Obesity remains one of the leading causes of death worldwide and is a well-known risk factor for a myriad of non-communicable diseases including diabetes, cardiovascular disease, and a variety of cancers  administered to older healthy males led to significant improvements in total body adiposity and lean body mass , and their associated carrier proteins and receptors, which are further regulated by nutritional status and hormones such as ghrelin and insulin (Renaville et al., GH is a potent anabolic hormone that plays a significant role in lipid metabolism at various sites including the liver, skeletal muscle, and adipose tissue (Dehkhoda et al., While GH is a powerful regulator of lipid metabolism, its role varies depending on the target site. For example, although GH has lipogenic effects within the liver, the opposite occurs in adipose tissue, particularly VAT, where GH elicits lipolytic effects through the suppression of LPL activity (Stanley and Grinspoon, via a negative feedback control, as nicotinic acid-mediated suppression of lipolysis, and consequently reduced circulating FFAs, led to a significantly greater GH response. This finding may help further explain why individuals with obesity and reduced CRF, who on average have elevated levels of FFAs (König et al., The bidirectional relationship between central obesity and impaired GH secretion has been widely reported despite being scantily understood (Stanley and Grinspoon, via diet modification. In fact, a previous study involving 18 adults with obesity and 18 age- and sex-matched controls showed that defects in GH secretion were ameliorated to near-normal function following significant diet-induced weight loss (Rasmussen et al., The obesity phenotype shares considerable overlapping risk factors with adult GH deficiency such as increased serum low-density lipoprotein cholesterol (Cordido et al., Regular aerobic exercise enhances the body's ability to transport and oxidize FFAs during exercise at varying work rates (Van Tienen et al., As mentioned earlier, GH promotes lipolysis within adipose tissue and increases mitochondrial oxidative capacity (Short et al., 2peak elicited greater GH response than when performing a single bout of 30-s sprint on a cycle ergometer. While the majority of research has been undertaken in men, a study by Sauro and Kanaley (2peak for a minimum of 10 min is sufficient to increase GH response in healthy young women. These findings highlight that while exercise intensity does appear to influence GH levels acutely, sufficient exercise volume may also be required.Acute aerobic exercise has been shown to increase GH levels, and these changes have been shown to be strongly associated with exercise intensity and volume, a function of exercise duration and frequency (Pritzlaff et al.,  Kanaley  also shoCurrently, it is unclear whether regular aerobic exercise can increase the GH-response to acute exercise. A study by Sasaki et al.  reportedWhile there are limited data pertaining to the effects of regular exercise on chronic changes in 24-h GH release and function, a study involving untrained eumenorrheic women showed that regular aerobic exercise above lactate threshold led to a two-fold increase in 24-h GH secretion (Weltman et al., Although the precise mechanisms driving exercise-induced improvements in cardiometabolic outcomes remain a matter of scientific debate, given the congruent metabolic effects of exercise, particularly aerobic exercise, and GH, it could follow that the behavior itself and ensuing endocrine and metabolic changes both improve cardiometabolic health, with the latter providing somewhat of an additive, but perhaps not essential, effect . Like GHBased on the current available literature, it appears that exercise-induced reductions in VAT are mediated by multiple factors, which may include acute and chronic exercise-induced change in GH levels. This could be due to the similar lipolytic effects of both GH and exercise on VAT. Furthermore, because CRF plays a significant role in GH response, partaking in regular exercise may ameliorate age-related reductions in GH response and action.As exercise has been shown to ameliorate obesity-related CRF and other cardiometabolic impairments, exercise should be incorporated into routine care for the treatment of adult-onset GH deficiency and associated metabolic perturbations. Importantly, increasing CRF and weight loss concurrently through exercise and dietary modification may yield greater restoration of GH function than either intervention on its own; however, there is limited evidence to confirm this. Consequently, further research is required to elucidate the relationship between somatotropic changes and exercise-induced cardiometabolic improvements.The conception and drafting of this article was led by AS. NJ and DC contributed to the drafting and critical revision of the article. All authors 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.


Hepatocellular carcinoma (HCC) is one of the most common malignancies globally. Despite aggressive and multimodal treatment regimens, the overall survival of HCC patients remains poor.Circular RNAs (circRNAs) are noncoding RNAs (ncRNAs) with covalently closed structures and tissue- or organ-specific expression patterns in eukaryotes. They are highly stable and have important biological functions, including acting as microRNA sponges, protein scaffolds, transcription regulators, translation templates and interacting with RNA-binding protein. Recent advances have indicated that circRNAs present abnormal expression in HCC tissues and that their dysregulation contributes to HCC initiation and progression. Furthermore, researchers have revealed that some circRNAs might serve as diagnostic biomarkers or drug targets in clinical settings. In this review, we systematically evaluate the characteristics, biogenesis, mechanisms and functions of circRNAs in HCC and further discuss the current shortcomings and potential directions of prospective studies on liver cancer-related circRNAs.CircRNAs are a novel class of ncRNAs that play a significant role in HCC initiation and progression, but their internal mechanisms and clinical applications need further investigation. HCC was the sixth most common malignancy and the third leading cause of cancer-related death globally in 2020, accounts for 75–85% of primary liver cancer cases and has been ranked second in terms of mortality in men . ChronicEmerging evidence indicates that noncoding RNAs (ncRNAs) are involved in many cellular biological and physiological processes and even pathological disease processes . Among tDrosophila, mice and humans, and is also found in the hippocampus , 2316, 2Adenosine deaminase-acting on RNA (ADAR1) was found to be an RNA-editing enzyme and to play a suppressive role in circRNA formation. This function was correlated with its adenosine-to-inosine (A-to-I) editing process, which frequently occurs near the location of reverse complementary matches (RCMs), a conserved feature of circRNA biogenesis . InteresThe emerging roles of circRNAs emphasize the importance of sequencing these circular transcripts. In the past decade, RNA sequencing (RNA-seq) technology has developed rapidly and has become an indispensable tool for analyzing the differential expression of genes and differential splicing events of mRNA at the transcriptome level .Next-generation sequencing  is currently the main way to sequence circRNAs. In next-generation sequencing, the coordination of gene cluster replication decreases with increasing read length, resulting in a decline in the quality of base sequencing. Therefore, next-generation sequencing is a high-throughput and short-read technique. Since most circRNAs are derived from exons, this alignment-based method is unable to distinguish circular reads from the overlapping regions of corresponding linear transcripts. The biggest drawback is that next-generation sequencing’s relatively short-read capacity considerably limits its detection ability in structural variant detection and genome assembly .Emerging long-read sequencing technologies have become a powerful participant in genomics. Compared with short-read approaches, long-read technologies can generate continuous ultralong sequences directly from native DNA. However, oligo(dT) primers are not suitable to use for circRNA sequencing because of their lack of poly(A) tails, and as a result, long-read sequencing technology cannot be widely applied in circRNA studies .Strikingly, a recent report presented a novel algorithm (CIRI-long) for detailed analysis of full-length circRNAs using nanopore sequencing technology. This technology utilized full-length circular reverse transcription (which was performed with random primers and SMARTer reverse transcriptase) to amplify circRNAs by producing long complementary DNA (cDNA) molecules containing multiple copies of full-length circRNA sequences. Then, a nanopore approach was used to directly sequence full-length circRNA sequences, and a specific algorithm was applied to quantify circRNA expression and recognize full-length mutant transcript sequences. The data showed that compared with Illumina RNA-seq , nanopore sequencing can enhance the detection efficiency for circular reads (with a 20-fold increase) and provide a fivefold increase in identifying alternative circularization events, indicating its higher sensitivity for circular isoform identification. Furthermore, nanopore sequencing can recognize circRNAs at a relatively low abundance and capture nonclassical circRNAs more sensitively, such as a new type of intronic self-ligated circRNA .Although many studies suggest that some circRNAs have the potential to be novel diagnostic or prognostic markers, the key problem is how to detect circRNAs in body fluids more efficiently. Very recently, our group reported a fully integrated electrochemical point-of-care testing (POCT) platform based on Au nanoflower (AuNF)/peptide nucleic acid (PNA)-modified carbon-fiber microelectrodes (CFMEs) . The plaThese new technologies provide more possibilities for the study of circRNAs. Undoubtedly, an increasing number of new technologies will emerge in the future, opening uncharted territory in circRNA research.With the development of RNA sequencing technology, the whole genome transcriptional map of circRNAs in HCC has been reported in several studies , 36, 37.To identify the circRNAs involved in HCC tumorigenesis, a recent study detected differential circRNA expression between HCC tissues and adjacent noncancerous liver (ANL) tissues. In this study, 13,686 distinct circRNAs were identified in total, excluding those with very low abundance, and the expression levels of the identified circRNAs were further analyzed. The results showed that 236 circRNAs were differentially expressed in HCC compared with matched ANL tissues, of which 108 were upregulated and 128 were downregulated [Another study identified 220 circRNAs that were differentially expressed between patients who experienced postsurgical pulmonary metastasis and those who did not. Among the identified circRNAs, 144 were upregulated and 76 were downregulated .HCC is characterized by a clear gender disparity, and AR is thought to be critical for this bias . To veriThese sequencing and bioinformatics analysis statistics demonstrate the possible involvement of circRNAs in HCC tumorigenesis and development. Many studies have reported that circRNAs play an important role in HCC, although their specific functions and internal mechanisms are still under investigation , 39–60 .Table 1OWith further developments in the study of circRNAs, the mechanisms of circRNAs have been gradually revealed. Through pre-mRNA back-splicing, circRNAs gain high stability to exert their important regulatory functions. It is universally acknowledged that circRNAs play a vital role as competing endogenous RNAs (ceRNAs) to regulate downstream signaling pathways –63. MeanThe primary location of circRNAs is the cytoplasm, which is a precondition for them to exert posttranscriptional regulation functions . NumerouOngoing investigations have reported that some circRNAs can function as miRNA sponges in HCC. CircSMARCA5 is a circRNA derived from exons 15 and 16 of the SMARCA5 gene and is downregulated in HCC . In HCC A large amount of evidence has confirmed the existence of the circRNA-miRNA pathway. However, it can be seen from the examples above that circRNAs usually sponge more than one miRNA to exert their function , 37, 52.RBP is a general term for a group of proteins possessing RNA recognition motifs that function by binding RNA to regulate its metabolic processes . RBPs plSeveral groups have reported that circRNAs may combine with RBPs to regulate gene expression , 70, 71.As mentioned above, circRNAs can physically combine with specific RBPs to facilitate or inhibit their function. Interestingly, one study revealed that a specific circRNA can alter the subcellular localization of its target RBP, providing a novel pattern for RBPs to perform specific functions under unusual cellular localization . HoweverEmerging evidence has demonstrated that some circRNAs may act as dynamic scaffolding molecules to regulate protein functions. Ongoing studies have shown that circRNAs can bind corresponding proteins in specific subcellular locations and facilitate the colocalization of relevant proteins, which may modulate protein–protein interactions .An outstanding study revealed that circACC1 combines with the regulatory β and γ subunits of AMPK to form a ternary complex and thereby facilitates the stabilization and activity of the AMPK holoenzyme . In HCC,Recently, our laboratory demonstrated that circSORE could bind the master oncogenic protein YBX1 to prevent YBX1 nuclear interaction with the E3 ubiquitin ligase PRP19 and inhibit PRP19-mediated YBX1 degradation, thereby inducing sorafenib resistance . In contAll these circRNAs serve as protein scaffolds. However, they exhibit different biological functions, suggesting that circRNAs can play different roles as protein scaffolds. Recent years have witnessed excellent progress in understanding of this novel mechanism in HCC, to which our group has made a contribution . Given tCircRNAs are regarded as ncRNAs due to the lack of essential components for cap-dependent translation, including the 5′ cap and the poly(A) tail. However, emerging evidence indicates that circRNAs may have the potential to encode proteins in a cap-independent manner . circRNACircZNF609 is one of the few endogenous circRNAs that have been reported to act as translation templates. Structurally, circZNF609 contains a 753-nt open reading frame (ORF) spanning from the start codon of the host gene to a stop codon created 3 nt after the splice junction . MechaniRecently, a novel circRNA, circβ-catenin, was identified as a protein-encoding circRNA whose translation can promote HCC cell growth through activation of the Wnt pathway . The stuThese results broaden our understanding of the human proteome. Currently, there is only one report on the translational function of circRNAs in HCC, indicating that the internal mechanisms of encoding circRNAs lack in-depth study. Our group suggests using a bioinformatics database to predict whether ORFs, IRESs or m6A modification sites exist in candidates. CircRNAs possessing these basic conditions may have the potential to encode proteins, which requires subsequent experimental analysis for further verification. The abundance and subcellular localization of circRNAs may lead to inferior translation efficiency compared with their cognate mRNAs. Hence, it remains controversial whether circRNA-derived proteins could achieve observable effects on the development of HCC .Emerging evidence has indicated that circRNAs play vital roles in HCC tumorigenesis and progression and are involved in cell proliferation, tumor metastasis, immune escape and drug resistance Fig.  36, 37,, 51–53. Investigations have reported that several circRNAs are expressed at an extremely high level in the early stages of HCC, suggesting that these specific circRNAs may promote tumorigenesis. A previous study demonstrated that circCDYL can regulate tumorigenesis by promoting the stem-like properties of HCC cells and showed that circCDYL-transduced HCC cells present a higher expression level of stem cell-associated genes with a markedly increased percentage of EpCAM + cells. Moreover, overexpression of circCDYL increased the expression of Ki-67 and alpha-fetoprotein (AFP), indicating that circCDYL promotes the malignant proliferation of HCC cells . AnotherOn the other hand, emerging evidence indicates that circRNAs can regulate tumor cell apoptosis through specific mechanisms . ApoptosHence, the important role played by circRNAs in cell proliferation and tumorigenesis is becoming even clearer, providing insights for a deeper understanding of HCC pathogenesis.Epithelial-to-mesenchymal transition (EMT) refers to a biological process by which epithelial cells are transformed into cells with a mesenchymal phenotype, and EMT plays a vital role in the ability of malignant tumor cells derived from epithelial cells to obtain characteristics that enable invasion and metastasis . MechaniRecent advances have shown that some EMT-related circRNAs can affect the EMT process in HCC. For instance, circARFGEF2 was found to be overexpressed in portal vein tumor thrombus (PVTT) and HCC tissues and to promote the EMT process via a miR-143-3p/FOSL2 axis and PCBP1/CD44v6 axis , leading to a remarkable acceleration in intrahepatic and pulmonary metastasis . MoreoveConsequently, circRNAs are considered key factors with well-characterized functions in regulating the EMT process, cancer cell invasion and metastasis in HCC.Immune dysfunction plays an important role in HCC development. Experimental evidence has shown that the circRNA expression profile changes during viral infection, which can regulate the function of the immune system . SubsequFor instance, a recent study found that HCC cells secrete circUHRF1 through exosomes and that circUHRF1 can inhibit NK cells from secreting IFN-γ and TNF-α by upregulating the expression of TIM-3 in NK cells. This study showed that the level of plasma exosomal circUHRF1 was negatively correlated with the infiltration level of NK cells in tumors . ResearcThese examples confirm that circRNAs can affect HCC development and prognosis by regulating the immune system of HCC patients, which prompted us to wonder whether circRNAs will become an ideal immunotherapy target in the future.Statistics indicate that most HCC patients are diagnosed at an advanced stage. At present, the main drug treatment for unresectable HCC includes chemotherapy  and targeted therapy , but conventional systemic chemotherapy lacks survival benefits.Currently, multikinase inhibitors, monoclonal antibodies and immune checkpoint inhibitors are the main targeted molecular therapies approved for treatment of advanced-stage HCC; however, the effects of these treatments are not satisfactory , 94. TheIncreasing evidence has indicated that ncRNAs are critical for the development of sorafenib resistance in HCC , 98. OurMeanwhile, ongoing studies have shown that circRNAs play a vital role in the development of drug resistance . Our groIn summary, we must further clarify the mechanism underlying drug resistance and explore how circRNAs function in resistance to molecular targeted drugs. Identifying a specific circRNA that could be used as a new therapeutic target to avoid drug resistance would be an important step forward.“Secondary prevention”  can improve the prognosis of HCC patients. The poor prognosis of most patients with HCC is due to the diagnosis of advanced HCC, and the reaction of advanced HCC to all treatment regimens is not satisfactory . At presEmerging evidence has demonstrated that circRNAs hold great potential as a novel attractive class of diagnostic biomarkers for HCC due to their resistance to RNase R digestion and their stability during circulation. Recent studies have indicated that circRNAs can not only be detected in tumor tissues but also in exosomes, blood, saliva and urine –107. TheCircRNAs have gradually been recognized as possible prognostic biomarkers. For instance, circSLC3A2, which is elevated in HCC tissues, plays an oncogenic role by sponging miR-490-3p to modulate PPM1F expression and could serve as a prognostic biomarker due to its positive correlation with poor survival in patients with HCC . In contHowever, the clinical feasibility of using circRNAs still needs to be verified in a large cohort of HCC patient samples. Meanwhile, inventing efficient circRNA detection methods, such as the POCT platform, is also of great significance.Recently, an increasing number of studies have focused on the significance of circRNAs in HCC and their correlation with HCC tumorigenesis and development, and some authors have proposed that circRNAs have the potential to serve as therapeutic targets in HCC Table . For exaCurrently, the main treatment for high-grade (middle and advanced) HCC is chemotherapy and molecular targeted therapy; however, the development of resistance to HCC treatments is an unavoidable problem, including resistance to traditional chemotherapy and even first-line molecular targeted therapies, such as sorafenib . As prevRecently, circRNA-based therapy has attracted increasing attention. Compared with other drugs, such as monoclonal antibodies or small molecule inhibitors, circRNA-based therapy has several advantages. First, circRNA drugs have a long half-life , while traditional therapeutic drugs are unstable once they enter the circulatory system; therefore, with circRNA drugs, the patient dosing frequency can be decreased compared with that for antibodies or small molecules . Second,On the other hand, proteolysis-targeting chimeras (PROTACs) are a burgeoning and promising field and can modulate protein concentrations at a posttranslational level by coopting the ubiquitin–proteasome system . As prevGenerally, whether circRNAs have clinical value merits further study and discussion. Although circRNAs have the potential to serve as diagnostic markers, prognostic markers, therapeutic targets or novel drugs, these roles are based only on theoretical assumptions and predictions; thus, more substantive studies are needed to verify these conjectures Table .Substantial progress has been achieved in specific circRNAs. These findings not only reveal a previously unexpected complexity of cellular regulatory mechanisms but have also identified many circRNAs with important physiological and clinical significance. Although the progress is exciting, it has led to many more questions than answers, which require future investigation.6A modification of circRNAs has been proposed to facilitate the cytoplasmic export of circRNAs [First, most studies have focused on a specific circRNA and its downstream mechanisms, while the upstream mechanisms remain largely unknown. For example, how exactly are circRNAs formed? Under what circumstances do pre-mRNAs form circRNAs rather than mRNAs? Are there any factors that are specifically required for back-splicing but not for splicing? How are circRNAs exported from the nucleus to the cytoplasm? These issues are rarely mentioned in most current studies. However, research groups have tried to answer these questions, and mcircRNAs . HoweverSecond, current biological tools and methods applied in circRNA-related studies still have some obvious shortcomings. Due to the high degree of sequence similarity between circRNAs and their cognate mRNAs, off-target effects of circRNA knockdown are difficult to avoid or completely eliminate. Similarly, the same dilemma has been observed in circRNA FISH and circRNA pull-down experiments. To successfully overexpress circRNAs, special sequence elements that promote circularization are usually inserted at both ends of the linear circRNA sequence. Inevitably, a large number of “linear circRNAs” will be produced due to the low efficiency of circularization. Whether this by-product will affect experiments remains to be further studied. Furthermore, the CRISPR-Cas9 system may be restricted in knockout studies of circRNAs because deletion of the whole genome sequence could disturb the expression of cognate mRNAs and impact specific biological functions. These challenges highlight the need for more advanced technologies for circRNA interference, detection and other functional studies. Very recently, Chen and colleagues reported that the CRISPR-Cas13 technique combined with guide RNA targeting sequences spanning junction sites featured in circRNAs can be applied to knock down circRNAs. Furthermore, compared with shRNA-mediated knockdown, RfxCas13d–BSJ-gRNA-mediated circRNA knockdown showed higher efficiency and specificity and lower rates of off-target effects on cognate mRNAs .Finally, the number of circRNAs that truly have cellular and physiological functions remains to be explored. In particular, our research has shown that the expression of circRNAs in HCC is widely reduced, suggesting that low expression levels will lead to nonfunctionalization . Given tIn recent years, an increasing number of studies have begun to focus on circRNAs; thus, circRNA-related research is progressing at a steady and fast pace. In the following section, we propose several challenging and innovative future research directions in the field of circRNAs.First, the current research remains at the cellular and animal levels, and how to facilitate translation toward clinical application will become a hot topic in the future. Because circRNAs are stable and have been detected in many types of body fluids, further studies are warranted to confirm their potential as biomarkers, therapeutic targets or novel drugs.Second, whether a circRNA with relatively low abundance can achieve measurable effects remains controversial. Future studies should focus on certain types of circRNAs with the same properties instead of a specific circRNA. For example, a striking study reported that endogenous circRNAs with 16–26 bp that form imperfect RNA duplexes can act as inhibitors of double-stranded RNA (dsRNA)-activated protein kinase (PKR) related to innate immunity .Generally, HCC is a multistep, multistage and multifactor comprehensive hereditary malignancy, and the specific pathogenic mechanisms remain unclear. Despite the availability of a comprehensive treatment regimen, including surgery, chemotherapy, targeted therapy and immune therapy, the overall survival of patients with advanced HCC is still unsatisfactory. Notably, circRNAs are a novel class of ncRNAs that play a significant role in HCC initiation and progression. As described in this review, circRNAs are aberrantly expressed in HCC and associated with the clinicopathological features and prognosis of HCC patients. Unexpectedly, there is a high degree of heterogeneity among HCC-related circRNAs, and individual circRNAs may have prooncogenic or antioncogenic roles and function through various pathophysiological mechanisms. Given the stability and polyfunctionality of circRNAs, circRNAs might serve as diagnostic indicators, prognosis predictors, therapeutic targets or novel nucleic acid drugs for precise treatment of HCC, thus improving the quality of life and extending the survival of HCC patients.


Vimentin expression contributes to cellular mechanoprotection and is a widely recognized marker of fibroblasts and of epithelial-mesenchymal transition. But it is not understood how vimentin affects signaling that controls cell migration and extracellular matrix (ECM) remodeling. Recent data indicate that vimentin controls collagen deposition and ECM structure by regulating contractile force application to the ECM and through post-transcriptional regulation of ECM related genes. Binding of cells to the ECM promotes the association of vimentin with cytoplasmic domains of adhesion receptors such as integrins. After initial adhesion, cell-generated, myosin-dependent forces and signals that impact vimentin structure can affect cell migration. Post-translational modifications of vimentin determine its adaptor functions, including binding to cell adhesion proteins like paxillin and talin. Accordingly, vimentin regulates the growth, maturation and adhesive strength of integrin-dependent adhesions, which enables cells to tune their attachment to collagen, regulate the formation of cell extensions and control cell migration through connective tissues. Thus, vimentin tunes signaling cascades that regulate cell migration and ECM remodeling. Here we consider how specific properties of vimentin serve to control cell attachment to the underlying ECM and to regulate mesenchymal cell migration and remodeling of the ECM by resident fibroblasts. Phenotypic regulation in many cell types is influenced by their interactions with the extracellular matrix (ECM), a major structural component of many organs and soft connective tissues that is comprised mainly of fibrillar collagens , glycoprThe physical reorganization of fibrillar proteins that accompanies the migration of fibroblasts through the ECM is a central feature of collagen remodeling , which iAs described in the Human Protein Atlas database , vimentiThe recent uptick of interest in vimentin originates in part from the growing appreciation of its diverse roles in a broad range of cellular functions that affect tissue and organ structure. Notably, alterations of vimentin expression are linked to diseases including lung and liver fibrosis and several types of cancer , all of Expression of vimentin in epithelial cells during EMT is associated with the adoption of a more mesenchymal cell shape, increased focal adhesion formation and enhanced cell motility . ConversFor many years VIFs were considered as very stable cytoskeletal structures whose principal functions provided resistance to mechanical stress  and partThe presence of VIFs in the cell periphery during mitosis is associated with the organization of cortical actin filament arrays, suggesting that vimentin filaments may help to strengthen the cortex during cell division . Electroin situ hybridization experiments demonstrate that ∼29% of total cytoplasmic poly (A) mRNAs co-localize with vimentin filaments (in situ hybridization experiments demonstrate a tripartite assembly between the 5′ untranslated region stem-loop (5′ SL) domain of collagen mRNAs, La ribonucleoprotein domain family member 6 (LARP6) protein, and vimentin filaments  complexes . Ultrastilaments . More diilaments . Specifiilaments . In thisilaments , these milaments . In addiilaments , bindingilaments , and binilaments . CurrentMore recently, proximity-based assays have shed light on vimentin’s involvement in cellular differentiation, homeostasis, and stress response through its association with RNA-binding proteins, misfolded aggregates, Stress Granules and Processing Bodies . These pVimentin is one of the main mediators of EMT and metastasis in a variety of cancers  and is iThe migration of cells through soft connective tissues depends in part on their ability to remodel the ECM through synthesis and degradation. Migration in turn is reliant on the ability of cells to attach to ECM proteins and to navigate through pores in the ECM, which, depending on their size, may require cell deformation. The deformability of a cell and its ability to return to its original shape are affected by the viscoelastic properties of cells and their constituents . VIFs exCell migration through ECMs like collagen networks often provokes nuclear rupture, which is related to constriction and deformation of the nuclear membrane as cells traverse pores in the ECM. VIFs form intricate networks such as the nuclear cage that extends from the perinuclear region to the sub-cortex. In these networks, vimentin filaments function as elastic springs, dissipating tensile forces when migrating cells squeeze through constricting pores. These properties of vimentin can prevent extensive fluctuations of nuclear shape . In addiPost-translational modifications (PTMs) are implicated in the spatiotemporal regulation of vimentin expression and organization of the VIF network, which consequently impacts the stability of the underlying cytoskeleton and remodelling of the ECM . CurrentVimentin, like other intracellular proteins, is prone to caspase-mediated proteolytic cleavage. Phosphorylation protects vimentin from caspase-mediated proteolysis , which occurs at vimentin D85 (via caspase 3 and caspase 7) and D259 (via caspase 6) to generate pro-apoptotic N-terminal fragments . AKT1 acvia distinct ubiquitin lysine residues   . Beclin and K63) . Beclin and K63) . Thus, aand K63) . This reand K63) .The search for causative mechanisms in rheumatic diseases and fibrosis, which involves extensive and often dysregulated ECM remodeling, has generated considerable interest in citrullinated vimentin, which involves the conversion of arginine residues to citrulline by the enzyme, peptidyl arginine deiminase (PAD) . VimentiThe acetylation of vimentin and other EMT-related proteins affects the migratory capacity and the metastatic properties of various types of cancer cells . AcetylaStreptococcus pyogenes  protein to lysine residues of acceptor proteins like vimentin can impact cell migration through poorly defined mechanisms . PIAS1 mReactive nitric oxide transfers nitrosyl moieties from donor to acceptor proteins, a process that involves nitric oxide synthase and modifies C328 of vimentin in response to mechanotransduction through the Akt pathway  and in sModifications of vimentin C328 protect against electrophilic and oxidative stress by preserving the flexibility of VIFs . But unlThis vimentin PTM is mediated by the addition of O-linked β-N-acetylglucosamine (O-GlcNAc) on serine and threonine residues mediated by O-GlcNAc transferase and O-GlcNAcase . GlycosyAs described in the discussion of PTMs above, some of the mechanisms by which vimentin regulate cell migration through ECM are now being defined. As vimentin interacts with actin filaments and microtubules to affect their structure and function , these iVimentin is essential for the elongation of invadopodia , and proArising from its involvement in several discrete cellular processes, VIF networks orchestrate cell spreading  Ostrows, single At higher resolution, long (>4 μm) vimentin filaments serve as a load-bearing scaffolds to distribute traction stress during single cell migration . As wounCollective cell migration is promoted by the elevation of traction forces in the migrating cell monolayer and by the preservation of intercellular contacts  De Pasc, which aVimentin promotes collective cell migration by restricting actin flow, aligning tractional stress  and suppIn highly motile epithelial cells, VIFs often co-distribute with keratin, the IF type first expressed in embryogenesis . In collVimentin expression accelerates cell migration through paxillin-dependent regulation of Cdc42 activation, which leads to PAK1-dependent vimentin phosphorylation and filament assembly . Thus, vCells organize their migratory activities partly through tightly controlled protein-protein interactions in cell adhesions to the ECM. Integrins are transmembrane receptors expressed by many cells and are obligate heterodimers comprised of α and β subunits. The β subunit contains a large extracellular domain, a transmembrane domain and a cytoplasmic domain, while the α subunit forms a similar structure, but contains different motifs within its extracellular domain . The intVimentin incorporates into nascent focal complexes and mature adhesions in a structure-dependent manner. While small vimentin oligomers  are abundant in nascent adhesions, mature adhesions exhibit fully organized vimentin filaments . In fibrAs vimentin can also be detected on the surface of cells (extracellular vimentin-ECV) and in the ECM after its release from activated macrophages , there hIn the context of ECM remodeling and cell migration, we considered how vimentin helps cells to sense, integrate, and respond to microenvironmental information. Vimentin can directly interact with focal adhesions and regulate cellular processes and discrete signaling pathways. These events in turn affect cell migration and ECM remodeling by spatially and temporally integrating arrays of external and internal signals. Cell-specific levels of vimentin expression, PTMs, and direct and indirect interactions with other cytoskeletal components, control the mechanical properties of cells during migration and ECM remodeling. It appears that the extraordinarily broad array of PTMs of vimentin and its structure in response to extracellular and intracellular mechanical signals are crucial for cell and tissue integrity . Express


This paper explores how biological responses can inform conservation strategies of threatened giant clams through global change, including climate change, global warming, marine heatwaves and ocean acidification. Understanding responses to environmental stressors can help project future distributions of suitable giant clam habitat for protection and use in selective breeding programmes. Giant clams are threatened by overexploitation for human consumption, their valuable shells and the aquarium trade. Consequently, these iconic coral reef megafauna are extinct in some former areas of their range and are included in the International Union for Conservation of Nature(IUCN) Red List of Threatened Species and Convention on International Trade in Endangered Species of Wild Fauna and Flora. Now, giant clams are also threatened by rapid environmental change from both a suite of local and regional scale stressors and global change, including climate change, global warming, marine heatwaves and ocean acidification. The interplay between local- to regional-scale and global-scale drivers is likely to cause an array of lethal and sub-lethal effects on giant clams, potentially limiting their depth distribution on coral reefs and decreasing suitable habitat area within natural ranges of species. Global change stressors, pervasive both in unprotected and protected areas, threaten to diminish conservation efforts to date. International efforts urgently need to reduce carbon dioxide emissions to avoid lethal and sub-lethal effects of global change on giant clams. Meanwhile, knowledge of giant clam physiological and ecological responses to local–regional and global stressors could play a critical role in conservation strategies of these threatened species through rapid environmental change. Further work on how biological responses translate into habitat requirements as global change progresses, selective breeding for resilience, the capacity for rapid adaptive responses of the giant clam holobiont and valuing tourism potential, including recognizing giant clams as a flagship species for coral reefs, may help improve the prospects of these charismatic megafauna over the coming decades. Global change has accelerated rapidly, particularly in the past 50 years, and we are tracking the worst-case emissions scenario—the business-as-usual Representative Concentration Pathway (RCP) 8.5  bony fishes, (ii) sea birds, (iii) giant clams, (iv) squids and octopuses, (v) sharks and rays, (vi) whales and sea cows and (vii) sea turtles  as one of the top five marine megafauna species threatened globally, based on a functional trait and extinction risk assessment  (see below). A recent analysis identified the giant clam (Hippopus and Tridacna) , a species that can weigh over 250 kg and measure over 1.3-m long  are large marine bivalves inhabiting coral reefs across the Indo-Pacific, with 12 recognized species in two genera (ridacna) . The lar3-m long , produciT. gigas) is one of the top three functionally unique marine megafauna species, along with the dugong and green sea turtle  by gleaning and free diving  Fig. 1bFig. 1b.Multicellular animals that have captured single-celled algae within their tissues include corals and giant clams. This ‘solar-power’ capability arises from photosynthesis by tiny algal protists—endosymbiotic dinoflagellates from the family Symbiodiniaceae, also known as zooxanthellae—within their bodies, providing the animal host with an additional energy source other than the ingestion of food items.T. gigas individuals  obtain 65% of their carbon from filter-feeding, compared to 34% for larger individuals   . Indeed,y source . The comy source . HoweverAt local and regional scales, giant clams face other human-induced pressures such as overfishing, habitat degradation and coastal urbanization ; Fig. 1cT. gigas, T. derasa, T. rosewateri and T. mbalavuana are listed as Vulnerable and Hippopus hippopus, H. porcellanus, T. maxima, T. squamosa and Tridacna crocea are listed as Lower Risk  have yet to be assessed, and due to lack of current knowledge about their biology and abundance, they will likely be classified as ‘Data deficient’.Giant clams are protected species under Appendix II of the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES), which consists of species that are not necessarily currently threatened with extinction but may become so unless trade is closely controlled. Of the 12 recognized giant clam species, only 9 have been assessed and listed in the International Union for Conservation of Nature (IUCN) Red List of Threatened Species: wer Risk . At locawer Risk . While twer Risk . For exaBreeding programmes have existed since the late 1980s to help restock over-exploited wild populations of giant clams e.g. . GametesIn recent years, we are recognizing that giant clams are not only threatened by overexploitation by humans, but are also threatened by a suite of environmental change drivers including (i) global change stressors, such as climate change, global warming and ocean acidification, and (ii) local and regional stressors from agriculture and urbanization, such as turbidity and sedimentation. Of the global pressures on marine life, pollution and climate change continue to occur at peak pressures . DHW are often used to indicate the likelihood of coral bleaching  (T. crocea)  , highlig2 from the atmosphere is absorbed by the oceans where it reacts with seawater and lowers pH in a process called ocean acidification. This process has caused a decline in the saturation state of seawater with respect to calcium carbonate polymorphs including aragonite  likely through mechanisms including disrupted functioning of ligand-gated chloride channels, such as GABAA receptors , further increasing the vulnerability of calcareous taxa. Behavioural changes could affect settlement choices in invertebrate planktonic larval stages and antipredator responses in planktonic and settled life stages.For animals with symbiotic microalgae, increased COynthesis . Ocean aT. squamosa, oxygen uptake exceeds total oxygen production and oxygen extraction in the dark is high relative to species without photosymbionts  in the water column, known as coastal ocean darkening. Since light regimes structure aquatic food webs, this water column light attenuation could be a key driver in coastal food web changes , whereas T. squamosa extends its depth range (down to 42 m) by heterotrophy  in seawater decreased gross production:respiration ratio by about one third through decreased photosynthesis ; changes in behaviour and/or physiology, including modifying symbiont communities (short- to mid-term), phenotypic plasticity (mid-term), genetic adaption (longer-term); and extinction. Distribution shifts can occur in response to a changing environmental condition that occurs along a gradient (e.g. temperature with latitude and light with depth). After considering (i) distribution shifts, we explore (ii) the capacity for rapid adaptive responses to environmental change from fast to slower timeframes.T. crocea, the boring clam, bores into large boulder-shaped coral colonies or other calcium carbonate-based structures.As global temperatures warm with climate change, species can take advantage of the decreasing planetary temperature gradient from equatorial to polar latitudes and shift their distribution poleward towards higher latitudes and cooler conditions . Global However, the saturation state of seawater with respect to calcium carbonate polymorphs such as aragonite decreases with increasing latitude. Light availability, a key metric for the success of giant clams, also decreases with latitude. Thus, the formation and persistence of giant clam shells  could be used to estimate the effect of reduced depth distributions on the total habitat availability within a species range. This modelling approach could be used to investigate the vulnerabilities of each giant clam species since they have different depth distributions, with certain species, such as Considering depth distribution in giant clams provides an example of how physiological responses may help predict ecological patterns and inform conservation management approaches of these threatened species. The principles of this approach, as well as other aspects of the current paper, could also be applied to other sessile photosymbiotic animals, such as reef-building corals.Although sessile beyond juvenile stages, giant clams have the ability to open and close their shell using their large adductor muscle. This immediate behavioural response to changing conditions is one way that clams can respond to environmental change , bacteria , Cladocopium (clade C) and Durusdinium (clade D)  and between generations  through parental provisioning, hormones and proteins and epigenetic marks . GeneticGiant clams are long-lived, and larvae that are produced in present-day oceans could survive until the end of the century. However, the opportunity for early life developmental acclimation in individuals will be limited to present-day conditions. Additionally, giant clams take a long time to reach reproductive maturity. They produce first male, then male and female gametes, when they reach a larger size. This means giant clam generation times are long, and reduced generation turnover time limits the potential for genetic adaption. Additionally, the selective exploitation of large giant clams from the wild will reduce the proportion of female clams. On the other hand, one positive characteristic of giant clam biology is that they can spawn millions of gametes, potentially producing high numbers of larvae and thus increasing the chances of selection acting to produce fitter individuals.2 emissions are stabilized, (ii) adaptive population management, (iii) selective breeding of tolerant giant clam holobionts for restocking programmes in selected areas and (iv) inoculation of tolerant strains of microalgae endosymbionts in giant clam early life stages.In addition to traditional breeding programmes, we should now view the conservation of giant clams through a global change lens to focus on protecting wild populations of these threatened species in the face of rapid environmental change. Conservation strategies and actions include (i) the management of local and regional conditions close to optimal while global CO2 emissions as soon as possible, and potentially implementing CO2 capture technologies, management of local–regional-scale stressors will involve water catchment management to maintain or improve water quality. Managing terrestrial run-off into the marine environment in areas of giant clam habitats will ensure associated impacts do not add further stress to giant clams. Additional impacts from stressors, such as reduced light levels  (2).Aquaculture breeding programmes often select for the best performing individuals, such as for growth or disease resistance. A recent mariculture study on ulation) , and thiBreeding programmes offer symbiotic microalgae to larval giant clams just before settlement, and the uptake of these symbionts are crucial for the survival of the larvae. Since giant clam offspring do not acquire their microalgae endosymbionts from their parents, larvae at a few days old gain their symbiotic microalgae from the environment independently. It is therefore plausible to help build resilience in giant clam holobionts by offering tolerant strains of microalgae symbionts at this stage.Symbiodinium, Cladocopium and Durusdinium, where each genus possesses unique ecological characteristics. For instance, Symbiodinium species are most adapted to living in shallow-water environments and tolerant to high irradiance stress  exclusively host Symbiodinium symbionts, suggesting that this specific host-endosymbiont interaction could be beneficial in the relatively extreme heat and irradiance conditions characteristic of the Red Sea  are sessile, unlike the majority of other iconic coral reef megafauna, (ii) inhabit shallow depths, providing good viewing opportunities on snorkel, (iii) are conspicuous, (iv) are long-lived, and (v) can be translocated into position. Aquacultured giant clams are well suited for translocation, ideally while small to medium sized. Giant clams can also be incorporated into citizen science programmes to enhance conservation efforts (e.g. The Great Barrier Reef injects AUD$6.4bn (USD$5.0bn) into the Australian economy every year , and recWhile we show how results from studies on giant clam physiology, morphology and behaviour can begin to inform conservation strategies, more work on global change stressors is required to understand the responses of giant clams to these pervasive changes in natural ecosystems. Elevated temperatures are already affecting giant clam wild populations. Further work on responses of individuals maintained at both elevated temperatures and simulated heat stress events could help determine any bleaching thresholds and other responses among different species to assist management and conservation efforts. The effects of ocean acidification on giant clams are still relatively little studied, and consideration of emerging threats, such as ocean deoxygenation, is an area for further work. Also, the interaction of global- and local–regional scale stressors, such as ocean warming and ocean darkening or pollution, need to be understood.For all these types of environmental change drivers, it will be important to assess a variety of biological responses, including physiology, morphology and behaviour, over various life stages to determine any life history bottlenecks. Additionally, the responses of communities within the holobiont, particularly the giant clam–Symbiodiniaceae association, as well as the response of the holobiont as a whole, will be important in assessing the overall resilience and adaptive capacity of giant clams to rapid environmental change. Transcriptomics could help unravel which genes are expressed at different environmental conditions and whether individuals or species have the capacity to upregulate critical genes to help cope with stressful conditions. Ecological modelling of biological responses of giant clams using physical and chemical data could help determine how the ranges of threatened species and populations may change over coming decades. Determining the economic value of giant clams for coral reef tourism and restocking activities could help incentivize and enhance their protection. Another opportunity to inform the conservation of these threatened species is to evaluate community assemblages using other types of information such as species richness, functional traits and phylogeny to identify species based on their functional and historical contributions. Such studies may also incorporate data on biological responses to holistically capture the species’ ecological standing within ecosystems.While new knowledge on the biological responses of threatened species is often urgently needed, particularly in respect to how they respond to rapid environmental change, research on threatened species can be challenging. Fortunately, giant clams can be successfully spawned in captivity, allowing experimental work to be undertaken on captive bred individuals, particularly in partnership with conservation breeding programmes. In interpreting results from captive bred individuals, one should consider that broodstock will be from a more limited genetic pool than wild stock, may be provisioned differently and may be acclimated to captive conditions, and that these factors could affect the responses of offspring in positive or negative ways to environmental drivers investigated.2 emissions as soon as possible and progress to beyond net zero CO2 emissions to give biodiversity the chance to survive the already locked-in changes to the Earth’s climate. Doing so will help to avoid lethal and sub-lethal effects of global change on threatened species, such as giant clams. In the meantime, this perspective article has (i) identified new environmental threats to giant clams, (ii) identified conservation management actions in the face of rapid environmental change, (iii) highlighted the importance of tourism in conserving giant clams and (iv) identified areas of further research on giant clams. While these actions are likely to allow the survival and appreciation of giant clams in the first half of this century, like coral reefs, their continued persistence in the world’s tropical oceans is ultimately dependent on rapid mitigation of global climate change.Enhanced global efforts are urgently needed to meet net zero CO


Transmission is a crucial step in all pathogen life cycles. As such, certain species have evolved complex traits that increase their chances to find and invade new hosts. Fungal species that hijack insect behaviors are evident examples. Many of these “zombie-making” entomopathogens cause their hosts to exhibit heightened activity, seek out elevated positions, and display body postures that promote spore dispersal, all with specific circadian timing. Answering how fungal entomopathogens manipulate their hosts will increase our understanding of molecular aspects underlying fungus-insect interactions, pathogen-host coevolution, and the regulation of animal behavior. It may also lead to the discovery of novel bioactive compounds, given that the fungi involved have traditionally been understudied. This minireview summarizes and discusses recent work on zombie-making fungi of the orders Hypocreales and Entomophthorales that has resulted in hypotheses regarding the mechanisms that drive fungal manipulation of insect behavior. We discuss mechanical processes, host chemical signaling pathways, and fungal secreted effectors proposed to be involved in establishing pathogen-adaptive behaviors. Additionally, we touch on effectors’ possible modes of action and how the convergent evolution of host manipulation could have given rise to the many parallels in observed behaviors across fungus-insect systems and beyond. However, the hypothesized mechanisms of behavior manipulation have yet to be proven. We, therefore, also suggest avenues of research that would move the field toward a more quantitative future. Entomophthora muscae  bassiana and several species of Metarhizium are among the relatively few fungal species that have been studied from a mechanistic perspective . Major fundamental differences between the life cycles and structures of Ascomycota and Zoopagomycota likely impact the molecular underpinnings of host manipulation. As such, our knowledge of the mechanisms that drive fungus-adaptive behavioral phenotypes is limited, especially with regard to the less lab-amenable Entomophthorales species  like  species . Despite species –22. Thisspecies –, 24, thespecies –. As suchMassospora, for example, produces infective spores while keeping its cicada hosts alive, coercing them to actively transmit the infection to conspecifics  appear to particularly benefit from increased locomotion activities in their hosts  are seemingly involved in causing enhanced locomotion and climbing behavior in its caterpillar host  infiltrate the brain tissue of their hosts  while the hosts are still alive . Lack of such direct access in hypocrealean fungi may indicate that behavior is altered through indirect routes . Alternatively, the position of fungal cells relative to host nervous tissue may be dispensable for behavior alteration .Ophiocordyceps-manipulated ants, the expression levels of genes implicated in response to starvation were found to be altered , while in E. muscae-infected fruit flies, metabolism Gene Ontology (GO) terms were significantly enriched among downregulated genes during late infection  have revealed a putative enterotoxin to be highly expressed during manipulated summiting and biting behavior (Ophiocordyceps species so far having been found to have the most), comparative genomic analysis found that orthologs of this specific enterotoxin are seemingly exclusively conserved among ant-manipulating Ophiocordyceps species  that impacts host physiology. There is mounting evidence for the importance of protein effectors in zombie ants: transcriptomic analyses of two distinct behavior . While e species . This su species .Ophiocordyceps spp. of which a significant number are upregulated during manipulated biting behavior  encoded by behavior , 21, 27.Massospora levispora-  analysis was performed to detect the presence of secreted compounds  and sphingosine, were found to be uniquely present in cocultures with the brain of the natural host, C. castaneus  and amphetamine cathinone in typediae ) and M.  cicadas . Analyse cicadas . Additiopulation , 27, furnization . Thus, tnization . Beyond ompounds . Resultaastaneus . A subsepulation . The stunfection .It is not yet clear which putative effector genes can be implicated in behavior manipulation by entomophthoralean fungi. Both a paucity of genomic data  and their much greater phylogenetic distance from well-studied Ascomycota (often impeding inference of function by homology) mean we currently know much less about the genes expressed by these fungi over the course of infection and during manipulation. The predominant hypothesis in the field is that entomophthoralean fungi refrain from producing effectors to elude immune detection from their host, as well as to avoid premature host death , 74, butE. muscae has found that house flies infected and housed in complete darkness  die without consistent timing  is essential for baculovirus-elicited ELA  is essential to elicit climbing behavior  and summiting comes, not from behavior-modulating fungi, but from baculoviruses. In ited ELA , 80; in behavior . While tbehavior , which dbehavior . Howeverbehavior , 84. ThiThe ability to manipulate host behavior has convergently evolved in an array of fungal entomopathogens and beyond. This complex trait is thought to be the result of a long, intimate coevolution between pathogens and hosts in which both organisms are in a constant arms race. Despite the independent evolutionary trajectories of each of these pathogens with their respective insect hosts, they have elicited similar types of behaviors. As discussed, fungi across two distinct phyla manipulate host locomotion activity, induce climbing behavior, trigger preference for or attraction to certain abiotic factors, activate mouthparts to adhere to vegetation, and do so with timed precision . This beC. floridanus, the ant host of O. camponoti-floridani, appears to be rather plastic and has been suggested to underlie at least some of the plasticity that gives rise to the behavioral division of labor in colonies of this species classification of many new species of zombie-making fungi in just the last decade , which are assembly averse. The advent of long-read sequencing and ever-improving future technological developments are promising options for solving this issue.Future research into the molecular underpinnings of host manipulation is also dependent on a complementary expansion in genomics, transcriptomics, and metabolomics work. For instance, the current relatively low availability of high-quality, annotated genomes, scattered across a few distantly related fungal species, complicates meaningful comparative analyses between these groups. Increasing the number of sequenced genomes within genera and families that harbor at least several zombie-making fungal species would allow for the identification of evolutionary patterns, genes, and gene clusters that give rise to behavior manipulation traits. This, too, however, poses various challenges: where the genomes of hypocrealean Ophiocordycipitaceae and Cordycipitaceae are relatively compact  andAs with any new field of study that ventures forward from descriptive to quantitative research on wild, nontraditional model systems, there are many hurdles to overcome before the mystery of mechanisms underlying fungal manipulation of insect behavior can be fully unraveled. Understanding these pathways will lead to valuable insight into the mechanisms of pathogen-host coevolution and genes underlying host specificity in animal models. Additionally, these efforts will shed new light on how and why animals behave in certain ways and may open the road toward the discovery of novel fungal biomolecules with potential pharmaceutical and industrial applications. Furthermore, characterization of biomolecules secreted by zombie-making fungi and their role in host manipulation at the molecular level will finally allow for a more holistic comparison between these animal pathogens and well-studied host-pathogen interactions in fungus-plant models.


Fat embolism syndrome (FES) occurs when fat particles are aberrantly distributed into the microcirculation, and it often manifests as either hypoxemia, neurological deficit, or petechial rash. Although cases have been reported in the literature since the twentieth century, no formal diagnostic criteria have been universally adopted, and FES remains a diagnostic challenge. We present a unique case of FES from a long bone fracture, leading to pulmonary embolism with paradoxical arterial embolization and cerebral infarction, and provide a review of the related literature. The incidence of subclinical fat emboli has been described in more than two-thirds of orthopedic trauma cases in a review by Bulger et al., with an even higher incidence when serum samples are drawn in close proximity to the trauma site -2. If thAn 83-year-old female presented to the hospital with generalized unresponsiveness after a mechanical fall at home. Past medical history was significant for coronary artery disease and transcatheter aortic valve replacement for severe aortic stenosis one year prior, which itself was complicated by an embolic left hemispheric stroke requiring dual antiplatelet therapy. After admission, the patient became increasingly hypoxic and hypotensive, prompting intubation, mechanical ventilation, and vasopressor support.Plain radiography on admission revealed right femoral neck and right clavicular fractures. Initial brain magnetic resonance imaging (MRI) demonstrated no acute findings, and lower extremity venous Doppler was negative for deep vein thrombosis. Chest radiograph, however, demonstrated bilateral infiltrates, and subsequent transthoracic echocardiogram (TTE) provided visualization of moderate right ventricular enlargement and moderate global hypokinesis with septal flattening in systole, consistent with right ventricular pressure overload. The left ventricular ejection fraction was normal Video .A continuous heparin infusion was initiated empirically; chest computed tomography (CT) with contrast was deferred due to poor renal function and its low likelihood to alter clinical decision-making. Due to a poor neurologic response off sedation, a second brain MRI was done on day three of hospitalization, which demonstrated new punctate focal lesions on T2 hyperintensity involving the gray-white matter junction of both cerebral hemispheres and within the left middle cerebellar peduncle Figures -1B.Additionally, the diffusion-weighted imaging (DWI) sequence revealed new, scattered punctate microhemorrhages at the gray-white matter junction of the frontal and parietal lobes bilaterally Figures -2B.A repeat TTE, instead of transesophageal echocardiogram (TEE) due to thrombocytopenia, was performed on day four of hospitalization. The repeat TTE showed no valvular vegetation and complete normalization of the right ventricular findings noted on the prior study; shunting was not appreciated on an agitated saline bubble study. The patient remained off sedation with poor neurologic responsiveness that included obtundation in addition to the absence of any purposeful movements; pupillary, corneal, oculo-cephalic, and gag reflexes were still present. The patient was eventually transitioned to comfort care.Although the differential for encephalopathy is broad, the star-field distribution pattern of lesions on MRI narrows the diagnoses to diffuse axonal brain injury, cardiogenic cerebral emboli, cerebral vasculitis, or hemorrhagic intracranial metastases. In the presented case, the patient was diagnosed with cerebral FES due to her clinical picture in the context of an MRI that demonstrated innumerable scattered punctate foci of faint restricted diffusion in the gray-white matter of bilateral cerebral hemispheres. In the absence of cutaneous changes on physical examination, she did meet two major criteria  of <60 mmHg and cerebral symptoms in non-traumatic brain injury) of Gurd and Wilson’s criteria for fat emboli syndrome Table  1]..1].We hypothesize that the fat emboli likely resulted in acute hypoxic respiratory failure and initial right ventricular overload with subsequent paradoxical embolization through pulmonary microcirculation, resulting in cerebral fat deposits and normalization of right ventricular pressure.The presentation of FES is variable and depends on the organ system whose microvasculature has been impacted by fat emboli. Damage to the retina, kidney, liver, and heart has been reported, although the triad of petechial hemorrhages, respiratory distress, and altered mental status is the most common clinical presentation -2. CutanSince the first reported incidence of fat embolism over 150 years ago, several theories have been proposed for the mechanism by which fat emboli are distributed into the arterial circulation . This alThe biochemical theory points to a local inflammatory response at the site(s) of fat emboli deposition, which itself is characterized by a surge in catecholamines as a stress response to microtrauma to the affected capillary bed. This surge then induces a liberation of free fatty acids, which interact with lung lipases, leading to the activation of the intrinsic apoptosis cascade and subsequent damage to both lung parenchyma and capillary endothelium. The specific disruption of capillary membranes is thought to be secondary to a loss of endothelial cells, which causes an increase in tissue permeability; inflammatory-mediated damage to surrounding tissues then results in interstitial hemorrhages and edema ,8-10.And although these two theories differ in mechanism, both pathways are likely contributory to the underlying injury FES. The mechanical phase causes blockage of capillaries and resultant veno-arterial shunting; the subsequent biochemical phase induces inflammation and apoptosis that causes damage to surrounding tissue . MoreoveMaking the diagnosis of FES can be challenging, and although Gurd and Wilson’s criteria Table  have beeThe clinical presentation and ultimate prognosis of CFE are variable and depend on the disease burden. Patients with acute respiratory distress syndrome generally have the worst prognosis, as most deaths are attributed to respiratory failure ,7. This Despite CFE traditionally being associated with poor neurologic outcomes, several reported cases have described full neurologic recovery with only minor residual deficits through prompt diagnosis and symptomatic care. The majority of patients who receive appropriate supportive care, in fact, can recover from both neurologic and pulmonary sequela, with an overall mortality of only 7-10% . In one Treatment in patients diagnosed with CFE begins with prompt diagnosis and general supportive measures. Proposed disease-directed therapies have been trialed but have largely been unsuccessful to date. Attempts to decrease free fatty acid mobilization and lipolysis have not shown any clinical benefit, and anticoagulation may increase the risk of bleeding ,3. A metFES with CFE, in summary, are potentially fatal sequelae of long bone fractures, and although there are correlative examination and imaging findings, the diagnosis remains clinical. For patients who experience traumatic long bone fractures and/or injury and then develop respiratory failure with encephalopathy, a diagnosis of CFE should be considered. If clinical suspicion remains high, an MRI of the brain with both diffusion and T2-weighted imaging should be obtained. And while disease-directed therapies are under development, the optimal management remains preventive and may be achieved through early operative fixation of long bone fractures and correction of hypoxemia to maintain end-organ and tissue perfusion.


Vaccines represent one of the major advances of modern medicine. Despite the many successes of vaccination, continuous efforts to design new vaccines are needed to fight “old” pandemics, such as tuberculosis and malaria, as well as emerging pathogens, such as Zika virus and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Vaccination aims at reaching sterilizing immunity, however assessing vaccine efficacy is still challenging and underscores the need for a better understanding of immune protective responses. Identifying reliable predictive markers of immunogenicity can help to select and develop promising vaccine candidates during early preclinical studies and can lead to improved, personalized, vaccination strategies. A systems biology approach is increasingly being adopted to address these major challenges using multiple high-dimensional technologies combined with in silico models. Although the goal is to develop predictive models of vaccine efficacy in humans, applying this approach to animal models empowers basic and translational vaccine research. In this review, we provide an overview of vaccine immune signatures in preclinical models, as well as in target human populations. We also discuss high-throughput technologies used to probe vaccine-induced responses, along with data analysis and computational methodologies applied to the predictive modeling of vaccine efficacy. Vaccines are the most effective preventive measure ever developed in the fight against diseases. They have led to the eradication of smallpox and to a major reduction in the incidence of diseases such as diphtheria, tetanus or poliomyelitis. Nevertheless, the need for new vaccines has never been so critical as demonstrated by the recent SARS-CoV-2 pandemic. Novel vaccines are also required to fight against “old” diseases like malaria and tuberculosis , which aOne of the main goals in vaccinology is to identify factors that reflect vaccine-induced immune responses and thus provide biomarkers of vaccine immunogenicity and efficacy. A biomarker can be defined as “a characteristic that is objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes, or pharmacological responses to a therapeutic intervention” . By exteThe emerging field of systems vaccinology aims to identify biomarkers and immune signatures that correlate with vaccine efficacy to decipher protective immune mechanisms. A correlate of protection is defined as a biomarker or an immune mechanism that is “statistically related to and responsible for protection” ,7. Thus,High-throughput technologies have rapidly expanded over the last several years and have been frequently employed in systems vaccinology studies, making it possible to extend the range of biomarkers included in vaccine signatures ,10,11,12Although systems vaccinology ultimately aims to develop predictive models of vaccine efficacy in human populations, applying the same approach to animal models, which allow the use of a wide range of tools in controlled study designs, empowers vaccine research and improves preclinical studies. Notably, high-throughput imaging technologies can be used in preclinical models to characterize immune responses at the whole-body and tissue levels ,14 and tHere, we review approaches to identify biomarkers and signatures of vaccine responses in preclinical models and humans. We provide an overview of high-throughput technologies used to probe vaccine-induced responses, including in vivo and in vivo imaging technologies. We also present data analysis and computational methodologies used to define signatures that correlate with and potentially predict vaccine efficacy.One of the main goals in systems vaccinology is to identify a strong and reliable vaccine signature that statistically differs between immunized and non-immunized individuals that can be easily measured in the blood and at a reasonable cost.However, protection conferred by vaccination results from complex interactions between innate and adaptive immunity and there are considerable differences between individuals in the response to immunization. Such variation, mediated by both host factors and vaccine properties, precludes the description of a universal marker of vaccine efficacy .Indeed, many studies have demonstrated the effects of sex on vaccine responses ,16,17, aSimilarly, vaccine composition influences the dynamics of immune parameters, such as immune-cell migration to the injection site or the immune-cell transcriptional profile ,27. In aConsequently, host- and vaccine-related factors, extensively reviewed by Zimmermann and Curtis , may infThe characterization of extensive vaccine signatures is further influenced by the different types and sources of biomarkers, as discussed hereafter.Antibody responses are widely used to assess vaccine responses . However+ T cells and CD4+ T helper cells, as well as immune memory. However, the role of innate cell populations has been recently re-evaluated to consider their influence in initiating and orienting the adaptive response. Consequently, innate cells are increasingly being studied as early biomarkers of vaccine efficacy. Notably, several studies have shown that innate myeloid cells, such as neutrophils, monocytes, and innate lymphoid cells (ILCs), such as natural killer (NK) cells, are of interest in defining vaccine signatures  adiolabeled NK cell-based vaccines in patients with renal carcinoma. The authors observed the accumulation of 50% of the activity in the lesions, but a high level of circulating activity was also observed, caused by the released Indium-111  in mice and NHPs [PET is mainly used in oncology for the visualization of sites of inflammation . To dateand NHPs . In addiand NHPs .18F]-FDG PET imaging after the administration of vaccines in mice [18F]-FDG, 2-deoxy-2-[fluorine-18]fluoro-D-glucose to track granuloma formation after Mycobacterium tuberculosis infection, as a correlate of active disease after BCG immunization. [18F]-FDG PET imaging can also be used for the monitoring of the inflammation related to vaccination, such as that induced by influenza vaccines -labeled 3′-fluoro-3′-deoxy-thymidine ([18F]-FLT), to assess the proliferative immune cell response in lymph nodes after vaccination with an antigen-loaded DC vaccine. The authors showed a better correlation of [18F]-FLT than [18F]-FDG PET imaging with immune reactivity. However, theses imaging methods, lack the specificity and discriminatory power that antibodies or their fragments provide to specifically target immune cells in vivo -oxine, in rhesus macaques. Several studies aiming to track immune cells have used ex vivo  imaging ,114. PET64Cu], [68Ga], or [89Zr]  ,116 for r [89Zr]  or fluorr [89Zr] ,118.Full-sized antibodies have been widely and successfully used for immuno-PET imaging ,120,121.64Cu]-labeled anti-CD11b for longitudinal monitoring of the mobilization of CD11b+ myeloid cells from the bone marrow to the spleen and to local inflammatory lesions in mice.Among the strategies for imaging innate myeloid inflammatory cells, entire anti-CD11b, anti-class II major histocompatibility complex (MHC), and anti-macrophage mannose receptor antibodies or antibody fragments have been widely used to characterize inflammation by immuno-PET, mainly in mice ,123,124.Imaging of macrophages has already been performed in various applications to study inflammatory processes by targeting folate receptors  with rad+ T cells has also been monitored by immunoPET in preclinical tumor models, specifically in the context of immunotherapies using checkpoint-blockade inhibitors against the PD-1/PD-L1 and CTLA-4 axes [The presence of CD8A-4 axes . StrategA-4 axes ,130,131.A-4 axes .Thus, whole-body immunoPET combines the sensitivity of PET with the high specificity and affinity of monoclonal antibodies. Furthermore, the use of antibody-derived fragments allows better tissue penetration, a lower background, and a smaller radiation burden for the patient.The complexity of the immune system, particularly when vaccines are involved, requires real-time, high-resolution imaging to visualize immune-cell interactions at the microscopic level. Intravital microscopy , two-photon imaging) provides the detailed visualization of vaccines and their behaviors in the injection sites or lymph nodes.Fibered confocal fluorescence microscopy (FCFM) was limited to preclinical applications due to the lack of human validated fluorescent tracers. FCFM is developed notably for the visualization of tumor growth and angiogenesis ,134, as + T cells and DCs in vivo in the lymph node before and during antigen recognition in mice [Two-photon microscopy (2PM) has been adopted to study single-cell dynamics at high spatial resolution at a depth of several hundred microns . The ava in mice . Althoug in mice ,141.To provide the most advanced technologies for characterization of the complexity of the immune response to vaccination in relevant tissues, it is necessary to complement and strengthen in vivo imaging methods with state-of-the-art technologies for in situ multiplexed characterization of cellular and subcellular markers of the immune response. Despite the considerable advantages of available imaging techniques, one of the main drawbacks is the small number of parameters that can be simultaneously analyzed. Immunohistochemistry (IHC) and immunohistofluorescence (IHF) are currently the most common and suitable techniques to gain insights on changes in the spatial immune phenotype in various tissues. Immunofluorescence is widely used to detect multiple immune and inflammatory cell populations, as well as pathogens or vaccines in ex-vivo samples. It allows the assessment of innate and adaptive immune responses, especially their effectors in lymphoid organs or infected sites after pathogen challenge or immunization. For example, Darrah and al.  observedEpifluorescence and confocal microscopy allow the observation of cellular and subcellular compartments. However, the number of observable markers is limited using such devices due to fluorophore spectra overlap. This can be partially resolved by the use of white laser confocal microscopy, spectral imaging, or cyclic immunofluorescence .Tissue clearing, by reducing light-scattering and light-absorbing components, overcomes the limits of light penetration and thus allows deep imaging . Three-dMass spectrometry imaging (MSI), the combination of molecular mass analysis and spatial information, can provide information on the spatial distribution of endogenous and exogenous species in tissue sections, without the need to disrupt sample integrity ,147. It One of the major recent advances of MSI is associated with the introduction of mass cytometry imaging (MCI), a label-based MSI technique to follow protein markers at the cellular and subcellular level that combines mass cytometry and immunocytochemistry (ICC), IHC or IHF techniques, a high-resolution laser ablation system, and a low-dispersion laser ablation chamber ,154. MCIProgress in the development of these high-multiplex techniques has allowed the identification of various cell populations that comprise the immune system, but they still present certain limitations. Gerner et al. developed an approach, histocytometry, which associates the spatial information that can be obtained using histology with the phenotypic data provided by flow cytometry . They shHigh multiplexing methods have prompted the need for the development of image processing and analysis tools using complex machine-learning algorithms.The ability to efficiently use or design analytical pipelines for interpreting -omics data has emerged as a critical element for modern vaccinology research. Such analytical pipelines are generally developed using one or more programming languages . Their aim is to handle a large panel of analytical steps, ranging from data preprocessing to data integration. Proper analytical pipelines should be sufficiently flexible to tackle various studies but also sufficiently focused on certain methods to answer immunological questions specific to each study. Bioinformatics pipelines used to interpret high-dimensional immunological data should follow the FAIR principles  . The usep-values) of differentially expressed genes. Once identified, the ability of one or multiple gene signatures to segregate the biological conditions of interest is tested using multivariate representations. Among them, heatmaps combined with dendrograms show the relative levels of gene expression for the gene signature in all samples using unsupervised hierarchical clustering at both the gene and sample level. Dimensionality reduction methods, such as PCA and MDS, are also useful in determining the quality of a signature and its ability to separate conditions in a multivariate manner. Venn diagrams are common representations that show the amount of overlap between multiple gene signatures. Due to their length , gene signatures cannot be interpreted manually, gene by gene, in relation to the literature and must be interpreted using specific methods. Functional enrichment analysis gathers a large set of methods and databases and aims to identify over-represented biological functions or pathways in a gene signature of interest. Statistical tests, generally based on Fisher’s exact test, make it possible to determine which pathways are significantly over-represented. The most widely used databases for functional enrichment analysis are Gene Onthology [Due to its development in the early 21st century, transcriptomics analysis now benefits from an outstanding variety of algorithms, methods, software, and dedicated databases. Despite the large set of available data analysis approaches, differential expression analysis is still the gold-standard for interpreting transcriptomic profiles . Differenthology , KEGG, anthology , are of nthology  is a metnthology , can be The development of algorithms for analyzing cytometry profiles is still an active area of research. Although automatic gating approaches mimic cytometry experts by positioning gates in cytogram plots (two-dimension (2D) representations in which each axis corresponds to the expression of one cell parameter), automatic cell clustering algorithms identify groups of cells that have similar phenotypes . SPADE  and FlowSingle cell sequencing will have for sure a pivotal role in modern biology to decipher both molecular and cellular events involved in vaccination. Thanks to this technique, key internal mechanisms responsible for cell activation, proliferation, and differentiation will be characterized at unprecedented level of detail allowing more rational when designing vaccines. While most of the recent efforts have been done for applying this technique on transcriptomics, the characterization of B and T cell repertoires at the single cell levels is of great interest. The analysis challenge for single cell sequencing data is important as methods created for the analysis of bulk transcriptomics and high dimensional cytometry must be combined for handling them. The Cell Ranger suite developed by 10X Genomics allow bioinformaticians a straightforward way to analyze single cell sequencing data, especially regarding the preprocessing steps. The first analysis step consists of the alignment of sequenced reads of a reference genome. The filtering of cell events with abnormal number of mapped transcripts or associated with aberrant mitochondrial activities is done at this step. Once the reads have been aligned on the reference and transcript expressions are quantified for each cell of each sample, an analysis step consisting in a dimensionality reduction is done. As for cytometry data, tSNE or UMAP algorithm are commonly used for that purpose. Of note, the Loupe browser also developed by 10X Genomics offers the possibility to graphically handle UMAP and tSNE representation for a processed dataset. Different R packages or approaches, such as Seurat  are compMachine learning is “the study of computer algorithms that improve automatically through experience”, as defined by Tom Mitchell , by learSeveral vaccines have been studied with the goal to identify predictors of immunogenicity after delineating signatures that correlate with immunogenicity, mainly in healthy adults . In mostThe most popular vaccine models for immunologists include the YF-17D and flu vaccines. YF-17D represents an ideal vaccine to understand and mimic because it induces life-long protection after a single injection. There are two major types of influenza vaccine: a live attenuated vaccine, which is delivered intranasally, and an inactivated vaccine, which is injected intramuscularly, both providing protection through distinct mechanisms. Adjuvanted versions or higher doses are also available for specific populations, such as the elderly, and thus represent the first personalized vaccines. However, these traditional seasonal flu vaccines do not provide long lasting and broad protection. They are based on yearly predictions of the circulating viruses, and they confer protection only when strains do match the circulating viruses. A key challenge is to develop pan-influenza viruses vaccines targeting conserved regions that would protect against seasonal, future drifted and pandemic strains. Anyway, not surprisingly, YF-17D and seasonal trivalent inactivated influenza vaccines (TIVs), likely because of practical reasons , are over-represented among studies to define predictors of immunogenicity. Of note, predictors of the antibody and T-cell responses ,180 and Camk4 [Gcn2  for YF-17D [It is expected that predictors of vaccine-induced antibodies could be clinically useful by predicting suboptimal immune responses to certain vaccines to stratify them, for example those requiring a booster. However, the robustness and predictive accuracy depend on the sample size and the identification of solid predictors requires extensive validation in multiple clinical trials. A cost-effective PCR-based ‘vaccine chip’ that focused on a set of predictive genes was successfully developed for flu vaccines . It is aCamk4  and apopCamk4  for flu r YF-17D .The purpose of mechanistic mathematical modeling differs from that of machine learning. It aims to mimic biological mechanisms through observations of and assumptions about the phenomenon of interest. This type of modeling uses mathematical formulations that seek to identify a mechanistic relationship between inputs and outputs of the phenomenon of interest ,198. TheAlthough reducing and refining animal experiments require permanent efforts from the scientific community, assessing vaccine responses in animal models is still, for now, a necessary step in the vaccine registration process . CurrentApplying systems vaccinology approaches to animal models assuredly provides a way to improve preclinical studies, accelerate vaccine development, and increase our knowledge of vaccine-induced protective responses, as discussed below.Through the use of diverse high-throughput technologies, systems immunology can lead to the identification of multiple biomarkers, combined into complex signatures that can be used to build predictive models of vaccine-induced responses. Being able to predict the efficacy of a vaccine with the identification of early biomarkers could considerably facilitate preclinical and clinical trials. Currently, vaccine development starts with an exploratory phase that addresses basic scientific questions and ideally leads to proof-of-concept studies that validate the efficacy of the vaccine in experimental—usually small animal—models. Translational research then follows with immunogenicity and efficacy studies in preclinical models, which guide subsequent clinical trials in humans .The evaluation of vaccine efficacy can be challenging and partially relies on the possibility to define reliable correlates of protection. According to Plotkin and Gilbert, a correlate of protection can be defined as a marker of immune function that statistically correlates with protection after vaccination . As mentDefining accurate correlates of protection is pivotal in the vaccine development process, as it allows the rapid assessment of vaccine efficacy. Hence, applying systems immunology approaches to clinical trials has been proposed to accelerate the discovery of early predictive markers of vaccine efficacy . AdditioApplying these approaches to human studies could therefore provide a way to reduce the time and cost of vaccine efficacy trials.Presently, validated correlates of protection are generally based on results obtained from cohorts of vaccinated healthy adults, whereas target human populations are often highly heterogeneous, and defining a universal response to vaccination is probably impossible . Indeed,In vulnerable populations, such as infants and young children, pregnant women, and elderly and immunocompromised individuals, immune responses to vaccination are often under characterized and assessing vaccine efficacy in these specific populations remains a major challenge. Preclinical models are well-suited for in-depth studies on population subsets with specific attributes, especially animal models with the most predictive value for human vaccine efficacy studies, such as NHPs. Although systems vaccinology approaches have already been used in human cohorts of aged individuals ,221,222,BCL2) transcript levels in T cells and lower interleukin-10 (IL-10) receptor alpha (IL10RA) transcript levels in T, B, and NK cells and monocytes of macaque neonates. This study illustrates how systems vaccinology in preclinical models can guide and help human vaccine efficacy trials. Moreover, identifying vaccine signatures in preclinical models of vulnerable populations would allow investigation of the influence of host factors on responses to immunization.Infant NHPs have been frequently used in studies to evaluate vaccines against Mycobacterium tuberculosis ,230,231 Pharmacogenomics and vaccinomics also contribute to improve personalized medicine and vaccination, respectively ,20. IndeConsequently, the various approaches of systems vaccinology empower personalized vaccination strategies, which should improve efficient vaccination coverage in target populations.Another way to increase global vaccine efficacy in highly diverse human populations is to improve vaccine formulation with adjuvants and refine administration routes and schedules. Systems immunology offers powerful tools to investigate the effects of adjuvants and vaccine regimens on human immune responses ,181 and Plasmodium falciparum [As previously stated, applying systems biology technologies in this context will further improve preclinical studies and enhance innovation and progress in the field of vaccinology. For example, transcriptomics was first used in mouse models to characterize the molecular and cellular signatures of clinically tested human vaccine adjuvants ,238. Thelciparum . In a hulciparum . This stThe mode of vaccine administration is another important parameter that modulates immune responses, as illustrated in a recent study of Cirelli et al. on the effect of slow delivery of HIV antigens in Rhesus macaques . They shOverall, these results illustrate how systems vaccinology can strengthen preclinical findings on vaccine formulation and administration, and thus support vaccine development processes.Finally, the use of systems vaccinology in preclinical models will provide mechanistic insights on immune responses triggered by vaccines. Because of the constraints on collecting tissue samples in human clinical trials, most studies have strived to define signatures of myeloid and lymphoid responses in the blood ,142,189.+ T cells differentiate into memory cells within lymph nodes and spleen GCs, and then migrate to the bone marrow, which represents their main homing site [As mentioned previously, early local events in the skin following immunization can shape subsequent immune responses and vaccine efficacy ,212,213.ing site ,243. Thuing site  or in huing site ,244. Howing site . In anoting site . They deing site  and to sing site .In addition, animal models are also particularly appropriate for assessing and dissecting immune responses to a combination of vaccines and/or pathogens. Indeed, exposition to pathogens and the vaccination history of animals are strictly monitored and recorded in laboratory settings. Such controlled experimental conditions facilitate the study of interactions between antigenically unrelated pathogens or immunogens. Recently, much attention has been given to the importance of non-specific effects of vaccines with a growing body of evidence suggesting that live-attenuated vaccines such as BGC, measles and oral polio vaccines overall improve childhood health . Indeed,+ T-cell responses to YF-17D vaccination in one of the earliest systems vaccinology studies [Gcn2 to demonstrate that GCN2 leads to increased autophagy and antigen presentation in DCs in response to YF-17D immunization and thus revealed a connection between a vaccine-induced stress response in DCs and adaptive immune responses [Finally, although NHPs most likely provide the best model of human vaccine responses in terms of prediction, small animal models can be used to further investigate the functional mechanisms underlying predictive biomarkers identified in preclinical and clinical studies. Notably, transgenic mice have been instrumental in elucidating the functions of genes implicated in immune responses to pathogens or vaccines ,252. Foresponses .To conclude, preclinical models are invaluable for expanding our knowledge of immune mechanisms underlying vaccine protection and for improving rational vaccine development.Systems immunology approaches used in preclinical studies and clinical trials are invaluable in vaccine biomarker discovery through the analysis and statistical modeling of large datasets.Despite the outstanding development of high-throughput technologies and computational methodologies, many challenges still remain to be tackled to realize the full promise of systems vaccinology. Indeed, most recent studies in the field of vaccinology have restricted their findings to a description of vaccine biomarkers without further exploring the correlative, predictive, or explanatory aspects of these signatures. Furthermore, being able to discriminate between markers of immunogenicity and protection is not an easy task as illustrated by many clinical trials of HIV vaccines . The appIn this review, we highlighted numerous technological and biological aspects useful to study vaccine responses and to the development of future vaccines. Nevertheless, limitations inherent to research techniques or to animal models question the methods currently employed in vaccine studies. First, the inherent differences in data formats generated by immunophenotyping platforms are a major impediment to the integration of large data sets. For instance, mRNA expression levels do not always represent intracellular protein expression levels and may not either reflect extracellular marker expression detected by cytometry . Second,Although human clinical trials of vaccine efficacy increasingly rely on systems immunology approaches, preclinical studies are lagging behind. Nonetheless, preclinical models harbor rare assets to explore vaccine-induced responses, including the possibility to investigate immune processes in tissues and the whole organism. The future of defining comprehensive vaccine signatures will likely rely on extended data analyses, including data obtained through imaging technologies, similarly to what has already been implemented in the field of cancerology ,259. In Overall, application of systems biology concepts to preclinical and clinical studies promises great advancements in our understanding of vaccine-induced immune protective responses and provides unprecedented opportunities for the development of new vaccines.