Extracellular vesicles (EVs) have been isolated in different body fluids, including urine. The cargo of urinary EVs is composed of nucleic acids and proteins reflecting the physiological and possibly pathophysiological state of cells lining the nephron and the urinary tract. Urinary EVs have been confirmed to contain low amounts of various types of RNA that play a role in intercellular communication by transferring genetic information. This communication through EV RNAs includes both continuation of normal physiological processes and conditioning in disease mechanisms. Although proteins included in urinary EVs represent only 3% of the whole-urine proteome, urinary EVs can influence cells in the renal epithelia not only by delivering RNA cargo, but also by delivering a wide range of proteins. Since urine is a readily available biofluid, the discovery of EVs has opened a new field of biomarker research. The potential use of urinary EV RNAs and proteins as diagnostic biomarkers for various kidney and urologic diseases is currently being explored. Here, we review recent studies that deal in identifying biomarker candidates for human kidney and urologic diseases using urinary EVs and might help to understand the pathophysiology. The discovery of extracellular vesicles (EVs) in human plasma dates back to 1967, when this subcellular fraction was identified by electron microscopy by Wolf and was shown to consist of small vesicles, with a diameter between 20 and 50 nm, originating from platelets and termed “platelet dust”. This “platelet dust” was capable to induce thrombin formation similarly to platelets [Sixteen years later, two independent groups observed that the transferrin receptors associated with 50 nm vesicles were jettisoned from maturing blood reticulocytes into the extracellular space ,3.In 1996, over a decade after the detection of exosomes in reticulocytes, other investigators  establisLater, EVs were purified from almost all mammalian cell types, including stem cells ,11,12,13The extracellular vesicles (EVs) are classified on the base of their cellular origin and/or biological function or on their biogenesis . The thrThe protein content of different types of EVs typically reflects that of the parent cells and is enriched in certain molecules, such as adhesion molecules, membrane trafficking molecules, cytoskeleton molecules, heat shock proteins, cytoplasmic enzymes, signal transduction proteins, cytokines, chemokines, proteinases and cell-specific antigens. Furthermore, EVs contain mRNAs, non-coding RNAs (ncRNAs), including microRNAs (miRNAs) and retrotransposon RNA transcripts, and even single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), mitochondrial DNA, and oncogene amplifications  ,32,33.Numerous mechanisms have recently been identified to control exosome biogenesis, thus facilitating protein and RNA cargo sorting to produce exosomes with a precise biochemical composition ,34,35,36Some investigators have studied EVs with a size less than 100 nm in diameter defining them exosomes, while others considered only EVs with a cut off value of 150–200 nm in diameter. This has become a main issue in many reports. Some papers use the term “microvesicles” instead of “exosomes” , others Exosomes and microvesicles are not one type of vesicles secreted by cells. Some agree that the term microvesicle covers a broader range of vesicles with a diameter from 100 nm up to 1 μm . On the Practically all cell types liberate EVs as well as tumour cells , antigenEVs were first found in human urine  and afteUrinary EVs can be obtained by a two-step differential ultracentrifugation process. This method allows efficient isolation of EVs with a high degree of purity, though it is laborious and time-consuming. However, density-gradient ultracentrifugation also cleans samples from other contaminants such as soluble proteins , thus coConsidering the limitations in the urinary EV isolation and the amount of variability between samples, strong normalisation methods are needed to allow valid studies. Human urinary EV proteins are normalized to urine creatinine that is the traditional variable, although creatinine excretion rate varies widely among humans. Alternatively, the excretion of specific exosomal proteins can be normalized to the excretion of proteins specifically associated with exosomes and often absent in EVs other than exosomes such as PDCD6IP or TSG101. They are part of the endosomal sorting complex required for transport (ESCRT) machinery that has been recently shown to play a direct role in exosome biogenesis . This asTypical populations of EVs are being studied using: (i) immunoblotting that shows the presence of EV proteins; (ii) transmission electron microscopy; and (iii) flow cytometry that provides information on the relative presence of surface proteins. It is important to note that the variability in number of EVs excreted in urine within and across people is not clearly defined and high volumes of urine are required in order to generate useful quantities of EVs. At present, counting a precise number of EVs in a given sample is a challenge. However, technological advances are promising for the future such as the Nanoparticle Tracking Analysis (NTA) that can rapidly size and directly count EVs in urine and identify surface marker proteins . A studyHuman urinary EVs contain mRNA that, like their protein cargo, derive from all regions of the nephron . Thus, uIt is important to note that the human urinary microvesicle nucleic acid load is dominated by ribosomal RNA reliable with a typical eukaryotic cell RNA profile together with the presence of prominent 18S and 28S rRNA peaks . This ismiRNAs are short (20–25 nucleotides) ncRNA molecules that inhibit a set of specific target mRNAs and regulate specific cellular proteins. They are a new class of disease biomarkers because certain miRNA species are organ specific. They are released from cells following injury  and are In addition, urinary EVs contain several kind of proteins secreted by the kidney tissue, some of which have sparked interest as potential diagnostic new markers of kidney disease in urinary EVs ,72,73.Urinary mRNA profiling has been proposed as a tool for biomarkers . HoweverFor clinical applications, EV diagnostics are required to be: (1) fast; (2) simple and highly sensitive; and (3) able to detect specific endogenous biomolecules contained in the EVs.Numerous studies in kidney and urologic diseases have described the presence of urinary biomarkers from EVs. ischaemia/reperfusion injury and in patients immediately after kidney transplantation [Proteomic analysis of urinary EVs has been used in a study investigating the changes in urine protein content associated with acute kidney injury . The anaantation .In the last years, several promising early biomarkers of acute kidney injury have been identified in urine, including neutrophil gelatinase-associated lipocalin (NGAL), interleukin-18 (IL-18) and kidney injury molecule-1 (KIM-1), that are in advanced stages of validation while despite the increasing number of studies on the EVs, urinary EV biomarker discovery remains in its infancy for insufficient number of patients involved in above mentioned studies.lupus nephritis and a positive correlation with urinary protein levels, suggesting its convenience as a predictive biomarker of lupus nephritis [Experiments have shown an increase in the levels of miR-26a in exosomes from patients of ephritis .IgA nephropathy. Many urinary exosome proteins were identified including cytoplasmic, membrane and vesicle components. Among different expressed proteins, 4 were selected  as candidate biomarkers potentially useful in the differential diagnosis of IgA nephropathy from congenital thin basement membrane nephropathy [Urinary exosomes have recently been utilized for biomarker discovery in hropathy .primary focal segmental glomerulosclerosis (FSGS) [Wilms Tumor 1 (WT1) is constitutively expressed on podocytes of healthy adult kidneys, and its expression decreases in kidney biopsies of patients with s (FSGS) . It has s (FSGS) . Unfortus (FSGS) . Other as (FSGS) .glomerular kidney disease such as IgA nephropathy and lupus nephritis showed elevated levels of ADAM10. Higher concentrations of this protein were detected in urine as well as in urinary exosomes [A study showed that urinary podocytes from patients with exosomes .diabetic nephropathy has demonstrated that in patients with microalbuminuria miR-130a and miR-145 were enriched, while miR-155 and miR-424 were reduced. This finding is of potential pathophysiological relevance because miR-145 is a glomerular marker of mesangial cells and is induced by TGF-β1 in this cell type [A study on microRNA expression in urinary exosomes from type 1 diabetic patients with and without incipient ell type . Dipeptiell type .The protein composition of urinary EVs is significantly different between patients and controls. Among the total amount of 254 different proteins analysed, 25 were significantly altered in diabetic nephropathy and validation studies confirmed that a panel of three of those proteins  could be markers of the disease .Until today markers in glomerulonephritis are miRNA  or chemoautosomal recessive polycystic kidney disease, analysis of the urinary exosome-like vesicles identified 552 proteins, of which polycystin-1 and 2, cystin-1 and ADP-ribosylation factor like protein 6 were implicated in cystic diseases [In a study using a patient with diseases .autosomal dominant polycystic kidney disease (ADPKD) [2+-activated signaling, cell division, cell differentiation, and Wnt signalling pathways. In particular, the ezrin/radixin/moesin family and CDC42 were significantly upregulated in ADPKD, reflecting the altered cytoskeleton regulation of tubular epithelial cells in ADPKD patients. Moreover, this study showed that expression of AQP2 reduced in ADPKD, consistent with the impaired urinary concentrating ability in these patients.A report described the multiplex quantitative proteomic analysis of urinary EVs from patients with  (ADPKD) . The autDespite their power in understanding functional aspects of EVs, investigation of urinary EV proteins show some limitations because they constitute only a subproteome.chronic kidney disease, including those with diabetic nephropathy, IgA nephropathy and ADPKD [tubulointerstitial fibrosis [fibrosis in lupus nephritis [renal fibrosis [In an exploratory study, osteoprotegerin was found in tubular cell-secreted exosome-like particles and urinary exosomes. This soluble protein was augmented in urinary exosome-like vesicles from patients with nd ADPKD . This find ADPKD . Recentlfibrosis . Moreoveephritis . Lv et afibrosis .These studies suggest that it is possible to find a match between cultured cells-derived EV content and body fluid-derived EV content.kidney transplantation. No correlation between exosomal mRNA levels and the seven-day creatinine reduction ratio (CRR7) was found, while the urinary proteins NGAL and IL-18 reflected the CRR7 [transplanted patients, CD133+ EVs were present at low levels in the first day after transplantation, to increase thereafter [A recent study assessed the urinary exosomal mRNA expression of NGAL, IL-18, KIM-1 and cystatin C in the urine of patients with the CRR7 . It has ereafter .These studies demonstrate that it is possible to select the type of EVs secreted according to the cell of origin, even if packaging of mRNA in EVs is selective, and is not necessarily representative of mRNA in the parent cells responsible for biomarker production.Several companies are developing dedicated commercial assays for the detection of EVs in plasma or serum samples and in urine of patients with neoplasia  .Other studies have also investigated on the proteomic cargo of prostate-derived extracellular vesicles from urine. Increased levels of integrin alpha-3 and integrin beta-1 have been found in urine exosomes of patients with metastatic prostate cancer, compared with patients with nonmetastatic disease or those with benign prostate hyperplasia . LikewisFew studies have addressed the potential use of urinary EVs as a diagnostic tool for renal cell carcinoma (RCC). A comparative analysis of urinary EVs performed using a hyphenated microLC-Q-TOF-MS  platform demonstrated a differential lipid composition between the exosomes of patients with RCC and those of healthy control subjects . ProteomBladder cancer is another important neoplasia of the urinary tract. A study investigated the potential application of the RNA content of urinary EVs in the diagnosis of bladder cancer. Using microarray technology followed by PCR validation, Perez at al. generated a list of differentially expressed genes in urinary EVs from patients with cancer compared with those from cancer-free controls. LASS2 and GALNT1, which encode proteins involved in cancer progression and metastasis, were found only in EVs from the urine of patients with bladder cancer .These studies show that currently the use of EV markers for tumour diagnosis can only be an initial useful clue but validation studies in large cohorts of patients are needed to determine their clinical use.In this review we have illustrated some current aspects of the urinary EVs. They are released from all nephron segments, and contain various biological molecules ranging from RNA to proteins.The detection of EVs is challenging because of their small and heterogeneous size range, high concentration, and heterogeneity in composition and morphology. At present, there is not one single method which can accurately phenotype, size, and enumerate the whole range of EVs. In addition, a more rigorous characterization of EVs using a combination of methods is likely needed . This will help to better explore the biology of EVs and their relationship to disease.Urinary EVs have been confirmed to contain low amounts of RNAs  whose quAlthough proteins included in urinary EVs represent only 3% of the whole-urine proteome , these sIn conclusion, these studies show the possibility of distinguishing kidney and urologic disease patients from healthy people by the identified EV biomarker candidates. Urinary EVs are a suitable material for diagnosis because they are easy to collect and represent the pathophysiological state of kidneys. A rapid urinary assay of predictive EV biomarkers could provide to the clinician a support for the diagnosis and may allow a faster choice for adequate therapy. They may replace in the future the kidney biopsy. Finally, their use may have important scientific and commercial perspectives.


The potential benefits of stimulating a patient's immune system to fight cancer are profound. Historically, immunotherapy has been a successful treatment approach for some cancer patients, and recent advances in immunotherapy highlight that the immune system can generate durable tumor responses and cures. However, a concern regarding immunotherapy in cancer patients is the possibility that the immune response will be compromised by the underlying cancer and/or previous anticancer therapies. A promising approach in immunotherapy is targeting pathogen-associated molecular pattern receptors, such as the Toll-like receptors (TLRs), to activate the innate immune system and enhance development of tumor-directed adaptive immune responses. To assess whether cancer patients exhibit immune insufficiency in response to the potent and selective TLR8 agonist motolimod, we compared the pharmacokinetic and pharmacodynamic (PK/PD) relationship defined in a Phase 1 study of motolimod in cancer patients to the response in healthy volunteers.® system (Myriad-RBM) defined the breadth and magnitude of immune mediators induced by TLR8 activation in vitro. In a Phase 1 dose-escalation study in patients with advanced solid tumors, many of these defined mediators of TLR8 activation were increased in the plasma of cancer patients treated with motolimod. Notably, multiple biomarkers of immune activation, including G-CSF, MCP-1, and MIP1-β increased in a dose-dependent manner and correlated with increasing plasma levels of motolimod. A second Phase 1 study of motolimod in healthy volunteers provided an opportunity to compare and contrast the immune response generated by treatment with motolimod in healthy volunteers to that observed in cancer patients.Stimulation of healthy volunteer human PBMC by motolimod using the TruCultureAt comparable dose levels, the PK profile and overall exposure (AUC) to motolimod were similar for cancer patients and healthy volunteers. Motolimod induced the same repertoire of circulating cytokines and chemokines, indicative of TLR8 activation, in both populations. The magnitude of the mediator response in cancer patients was highly comparable to the response in healthy volunteers that received a similar dose.In summary, advanced cancer and prior treatment with cytotoxic agents did not appear to blunt the response to motolimod based on the PK/PD relationship using predictive biomarkers. This comparison demonstrates that the immune system of cancer patients with advanced disease remains highly responsive to TLR8 activation by motolimod.


The Nkx factors are a family of homeodomain containing transcription factors known to have tissue specific expression and thought to play a regulatory role in organ development. Certain members of this family have been studied in detail and found to play a role in disease. For example, NKX6-1 is involved in pancreatic development and serves as a marker for pancreatic and duodenal neuroendocrine tumors . EvidencGastric cancer has one of the highest mortality rates of all cancers . ContribEBioMedicine by The research presented in this issue of tumor, regional lymph node, and metastasis) stage. Mining publically available data from the Gene Expression Omnibus (GEO), demonstrated a strong correlation between reduced expression of NKX6.3 and worse patient outcomes.Previous studies by the same authors had found NKX6.3 expression to be reduced in gastric cancers as a result of decreased DNA copy number and allelic loss . BuildinIn dissecting the molecular basis for these important disease related findings, Yoon et al. demonstrate the role of NKX6.3 in regulating proteins involved in epithelial-mesenchymal transition (EMT). EMT is important in wound healing, organ fibrosis, and — of key relevance here — tumor metastasis. Previous studies implicate EMT in gastric cancer, particularly relating to cell motility and invasiveness (recently reviewed by Yoon et al., also show NKX6.3 inhibits Rho-GTPase family proteins which are known to contribute to cancer progression and development. This is important as the Rho-GTPase family proteins RhoA and Rac1 have been shown to be up-regulated in gastric cancers . This suPlacing this in the context of broader studies, analysis of gastric cancers by The Cancer Genome Atlas Research Network discovered four subtypes; microsatellite unstable tumors, genomically stable tumors, Epstein-Barr virus, and tumors with chromosomal instability . InteresIn sum, the studies of None.


Togaviridae family, is capable of causing severe febrile disease in humans. In December of 2013 the Asian Lineage of CHIKV spread from the Old World to the Americas, spreading rapidly throughout the New World. Given this new emergence in naïve populations we studied the viral genetic diversity present in infected individuals to understand how CHIKV may have evolved during this continuing outbreak.Chikungunya virus (CHIKV), an alphavirus and member of the We used deep-sequencing technologies coupled with well-established bioinformatics pipelines to characterize the minority variants and diversity present in CHIKV infected individuals from Guadeloupe and Martinique, two islands in the center of the epidemic. We observed changes in the consensus sequence as well as a diverse range of minority variants present at various levels in the population. Furthermore, we found that overall diversity was dramatically reduced after single passages in cell lines. Finally, we constructed an infectious clone from this outbreak and identified a novel 3’ untranslated region (UTR) structure, not previously found in nature, that led to increased replication in insect cells.Here we preformed an intrahost quasispecies analysis of the new CHIKV outbreak in the Caribbean. We identified novel variants present in infected individuals, as well as a new 3’UTR structure, suggesting that CHIKV has rapidly evolved in a short period of time once it entered this naïve population. These studies highlight the need to continue viral diversity surveillance over time as this epidemic evolves in order to understand the evolutionary potential of CHIKV. Chikungunya virus is a re-emerging and rapidly spreading arbovirus that has caused several outbreaks in the last decade with the most recent in the Caribbean islands and the Americas beginning in 2013 infecting over 1 million individuals. The ability to monitor such epidemics would be enhanced by characterizing the viral populations that circulate within infected individuals. To do this, we deep-sequenced viral populations from infected individuals and identified minority variants present at high frequencies, as well as the presence of a novel 3’ untranslated genome region (UTR) structure, a key determinant of chikungunya virus infectivity and evolution never before described in nature. Finally, we genetically engineered an infectious clone from this outbreak strain and established that the novel 3’UTR structure increases viral replication in mosquito cells. Taken together these studies highlight the vast diversity of viral populations in infected individuals, reveal potential novel determinants of chikungunya virus biology, and provide an indispensable tool for future studies involved in viral evolution and adaptation. Aedes albopictus, and thus increase its transmission throughout areas of the world harboring this mosquito species. Interestingly, the CHIKV strain that has arrived to the Americas is from the Asian lineage and does not contain these adaptive mutations as of yet. However, both Aedes aegypti and albopictus are prevalent throughout many parts of North and South America  4]. [4].3] [. [4].3]  America  that, alSamples involved in this study were chosen among human serum specimens received as part of standard diagnostic and expertise activities of the arboviruses National Reference Center for French Departments of the Americas located in French Guiana. The donor samples were rendered completely anonymous and renumbered prior to preparation of extracted RNA for sequencing with only the week of sampling and island of origin retained. Of the 100 samples, 20 gave whole-genome deep sequence coverage and five others . One third of these samples were from Guadeloupe and two thirds from Martinique. The consensus sequences of the 20 whole-genome samples were deposited in the European Nucleotide Archive, with the accession numbers indicated in http://www.ebi.ac.uk/ena/data/view/LN898093-LN898112.100 human sera positive for CHIKV qRT-PCR were randomly selected amongst (1) those sampled between week 52 of 2013 and week 5 of 2014 around the beginning of epidemic phase in the French Caribbean islands, and (2) those having a high viral load (mostly between 10occurred . One thiTable 2) (http://www.european-virus-archive.com/article147.html). Amplicons were subsequently purified via a nucleospin PCR purification kit (Macherey-Nagel), quantified by picogreen, and fragmented as described previously , pla, plaTablTable 4, bold).Furthermore, deep-sequencing analysis identified a variety of unique high-frequency intra-host minority variants (at greater than 0.5% frequency) in infected individuals, as well as five synonymous consensus sequence differences with respect to the initially reported strain from St. Martin, which were common to all patients  . We found that the nonstructural proteins presented many more variants (56 at a frequency of at least 10% of the population) than the structural proteins (5 variants at a frequency above 10% of the population). In particular, within nsP1, which functions as the methyltransferase and is necessary for RNA replication, we observed eight amino acid changes and three synonymous changes in individual patients and localized variation concentrated in the methyltransferase and D3 domains. Nsp2 is a multifunctional protein serving as the viral helicase, protease, and NTPase. Although we observed considerable variation scattered across nsP2, this protein contained six synonymous and six amino acid changes including one mutation (G772D) present at roughly 50% of the viral population in one individual. The nsP3 gene, which encodes a phosphoprotein required for RNA replication, contained the largest number of minority variants, with 23 total changes. These include only five synonymous changes and 18 coding changes , with several minority variants making up a considerable portion of the viral population. We noted the mutations of several serine residues in nsP3  that were present at nearly 100% in one individual that could change the phosphorylation state of the protein, as well as several charge changes  that may have functional roles in RNA binding or viral replication. The viral RNA dependent RNA polymerase, nsP4, contained 10 sequence changes with three synonymous and seven coding changes. Interestingly, one patient presented a stop codon (L441Stop) at 63% of the viral population that is coupled with the R178Q mutation of nsP3 mentioned above. It is possible that these mutations may function together to oblate the large amount of truncated nsP4 in this individual.We next analyzed the specific variants in each gene . Given the role of the viral glycoproteins E2 and E1 in viral evolution and transmission, we looked at the overall genetic variation within these two proteins. Here, we found portions of the protein involved in protein-protein interactions  and the transmembrane domains required for membrane binding to contain variation. Interestingly, regions flanking the fusion-loop (amino acids 83–101) of E1 contained more variation then the fusion-loop itself indicating that changes directly in the fusion loop are poorly tolerated. Importantly, no samples contain any significant levels of previously observed vector-adaptive mutations such as E2 L210Q, E1T98A, or E1 A226V, which could potentially facilitate dissemination of this virus in Aedes albopictus mosquitoes . In. Inde noPhylogenetic analysis revealed that the strains collected in Guadeloupe and Martinique islands belong to the Asian genotype circulating in the Caribbean. Furthermore, the strains sequenced in this study show a closer phylogenetic relationship, which can be attributed to the short genetic distances depicted in their tree branches. We observed strains from Brazil (TR206 and AMA2798) and Mexico (InDRE_4CHIK and InDRE_51CHIK) to cluster with our cohort as well. Interestingly, the strain InDRE_4CHIK in Mexico was obtained from an imported case from Antigua and Barbuda, islands in close geographic proximity to Guadeloupe and Martinique  23] and and23] aMoreover, whereas previous analyses focused on amino acid changes that may impart a functional or enzymatic impact, we also analyzed variation in the noncoding, untranslated regions of the genome. In this analysis, we identified a novel 3’UTR duplication that had not previously been observed in nature, yet is present in several viruses circulating in the Americas including Mexico, the Dominican Republic and Trinidad. The fact that we have found this duplication in clinical samples from 2013 and again in 2015 suggests this duplication is fixed in the current circulating Caribbean strain. Importantly, we found this new UTR structure to provide no disadvantage for the virus and interestingly, it led to increases in viral titers in mosquito cells when compared to a similar Asian strain and a Caribbean strain clone lacking the duplication. As the 3’UTR has been shown to play essential roles in arbovirus replication, evolution, and host adaptation  24], it, it24], These studies highlight the need to carefully re-analyze deep-sequencing assemblies, such as sudden increases in read coverage and the inability to map unfiltered reads that still contain virus-specific sequences, which may be indicative of duplications and insertions. The origins of this duplication still needs to be determined to understand if it first originated in Asia prior to arriving in the Caribbean, or was generated just before or after December 9, 2013, the start of the epidemic in St. Martin  and has Nonetheless, this study provides an in depth look at the minority variants present during an ongoing chikungunya virus epidemic, identifying novel variants and structural elements, and the construction of an infectious clone of this virus to be used to future study the pathogenesis, adaptation and evolution of chikungunya virus in the Americas. The study of these evolutionary elements, which may play crucial roles in chikungunya virus evolution and adaptation, will be necessary to address potential future public health issues both in disease and viral dissemination.


Recent advances in basic cardiovascular research as well as their translation into the clinical situation were the focus at the last “New Frontiers in Cardiovascular Research meeting”. Major topics included the characterization of new targets and procedures in cardioprotection, deciphering new players and inflammatory mechanisms in ischemic heart disease as well as uncovering microRNAs and other biomarkers as versatile and possibly causal factors in cardiovascular pathogenesis. Although a number of pathological situations such as ischemia–reperfusion injury or atherosclerosis can be simulated and manipulated in diverse animal models, also to challenge new drugs for intervention, patient studies are the ultimate litmus test to obtain unequivocal information about the validity of biomedical concepts and their application in the clinics. Thus, the open and bidirectional exchange between bench and bedside is crucial to advance the field of ischemic heart disease with a particular emphasis of understanding long-lasting approaches in cardioprotection. Ischemic heart disease (IHD) is the leading cause of death and disability worldwide . Thus, nDerek Yellon (UK) opened the meeting by providing an expert overview of cardioprotection from bench-to-bedside with focus given to new therapeutic targets for cardioprotection and the challenges facing the translation of new cardioprotective therapies from the laboratory to the clinical arena; a topic which has been extensively discussed in recent literature, and which has been attributed to a number of factors including the use of inappropriate animal models and poor clinical trial design The heart can be protected from acute IRI, both experimentally and in the clinical setting, by subjecting it to brief episodes of ischemia and reperfusion, a phenomenon termed “ischemic preconditioning” . HoweverThe results of the recently published ERICCA  and RIPHMitochondrial dysfunction lies at the heart of a number of cardiovascular diseases including acute IRI, and treatments aimed at preserving mitochondrial function represent an important cardioprotective strategy for limiting MI size and preserving cardiac function . GlycogeThe phenomenon of “mitochondrial dynamics” was introduced by Derek Hausenloy (UK and Singapore) as a novel target for cardioprotection—this refers to the ability of mitochondria to move and change shape by undergoing fission and fusion to generate fragmented and elongated mitochondria, respectively , 74, 75.The impact of advanced age on mitochondrial dysfunction, which occurs in the context of acute myocardial IRI, was discussed by Marisol Ruiz-Meana (Spain). Preclinical data have suggested that mitochondria play a causative role in the increased susceptibility of the senescent myocardium to ischemic damage. The existence of an age-dependent disruption of the molecular communication between SR and mitochondria appears to have consequences on calcium handling and bioenergetics in response to acute myocardial IRI . MoreoveFinally, translating a therapeutic cardioprotective approach that targets mitochondria into the patients´ benefit has recently been illustrated with the neutral results of the CIRCUS trial: This trial failed to demonstrate improved clinical outcomes at 1 year  in STEMI patients who received an intravenous bolus of the MPTP inhibitor cyclosporine-A prior to PPCI . AlthougWhile several single-target treatment strategies failed to improve clinical outcomes in patients with ischemic heart disease , 33, 52,Low levels of high-density lipoprotein (HDL) cholesterol constitute a major risk factor for cardiovascular disease; however, recent therapies aimed at raising HDL levels have failed—the underlying reasons remain unclear. Native and/or synthetic HDL-subtypes were studied in experimental models of IHD to characterize their possible role in cardioprotection, and Sandrine Lecour (South Africa) reported that HDL subtype 3, containing a high level of sphingosine-1 phosphate, may be superior against acute IRI than HDL subtype 2 , indicatThe close proximity of epicardial adipose tissue toward the media of arteries and its colocalization with atheroma lesions commonly observed in patients submitted to revascularization strongly suggests an active role of epicardial adipose tissue in the genesis and progression of atherosclerosis . EpicardAtherosclerosis is a chronic inflammatory disease and is one of the major underlying causes of an acute myocardial infarction . At the The left ventricular (LV) remodeling, which takes place following an acute MI, is a critical determinant of final LV systolic function, the onset of heart failure and adverse clinical outcome. In this regard, Elisa Liehn (Germany) discussed the role of immune cells which are able to modify collagen synthesis –58 and tThe ability to detect the presence of myocardial necrosis in patients presenting with chest pain as a criterion for diagnosing an acute MI depends on the ability to accurately quantify the extent of damage to the myocardium . The useDifferent endogenous adaptive responses to acute IRI in the heart may mobilize as yet unrecognized pathogenetic factors and help to identify novel therapeutic targets for cardioprotection. The hypoxia-inducible factors HIF-1α and HIF-2α orchestrate the body’s adaptive response to hypoxia (a major component of IRI) through the transcription and translation of several hundred proteins. While HIF-1α has been suggested as a therapeutic target for cardioprotection , 77, litWhile hypoxia is known as a stimulus for the increase in sympathetic tone, observed in patients with arterial hypertension, Nephtali Marina (UK) presented evidence that the brainstem in hypertensive animals  was relatively hypoxic as well, compared to normotensive Wistar rats . As a coA variety of inflammatory processes has been shown to initiate and/or sustain molecular processes culminating in CVD , 80. As It has been recently shown that eRNA can exert pro-thrombotic and pro-inflammatory properties in the cardiovascular system and provoke cytokine mobilization as well as macrophage polarization, independent of Toll-like receptor-3 activation , 92. Hec2+)-permeable channel with an adenosine diphosphate ribose pyrohydrolase activity [2+ influx into the cell and/or Ca2+ release from lysosomes, contributing to cell migration, cell death, cytokine production, as well as degranulation in monocytes, dendritic cells, neutrophils, and mast cells [The bi-functional “Transient receptor potential melastatin-2” (TRPM2), which combines a nonselective calcium  -cadherin/catenin complex, the structural and functional backbone of endothelial cell junctions, and actin filament dynamics including actin polymerization and actin/myosin-mediated contractility, simultaneously maintains integrity and dynamics. Due to the lifelong shear stress-dependent biomechanical forces at specific sites of the vascular tree, the affected endothelial cells become continuously stimulated and acquire a dysfunctional phenotype that resembles the inflammatory endothelium. Such chronic conditions are associated with the disturbance of the vascular barrier properties and allow uncontrolled adhesion and transmigration of pro-inflammatory cells into the neointimal region of the inflammatory vessel wall to feed the atherogenic process. To follow the time-related changes and molecular arrangements of endothelial cell junction dynamics, life cell imaging using fluorescent tagged proteins has been very instrumental, as addressed by Hans Schnittler . Here, the formation of “Junction associated intermittent lamellipodia” (JAIL) has recently been reported to be one of the key mechanisms that control junction remodeling by largely maintaining cell-junction integrity , 2, 88. 2+ signaling and the activation of intra-platelet calpains, which constitute Ca2+-activated proteases that are required for the limited proteolysis of several substrate proteins and subsequent alterations in platelet signaling. Ingrid Fleming (Germany) has found that the activation of µ- and m-calpain in patients with type 2 diabetes has profound effects on the platelet proteome and identified septin-5, the integrin-linked kinase (ILK) and Dicer as novel calpain substrates [Platelets from patients with diabetes are hyper-reactive and demonstrate increased adhesiveness, aggregation, degranulation, and thrombus formation as well as conjugate formation with leukocytes: processes that contribute to the accelerated development of vascular disease. Part of the problem seems to be dysregulated platelet Cabstrates , 82. TheFinally, Manuel Mayr (UK) provided an overview from a systems biology perspective to help integrate the diverse topics in cardiovascular research for combined technological approaches in finding e.g., new biomarkers or causative relations. This can be achieved using proteomics in combination with other postgenomics technologies, such as lipidomics or microRNA profiling as well the integration of biological information in disease-specific networks for CVD. Unlike genomics, postgenomics approaches are not “off-the-shelf technologies” to identify novel biomarker candidates. Rather than evaluating individual biomarkers by routine clinical measurements, the linkage of cutting-edge postgenomics technologies with population studies aims to explore biomarker panels derived from a multi-omics approach  in a primary preventive setting , 110. ThIn summary, recent advances in cardiovascular research were discussed at this New Frontiers in Cardiovascular Research meeting covering a range of topics including cardioprotection, inflammation research, and vascular biology. A number of novel therapeutic targets for mediating cardiovascular protection in relation to a number of conditions including acute ischemia/reperfusion, heart failure, and atherosclerosis were introduced at the meeting. The important issue of translating new therapeutic strategies from the benchside to the bedside was highlighted given the challenges facing this process—these will have to be improved should we wish to impact on clinical outcomes in our patients with CVD.


The placenta is the primary interface between the fetus and mother and plays an important role in maintaining fetal development and growth by facilitating the transfer of substrates and participating in modulating the maternal immune response to prevent immunological rejection of the conceptus. The major substrates required for fetal growth include oxygen, glucose, amino acids and fatty acids, and their transport processes depend on morphological characteristics of the placenta, such as placental size, morphology, blood flow and vascularity. Other factors including insulin-like growth factors, apoptosis, autophagy and glucocorticoid exposure also affect placental growth and substrate transport capacity. Intrauterine growth restriction (IUGR) is often a consequence of insufficiency, and is associated with a high incidence of perinatal morbidity and mortality, as well as increased risk of cardiovascular and metabolic diseases in later life. Several different experimental methods have been used to induce placental insufficiency and IUGR in animal models and a range of factors that regulate placental growth and substrate transport capacity have been demonstrated. While no model system completely recapitulates human IUGR, these animal models allow us to carefully dissect cellular and molecular mechanisms to improve our understanding and facilitate development of therapeutic interventions. In eutherian mammals, the placenta is the primary interface between the fetus and mother. One of the main functions of the placenta is to deliver nutrients and oxygen to the fetus. Failure of the placenta to deliver an adequate supply of nutrients to the fetus is termed placental insufficiency and results in intrauterine growth restriction (IUGR), affecting up to 5%–10% of pregnancies in developed countries ,3. IUGR In mammals, the placenta is the primary interface between the fetus and mother and plays an important role in maintaining fetal growth by performing several physiological functions, which, following birth, are controlled by the kidney, gastrointestinal tract, lungs and endocrine glands. The main functions of the placenta include modulation of the mother’s immune response to prevent immunological rejection termed tolerance, facilitating the exchange of respiratory gases, water, ions, nutrients and wastes between the maternal and fetal circulations, and producing and secreting hormones, cytokines and other signalling molecules required to maintain pregnancy and to ensure placental and fetal development and growth .Maternal blood supply to the placenta is established at the end of the first trimester of human pregnancy, with maternal blood entering the intervillous space of the placenta from the transformed spiral arterioles for substrate transport to the fetus ,10,11. Tin vivo studies of the human placenta are difficult as the methodology presents risks to both the mother and fetus. Therefore, the majority of the body of knowledge about the placenta and its function is a result of ultrasound studies across gestation or those performed in placentas collected at delivery or in animal models. In terms of morphological similarities, the placenta of higher primates is the most comparable to that of the human, with a discoid hemomonochorial structure, where fetal trophoblast cells are in direct contact with the maternal blood supply [It is important to understand the role of placental function in the IUGR fetus from a clinical and scientific point of view. However, d supply . Howeverd supply . Therefod supply ,19,20. Hd supply ,22,23.in utero [One of the advantages of working with sheep compared to other animal models of pregnancy is that the functional responses of the sheep fetus to short or long periods of placental insufficiency can be studied in utero ,26,27,28in utero . In addiin utero . Therefoin utero ,30.In sheep, implantation begins at ~14–16 days gestation (term = 150 ± 3 days gestation) and is complete by ~22 days gestation, marking the initiation of placentation , which iPlacentomes are classified into four types (A–D) based on their gross morphology . The fetin utero and the extent to which placental adaptations during intrauterine development have occurred in order to meet fetal growth demands. These morphological and/or functional adaptations determine placental substrate transport capacity and efficiency to the fetus. The major substrates required for fetal growth include oxygen, glucose, amino acids and fatty acids [Birth weight depends not only on maternal nutrition but also on the placenta’s ability to transport substrates from the maternal to the fetal circulation . Placentty acids ,12,13.While the placenta regulates the transport of nutrients to the fetus according to the mother’s ability to deliver them, fetal demands as well as hormones and growth factors secreted by the placenta, the weight, size and shape of the placenta also affect its ability to transfer nutrients ,39,40. T2) plays a critical role in the development and function of the placenta and fetal hypoxaemia is a common condition of complicated pregnancy. Oxygen diffuses from the maternal to the fetal circulation across a placental epithelial layer that consumes O2. This O2 consumption generates a transepithelial oxygen partial pressure (PO2) difference whose magnitude depends upon the rate of umbilical and uterine blood flow, fetal and maternal blood oxygen carrying capacity, haemoglobin oxygen binding affinity, placental surface area and placental permeability [2 gradient across the placenta remains constant in order to sustain fetal oxygen delivery at a normal rate [Simple molecules such as oxygen and carbon dioxide are transported by diffusion and bulk flow. Oxygen  and amino acid transporter (SNAT2) during mid gestation, however IUGR still develops near term [Insulin-like growth factors (IGFs), IGF-1 and IGF-2, are single chained polypeptides that promote fetal and neonatal growth, acting in response to fetal, maternal and placental signals such as nutrients, oxygen and hormones via the IGF receptors, IGF-1R and IGF-2R, and the insulin receptor (InsR). IGF-2 is the most abundant of the fetal IGFs, however, deletion of either h weight . IGF-1 rh weight . Furtherh weight . In humah weight . Elevateh weight ,28. In sh weight . Furtherh weight . IGF-1 sh weight . Interesear term ,60,61.The role of the apoptosis cascade has been characterized in human villous trophoblast turnover and syncytium formation . ApoptosAutophagy is a highly regulated and dynamic process involving invagination and degradation of cytoplasmic components that maintain cellular homeostasis and promote cell survival in response to environmental stresses such as starvation and hypoxia . AutophaIn utero, the placenta and embryo/fetus are each exposed to physiological glucocorticoids arising from either the maternal or fetal adrenal glands. Glucocorticoid actions are moderated by the glucocorticoid receptor and two isoforms of intracellular 11β-hydroxysteroid dehydrogenases (11βHSDs) [11βHSDs) . 11βHSD111βHSDs) . 11βHSD111βHSDs) . In huma11βHSDs) . In cont11βHSDs) . The fet11βHSDs) .The placenta’s ability to transport substrates for fetal growth is also influenced by the abundance, activity and localization of transporters in the placental membranes . FurtherGlucose is the primary nutrient required for the fetus and placenta ,74. HoweGLUT1 is insulin independent and ubiquitously expressed in a variety of human tissues including muscle, adipose, brain and endothelial cells . GLUT1 iGLUT3 is insulin independent and its expression is cell-specific, depending on the stage of pregnancy . In bothGLUT4 is insulin dependent and its immunostaining was detected in fibroblasts from amnion and chorion . A stronThe human placenta expresses more than 20 different amino acid transporters . Fetal aSystem A amino acid transporters are an example of neutral amino acid transporters that facilitate the uptake of small non-essential neutral amino acids such as alanine, glycine and serine. Uptake occurs against their concentration gradient simultaneously with the transport of extracellular sodium into the cell. While system A transporters are present on both placental membranes, expression is greater on the MVM. System A activity contributes to the high intracellular concentration of amino acids such as glycine, which is exchanged for extracellular essential amino acids by system L transporters . TherefoSystem L transporters are sodium independent exchangers of neutral amino acids. Non-essential amino acids are exchanged for essential amino acids with aromatic or branched side chains such as leucine or phenylalanine to allow for transport against their concentration gradient . System et al. also found that CAT1 (SLC7A1), CAT2 (SLC7A2), and CAT4 (SLC7A4) are expressed in cultured trophoblast cells and in BeWo choriocarcinoma cells [It is well established that the cationic amino acid transporter (CAT) is the main transporter for cationic amino acids in MVM . Kamath ma cells , supportAG amino acid transporters are capable of sodium dependent and d-aspartate-inhibitable glutamate/aspartate transport activity. A family of five anionic amino acid transporters (excitatory amino acid transporters EAAT1-5) have been cloned and, of these, EAAT1-4 are expressed in the human and rat placenta [Anionic or system Xplacenta ,104,105.placenta .Fatty acids are important precursors of bioactive molecules, which are structural components of cells and provide a major source of energy . Two souFatty acid transfer from the mother to the fetus can be accomplished via simple diffusion, as with NEFAs . HoweverWe have summarized the major placental changes in morphology and substrate transport capacity that regulate placental efficiency and fetal growth in normal pregnancies. As we know, placental insufficiency results in a failure of the placenta to deliver an adequate supply of substrates to the fetus and IUGR develops. Therefore, several different experimental methods used to produce placental insufficiency and induce IUGR in animal models and a range of factors that regulate placental growth and substrate transport capacity have been discussed in human and animal IUGR studies below.Intrauterine growth restriction is clinically defined as a birth weight below the tenth centile for gestational age where the fetus does not meet its growth potential ,112. IUGSeveral different experimental methods have been used to produce placental insufficiency and induce IUGR in both small and large animal models, and previously discussed in detail . For exa2 < 17 mmHg), but not acidotic in late gestation [Single umbilical artery ligation (SUAL) involves the isolation and ligation of one umbilical artery close to the fetal abdomen . SUAL caestation .The hyperthermia-induced model of placental insufficiency induces fetal growth restriction by exposing pregnant ewes to an environment with an increased ambient temperature with a diurnal cycle of 40 °C for 12 h and 35 °C for 12 h, from ~38 days gestation until ewes are sacrificed at post mortem . Ewes caPlacental embolism of the placental vasculature aims to mimic the onset of IUGR in late gestation. This procedure involves repeated injection of microspheres (15 μm) into the placenta via the umbilical artery through a catheter implanted in the descending aorta or fetal umbilical vein ,125,126.Uterine carunclectomy limits the number of placentomes after surgical removal of the majority of the endometrial caruncles from the uterus of nonpregnant ewes prior to conception. This restricts the number of placentomes that form during pregnancy, consequently limiting placental and fetal growth . ExperimFetal growth is dependent on substrate supply, which is dependent on substrate transport and its regulation. These processes also depend on morphological characteristics of the placenta, such as placental size, morphology, blood flow and vascularity. Therefore placental nutrient transfer capacity is specifically regulated by signals of fetal, maternal and placental origin in an effort to control fetal growth. However, in late gestation, when fetal nutrient demand is at its greatest, the compensatory upregulation is no longer sufficient to meet fetal demand and thus IUGR develops ,130. HerIn human pregnancies, Chen and coworkers have found a significant decrease in villi vascular density and cell proliferation in both trophoblast and stromal cell compartments within the IUGR placentas compared with control placentas at 25 to 41 weeks of gestation . Other rExperimental restriction of placental size by surgical removal of implantation sites prior to conception, multiple pregnancy or adverse conditions in early gestation restricts fetal growth, but increases fetal to placental weight ratio and placental efficiency in late pregnancy ,53. Conv2 diffusion reducing the surface area of exchange between the uterine and umbilical circulations, and resulting in decreased fetal umbilical oxygen supply [In vitro studies have demonstrated that hypoxia can affect the proliferation, differentiation, and invasion of cytotrophoblast cells [In situations of placental insufficiency and IUGR, there appears to be an underdevelopment of the placental epithelial sites, with low hindrance to transplacental On supply ,132. Undn supply . In vitrst cells . Followist cells . Hypoxiast cells . Low oxyst cells .Morphological studies show that physiological remodelling of the maternal uterine vasculature into spiral arteries is deficient in IUGR pregnancies due to inadequate trophoblast invasion ,147. ThiIgf2 gene, placental surface area is reduced and placental thickness increased, leading to reduced placental growth and fetal growth restriction [In knockout mice with a deletion of the placental-specific transcript (P0) of the triction ,61. Plactriction . Similartriction . Howevertriction . Cotyledtriction . In conttriction .Placentas from women with IUGR pregnancies show enhanced apoptosis when compared with those from normal pregnancy ,155. CulCompared with normal pregnant women, women with IUGR had increased placental levels of autophagy-related proteins including LC3B-II, beclin-1, DRAM, and p53 . Furtheret al found a lower expression and activity of 11βHSD1 in both chorionic and basal plates of the placentas from full term small for gestation age newborns compared with those in the placentas from appropriate for gestational age newborns, suggesting a possible compensatory mechanism to diminish the higher cortisol concentrations in fetuses with IUGR [Placental 11βHSD2 activity was significantly reduced in deliveries complicated by IUGR compared with the term deliveries and with appropriately grown preterm deliveries, suggesting that glucocorticoids may, in part, contribute to impaired fetal growth closely controlled through placental 11βHSD2 expression . There iith IUGR .in vivo with high altitude pregnancies also resulted in a decrease in GLUT1 expression in the BM but not MVM of the placenta, leading to reduced nutrient supply and fetal growth [in vivo and in vitro studies.Alterations in placental glucose transport have been implicated in adverse perinatal conditions such as IUGR and fetal hypoxia . As glucl growth . In micel growth . Anotherl growth . Placentl growth . Taken tIn the IUGR placenta the different amino acid transport systems are affected. Tracer studies of placental amino acid transport have demonstrated that there is a significant reduction in the transplacental flux/fetal turnover ratios of essential amino acids ,177,178.In normal pregnancy, system A transporter activity was shown to be the highest in the smallest babies per milligram of microvillous protein . In contHypoxia enhances the expression of FABP1, -3, and -4 in term human trophoblasts, suggesting that FABPs play a role in fat accumulation in the hypoxic placenta . Similarin vivo or in vitro studies. While no model system completely encapsulates complete human IUGR, these model systems do allow us to carefully dissect aspects of the issue so as to further expand our understanding of the cellular and molecular mechanisms involved, and promote the development of therapeutic interventions.The placenta is the main interface between the fetus and mother. Placental insufficiency results in a failure of the placenta to deliver an adequate supply of substrates to the fetus and IUGR develops. A range of human and animal studies in IUGR pregnancies have suggested that placental restriction and insufficiency may result in a series of placental changes such as altered placental growth and placental substrate transport capacity, increased apoptosis and autophagy and increased glucocorticoid action . Such pl


We attempted to adopt this strategy for breast cancer PDXs. We studied five breast cancer PDXs, with the emphasis on two, called HCI-001 and HCI-002, both derived from triple negative breast cancer patients. However significant technical obstacles were encountered. These include the inherent slow growth rates of PDXs, the rarity of overt spontaneous metastases (detected in only 3 of 144 mice), very high rates of tumor regrowths at the primary tumor resection site, the failure of the few human PDX metastases isolated to manifest a more aggressive metastatic phenotype upon re-transplantation into new hosts, and the formation of metastases which were derived from de novo mouse thymomas arising in aged SCID mice that we used for the experiments. We discuss several possible strategies that may be employed to overcome these limitations. Uncovering the basis of the failure to detect a high rate of overt spontaneous distant metastases having a heritable phenotype in PDX models may reveal new insights into the biology and treatment of advanced metastatic disease.Several approaches are being evaluated to improve the historically limited value of studying transplanted primary tumors derived by injection of cells from established cell lines for predicting subsequent cancer therapy outcomes in patients and clinical trials. These approaches include use of genetically engineered mouse models (GEMMs) of spontaneous tumors, or patient tumor tissue derived xenografts (PDXs). Almost all such therapy studies utilizing such models involve treatment of established primary tumors. An alternative approach we have developed involves transplanted human tumor xenografts derived from established cell lines to treat mice with overt visceral metastases after primary tumor resection. The rationale is to mimic the more challenging circumstance of treating patients with late stage metastatic disease. These metastatic models entail prior  The my tumors . An examy tumors  In addity tumors . These ry tumors –11. The y tumors , 13, siny tumors .Also noteworthy is our finding that if prolongation of survival of mice with systemic metastatic melanoma can be achieved using a therapeutic intervention, a significant proportion of the mice relapse with spontaneous brain metastases , a cliniWe therefore decided to develop similar models of overt spontaneous metastasis using patient derived xenografts (PDXs), in this case, breast cancer PDXs. PDXs are being used increasingly to evaluate anti-cancer drug activity instead of human tumor xenografts derived from established cultured cell lines . The ratin vivo serial selection method adopted to successfully isolate variants with increased aggressiveness for spontaneous overt metastasis, not just micrometastasis after primary tumors resection, but using established cell lines—as described above and elsewhere [We studied several breast cancer PDXs originally isolated and characterized by Welm and colleagues  to initilsewhere , 2, 4, 5lsewhere , 2, 4, 5lsewhere . Moreovelsewhere .de novo tumors in the mice which upon analysis proved to be thymic lymphomas of mouse origin, spreading outside the thymus.Here we report our experience using this approach employing several breast cancer PDXs, called HCI-001, HCI-002, HCI-004, HCI-008 and HCI-009 where we encountered several technical limitations that may restrict, or even preclude in some cases this approach as a practical strategy for studying metastasis of PDXs, at least using breast cancer PDXs, and thus utilizing such an approach for investigational therapeutics. These limitations include: i) an extremely low rate of distant overt metastases of human origin in SCID mice even after prolonged periods of observation; ii) very long latency periods for rare human metastases (in the lungs) to be detected; iii) emergence of spontaneous 3, were implanted in the mammary fat pads of new animals, as described previously [3 tumour piece was implanted orthotopically in the mammary fat pad and the skin was closed with wound clips. 24 hours after surgery another dose of buprenorphine was administered. The mice were daily monitored for any clinical signs  and weight and tumour size was recorded once a week. Mice were humanly killed by cervical dislocation when their tumor volume reached 1500 mm3 or they showed any clinical sign, as dyspnea, hind limb immobility, jaundice, body weight loss of 20% or presented a distended abdomen (a sign of ascitis).All animal procedures, including maintenance and determination of experimental endpoints, were performed in strict accordance with the guidelines of the Sunnybrook Research Institute Animal Care Committee and the Canadian Council on Animal Care. The protocol was approved by the Animal Research Ethics Committee at Sunnybrook Research Institute. 144 female yellow fluorescent protein (YFP) severe combined immunodeficient (SCID) mice  were brea2b/2 where a is the width and b is the length. All tissue samples were originally collected with informed consent from individual being treated at the Hunstman Cancer Institute and the University of Utah under a protocol approved by the University of Utah Institutional Review Board, as noted in the Online Methods section of the study by DeRose et al [Most of the experiments involved the use of the HCI-001 and HCI-002 PDXs. These were derived from primary tumors of two triple negative breast cancer patients . The donse et al .Tumors were fixed in 10% buffered formalin and embedded in paraffin. Sections were stained with H&E. Antibodies used for specific tissue immunostaining included mouse monoclonal anti-HLA , rat anti-mouse CD34 (1:100 LSBio), and rabbit anti-mouse Ki67 . The LSAB + HRP-system from Dako was used as a secondary antibody.3, they were resected and mice observed afterwards. When a spontaneous metastasis of human origin developed  and could be detected by gross inspection., 2–5 mm3 pieces of the metastatic nodule (which only arose in the lungs) were implanted orthotopically in the mammary fat pad of 5 new SCID females, designating the new variant HCI-002 LM2. All 5 mice developed tumors in the mfp. When this variant, with an accelerated growth rate, developed spontaneous metastases large enough to be detected by gross morphology at time of necropsy , they were orthotopically implanted in the mammary fat pad of new SCID mouse recipients, and a new serial variant was designated, HCI-002 LM2-1, with the aim of developing a uniform and reliable metastatic tumor model. We focused our efforts mainly in the two TN PDX tumors that had more rapid growth rates: HCI-001 and HCI-002 (shown in 3). From the group of mice that did not manifest such tumor regrowth (19 implanted with HCI-001 and 13 implanted with HCI-002), and had longer survival times, only one from the HCI-001 PDX and two from the HCI-002 PDX, eventually developed lung metastases of human origin visible at naked eye at the time of necropsy and which could then be implanted orthotopically in new SCID females. As shown in In an attempt to develop a metastatic variant from several PDX breast tumors, tissue fragments from five different PDXs  were fishown in , both ofWe also detected mouse-derived tumors (thymic lymphomas) when implanting PDXs in SCID mice that we had not previously encountered to such an apparent high degree when studying established cell lines. As shown in Taken together our results highlight several potential issues to consider when trying to establishing post-surgical metastatic PDX models, which in this case apply to breast cancer PDXs in particular but which could conceivably be relevant to other types of PDXs. These include the slow tumor growth rates, and the unexpected rarity of spontaneous overt metastases of human origin as such metastases were observed in only 3 of 144 mice examined, and more specifically, in 3 out of 32 mice without evidence of tumor regrowth at the site of primary tumor resection. This extremely low rate of overt spontaneous metastasis was not expected given an impressive and interesting body of work by Hoffman and colleagues, much of it published two decades ago , 21. The-/- (NSG/NOG) mice may result in a much greater level of distant spontaneous metastases developing at a more rapid rate. In this regard, we reported that the metastatic properties of a triple-negative breast cancer cell line such as MDA-MB-231, after surgical resection of orthotopically grown/transplanted primary tumors, was remarkably more aggressive for spontaneous metastasis formation in such mice compared to recipient control SCID or NOD-SCID mice [-/- (NSG/NOG) mice.Another unexpected observation was the failure of spontaneous lung metastases of human origin arising in mice to manifest a more aggressive metastatic phenotype upon re-transplantation into new SCID mouse recipients. This stands in marked contrast to the results we have observed using established cell lines, as summarized in the Introduction and elsewhere , 2, 5, 6CID mice ; iv) addCID mice , 34; v) CID mice –37. HoweWhen considering the possibility of developing metastatic PDX models for future studies, awareness of one or more the aforementioned factors might be considered as a pro-active strategy to improve the likelihood of generating distant spontaneous metastases in clinically relevant organ sites, in a practical manner, which would facilitate studies of the biology and treatment of human metastatic disease in an experimental mouse setting. Finally we would note that if our observation of failure or low success rate of developing PDX models showing evidence of overt spontaneous metastases can be reproduced, identifying the basis for the lack of such metastases, despite robust growth as primary tumors, may reveal valuable new insights into the biological basis and control of metastases, as well as treatment of metastatic disease.


Glutamate, a nonessential amino acid, is a major bioenergetic substrate for proliferating normal and neoplastic cells on one hand and an excitatory neurotransmitter that is actively involved in biosynthetic, bioenergetic, metabolic, and oncogenic signaling pathways on the other. It exerts its action through a family of receptors consisting of metabotropic glutamate receptors (mGluRs) and ionotropic glutamate receptors (iGluRs), both of which have been implicated previously in a broad spectrum of acute and chronic neurodegenerative diseases. In this review, we discuss existing data on the role of glutamate as a growth factor for neoplastic cells, the expression of glutamate receptors in various types of benign and malignant neoplasms, and the potential roles that GluRs play in cancer development and progression along with their clinical significance. We conclude that glutamate-related receptors and their signaling pathways may provide novel therapeutic opportunities for a variety of malignant human diseases. Glutamate (Glu) is one of the key neurotransmitters in the mammalian central nervous system responsible for fast excitatory synaptic signaling in the brain. It affects several physiological processes such us learning, memory or behavior. High levels of Glu resulting in activation of respective receptors lead to neuronal cell death called excitotoxicity that is involved in pathophysiology of various neurodegenerative diseases, epilepsy, hypoxia or stroke  receptors (NMDARs), amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors (AMPARs), and 2-carboxy-3-carboxymethyl-4-isopropenylpyrrolidine  receptors (KARs). NMDARs are assembled from seven subunits: NR1, NR2A/B/C/D, NR3A/B, each one being a product of a separate gene. Alternative splicing results in additional heterogeneity of NMDAR subunits. NMDARs consist of two obligatory NR1 subunits and two of four types of regulatory subunits, NR2A, B, C and D, which assemble as a dimer of dimers. The resulting complex might eventually join with either NR3A or NR3B, which, in such a case, replaces one of the NR2 subunits. Moreover, glycine is a requisite natural agonist for NMDARs  residue at amino acid 607, but during the process of nuclear RNA-editing glutamine is replaced by arginine (R) in the vast majority of neuronal cells, which results in a very low calcium permeability for receptors containing the GluR2 subunit , which are responsible for glutamate uptake and are amply expressed in nonmalignant glial cells  or edited (R) GluR2 (Q/R site) subunit is critical for calcium permeability of AMPA receptor. Developmentally controlled replacement of the arginine with glutamine at this critical site (Q/R site) during an RNA-editing process renders AMPA receptors permeable (unedited GluR2) or non-permeable to CaAs mentioned above, in contrast to AMPARs, NMDARs expression is reported mainly in peripheral cancers. Moderate to high expression of NR1 subunit of NMDAR has been demonstrated in prostate cancer samples, whereas its expression in normal prostate tissue and benign prostate hyperplasia was very low or absent. Similar expression pattern was found in normal colon or cancer specimens  cell lines application of glutamate (10 mM) resulted in inward currents that were almost completely blocked by application of NMDA and AMPA receptor antagonists. Interestingly, the evoked currents were small, which is consistent with the low expression of these receptors in examined cancer cells  subunit at mRNA and protein levels inhibited proliferation of glioma cells in vitro and in vivo  formation, and altered ERK1/2 kinases activity  contributes to human melanoma susceptibility, especially in a subgroup of patients with a low level of sun exposure and tumors located on the trunk and extremities  during breast cancer progression was reported, showing higher methylation levels and frequencies in ductal carcinoma in situ when compared with preinvasive lesions such as flat epithelial atypia or atypical ductal hyperplasia; significantly higher methylation frequencies in grade III than in grade I of invasive ductal carcinoma have also been shown which suggests that CpG island methylation of GRIN2B might be an early event in breast cancer progression , which may result in distinct isoforms (for example eight in case of NR1 subunit) due to the presence of independent sites of alternative splicing. On top of that receptor subunits might undergo further posttranslational and epigenetic modifications resulting in even more complex glutamate signal transduction. Thus, the clinical significance of glutamate receptor expression may differ among tumor entities and it is difficult to predict how the expression of a particular subunit will influence cancer behavior. Still, it is tempting to speculate that GluRs and their signaling pathways render promising targets for therapeutic interventions. As a matter of fact, in recent years, there have been multiple attempts to implement iGluR and mGluR antagonists, in particular in malignant glioma treatment. Drugs such as AMPAR/KR inhibitors, ZK 200775 and GYKI 52466, despite having little effect on glioma growth in vitro, have been shown to exert anti-proliferative and anti-excitotoxic effects in rat hippocampal glioma models. Similar results were obtained for the NMDAR antagonists, norketamine 72 and MK801 (memantine). In fact, memantine has even been employed in a Phase II clinical trial to determine its safety/efficacy in glioma patients; however, results are not yet available . Similarly, the effectiveness of talampanel (an AMPAR antagonist) against glioma has been explored in the clinic. This study was completed with results that were not encouraging though (de Groot and Sontheimer Research into the role of Glu signaling in cancer development and progression is still in its infancy; however, important progress has been made in recent years. Considerable evidence exists and indicates that glutamate plays an important role in tumor development, acting as a growth factor and a signal mediator in neural as well as non-neuronal tumors tissues, in both autocrine and paracrine fashions. It has been proven that its actions involve mainly a family of receptors consisting of metabotropic glutamate receptors and ionotropic glutamate receptors whose presence has been proven in a variety of benign and malignant lesions throughout the body. Their actions, however, might differ significantly due to the fact that GluRs are combined from various subunits, which result in a large diversity of intracellular signaling and distinct pharmacological properties. Moreover, receptor subunits such as the It is clear that more research is needed to define the clinical significance of Glu–GluR expression and signaling in various cancers. Given existing preliminary studies, it will be extremely interesting to follow the field of glutamatergic signaling in cancer in future years.


Integrated transcriptomic and high-resolution whole genome methylation analysis in a myeloid leukemia cell line defines genes that respond to clinically relevant DNA methyltransferase inhibitors. Nucleotide sequence serves as the essential text of the genetic ‘book’ known as the human genome, but this book also comes with ‘reading instructions’, ‘bookmarks’ and ‘highlighted passages’ in the form of epigenetic patterns. Not surprisingly, the initiation and progression of cancer are governed not only by the accumulation of genetic lesions in the text itself but also by perturbations in the epigenome. Indeed, epigenetic alterations that dysregulate the ability of cancer cells to utilize genomic information are increasingly being appreciated as an important contributing factor in the development of different malignancies .Genome Biology, Lund et al. [Unlike genetic lesions, epigenetic patterns are relatively fluid and might be correctable by therapeutic perturbations. Such malleability has made epigenetic therapies an attractive alternative to cytotoxic chemotherapies. Rather than trying to destroy rapidly dividing cells instantly, epigenetic therapies aim to reprogram cancer cells by attenuating cancer-promoting transcriptional networks . These td et al.  employ ad et al. .IDH1/2, TET2, and DNMT3A [Myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) are malignant disorders of hematopoietic progenitor cells that lead to defective hematopoiesis. MDS is characterized by cytopenia (a reduction in the number of blood cells); AML, on the other hand, is typified by leukocytosis, which primarily results from the expansion of immature myeloid blasts. Both types of malignancies are most commonly diagnosed later in life (median age at AML diagnosis is 68 years). An intense interest in demethylating therapies for AML and MDS has been fueled by the presence of hallmark focal DNA hypermethylation that distinguishes leukemic blasts from normal hematopoietic progenitors, and by the high frequency of somatic mutations in genes that regulate the methylation of genomic DNA, including d DNMT3A ,5, in myde novo DNA methyltransferases  over the course of cellular differentiation, starting in embryogenesis. After each round of replication, the methylation pattern can be copied from the (methylated) parent strand to the (unmethylated) daughter strand by the maintenance methyltransferase DNMT1 because of its preference for hemimethylated DNA. The TET family of enzymes can mediate active DNA demethylation, although the steps subsequent to TET enzymatic activity have not been fully elucidated. In replicating cells, DNA methylation also decreases passively in the absence of re-methylation [The classical view of the establishment and maintenance of site-specific DNA methylation posits that methyl groups are introduced at CpG palindromic sites by hylation ,6.Cancer-associated focal DNA hypermethylation, especially in the promoter regions of key effector genes, is believed to be responsible for silencing critical tumor suppressors, such as pro-apoptotic genes and regulators of the cell cycle and differentiation commitment ,6,7. Treet al. [et al. [As an alternative hypothesis to explain why there was no correlation between gene expression and DNA methylation changes, Lund et al.  reasoned [et al.  investigNPM1 and DNMT3A genes. The nucleophosmin gene NPM1 is one of the most frequently mutated genes in AML. Mutations in NPM1 most often disrupt its nuclear localization signal and are associated with favorable prognosis [DNMT3A is another gene that is frequently mutated in AML. In contrast to NPM1 mutations, DNMT3A mutations predict poor survival rates [NPM1 and DNMT3A in AML, the co-occurrence of mutations in both of these genes in the absence of FLT3 internal tandem duplications (FLT3-ITD) is relatively uncommon [The study focused on the OCI/AML-3 leukemic cell line, which is known to carry mutations in the rognosis ,5. Even rognosis . DNMT3A uncommon . ConsequDNMT3A mutant AML patient-derived samples show a small but significant decrease in mean methylation relative to non-mutated samples [DNMT3A mutations with respect to the response to hypomethylating agents has not been conclusively established. Overall, four small-scale clinical trials that investigated the relationship between DNMT3A mutations and response to hypomethylating agents showed a trend towards prolonged progression-free survival (PFS) and higher complete remission (CR) rate in those carrying the mutation [DNMT3A is directly involved in the establishment and maintenance of DNA methylation patterns, and  samples . Despitemutation .The authors’ general characterization of the DNA-methylation landscape by WGBS confirmed previous findings ,7,9: metet al. [WIMSi analysis relies on the identification of gene clusters that share similar DNA methylation profiles by shape-curve and concordant changes in gene expression before and after treatment with Aza to identify specific genes or loci of interest. This approach yielded a list of 246 derepressed genes that have functions in cell death and survival or in cell movement and proliferation, all presumably regulated directly by DNA methylation. Cross-validation with other published datasets showed that WIMSi genes were enriched among genes whose expression was downregulated in AML compared to normal hematopoietic stem and progenitor cells (HSPCs). Many WIMSi genes were also found as upregulated in a previous study investigating the impact of DNMT inhibitors on methylation and gene expression in primary AML samples that failed to detect a relationship between DNA hypomethylation and gene expression by more general methods [et al.  demonstrDNMT3A mutational status on predicting response to hypomethylating agents? Extension of these studies to cellular models (cell lines or primary AML samples) with more relevant and comprehensive mutational landscapes will be eagerly awaited.As with any novel methodology, this study raises many questions for subsequent investigation. First, the question of whether other epigenetic marks are involved in early regulation of gene expression is the most important and the most informative. Are DNA methylation and histone modifications co-regulated, or does one direct the other? What is the timing of the chromatin reorganization that is necessary to allow gene de-repression, and what is the optimal time point at which to measure it? Would histone modifications be more directly related to changes in gene expression than changes in DNA methylation? Comprehensive epigenetic profiling that integrates analysis of the DNA methylome, the epigenome and the transcriptome will further our understanding of the mechanisms behind the efficacy of hypomethylating therapies. Such studies will be welcomed with great enthusiasm in the field of clinical cancer epigenetics. Second, what are the implications of the apparent reprogramming of OCI/AML-3 cells into a state more similar to HSPCs after Aza exposure? Are the effects on self-renewal genes distinct from those on differentiation and lineage-commitment genes? How similar are the reprogrammed profiles to those of more mature populations? Detailed studies involving additional leukemic and normal samples will be needed to shed light on the complex processes that guide epigenetic reprogramming. Finally, how generalizable are these findings, given the specific genetic make-up of the OCI/AML-3 cells and the uncertain impact of In the book of life that is the genome, aberrant DNA hypermethylation that silences tumor suppressor genes can be likened to pages that are stuck together, concealing important plot twists. Hypomethylating therapies can peel these pages apart and open the book at the right chapter. Whether it will be fully readable and able to guide the reprogramming of malignant cells, and what other signals or epigenetic marks are needed to promote proper genome reading, are questions that we are just beginning to answer.


Mobilization of defense mechanisms against this toxic agent requires rapid activation of signaling pathways. The article presents recent advances in the research concerning cadmium signal transduction in plants. New insights into the involvement of reactive oxygen species (ROS), nitric oxide (NO), plant growth regulators, and Cd-induced protein modifications are reviewed. Moreover, the role of recently recognized Cd-associated signal elements, including micro RNAs and several  Contamination of the environment with heavy metals, including cadmium, is a serious problem of the modern world. It is estimated that annually around 30,000 tones of cadmium are released into the environment, of which 13,000 tones result from human activity  acts as the primary messenger, in part because of its relative stability and in part because it can cross membranes through aquaporins  in mitochondria, which in turn, causes fatty acid hydroperoxide accumulation . Strong superoxide accumulation , but also CDPK and PI3 kinase, and may be triggered by mitochondrial dysfunction resulting from the closure of the mitochondrial permeability transition pore , biosynthesis (OsYUCs), and transport (OsPINs), auxin-responsive (OsARFs/OsIAAs) gene expression, and/or cell division (cell-cycle genes). However, the possibility that auxin functions in parallel to H2O2 cannot be excluded  are required to induce signal transduction leading to cell death in plants exposed to Cd   and, in consequence, amplifying Cd accumulation and the subsequent inhibition of root growth  and the activity of the antioxidant system after SA pretreatment was observed in many different species, including maize , it may induce changes usually observed under heavy metal stress, such as growth reduction, chlorophyll degradation, and inhibition of various photosynthetic parameters  of the metal  is crucial plant growth hormone controlling physiological and developmental processes, but its involvement in cadmium response is still poorly recognized. An increasing body of evidence indicates that cadmium disturbs auxin homeostasis by affecting its level, distribution, metabolism, transport, and balance with other phytohormones  such as putrescine (Put), spermidine (Spd), and spermine (Spm) have been proven to play a crucial role in the signaling network and plant defense to cadmium. The pathways of the PA metabolism may crosstalk with other signaling molecules, such as phytohormones, ROS, and NO  TF isolated from Arabidopsis. The described substitution leads to changes in SUP37 conformation which can alter its DNA binding ability (Malgieri et al., Plants possess an average of 590 TFs grouped in various families usually named after their DNA-binding motifs (Charoensawan et al., cis-acting elements. In bean plants, a cis-acting sequence called PvSr2 was shown to be associated with heavy metal stress. Transgenic tobacco plants containing the PvSr2 sequence exhibited an increased expression of the reporter gene in response to Cu2+, Zn2+, Hg2+, and Cd2+ (Qi et al., cis-acting elements is useful not only in the search for metal-responsive sequences, but also in uncovering the signaling molecules involved in Cd-dependent gene regulation. In soybean, Cd-responsive genes contained in their promoter region elements associated with ethylene and ABA signaling suggesting that these plant hormones are involved in the response to cadmium stress (Chmielowska-Bąak et al., TFs bind to defined DNA sequences called The levels of transcribed mRNA can be regulated by micro RNAs. Recent research shows that cadmium stress affects the levels of miRNAs in rice, soybean, and rape plants (Ding et al., Posidonia oceanic. In the latter case, increased methylation was correlated with elevated expression of methylotrasferase, indicating the de novo methylation process (Yang et al., Gracilaria dura exposure to cadmium resulted in a decrease in methylated DNA (Kumar et al., The expression of genes can be influenced by changes in chromatin, including histone modifications and DNA methylation. Increased levels of methylated DNA is associated with the repression of genes activity and vice versa—hypomethylation loosens the chromatin structure and facilitates genes activation (Chinnusamy and Zhu, 2+ or Zn2+ (Chmielowska-Bąak et al., 2+ by Cd2+ in calmodulin resulted in inhibited activity of this sensor protein (Rivetta et al., Protein activity can be influenced not only by changes in the expression levels of encoding mRNAs, but also by post-translation modifications. Heavy metals, including cadmium, can bind to the functional groups of biological molecules, leading to changes in their structure and activity (Latowski et al., As has also been mentioned, NO can modify proteins by binding to critical Cys residues, leading to their S-nitrosylation (Arasimowicz-Jelonek and Floryszak-Wieczorek, In summary it can be concluded that plants' response to cadmium involves various signaling elements, such as plant hormones, polyamines, calcium ions, ROS, NO, MAPK cascades, TFs, and microRNAs. The mentioned elements are often interrelated with one another and form a complex signaling network. Although significant progress has been made in recent years in the uncovering of the role of compounds participating in this network, there are still many ambiguities:➢ It is postulated that peptides derived from oxidatively damaged proteins may act as secondary ROS messengers and regulate specific genes; however, it has not been proven that such an ROS signaling pathway is involved in the response to cadmium stress.➢ The role of endogenous NO and other RNS during Cd stress is still very puzzling; therefore, the recognition of the molecular targets of RNS will be an exciting challenge for future research.➢ Plant growth regulators are substantially involved in the signaling pathways of plant response to cadmium, but their mutual interaction and exact crosstalk with the overall signaling network is still not fully recognized.➢ Recent research implies that cadmium stress leads to the induction of various TFs; however, information concerning their role in plant response to this heavy metal is still scarce.➢ There is a lack of research examining whether Cd-dependent changes in the levels of DNA methylation are associated with acquiring long-term resistance to this stress factor that can be memorized and passed to the offset.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.


In eukaryotic normal diploid cells, transcription must be accomplished in an efficient and faithful manner. The three nuclear RNA polymerases, transcription factors, chromatin regulators, and signaling pathways are among the key components that control gene expression. The molecular mechanisms by which these regulators control expression of individual genes have been studied extensively and are reviewed elsewhere ,2. RecenIn this issue of Cell & Bioscience, we have provided some evidences that deregulation of ribosomal gene (rDNA) transcription is a feature of cancer and other human disorders . Criticaet al. presented in their review entitled “Perturbations at the ribosomal genes loci are at the centre of cellular dysfunction and human disease” multiple aspects of rDNA functions. These functions not only include the relationship between RNA polymerase I (Pol I) transcription and proliferative property, but also the role of Pol I transcription in mediating heterochromatin structure and function, global gene expression and DNA damage response. Their review casts nucleolus as a central hub for coordination of stress responses. Notably, deregulation of Pol I transcription is required for malignant transformation and can be targeted with small molecule inhibitors to treat cancer [Diesch t cancer .et al. reviewed the principles and techniques of the recent NGS-based studies that link RNA editing to various aspects of post-transcriptional RNA processing, alternative splicing, transcript stability and localization. Their review also pointed out the possibility that NGS data may be applied for further interrogation of the “RNA editing codes”, at which functional outcome could be explored in a global manner [Liu and associates summarized current technological advances in genomics methods in the study of functional impact of RNA editing and the adenosine deaminse acting on RNA (ADAR) family of enzymes on the regulation of gene expression in their review. In the past few years, research in the field of A-to-I RNA editing has been accelerated dramatically by the development of next generation sequencing (NGS) technology, which has expanded the landscape of RNA editome as well as our understanding of its regulation. Liu l manner .et al. emphasized on the transcriptional activity of HIF-α modulated by the histone methyltransferase G9a mediated methylation of non-histone proteins such as Reptin and Pontin in hypoxia. This review provides useful information for future therapeutic potentials by targeting the methylation of non-histone proteins under hypoxic conditions [Baxter and colleagues systematically reviewed the alterations and mechanisms of DNA methylation and histone modifications in cancer. They described major findings on DNA demethylation resulting from replication-dependent passive dilution of 5-methylcytosine and active DNA demethylation catalyzed by Ten-eleven translocation (TET) family proteins. Baxter nditions .DNMT genes by p53, RB and FOXO3a transcriptional regulators and corepressors. Interestingly, overexpressed MDM2 may induce DNMT1, DNMT3A, and DNMT3B expression by negative control over p53, RB and FOXO3a. In addition, the low expressions of miRNAs 29 s, 143, 148a and 152 are associated with overexpression of DNMTs in various cancers. In this review, several important post-translational modifications including acetylation and phosphorylation in the control of protein stability and activity of the DNMTs especially DNMT1 are discussed [Lin and Wang evaluated in their review entitled “Dysregulated transcriptional and post-translational control of DNA methyltrasferases in cancer” the deregulation mechanisms of DNMTs. They focused on the transcriptional repression controls on Due to the broad nature of transcriptional regulation and its misregulation in diseases, it is not our intention to cover all major aspects of this active research area in this single issue. However, we hope that these articles provide readers with insightful information of how alterations of Pol I transcription, RNA editing, non-histone methylation, and DNA methylation regulations may be used as disease biomarkers and for targeted therapies.