PITX2 and ZFHX3 genes), that in the future, might allow rapidly guiding other diagnostic tests and accelerating the onset of an optimal secondary prevention.One promising field in neurovascular diseases investigation is the use of biomarkers to guide stroke etiology diagnosis and classification. Since treatment differs among etiologic subtypes and nowadays many patients receive a diagnosis of undetermined stroke, biomarkers might become an important additional diagnostic tool. In this review we update current knowledge about biomarkers related with cardioembolic stroke etiology (such as BNP and D-dimer proteins, or  The use of plasma biomarkers is getting increasingly popular in several fields of medicine. In fact, decision making processes using biomarkers are widely accepted in medical situations such as initiating lipid lowering therapies (by means of low-density lipoprotein plasma levels), diagnosing acute myocardial infarction (with troponins), ruling-out pulmonary embolism suspicions (by D-dimer levels), etc. Interest has also arrived to cerebrovascular diseases, since biomarkers might aid physicians in several steps of stroke evaluation .At present, the diagnosis of stroke remains based on clinical grounds and neuroimaging assessment. As neuroimaging is not a widely available tool, other methods might be useful to early differentiate between hemorrhagic and ischemic stroke or to discard pathologies that simulate cerebral ischemia, mainly in the pre-hospital setting. Biomarkers use might aid in ruling out these “mimics”, such as tumours or epileptic seizures, that account for up to a third of patients with stroke-like symptoms , thus avNowadays intravenous thrombolysis with recombinant tissue-type plasminogen activator (rt-PA) remains the only approved acute treatment for ischemic stroke. However, despite great improvements in the field, still very few stroke patients (1-5%) benefit from these therapies . That smOnce the diagnosis and hyperacute treatment of ischemic stroke have been done, etiologic classification is critical to discern the best treatment offered both in the stroke units during the acute phase and at the outpatient clinics for the secondary prevention. However, even with a thorough evaluation, the etiology of ischemic stroke remains undetermined in 25-39% of patients ; that was evaluated in 253 consecutive patients with acute ischemic stroke in whom Lp(a) levels >30 mg/dL were more frequent among the LAA subgroup than among CE  . Moreover, as there exist also white platelet-rich thrombi, platelet activation is another phenomenon that could provide with some good candidates, like CD40 ligand (CD40L), CD63 or P-selectin mainly associated with LAA strokes although these biomarkers have only been compared among non-CE strokes , 28. HowA proteomic analysis in serum samples of 24 patients with ischemic stroke (12 LAA and 12 CE patients) identified four spots whose expression intensity was at least four times stronger in LAA patients than in CE patients: haptoglobin (Hp) related protein, serum amyloid A (SAA) (two spots) and Hp alpha chain. In a larger series of patients (n=262) with ischemic stroke using ELISA techniques, Hp levels >1,040 microg/mL identified LAA patients with 95% sensitivity and 88% specificity whereas SAA levels > 160microg/ mL identified LAA patients with 91% sensitivity and 83% specificity . The same group also studied 15-deoxy-Delta prostaglandin J2 (15-d-PGJ2) in 552 patients with an acute stroke admitted within 24 hours after symptoms onset . Levels The involvement of some pathways in several stroke etiologies reflects the complexity of finding good candidate biomarkers to identify CE strokes. Moreover, the characteristics of the different studies performed  make hard to reach a consensus about the most suitable biomarkers involved in each etiology. In fact, some etiologies may share mechanisms (and therefore biomarkers) leading to stroke, such as a coronary disease that use to be atherothrombotic but may produce a cardiac discinesia or other heart disfunctions that generate an embolic stroke. The measurement of those markers at different time-points (acute versus subacute or chronic stages of neurovascular disease), includes also some other bias in these studies. To overcome all those limitations, more studies involving a large number of ischemic stroke patients to compare biomarkers among etiologies should be done in the next future and it is likely that well-designed diagnostic trials will lead to clinical validation of selected stroke biomarkers. Although blood proteins have been the main molecules evaluated as biomarkers, recently some investigations support the usefulness of nucleic acids in the diagnosis of stroke. In 2008, Xu and colleagues presented the first study showing differences of gene expression when they compared CE versus LAA strokes . Using RAnother approach to use nucleic acids as biomarkers is by studying the microRNAs (miRNA), small non-coding RNAs, which control gene expression by both inhibition and activation. In a young Asian cohort, when comparing the expression profile of 836 miRNAs among CE, SVD and LAA strokes using microarrays, 132 miRNAs were useful in predicting etiology . The miREven though there are few reports in this field, the promising results obtained by means of these screening techniques using arrays should be analyzed with more detail in the future to find some combination of genes that could characterize each stroke etiology. Furthermore, these studies show how different the scenarios of the stroke pathophysiology are among etiologies and also which processes are altered depending on the cause of stroke. At present there are not many studies trying to identify genetic markers of etiology in ischemic stroke. Few analysis have attempted to find these biomarkers but with poor results  or with PITX2 gene) associated with ischemic stroke but only in CE etiology. This gene encodes a catenin-regulated transcription factor associated with AF [ZFHX3 gene has been associated with AF and with CE stroke [ZFHX3 gene was found after the analysis of 2,385 AF cases and 33,752 controls. In a second phase the SNP was genotyped in five ischemic case-control sample sets of European descent comprising 1,036 cases and 3,468 controls and reaching significant association. The most promising results until now are referred to CE stroke and the use of Genome Wide Analysis (GWA) approaches. Several studies using GWAs, that detect over 1 million of single-nucleotide polymorphisms (SNPs) across the genome, have observed significant signals in CE stroke. All these studies have identified SNPs that were associated also with AF, the main risk factor for CE stroke. The DeCODE group found a SNP in the Chromosome 4q25 (rs2200733, near  with AF . In this with AF . Very reE stroke . This geAlthough there are not specific designed studies developed to find genetic differences between the etiologies of ischemic stroke, the second wave of GWAs in ischemic stroke that will be carried out next year might find these genetic differences through the promising use of endophenotypes. Although there are many biomarkers that have been associated with stroke diagnosis, etiology or prognosis, information regarding clinical utility is extremely limited by the scarcity of large, prospective, rigorous, randomized clinical trials. Massive sequencing of high quality, full-length cDNA libraries, coupled with proteomics and functional genomic approaches will bring a revolution in biomarkers discovery in the next years. Proteomics seems a promising tool for massive biomarkers identification in the stroke field, mainly directed to diagnosis and treatment response. Also transcriptomics and GWAs studies are recently incorporating new candidate genes that might generate diagnostic and therapeutic targets for specific stroke etiologies. All generated candidates should be carefully validated before they are implemented in clinical routine. The ideal biomarker should be very sensitive, specific, reliable, accessible, standardized, cost-effective and easy to interpret. Due to the complex pathophysiology of the stroke, a combined biomarker panel seems more feasible that a single biomarker for a diagnostic test . IncludiAlthough point-of-care tools seem ideal in the stroke units setting, information would be useful even if obtained during the first days after stroke onset, allowing the use of standard platforms to get etiological biomarkers results. The use of such biomarkers would allow to drastically reducing the number of undetermined strokes. The identification of CE strokes in cases of paroxistic arrithmias is one of the main indications since this might produce an intensification of the secondary prevention and might be easily missed in routine examinations. For doing this therapeutical decisions we need a very specific test and for guiding other diagnostic tests a very sensitive biomarker would be needed. Models of clinical data plus biomarker information and easy interpretation algorithms for clinicians would be mandatory if these markers have to be applied in daily practice. It is likely that future well-designed diagnostic trials will lead to clinical validation of selected stroke biomarkers.


Methylphenidate is a psychostimulant that inhibits the neuronal dopamine transporter. In addition, methylphenidate has the intriguing ability to provide neuroprotection from the neurotoxic effects of methamphetamine and perhaps also Parkinson’s disease; both of which may likely involve the abnormal accumulation of cytoplasmic dopamine inside dopaminergic neurons and the resulting formation of dopamine-associated reactive oxygen species. As delineated in this review, the neuroprotective effects of methylphenidate are due, at least in part, to its ability to attenuate or prevent this abnormal cytoplasmic dopamine accumulation through several possible neuropharmacological mechanisms. These may include 1) direct interactions between methylphenidate and the neuronal dopamine transporter which may attenuate or prevent the entry of methamphetamine into dopaminergic neurons and may also decrease the synthesis of cytoplasmic dopamine through a D2 receptor-mediated signal cascade process, and 2) indirect effects upon the functioning of the vesicular monoamine transporter-2 which may increase vesicular dopamine sequestration through both vesicle trafficking and the kinetic upregulation of the vesicular monoamine transporter-2 protein. Understanding these neuropharmacological mechanisms of methylphenidate neuroprotection may provide important insights into the physiologic regulation of dopaminergic systems as well as the pathophysiology of a variety of disorders involving abnormal dopamine disposition ranging from substance abuse to neurodegenerative diseases such as Parkinson’s disease. It was first synthesized in 1944 dihydrotetrabenazine (DHTBZ), and vesicular DA content in a rat model of METH neurotoxicity DHTBZ, in cytoplasmic vesicles purified from mice striata [3H]DHTBZ binding in whole striatal homogenates prepared from treated mice. These and other vesicle trafficking data suggest that multiple high-dose METH administrations cause a subcellular redistribution of VMAT-2 containing vesicle out of the cytoplasm such that fewer VMAT-2, and presumably associated cytoplasmic vesicles, are available to sequester DA DHTBZ is affected similarly [max of DA transport and the density of kinetically active VMAT-2 without changing the Km, the catalytic rate constant, or the rate constant for DA binding to the VMAT-2 [This possible mechanism of neuroprotection is supported by the experimental findings that MPD increases and decreases VMAT-2 immunoreactivity in the cytoplasmic and membrane-associated vesicle subcellular fractions, respectively, without altering total synaptosomal VMAT-2 content . Bindingimilarly . The traimilarly , 47, 65.e VMAT-2 . MPD alse VMAT-2 , 65 whice VMAT-2 . This sue VMAT-2 , 62. In the second possible neuropharmacological mechanism of neuroprotection, MPD actually alters the functioning of VMAT-2 in the membrane-associated vesicles to transport more DA. This mechanism is supported by the findings that, in addition to redistributing vesicles away from membranes and into the cytoplasm, MPD also increases vesicular DA sequestration into the remaining membrane-associated vesicles . MPD thuThese MPD-induced increases in vesicular DA sequestration may have several additional interesting functional consequences. The increase in vesicular DA transport increases vesicular DA content with no change in whole striatal tissue DA content , 65. By 3), attenuate or prevent the abnormal accumulation of cytoplasmic DA and the resulting formation of potentially neurotoxic DA-associated reactive species. At present, it is difficult to quantify the amount that any individual mechanism contributes to MPD neuroprotection in vivo. Further studies investigating the neuropharmacology, biochemistry, and molecular biology underlying these mechanisms are warranted as these will help to determine the in vivo contribution of each mechanism. These additional studies may also provide insight into the physiologic regulation of dopaminergic systems as well as the pathophysiology of a variety disorders involving abnormal DA disposition ranging from substance abuse to neurodegenerative diseases such as Parkinson’s disease. Ultimately, this may suggest leads for developing related novel and more effective neuropharmacologic therapeutic treatment strategies.The studies reviewed above help us to understand how MPD has the ability to provide neuroprotection against METH-induced neurotoxicity and perhaps Parkinson’s disease through possible mechanisms involving direct interactions with the DAT and additional mechanisms involving indirect effects upon the VMAT-2. These mechanisms, summarized in Fig. (


A 51-year-old woman with severe asthma underwent bronchoscopy and endobronchial ultrasound (EBUS) for investigation of a 15-mm peripheral lung nodule. Histology demonstrated a typical carcinoid tumor. Pulmonary location is the second commonest site for carcinoid tumors. Diagnosis of peripheral carcinoid tumor of the lung is difficult due to its small size, poor accuracy of cytologic diagnosis, and low sensitivity of positron emission tomography in detecting it. EBUS has a high diagnostic yield and a low complication rate in the evaluation of small solitary pulmonary nodules. The ultrasound appearance of carcinoid tumors is identical to that of lung carcinomas. Prompt diagnosis of carcinoid tumor is desirable as regional lymph node metastasis is seen in 10% of patients and is associated with a reduced 5-year survival. We feel that, where possible, all patients presenting with solitary pulmonary nodules should be investigated initially using EBUS due to its high diagnostic rate and the very low incidence of adverse events. CT scanning of the chest was performed to exclude any factors that may have been responsible for exacerbating her illness. A lobulated, well-defined, soft tissue density nodule, measuring 15 mm was identified in the left lower lobe [A 51-year-old lady presented with persistently poor control of her asthma symptoms. She had a long-standing history of difficult asthma, frequently requiring oral prednisolone therapy. Respiratory function testing demonstrated moderate fixed airflow obstruction with a forced expiratory volume in 1 second . Examination in the posterobasal segment of the left lower lobe demonstrated a solid lesion . FluorosHistologic examination of biopsy specimens showed soft tissue infiltrated by sheets and cords of cells with slightly irregular nuclei, intranuclear inclusions, and fine granular chromatin. ImmunohCarcinoid tumors are low-grade malignancies comprising neuroendocrine cells. The commonest site for such lesions is the gastrointestinal tract, with a pulmonary location being the second commonest site. They are2The majority of pulmonary carcinoids are centrally located; when loEBUS has been demonstrated by numerous groups to improve the diagnostic evaluation of solitary pulmonary nodules (SPNs). Its value is most apparent with smaller lesions,8 where tEven large series examining the efficacy of EBUS report only very rare diagnosis of carcinoid tumour. Only one carcinoid was diagnosed in a cumulative 577 EBUS procedures.–13 Carci14Without the histological diagnosis that was made possible by EBUS our patient may have gone undiagnosed for some more time. Firstly, over one-third of carcinoid tumors are diagnosed only at thoracotomy, and this17While 5-year survival from typical carcinoid tumors is as much as 91%, delay inThis case illustrates the significant value of bronchoscopy using EBUS in the investigation of small peripheral pulmonary nodules. Peripheral pulmonary carcinoid tumors may reliably be diagnosed using transbronchial biopsy, and their ultrasonographic appearance seems to be similar to other bronchogenic carcinomas. We feel that, where possible, all patients presenting with solitary pulmonary nodules should be investigated initially using EBUS due to its high diagnostic rate and very low incidence of adverse events.


One of the major instigators leading to neuronal cell death and brain damage following cerebral ischemia is calcium dysregulation. The neuron's inability to maintain calcium homeostasis is believed to be a result of increased calcium influx and impaired calcium extrusion across the plasma membrane. The need to better understand the cellular and biochemical mechanisms of calcium dysregulation contributing to neuronal loss following stroke/cerebral ischemia is essential for the development of new treatments in order to reduce ischemic brain injury. The aim of this paper is to provide a concise overview of the various calcium influx pathways in response to ischemia and how neuronal cells attempts to overcome this calcium overload. Cerebral ischemia results in a reduced blood supply to brain tissue, causing oxygen-glucose deprivation and ATP production failure. The resulting energy crisis can trigger a cascade of detrimental biochemical and physiological events leading to acute or delayed cell death . The lacAlthough the exact mechanisms underlying how neuronal calcium disturbances lead to cell death have not been fully elucidated, the major pathways responsible for calcium overload during and following ischemia are better characterised. Ultimately, calcium dysregulation and overload occur when there is a disequilibrium concerning homeostatic pathways controlling calcium influx, efflux, and release from intracellular organelles. IEIC)–calcium-permeable channels [The major pathways involved in ischemia-associated neuronal calcium influx are the glutamate receptor channels , voltage-dependent calcium channels (VDCCs), and the sodium calcium exchanger (NCX) , 3. Morechannels  have alschannels , 8.With respect to calcium efflux, there are only two known mechanisms: via the calcium ATP-ase pump ) and through sodium calcium exchanger (NCX) exit mode activity  Figure . Taken tα-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors and kainic acid (KA) receptors; and (2) the metabotropic glutamate receptors (mGluR), which are activated selectively by quisqualate (an agonist of mGluR). Metabotropic receptors, unlike ionotropic receptors, do not form an ion channel but rather interact with other receptors on the cell membrane and are linked to G-protein activation of phospholipase C, which converts phosphotidylinositol bisphosphate in the cell membrane to inositol trisphosphate and diacylglycerol. Inositol trisphosphate acts to release calcium from the ER, whilst diacylglycerol activates protein kinase C (PKC) which mediates many effects. The release of calcium ions is required for activation of calcium-dependent PKC isoforms; likewise, PKC can phosphorylate proteins that can then alter calcium signalling.Glutamate receptors are located on the cytoplasmic membrane of neurons and are activated following the binding of the neurotransmitter glutamate. Their main function following glutamate binding is to cause postsynaptic excitatory transmission; however, following ischemia their over stimulation can be damaging to neurons. Glutamate receptors can be divided into two broad groups based on selective affinity for different agonists: (1) Ionotropic glutamate receptors which include N-methyl-D-aspartic acid (NMDA) receptors, With respect to ionotropic glutamate receptors during ischemia, excessive activation of NMDA and AMPA glutamate receptors is a major source of calcium influx –14. LikeThe NMDA receptor is a nonspecific cation channel with a high affinity for calcium ions. It is comprised of a four peptide subunit structure with several different subunits being identified  . ActivatRecent evidence has shown that activation of synaptic NMDA receptors is associated with a prosurvival response in neurons. This response, which has mainly been characterised using cultured neurons, is trigged by a mild nondamaging level of NMDA receptor activation. The prosurvival response is associated with the upregulation of prosurvival proteins  and downregulation of prodeath proteins  .In contrast to synaptic NMDA activation, overstimulation of extra-synaptic receptors triggers a neuronal damaging signalling response. For example, stimulation of extra-synaptic NMDA receptors can mediate upregulation of the CLCA1 and activation of p38 (mitogen-activated protein kinase p38) both of which contribute to neuronal death –20.In addition to NMDA receptor subcellular location, receptor subunit composition is also important in determining neuronal fate following cerebral ischemia . It has AMPA receptors are nonspecific cation channels that consist of four subunits (GluR1-4), with receptor permeability to calcium dependent on the configuration of the subunits , 30. AMPγ-aminobutyric acid (GABA). For example, it is considered that the post-synaptic KA receptor-mediated release of GABA activates GABA receptors, causing inhibition of ischemia-induced NMDA overactivation [KA receptors consist of a four subunit structure containing a combination of one or more of five different subunits . Receptor subunit composition determines receptor permeability and function. KA receptors are normally permeable to sodium and potassium ions and generally not permeable to calcium . Their rtivation –36.Metabotropic glutamate receptors can be divided into three different families with subtypes for each group consisting of: Group I , Group II , and Group III . Due to metabotropic receptor-mediated release of calcium from the ER these receptors can contribute to increased intracellular calcium following ischemia. However, it has also been demonstrated that metabotropic glutamate receptor agonists can be protective following ischemia –41.α1 transmembrane subunits which form a calcium-permeable pore along with α2δ, β1-4, and γ auxiliary subunits which function in modulating the channel complex [Voltage-dependent calcium channels (VDCCs) are a type of transmembrane ion channel found in excitable cells and are composed of four homologous  complex . There eL-type VDCCs (otherwise known as long-lasting or DHP receptors) are commonly found on dendritic neurons and, when activated, trigger calcium influx and the expression of genes leading to cell survival . Howeverώ-conotoxin is a specific blocker of these channels and is regularly used to study their function and mechanisms. Early studies [N-type VDCCs  play a primary role in neurotransmitter release from the presynaptic terminal via the influx of calcium after depolarisation. The toxin  studies –49 reveaώ-agatoxin has been shown to reduce brain infarcts following focal cerebral ischemia [P/Q-type VDCCs (or Purkinje) are found mainly in the cerebellum and are involved in presynaptic neurotransmitter release. Blockade of these channels with the ischemia .T-type VDCCs (or transient) are associated with low-voltage activity in the brain and activate when the neurons are at rest (~−60 mV) allowing small amounts of calcium influx, which have been shown to benefit signal amplification. Specific inhibitors of these channels have been shown to dramatically reduce neuronal damage in hippocampal slice cultures following oxygen-glucose deprivation .Transient receptor potential (TRP) channels are a family of cation channels which are nonselective for ions such as magnesium, sodium, and calcium. TRP channels consist of six transmembrane segments with pore formation between segments 5 and 6 . In mammOf particular interest for this paper are the TRPM and TRPC family, members. Within the TRPM family there exists eight subtypes (TRPM1-8). Of these subtypes the TRPM7 and to a lesser extent TRPM2 members are the most important with respect to ischemia-induced neuronal calcium influx , as thesTRPC channels can be divided into four main subgroups , and they are thought to play an important role in regulating the refilling of the intracellular calcium stores after phospholipase C-induced calcium release. After calcium release, a proportion of the released calcium is pumped out of the neuron due to the activity of the plasmalemmal calcium extrusion systems, making it unavailable for reuptake by the calcium store. The TRPC channels are activated by calcium store depletion, and the ensuing calcium entry provides the calcium necessary for complete refilling of the calcium store , 58. It Acid-sensing Ion Channels (ASIC) are nonselective ion channels, which are activated in response to decreased extracellular pH. These trimeric channels consist of one or more of six different subunits , which vary in their response to pH levels. In the event of cerebral ischemia, the resulting extracellular pH decrease triggers ASIC channel opening, allowing calcium to enter neurons . It has The sodium-calcium exchanger (NCX) is a bidirectional ion transporter, with a low affinity but high transporting capacity for calcium. Its structure consists of nine transmembrane segments which are involved in binding and transportation of sodium and calcium ions and a large intracellular hydrophilic loop which functions to regulate NCX activity . There eOne working model is that under severe ischemic conditions, the NCX operates in calcium entry mode and facilitates calcium-induced acute neuronal cell death. Under these conditions-blocking NCX activity is neuroprotective –70. In cEIC), which the authors believe is also responsible for glutamate-induced extended neuronal depolarisation (END) and calcium-mediated excitotoxicity. Based on in vitro experimental studies, the IEIC is activated after an excitotoxic insult, and once activated results in sustained neuronal calcium entry. Further investigations showed that blocking of the IEIC by gadolinium following excitotoxicity attenuated sustained calcium influx and prevented neuronal death. Additional studies, including in vivo experiments, are needed to clarify the characteristics, structure, and exact function of this channel in neurons following cerebral ischemia.A recent study  has descMITO), located in the inner mitochondrial membrane [MITO suggesting that calcium is recycled across the mitochondrial membrane of neurons via the NCXMITO in response to overstimulation of the NMDA receptors.In excitable cells such as neurons, mitochondria play a role in regulating intracellular calcium levels , 76. In membrane , 78. TheHowever, mitochondria have a limit to the amount of calcium that can be sequestered and this limitation is also influenced by the metabolic status of the cell. Thus, while mitochondrial calcium sequestration is a protective response, once this system becomes overwhelmed, it has severe consequences for the cell, leading to the activation of proapoptotic proteins and eventual cell death. Therefore following cerebral ischemia, the inability of mitochondria to adequately buffer neuronal intracellular calcium can result in calcium dysregulation , 79. In 2+-ATPase pump located on the ER membrane, but during and following ischemia, its function in neurons is compromised due to declining ATP levels. Furthermore, cerebral ischemia activates phospholipase C causing hydrolysis of PIP2  bisphosphate) to release the signalling molecule IP3  trisphosphate). Receptors for IP3 trisphosphate receptors IP3Rs) are located on the ER membrane and function as ligand-gated channels [3Rs by IP3 results in a rapid release of calcium from the ER. Similarly, ryanodine receptor (RyRs) calcium release channels located on ER membranes are also likely to be activated following cerebral ischemia resulting in additional release of calcium via these channels [The ER serves as a storage facility for calcium in neurons and other cells. The ER plays a fundamental homeostatic role during and following cerebral ischemia, by sequestering excess cytosolic calcium, which is thought to prevent ER stress and thus provide a protective mechanism against cell death . Howeverchannels , 82. ActStore-operated calcium entry  refers to an influx of extracellular calcium across the plasma membrane via store-operated calcium channels  in response to ER intracellular calcium release and store depletion . RecentlThe calcium ATPase pump  serves to regulate intracellular calcium levels by actively expelling calcium out of the cell. It has a high affinity but low transporting capacity for calcium. Its structure consists of ten transmembrane domains which forms the calcium-permeable pore and three intracellular loops which regulate its activity . It is dUnder normal physiological conditions NCX acts as a calcium extrusion transporter by operating in the forward or calcium exit mode . As mentExcessive intracellular calcium influx is a major instigator of neuronal cell death following cerebral ischemia. This influx is mediated by a number of important channels and transporters. In addition, the overload of calcium in intracellular stores and the subsequent release of calcium from these stores can further exacerbate the problem. In contrast, there are only two known mechanisms to allow calcium exit (PMCA and NCX), both of which are susceptible to biochemical and/or proteolytic inactivation caused by intracellular disturbances associated with ischemia. This imbalance proves to be a major downfall in a neuron's ability to maintain calcium homeostasis following ischemia, which ultimately contributes to its demise. While researchers have made significant progress in understanding these important calcium influx and efflux pathways, especially under pathological conditions such as cerebral ischemia, it has not translated into any clinical therapeutic neuroprotective agents. However, it is anticipated that further investigation of calcium influx and efflux pathways will eventually enable the design of drugs to manipulate neuronal intracellular calcium levels following ischemia and lead to new neuroprotective therapies.


Ovarian cancer is the fifth largest cancer killer in women. Improved understanding of the molecular pathways implicated in the pathogenesis of ovarian cancer has led to the investigation of novel targeted therapies. Ovarian cancer is characterized by an imbalance between pro- and antiangiogenic factors in favor of angiogenesis activation. Various antivascular strategies are currently under investigation in ovarian cancer. They can schematically be divided into antiangiogenic and vascular-disrupting therapies. This paper provides a comprehensive review of these new treatments targeting the tumor vasculature in this disease. Promising activities have been detected in phase II trials, and results of phase III clinical trials are awaited eagerly. Ovarian cancer is the fifth largest cancer killer in women. Primary surgical cytoreduction followed by platinum-based chemotherapy is the standard treatment for patients with advanced epithelial ovarian cancer. However, despite this aggressive approach, all stages combined, the 5-year survival rate remains only around 45% . Novel aThere is a strong rationale to use antivascular therapies in epithelial ovarian cancer. Ovarian cancer is characterized by an imbalance between pro- and antiangiogenic factors in favor of angiogenesis activation, with an increase in the tumor levels of proangiogenic factors , fibroblast growth factor (FGF), platelet-derived growth factors (PDGFs), tumor necrosis factor (TNF)-alpha, angiopoietins, interleukin  and a decrease in anti-angiogenic factors  . AngiogeVarious antivascular strategies have been investigated in ovarian cancer. They can schematically be divided into antiangiogenic therapies and vascular-disrupting therapies. Given the important role of vascular biology in ovarian cancer, it is not surprising that these new treatment approaches have shown promising activity in this disease, even when administered as a single agent.The most studied antiangiogenic strategies target the VEGF/VEGF receptor (VEGFR) pathway through inhibition of its ligands and/or receptors. The VEGF family includes 6 glycoproteins  and 3 tyrosine kinase receptors (VEGFR1 to 3). VEGF-A promotes angiogenesis through enhancement of permeability, activation, survival, migration, invasion, and proliferation of endothelial cells . VEGFR1 bevacizumab (BEV). BEV is a recombinant humanized monoclonal antibody that binds and neutralizes all biologically active isoforms of VEGF. Published studies are presented in this section, while ongoing trials are summarized in (1) The most widely investigated anti-VEGF ligand agent is In 2005, Monk et al. reported an objective response lasting more than 5 months in a patient treated with BEV monotherapy after failing eleven lines of chemotherapy and radiation therapies . Later, In the phase II GOG 170-D trial, Burger et al. reported a partial response (PR) rate of 18% (11 out of 62) and a complete response (CR) rate of 3% (2 out of 62) in patients with persistent or recurrent epithelial ovarian cancer or primary peritoneal cancer having received 1 or 2 prior cytotoxic regimens and treated with BEV monotherapy. Median progression-free survival (PFS) was 4.7 months .BEV maintenance therapy after complete response to cisplatin-based chemotherapy is an interesting concept and showed promising results in xenograft models of ovarian cancer by prolonging survival . This apThe vessels formed during tumor angiogenesis are structurally and functionally abnormal. This leads to an impaired tumor blood supply that may interfere with the delivery of therapeutics. Hypoxia also renders tumor cells more resistant to both radiation and cytotoxic drugs . It has In a retrospective analysis of 23 patients with recurrent platinum-refractory epithelial ovarian cancer progressing after 2 to 15 prior cytotoxic regimens, Wright et al. observed PR in 35% with a combination of BEV associated with various chemotherapy regimens . Median PFS was 5.6 months in the patients who achieved a PR . RichardMetronomic administration of chemotherapy, defined as the frequent administration of doses substantially lower than the maximum tolerated dose, can suppress tumor growth, probably through stimulation of the release of thrombospondin 1, a potent and endothelial-specific inhibitor of angiogenesis . Shortentested; these studies are briefly described in Various combinations of BEV and chemotherapy are currently being Combination therapy in this context can be divided into horizontal and vertical molecular pathway blockade. The horizontal approach involves the association of targeted agents to inhibit two or more different pathways simultaneously, while the vertical approach involves the inhibition of various molecular steps of the same pathway, thus counteracting negative feedback loops. By inhibiting the activation of alternate molecular pathways, these combinations could theoretically decrease treatment resistance .Epithelial growth factor receptor (EGFR) is overexpressed in up to 70% of advanced epithelial ovarian cancers  and an iSorafenib inhibits, among others, the VEGFR2 and Raf kinases. In a phase I dose-escalation study with a combination of BEV and sorafenib, 6 of 13 (46%) patients with ovarian cancer had a PR . A phaseOther combinations of BEV with targeted therapies have been tested in preclinical models. Since the mammalian target of rapamycin (mTOR) pathway regulates VEGF expression in cancer cells , researccomplications following treatment with BEV in colorectal cancer, where this drug is widely used, include hypertension , proteinuria , bleeding , wound-healing complications , arterial thrombo-embolic events , and gastrointestinal (GI) perforations  FDG PET imaging could predict tumor response as early as 2 hours after therapy [Tracers focusing on VEGF and VEGFR2 have been developed to visualize angiogenesis-related events with noninvasive  imaging , 155. In therapy .Antiangiogenic therapy in ovarian cancer is very promising so far, at least in phase II trials. This is probably due to the highly angiogenesis-dependent pathophysiology of this disease. We should however keep in mind that angiogenesis might not be the driving force behind all cases of epithelial ovarian cancer and that we are still missing large placebo-controlled phase III trials that show a benefit in term of PFS or overall survival. Tools to detect the patients that are likely to benefit from antiangiogenic treatment have yet to be validated in the clinic. This would allow us to restrict the use of these very potent but also onerous new drugs to those who are most likely to benefit. A better selection of patients would also help to reduce the high complication rate seen with these agents, in particular GI perforations. The optimal duration of maintenance treatment with BEV will also have to be evaluated, and pharmaco-economic considerations will have to be addressed. Finally, combined targeting of tumor cells, endothelial cells, and pericytes  is a very interesting approach that warrants further studies.In conclusion, antivascular treatment for epithelial ovarian cancer is a very promising approach that still needs to be validated in the phase III setting. As many patients as possible should be encouraged to take part in well-designed clinical trials.


Human immunodeficiency virus disease involves progressive destruction of host immunity leading to opportunistic infections and increased rates for malignancies. Both depletion in immune cell numbers as well as defects in their effector functions are responsible for this immunodeficiency The broad impact of HIV reflects a similarly broad pattern of cell depletion including subsets that do not express viral receptors or support viral replication. Indirect cell killing, the destruction of uninfected cells, is due partly to activation of the Fas/FasL system for cell death. This death-signaling pathway is induced during HIV disease and contributes significantly to viral pathogenesis and disease. Changes in CD4 cell count and viral RNA burden are the most commonly used markers for HIV disease progression. However, evidence has existed for several years that many patients with HIV disease experience a broad loss of leukocyte subsets without an apparent preference for depleting CD4 T cells -3. EffecBesides depletion of immune cells due to cell death, HIV infection is also characterized by defects in effector cell functions. T cell exhaustion or low HIV-specific T cell cytotoxicity has been attributed to loss of zeta chains  or to hiin vivo. Cell loss is accelerated during elevated viremia and a broad recovery of leukocytes attends highly active antiretroviral therapy (HAART) [Despite overwhelming evidence for indirect cell depletion, little is known about how it occurs  (HAART) . Some of (HAART)  as viral (HAART) . While H (HAART) ,16 argueA process described as "chronic immune activation" or "hyperactivation" occurs during HIV disease and is accompanied by higher expression of TNF superfamily ligands and their receptors, e. g. Fas/FasL and TRAIL-DR5 -20. ChroEarly investigators  proposedApoptosis is observed consistently among uninfected cells in SIV+ macaques or HIV+ humans . Chronicin vitro [Many studies have explored the mechanisms connecting HIV/SIV antigen expression and apoptotic cell death. Human macrophages express FasL after exposure to HIV , creatinin vitro . Cross-lin vitro ,42.The patterns of CD4 T cell depletion appear to be antigen-specific, as perturbation of the CD4 receptor repertoire is significantly associated with higher plasma viremia . This caNeuronal cells that are depleted during chronic HIV infection comprise an important example of bystander depletion since neuronal cells are not infected by HIV. Neuronal cell loss has been attributed to interaction with viral proteins such as gp120, vpr, nef, and tat, and to soluble neurotoxic factors released by infected macrophages . The priBroadly acting immune stimulation during HIV or SIV infection especially implicates the Fas death pathway since activated T or B cells express Fas. Acute SIV infection triggers rapid increases in Fas and FasL expression in peripheral blood  as well Another recent publication from Mattapallil's laboratory notes that early mucosal HIV/SIV infection (2-4 days after inoculation) there are high level of infected CD4+ TH-17 T cells that are depleted during the course of infection . TH-17 cSeveral important studies have implicated apoptosis in uninfected cells as a major mechanism for leukocyte depletion. Fas ligation was a probable cause of apoptosis in T cells from SIV infected macaques . HoweverA recent publication describes gene expression profiles of three stages of HIV infection: acute, chronic, and AIDS . The acuCuriously, infected cells are more resistant to apoptosis than uninfected cells ,59. The in vitro rises and falls with changes in viremia, making it difficult to separate direct from indirect killing mechanisms in terms of their contribution to disease progression. In murine systems, these problems are addressed readily by the use of knock-out mutations eliminating Fas, FasL or both molecules. For AIDS-related questions, the initial studies are done most appropriately in nonhuman primates using SIV or SHIV to establish persistent infection and then applying interventions to modulate the Fas/FasL pathway.Substantial data have been accumulated on HIV induction of Fas or FasL, the susceptibility of PBMC from HIV patients to apoptotic cell death and the reversal of these conditions by HAART. When viremia was suppressed by effective HAART, CD4 cells in PBMC had significantly reduced apoptosis that correlated with increasing CD4 counts in blood even though lymphoid tissue FasL levels were unchanged ,62. SimiUsing a recombinant humanized anti-FasL monoclonal antibody  we develin vitro apoptosis among PBMC and remain disease-free [Another indication of the importance for indirect cell killing comes from studies of "naturally-infected" macaques i.e., sooty mangabeys infected with SIVsmm that maintain plasma viremia, do not show high susceptibility to ase-free . Here, aase-free  and indiWe reported  that halin vitro and tissue studies on HIV agree with these findings. Disagreements remain about the mechanisms for cell death, the roles for viral proteins and the relative importance of Fas versus non-Fas death pathways. In addition to direct infection and cell destruction by viral cytopathic effects or antigen-specific cytotoxicity, indirect cell killing mechanisms have broad impact on host immune capacity and are important in the pathogenesis of HIV/AIDS.Reports showing lower levels of indirect cell killing/apoptosis among naturally-infected macaques despite similar viremia ,67,70, tKnowing the role for uninfected bystander cell killing in disease, it remains a challenge to apply this information to treating HIV in man. An anti-Fas treatment during acute infection would be difficult to deliver. Since it may not modulate vRNA, the main marker for HIV therapy, lengthy studies would be needed to document treatment effects. The use of anti-retroviral therapy during this time would obscure the impact of blocking FasL. A combination of anti-FasL plus active immunization during interrupted HAART is conceivable, but unlikely to be pursued given the treatment choices and durable virus suppression achievable with available drugs. The most likely application of this knowledge may come in the evaluation of prophylactic or therapeutic vaccines. If we define viral proteins that are responsible for inducing bystander cell killing or identify particular motifs within these proteins that trigger killing mechanisms, vaccine-elicited antibodies may block these effects and preserve immunity even if vRNA levels appear unchanged. For example, gp41 peptide was shown to induce NKp44L on CD4+ T cells during HIV infection, making them highly susceptible to NK lysis. Immunizing against this peptide reduced ligand expression on CD4 lymphocytes and decreased apoptosis rates in SHIV-infected macaques  thereby HIV infection depletes a broad profile of leukocyte subsets including many that do not express CD4 and are not susceptible to direct virus infection. Models for the loss of non-CD4+ subsets including activation-induced cell death, explain the antigen-specific pattern of cell loss and frequently invoke Fas/FasL interactions as a principal mediator of cell death. The role for FasL was tested in the SIV/macaque model for AIDS using recombinant humanized monoclonal antibodies to neutralize this cell death ligand. Treated macaques modulated SIV disease and increased virus-specific immunity without consistent reduction in vRNA burden. The status of animals treated with anti-FasL was similar to natural SIVsmm infection of sooty mangabeys that have no overt disease despite high chronic viremia and show minimal apoptosis and immune hyperactivation.The Fas/FasL death pathway is an important component of SIV or HIV disease. Whether this pathway for cell death is driven by viral proteins, virus particles or induced host factors remains unknown, although compelling examples of each exist in literature. The capacity to control activation of this cell death pathway may be important for the ultimate success of preventive vaccine strategies. The magnitude and duration of protective immunity, once viral exposure has occurred, are key to controlling infection and disease. Cell death pathways like Fas/FasL may be exploited by HIV to reduce the level of protective immunity and to establish a persistent infection with progressing disease. There is a continuing need to understand these mechanisms and develop effective interventions to improve the impact of antiretroviral therapy.The authors declare that they have no competing interests.PB wrote the manuscript and approved its content. CDP wrote the manuscript and approved its content. MSS wrote the manuscript and approved its content.


Here we report a computational method to mechanistically identify cis-acting RNA elements that contribute to the tissue-specific alternative splicing pattern. This method is an extension of our previous model, SplicingModeler, which predicts the significant CAEs that contribute to the splicing differences between two tissues. In this study, we introduce tissue-specific functional levels estimation step, which allows evaluating regulatory functions of predicted CAEs that are involved in more than two tissues.Human genes undergo various patterns of pre-mRNA splicing across different tissues. Such variation is primarily regulated by ® Human Exon Array dataset, our method identifies 652 cis-acting RNA elements (CAEs) across 11 human tissues. About one third of predicted CAEs can be mapped to the known RBP (RNA binding protein) binding sites or match with other predicted exonic splicing regulator databases. Interestingly, the vast majority of predicted CAEs are in intronic regulatory regions. A noticeable exception is that many exonic elements are found to regulate the alternative splicing between cerebellum and testes. Most identified elements are found to contribute to the alternative splicing between two tissues, while some are important in multiple tissues. This suggests that genome-wide alternative splicing patterns are regulated by a combination of tissue-specific cis-acting elements and "general elements" whose functional activities are important but differ across multiple tissues.Using a publicly available Affymetrix Genechipcis-acting RNA elements by considering the exon splicing variation as the combinatorial effects of multiple cis-acting regulators. This methodology provides a novel evaluation on the functional levels of cis-acting RNA elements by estimating their tissue-specific functions on various tissues.In this study, we present a model-based computational approach to identify potential  According to studies using large-scale expressed sequence tags (EST), as high as 60% of human genes are estimated to undergo alternative splicing . Moreovecis-acting RNA elements (CAEs) contribute to tissue–specific patterns of alternative splicing. These types of analytical tools are important for elucidating the combinatorial code governing splice site selection. We previously reported a model-based computational approach called SplicingModeler. This was successfully implemented to predict cis-acting RNA elements between heart and liver tissues [SplicingModeler regards the splicing variation between two tissues as the combinatorial activities of multiple CAEs. Given the splicing index (SI) [SplicingModeler predicts the CAEs as well as their relative functional levels (RFL) by selecting the most significant motifs with the highest Exon Inclusion Contribution (EIC) scores. However, when being placed in a large set of tissues, we are not able to estimate CAEs' functions across tissues directly. In this study, we attempt to determine the cis-acting elements that contribute to tissue-specific alternative splicing and their functions across multiple tissues. Our method consists of the following steps  A two-step procedure is then performed to estimate the tissue-specific functional levels (TSFL) of predicted elements across tissues. The TSFL is a direct measure of the contribution of each CAE to inclusion or exclusion of an alternative cassette exon from an mRNA isoform in certain tissue.Despite an increased focus on the factors regulating tissue-specific alternative splicing, these mechanisms still remain largely unclear. The advent of genome-wide splicing-sensitive microarrays provides a new perspective to address issues of combinatorial control of alternative splicing. Evaluation of global splicing patterns using statistical approaches has the potential to reveal how combinations of  tissues . Splicindex (SI)  of each s Figure : 1) For ® Exon Array data from 11 human tissues [cis-acting RNA elements (CAEs) to be involved in tissue-specific alternative splicing. Of all the predicted CAEs, the majority are located within intronic regions close to 5'splice site (ss) or 3'ss. Nearly one-third of total predicted CAEs can be mapped to known RBPs' binding sites or exonic splicing prediction databases, and another two-thirds of CAEs may be potential splicing factors. Bipartite network demonstrates that the predicted elements can be classified into two different categories; one group of CAEs functions with RBPs only in a small set of tissues, while another group plays general but differential activities across a large number of tissues.We applied this method using Affymetrix GeneChip tissues . Without® Exon 1.0 ST Array is designed to monitor alternative splicing using more than 1.4 million Probe Selection Regions (PSR) within 1 million exon clusters, which are constructed from various exon annotations [The Affymetrix GeneChipotations . This stotations . We compCerebellum, spleen and testes have the largest diversities in exon expression when compared to other tissues , indicating the importance of the specific hexamer; and Relative Functional Levels (RFL), where a positive or negative value suggests its role in exon inclusion in one of two comparing tissues. The most significant hexamers, which receive EIC scores that are more than 5 × IQR (interquantile range) away from median EIC score, are selected as the potential cis-acting elements that contribute to the splicing variations between two tissues.In this study, we focus our analysis on one of the most important splicing variants, cassette exons , where eeveloped  to identFor each pair of tissues, significant hexamers can be separated into "relative splicing enhancers" (RFL > 0) and "relative splicing silencers" (RFL < 0) depending upon their estimated functional levels. It is important to note that enhancer and silencer are relative concepts here, which describe the relative function on exon inclusion in one tissue comparing to another tissue. Figure cis-acting elements. For each pair of tissues, we randomized the order of observed SI values, and conducted prediction using SplicingModeler. This approach not only effectively disconnects the functional relationships between exons and their regulatory sequences, but also preserves the regulatory element contents and the distribution of the levels of splicing variation. Wilcoxon test was then conducted on the predicted EIC scores (Exon Inclusion Score) with the alternative hypothesis that the predicted cis-acting elements from the original data have greater EIC scores than those from the randomized data. Permutation tests between all the tissue comparisons resulted in significant p-values lower than 2.2e-16, which suggested that SplicingModeler predictions are biologically meaningful.Permutation tests are applied to validate the statistical significance of predicted cis-acting elements, we compared the sequence similarities between the predicted elements with the known binding sites of RNA binding proteins, from documented binding sites for 14 proteins [In order to test the biological relevance of predicted proteins , and preproteins ,25. OverIn order to estimate the functions of predicted elements in each tissue (not the relative functions in each tissue pair), we define Tissue-Specific Functional Level (TSFL), which is derived from the Relative Functional Levels (RFL) that are calculated for each hexamer (Equation 4) in each tissue pair. For each hexamer, the TSFL for each tissue is achieved by solving a reduced linear equation with 55 equations  restricted by the relative relationships of 11 parameters, where each parameter models its function in the respective tissues (See Method section). The TSFL represents each hexamer's relative contribution to exon inclusion or exclusion in a specific mRNA isoform. For each element, the median value of their functions (TSFL) on exon inclusion among 11 tissues is set as baseline (0)., version 2.7.1) based upon Pearson's correlation between each two rows and columns  are enhancing the inclusion of exons in cerebellum, and 46.6% of them contribute more exon inclusion in cerebellum than any other tissue. These data is consistent with the splicing pattern observed in Figure We further conducted hierarchical clustering for the 11 tissues based upon the tissue-specific functional level identified for each significant element. In principle, the splicing regulatory factors binding on these elements control global splicing pattern. Clustering tissue types based upon the functions of these regulatory elements provides unique insight in the relationship of alternative splicing across multiple tissues. The tissues were clustered using gplots package  of R pros Figure . Two majcis-acting elements in regulating the splicing patterns across multiple tissues, a bipartite network is constructed where predicted elements and regulated tissues are considered two types of nodes  and testes (47/223) including PTB, hnRNP-B and hnRNP-C binding sites. In addition, a Nova binding site is found at 5'ss intronic region in cerebellum regulators. Overall, 23% of all predicted elements regulate multiple (>= 3) tissues, although most of the elements only connected to two tissues  in different tissues  with 3 replicates for each, were obtained from Affymetrix Exon 1.0ST Array Sample Dataset . AffymetFor each pair of tissues ; Sk, r is the total set of functional candidate elements in regulatory region r; Sk, i, r is the number of binding sites of candidate element i in regulatory r-th region on the k-th exon; RFLi, r stands for the relative functional level of motif i in regulatory region r; a positive and negative value implies its function on exon inclusion in one of the two comparing tissues.where, Sk, i, r can be obtained from the regulatory sequences, SIk stands for the splicing index of the k-th exon, which will be measured by the exon array experiment, and calculated based upon Equation 1. The Relative Functional Level (RFL) can then be estimated by fitting k equations with nr parameters using least-squares procedure. The significant cis-acting elements are selected in an iterative fashion. In brief, n = 20 candidate elements were randomly selected from all the candidates. The adjusted reciprocal of sum square error, calculated following least-squares procedure, is equally deposited to exon inclusion contribution (EIC) score of each motif candidate . The estimated RFL values are also accumulated into the final RFL scores. A detailed description on the SplicingModeler procedure can be found in [found in .cis-acting elements.In this study, to speed up the modeling, motif candidates whose binding sites occurrences are less than 5% of all alternatively spliced exons are filtered out. For each tissue pair comparison, we repeat the procedure for 10 million times. Motif candidates whose EIC scores are higher than median + 5 × IQR (interquantile range) are selected as significant Functional levels of predicted elements in each tissue, or Tissue-Specific Functional Level (TSFL), is derived from the Relative Functional Levels (RFL) that are calculated for each hexamer (Equation 4) in tissue pairs. The TSFL is calculated through the following two steps:m-th element is proportional to the instances that motif candidate m is selected in the SplicingModeler procedure. This number could be different since the hexamers whose binding sites occurrences are less than 5% of all alternatively spliced exons are pre-filtered out. The normalization procedure is conducted to eliminate this bias. If there are totally Mi, j candidate motifs were used in the SplicingModeler when comparing paired tissues i and j, the normalized relative functional level for motif candidate m can be calculated using following formula:For each tissue pair comparison, the derived RFL for the TSFLm, i represents the functional level of motif m for tissue i, the relative functional level (RFL) of the same motif between tissues i and j can be reconstructed as:If nRFLm, i, j score estimated from SplicingModeler, TSFL value was solved using least-squares procedure by randomly selecting one tissue as the standard and assigning 0 to its corresponding TSFL. For every predicted motif, the derived TSFL score is further normalized assuming standard normal distribution.For any motif candidate , such equation exists for every tissue comparison. Based on cis-acting RNA elements (CAEs) on regulated tissues, a bipartite network is constructed using Cytoscape [To illustrate regulatory relationships of predicted ytoscape . PredictThe authors declare that they have no competing interests.XW and YL contributed to the design of the study. XW and YL designed and performed the computational modelling and drafted the manuscript. KW, MR, JS, YW, WF and GW participated in coordination, discussions related to result interpretation and revision of the manuscript. All the authors read and approved the final manuscript.


This method utilizes a cDNA library normalization step to diminish the representation of highly expressed transcripts and biology-oriented bioinformatic analyses to facilitate detection of rare and novel transcripts.The cancer transcriptome is difficult to explore due to the heterogeneity of quantitative and qualitative changes in gene expression linked to the disease status. An increasing number of "unconventional" transcripts, such as novel isoforms, non-coding RNAs, somatic gene fusions and deletions have been associated with the tumoral state. Massively parallel sequencing techniques provide a framework for exploring the transcriptional complexity inherent to cancer with a limited laboratory and financial effort. We developed a deep sequencing and bioinformatics analysis protocol to investigate the molecular composition of a breast cancer poly(A)MALAT1, which is highly expressed in many human carcinomas.We analyzed over 132,000 Roche 454 high-confidence deep sequencing reads from a primary human lobular breast cancer tissue specimen, and detected a range of unusual transcriptional events that were subsequently validated by RT-PCR in additional eight primary human breast cancer samples. We identified and validated one deletion, two novel ncRNAs (one intergenic and one intragenic), ten previously unknown or rare transcript isoforms and a novel gene fusion specific to a single primary tissue sample. We also explored the non-protein-coding portion of the breast cancer transcriptome, identifying thousands of novel non-coding transcripts and more than three hundred reads corresponding to the non-coding RNA Our results demonstrate that combining 454 deep sequencing with a normalization step and careful bioinformatic analysis facilitates the discovery and quantification of rare transcripts or ncRNAs, and can be used as a qualitative tool to characterize transcriptome complexity, revealing many hitherto unknown transcripts, splice isoforms, gene fusion events and ncRNAs, even at a relatively low sequence sampling. The classic image of the mammalian transcriptome is composed of a large assembly of spliced mRNAs, each structured with a capped 5' end, a 5' untranslated region, a coding sequence, a 3' untranslated region and a polyA tail, together with a relatively well-defined set of non-protein-coding RNAs with different functions , with most of the genome thought to be genetically inert. Transcriptome sequencing and annotation initiatives have challenged this view by discovering that most of the genome is actively transcribed to yield complex patterns of interlaced and overlapping transcripts, including tens of thousands long (>200 nt) non-protein-coding RNAs (ncRNA) -3.Non-coding RNAs (ncRNAs) have emerged as a diverse and important class of functional transcripts, accounting for approximately the 1.5% of the transcriptional output of mammalian genomes ,5. The ri.e. drastic changes in expression levels of specific transcripts), and recent RNA-seq studies have identified a set of cancer-specific transcripts and transcriptional variants in tissues and cell lines [CD44, BRCA1, survivin etc), whose expression seem to correlate with the disease, have been described in literature [Compared to wild-type, the cancer cell transcriptome is grossly altered. Microarray studies have revealed a host of aberrations , having a purity of 85–90%. cDNA was synthesized using Super SMART™ PCR cDNA Synthesis Kit . Prior informed consent for the research use of biological material from surgery was obtained for this sample. The ethics committee of the Institute for Biomedical Technologies – National Research Council approved the use of this biological sample for the study presented here. After reverse transcription, the cDNA library was normalized to obtain an equilibrated mix of low and high abundance mRNAs using Kamchatka crab double-strand nuclease (DSN) [Polyadenylated RNA was isolated from a breast invasive tumor sample (se (DSN) , as desc2.1 μg of normalized double stranded cDNA was sheared by nitrogen nebulization following the manufacturer's instruction . Ligation of the nebulized sample to specific adaptors and preparation of the single strand libraries (sstDNA) was performed as previously described . After pThe sstDNA library was then amplified by emulsion PCR performed in water-in-oil microvescicles. Each PCR reaction was recovered by propanol emulsion breaking and buffer washing and enriched for positive reaction beads. The beads were then washed; the primers were annealed and then counted using the Multisizer™ 3 Coulter Counter . The kits for DNA fragmentation, polishing, capture on beads, emulsion PCR and sequencing were purchased from Roche Diagnostics. Samples were loaded onto 70x75 PicoTiterPlate (PTP) and inserted in the 454 – Roche GS 20 Genome Sequencer for the pyrosequencing reaction.. After mapping the remaining reads to the genome, we employed a second sequence redundancy reduction step for the analyses investigating the overlap between our reads and genomic features such as ENCODE regions, ncRNAs or genes. For this purpose, we used the CleanUp Algorithm [Sequence reads were extracted from the raw pyrosequencing data following the manufacturer's technical documentation. The technical redundancy in the dataset (perfect sequence duplication) was removed using the NCBI nrdb program, included in the downloadable Blast suite lgorithm  to generlgorithm  to perfoA detailed description of the bioinformatics methods used in this part of the work can be found in Additional file A collection of Conserved Sequence Tags (CSTs) ,35, obtaThe details of the bioinformatics strategy used for detection of gene fusions and deletions is described in detail in Additional file To analyze cancer-associated splicing events we used the ASAP II database , which cThe breast cancer cDNA library reads were aligned to UCSC Known Genes FastA sequences  using BLAST, and were classified on the basis of their genomic location. The conservation profile of non-exonic reads was assessed using the UCSC PhastCons17way conservation score. A total of four different datasets were generated: intronic, extragenic, desert conserved and desert non-conserved. These datasets were subsequently cross referenced against CRITICA ncRNA predictions , a subse®II-TOPO TA vector . Additional file five lists all the PCR primers and their annealing temperatures, together with the results of all validations experiments. Since we were investigating rare transcripts detected from a normalized cDNA library, we reasoned that RNA extracted directly from primary samples could be the best source of genetic material for validation.Validation was performed by direct sequencing of the cDNA library and RT-PCR. We used RNA obtained from the original lobular breast cancer sample and from other eight tumors and performed RT-PCR using an oligo (dT) primer and SuperScriptTM II Reverse Transcriptase  according to manufacturer's instructions. For fusion transcripts we sequenced individual PCR products after cloning them into the pCRGAPDH  is reduced and the expression of weakly expressed HPRT1 (Hypoxanthine phosphoribosyltransferase 1) is increased.Aiming to detect rarely expressed transcripts, we complemented the standard deep-sequencing protocol with a normalization step. Reference genes, which are often referred to as 'housekeeping genes', are frequently used to normalize mRNA levels between different samples . In ordeACTB (Beta-actin), GAPDH and HPRT and compared them with the abundance of these transcripts  in a public EST library  . The 'unspliced' dataset was split into intragenic or intergenic. Intragenic reads were then assigned to 4 different classes: exon, intron, extended 5' and extended 3'. The 'spliced' dataset was also classified by location within a gene. In order to detect potentially novel transcriptional features we excluded the entire unspliced-exon dataset from further analyses, as this will mostly contain well-known entities. We noticed that there are a significant number of matches in the intragenic non-exonic portion of genes, which we attribute to new exons, retained introns or intronic transcripts.Genome-wide identification of coding and non-coding Conserved Sequence Tags (CST) in human and mouse genomes ,35 proviDetailed bioinformatic analyses were performed on our breast cancer library to identify fusion transcripts, aberrant or novel splicing isoforms, as well as known cancer-related splice variants  on chromosome 1 and GLB1  on chromosome 3. UBR4, commonly known as p600 or retinoblastoma protein-associated factor 600, is a cellular target of the human papillomavirus type 16 E7 oncoprotein, contributing to anchorage-independent growth and cellular transformation. UBR4-E7 interaction strongly contributes to cellular transformation [GLB1 gene encodes beta-galactosidase-1 (EC 3.2.1.23), a lysosomal hydrolase that cleaves the terminal beta-galactose from ganglioside substrates and other glycoconjugates. The predicted fusion, verified by direct sequencing of the original cDNA library, links exon 16 of the gene UBR4 with the terminal exon (composed of coding and 3'UTR sequence) of the GLB1 gene. Our sequence is colinear with both transcripts and exon-exon junctions are clear in the hybrid sequence. The predicted final processed fusion cDNA UBR4/GLB1 would be 14,022-bp long and would produce a large protein of 4,526 residues, which is shorter than the original UBR4 protein . The UBR4/GLB1 fusion protein is identical to UBR4 up to residue 4,433, and then diverges significantly  domains. Two alternatively spliced WHSC1L1 variants have been described. The long isoform contains a PHD-finger domain (an interleaved type of Zn finger chelating 2 Zn ions) and a SET domain (protein-protein interaction domain); however, the function of the protein has not been determined yet and hence the relevance to cancer aetiology of this deletion is uncertain.An example of a transcriptional or genomic deletion event is provided by sequence read 1B (167378_1645_3303), located on chromosome 8. We interpret this sequence as a deletion, probably due to a loop that causes the inclusion of exons 2 and 7 of the a priori' processing of the RNA, apart from library normalization. This approach may be particularly useful to researchers investigating the transcriptome of species with poor genome annotation.The median length of the sequence reads in this sequencing run was around 85 nt, which is considerably longer than what can be achieved with other deep sequencing technologies, although much shorter than the length achievable with the latest generation of 454 sequencers (FLX and Titanium). This read length made it possible to identify interesting transcripts  without any sophisticated molecular 'BC031316 and is supported by three spliced ESTs but by no full-length mRNA. This isoform was confirmed by direct sequencing on the original cDNA library  gene  identified by deep sequencing contains a distinct 5'UTR and lacks a portion of the 5' coding region, compared to variant a. These differences cause translation initiation at a downstream AUG and a protein with a shorter N-terminus. The predicted association with cancer is strongly supported by the ASAPII LOD score for this transcript [HIGD1A are expressed in breast cancer and may be associated with the disease state.Specific cancer-associated transcript isoforms have been predicted based on differential representation of ESTs between tumor and healthy tissues. When we queried the ASAPII database of alternative splicing with our deep sequencing reads we identified five putative cancer-associated isoforms, one of which has a plausible role in breast cancer biology. Isoform b of e Figure  encodes anscript . Our datA subset of the above-described transcripts, including some potential cancer-associated variants, were selected for biological validation by RT-PCR on RNA derived from the same tissue sample that was used for deep sequencing, as well as from other eight lobular breast cancers. Amplification products from the original sample were assessed by Sanger sequencing. We obtained experimental confirmation of one deletion, one novel intergenic transcript, one novel intragenic transcript, nine previously unannotated isoforms – two associated with exon skipping and seven with novel splice site usage – and one known, but rare isoform. A gene fusion event which we detected by deep sequencing could be validated only in the original sample. These results, the primer sequences, the transcript labels and the PCR product sizes are described in Additional file SRA1 and MALAT1, have previously been associated with other tumor types and might also play a role in breast cancer pathogenesis. The steroid receptor RNA activator (SRA) is a unique modulator of steroid receptor transcriptional activity that functions as a regulatory RNA assembled in a ribonucleoprotein complex. Recent findings, however, have shown that the SRA1 locus can produce both protein-coding and non-coding transcripts which are involved in the regulation of estrogen and androgen receptor signaling pathways. Moreover, several reports have shown increased SRA expression in breast, uterus and ovarian cancers, and a possible direct involvement of SRA transcript in the mechanisms underlying breast tumorigenesis and tumor progression has been proposed [A number of approaches are currently available to discriminate protein-coding transcripts from ncRNAs . The mosproposed .MALAT1 (metastasis associated lung adenocarcinoma transcript 1). MALAT1 is a conserved 8-kb ncRNA whose expression correlates with the risk of developing metastasis in non-small-cell lung cancer (NSCLC) patients. Recent studies have also reported the overexpression of MALAT1 in uterine endometrial stromal sarcoma and hepatocellular carcinoma [MALAT1 which is cleaved in the nucleus and generates a cytoplasmic tRNA molecule [MALAT1 is abundantly expressed in our primary sample, in accordance with previous results [The most abundant ncRNA we detected was arcinoma ,26. We farcinoma , gave rimolecule . This obMALAT1 is abundantly expressed in all the publicly available annotated breast cancer samples retrieved from the CleanEx database [MALAT1 transcript abundance, including a few outliers with very high expression of this ncRNA. Further analysis of cDNA microarrays, probed with total polyA+ RNA from ER+ lobular and ductal breast cancers treated with Tamoxifen, showed the same expression patterns [MALAT1 expression may be episodically associated with single breast tumors and that the sensitivity of our deep sequencing approach facilitated the detection of this ncRNA in our sample. We also noticed that the Coefficient of Variation of gene expression values was higher in Tamoxifen-treated versus Tamoxifen-untreated breast cancer samples (84% versus 43% for the Affymetrix array experiments dataset) , which are thought to be selectively expressed in male and female gonads and are important for the control of transposable elements during germline development . HoweverSequences that mapped to 'gene deserts', which lie at least 2 kb from the boundaries of any known transcript, and which resulted highly conserved according to UCSC PhastCons17, were manually examined for overlap with ESTs, transcript predictions and non-coding RNAs annotations derived from CRITICA ,58. ElevThe ENCODE annotation of transcriptionally active regions  Transcr.AKAP9, which interacts with multiple signal transduction pathways, had the highest number of counts, followed closely by the ncRNAs MALAT1 and XIST. Among transcripts with very few counts we identified a number of annotated pseudogenes.Functional annotation of transcripts has become an important aspect of microarray studies, and many tools are now available to assess gene expression biases . Using tWe applied the DAVID on-line analytical tool  to identQuantitative transcriptional analysis of all the genes expressed by breast tumors has provided the first steps towards defining a molecular signature for the disease, and might ultimately make conventional diagnostic techniques obsolete. The qualitative analysis of the breast cancer transcriptome – such as the one obtained by massive cDNA sequencing and presented here – should instead contribute different and complementary information: the identification of novel possible pathogenic determinants (gene fusions and genome deletions) or biomarkers .We demonstrated in this work that 454 deep sequencing of a normalized cDNA library, coupled with detailed biology-oriented bioinformatic analyses, has the potential to identify transcripts that may further our understanding of the breast cancer transcriptome, even starting from a relatively small number of sequences. In our primary breast cancer cDNA library and in a number of additional samples with a matching histotype, we have identified and validated several unusual transcriptional events that could be suitable for subsequent functional studies: gene fusions, gene deletions, novel or cancer-associated isoforms and putative novel ncRNAs.MALAT1 ncRNA and we replicated this observation in two different gene expression profiling experiments of well-annotated ER+ breast cancer patient cohorts, finding also an high variance between Tamoxifen treated and untreated patient samples. Although further technical refinements, such as controlled hydrolysis of RNA samples before cDNA synthesis and paired-end or di-tags sequencing, can increase significantly the number and diversity of sequences which can be annotated, our protocol has proved to be very effective in detecting rare or novel transcriptional events. Based on the results presented here, we are confident that further deep sequencing experiments and a similar bioinformatic analysis strategy will yield an even more comprehensive and detailed picture of the breast cancer transcriptome.We have also identified from our sequences a very high expression of the cancer-associated AG planned and coordinated all the bioinformatic analyses, performed the statistical analysis and functional characterization part and wrote the manuscript. MI contributed the genome mapping and read classification. PP and IZ prepared the normalized cDNA library and performed the biological validation. MAA performed experimental validations on the ncRNAs part. NK performed the bioinformatic search for gene fusions, deletions and cancer-associated isoforms. LJC and RJT performed the analyses on novel ncRNAs and contributed to the manuscript. GS performed the analyses on known ncRNAs, and contributed to the manuscript. ER performed the deep sequencing. MC contributed the bioinformatic analysis of breast cancer cDNA array data. RJB contributed computational and bioinformatic support to this project. FM and GP contributed the CST bioinformatic analysis. GB contributed the biological sample and pathological characterization. LRB and AA conceived this research project and established the deep sequencing laboratory. CL contributed to the cancer isoforms detection analysis. JSM contributed to the manuscript and coordinated the ncRNA analysis part. GdB planned and coordinated the deep sequencing work and contributed to the manuscript.Supplementary methods. document detailing the methods for the assessment of library normalization; mapping to transcriptome and genome; identification of cancer-specific splice sites and fusion/deletion transcripts; analysis of non-protein coding transcripts.Click here for fileGenomic classification of cDNA reads. Excel document containing the identifiers and annotations of all the mapped cDNA reads classified as described in Table Click here for fileMALATIFASTA sequences of cDNA reads corresponding to fusions, deletions, new transcripts and . WinRAR zip archive containing three multifasta text files: (1) Fusion_Deletions: cDNA reads corresponding to fusions and deletions described in Additional file MALATI ncRNA.Click here for fileAnnotation of cDNA reads corresponding to fusions, deletions and a rare isoform. Word document containing sequence analysis details of the all fusions and deletions  predicted from the analysis of cDNA reads, plus an example of a  rare isoform.Click here for fileBiological validation of selected interesting transcripts. Word document containing the description of the RT-PCR validations for potential cancer-related transcripts identified in this study; the reanalysis of two Affymetrix and cDNA array breast cancer patients datasets for the investigation of the MALAT1 ncRNA expression pattern.Click here for fileAnalysis of cDNA reads corresponding to known and novel ncRNAs. Excel document containing the list, genomic coordinates and annotation of reads corresponding to known ncRNAs supported by ESTs; 'desert' and 'intronic' reads overlapping with CRITICA non-coding RNAs; conserved extragenic cDNA reads corresponding to novel or extended transcripts supported by ESTs.Click here for fileExtragenic cDNA reads overlapping with CSTs. Excel document containing the list and genomic coordinates of extragenic cDNA reads overlapping CSTs with or without coding potential.Click here for filecDNA reads overlapping with ENCODE transcripts. Excel document containing the identifiers and genomic coordinates of the cDNA reads overlapping the ENCODE transcripts subset supported by microarray evidence .Click here for fileAnnotation and count of cDNA reads corresponding to known genes. Excel document containing the HUGO gene identifiers corresponding to the mapped cDNA reads and the relative count.Click here for file


A 62-year-old male presented with symptoms of urinary obstruction and elevated serum prostate-specific antigen level of 3.61 ng/mL. Prostate needle biopsies showed benign prostatic tissue with a focus of crowded glands with minimal cytological atypia, fairly well-circumscribed with infiltrative appearance of glands at the edges. This focus had both small and larger glands with similar histological features. This focus was strongly positive for alpha-methylacyl-coenzyme A-racemase (AMACR), but showed scattered patchy staining with basal cell markers (p63 and CK903/34βE12). Hence, the final histologic diagnosis was benign prostatic tissue with a focus of florid adenosis. Two subsequent follow-up prostate needle biopsies performed six and 12 months later both showed benign prostatic tissue with atrophic changes. This case highlights the utility of these three immunostains  in the accurate diagnosis of adenosis of the prostate on needle biopsy, and avoiding its misinterpretation as prostate adenocarcinoma. Atypical adenomatous hyperplasia (AAH) or adenosis of the prostate is a pseudoneoplastic lesion that can mimic prostate adenocarcinoma because of its cytologic and architectural features -3. For mA 62-year-old male was referred to his urologist for symptoms of urinary obstruction and an elevated serum prostate-specific antigen (PSA) level of 3.61 ng/mL. Digital rectal examination and ultrasound revealed an ill-defined nodule in the prostate suggestive of malignancy. A needle biopsy was performed.The histologic section of one needle biopsy was characterized at low-power examination by the replacement of normal prostatic tissue by a proliferation of haphazardly arranged glands, partially arranged in an ill-defined nodule. The lesions had an infiltrative aspect at their edge, but the glands were not admixed with normal prostatic acini. At medium power, the proliferating glands were often slit-like, variably sized and shaped, alternating small and rounded acini with elongated and branching ones Figure  &1B. OccImmunohistochemical (IHC) stains revealed strong reactivity for alpha-methylacyl-coenzyme A-racemase (AMACR) in both small and large glands Figure  &2B, patThe final histologic diagnosis was benign prostatic tissue with a focus of florid AAH. Two subsequent follow-up prostate needle biopsies performed six and 12 months later both showed benign prostatic tissue with atrophic changes.AAH of the prostate is a microglandular lesion and a recognized mimicker of small acinar adenocarcinoma -3. AAH iMicroscopically, AAH is a localized proliferative lesion consisting of small amounts of atypical epithelial cells arranged in irregular glandular patterns, often within or adjacent to typical hyperplastic nodules . At low in situ hybridization analysis, chromosomal anomalies were seen in only 9% of AAH cases, compared with 55% of prostatic adenocarcinoma cases [While circumstantial evidence exists, there is lack of proof of a relationship between AAH and adenocarcinoma. It has been suggested that AAH is a precursor of some low-grade transition zone carcinomas but the lack of an increased prevalence of AAH in prostate glands with transition zone carcinoma argues against this hypothesis. Clearly, there is less evidence linking AAH to carcinoma than there is for high-grade PIN and cancer. Therefore, the major importance of AAH is its potential for being misdiagnosed as adenocarcinoma. Biochemical and molecular analyses of AAH have generated inconclusive results. There is limited data that AAH has a proliferation rate higher than hyperplasia but lower than adenocarcinoma ,7,8,13. ma cases . Recent ma cases ,13,14.The widespread use of PSA screening has led to an increase in prostate needle biopsies and, subsequently, an increase in earlier detection of prostate carcinoma. This trend has also led to an increase in the number of equivocal diagnoses on prostate biopsy specimens. Surgical pathologists must make critical decisions on an increasing number of prostate needle biopsy specimens with only small foci of atypical glands. In this setting, the mimics of prostate cancer must be distinguished from a small focus of adenocarcinoma. The distinction of benign small acinar proliferations (benign mimickers of cancer) from atypical acinar proliferations suspicious for cancer is crucial, since the subsequent clinical approach is different. Biopsies harboring a small focus of atypical glands frequently represent an under-sampled cancer and a subsequent biopsy will show cancer in up to 50% of cases . In contAAH is a transition zone lesion of the prostate that can simulate small acinar carcinoma. By itself positive immunostaining for AMACR is not diagnostic for carcinoma because the latter is also positive in high-grade PIN and some benign mimickers of adenocarcinoma. The case herein presented highlights the utility of AMACR, p63 and CK903/34βE12 immunostaining in the accurate diagnosis of adenosis of the prostate, a benign mimicker of prostate adenocarcinoma.The authors declare that they have no competing interests.HBA participated in the histopathological evaluation, performed the literature review, acquired photomicrographs and drafted the manuscript. AVP conceived and designed the study, gave the final histopathological diagnosis and revised the manuscript for important intellectual content. Both authors read and approved the final manuscript.


The Choline-PET/CT was repeated after 10 months of continuous therapy and demonstrated a partial remission of the bone lesions and a regression of all involved lymph nodes but one. Taken together we found an astonishing and durable activity of the alkylating agent trofosfamide given in a metronomic fashion. We rate the side effects as low and state an excellent therapeutic ratio of this drug in our patient.The treatment of metastatic prostate cancer patients refractory to androgen withdrawal and docetaxel therapy is currently discouraging and new therapeutic approaches are vastly needed. Here, we report a long-term remission over one year in a 68-year-old patient with metastatic docetaxel-refractory prostate cancer employing low-dose trofosfamide. The patient suffered from distant failure with several bone lesions and lymph node metastases depicted by a (11) C-Choline positron emission tomography/computerized tomography (PET/CT). After initiation of trofosfamide 100 mg taken orally once a day we observed a steadily decreasing PSA value from initial 46.6 down to 2.1  In hormone-refractory prostate cancer (HRPC) the treatment of choice is chemotherapy with docetaxel plus prednisone since this combination has shown a survival benefit in a large phase III trial. However, this therapy is often associated with considerable toxicity. After docetaxel failure currently no standard of care exists. In such a palliative setting a treatment strategy is highly desirable, which provides antitumoral activity without causing a significant amount of toxicity. In the last years the concept of metronomic chemotherapy has emerged, which fulfills these requirements . Metrono2, qd21, six cycles). After the beginning of docetaxel application PSA was initially declining then increased again. Moreover, after six cycles of docetaxel the patient developed a polyneuropathy grade II. Directly after docetaxel treatment the PSA value rose continuously and antihormonal treatment was ongoing. Before and after treatment with docetaxel, alkaline phosphatase, haemoglobin, and lactate dehydrogenase were in normal ranges. Intravenous bisphosphonate treatment with zoledronic acid  was already started one year before the initiation of trofosfamide therapy. In this paper, we describe a long-term partial remission over one year of a 68-year-old man treated with oral trofosfamide. Diagnosis was established in January 2005  and prostatectomy, extended field radiation, and orchiectomy were performed. After complete androgen depletion with bicalutamide (casodex) the patient relapsed one year later. This patient showed multiple bone lesions and lymph node metastases and was refractory to standard dose docetaxel treatment  showed for the first time a survival benefit in men with metastatic HRPC. In particular, docetaxel-based chemotherapy demonstrated a median improvement in overall survival of 2.5 months as compared with mitoxantrone and prednisone (MP) in metastatic HRPC . If a paA promising new treatment strategy in prostate cancer is the targeting of tumor angiogenesis. Antiangiogenic strategies seem to be rationale in HRPC, because microvessel density is an independent prognostic factor for progression and survival in clinically localized prostate cancer. Moreover, tumor angiogenesis correlates with metastases and progression also in advanced HRPC , 7. SeveBeside targeted drugs, thalidomide and low-dose trofosfamide are also effective through an antiangiogenic manner; however, they employ a different mode of action . In a stTaken together, we found high activity of metronomically administered trofosfamide in a situation beyond standard treatment algorithms. We conclude, that low-dose trofosfamide could be a treatment option for patients with advanced prostate cancer, also in a docetaxel-resistant setting. Since our patient improved with a lowered dose of trofosfamide, we suggest that the optimal antiangiogenic dose and schedule of trofosfamide in this setting has yet to be determined.