 4-OHT treatment and DNA repair accurately monitored following auxin treatment. AsiSI induces 100-200 DSBs across the human genome [as determined by BLESS  These systems represent powerful tools to compare DNA repair events that occur at different genomic loci and, because they induce DSBs at annotated positions in a homogenous manner in the cell population, are compatible with all high resolution, high throughput sequencing-based techniques such as ChIP-seq, Hi-C, etc.(−) For all these systems, as for the above-mentioned I-SceI based systems: (i) DSB production is not immediate nor synchronized in the cell population and (ii) accurately repaired DSB can be re-cleaved. Hence while being powerful to analyze the spatial distribution of repair protein and chromatin changes around DSBs, they preclude a fine temporal resolution of these events. Moreover, the position of the DSBs is dictated by the target site of the chosen enzyme, which can represent a limitation to the number of different loci analyzed.A number of specific tools have more recently allowed to induce DSBs at chosen endogenous genomic loci.FokI endonuclease to a protein able to specifically target a particular locus. This approach was implemented for example to induce DSBs at telomeres by fusing FokI to the shelterin protein TRF1  (Zinc finger nucleases (ZNF) are chimeric proteins comprised of both a zinc finger domain designed to recognize a specific locus and the p84-ZNF)  were usep84-ZNF)  and tranp84-ZNF) .FokI, this system provides a rapid and easy way to design sequence-specific nucleases called TALEN. TALEN have been used to investigate DNA repair in a large number of organisms and genomic contexts, such as in CTG trinucleotide repeats in budding yeast  Methods to induce DNA breaks at specific endogenous loci in the genome are particularly powerful in that they provide the liberty to choose the locus to be analyzed. As for the other endonuclease-mediated DSB induction systems, they are amenable to both imaging and molecular high throughput sequencing-based technologies such as ChIP-seq.(−) Yet, similarly to the other endonuclease-mediated DSB induction, they are less suited for thorough, careful kinetics analyses since they rely on the controlled expression of the Cas9, or transient transfection of the sgRNA and accurately repaired DSBs may be re-cleaved by Cas9. Moreover, the fact that Cas9-induced DSBs may be particularly refractory to repair, and hence biased in terms of repair pathway choice, call for caution when using these systems.Coating of telomeres with shelterin factors including telomeric repeat-binding factor 2 (TRF2), prevents fusions of linear chromosome ends and suppresses local DNA damage responses . DysfuncFokI fusion with TRF1 described above, DSB-signaling at telomeres can be activated upon TRF2 deletion  Using dysfunctional telomeres as surrogates for DSBs is easy to implement and can be coupled with different imaging and biochemical approaches to directly inspect the molecular details of the DDR signaling.(−) Telomeres possess several specific features that render them particularly refractory to repair, and, when uncapped through TFR2 deletion, show a strong bias in terms of repair pathway choice toward NHEJ. The number of dysfunctional telomeres may vary considerably between cells and this heterogeneity may raise issues related with cells viability.Our capacity to create DSBs in a programed manner and in such a way that is compatible with a set of diverse methodologies to investigate the events that follow DNA damage, has led to our current deep understanding of the DDR. The induction of DSBs at random locations using different sources of radiation or genotoxic compounds, provides the easiest approach to analyze the recruitment kinetics of proteins to sites of DNA damage and is a powerful strategy to temporally resolve the sequence of DNA repair events. The development of methods to induce annotated DNA breaks at transgenic loci inserted in the genome, or at endogenous loci  allowed the analysis of the DDR at molecular resolution and were instrumental in disclosing functional links between the DDR and processes such as transcriptional, chromatin dynamics, and DNA replication. Yet all the tools described here display significant drawbacks. For instance, nucleases-induced DSBs undergo consecutive cycles of repair/cleavage until these have been mutated, calling for caution when investigating DNA repair using these tools. A major challenge is now to refine these DSB-inducible systems and the subsequent methodologies to analyze repair in order to overcome these limitations.All authors contributed to discussing the review contents and to writing 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.