PDI inhibition caused accumulation of unfolded or misfolded proteins, which led to ER stress and the unfolded protein response (UPR), and in turn resulted in cell death. Nitazoxanide [NTZ: 2-acetyloxy-N-(5-nitro-2-thiazolyl)benzamide] is a thiazolide antiparasitic agent with excellent activity against a wide variety of protozoa and helminths. In this article, we propose that NTZ, acting as PDI inhibitor, may be a new and potent addition to the chemotherapeutic strategy against ovarian cancer.Among gynecological malignancies epithelial ovarian cancer (EOC) is the leading cause of death. Despite improvements in conventional chemotherapy combinations, the overall cure rate has remained mostly stable over the years, and only 10%–15% of patients maintain a complete response following first-line therapy. To improve the efficacy of ovarian cancer chemotherapy it is essential to develop drugs with new mechanisms of action. Compared to normal tissues, protein disulfide isomerase (PDI) is overexpressed in ovarian tumors. PDI is a cellular enzyme in the lumen of the endoplasmic reticulum (ER) of eukaryotes or the periplasmic region of prokaryotes. This protein catalyzes the formation and breakage of disulphide bonds between cysteine residues in proteins, which affects protein folding. Selective inhibition of PDI activity has been exhibited both  Epithelial ovarian cancer (EOC) is responsible for 4% of cancer-deaths in women and continues to be the leading cause of death from gynecologic malignancies in the United States . The incPrimary treatment of ovarian and ovarian-related cancers should consist of optimal cytoreduction. After surgery, the standard treatment is a combination of platinum and taxane chemotherapy. Unfortunately, the majority of patients (up to 70%) who present with advanced stages of disease exhibit recurrent or persistent disease following primary treatment. Despite advances in surgical techniques and chemotherapeutic agents, mortality rates from EOC have decreased only slightly over the last 30 years .Drug resistance is thought to cause treatment failure and death in more than 90% of patients with metastatic disease . Given tin vitro and in vivo anticancer activity by targeting protein disulfide isomerase (PDI), a superfamily of oxidoreductase proteins localized in the endoplasmic reticulum (ER), nucleus, cytosol, mitochondria and cell membrane.Recently, propionic acid carbamoyl methyl amides (PACMAs) have shown a broad spectrum of cytotoxicity in a panel of human cancer cell lines, with relatively selective potency in ovarian cancer cells resistant to doxorubicin and paclitaxel . PACMAs Currently, the PDI gene family contains 21 members, varying in domain composition, molecular weight, tissue expression, and cellular processing. PDI is the most abundant chaperone/isomerase in the endoplasmic reticulum and plays pivotal role in protein folding through isomerase and chaperone activity. Given their vital role in protein-folding, loss of PDI activity has been associated with the pathogenesis of numerous diseases, most commonly related to the unfolded protein response (UPR) including malignancy. During tumor development, PDI is involved in the ER stress response that allows cancer cells to survive outside their normal environment. Increased PDI levels have been documented in a variety of human cancers, including ovarian , prostatThe activation of cellular stress responses, mediated by the ER, promotes survival of cancer cells . UPR redet al., the antibiotic bacitracin was shown to increase cell death of melanoma cells; cell death was a consequence of ER stress [Bacillus licheniformis and Bacillus subtilis. This antibiotic’s ability to inhibit PDI activity led to the effects it had on melanoma cells [In a study conducted by Lovat R stress . Bacitrama cells . HoweverGiardia lamblia and Cryptosporidium parvum, and has also been found efficacious in the treatment of other anaerobic bacteria and parasites residing in the human bowel [Unlike the antibiotic mentioned above, nitazoxanide (NTZ)—of the thiazolide family—is a safe and inexpensive broad-spectrum, FDA approved, drug. NTZ is traditionally utilized for the treatment of anaerobic intestinal parasites an bowel . Studiesan bowel . The phaan bowel . Similaran bowel .in vitro when bound to the protein glutathione S-transferase P1-1 (GSTP1) [Although NTZ was initially designed as an anti-microbial drug, anti-cancer properties have also been observed. A recent study accessing the development of drugs regulating c-Myc validated NTZ’s anti-Myc activity . Another (GSTP1) . A corre (GSTP1) . Specifi (GSTP1) .The purpose of this article is to propose, for the first time, the intriguing hypothesis that NTZ may sensitize cancer cells to stress-induced cell demise by blocking the PDI that under typical physiological circumstances restores ER homeostasis. In this fashion, we suggest that NTZ might enhance the efficacy of standard chemotherapy regiments in ovarian cancer patients and ultimately improve survival.The endoplasmic reticulum (ER) is the first compartment of the secretory pathway. It plays a major role in ER chaperone-assisted folding and quality control, including post-translational modification like the disulfide bond formation of newly synthesized secretory proteins . ProteinER stress can be provoked through a variety of physiological conditions, including perturbations in calcium bhomeostasis, glucose/energy deprivation, redox changes, ischemia, hyperhomocystinemia, viral infections and mutations that impair client protein folding . LikewisAs a consequence of ER stress, the cell has evolved an adaptive, coordinated response to limit accumulation of unfolded proteins in the ER. This signaling pathway is termed the unfolded protein response (UPR) . The molMammalian PDI is a multifunctional protein with both enzymatic oxidoreductase and chaperone properties . PDI catThe physiological responses ultimately regulated by the UPR are induction of molecular chaperones and upregulation of ER-associated Protein Degradation (ERAD). ERAD is responsible for the degradation of aberrant or misfolded proteins, which are potentially toxic for the cell . In factIf ERAD fails to promote clearance of unwanted proteins while the cell is under extreme ER stress, cell death may be triggered. This process involves caspase-dependent apoptosis and caspase-independent death . MountinAlthough apoptosis is the primary form of programmed cell death, there are some instances wherein cell death is associated with the presence of autophagic vacuoles. These are vacuoles indicative of autophagic cell death. Autophagy mediated cell death is purportedly linked to the apoptotic pathway through alterations in mitochondrial function. Both apoptotic and autophagic pathways are reported to share mediators, which supports the thesis that there is crosstalk between them . AutophaAutophagy is instrumental in a variety of physiological and pathophysiological processes, including cellular homeostasis, survival, development, and differentiation. Autophagy accomplishes the degradation and recycling of redundant and aged molecules and organelles, and destroys potentially harmful molecular and cellular components. For these reasons, autophagy has consistently been observed in response to ER stress as a complementary mechanism to the UPR whose function is to alleviate protein stress .Autophagy is ongoing at basal levels in eukaryotic cells and allows the cell to function optimally by removing unwanted substrates which may otherwise lead to cellular toxicity . EukaryoGenetic evidence for tumor suppressing function has been demonstrated in the autophagy-related gene Beclin1 . Beclin1High grade ovarian cancer cells express very low levels of the autophagy protein LC3, indicating that LC3-labeled autophagosomes do not accumulate. Moreover, the impairment of autophagic function has been correlated with alteration of the oncosuppressors PTEN, ARHI and p53 in ovarian cancer cells. For instance, deficient p53 mutants are unable to sequester bcl-2 which inhibits the formation of the autophagy interactome by interacting with beclin1 . Over-exOther indirect evidence that autophagy may act as a tumor-suppressant can be observed in the instigation of the PI3K/Akt pathway via PI3K mutations, AKT amplifications, or PTEN loss, which leads to decreased autophagy in many settings, largely through mTOR activation ,45. TOR mTOR negatively regulates autophagy by causing phosphorylation of Atg13, which reduces its interaction with ULK1 and inhibits formation of a trimeric complex required for autophagosome formation . The facIn addition to the cytoprotective function of autophagy, it has been proposed that autophagy is a mechanism for cell death. This conclusion was drawn after morphological features of autophagy have been observed within dying cells.Programed cell death (PCD) is an evolutionarily conserved essential for several vital functions, including developmental morphogenesis, tissue homeostasis, and defense against pathogens . ApoptosPCD does not seem to be restricted to apoptosis. Another classification of cell demise was identified by Clark and is known as type II cell death. He observed morphological change such as multiple‐membrane enclosed vesicles in the cytoplasm that engulf portions of cytoplasm and/or organelles in cell death . It has The antineoplastic effects of protease inhibitors include inhibition of AKT signaling, inhibition of angiogenesis and the provocation of autophagy following induction of ER stress. The authors draw the conclusion that ER stress and autophagy are important mechanisms of protease inhibitor-mediated cell death in ovarian cancer cells. Similarly, Bursch and colleagues reported that most MCF7 breast carcinoma cells undergo autophagic cell death, rather than apoptosis, after treatment with the anti-estrogen agent tamoxifen . Thus, iIndeed it is a reasonable conclusion that autophagic activity above a certain threshold may destroy a major portion of the cytosol and organelles, and in turn lead to an irreversible collapse of cellular functions. During extensive autophagy, the total area of the autophagic vacuoles and dense bodies are roughly equal to or greater than that of cytosol and organelles outside the vacuoles . In thisER stress autophagy and death cell may be triggered by several pathways. Nevertheless it is not yet completely understood if cell death is a consequence of the severity and/or extended duration of autophagy or if it is a deliberate mechanism of programmed cell death such as apoptosis.extreme autophagic process in order to cope with misfolded proteins and to relieve stress. Yet this prolonged and irreversible ER stress leads ultimately to cell death which is the main goal of any anti-cancer chemotherapy.In our paper we postulate that the inhibition chaperone function of PDI, whose overexpression allows cancer cells to survive under harsh conditions, pushes the cells into an Cryptosporidium species and Giardia intestinalis in pediatric patients 1–11 years of age. In July 2004, NTZ was approved for treatment of diarrhea caused by G. intestinalis in adults. NTZ is generally well tolerated, and no significant adverse events have been noted in human trials [Nitazoxanide (NTZ) is a main compound of a class of broad-spectrum anti-parasitic compounds named thiazolides. It is composed of a nitrothiazole-ring and a salicylic acid moiety which are linked together by an amide bond . NTZ wasn trials .In vitro, NTZ and tizoxanide function against a wide range of organisms, including the protozoal species Blastocystis hominis, C. parvum, Entamoeba histolytica, G. lamblia and Trichomonas vaginalis [in vitro activity of anaerobic bacteria and rotavirus are also inhibited by NTZ. In regards to its antiparasitic activities, NTZ has been shown to interfere with the pyruvate ferredoxin oxidoreductase, an enzyme essential to the anaerobic energy metabolism of the parasites. These enzymes are found in the amitochondriate eukaryotic human parasites , including Cryptosporidium parvum, most anaerobic bacteria (Clostridium spp.), Archaea, and microaerophiles of the epsilon proteobacterial group [aginalis . In addial group . Other dal group . HoweverNeospora caninum tachyzoite activity in vitro [Neospora caninum is an important intracellular protozoan parasite closely related to Toxoplasma gondii. Muller et al.’s work demonstrated, for the first time, that the activities of NTZ can be attributed to the inhibition of protein disulfide isomerase in the test parasite [NTZ has exhibited considerable anti-in vitro . Neosporparasite . Even moparasite . BecausemTORC1 is a major negative regulator of autophagy, its inhibition by NTZ contributes to the prevention of M. tuberculosis proliferation in infected macrophages . NTZ strNTZ has also shown significant immunomodulation properties, inhibiting lipopolysaccharide (LPS)-induced pro-inflammatory cytokine production in macrophages . ChronicInterleukin (IL-6) is a proinflammatory cytokine whose irregular production has been implicated in the development of various inflammatory and autoimmune diseases including cancer. IL-6 has had tumor-promoting effects on both malignant and stromal cells in a range of experimental cancer models. Specifically in ovarian cancer, IL-6 enhances tumor cell survival and increases resistance to chemotherapy via JAK/STAT signaling in tumor cells . IL-6 isin vitro and in vivo mice models with LPS-stimulated macrophages [The first report of NTZ having direct inhibitory ability on the production of IL-6 was recently published in a non-oncological study evaluating rophages . The mecin vitro. The enzymatic activity of recombinant glutathione-S-transferase (GSTP1) was strongly inhibited by thiazolides when evaluated through affinity chromatography and mass spectrometry. High levels of GSTP1 in tumor cells may substantially limit the efficacy of antitumor chemotherapy, much like the PDIs discussed above especially in regards the similar and cross-related function between them.From an oncological perspective, NTZ and select derivatives shown to induce apoptosis in colon cancer cells In a very recent study, NTZ was observed to inhibit c-Myc. The c-Myc oncogene is overexpressed in the majority of human cancers and contributes to the cause of at least 40% of tumors. c-Myc drives cell proliferation and is critically involved in the regulation of many growth-promoting signal transduction pathways . In partIn this concept paper we focused on PDI as an anti-cancer target for NTZ and aimed to draw organized connections between ER stress, URP and autophagy. Since our speculative area of interest is ovarian cancer, our hypothesis began with the finding that the anti-cancer properties of PACMAs worked by inhibiting the PDI in ovarian cancer cells. PDI is a dithiol-disulfide oxidoreductase chaperone from the thioredoxin superfamily and regulates a broad range of processes including protein folding, signal transduction, protein traffic, cell communication, and extracellular cell surface events related to thrombosis and immune functions. These regulatory behaviors converge to significantly affect cell redox status and regulate cell survival or death.Once we recognized the broad spectrum of action that PDI has, we investigated the possibility that there might be drugs currently used in clinical practice that could mimic the anti-PDI effect of PACMAs. We determined, based on previous publications, that the antiparasitic drug NTZ may show anti-PDI activity similar to that of the PACMAs. We restricted our focus to antiparasitic agent on the basis of the theoretical assumption that cancer cells, during their development display some unicellular/prokaryotic behavior reminiscent of robust ancient life forms . The regHowever, we do not exclude that there could be other mechanisms of action behind PDI inhibition and the ER stress cascade pathways. Further studies evaluating the preclinical, antitumor properties of NTZ are imperative, especially for ovarian cancer which is among the top four most lethal female malignant diseases in United States. Nevertheless, we endorse the idea that NTZ might have anti-cancer activity toward other solid epithelial cancers. It is worth recognizing that NTZ is an effective, safe, orally bioavailable and inexpensive drug, already approved for human use. To the best of our knowledge, there has been no report about the relationship between NTZ and ovarian cancer treatment.. ER stress can switch the cytoprotective functions of UPR into a cell death promoting pathway. Cancer cells under extreme or prolonged ER stress, by way of autophagic cell death pathways, can execute a cellular demise.The decision to live or die marks cell fate. Understanding how cells make that decision is essential for the development an anti-neoplastic strategy meant to overcome current treatment failure. An increasing number of studies are supporting the role of PDI in the development of malignancy. PDI inhibition causes accumulation of unfolded or misfolded proteins that leads to ER stressAutophagy is an evolutionarily conserved, lysosomal-mediated catabolic response pathway that is induced by metabolic stress particularly under nutrient-poor conditions. During cancer development, growth factors should be signaling for stimulation of anabolism and suppression of catabolism because replicative cell division requires a doubling of biomass. This may also explain the down-regulation of autophagy in cancer cells. Hence, the PDI inhibitor strategy is becoming an interesting therapeutic approach to killing cancer cells, particularly in the context of impairing apoptosis pathways. In this context, we propose NTZ as a viable tool for the inhibition of PDI.Ovarian carcinoma remains one of the most lethal female malignancies. A lack of available treatment against advance metastatic disease is responsible for the high mortality. Unfortunately, due to an increase in drug resistance, it is the therapies themselves that represent a major barrier to the long-term efficacy of the ovarian cancer treatment. The development of new cancer drugs is usually a slow and costly process, while new, more effective anti-cancer therapies are critical and urgent. During 2012, in the United States, approximately 22,000 women were diagnosed with EOC. In conclusion, future preclinical studies focusing on the ability of NTZ, either acting alone or as coadjuvant in the treatment of ovarian carcinoma, are compelling.


Lenalidomide is a synthetic compound derived by modifying the chemical structure of thalidomide. It belongs to the second generation of immunomodulatory drugs (IMiDs) and possesses pleiotropic properties. Even if lenalidomide has been shown to be active in the treatment of several hematologic malignancies, this review article is mostly focalized on its mode of action in multiple myeloma. The present paper is about the direct and indirect antitumor effects of lenalidomide on malignant plasmacells, bone marrow microenvironment, bone resorption and host's immune response. The molecular mechanisms and targets of lenalidomide remain largely unknown, but recent evidence shows cereblon (CRBN) as a possible mediator of its therapeutical effects. Lenalidomide and pomalidomide are synthetic compounds derived by modifying the chemical structure of thalidomide . In partLenalidomide and its parental molecule thalidomide have shown therapeutical activity in various malignancies –21.P < 0, 001), overall survival  and overall response rate . At a median followup of 48 months for surviving patients, a pooled update analysis of these studies has shown a significant benefit in overall survival  for those patients initially randomized to be treated with lenalidomide-dexamethasone combination , which possess pro-osteoclastogenic activity, as previously mentioned, are present in the BM milieu. Other molecules, as SDF-1α, IL-3 and hepatocyte growth factor (HGF), secreted by both malignant PCs and BMSCs, stimulate the expression of RANKL by BMSCs and thus enhance osteoclastogenesis. In MM, OPG production is downregulated. In addition, malignant PCs internalize and degradate OPG. This vicious cycle determines an increased RANK-RANL binding, augments OCL differentiation and proliferation and favours bone resorption [β, overexpressed in MM BM milieu, also downregulate the OBL maturation [ Bone remodelling is a tightly regulated process. The binding of receptor activator of NF-kB ligand (RANKL), on BMSCs and OBLs, to its receptor RANK, on mature OCLs and their precursors, stimulates OCL late differentiation and activity. Osteoprotegerin (OPG), a decoy receptor for RANKL, is produced by OBLs. OPG inhibits RANK-RANKL interaction, thus suppressing osteoclastogenesis . Severalsorption . Moreoveturation .P < 0, 05), whereas RANKL production was inhibited, so lenalidomide has been confirmed to reduce the serum markers of bone lytic disease.Lenalidomide has been reported to reduce osteoclastogenesis in MM . This efAlthough all the above mentioned mechanisms explain the direct and indirect anti-myeloma effect of lenalidomide, the precise molecular mechanisms and targets through which this molecule exerts its effects remain not completely understood.A seminal paper has recently identified cereblon (CRBN) as a primary target of thalidomide teratogenecity  and moreSeveral in vitro studies have shown that CRBN is also the target molecule of lenalidomide activity. Zhu et al. have clearly demonstrated in human MM cell lines (HMMCLs) the central role of CRBN in sensitivity and resistance to lenalidomide and have identified interferon regulatory factor-4 (IRF-4) as one of the downstream targets of CRBN. IRF-4 has previously reported to also be a target of and downregulated by lenalidomide .α production by these cells, thus suggesting that some immunomodulatory effects of lenalidomide are mediated by CRBN complex. This study has also shown that induction of p21/waf1 cyclin-dependent kinase inhibitor protein is prevented in absence of CRBN expression, indicating a role of CRBN in mediating antiproliferative effects of lenalidomide [Lopez-Girona et al. have demonstrated that lenalidomide binds to CRBN-DDB1 complex in a dose-dependent manner and with a ten-fold higher affinity than thalidomide. Moreover, after reducing CRBN expression by short interfering RNAs (siRNAs) in activated human T cells, lenalidomide has increased IL-2 and TNF-lidomide .Heintel et al. have found a significant relationship between CRBN expression and response to lenalidomide in 44 MM patients. In fact, CRBN expression resulted three times higher in responding patients compared to non-responders. Moreover, this study has shown a clear correlation between CRBN levels and quality of response. CRBN expression was lower in patients with stable or progressive disease and higher in patients with complete remission or partial responses . The data emerging from in vitro studies as well as the in vivo findings about the role of CRBN in lenalidomide action wait to be confirmed. IMiDs including lenalidomide have proven therapeutically effective molecules in several malignant diseases characterized by different hystogenetic origin of neoplastic cells, as well as by distinct phatogenetic pathways. Notwithstanding these differences IMiDs activity in the diverse neoplasia can be traced back to the pleiotropic mechanism of these molecules.Lenalidomide exerts a direct antitumor effect, interferes with the tumor microenvironment and enhances the host's antitumor immune responses. In MM, because of the complex bidirectional cross-talk between malignant PCs and the BM milieu including the BMSCs, the ECM proteins and the multitude of cytokines secreted in the BM milieu, the final effects of lenalidomide are the results of additional or synergic actions on different relevant pathogenetic events operating in this disease.In addition, lenalidomide activates caspase 8 and downregulates NF-kB activity induced by cytokines secreted in the BM milieu. This in turn determines reduced expression of antiapoptotic proteins. Thus, relevant in the lenalidomide apoptosis is also the modulation induced by this drug on adhesion molecules on PCs and BMSCs as well as on cytokines production. The anti-angiogenetic well known properties of IMiDs, including lenalidomide, might be relevant in MM as increased microvascular density has been reported to be associated with disease progression. Furthermore, lenalidomide acts on different host's effector immune cells. However the immune-mediated antitumor activity well defined in vitro are not completely correlated with the clinical outcome because of the complex immunosuppressive activity of underlying disease as well as of conventional antitumor drugs.Finally, lenalidomide downregulates bone resorption. The molecular mechanisms and targets of lenalidomide remain largely unknown. However, CRBN has recently been identified as the possible central mediator of lenalidomide activity and IRF-4 as a downstream molecule of CRBN action. Lenalidomide resistance in MM cells which, despite CRBN depletion, are able to restore their IRF-4 levels, suggest the existence of alternative pathways.


Severe eosinophilic syndromes related to the administration or use of unsuspected immunogenic substances have been described previously. Many of these diseases presented initially as clusters or isolated cases. The spanish toxic oil syndrome, the eosinophilia myalgia syndrome, and nephrogenic systemic fibrosis are examples of such diseases. We describe 2 cases of a severe eosinophilic syndrome characterized by marked peripheral blood eosinophilia , mononeuritis multiplex, and necrotizing vasculitis which developed in a close temporal association with the recent onset use of nonprescription probiotics. There was no history of a prior autoimmune disease. Although both cases had prompt response to immunosuppression with rapid resolution of peripheral blood eosinophilia and accompanying constitutional symptoms, they remained with permanent neurological deficits. Lactobacillus or Bifidobacterium species as well as a range of lactic acid producing bacteria. Being considered a dietary supplement in the USA, the Food and Drug Administration (FDA) regulation of probiotics is not as strict as that for prescription drugs [Lactobacilli, Saccharomyces Boulardii fungemia and endocarditis in postoperative patients, as well as Saccharomyces Cerevisiae fungemia in immunocompromised patients [Probiotics are defined by the World Health Organization/Food and Agriculture Organization (WHO/FAO) as live microorganisms that when administered at appropriate doses confer a health benefit to the host by regulation of the gastrointestinal flora , 2. Moston drugs . On the patients –6. This A 55-year-old female with no significant past medical history, presented with sudden onset of arthralgias, myalgias, malaise, night sweats, lower extremity weakness, and paresthesias of 1-week duration. Physical and neuromuscular examination revealed bilateral moderate-to-severe lower extremity weakness, asymmetrical in severity and predominantly distal with right foot drop. She was found to have leukocytosis  with 56% eosinophils . Electromyography (EMG) and nerve conduction studies revealed mononeuritis multiplex. Right quadriceps muscle and right sural nerve biopsies performed before the initiation of IV steroids or cyclophosphamide showed severe necrotizing eosinophilic vasculitis . The surA 65-year-old female with a history of inactive mild episodic asthma, sinusitis, and a remote history of Hashimoto's thyroiditis presented with sudden onset of abdominal pain, left arm weakness, numbness, right foot drop, diplopia, and profound general malaise. Physical and neuromuscular examination revealed mild left arm weakness along with profound distal right lower extremity weakness and foot drop. No skin lesions were noticed. Laboratory studies showed leukocytosis of 24,300 cells/mL with severe eosinophilia of 76% . EMG and nerve conduction studies showed evidence of mononeuritis multiplex and in addition, a mild underlying diffuse peripheral neuropathy. A right quadriceps muscle biopsy performed 1 week after initiation of IV steroids showed necrotizing vasculitis without eosinophils . By the Extensive questioning did not disclose the use of any other over-the-counter products, including L-tryptophan, recent use of leukotriene inhibitors, or any other new medication. However, both patients recalled the use of a new brand of probiotics described as an “extra-strength concentration” in a boxed blister pack, purchased over-the-counter in a Philadelphia metropolitan area pharmacy. Unfortunately we were unable to unequivocally identify the specific brand. None of the patients had gastrointestinal complaints, but they used the probiotic to “improve their immune system”.Both patients were negative for FIP-1 (PDGF) mutations which are commonly present in myeloproliferative forms of eosinophilic syndrome. Furthermore, there were no abnormalities detected by peripheral blood or bone marrow flow cytometry, nor abnormal PDGF-related translocations or other cytogenetic abnormalities. None of the patients developed respiratory symptoms or had pulmonary infiltrates on high-resolution chest-computed tomography scans. Response to IV corticosteroids followed by immunosuppressive therapy was optimal in both cases, with rapid resolution of peripheral blood eosinophilia and of the generalized symptoms. Although there was some degree of neurological improvement, to date, both patients have remained with permanent motor and sensory deficits in the lower extremities requiring orthotic devices for ambulation. We describe here the clinical manifestations and extensive laboratory, histopathologic, and radiological studies of two patients who developed an abrupt onset of a severe eosinophilic syndrome with prominent constitutional symptoms, necrotizing vasculitis, and mononeuritis multiplex, occurring in close temporal association with the ingestion of a probiotic supplement. Both of the patients were initially diagnosed with Churg-Strauss vasculitis, and one of them fulfilled the American College of Rheumatology diagnostic criteria proposed for Churg-Strauss . There w The cases presented differ from eosinophilia myalgia syndrome (EMS) because of the absence of skin edema or fibrosis, the lack of a history of L-tryptophan ingestion, and the presence of acute neurological involvement as opposed to the subacute or chronic course of neurological manifestations typically described in EMS .  Numerous severe eosinophilic syndromes related to unsuspected immunogenic substances have been well described in the past presenting initially as either clusters or isolated cases which subsequently reached epidemic proportions. The Spanish toxic oil and EMS are good examples , 9. Alth Prior experience in unveiling the etiology of the eosinophilic syndromes has shown that a high index of suspicion is required. The description of these two cases may allow the recognition of similar cases and remind the medical community that the widespread use of agents seemingly considered to be beneficial and free of side effects may occasionally have serious consequences. In this paper, we welcome government efforts focused on the establishment and enforcement of regulations promoting “good manufacturing practices” for dietary supplements.


The hazard rates for ipsilateral breast tumour recurrence (IBTR), distant metastasis (DM) and mortality throughout 10 years of follow-up were assessed.DM dynamics displays the same bimodal pattern  for both estrogen receptor (ER) positive (P) and negative (N) tumours and for all local treatments and metastatic sites. The hazard rates for IBTR maintain the bimodal pattern for ERP and ERN tumours; however, each IBTR recurrence peak for ERP tumours is delayed in comparison to the corresponding timing of recurrence peaks for ERN tumours. Mortality dynamics is markedly different for ERP and ERN tumours with more early deaths among patients with ERN than among patients with ERP primary tumours.DM dynamics is not influenced by the extent of conservative primary tumour resection and is similar for both ER phenotypes across different metastatic sites, suggesting similar mechanisms for tumour development at distant sites despite apparently different microenvironments. The IBTR risk peak delay observed in ERP tumours is an exception to the common recurrence risk rhythm. This suggests that the microenvironment within the residual breast tissue may enforce more stringent constraints upon ERP breast tumour cell growth than other tissues, prolonging the latency of IBTR. This local environment is, however, apparently less constraining to ERN cells, as IBTR dynamics is similar to the corresponding recurrence dynamics among other distant tissues. Our previous work  providesThe distinct and predictable recurrence and subsequent death risk patterns indicate a likely synchronizing effect of the surgical resection upon subsequent metastatic cancer development, apparently similar in all seeded distant organs . This syOur earlier findings  were desThe data derived from patients undergoing conservative surgery within a series of randomized clinical trials carried out at the Milan National Cancer Institute, investigating the role of different approaches for the primary tumour treatment, were scrutinized. In the Milan Institute, since the preliminary results of the first trial on the conservative treatment of early breast cancer  were repIn one trial, patients with invasive breast carcinoma 2,5 cm or less were randomized to either quadrantectomy, complete axillary dissection and radiotherapy (QuaRT) (360 women) or tumourectomy plus axillary dissection and radiotherapy (TaRT) (345 women). Quadrantectomy involved radial breast resection with excision of 2-3 cm of normal tissue around the tumour plus the removal of a sufficiently large portion of overlying skin and underlying fascia whilst lumpectomy removed only the tumour mass with a margin of normal tissue of 1 cm. In a succeeding trial eligibility criteria were identical and patients were randomized to QuaRT (294 women) or quadrantectomy plus axillary dissection without radiotherapy (Quad) (273 women). The full series of out-trial patients, who received QuaRT for unilateral primary breast cancer, amounts to 1652 patients. All analyzed trials were performed with the approval of the ethics committee of the Milan National Cancer Institute and patients were enrolled following informed consent.With the exception of patients allocated to the Quad arm, who did not receive radiotherapy, all patients received a radiation dose of 60 Gy to the ipsilateral breast. All axillary node positive (N+) patients were offered systemic adjuvant treatment [Cyclophosphamide plus Methotrexate plus Fluorouracil (CMF) or CMF plus Doxorubicin (Dx)], while no further post-surgical systemic treatment was recommended to axillary node negative (N-) patients. Adjuvant hormone therapy was not utilized within the randomized clinical trials and infrequently employed for out-trial patients, as it was not considered mandatory at that time. Details of the two trials and of the out-trial series have been reported elsewhere -15.All baseline data, treatment features and relevant clinical events were collected in standard format and stored in a clinical database, from which, after excluding 99 patients for whom the pathological T classification could not be determined, data of the 2552 patients undergoing conservative surgery plus RT and the 273 patients receiving quadrantectomy without RT were extracted for the present analysis. ER status was measured by the dextran-coated charcoal method and tumours with ER values >10 fmol/mg protein were considered ERP.Ipsilateral breast cancer recurrence (IBTR) and distant metastasis (DM) as first events and death from any cause were considered in the analysis. IBTR was defined as any new breast cancer focus appearing in the operated breast. DM was defined as any breast cancer manifestation(s) in areas other than that of IBTR with the exception of the contralateral breast. All diagnosed primary tumours, including contralateral breast cancers, were considered as competing events. In order to avoid the usual uncertainties related to the cause of death, deaths from any cause were studied instead of breast cancer related deaths only.Flexible piecewise exponential regression models for the hazard function were performed by subdividing observed time data in three months intervals . For a sCrude cumulative incidence for IBTR and DM was non-parametrically estimated to account for the presence of competing events.The main characteristics of patients are reported in Table The median follow-up time is 121 months. The number of recurring patients and the number of deceased patients at 5 and 10 years of follow-up are reported in Table per se did not influence the cumulative rate of recurrence. Also, the late outcome for ERP and ERN tumours was similar with global survival of 76.6% and 72.7% respectively.At 10 years of follow-up, ERP and ERN patients showed the same crude cumulative incidence of total recurrence (36%) as well as similar frequency of IBTR  or DM ; suggesting that ER status The patterns of the hazard rate for DM for patients undergoing different local treatments are reported in Figure When the DM dynamics was related to the ER status, patients with ERP tumours receiving different local treatments displayed hazard rates for DM with remarkably similar patterns Figure . RegrettFinally, the DM dynamics in different metastasis sites  was analyzed in the subset of patients for whom the treatment protocol required recording this information. Such patients received conservative surgery plus RT. The model allowing for the cause specific hazard to have a different behaviour according to ER group, but only a different scale level according to the different metastatic sites, was actually selected in agreement with previous results . For ERPIBTR dynamics was analyzed in patients undergoing conservative surgery plus RT. The general bimodal pattern emerges for both ER levels Figure . HoweverIn spite of the fact that the cumulative mortality is quite similar for both ER categories, the mortality chronology is definitely different for ERP and ERN tumours Figure . For patThis explorative investigation of a large and mature database provides evidence that the bimodal DM dynamics, previously observed in patients undergoing mastectomy for early breast cancer, is also present in patients undergoing more conservative resections with or without RT. The peak recurrence timing for these patients Figure  is compaThe similarity between the hazard rate patterns for all patients and for patients with ERP tumours Figures  and 2, iAn additional result supporting the lack of relevance of the local treatment on the metastatic process is provided by the analysis of DM dynamics by site Figure  that yieAlthough the analysis of the IBTR dynamics yields a bimodal hazard rate pattern for both ERP and ERN tumours, there is a profound difference in the peak timing between the two ER categories Figure . This fiAt least three mechanisms could be involved in IBTR origin. A) Over 60% of all primary breast cancer specimens contain additional foci of in-situ or micro-invasive disease most of which are outside the index quadrant ,18, reprIBTR may also be produced by tumour cells lodged within the breast to which adjuvant RT is administered. RT causes both a conventional short-term tumouricidal effect and the so called "tumour bed effect", in which the radiation-induced vascular damage of the surrounding tissue more indirectly impairs tumour growth . We cannper se is related to delayed IBTR appearance.Summarizing, different mechanisms could account for IBTR timing namely: a mechanism identical to a new primary, a mechanism equivalent to metastatic re-seeding, a mechanism related to the outgrowth of residual disease (unique for each hormone receptor phenotype) and finally the tumour bed effect. None of them seems to yield a persuasive explanation. Therefore, we favour a more general hypothesis, namely that the ERP status To colonize a new organ, disseminated tumour cells must have the capacity to effectively interact with the new microenvironment , both duThis picture is not unreasonable. Besides reports revealing the role of activated stroma on promoting tumour growth -26, seveThe hazard rate curves for mortality reveal differences between ERP and ERN tumours in the clinical course of the disease during the critical span from clinical recurrence to death. As suggested for a similar delay observed in patients undergoing mastectomy , both thIt should be acknowledged that, for analyses like the present one, there may be potential selection bias in the data. For example, there may have been differences in surgical practice or systemic therapy, since the patients were enrolled during a time span of about a decade. It is to be noticed, however, that surgery, RT and systemic treatments were performed according to constant guide-lines by the same therapy units throughout the accrual of all examined patients. Moreover, as detailed in the section "Patients and methods", the eligibility criteria of all examined series were virtually identical. In our opinion, therefore, the potential selection bias related to merging consecutive trials, which could account for differences in clinical outcome, particularly in local recurrence, is quite small.If the results of the present study are taken together with the results of similar analyses on patients undergoing mastectomy ,4,16, itLocal breast cancer recurrence dynamics are similar to DM dynamics for both ERP and ERN tumours following mastectomy while following conservative surgery plus RT such a similarity is restricted to ERN tumours only. Therefore, delays observed in the IBTR risk peaks for ERP tumours following conservative surgery and postoperative RT are a remarkable exception to the otherwise common recurrence risk rhythm. This clinical behaviour is likely to reveal a ER related relationship between tumour cells and the breast tissue, which may play a dominant regulatory role upon ERP breast tumour cells with more stringent constraints than upon ERN cells, for which the time to overt recurrence is similar to the corresponding time of the other tissues.All observed regular patterns in the clinical course of a disease that conversely shows a high degree of heterogeneity modulated by host vs tumour interactions should guide translating into clinical behaviours findings from investigations on prognostic factors (e.g. studies on tumour gene expression profiles). More in general, since life is by its very nature, a terminal condition, we believe and our data support that more careful attention should be paid to the temporal patterns of cancer recurrence and death over the span following diagnosis and therapeutic intervention. Being or becoming a member of a subgroup whose death is more likely to occur very early or much later is important for the discovery and testing of therapies designed to delay these events, as well as, for planning one's life, in general. Timing is, after all, everything in all such human events.ER: estrogen receptor; ERP: estrogen receptor positive; ERN: estrogen receptor negative; RT: radiotherapy; QuaRT: quadrantectomy plus radiotherapy; TaRT: tumourectomy plus radiotherapy; Quad: quadrantectomy; CMF: Cyclophosphamide plus Methotrexate plus Fluorouracil; Dx: Doxorubicin; IBTR: ipsilateral breast tumour recurrence; DM: distant metastasis; AIC: Akaike Information Criterion.The authors declare that they have no competing interests.RD: conception, design, analysis, interpretation of data, manuscript drafting, manuscript revising, final approval. IA: analysis of data, manuscript drafting, manuscript revising, final approval. PB: analysis of data, manuscript revising, final approval. DC: acquisition, analysis of data, manuscript revising, final approval. RA: acquisition of data, manuscript revising, final approval. FC: acquisition of data, manuscript revising, final approval. MG: acquisition of data, manuscript revising, final approval. WJMH: interpretation of data, manuscript drafting, manuscript revising, final approval. EB: analysis, interpretation of data, manuscript drafting, manuscript revising, final approval.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/10/656/prepub


A hallmark of many ciliopathies are renal cysts, ultimately disrupting kidney architecture and resulting in end-stage renal disease as the most common cause of mortality. Yet despite being the largest demand for renal replacement therapy  in young patients, the exact etiology of nephronophthisis (NPHP) and polycystic kidney disease is still largely unknown. Many gene products associated with NPHP and related disorders do not directly affect cilia, but appear to be important in tubular architecture, lumen formation, and polarized exocytosis. Personalized therapy to delay disease progression is beginning to explore gene replacement therapy, through use -for example- of induced pluripotent stem cells (iPSCs). For patients with nonsense mutations in ciliopathy genes, read-through drugs such as PTC124 and modified aminoglycosides might hold future promise. Furthermore, several drug screens in cell-based, zebrafish, and murine models have suggested new pathways preserving kidney function. Although much of this work has yet to be validated in human patients, this overview will discuss the approaches currently being employed in the context of improving ciliopathy treatment options.


The appeal of simple, sweeping portraits of large-scale brain mechanisms relevant to psychological phenomena competes with a rich, complex research base. As a prominent example, two views of frontal brain organization have emphasized dichotomous lateralization as a function of either emotional valence (positive/negative) or approach/avoidance motivation. Compelling findings support each. The literature has struggled to choose between them for three decades, without success. Both views are proving untenable as comprehensive models. Evidence of other frontal lateralizations, involving distinctions among dimensions of depression and anxiety, make a dichotomous view even more problematic. Recent evidence indicates that positive valence and approach motivation are associated with different areas in the left-hemisphere. Findings that appear contradictory at the level of frontal lobes as the units of analysis can be accommodated because hemodynamic and electromagnetic neuroimaging studies suggest considerable functional differentiation, in specialization and activation, of subregions of frontal cortex, including their connectivity to each other and to other regions. Such findings contribute to a more nuanced understanding of functional localization that accommodates aspects of multiple theoretical perspectives. Across decades of research to identify the functions served by various brain regions, using non-invasive, low-density scalp EEG recording, emphasis on large brain regions was understandable, because more localized inferences were rarely feasible. Proposals about broad functional differences in the cerebral hemispheres were common. A prominent literature in that tradition attempted to assign functions differentially to left- and right-frontal or posterior cortex, and much attention was spent on frontal lateralization and frontal specialization with respect to emotion and emotion-related psychopathology such as depression.response to emotion, but some have proposed that the physiology is part of emotion . Consensus has not been reached on how to segment even the gross structural or functional anatomy of the brain, particularly in the face of individual differences  index of neural activity in those units. Accordingly, differences in EEG recorded over left- and right-frontal cortex have been used to infer lateralized specialization or function. The following discussion examines a particular line of research by the authors and their colleagues for evidence about the appropriate granularity for the literature on frontal lateralization in emotion and suggests that hemisphere-level models of functional differentiation are no longer viable.As noted above, implicitly and sometimes explicitly the literatures arguing for valence or approach interpretations have often treated the left- and right-frontal lobes as single functional units. This assumption was understandable given that, until recently, much of the research involved scalp EEG studies relying on what are now considered low-density montages or, less commonly, lesion patients with uncertain or inconsistent trauma. Dense-array EEG recording montages can provide more precise localization of activity. However, the frontal lobes pose particular challenges to EEG source localization, especially in the absence of hypotheses about specific, dipolar sources, typically leading to reliance on the relatively low spatial localization precision of methods aimed at identifying distributed sources. Many studies were undertaken without individual structural MRI  and before dense-array EEG recording was widely available. EEG source analysis was rarely attempted, and inferences beyond the level of hemisphere, cortical quadrants, or gross superior/inferior distinctions were rarely advanced.The frontal-laterality EEG literature faces additional challenges, including reliance on resting data with little knowledge of or control over what subjects are doing as well as methodological disputes about choice of reference site, inter-session replicability, and laterality quantification metrics  one faces choices. One cannot surround the head with sites. There are times when local electrode density may be more important, such as distinguishing finger locations in motor cortex, and times when spatial extent may be more important, such as localizing deeper sources (e.g., Hanlon et al., It has become clear that traditional hemisphere and quadrant models of regional brain function in emotion and psychopathology are generally too coarse. Network accounts of brain function are growing in prominence, though challenging to operationalize and test. For example, Spielberg et al.  offered 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.


Preterm birth occurs in 11% of live births globally and accounts for 35% of all newborn deaths. Preterm newborns have immature immune systems, with reduced innate and adaptive immunity; their immune systems may be further compromised by various factors associated with preterm birth. The immune systems of preterm infants have a smaller pool of monocytes and neutrophils, impaired ability of these cells to kill pathogens, and lower production of cytokines which limits T cell activation and reduces the ability to fight bacteria and detect viruses in cells, compared to term infants. Intrauterine inflammation is a major contributor to preterm birth, and causes premature immune activation and cytokine production. This can induce immune tolerance leading to reduced newborn immune function. Intrauterine inflammation is associated with an increased risk of early-onset sepsis and likely has long-term adverse immune consequences. Requisite medical interventions further impact on immune development and function. Antenatal corticosteroid treatment to prevent newborn respiratory disease is routine but may be immunosuppressive, and has been associated with febrile responses, reductions in lymphocyte proliferation and cytokine production, and increased risk of infection. Invasive medical procedures result in an increased risk of late-onset sepsis. Respiratory support can cause chronic inflammatory lung disease associated with increased risk of long-term morbidity. Colonization of the infant by microorganisms at birth is a significant contributor to the establishment of the microbiome. Caesarean section affects infant colonization, potentially contributing to lifelong immune function and well-being. Several factors associated with preterm birth alter immune function. A better understanding of perinatal modification of the preterm immune system will allow for the refinement of care to minimize lifelong adverse immune consequences. Preterm birth is defined as birth prior to 37 completed weeks of gestation  and to directly kill pathogens through their antimicrobial properties. There is limited production of soluble factors, such as immunoglobulin (Ig), by the fetus so it must rely on maternal supply. Antigen-specific IgG is transferred across the placenta from the maternal circulation in large amounts after 32 weeks of gestation  are released from leukocytes, including neutrophils, monocytes/macrophages and lymphocytes, and can bind to microorganisms and destroy them. APPs act through various mechanisms to destroy pathogens, such as iron binding (using the bacterium's source of iron), enzymatic destruction, zinc depravation and membrane pore formation Levy, . The proThe classical, alternative and lectin complement pathways are all reduced in their pathogen-killing abilities in preterm infants . Preterm infants have a reduced pool of neutrophils and monocytes, and their precursors, due to reduced granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) levels , is pathogen-specific and requires acquisition of immunological memory. Genetic recombination of lymphocyte cell surface receptors results in an increased repertoire of pathogen recognition. Recognition of a pathogen results in lymphocyte activation, pathogen clearance and memory lymphocyte production, leading to swifter adaptive immune responses on subsequent encounters with the same pathogen.Maturation of adaptive immunity occurs mostly after term birth, so all newborn infants have deficiencies in T cell activation and cytokine production, B cell immunoglobulin production, and interactions between T and B cells, relative to adults.Cell-mediated immunity involves two main types of T cells, cytotoxic T lymphocytes  and T helper lymphocytes . T cells recognize pathogens when their T cell receptor (TCR) is presented peptide segments of a pathogen on MHC by an antigen-presenting cell  , interleukin (IL)-2 and tumor necrosis factor (TNF). The Th2 phenotype is anti-inflammatory, producing cytokines such as IL-4, IL-5, IL-13, and IL-10.At birth lymphocyte subpopulations are lower than in adults, and require clonal expansion. This occurs in the first few weeks of postnatal life, with both term and preterm infants following the same pattern of expansion; but preterm infants have lower absolute numbers of circulating lymphocytes  in fetal sheep lung tissue significantly increase in response to intra-amniotic lipopolysaccharide (LPS) infusion  axis regulates many physiological processes, including the immune system. An increase in pro-inflammatory cytokines causes activation of the HPA axis, which increases cortisol. Cortisol suppresses the immune system, by acting on glucocorticoid receptors of leukocytes, which causes translocation to the nucleus  or maternal administration of exogenous glucocorticoids on the developing immune system has not been extensively studied in humans or other animals but the limited data show lasting effects. Glucocorticoid exposure results in long-lasting alterations to physiological and cellular responses to infection and inflammation in the offspring. In pigs, maternal cortisol treatment during pregnancy caused an increased febrile response of female offspring to LPS challenge  caused a reduction in lymphocyte proliferation and IL-2 production in late-gestation fetuses and for up to six days after birth . In preterm infants, VILI is believed to be a major contributor to the development of the chronic respiratory disease BPD. The shear stress, inspiratory volume, air pressure, and oxygen concentration of ventilation are believed to cause epithelial cell damage, which contributes to protein leak into the airways, inhibiting the function of surfactant and increasing inflammatory cell infiltration (Attar and Donn, Ventilation of the preterm lungs also results in a systemic inflammatory response. Circulating phagocytes undergo activation in mechanically ventilated infants, resulting in increasing expression of the cell adhesion molecule CD11b on both neutrophils and monocytes (Turunen et al., The changes in the activation of circulating leukocytes are coupled with increased cytokine production. Term infants that require mechanical ventilation have an increased serum plasma level of IL-8, IL-1β, and TNF-α, no change in IL-6, and a decrease in IL-10 (Bohrer et al., Rates of birth by Caesarean section have risen in developed countries in recent years, such that this mode of delivery now accounts for almost one-third of births (Li et al., A relationship between birth by Caesarean section and increased risk of asthma was first demonstrated just over a decade ago (Xu et al., Relationships between Caesarean delivery and increased risk of asthma and allergic disease have been attributed to differences in microbial colonization of infants at birth (Gronlund et al., The systemic immune function of neonates may also be impacted by Caesarean section because more than half of Australian women who deliver in this way do so without labor (Li et al., Advances in obstetric and neonatal medicine have enabled profound reductions in perinatal mortality in recent years but this benefit has not come without cost. With increased rates of survival of preterm infants has come growing numbers of babies with illness and long-term disability, even for those born close to term (Cheong and Doyle, 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.


Genetic and behavioral screening of mice play important roles in sleep research, but the need for invasive electrophysiological (EEG/EMG) measurements for determination of sleep-wake and behavioral state limits the scope and rate of experimentation.In this study we explore the utility of a noninvasive method based on the signal from a piezoelectric sensor on the cage floor for scoring sleep-wake behavior in mice. It was previously demonstrated that the piezo signal can accurately discriminate sleep from wake activity; however, this was verified mostly by visual observation. Here we perform a more objective validation by correlating piezo measurements with EMG activity, which is dramatically suppressed during sleep. Furthermore, the piezo sensor is sensitive to respiration-related thoracic movements. Since breathing is relatively irregular in REM sleep compared to non-REM, we extract piezo features that reflect breathing regularity to try to distinguish between these sleep states.We validate our methods against simultaneous video/EEG/EMG measurement, which constitute the gold standard for scoring sleep. But rather than rely on subjective visual scoring to determine state, we use an unsupervised probabilistic model, the hidden Markov model (HMM), to automatically partition time series of extracted EEG/EMG features into REM, non-REM and wake states. A similar HMM, estimated exclusively from piezo features of instantaneous energy and breathing regularity, displayed dynamical stages with a similarity to REM/non-REM sleep, transient arousal, and wakefulness. These preliminary results suggest that a combination of piezoelectric measurements and computational modeling could yield a novel noninvasive method for analysis of sleep and sleep-related disorders.


Recent technological developments have brought forth a new era of RNA research in which large sets of data are collected rapidly using the high-throughput next generation sequencing technology. Growing evidence suggests that only around 5% of nucleotides in the mammalian genomes are transcribed into protein-coding RNA, and large amount of transcripts are non-protein-coding RNA (ncRNA). During the last decade, much information has been generated from the studies of one type of ncRNA, namely microRNA . miRNA modulates the expression of target genes through repression of mRNA translation or mRNA degradation. Its dysregulation has been implicated in various biological disorders and human diseases. Meanwhile, the long-non-coding RNA  has recently emerged to catch significant attention. lncRNA is involved in chromatin modification, epigenetic regulation, transcription control, and pre- and post-translational mRNA processing. The functions of lncRNA are believed to be associated with development, imprinting, mental and psychiatric disorders, and tumor growth.Bioinformatics is a pivotal component of this new RNA research revolution. It utilizes mathematical models and computer simulations to form, extract and analyze RNA data, and to search new ncRNA gene sequences and predict their targets. Assumptions in this computational modeling are derived from the observations that ncRNAs are produced following step-wise processes from precursors to functional end products. Based on miRNA biogenesis, criteria in searching for new miRNAs from sequencing data include that the precursors fold into a stable stem-loop structure, mature miRNAs are found on one arm of the stem, and these sequences are usually evolutionarily conserved  have offered much improved avenue for ncRNA discovery  accompanied by a quality score for each nucleotide in each sequence. Because of the high sensitivity of the technique, the “raw” data will also contain sequencing primers and contaminants which can potentially produce sequence bias that requires more sophisticated computational approaches to sieve out miRNA transcripts (Mendes et al., Computational approaches developed so far make extensive use of evolutionary conservation information either to predict ncRNA genes or ncRNA-target associations, sometimes ignoring the subtle rules presiding ncRNA biogenesis and target specificity. Thus, approaches combining high-throughput sequencing biochemical techniques and bioinformatic analyses that emphasizes the synergy of genome-wide approaches are essential (Mendes et al.,


Recent advances in high-throughput technologies have transformed methodologies employed to study cell-specific epigenomes and the approaches to investigate complex cellular phenotypes. Application of next-generation sequencing technology in the skeletal muscle differentiation field is rapidly extending our knowledge on how chromatin modifications, transcription factors and chromatin regulators orchestrate gene expression pathways guiding myogenesis. Here, we review recent biological insights gained by the application of next-generation sequencing techniques to decode the epigenetic profile and gene regulatory networks underlying skeletal muscle differentiation. During developmental processes, in response to external cues, changes in chromatin involving activation and repression of transcription factors and chromatin regulators  underlie commitment of specific cells to defined lineages. Importantly, transcription factors and chromatin modifiers are also able to alter the cellular expression program to maintain cell identity even upon removal of the initiating differentiation stimuli . During  Myogenic lineage commitment and execution of the terminal differentiation program relies on the activity of the paired-box transcription factors Pax3 and Pax7 and of the muscle regulatory factors (MRFs) MyoD, Myf5, MRF4, and myogenin [ In response to extrinsic signals, Pax3+ cells are committed to myoblasts and undergo terminal myogenic differentiation through the transcriptional network activated by MRFs. During postnatal life, muscle growth relies on satellite cells, which are a subpopulation of somite-derived cells that reside between myofibers and the basal lamina . Satelli MRFs achieve the task of transcriptional activation through the participation of nonmuscle restricted transcriptional activators, such as the Mef2, Six, and Runx family members . The pro Moreover, other sets of epigenetic regulators exert their function by repressing gene transcription: HDACs   and hist Several lines of evidence showed that enhancer of Zeste homolog 2 (Ezh2), the catalytic subunit of the PRC2 complex, is recruited to a subset of muscle-specific promoters in myoblasts , 20, 21 In summary, PcG role in satellite cells is to prevent ectopic differentiation, thus maintaining quiescence and proliferation states and safeguarding the mis-expression of non-myogenic genes in skeletal muscle cells. Another layer of control in myogenesis and muscle homeostasis is exerted by microRNAs (miRNAs) and long noncoding RNAs (lncRNAs) , 24. miR In this paper we will focus on recent findings depicting the epigenetic landscape of myoblasts cells, obtained through the application of Next Generation Sequencing (NGS) technology. The most recent results presented by three independent research groups shed new light on the genomewide epigenome of myoblast cells, focusing on histone modifications signature, the genomewide location of the MyoD transcription factor and of chromatin modifiers, for example, PcG proteins. In recent years, high-throughput technologies have been developed and rapidly improved to interrogate several aspects of cellular processes. Likewise, traditional approaches employed to interrogate the epigenome have undergone profound transformation since their coupling to massive parallel sequencing. As a result, approaches that investigated restricted groups of genomic loci have been adapted to genomewise analysis, with unprecedented resolution and specificity, and with dramatically decreasing sequencing costs.Methodologies currently employed to investigate global epigenetic signature are summarized in (i) 4C-Seq (circular chromosome conformation capture) is used to map long-range chromatin interactions and relies on proximity-based DNA ligation and sequencing of chromosomal regions contacting a bait DNA region .Hi-C-Seq is also employed to define chromosome architecture but it is free of bias for a bait locus .(ii) ChIA-PET-Seq (chromatin interaction through DNA-binding protein) combines chromatin immunoprecipitation (ChIP), proximity ligation, pair-end tag, and deep sequencing to detect chromatin interactions mediated by a protein, at the genomewide scale , 35.(iii) MNase-Seq exploits the property of micrococcal nuclease (MNase) to preferentially cleave linker region DNA and the MNase-digested DNA can therefore be used to annotate nucleosomes distribution . DNase s(iv) Chip sequencing (ChIP-Seq) couples the conventional chromatin immunoprecipitation technique with massive parallel sequencing, and it is used to profile histone modifications, and map transcription factors, core transcriptional machinery and chromatin modifiers recruitment in a genomewide scale. The genomewide map of generated reads provides information on the location and the intensity of the protein-DNA interaction , 39.(v) Bisulfite-Seq relies on the ability of bisulfite to convert unmethylated cytosines to uracils so that massive parallel sequencing can identify cytosine methylation status at the single base pair resolution . Alterna(vi) Furthermore, global mRNA expression profiling can now be achieved through a deep-sequencing approach (RNA-Seq) that sequences steady-state RNA (known and novel ones) in a sample, offering larger than microarrays dynamic range and sensitivity. However, a gene list is not the final step of this approach, deeper insight is gained by bionformatics resources such as gene enrichment analysis, pathways, and regulatory network analysis . One key Exploiting Chip assays coupled to massive parallel sequencing, Cao et al. identified the genomewide binding of MyoD, in C2C12 myoblast cells, satellite cells, and in mouse embryonic fibroblasts (MEFs) converted to myotubes by the stable expression of MyoD . As pred Most of MyoD bound regions regulate genes expressed both in undifferentiated C2C12 and differentiated myotubes, while, in approximately 10% of MyoD targets, the binding is associated with distinct peaks in myotubes versus proliferating cells. Therefore, MyoD constitutively binds the majority of identified sites in both proliferating and differentiated C2C12 cells. Most of MyoD binding sites do not represent classical enhancer elements, since out of 25 MyoD bound regions only 10 showed enhancer activity in a transient transfection assay. Interestingly, MyoD broad binding throughout the genome correlates with chromatin regions with overall increase in their histone H4 acetylation levels in MyoD-transduced MEF cells versus control cells. Conversely, MyoD expression in MEF cells marginally impacts the distribution of the H3K4me3 marks. Thus, MyoD may play a crucial role in broadly reprogramming the epigenetic architecture of the lineage-specified cells. The latter findings suggest that MyoD association throughout the genome leads to the recruitment of histone acetyltransferases and most likely of chromatin remodeling factors , possibl Asp et al. performed an initial comprehensive description of myoblasts and myotubes histone modifications profile . The aut Furthermore, genes involved in nonmuscle lineage commitment can be grouped in a distinct cluster characterized by the H3K27me3 mark and a transcriptionally silent state, both in myoblasts and myotubes. Notably, mesoderm-related genes, such as genes involved in myogenesis, adipogenesis, and osteogenesis are not present in this cluster. Interestingly, as shown for other lineage committed cells , only a  Because every cell lineage displays a specific genomewide profile of histone modifications, a comprehensive description of the chromatin signature of skeletal muscle cells will represent a precious resource for scientists investigating mechanisms underlying myogenic transcriptional regulation. Genomewide mapping of chromatin factors has provided insights into the gene networks regulated by histone acetyltransferases, HDACs, and chromatin modifiers in ESCs and hematopoietic lineages , 48, whi NGS technologies open novel avenues in the study of myopathies: the new methodological approach will shed new light on the biological pathways involved in skeletal muscle diseases and become an essential tool to study genetic causes of myopathologies. Furthermore, it will likely become a valuable technological approach in molecular diagnosis . A recent report by Geng et al. highlights the role of ChIP-Seq technology in revealing the molecular pathways modulated by the homeobox protein DUX4, a candidate gene misexpressed in facioscapulohumeral dystrophy (FSHD) . The aut NGS approaches may also represent a suitable diagnostic tool for Duchenne and Becker muscular dystrophy, because of the heterogeneity in the mutational spectrum of the disease and the large size of the dystrophin gene . Therefo Comprehensive understanding of the myoblast/myotube epigenome will extend our fundamental knowledge of molecular mechanisms orchestrating biological processes, such as myogenic differentiation, muscle development, maintenance, and regeneration, in physiological and myopathological conditions. Deep sequencing methodologies will assist stem cell research toward the comprehension of mechanisms underlying muscle cells identity. This knowledge has important implication for regenerative medicine, underscoring new paradigms for satellite cells biology and offering novel targets of pharmacological intervention.Recent advances in NGS technology allow addressing several epigenetic questions at the genomewide scale . To dateCharacterization of the histone modifications landscape in skeletal muscle cells can be exploited further to annotate lineage-specific functional genomic elements, such as enhancers and insulators –57. Chro Potential drawbacks of NGS techniques are the amount of starting material that frequently is a limiting factor. Isolation of quiescent satellite cells by cell sorting greatly reduces the number of cells one can use for RNA-Seq or ChIP-Seq experiments. Nevertheless, new protocols for chromatin immunoprecipitation assays coupled with massively parallel sequencing have been developed and optimized for limiting sample material . Further Another challenge introduced by NGS technology is the growing need for bioinformatics tools to analyze the vast amount of data generated by each experiment. Moreover, large-scale data obtained from NGS platforms need to be made available to the broad scientific community in a standardized and simple annotation form, as these data represent a precious framework to infer biological information. Bioinformatics pipelines require further refinements to enable expansion of mapping capabilities to the genome , improve Besides implementing computational methods in biological studies, successful application of NGS technologies imposes a more profound revolution related to experimental design and forces a transition from single-gene hypothesis-driven experiments to a genomewide view, in which understanding of the integration and the cross-talk of different circuitries is at the forefront. The achievements of these remarkable research capabilities lie in the combination of conventional molecular and cellular biology approaches with a systemwide genetic view, which enables us to explore so far not addressable questions and will help us to gain a more comprehensive insight into mechanisms guiding skeletal muscle cells during differentiation, its maintenance and response to pathological insult.


Metastasis, not the primary tumor, is responsible for the majority of breast cancer-related deaths. Emerging evidence indicates that breast cancer stem cells (CSCs) and the epithelial-to-mesenchymal transition (EMT) cooperate to produce circulating tumor cells (CTCs) that are highly competent for metastasis. CTCs with both CSC and EMT characteristics have recently been identified in the bloodstream of patients with metastatic disease. Breast CSCs have elevated tumorigenicity required for metastatic outgrowth, while EMT may promote CSC character and endows breast cancer cells with enhanced invasive and migratory potential. Both CSCs and EMT are associated with a more flexible cytoskeleton and with anoikis-resistance, which help breast carcinoma cells survive in circulation. Suspended breast carcinoma cells produce tubulin-based extensions of the plasma membrane, termed microtentacles (McTNs), which aid in reattachment. CSC and EMT-associated upregulation of intermediate filament vimentin and increased detyrosination of α-tubulin promote the formation of McTNs. The combined advantages of CSCs and EMT and their associated cytoskeletal alterations increase metastatic efficiency, but understanding the biology of these CTCs also presents new therapeutic targets to reduce metastasis. In the United States, improved early detection and new treatments have led to a decline in the overall mortality due to breast cancer, but the survival rates for patients with metastatic disease have not improved significantly . As a coMetastasis is a multi-step process whereby cancer cells face numerous challenges. They must: (1) detach from the primary tumor, (2) invade through the basement membrane, 3) enter the bloodstream or lymphatic system, 4) resist anoikis (detachment-induced apoptosis), 5) evade immune surveillance, 6) exit circulation, 7) persist and (8) eventually colonize a distal site [ enter th resist a evade im exit cir persist Animal models of metastasis are critical tools for improving our understanding of the biology of metastasis and for developing pharmaceutical compounds that can prevent or reduce metastatic spread. Murine modeling remains the mainstay for studying cancer metastasis. Multiple animal models for mimicking human cancer metastasis exist, with varying degrees of success in recapitulating the features of human metastasis. There are three general classes of mouse models of human cancer cell metastasis: (1) ectopic modeling implanting human tumor cells subcutaneously in nude mice; (2) orthotopic modeling implanting human tumor cells into the murine equivalent organ of their primary site; and (3) experimental metastasis models directly injecting human cancer cells into the murine circulation, either via the tail-vein or by intracardiac injection .Importantly, mouse modeling can also be used to study the CTC phase of metastasis, where cancer cells are suspended in the circulatory system. Technology for imaging and modeling CTCs in mice has rapidly developed. The use of human cancer lines labeled with fluorescent proteins such as GFP provides a powerful tool for non-invasive imaging to study the real-time behavior of cancer cells during the process of metastasis .et al. demonstrated that orthotopic xenografts generated viable CTCs that could be expanded after FACS sorting for GFP-positive cells from cardiac puncture [Using green fluorescent protein (GFP)-labeled PC-3 prostate cancer cells, Glinskii puncture . This orpuncture . Importapuncture . Taking puncture . Importapuncture . These Cpuncture . Transfepuncture . Notablypuncture .in vivo, which is particularly useful because the captured cells readily proliferate for further biological studies. Similarly, the CellTracks System  was adapted for use in enumerating and fluorescently-analyzing CTCs from breast cancer xenografts, although this system requires a fixation step that precludes further functional study [Similar to methods used to detect CTCs in human patients, immunomagnetic beads can capture CTCs in mouse models of CTCs from orthotopic injection of GFP-PC-3 cells, confirmed by fluorescence microscopy . This meal study .Up to 40% of patients with occult metastases detected in the bone marrow had no clinical symptoms or signs of metastatic disease by imaging . To imprSomatic stem cells serve to populate the tissues of the body but are generally constrained by their ability to differentiate only into organ-specific cell types. Parallels have been drawn between the characteristics of these tissue-specific stem cells and tumor cells, leading to the cancer stem cell (CSC) hypothesis that tissue stem cells are the cell of origin for tumor development and the target for oncogenic transformation ,23. Tiss−/CD29hi/CD24+ to enrich for murine mammary stem cells, a single isolated cell can reconstitute an entire functional mammary gland, providing convincing evidence for the existence of mammary stem cells [The identification of surface markers to enrich for mouse mammary stem cells paved the way for studies supporting the existence of these mammary stem cells. Using the surface markers Linem cells ,26. Thesem cells . Stem ceem cells . AsymmetSubversion of the normal stem cell signaling pathways by a mutated tissue stem-cell may drive both dysregulated proliferation, leading to tumorigenicity, and aberrant differentiation, leading to tumor heterogeneity. Traditional models suggesting that any of the cancer cells in a heterogeneous tumor may be continuously replicating and are equally capable of recreating a tumor or metastasis are not supported by studies showing that many thousands of these cancer cells must be transplanted into mice in order to generate tumors . In conthi/CD24lo and lineage-marker-absent population of cells from breast cancer patients, but not CD44lo/CD24hi cells, was enriched in the ability to form tumors in NOD/SCID mice [hi/CD24lo/lin− cells could form tumors in mice, while tens of thousands of unenriched cells failed to form tumors [hi/CD24lo/lin− subpopulation of cells, which remained tumorigenic when transplanted again into mice a second time while the unsorted tumor bulk cells were not tumorigenic, providing support for the existence of breast CSCs that are able to divide to produce both stem cells capable of further division and differentiated progeny that lack such capability.The identification of CSCs on the basis of unique surface marker phenotypes began in leukemia and brain cancers, and this technique for identifying CSCs continued in the hunt for breast CSCs. Based on the premise that only the CSC subpopulation is tumorigenic, studies beginning with the well-validated leukemia CSC model frequently use the number of cells needed to form a tumor in mice as an indication of the proportion of stem cells in a population, with populations that form tumors from fewer cells more representative of a stem cell population ,30. FlowCID mice . As few m tumors . The comm tumors . These thi/CD24lo/lin− population; however, most cell lines and tumors possess their own unique ratio of stem cell markers and populations. The observation that different sets of markers identify distinct and separate populations of self-renewing cells demonstrates a limitation in the ability of surface markers to accurately and consistently define CSC populations. Thus functional determinants of the CSC population, such as the ability to recapitulate heterogeneous tumors similar to the phenotype of the original tumor upon limiting dilutions in mice, remain the most persuasive evidence for the existence of breast CSCs.Functional assays for stem cell character are important for validating the use of phenotypic surface markers. Based upon the model in which stem cells, including cancer stem cells, have innate chemotherapeutic resistance, a functional assay was developed initially in hematopoietic cells to select for CSCs based upon the expression of one mechanism of chemotherapeutic resistance, aldehyde dehydrogenase ,32,33,34in vitro have been hampered by the inability to grow progenitor cells in large enough quantity for experimentation without inducing differentiation because traditional, adherent cell culture conditions promote differentiation of progenitor cells. Pioneering work by Dontu et al. is significant because it marks the first development of an in vitro system for the continuous propagation of non-adherent human mammary epithelial cells without differentiation [Attempts to study mammary progenitor cells ntiation . The mamntiation . In addintiation . Furtherntiation .in vitro and in vivo models for cancer stem cells, as it used the NOG mouse as a model host, which is further immunocompromised than the NOD/SCID mice used in most of the preceding CSC studies, and thus more easily accepts heterologous cells. The increased tumorigenicity seen using the NOG mouse model suggests that the host immune system interacts in crucial but yet undefined ways with the cancer stem cell population. Indeed, a recent study has shown the potential for immune targeting of breast CSCs [While evidence has been accumulating in support of the CSC hypothesis in breast carcinogenesis, it is far from becoming widely accepted. One troubling aspect of the cancer stem cell theory is that it is unclear whether or not the cancer stem cell population is indeed a very small subset of the tumor population, as some studies in lymphoma and melanoma have shown that a much larger than expected proportion of the population is tumorigenic ,40. Thisast CSCs . Additioast CSCs . While fin vivo stem cell activity by tumorigenicity assays in mice. Importantly, these breast CSCs were shown to have increased metastatic potential by intracardiac injection into NOD/SCID mice and subsequent luciferase assays for metastasis formation [hi/CD24lo population may be more associated with metastasis than primary tumor development [hi/lin− human patient tumor cells in a mouse xenograft model, where they saw an enrichment of breast cancer stem cells in spontaneous metastases, suggesting that breast CSCs are more capable of metastasis [hi/CD24lo [Most research has focused on the role of breast CSCs in primary tumor development, but recent studies suggest that breast CSCs may also play a critical role in metastasis. If the cancer stem cells are the only cells in a tumor with inherent tumorigenicity, then it is reasonable to conclude that the cancer stem cells are involved in the metastatic process, as metastatic outgrowth requires many of the same characteristics as primary tumor development . Emerginormation . In suppelopment . A recentastasis . Importai/CD24lo . Additioi/CD24lo . Interesi/CD24lo . These fDynamic cytoskeletal alterations are one of the most critical requirements for metastasis, as breast cancer cells must dissociate from the primary tumor site, migrate towards the bloodstream, and intravasate to become circulating tumor cells (CTCs). The epithelial-to-mesenchymal transition (EMT) has been implicated as a mechanism responsible for endowing epithelial cancer cells with the necessary traits and cytoskeletal alterations for successful metastasis. EMT is well characterized as an embryonic process whereby epithelial-like cells in the developing embryo convert into motile mesenchymal cells that can migrate to alternative sites in the embryo. This developmental program is thought to occur in a deregulated manner in epithelial cancers, allowing stationary, polarized breast cells tightly connected to adjacent cells to disband their cell-cell junctions and invade as non-polarized single cells ,51,52. AThe majority of the studies of EMT’s effects on the cytoskeleton focused on cancer cells attached to the extracellular matrix (ECM), which is a dramatically different environment than a CTC in circulation. This research on attached cells heavily stresses the role of actin reorganization and actin-based motility structures. How the EMT program may help cancer cells resist anoikis and survive in circulation as CTCs is a currently developing area of research. In the bloodstream, the mechanical forces on a CTC are vastly different from those when the cell is migrating or invading within tissues. In circulation, epithelial cells tend to die from anoikis, an apoptotic cell death that occurs in response to detachment. Prior activation of the EMT program during the initial invasion steps of metastasis may provide resistance to anoikis once in the bloodstream. EMT-induced loss of cell polarity in metastatic cancer cells can help downregulate the Hippo pathway, leading to resistance to anoikis ,54. One Many studies suggest that EMT occurring in metastasizing cancer cells is induced by microenvironmental factors such as TGF-β, leading to reversible changes in master regulators of the EMT program, such as transcription factors Twist and Snail, or epigenetic modulation by changes in microRNA expression of the mir200 family, rather than by permanent genetic changes ,51. The In addition to aiding in detachment from the primary tumor, invasion, and anoikis resistance in circulation, EMT also has specific effects on the cytoskeleton of breast cancer cells that affect the tumor cell during the CTC phase of metastasis. The environment of the circulatory system presents a unique challenge for circulating epithelial tumor cells, and they must develop a more flexible cytoskeleton to avoid fragmenting in small capillary beds due to shear forces in circulation . The celin vivo studies of CTC retention [After surviving in circulation, a CTC must first attach to the capillary endothelium and then extravasate into the tissue to effectively develop into a metastatic lesion. The mechanism by which CTCs reattach to the capillary endothelium is poorly characterized. Intravital imaging studies using colon carcinoma cells revealed that CTCs attach to the liver sinusoid capillaries in a microtubule-dependent, but not actin-dependent manner . Recent etention ,63,64. Fetention ,66. Liveetention . Importain vivo CTC reattachment, inhibitors of actin polymerization increase the formation of McTNs, while inhibitors of tubulin polymerization decrease McTNs [in vivo, persisting for hours rather than the 3 to 5 minutes seen in microtubules composed of Tyr-tubulin [McTNs are promoted by specific cytoskeletal modifications. Matching the mechanism of se McTNs ,63. Micrse McTNs ,69. The -tubulin . Interes-tubulin ,72. In a-tubulin .Microtubules can increase their stability by associating with intermediate filaments, an association that also enhances McTN formation. Western blotting and immunofluorescence of suspended breast carcinoma cells revealed that vimentin intermediate filaments, but not cytokeratins, extend into McTNs and that vimentin-expressing breast cancer cell lines had higher McTN frequencies . These din situ, suggesting that EMT can promote microtubule stability as breast cancer cells escape the tissue and become CTCs [The cytoskeletal alterations promoting McTN formation, increased vimentin expression and increased tubulin detyrosination, are enhanced in EMT. Vimentin is an intermediate filament strongly associated with EMT, and is a poor prognostic indicator in triple negative breast cancer ,76. Induome CTCs . Since Eome CTCs ,78. hi/CD24lo breast cancer stem cell phenotype and increased mammosphere formation, a measure of stem cell self-renewal [The CSC hypothesis and EMT both provide compelling insight into the mechanisms of metastasis; recent studies revealing the convergence of these two research fields has considerable ramifications for our understanding of breast cancer progression. When human mammary epithelial cells undergo a forced EMT by the expression of transcription factors Twist or Snail, they acquire the CD44-renewal . Importa-renewal . In comp-renewal . The pre-renewal ,81. Epig-renewal . Importa-renewal . This dihi/CD24lo CSCs in the bone marrow was identified for the first time as an independent predictor for reduced disease-free survival [hi/CD24lo CSC phenotype from the peripheral bloodstream, providing further evidence that CSCs are important in breast cancer metastasis [hi/CD24lo cells disseminated in the bone marrow [in vivo by expanding the CTCs in culture and injecting them into the tail-vein or intracardiacally into immunodeficient mice to find metastases to the brain and lung [+/MET+/CD47+ CTCs paralleled clinical progression and was a better indicator of overall survival than the frequency of bulk CTCs [While EMT and CSCs have been implicated in the metastatic process, definitive evidence in cancer patients has been limited by available technology to observe CTCs during the process of metastasis. Recent technology to isolate CTCs from the peripheral blood of breast cancer patients has created a unique opportunity to assess CTCs during the metastatic process. Multiple studies of metastatic breast cancer patients using the CellSearch System  have found that the presence of CTCs was a robust independent prognostic marker of progression-free survival and overall survival ,83. CTC survival . Differetastasis ,86,87. Ce marrow ,88,89. Tand lung . A recenand lung . The metand lung . The levulk CTCs .et al. used dual-colorimetric RNA-in situ hybridization (RNA-ISH) to assess breast tumor cells for EMT markers in circulation and at the primary tumor site and draining lymph nodes [In addition to detecting CTCs with CSC character, many breast CTCs display evidence of EMT. Immunostaining of breast cancer patient CTCs revealed EMT markers Twist and vimentin in 73% and 77%, respectively, of CTCs from patients with early disease, and in 100% of the CTCs from patients with metastatic disease . Interesph nodes . They reph nodes . In a clph nodes . These set al. isolated CTCs from the peripheral blood of patients with invasive breast cancer by multiparametric fluorescence-activated cell sorting. Interestingly, Zhang et al. relied on cancer stem cell marker ALDH1 in their CTC isolation, further emphasizing the role of CSC character for metastasis. After culturing these isolated CTCs, they found that these CTCs had high levels of the other breast CSC markers, CD44hi/CD24lo, and high levels of mesenchymal marker vimentin [Providing further support for the connection between EMT and CSCs, patient CTCs with CSC characteristics were also found to have EMT markers . Patientvimentin . + patients with metastatic breast cancer, Giordano et al. isolated CTCs from the peripheral bloodstream and found a heterogeneous CTC population, where some CTCs expressing an EMT or CSC-like phenotype did not express EpCAM and thus would be missed by CellSearch [While CellSearch remains the only U.S. Food and Drug Administration-approved test for CTC enumeration, this test relies on the EpCAM antibody to identify CTCs and thus is unable to capture CTCs that have become more mesenchymal than epithelial and have downregulated EpCAM expression. If EMT increases the chances of successful metastasis, then the CellSearch method may not be the best choice for metastasis studies, as it would miss a significant fraction of CTCs having undergone EMT. In a prospective study of HER2llSearch . These fCurrent clinical trials for cancer chemotherapeutics assess the ability to shrink existing tumors, rather than prevent metastatic spread and outgrowth. This approach neglects the fact that the biology of metastasis may be very different than that of primary tumor development . Indeed,. The EMT program has been widely studied as a mechanism that enhances cancer cell motility and escape from the primary tumor, but recent studies using developing technology to isolate CTCs suggest that the EMT program provides additional advantages to cancer cells in the very different microenvironment of the circulatory system. The EMT program can be particularly advantageous for breast CTCs since it appears to increase invasiveness to aid in the generation of CTCs, confers resistance to anoikis once the cells are in circulation, and promotes McTN formation and CSC character, giving the CTCs a metastatic advantage in exiting the bloodstream and surviving to emerge later as dangerous metastatic lesions.In order to enter the circulatory system, breast cancer cells must undergo extensive cytoskeletal alterations