It was 36 years ago when John Bennett first discovered chloroplast protein phosphorylation Bennett, . The mosWith more than 25 protein subunits and 350 kDa in size, the PS II core monomer is a massive structure  pool  in high light (Betterle et al., As discussed earlier, our model predicts that PS II core phosphorylation drives monomerization of the dimeric cores. Monomerization has also been suggested to result from the damage or rearrangement of the extrinsic PsbO subunit of PS II (Nixon et al., During the PS II repair cycle, the following scenario can be envisioned. PS II is phosphorylated, whether they are damaged or not. This will break up supercomplexes into monomeric cores. Monomeric cores in the grana, with their smaller particle size, will be able to diffuse faster toward the stromaly-exposed regions of the thylakoid membrane where the repair machinery has access to the damaged photosystems. A swift migration of the monomeric cores may also result from the smaller diffusion area of the grana as they shrink in diameter in high light (Herbstova et al., State transitions correct imbalance in excitation of individual photosystems by redistributing excitation energy to the rate-limiting photosystem (Bonaventura and Myers, This functional similarity of LHC II and PS II core phosphorylation is even deeper. The protein kinases, Stn7 and Stn8, which underpin state transitions and PS II repair, are paralogs, sharing 43% identity at the amino acid sequence level. They both require reduced PQ for activation (Allen et al., Sujith Puthiyaveetil and Helmut Kirchhoff wrote the paper.


Drosophila, has been identified as dominant determinant of liver growth. Defects in this pathway in mouse liver lead to sustained liver overgrowth and the eventual development of both major types of liver cancer, hepatocellular carcinoma and cholangiocarcinoma. In this review, we discuss the role of Hippo signalling in liver biology and the contribution of this pathway to liver cancer in humans.The mechanisms controlling mammalian organ size have long been a source of fascination for biologists. These controls are needed to both ensure the integrity of the body plan and to restrict inappropriate proliferation that could lead to cancer. Regulation of liver size is of particular interest inasmuch as this organ maintains the capacity for regeneration throughout life, and is able to regain precisely its original mass after partial surgical resection. Recent studies using genetically engineered mouse strains have shed new light on this problem; the Hippo signalling pathway, first elucidated as a regulator of organ size in  Liver cancer is the fifth most common cancer worldwide and the third leading cause of cancer death . The twoMst1, Mst2, Sav1 , and Yap genes are important for growth control and tumourigenesis in the liver. Notably, each of these genes is orthologous to a member of the Drosophila Hippo tumour suppressor pathway, a gene network that seems to monitor cell–cell contact and cell polarity, and thereby restrict organ overgrowth  and nuclear exit, whereas depletion of Lats1/2 in some cancer cell lines inhibits Yap phosphorylation. In vitro, Mst2 can phosphorylate purified Lats1/2, and the latter directly phosphorylates Yap (Ser127) .In addition to Mst1/2, Sav1 and Yap, the other core components of the ‘Hippo pathway’ are conserved in mammals  and each can rescue the Growth control in the liver has a number of unusual features compared with that in other organs. Adult liver cells are largely quiescent, dividing approximately once/year; nevertheless, differentiated adult hepatocytes, rather than multipotent stem cells, are the source for tissue replenishment of cell turnover in the undamaged liver . The livAlthough numerous models of HCC have been described, no master regulators of either hepatocyte or oval cell quiescence had been identified until recently. As regards oval cells, selective expansion of these cells is observed in mice that overexpress TWEAK (Drosophila Yorkie) desensitises liver cells to apoptosis. Yap overexpression also led to aberrant proliferation in the intestinal epithelium, pancreas and skin, although neither gross organ enlargement or tumours were reported in these organs  engineered to overexpress the c-Myc oncogene . Hence, −/−Mst2−/− double-knockout mice exhibit early embryonic lethality –Cre strain enabled study of liver specific homozygous Mst1/Mst2 inactivation  loss-of-function phenotypes. Consistent with this, acute inactivation of Mst1/Mst2 in the liver is associated with rapid loss of Yap(Ser127) phosphorylation, increased Yap nuclear localisation and polypeptide abundance  phosphorylation promotes Yap degradation  phosphorylation persists, the nuclear abundance of Yap is nevertheless also increased. The functional importance of Yap to the hyperproliferative behaviour of Sav1 null oval cells, although likely, remains to be demonstrated. It is notable that despite the evidence that Yap is activated in oval cells harbouring Hippo pathway defects, oval cell expansion in mice overexpressing Yap in the liver has not been described , an inhibitor of the EGFR (and other) kinases, or by heterozygous inactivation of Yap1  gene results in the massive expansion of a periportal, CK2 positive cell population, variously called oval cells . Thus, Yap is probably a significant oncogene is all subtypes of liver cancer.The contributions of the core Hippo pathway components to human malignancies has received limited attention thus far. Mutations in Mst1/2, Lats1/2 and Sav1 in human cancers are very infrequent in the COSMIC catalogue, however there is evidence of promoter silencing by hypermethylation in some cancer types (e.g., Lats1/2 in breast cancers; As regards the mechanisms underlying aberrant Yap activation, our preliminary immunoblot analysis of lysates from human HCC and adjacent non-neoplastic liver , is greaDrosophila Hippo pathway (reviewed by Mst1/2, Yap, and Sav1 are now clearly established as regulators of liver cancer pathogenesis, yet many questions remain. Of primary importance is the need to understand the mechanisms that underlie Mst1/2 activation in hepatocytes and oval cells, that is, the nature of the activating signals and the role of cell surface proteins such as the atypical cadherin Fat and sub-membrane FERM domain scaffold proteins, such as Merlin/NF2 and Expanded, elements known to be functionally associated with the In terms of human liver cancers, it will be important to identify clinically useful biomarkers for deregulation of Mst1/2 signalling in human HCC and CHC. At present, Yap IHC staining of liver biopsy is commonly used (


The recently emerging atypical bovine pestiviruses have been detected in commercial foetal bovine serum (FBS) of mainly South American origin so far. It is unclear how widely the viruses are presented in commercial FBS of different geographic origins. To further investigate the possible pestivirus contamination of commercially available FBS batches, 33 batches of FBS were obtained from ten suppliers and analysed in this study for the presence of both the recognised and the atypical bovine pestiviruses. All 33 batches of FBS were positive by real-time RT-PCR assays for at least one species of bovine pestiviruses. According to the certificate of analysis that the suppliers claimed for each batch of FBS, BVDV-1 was detected in all 11 countries and BVDV-2 was detected exclusively in the America Continent. The atypical pestiviruses were detected in 13 batches claimed to originate from five countries. Analysis of partial 5′UTR sequences showed a high similarity among these atypical bovine pestiviruses. This study has demonstrated, for the first time that commercial FBS batches of different geographic origins are contaminated not only with the recognised species BVDV-1 and BVDV-2, but also with the emerging atypical bovine pestiviruses. Pestivirus consists of four recognized species: Bovine viral diarrhoea virus 1 (BVDV-1), Bovine viral diarrhoea virus 2 (BVDV-2), Border disease virus (BDV) and Classical swine fever virus (CSFV); and a tentative species, Pestivirus of giraffe The genus All 33 batches of FBS were found containing at least one species of bovine pestiviruses. Twenty-nine batches were found positive for BVDV-1, 11 batches positive for BVDV-2, and 13 batches positive for the atypical bovine pestivirus . AccordiThe newly determined sequences have been deposited in GenBank with accession numbers JN967700–JN967748. By studying the genetic relationship of the detected BVDV-1 and BVDV-2 viruses, the neighbour-joining analysis of 49 partial 5′UTR sequences revealed a certain grouping, but no particular clustering pattern in general in relation to the country of origin , with thThere are many technical questions here, which need careful investigations and openness from the side of the producers. For example, a possible scenario is that mixing of raw serum materials of different origin might have occurred at certain companies, intentionally or unintentionally, during manufacturing steps. As companies may collect raw serum materials from different geographic locations and transport the frozen sera to a manufacturing site for further processing, including mixing, heat inactivation, gamma irradiation, sterile filtration, and/or centrifugation. It is a possibility that the facility is not cleaned enough after producing one batch, so a small amount of raw sera collected from one location (e.g. South America) was mixed with sera collected from another location , and even such a small amount of residue sera is tested positive by the highly sensitive real-time RT-PCR method. Another possible scenario is that the FBS batches may be mistakenly labelled with country of origin different from its real origin. This could happen but it is unlikely all six suppliers had the same conduct. A further alternative is that spreading of the atypical bovine pestiviruses in the above-mentioned countries could have occurred very recently such that viruses had not evolved into distinct groups. This could explain the high sequence similarity among these atypical pestiviruses. Such a scenario would suggest that it is likely that the atypical bovine pestiviruses have a much wider geographic distribution than previously described. Although the origin and the time of emergence of these novel viruses are unknown, a recent study estimated that the emerging of the atypical bovine pestiviruses (BVDV-3a) might occur very recently, around 50 years ago rns protein by a commercial Antigen ELISA test. Nevertheless, the possible role and importance of the bovine pestiviruses should be thoroughly investigated from several aspects, including veterinary and human medicine and the safety of biological products, such as cell lines and vaccines. In addition, this study also has important implications in the possible problems of pestivirus contamination in research and in diagnostic laboratories, and effect of the emerging pestiviruses on the BVD control programs that are currently implemented in several countries. The occurrence of a severe respiratory disease in Italy caused by the atypical bovine pestiviruses This study has demonstrated, for the first time that commercial FBS products of different geographic origins are contaminated not only with BVDV-1 and BVDV-2, but also with the emerging pestiviruses. Attempt to isolate bovine pestiviruses from other batches of FBS had been unsuccessful, even though these batches were positive for the presence of BVDV EA total of 33 batches of FBS were purchased from ten commercial suppliers . RNA was


The role of gene-gene and gene-environment interactions, as well as epigenetics, has the potential to further elucidate and improve understanding of the underlying mechanisms or pathways of spontaneous preterm birth. Refinement in study design and methodology is needed in future research for the development and validation of individual or group biochemical marker(s) for use independently or in conjunction with other potential risk factors such as genetic variants and environmental and behavioral factors in predicting spontaneous preterm birth across diverse populations.Preterm birth is a delivery that occurs at less than 37 completed weeks of gestation and it is associated with perinatal morbidity and mortality. Spontaneous preterm birth accounts for up to 75% of all preterm births. A number of maternal or fetal characteristics have been associated with preterm birth, but the use of individual or group biochemical markers have advanced some of the understanding on the mechanisms leading to spontaneous preterm birth. This paper provides a summary on the current literature on the use of biochemical markers in predicting spontaneous preterm birth in asymptomatic women. Evidence from the literature suggests fetal fibronectin, cervical interleukin-6, and  Preterm birth is defined as birth at less than 37 completed weeks of gestation and it accounts for approximately 75% of perinatal mortality and nearly half of the cases of long-term neurologic morbidity . The ratThe onset of preterm labor is thought to be brought on by multiple mechanisms or pathways that may have been initiated weeks to months before the actual presence of clinical symptoms , 8. VariThe identification of risk factors for predicting preterm birth is advantageous because it allows for the initiation of risk-specific treatment for at-risk women and these risk factors may provide insights into a better understanding of the mechanisms leading to preterm birth , 23. HowBiomarkers of sPTB are divided into mechanistic pathways and they include intrauterine infection and inflammation, extracellular matrix degradation, fetal stress, fetal anomalies, and estrogen metabolism pathways .Bacterial vaginosis (BV) is an alteration of the maternal vaginal flora in which normal lactobacilli are replaced by Gram-negative anaerobic bacteria  and diagα [TNF-α], and C-reactive protein [CRP]) have been evaluated as biomarkers in subsequent sPTB.Intrauterine infection is an important mechanism leading to preterm birth . BacteriInterleukin-6. IL-6, a proinflammatory cytokine, is a major mediator of host response to inflammation and infection. It can be analyzed from samples of maternal cervical fluids or serum and to date, IL-6 is one of the most well-studied biomarkers of sPTB [ of sPTB . In a ne of sPTB , cervica of sPTB , an incr of sPTB  further Evidence in the literature to date has not shown serum IL-6 to be a useful biomarker in predicting sPTB for asymptomatic women. Paternoster et al.  and KramC-Reactive Protein. C-reactive protein (CRP) is a maternal systemic inflammatory marker that has been evaluated in the literature as a potential marker for preterm birth. In a prospective nested case-control study, Hvilsom et al. [μg/mL (at 75th percentile) to 16.4 μg/mL (at 95th percentile), and elevated CRP concentrations in maternal serum (≥85th percentile) increased the risk of sPTB  compared to lower CRP concentrations (<85th percentile). In a subgroup analysis, women without a previous preterm birth but with CRP levels at the 90th percentile and the 95th percentile had an increased risk of sPTB  compared to women with CRP levels at <75th percentile. Catov et al. [μg/mL obtained <21 weeks of gestation) in asymptomatic women. Likewise, Riboni and colleagues [μg/mL) collected in mid-second trimester. Other studies, in contrast, showed weak [m et al.  examinedv et al.  reportedlleagues  found anwed weak  to no efwed weak  of elevaOther Inflammatory Cytokines, Tumor Necrosis Factor- α. In addition to the evaluation of IL-6 and CRP in predicting subsequent preterm birth, few earlier studies also evaluated the association between other common cytokines IL-1, IL-2, IL-8, and TNF-α but there was no evidence suggesting that elevated cytokine values were associated with an increased risk of sPTB [ of sPTB –31, 34.The presence of fetal fibronectin, thus far, is the most effective biochemical marker for predicting preterm birth , 35–39. The association between the presence of fetal fibronectin and sPTB in asymptomatic women was further examined in a meta-analysis by Honest et al. . The likThe fetal fibronectin test can be performed as a single test or in series. A sample is collected from the posterior vaginal fornix and cervix with a sterile swab and the sample is then placed in a buffer solution and shipped to the laboratory for assay. Ideally, this test should be used in clinical settings in conjunction with patient medical history and other clinical information to assess overall risk of sPTB.Corticotropin-releasing hormone (CRH) is expressed by the human placenta and the fetal membranes, and its highest level of production is seen during the third trimester . In a reIn contrast, analysis from a nested case-control study of 254 asymptomatic women showed that elevated maternal plasma CRH (>90th percentile) at 28 weeks of gestation was associated with sPTB at <35 weeks  but high levels of CRH at 24 or 28 weeks of gestation did not predict sPTB at <32 weeks of gestation or <37 weeks of gestation . A studyβ-human chorionic gonadotropin (hCG) and α-fetoprotein to be associated with an increased risk of sPTB in cervicovaginal fluids among asymptomatic pregnant women [α-fetoprotein were also associated with predicting early and late preterm births. Moawad et al. [α-fetoprotein at 24 and 28 weeks of gestation and subsequent sPTB. For α-fetoprotein measured at 24 weeks of gestation, elevated values (>90th percentile) were found to increase the risk of spontaneous preterm birth at <32 weeks  and for sPTB at <35 weeks . The levels of α-fetoprotein measured at 28 weeks of gestation were also associated with subsequent sPTB, but the effect estimates were weaker for subsequent sPTB.Several earlier studies have identified elevated levels of nt women , 31, 48.nt women . Elevated et al.  conducteEstriol is the major form of circulating estrogen during pregnancy  and measα-fetoprotein, and granulocyte colony-stimulating factor) had a collective sensitivity of 81% and specificity of 78% for the prediction of spontaneous preterm birth at <32 weeks of gestation and 60% sensitivity and 73% specificity for the prediction of spontaneous preterm birth at <35 weeks of gestation.In addition to examining individual biochemical markers for predicting preterm birth, there is a growing interest in developing assessment tools using multiple markers. Given the heterogeneity of pathways leading to spontaneous preterm birth, multiple markers increase sensitivity of prediction by combining risk predictors that address diverse causes of spontaneous preterm birth . In a neAlthough a number of individual or set of multiple biomarkers for sPTB have been explored and examined, high intra-individual variability makes accurate prediction and subsequent prevention of preterm birth challenging. Advances in molecular biology and improved sophistication in methodology and technology of genomics have allowed for many investigators to turn their focus on the functional contributions of genetic variants to better understand preterm birth overall, but more importantly, the racial disparity of preterm birth –58. AccoAlthough medical advances have improved the survival of preterm infants and the treatment of short- and long-term morbidities, little success has been attained in understanding and preventing preterm birth. Great efforts have been spent to characterize and define the utility of biologic fluids from various sources in predicting sPTB. Not only does identifying biochemical markers that are associated with sPTB allow researchers to gain a better understanding on the underlying mechanisms or pathways leading to preterm birth, but also, this valuable tool can guide in designing the most effective targeted intervention strategies aimed at women at risk for preterm birth.α-fetoprotein, in addition to fetal fibronectin, as potentially promising biochemical markers; however, little information on the sensitivity, specificity, and positive predictive value is known at this time to determine the clinical usefulness of these markers.Many studies have evaluated the association between individual or group biochemical marker(s) and sPTB among asymptomatic women; to date, results of a number of biochemical markers remain inconsistent. Understanding sPTB is challenging due to its multifactorial etiologies and pathophysiologic heterogeneity. Limitations from past studies on biomarkers of sPTB including the identification of appropriate study population (low risk versus high risk asymptomatic women), gestational age at the time of specimen collection, timing of collection relative to time of pregnancy outcome, and the definition of study outcome  may lead to discrepant findings. Furthermore, practicality and acceptability and maternal and fetal risks also are of great concern. In reviewing the literature, fetal fibronectin , thus far, is most effective in predicting sPTB in asymptomatic women. Along with its relatively high sensitivity, the required specimen collection procedure is minimally invasive and poses little to no maternal and fetal risks; the reliance of fetal fibronectin as a screening tool in clinical settings is justifiable in the management and for providing timely interventions for women with positive results. Cervical length, a biophysical marker measured by transvaginal ultrasound (not reviewed here), has been promoted by the medical community as a valuable screening tool for sPTB. While no evidence to date has suggested one marker outperforming another, fetal fibronectin and cervical length can be useful independently or jointly according to woman's a priori risk status for sPTB. Evidence in the current literature also has indicated cervical IL-6 and Additional research that improves understanding of the mechanisms of preterm birth is important. In addition to the development and validation of multiple biochemical markers to be used independently or in conjunction with other clinical and biophysical markers, demographic, and behavioral risk factors, future work in this area should include further refinement of study design and methodology in the evaluation of gene-gene and gene-environment interaction studies and the role of epigenetics in predicting sPTB across diverse populations.


The concept of the heart as an endocrine organ arises from the observation that the atrial cardiomyocytes in the mammalian heart display a phenotype that is partly that of endocrine cells. Investigations carried out between 1971 and 1983 characterised, by virtue of its natriuretic properties, a polypeptide referred to atrial natriuretic factor (ANF). Another polypeptide isolated from brain in 1988, brain natriuretic peptide (BNP), was subsequently characterised as a second hormone produced by the mammalian heart atria. These peptides were associated with the maintenance of extracellular fluid volume and blood pressure. Later work demonstrated a plethora of other properties for ANF and BNP, now designated cardiac natriuretic peptides (cNPs). In addition to the cNPs, other polypeptide hormones are expressed in the heart that likely act upon the myocardium in a paracrine or autocrine fashion. These include the C-type natriuretic peptide, adrenomedullin, proadrenomedullin N-terminal peptide and endothelin-1. Expression and secretion of ANF and BNP are increased in various cardiovascular pathologies and their levels in blood are used in the diagnosis and prognosis of cardiovascular disease. In addition, therapeutic uses for these peptides or related substances have been found. In all, the discovery of the endocrine heart provided a shift from the classical functional paradigm of the heart that regarded this organ solely as a blood pump to one that regards this organ as self-regulating its workload humorally and that also influences the function of several other organs that control cardiovascular function. The phenotype of ventricular cardiomyocytes reflects functions associated with properties inherent to their mechanical task and the conduction of electrical excitation arising from the sino-atrial node. The advent of the electron microscope demonstrated in mammalian atrial cardiomyocytes elements that were previously associated with polypeptide hormone-producing cells. These included abundant rough endoplasmic reticulum, highly developed Golgi complex and storage granules referred to as atrial-specific granules  .The function of these differentiations was not evident and so these findings remained functionally an enigma for 2 decades. In 1981, after developing a number of investigative techniques 99–126 and BNP77–108 are the biologically activepeptides derived from processing proANF and proBNP by cardiomyocytes before release into the circulation.The NPs share a 17 amino acid ring structure formed by an intra-molecular disulphide linkage , Table 1Plasma levels of ANF and BNP in normal subjects are 3.2–19.5 and 1.4–14.5 pmol/l respectively Although it is often mentioned that BNP is a ventricular hormone while ANF is viewed as an atrial hormone, this concept needs to be reconsidered. In certain pathophysiological conditions, ANF and BNP plasma levels increase significantly and while ANF remains higher than BNP, the latter increases only relatively more than ANF The biochemical evidence supports an overwhelming atrial source of cNPs. The ANF concentration in normal subjects in atrium and ventricle is 9600 and 37 pmol/g respectively, while the BNP concentration of normal subjects in atrium and ventricle is 250 and 18 pmol/g respectively Cardiac catheterisation data in humans with left ventricular hypertrophy showed that the abnormal circulating levels of cNPs are significantly derived from atrial sources Both ANF and BNP are synthesised in and secreted from the atria in a regulated manner. In ventricles, given the practical absence of granules in the bulk of the muscle, the minute amounts secreted are said to occur via the constitutive pathway ANF and BNP are continuously secreted from the atria under basal conditions. Mechanical stretch of atrial muscle increases the rate of peptide secretion rapidly followed by a sharp decline to baseline. This transient increased secretion of ANF and BNP is not due to the cNPs pool depletion but due to the decreased sensitivity to the stimulating signal for regulated release Many of the agonists of cNPs secretion, such as ET1, angiotensin II (AII) and PE, signal through the receptors coupled to Gq proteins, although other subclasses may also be used An involvement of glucagon-like peptide-1 (GLP1) has recently been implicated in the regulation of ANF secretion from the heart. GLP1 is one of the incretin hormones secreted from endocrine cells located in the epithelium of the small intestine. Glucose-stimulated incretin is carried through the circulation to the pancreatic β cells where it stimulates insulin secretion. Blood pressure reduction has been reported in type-2 diabetic patients treated with GLP1 analogues or dipeptidyl peptidase-4 enzyme inhibitors The biological effects of ANF and BNP are predominantly mediated through the NPR-A receptor, a guanylyl cyclase-coupled receptor, that is widely distributed throughout the body, including the kidneys, vascular smooth muscle, adrenals, brain and heart + reabsorption in renal inner medullary collecting ducts and in various other effects such as modulation of steroidogenesis in the adrenal glands and phototransduction Intracellular cGMP targets several enzymes and ion channels including cGMP-dependent protein kinases, cGMP-gated ion channels and cGMP-regulated cyclic nucleotide phosphodiesterases A third natriuretic peptide receptor, NPR-C, is considered to be a clearance receptor, although G-protein signalling has been associated with it The binding potency of the NPRs is ranked as follows: NPR-A=ANF≥BNP>CNP; NPR-B=CNP≥ANF>BNP; NPR-C=ANF≥CNP>BNP From the haemodynamic point of view the endocrine heart serves to regulate cardiac preload and afterload due to its influence in the regulation of extracellular fluid volume mediated by targets such as the kidney, the renin–angiotensin–aldosterone system and arterial and sympathetic tones. However, many other targets have been characterised , subacute (24 h to 1 week) or chronic (longer than a week).The increased secretion of cNPs following acute mechanical atrial stretch is based on a phenomenon referred to as stretch-secretion coupling Subacute haemodynamic load is observed during mineralocorticoid escape, which is characterised by a transient period of positive sodium balance resulting from chronic exposure to mineralocorticoid excess followed by a vigorous natriuresis leading to a new steady-state of sodium balance. The increase in intravenous volume and central venous pressure leads to increased ANF and BNP production in atria but not in ventricles, in which plasma ANF but not BNP is significantly elevated. In subacute haemodynamic load, the predominant circulating cNP source is a stored hormone plus newly synthesised hormone Both, atria and ventricles increase synthesis and secretion of ANF and BNP during chronic haemodynamic overload. This condition is seen in chronic arterial hypertension and chronic congestive heart failure and is accompanied by the ventricular re-expression of the cardiac foetal gene programme, which includes the re-expression of both ANF and BNP 3, glucocorticoids, thyroid hormones, several growth factors, thrombin, AII, prostaglandins and α1-adrenergic agents In addition to mechanical stimuli, circulating neuroendocrine agents can act on atrial cardiomyocytes to modulate cNP synthesis and secretion. These include ET1, hydroxyvitamin D99–126 and BNP77–108, the biologically inactive N-terminal fragments of the cNP prohormones, NT-proANF and NT-proBNP, also circulate in plasma. As these N-terminal fragments are more stable in the circulation and their half-lives are longer than those of the C-terminal prohormones cNPs blood levels are increased in various pathological conditions such as heart failure, myocardial infarction, hypertension, left ventricular hypertrophy and pulmonary hypertension An immunoassay that detects the midregional fragment of proANF (MR-proANP) has been developed Because the cNPs possess diuretic, natriuretic and hypotensive activity, ANF and BNP have been used in the treatment of various cardiovascular conditions. As ANP and BNP bind to the same receptor, NPR-A, it would be expected that the therapeutic effects of these peptides would be similar, but some differences between ANF and BNP are apparent in the clinical settings. The infusion of carperitide, a recombinant form of human ANF, improved congestive heart failure and acute myocardial infarction, and also had renal protective effects on contrast-induced nephropathy CNP gene expression is found mainly in endothelial cells and to some extent in cardiomyocytes and cardiac fibroblasts CNP plasma levels in the coronary sinus is significantly higher than that in aorta, suggesting that CNP is synthesised in the human heart CNP secretion from endothelial cells is stimulated by various cytokines, such as transforming growth factor, tumour necrosis factor and interleukin-1 CNP in endothelial cells The third member of NP family, CNP, is produced within the heart in a constitutive-like manner. Plasma CNP levels are 1–6 pmol/l, which is one-third of those for ANF NPR-B gene expression is observed in vascular smooth muscle, suggesting that CNP acts locally to inhibit vasoconstriction and vascular growth. CNP also has direct effects on cardiac function where it acts initially as a positive inotropic and lusitropic agent followed by a negative inotropic effect in the isolated heart CNP possesses potent vasodilating properties on isolated blood vessels and produces growth inhibition of vascular smooth muscle cells I.v. administration of CNP to healthy volunteers decreased blood pressure with an increase in diuretic and natriuretic activities in doses ten times higher than those used to compare it with ANF Adrenomedullin (AM) is a 52-amino acid peptide first isolated from human pheochromocytoma in 1993 A unique 20 residue sequence followed by Gly-Lys-Arg, a typical amidation signal, exists in the N-terminal region of preproadrenomedullin that was found in human pheochromocytoma AM is seen in adrenal medulla, which is more than ten times higher than in ventricle or atria AM gene expression is also seen in cardiomyocytes, cardiac fibroblasts, vascular endothelial cells and smooth muscle cells AM originates from the endothelial cells in various organs Plasma level of AM is 2–10 pmol/l, which is comparable with ANF plasma levels AM binds to three types of receptors structurally made of two components: one is a calcitonin receptor-like receptor and the other one is denominated receptor activity-modifying protein AM possesses a wide spectrum of biological actions such as vasodilation, natriuresis and diuresis Plasma AM is increased in various pathological condition such as essential hypertension, acute coronary syndrome, congestive heart failure and septic shock Although both the cNPs and AM possess hypotensive, diuretic and natriuretic properties, the precise regulation of circulating levels of these peptides might be different because cNPs are synthesised mainly in heart, whereas the principal production sites of AM are blood vessels and adrenal medulla.Plasma level of PAMP is 0.1–1.0 pmol/l, which is 10–20% of plasma AM concentration PAMP is a potent hypotensive peptide. The blood pressure lowering mechanism is different from that of AM Similarly to AM, plasma levels of PAMP are increased in patients with a variety of diseases such as essential hypertension, congestive heart failure, chronic renal failure and septic shock The 21-amino acid peptide ET was first isolated from the conditioned medium of cultured endothelial cells and then sequenced in 1988 ET1 produces a potent and long-lasting vasoconstriction resulting in an increase in blood pressure There are two types of G-protein-coupled receptors for ET: ETA and ETB ET1 production is stimulated by hypoxia, angiotensin II, cytokines, growth factors and reduced by nitric oxide and ANF The heart as an endocrine organ owes its designation to the secretory phenotype of mammalian atrial cardiomyocytes that is associated with the production of the natriuretic polypeptide hormones ANF and BNP. In non-mammalian vertebrates, as well as in the mammalian fetus, the secretory phenotype is displayed by both atrial and ventricular cardiomyocytes. It should be noted that cardiomyocytes in the ventricular conduction system of mammals also display a secretory phenotype.Cells in the heart also express other polypeptide hormones and their receptors, such as AM and ET1. The expression levels of these hormones in the heart are increased in various cardiovascular pathologies such as hypertension, cardiac hypertrophy, congestive heart failure and acute coronary syndrome. Whether these hormones spill over to contribute to the observed increases in plasma levels is often not clear so that the locally synthesised hormones may be viewed as acting locally within the heart in a paracrine and/or autocrine fashion.In terms of overall properties of hormones found in the heart, those belonging to the natriuretic peptide family or AM-related peptides have hypotensive, natriuretic and diuretic properties. Many of these peptides also have anti-proliferative and anti-fibrotic properties and thus may be involved in preventing cardiac remodelling. On the other hand, ET1 has hypertensive, volume retentive, and hypertrophic properties that while essential for maintaining cardiovascular homeostasis in cases of massive haemorrhage or hypotensive conditions, can lead to hypertrophic or fibrotic changes in the heart under protracted action.Regardless of whether their main site of production is cardiac or not, the polypeptide hormones mentioned above appear to contribute cooperatively to regulate cardiovascular haemodynamics through their actions on vascular tone, autonomic nerve activity and cardiac mass in both health and disease.


Due to human activities, marine and terrestrial ecosystems face a future where disturbances are predicted to occur at a frequency and severity unprecedented in the recent past. Of particular concern is the ability of systems to recover where multiple stressors act simultaneously. We examine this issue in the context of a coral reef ecosystem where increases in stressors, such as fisheries, benthic degradation, cyclones and coral bleaching, are occurring at global scales. By utilizing long-term  monitoring programs, we examined the combined effects of chronic  and pulse  disturbances on the trophic structure of coral reef fishes at two isolated atoll systems off the coast of northwest Australia. We provide evidence consistent with the hypothesis that the loss of sharks can have an impact that propagates down the food chain, potentially contributing to mesopredator release and altering the numbers of primary consumers. Simultaneously, we show how the effects of bottom-up processes of bleaching and cyclones appear to propagate up the food chain through herbivores, planktivores and corallivores, but do not affect carnivores. Because their presence may promote the abundance of herbivores, the removal of sharks by fishing has implications for both natural and anthropogenic disturbances involving the loss of corals, as herbivores are critical to the progress and outcome of coral recovery. Marine and terrestrial ecosystems are assailed by disturbances that operate as regulators of system structure and function Acanthaster planci) outbreaks and coral disease are commonplace Coral reef ecosystems offer an ideal model to explore this issue. Reefs are dynamic environments, where pulse disturbances such as coral bleaching, cyclones, crown-of-thorns  and the bottom-up, pulse disturbances of cyclones and bleaching as processes structuring reef fish communities on remote atolls in the eastern Indian Ocean. Because long-term monitoring of fish and coral communities has been conducted on these reefs for over a decade, they provide an ideal ecosystem-scale (hundreds of kilometres), natural experiment to investigate this subject. Our objectives were to examine (1) how fishing changed shark communities in coral reef ecosystems, (2) if such changes impacted the trophic structure of other fish communities, (3) the role of benthic disturbances in structuring fish communities and (4) whether there were any combined impacts of fishing and benthic disturbances on the community structure of fishes.2, where Indonesian fishermen are granted access to the Australian exclusive economic zone to pursue fishing for sharks using traditional techniques A unique combination of circumstances allowed our study to examine effects of shark removal and benthic disturbances on the trophic structure of coral reef fishes. Since 1994, changes in the abundance and diversity of benthic habitats and fishes have been quantified on the outer reef slopes of two groups of uninhabited, atoll-like coral reefs that lie off the coast of north-western Australia. As this has only involved passive long-term monitoring, no ethical considerations applied in this case. The first of these, the Rowley Shoals  and trochus  in a fishery that has historical origins dating to well before European settlement of Australia Indonesian fishermen provide a chronic disturbance on the reefs by targeting “banquet” species of high economic value, principally shark (for the shark fin trade), trepang (Carcharhinus amblyrhynchos) at the Rowley Shoals have shown that there is little to no movement among reefs within the Shoals The Rowley Shoals and Scott Reefs are atoll-like reefs without any significant emergent land that lie over 300 km from the nearest coast. Distances between these groups of reefs and the coast limits any likelihood of larval exchange and genetic evidence suggests that fish communities on the reefs can be largely dependent on self-recruitment c. 60% to <10%, while similar reductions in coral cover occurred at two of three reefs of the Rowley Shoals after a Category 5 cyclone  and Scott Reefs  during June 2003 and at Clerke (n = 24) and Imperieuse (n = 42) Reefs in the Rowley Shoals in October 2004  usually in October of 1994–2008 Isopora, soft coral, corymbose Acropora, digitate Acropora, Pocilloporidae, sponge, massive coral, foliose, macro algae, turf-coralline algae, other algae, sand/rubble, other coral, and other) in order to create a habitat model (see n = 999) and variance partitioning to estimate the amount of variation in community structure that could be accounted for by differences in habitat, fishing and an interaction between these factors R2anova, packfor, varpart and rda libraries in the vegan package of R Statistical Computing To determine the extent to which differences in habitats between reef systems contributed to patterns in fish communities, we used a forward selection procedure to select 15 of 19 habitat categories , non-fished/disturbed (n = 21), fished/non-disturbed (n = 46) and non-fished/non-disturbed (n = 29). In this case, fishing represented a chronic disturbance at the Scott Reefs that has been occurring for centuries, while the reef fishes at the Rowley Shoals are protected from fishing  and RDA were used to investigate differences in benthic and fish assemblages among four treatment groups: fished/disturbed ( fishing . A distu fishing . Speciesn = 9999) t-test that accommodated non-parametric data with unequal sample sizes n = 46), another fished reef within the MoU74 box using a two-tailed permuted t-test . As there was no significant difference in shark abundance between these reefs, we used data from Scott Reefs for subsequent analyses.Comparisons of shark abundance between fished (Scott Reefs) and non-fished  reefs were conducted using R Statistical Computing adonis function in the vegan package of R Statistical Computing n = 9999). Further pairwise comparisons using two-tailed permuted t-tests (n = 9999) were conducted to test for fishing and disturbance effects between the four treatment groups. As Euclidean distances were used in permuted ANOVAs, abundances were Hellinger transformed prior to testing Permuted ANOVAs were conducted using Isopora, corymbose Acropora). These groups made only a small contribution to patterns in benthic composition , which was associated with the pulse disturbance events observed on all reefs . Howeverp<0.001) and uniquely explained 23.4% of variation in the fish community across all sites. Further, the presence or absence of fishing significantly (p<0.001) and uniquely explained 13.8% of variation in the fish community across all sites. Finally, the model that combined habitat and fishing was significant (p<0.001) and was able to explain 60.3% of variation in the fish community . Thus, both habitat and fishing uniquely and interactively contributed to patterns in reef fish communities across the sites.Variance partitioning and permutation tests revealed that habitat (i.e. benthic cover) significantly  at the protected Rowley Shoals were approximately three times those occurring on the fished Scott Reefs  were significantly more abundant on the fished Scott Reefs after the impact of bleaching than during undisturbed phases. In contrast, they did not differ in abundance during impact and undisturbed phases on the unfished Rowley Shoals. This result implies that the reduction in shark numbers may directly or indirectly allow these species to take advantage of the increase in detrital material trapped by turfing algae that overgrew dead corals during the disturbance at the Scott Reefs.Disturbances can act synergistically, additively or antagonistically on the abundances of animals within an ecosystem Isopora and corymbose Acropora as benthic groups that differed between reef systems  we cannot completely discount these as alternative explanations for some of the patterns we found in fish communities on reefs with and without reduced numbers of sharks. For the most part, such habitat differences were relatively small. While our PCA identified macro algae, sponges,  systems , these c systems , resultiSome of our results might also reflect other, more fundamental differences in the nature of the disturbances between reef systems. For example, wave action caused by cyclones breaks up coral skeletons, reducing three-dimensional structure of the reef Finally, another possibility is that differences in numbers of some trophic groups between fished and unfished reefs may be simply a result of random variation in patterns of larval supply. For surgeonfishes, rare strong pulses in recruitment (greater by orders of magnitude than background levels) can be a feature of their biology on isolated reefs and atolls Scarinae) to damselfishes (Pomacentridae).One interpretation of the correlation between low numbers of sharks and herbivorous fishes on our study reefs is that this is evidence of a trophic cascade. These occur when changes in the abundance of higher-order predators directly and/or indirectly affect species at a number of lower trophic levels in a food web. Such cascades are well-recognised in marine systems, with examples involving reductions in the numbers of sharks, lobsters, seastars and sea otters as top-order predators causing fundamental changes in the structure and function of temperate marine ecosystems where they formerly occurred Irrespective of the mechanism involved, if a link does exist between the abundances of sharks and herbivores, then this has important implications for coral reef ecosystems. Herbivorous fishes are fundamental to the dynamics of communities on reefs, since their feeding reduces algal cover and allows corals more space to colonize and grow in benthic habitats Although we may soon lack any practical ability to affect the frequency of bottom-up disturbances to coral reefs where these are driven by climate change, this is not the case with the loss of reef sharks. Tracking studies show that reef sharks can maintain a high degree of site fidelity around coral reefs Figure S1Principal components analysis of the benthic composition of 19 different classes of coral, algae, sponge, and other benthos among sites. Benthic cover types contributing the most to patterns are denoted in black, while others are shown in the middle of the plot in grey. Sites have been coded by the four treatments (see key). The amount of variation explained by each axis is shown.(TIF)Click here for additional data file.Figure S2PCA biplot of fish abundances by genus in the carnivore trophic group. The sites were coded by each of the four treatments and the 15 genera that made up the carnivore group are shown. The amount of variation explained by each axis is shown.(TIF)Click here for additional data file.Figure S3PCA biplot of abundances of fish by genus in the herbivore trophic group. The 12 genera that make up the herbivore group are shown on the figure and the sites were coded by each of the four treatments. The amount of variation explained by each axis is shown.(TIF)Click here for additional data file.Table S1A summary of anthropogenic, reef metrics, environmental and biotic factors at protected and fished reefs. Protected sites included Mermaid, Clerke and Imperieuse Reefs. Fished sites included South and North Scott, Seringapatam and Ashmore Reefs.(DOCX)Click here for additional data file.Table S2Species composition of the five trophic groups  used in our study. The list is alphabetical by family and species. Those classified as corallivores included both obligate and facultative coral feeders (DOCX)Click here for additional data file.Table S3Summary of statistical tests to evaluate fishing, disturbance and interactive effects on densities of trophic groups. Fishing, disturbance and interaction effects were evaluated using a permuted two-way ANOVA. Permuted t-tests were used to conduct contrasts. p-values were Bonferroni corrected.(DOCX)Click here for additional data file.Text S1(DOCX)Click here for additional data file.


To the Editor: We have read with interest many of the articles concerning West Nile virus (WNV) published in the July 2003 issue of Emerging Infectious Diseases. Last summer Ohio was one of the leading states with WNV infection in humans. Consequently, requests for tests for this pathogen have increased. Unfortunately, the turnaround time for testing these specimens may be delayed because of shipping difficulties secondary to associates, the limited number of laboratories that can perform these assays, and an increase in requests at testing facilities.Cytologic examination of cerebrospinal fluid (CSF) from patients with WNV has not been studied. Although cytologic examination of CSF from patients with encephalitis is likely nonspecific, it may provide supportive information of the suspected disease process, and is useful for excluding other conditions, such as neoplasia. Of the 22 patients that were hospitalized at our institution last year with WNV meningoencephalitis, documented by serologic tests and/or reverse transcription-polymerase chain reaction, CSF of 4 of these patients was submitted for cytologic examination. Of these 4, 3 had a sufficient number of cells in the CSF specimen  to afford cytologic examination, whereas one had a paucicellular CSF, with only 2 white blood cells/μl. The cytologic features from the 3 patients >10 cells/μL consistently demonstrated a mixture of lymphocytes at various stages of activation and occasional large monocytic-like cells with cerebriform nuclei reminiscent of the Mollaret cells described in CSF of patients with recurrent meningitis  (1).Mollaret described cells with enlarged nuclei and cerebreform nuclear contours in CSF of patients with recurrent, aseptic meningitis (One of the patients infected with WNV meningoencephalitis who had Mollaret-like cells in CSF died. Postmortem neuropathologic examination showed an extensive perivascular lymphocytic infiltrate which contained mononuclear cells consistent with the Mollaret-like cells in CSF. These mononuclear cells were stained with an immunohistochemical stain directed against the CD68 antigen, which supports a monocytic origin (


These data strongly suggest that SnoN can function as a tumor suppressor at early stages of tumorigenesis in human cancer tissues.SnoN is a negative regulator of TGF-β signaling and also an activator of the tumor suppressor p53 in response to cellular stress. Its role in human cancer is complex and controversial with both pro-oncogenic and anti-oncogenic activities reported. To clarify its role in human cancer and provide clinical relevance to its signaling activities, we examined SnoN expression in normal and cancerous human esophageal, ovarian, pancreatic and breast tissues. In normal tissues, SnoN is expressed in both the epithelium and the surrounding stroma at a moderate level and is predominantly cytoplasmic. SnoN levels in all tumor epithelia examined are lower than or similar to that in the matched normal samples, consistent with its anti-tumorigenic activity in epithelial cells. In contrast, SnoN expression in the stroma is highly upregulated in the infiltrating inflammatory cells in high-grade esophageal and ovarian tumor samples, suggesting that SnoN may potentially promote malignant progression through modulating the tumor microenvironment in these tumor types. The overall levels of SnoN expression in these cancer tissues do not correlate with the p53 status. However, in human cancer cell lines with amplification of the  In recent years, however, more and more evidence have emerged indicating that SnoN can also act as a tumor suppressor. Heterozygous mice lacking one copy of the snoN gene show an increased susceptibility to carcinogen-induced tumorigenesis, suggesting that SnoN may protect mice from carcinogenesis in vitro and block tumor development in vivo in a two-step skin carcinogenesis mouse model in vitro and tumor metastasis in vivoSnoN (Ski-novel protein) was fist discovered as an oncoprotein that can induce transformation of chicken and quail embryonic fibroblasts when overexpressed snoN gene is located at chromosome 3q26.2, a locus frequently amplified in many cancer types, including that of esophagus, ovary and many others Given the complex roles of SnoN in tumorigenesis, it is important to determine its expression pattern in normal human tissues and monitor how it changes during malignant progression. SnoN is ubiquitously expressed in all embryonic and adult tissues. Its expression is upregulated during specific stages of embryogenesis and organ morphogenesis SnoN gene as well as SnoN transcript levels have been found to increase in these cancers In this report, we examined SnoN expression in four types of normal human tissues and matching cancer tissues of various clinical stages of malignancy to assess whether alterations of SnoN expression correlate with tumor malignancy and/or status of p53 inactivation. These four types, esophageal, ovarian, breast and pancreatic tissues, were chosen because SnoN has been implicated in the development of these cancers and/or in the normal functions of these tissues. Esophageal and ovarian cancers often involve amplification of the 3q26 amplicon, and the copy number of the All arrays were purchased from Biomax (Cybrdi Inc). These arrays come with information on clinical stages, pathology grades and TNM. The esophageal array contains pairs of tumor biopsy samples and matched normal samples from 39 patients with histologically and clinically classified adenocarcinoma: 8 patients with grade I, 19 patients with grade II, and 11 patients with grade III tumors.The ovarian tumor array contains tumor biopsy samples and 10 normal matched samples from 69 women aged 50+/−10. The tumors were classified to be ovarian adenocarcinomas: 3 patients with ovarian clear carcinoma, 12 patients with grade I, 29 patients with grade II, and 25 patients with grade III adenocarcinomas .The pancreatic tumor array contains samples from 90 patients and five normal pancreatic tissue samples. The tumor types on this array include ductal adenocarcinoma, mucinous adenocarcinoma, neuroendocrine carcinoma, adenosquamous carcinoma, carcinoid, and solid pseudo-papillary carcinoma. They have been classified into different clinical classes based on histological evaluation: 21 patients with grade I, 59 patients with grade II, 8 patients with grade III (many with lymph node metastasis), and 2 patients with grade IV tumor with distant metastasis.The breast cancer array contains duplicated tumor biopsy samples from 31 patients, with six normal samples from six matching patients, and 9 metastatic samples from 9 matching patients. These tumors were clinically classified by histological analysis to be infiltrating ductal carcinoma: 9 patients with grade IIA, 4 patients with grade IIB, 10 patients with grade IIIA, 5 patients with grade IIIC, 9 matching patients with lymph node metastatic, and 1 patient with metastatic adenocarcinoma to the ovary. Because no difference in SnoN expression was detected between grade IIA and IIB or between grade IIIA and IIIC tumors, we grouped these samples together as grade II or grade III tumors. In addition, 24 pairs of breast ductal carcinoma in situ (DCIS) samples with matching normal tissues were obtained from UCSF cancer center.The thickness of all the arrays is 5 µm. The breast cancer array contains duplicate samples from each patient and esophageal, ovarian and pancreatic arrays have one section from each patient.Paraffin-embedded arrays were deparaffinized in xylenes then rehydrated in a series of ethanol gradient. Sections were immersed in 0.85% Nacl for 5 minutes, then in PBS for 5 minutes, and fixed with 4% paraformaldehyde (PFA). Permeabilization of the slides was carried out with 20 µg/ml proteinase K for 10 minutes at room temperature (RT), followed by washing in PBS for 5 minutes. Samples were fixed a second time with 4% PFA, and blocked overnight in 1%BSA/10% newborn calf serum/0.02% Triton X-100 in PBS in a humidified chamber at 4°C. The staining was performed with an anti-SnoN antibody recognizing a C-terminal peptide of SnoN The sections from the same tissue types  were all stained in the same experiment under identical conditions, and images were taken under the confocal microscope in one session. In addition, the same antibody lot and dilutions were used for all stainings described in this paper. Under no circumstances was the antibodies re-used in any of the experiments.2O2 for 10 minutes to quench endogenous peroxidase activity. Then sections were microwaved at 95°C for 30 minutes in sodium citrate buffer (pH 6) Paraffin-embedded sections were deparaffinized in xylenes, rehydrated in ethanol, and incubated with 3% HFor each SnoN-stained tumor sample, at least three images were captured using the LSM 510 confocal microscope under the 20x objective to make sure that the major areas for each sample are included in the quantification. SnoN intensity (the number of pixels/area) of each of the three areas was calculated using the image J software, and the size of the area was kept constant throughout the quantification process. The numbers were then averaged for each tumor sample. The negative control and peptide competition background signals were substracted from all the samples during quantification. The p53 images are taken with the Zeiss AxioImager fluorescence microscope. Nuclear p53 signal intensity was quantified in a 0 (negative) to 5 (highly intense staining) scale.For analysis of potential association of TP53 mutation and SKIL amplification, we performed data mining of the Novartis cell line encyclopedia (CLE) that contains 947 human cancer cell lines Statistical analysis was performed using the student’s t-test function in bioconductor R package as 2-tailed t-tests assuming unequal variances. Pearson’s correlation coefficient was calculated using the R package, and the significance of the observed correlation coefficient was determined using one-sample t-test against the population of correlation coefficients in-between p53 and all Entrez genes in the human genome. Potential correlation between SnoN expression and p53 inactivation in cancer tissues was evaluated using the Kruskal-Wallis test.To determine a normal pattern of SnoN in various human tissues and establish a baseline for further analysis of cancer tissues, we first examined SnoN expression in 39 normal esophageal tissue samples, 10 normal breast samples, 6 normal ovarian and 5 normal pancreas samples by immunohistochemistry with anti-SnoN. This affinity-purified antibody was raised against a C-terminal peptide of human SnoN and has been previously characterized for imunofluorescence and immunohistochemistry studies esophagus contains three layers: the non-keratinized stratified squamous epithelium, the lamina propria consisting of loose connective tissue, and the muscularis mucosa consisting of smooth muscle. In the esophageal stratified squamous epithelium, SnoN was expressed at moderate to high levels in the cytoplasm, with a stronger staining in the upper surface part of the flattened epithelium than in the lower layer of cuboidal cells  and to a lesser extent, grade II tumors (p = 0.142) . InteresSimilar to what have been observed in esophageal cancer, SnoN expression was downregulated in grade I and to a lesser degree, grade II ovarian adenocarcinoma samples in both tumor epithelia and stroma , but recThe pancreatic cancer tissue array contains multiple types of adenocarcinoma of all three clinical grades (I-III), including ductal adenocalarcinoma, mucinous adenocarcinoma, neuroendocrine carcinoma, adenosquamous carcinoma, carcinoid, and solid pseudo-papillary carcinoma. In all pancreatic cancer samples across all three tumor grades, SnoN was cytoplasmic and was expressed at a lower level than that in the normal age-matched samples . Across The breast cancer tissue array contains duplicated biopsy samples from patients with infiltrating breast ductal carcinoma of stages II and III and metastatic carcinoma to the lymph nodes. To our surprise, SnoN levels were not elevated in any of the tumor samples . On the To test the possibility that SnoN may be elevated during early stages of tumorigenesis but reduced at late stage of malignant progression, we collected and stained 24 human DCIS samples and their matching normal tissues for SnoN expression. Interestingly, although in any individual cell in the DCIS tissues, the level of SnoN expression was similar to or in a few cases, lower than that in cells in normal tissues, due to a markedly increased epithelial content in DCIS samples, the overall SnoN level in the whole tissue was much higher than that in normal tissues . Thus, tTaken together, unlike previously proposed, SnoN expression is reduced in lower grade malignant tumors of esophagus, breast, pancreas and ovary . In esopSnoN gene also tend to show inactivation of p53 as indicated by a loss of p53 copy number or the presence of known inactivating mutations. Based on the mutation status of p53, we classified cell lines into mutant or wild type, and copy number of SnoN were compared among these two groups. As shown in In contrast to its reduced expression in primary tumor samples, SnoN have been shown previously to be elevated in esophageal, ovarian and breast cancer cell lines Next we asked whether there was any correlation between SnoN expression and the status of p53 in the four types of human cancer tissue samples that showed reduced SnoN expression by staining the cancer tissue arrays with anti-p53. In all normal tissue samples, p53 was absent as expected (data not shown). In contrast, majority of ovarian tumor samples exhibited a strong p53 nuclear staining, indicating the presence of inactivating mutations Together our study suggests that SnoN is likely to play a tumor suppressor role in the initial stages of human cancer development, and inactivation of this pathway by targeting either SnoN itself or p53 is necessary for malignant progression.In this report, we found that SnoN is expressed in the normal human esophageal, ovarian, pancreatic and breast tissues predominantly in the cytoplasm of both the epithelium and stroma. The intracellular localization of SnoN did not alter significantly in tumor samples, but its expression is downregulated in all four types of tumor epithelia at lower malignant grades, but gradually recovered in higher grade tumors and markedly upregulated in the stroma of esophageal and ovarian cancer tissues. These results suggest that SnoN expression may vary at different stages of tumor progression and that SnoN may play different roles at different stages of malignancy. In particular, the initial downregulation of SnoN in the tumor epithelium is consistent with its anti-tumorigenic activity. The later upregulation of SnoN in the stroma microenvironment in esophageal and ovarian cancers suggests that it may play a role in promoting tumorigenesis in an indirect manner. Thus, the pro-oncogenic activity of SnoN in human cancer may be non-tumor cell autonomous and through affecting the tumor microenvironment.Our results differ somewhat from those published previously, in particularly those that reported an increase in SnoN levels in cancer tissues Our study has revealed a previously unappreciated role of SnoN in the tumor microenvironment. Especially in esophageal and ovarian tissues, multiple stromal cell types showed high levels of SnoN expression, including smooth muscle cells, blood cells, fibroblasts and infiltrating lymphocytes. Interestingly, the cancers of esophagus and ovary tend to harbor 3q26 amplification and show SnoN copy number increases, and they also display a stronger increase in stroma SnoN levels. Thus, the high SnoN expression in the stroma may be related to gene amplification, and this in turn may enhance malignant progression in these tissues. More and more studies have indicated that tumor-associated stromal cells and infiltrating lymphocytes promote epithelial tumorigenesis, invasion and metastasis, and cancer inflammation High levels of SnoN have been shown to directly stabilize and activate p53. Thus, tissues or cells that show elevated SnoN expression (either due to gene amplification or other means) may cease proliferation due to p53-mediated senescence or cell cycle arrest. Malignant cells have to overcome this barrier in order to maintain their survival and expansion and may do so by either inactivating/deleting SnoN itself or any downstream steps in the SnoN-p53 pathway. Indeed, our analysis of CLE cell lines has indicated a significant correlation between increased SnoN copy number and p53 inactivation, supporting the idea that the tumor suppressive SnoN-p53 pathway must be inactivated to allow malignant progression. A previous study of human breast cancer tissue samples also note a correlation between SnoN upregulation and elevated p53 expression, which is indicative of inativating mutation of p53 In summary, we have found that SnoN is expressed in both epithelial and stromal cells in normal human esophageal, ovarian, pancreatic and breast tissues. Unlike what has been depicted in the current models, SnoN expression is downregulated at the protein level in the tumor epithelium in low grade tumors in all four tissues, consistent with its anti-tumorigenic activity. In contrast, SnoN expression is markedly upregulated in tumor stroma in some cancer types, especially at late stages of malignancy. Giving the importance of tumor stroma in influencing malignant progression, this result promopted us to speculate that SnoN may also regulate tumorigenesis through modulating tumor microenvironment.


The study of the ovarian proteomic profile represents a new frontier in ovarian cancer research, since this approach is able to enlighten the wide variety of post-translational events (such as glycosylation and phosphorylation). Due to the possibility of analyzing thousands of proteins, which could be simultaneously altered, comparative proteomics represent a promising model of possible biomarker discovery for ovarian cancer detection and monitoring. Moreover, defining signaling pathways in ovarian cancer cells through proteomic analysis offers the opportunity to design novel drugs and to optimize the use of molecularly targeted agents against crucial and biologically active pathways. Proteomic techniques provide more information about different histological types of ovarian cancer, cell growth and progression, genes related to tumor microenvironment and specific molecular targets predictive of response to chemotherapy than sequencing or microarrays. Estimates of specificity with proteomics are less consistent, but suggest a new role for combinations of biomarkers in early ovarian cancer diagnosis, such as the OVA1 test. Finally, the definition of the proteomic profiles in ovarian cancer would be accurate and effective in identifying which pathways are differentially altered, defining the most effective therapeutic regimen and eventually improving health outcomes. Ovarian cancer represents one of the four main gynecological cancers, counting worldwide about 225,000 new cancer cases diagnosed in 2008  and about 140,000 deaths  . N. N9]. NoPTEN, PI3K catalytic subunit-α (PIK3CA), KRAS, BRAF and b-catenin (CTNNB1) mutations, along with genomic stability and TP53 mutations in only a small fraction of cases. Type II ovarian cancers include high-grade serous carcinomas, mixed malignant mesodermal tumors, carcinosarcomas and undifferentiated cancers. These tumors have TP53 mutations in up to 80% of cases, along with marked genomic instability and arise more frequently from the fallopian tubes and the peritoneum and in BRCA1 and BRCA2 mutation carriers . . 12]. ThBRCA1 and BRCA2, confer an increased lifetime risk of ovarian cancer. However, BRCA mutations do not account for the entire range of hereditary ovarian cancer syndromes. Other hereditary epithelial ovarian cancers are attributed to Lynch syndrome. Lynch syndrome is an autosomal dominant disorder, which predisposes to colorectal, endometrial, ovarian, gastric, small bowel, biliary/pancreatic, urothelial, skin and central nervous system cancers. The cumulative risk of ovarian cancer is estimated to be 8%–10%, with an average age of onset of 42 years [TP53 and PTEN or CHEK2 and PALB2, confer a low to moderate risk for breast and ovarian cancer [RAD51 and BARD1[FA-BRCA pathway and the mismatch repair system. However, many families with suspicious pedigrees do not have a specific mutation identified through clinical testing, due to a currently undetectable BRCA1/2 mutation or a mutation in another susceptibility gene.The most important risk factor for ovarian cancer still remains a family history of breast or ovarian cancer. Up to 10% of ovarian cancer patients may have inherited a germline mutation that places them at increased risk for the disease. Mutations in the breast and ovarian cancer-susceptibility genes, 42 years . Moreoven cancer ,15. Receand BARD1. To dateBRCA 1/2 genes. Women who have inherited genetic mutations have substantially increased risk of ovarian cancer, with a lifetime risk that varies with the genetic defect  [BRCA1/2 genes can be involved even in the pathogenesis of BRCA1-like and BRCA2-like sporadic ovarian cancer [Inheritance of DNA repair defects contributes to as many as 10%–15% of ovarian cancers. About 90% of hereditary ovarian cancer derives from germline mutations in cer, 7%) . ParticuTraditionally, proteomic analyses have been performed using two-dimensional gel electrophoresis (2DE), which separates proteins according to two distinct protein characteristics, size and charge. The introduction of immobilized pH gradients and advanced bioinformatic approaches have recently improved the reproducibility and comparability of this technique . In seveThe analysis of the proteome started out with techniques based on 2DE and then extended with liquid chromatography–mass spectrometry (LC–MS)-based techniques. However, despite the rapid advance of MS-based proteomics, 2DE still plays a crucial role in several fields of protein and molecular biology. There is an important niche for 2D gel-based proteomics, which complete traditional LC-MS techniques, in the areas of protein identification from organisms with no or incomplete genome sequences available, alternative detection methods for modification of specific proteomics and identification of protein isoforms and modified proteins .m/z) of proteins and, thus, identifying the actual precursor proteins or protein profiles. In recent years, MS instruments have been greatly modified and improved, becoming sensitive to the picomole to femtomole range required for the detection of oligonucleotides, small polar molecules, peptides, proteins and post-translationally modified proteins, such as glycoproteins and phosphoprotein [Mass spectrometry (MS) provides a snapshot of a proteome in time and space, determining the precise mass and charge  has been widely introduced in recent years. A shotgun-MS strategy consists in the separation of peptides produced from protein digestion in complex mixtures followed by tandem mass spectrometry (MS/MS) analysis. Traditional shotgun proteomics is a useful tool for the analysis and identification of proteins and their variations in biological samples with a non-targeted approach.m/z and relative abundance. These ions can then be processed through databases to identify the corresponding peptide sequences [Biological samples are often heterogeneous and contain several different molecules; thus, methods of separation are often necessary to select the target to analyze from the other molecules in the sample. Gas (GC) or liquid chromatography (LC) are common methods of pre-MS separation used when analyzing complex gas or liquid samples. High performance liquid chromatography (HPLC) is the most common separation method to study biological samples by MS or MS/MS . Traditionally, the first step is sample lysis and protein extraction from the cells, tissues or body fluids, followed by digestion into peptides. This process generates thousands of peptides, which can be pre-fractionated according to their physicochemical properties (such as charge or isoelectric point) or enriched by different means . These pre-fractionated or enriched samples are then analyzed one-by-one by reversed-phase liquid chromatography coupled to mass spectrometry (LC–MS), in which selected peptides are fragmented by tandem mass spectrometry (MS/MS). MS and MS/MS are connected to computer software that analyzes the detected ions by their individual equences ,23.Among the MS-based proteomics approaches, matrix assisted laser desorption/ionization mass spectrometry (MALDI MS) and surface enhanced laser desorption/ionization mass spectrometry (SELDI MS) have been employed for large scale polypeptide profiling in clinical proteomics . ProteomC-terminal fragment of the activator of the immunoproteasome, PA28 alpha), it is possible to detect the protein and the mRNA encoding this protein in the same sample. Since the result showed that the level of transcription is related to the abundance of the protein fragment, cross-validation between the transcriptome and the proteome can be performed on the same tissue. To conclude, targeted MSI associated with classical immunohistochemistry (IHC) may offer the possibility to obtain a quick answer and a better molecular diagnosis for clinical surgery [MS-based proteomics is a universally used technique for analyzing complex protein samples. To visualize the spatial distribution of specific molecules in biological systems, an increasing number of groups are looking to combine the sensitivity and specificity of mass spectrometry with imaging capabilities. This involved both of the macromolecular ionization techniques, MALDI and ESI and provided a foundation for secondary ion mass spectrometry (SIMS) imaging. Since MALDI provides proteomics information and SIMS that of lipids and other surface active species, several groups now use the complementary information provided by SIMS and MALDI to obtain proteomics, lipid and vitamin distributions within the same tissue section . However surgery .These technologies enabled the detection of low-abundance proteins, identifying hundreds of potential biomarkers with diagnostic, prognostic and therapeutic value. However, there is a lack of analytical validation of platforms for the precise measurements of the candidate markers in a smaller set of samples before lengthy and costly large-scale clinical trials. In this context, targeted proteomic technologies, such as multiplex MS, protein arrays and several variations of enzyme-linked immunosorbent assays (ELISAs) have provided a sensitive and specific quantitative measurement of protein targets in biological systems. The accuracy and statistical rigor of these technologies may be used to investigate the molecular mechanism of cellular function through the analysis of protein networks and pathways and to confirm and triage hundreds of potential biomarkers prior to large-scale clinical trials. Stable isotope dilution (SID) multiple reaction monitoring MS (SID-MRMMS) has emerged as one of the powerful targeted proteomic tools in the past few years .in situ hybridization (FISH), etc., to detect the genetic alterations occurring in the cancerous state. In the last decade, microarray technology has allowed for the measurement of the expression of tens of thousands of genes in a given tissue sample and to compare gene expression between normal and malignant cells, identifying genes that are differentially regulated during cancer development [Malignant transformation of ovarian epithelial cells is due to genetic mutations that compromise regulation of proliferation, differentiation and apoptosis. Since a close connection exists between genetic alterations and tumorigenesis, research on gene level  could provide important biological, diagnostic and prognostic information. DNA or RNA-based cancer biomarkers utilize microarrays, polymerase chain reaction (PCR), reverse transcriptase polymerase chain reaction (RT-PCR), DNA sequencing, fluorescent elopment .Protein microarrays, similar to gene arrays, emerged as a promising technique to analyze the abundance of proteins and their alterations . ProteinProteomic strategies are able to provide more detailed information about biological systems than the determination of the static genome or expression profiling based on mRNA. In contrast to the genome, the complexity of the proteome is increased by alternative splicing of mRNAs and a wide variety of post-translational events . For all these reasons, genomics and mRNA analysis do not correlate with protein content, while proteomics may study the presence of a specific protein and provide a measure of the quantity present.et al. studied the differences in the transcripts of a restricted set of glycosyltransferases involved in N-linked glycosylation in ovarian cancer tissues and normal ovarian tissues, identifying candidate biomarkers with cancer-specific glycosylation, such as periostin and thrombospondin [et al. found that several proteins are aberrantly sialylated in N-linked glycopeptides in ovarian cancer tissue, and detection of glycopeptides with abnormal sialylation changes may be exploited as biomarkers for ovarian cancer [et al. identified the sialome  utilizing tandem mass spectrometry as a potential pool of novel biomarker candidates. In all of the samples analyzed, 579 glycosylation sites on 333 proteins were identified [Glycosylation is one of the most common post-translational modifications. The mechanism of glycosylation is involved in several biological processes, including cell communication, adhesion and signaling. Moreover, the association between aberrant glycosylation and disease development has promoted a number of research activities directed to provide reliable diagnostic or prognostic information . MS glycospondin . More ren cancer . Finallyentified . To concReversible protein phosphorylation represents one of the most dynamic post-translational modifications. Rapid changes of protein phosphorylation largely influence signaling networks, playing a crucial role in the regulation of many cellular processes, including metabolism, differentiation, migration, proliferation and apoptosis. Phosphoproteomic strategies represent the optimal choice for studying phosphorylation-based signaling networks. The proteomics development in the area of MS has been improving with the breakthrough of quantitative MS through ICAT (isotope-coded affinity tag), iTRAQ (Isobaric tags for relative and absolute quantitation), isobaric peptide termini labeling (IPTL) and SILAC (stable isotope labeling by amino acids in cell culture). SILAC, the most extensively used method during the past years, represents an accurate and inexpensive procedure that can be used as a quantitative proteomic approach in any cell culture system . PhosphoOne of the main goals of proteomics is the identification of biomarkers for diseases from tissues and body fluids. Current diagnostic tools have had very limited success in early detection of ovarian cancer. The search for an ovarian cancer screening modality with improved specificity and sensitivity has led to the examination of serum biomarker patterns using new “omic” technologies –41. SeveA recent comparative proteomic study investigated and defined protein expression patterns associated with advanced stage ovarian cancer, to identify a panel of diagnostic or prognostic markers. The study also investigated proteins secreted by the cancer cell into the interstitial fluid, as cancer growth and progression also depends on stromal factors present in the tumor microenvironment. Moreover, many biomarkers present in biopsied cancer tissues can also be found in blood serum, representing potential biomarkers of the disease. Proteomic profiling of differentially expressed proteins in cancer ovarian tissue, tumoral interstitial fluid (TIF) and ascitic fluid, compared with healthy tissue sample and normal interstitial fluid (NIF), allowed for the identification of protein spots consistently differentially expressed between normal and cancer samples  and 4.Protein expression/identification was evaluated by 2DE  and MS (mass spectrometry) analysis and were confirmed by immunohistochemistry. Six proteins showed differential expression in tumoral interstitial fluid and tumor tissue compared to normal interstitial fluid and healthy tissue. Protein different expression between tumoral and normal ovarian tissue is presented in S-transferase A-2, retinol binding protein 1, phosphatidylethanolamine-binding protein and annexin 5, while the calgranulin was significantly upregulated in all pathological samples, including the ascitic fluid. This is the first study to report an overexpression of calgranulin by 2DE associated with MS/MS analysis on surgical biopsy. As previously reported, the reduced expression of galectin 3 and retinol binding protein 1 in cystic fluid and serum of patients with early stage disease is confirmed in this study. The results highlight alterations in proteins that control cell-cycle progression and apoptosis, as well as factors that modulate the activity of signal transduction pathways [Five were found to be downregulated and identified as galectin 3, glutathione pathways .et al. [p < 0.001) were visualized, and the three most significant peaks were identified as the fibrinogen alpha fragment, collagen alpha 1 (III) fragment and fibrinogen beta NT fragment. These results supported the feasibility of using urine as a diagnostic tool and suggested the enhanced prediction performance of combined marker analysis.Petri et al.  examinedet al.[Li et al. performeet al.[Jackson et al. identifiet al.. Recentlet al..et al.[N-acetylglucosamine kinase).An et al. identifiet al. published a series of papers that used a combination of CA125, HE4 and menopausal status to predict the presence of a malignant ovarian tumor and developed the Risk of Ovarian Malignancy Algorithm (ROMA), a simple biomarker-based algorithm, which requires ultrasound [In 2008, the FDA approved human epididymis protein 4 (HE4) to monitor recurrence or progression of epithelial ovarian cancer. Moreover, reliable clinical evidence demonstrates that HE4, alone or in combination with CA125, improves the accuracy of screening . HE4 is trasound ,53.et al. evaluated several biomarkers alone and in combination with CA 125 in prediagnostically collected sera from women in the Prostate, Lung, Colorectal and Ovarian Cancer Screening trial. In contrast to the prior findings on post-diagnostically collected samples, the addition of these biomarkers to CA125 did not improve sensitivity for preclinical diagnosis [et al. created a large scale database of 1129 proteins, combining information from ovarian cancer tissues and normal tissues, to enable tumor marker discovery [Since ovarian cancer represents a complex and heterogeneous disorder, it is unlikely that a single biomarker could be able to discriminate between healthy women and patients with ovarian cancer. The use of a combination of biomarker candidates could provide higher specificity and sensitivity for early detection of ovarian cancer ,55. On tiagnosis . In addiiscovery . In 2007iscovery . Despitein Vitro Diagnostic Multivariate Index Assay (IVDMIA) of the following five proteomic biomarkers: CA125, transthyretin , apolipoprotein A1, beta 2 microglobulin and transferrin. These biomarkers, other than CA125, were part of seven biomarkers measured by SELDI MS analysis in serum specimens from multiple centers [In 2009, the FDA cleared the OVA1 test to assess preoperatively the risk of ovarian cancer among women with known pelvic masses. The test represents a useful tool to assist physicians in determining which patient would benefit from referral to a specialist surgeon, who would be able to perform debulking and staging surgeries that form the basis of optimal care for ovarian cancer. The OVA1 test is an  centers ,63. Unfo centers ,65.To conclude, critical assessment of the results has shown significant shortcomings and uncertainties in regard to the reproducibility of the findings and identity of the proteins behind the pattern peaks; thus, the validation of the newly discovered biomarkers still remains the most challenging aspect of clinical proteomics, whereas utilizing pre-diagnostic samples for discovery may be helpful in developing validated early detection panels .Proteomic technologies may help to analyze protein signaling pathways to define the preferred targets of patient-tailored molecular therapy. Assessing quantity and functionality of proteins, taking into account the genetic, epigenetic and proteomic features of ovarian cancer cells, may guide treatment decisions. Target therapies may be effective for the subset of patient who harbor tumors with specific protein network defects; thus, the proteomic approach may help to select patients who truly benefit from select molecular therapy.et al. examined epidermal growth factor receptor (EGFR) and downstream pathways in a phase II clinical trial of gefitinib in ovarian cancer. Notably, the author observed a lack of association between biochemical and clinical effects, since a decrease in the quantity of both total and phosphorylated EGFR in tumor tissue with the administration of gefitinib was not associated with a clinical benefit [Posadas  benefit . Even c- benefit , and sev benefit .et al. found protein expression changes in ovarian cancer cells in response to HSP90 inhibitor 17AAG [n = 3). These results showed that acetylation levels are altered following HSP90 inhibition, suggesting that effects on protein acetylation may potentially play a role in the mechanism of action of HSP90 inhibitors. They also suggested that care should be taken in combining histone deacetylase and HSP90 inhibitors to decrease cell proliferation.Maloney or 17AAG . The hisBRCA 1/2 genes are the best validated genetic molecular target in ovarian cancer. The poly (ADP-ribose) polymerase (PARP) inhibitors represent a considerable promise for the treatment of cancers in BRCA 1/2 mutation carriers. This therapeutic approach exploits a synthetic lethal strategy to target the specific DNA repair pathway in these tumors [BRCA mutations carriers showed favorable responses to the PARP inhibitor olaparib compared with patients without BRCA mutations [et al. showed that the development of a subset of sporadic tumors is similar in genomic alterations to that of BRCA1/2-related tumors. The importance of these observations lies in the potential benefit from targeted therapy, through the use of PARP inhibitors, for a much larger group of patients than the BRCA1/2 germline mutation carriers [Genetic aberrations in e tumors . Particuutations . Interescarriers .However, despite the good premises, this wealth of information has yielded little in changing the clinical care from a treatment perspective .Ovarian cancer represents one of the four main gynecological tumors with an increasing incidence and the poorest survival of all gynecological cancer sites. In the last few decades, new approaches at the molecular and cellular level have given useful insights to improve early detection and to personalize treatment strategies. Ovarian cancer represents a heterogeneous disorder with different epidemiology, precursor lesions, gene expression profiles, biomarkers, therapy responses and prognosis, according to each distinct histotype. In order to identify a gene-proteomic signature that may bring to the definition of specific profiles, gene and protein expression analyses have been widely introduced. Particularly, the development and implementation of advanced investigation technologies lead to a substantial change of research objectives toward a genetic and proteomic setting.Proteomic techniques characterize all proteins in a biological system. One of the main goals of proteomics is the identification of biomarkers for diseases from tissues and body fluids. The advancing techniques for proteomics have shown promise in a variety of studies identifying several potential candidate biomarkers and prognostic indicators of outcome and response to therapy, but few have turned out to be useful in the clinical setting. The use of a combination of biomarkers may improve specificity and sensitivity for early detection of ovarian cancer. Thus, in the last few years, several multiplex assays integrating different candidate biomarkers, sometimes associated with radiological and clinical features, have been developed (such as ROMA). However, a number of questions about the current paradigm for biomarker development in the diagnostic setting still remain open, and in future research, utilizing pre-diagnostic samples for discovery may be necessary to develop validated early detection panels. Despite the wide abundance of candidate-biomarkers, only a few proteins have been validated for clinical use by the FDA. Among these, HE4 has been approved to monitor recurrence or progression of epithelial ovarian cancer, and the OVA-1 test has been cleared to assess preoperatively the risk of ovarian cancer among women with known pelvic masses. At present, the development of an effective strategy for early detection of ovarian cancer is still a work in progress. On the other hand, proteomics have given useful insight into the perturbations of signaling pathways within tumor cells, allowing for the discovery of new drug targets and helping to select patients who truly benefit from select molecular therapy. However, this wealth of information has yielded little in changing clinical care from a treatment perspective.In conclusion, parallel to the increasing optimism for proteomic expression profiling, an increasing need for accurate and rigorous research is required to answer clinically relevant questions, since, to date, integrating the biochemical information into clinical management still remains an unresolved challenge.


It is caused by protozoan parasites of the genus Plasmodium, which have a complex life cycle in a vertebrate host and a mosquito vector. Malaria parasites are haploid throughout most of this life cycle, replicating by asexual multiplication twice in a mammalian host: in liver hepatocytes (pre-erythrocytic schizogony) and within red blood cells (blood stage schizogony), and once in the mosquito (sporogony). The essential sexual stage occurs at the transmission from vertebrate to insect. Some asexual blood stage parasites develop into either male or female gametocytes (the precursor sex cells) and following ingestion in a mosquito blood meal differentiate further into gametes in the lumen of the mosquito's gut, where fertilization takes place. The core processes of gametocytogenesis, gamete activation, exflagellation, fertilization, and zygote formation are conserved across the species from the human parasite Plasmodium falciparum to the rodent parasite Plasmodium berghei, which is an attractive laboratory model, in part because of its ease of genetic manipulation and the shorter time for the maturation and differentiation of the sexual stages. Here, we focus largely on recent functional studies using reverse genetics that have uncovered many aspects of the parasite's sexual development.Malaria is a devastating global disease with several hundred million clinical cases and just under 1 million deaths each year , and then cytokinesis produces daughter merozoites that burst out and invade fresh erythrocytes. However, a sub-population of intracellular parasites that forego mitosis undergoes gametocytogenesis (the production of gametocytes) in preparation for the sexual phase. The master regulator(s) of this commitment are completely unknown, but gametocytogenesis can be induced chemically or by culture conditions in vitro. The process involves environmental stress responses, depends on parasite density and genetic variation, and is mediated by a number of signaling pathways pfs16, pfg27, and SET) have all implicated the previous schizont stage in commitment to gametocytogenesis pfg377, expressed only in females and with a role in egress from the erythrocyte, and α-tubulin II, expressed only in males. Gene disruption studies of other putative gametocyte developmental markers in P. falciparum  include gene implicated in gametocytogenesis (pfgig) (deletion resulted in reduced gametocyte production) and male development gene 1 (pfmdv-1)   controlled by gametocyte-specific promoters  have identified many genes expressed in sexual stages, and many of these have a function specific to these stages , mitogen-activated protein kinase 2 (map2), and SR protein kinase (srpk) have vital roles in DNA replication and axoneme assembly, cytokinesis and axoneme-mediated motility, and exflagellation, respectively P. falciparum, map2 is essential for asexual replication in the bloodstream, so there may be species-specific differences in the roles of different kinases Ingestion of the blood meal by the mosquito activates the gametocytes to produce gametes in the midgut. Activation of both male and female gametocytes is due to a drop in temperature, a rise in pH and calcium concentration, and the presence of the mosquito-derived metabolic intermediate xanthurenic acid p48/45, pf16, hap2/gcs1, and actin II. Deletion of pf16, a gene coding for an armadillo repeat motif (ARM) protein of the flagellum central apparatus, results in abnormal movement and reduced fertility, but does not lead to complete sterility p48/45 coding for a 6-Cys repeat domain protein, or hap2/gcs1 coding for a conserved plant-sterility gene, results in sterility due to the male gamete being unable either to attach (p48/45) hap2/gcs1) actin II is shown to affect egress of the male gamete from the host cell Gene disruption studies have implicated other specific proteins in male gametogenesis and gamete motility and fertility, including those coded by the genes As with the male, the female gametocyte is activated by exposure to mosquito factors. DNA replication does not occur but the gamete exits the erythrocyte, allowing the male gamete to attach and fuse. Nuclear fusion in the zygote is followed by DNA replication and meiosis dozi (development of zygote inhibited) and cith (homolog of worm CAR-I and fly Trailer Hitch) resulted in hundreds of normally translationally repressed transcripts to be targeted for degradation, with deleterious effects on zygote formation Aspergillus (NIMA)-related protein kinases (nek2 and nek4) have also been shown to be vital for meiosis in zygote development and ookinete maturation. Other protein kinases that play a role in ookinete development and maturation include those coded by the genes gak and pk7Gene deletion studies have defined a number of genes that are vital to zygote and ookinete development Functional studies have uncovered a number of candidate targets for drug and vaccine development; however, parasite resistance to current antimalarial drugs will develop, and the first potential malaria vaccine is only in Phase 3 trials. Nonetheless, the sexual stage of parasite development is increasingly being considered to be a key component in future campaigns to block transmission, and to eliminate and eventually to eradicate malaria.cdpk4 and map2), regulators of DNA replication prior to meiosis (nek2 and nek4), or PKG. Proteins on the gamete surface such as p48/45 or hap2/gcs1 may be vaccine candidates, since antibodies to them induced by immunization could interfere with fertilization. The studies reviewed here are part of the ongoing intensive research to understand sexual development in the malaria parasite with the potential to make a significant contribution in the fight against malaria through the design of intervention strategies for blocking malaria parasite transmission.Potential drug targets include proteins coded by genes described in this review, such as the kinase regulators of exflagellation (