E(spl)D mutation encoding the truncated protein M8*, which potently inhibits Ato at stage-1 of R8 birth. We also provide evidence that PP2A likely targets the MAPK site. Thus multi-site phosphorylation controls timely onset of M8 repressor activity in the eye, a regulation that appears to be dispensable in the bristle. The high conservation of the CK2 and MAPK sites in the insect E(spl) proteins M7, M5 and Mγ, and their mammalian homologue HES6, suggest that this mode of regulation may enable E(spl)/HES proteins to orchestrate repression by distinct tissue-specific mechanisms, and is likely to have broader applicability than has been previously recognized.The specification of patterned R8 photoreceptors at the onset of eye development depends on timely inhibition of Atonal (Ato) by the Enhancer of split (E(spl) repressors. Repression of Ato by E(spl)-M8 requires the kinase CK2 and is inhibited by the phosphatase PP2A. The region targeted by CK2 harbors additional conserved Ser residues, raising the prospect of regulation via multi-site phosphorylation. Here we investigate one such motif that meets the consensus for modification by MAPK, a well-known effector of Epidermal Growth Factor Receptor (EGFR) signaling. Our studies reveal an important role for the predicted MAPK site of M8 during R8 birth. Ala/Asp mutations reveal that the CK2 and MAPK sites ensure that M8 repression of Ato and the R8 fate occurs in a timely manner and at a specific stage (stage-2/3) of the morphogenetic furrow (MF). M8 repression of Ato is mitigated by halved EGFR dosage, and this effect requires an intact MAPK site. Accordingly, variants with a phosphomimetic Asp at the MAPK site exhibit earlier (inappropriate) activity against Ato even at stage-1 of the MF, where a positive feedback-loop is necessary to raise Ato levels to a threshold sufficient for the R8 fate. Analysis of deletion variants reveals that both kinase sites (CK2 and MAPK) contribute to ‘cis’-inhibition of M8. This key regulation by CK2 and MAPK is bypassed by the  The external sensory organs of Drosophila include the compound eye and the bristles, both of which have served as paradigms to understand mechanisms underlying neurogenesis (reviewed in –5). Studato auto-activation), from which arise groups of equipotential cells akin to proneural clusters C-encoded bHLH repressors [senseless (sens) [R8 specification involves biphasic Notch signaling . At stags . At stapressors . The E , 24. Thes (sens) .E(spl)C, a genetically dense locus [E(spl)C remains largely unchanged over ~100 MYR [Given that lateral inhibition is key to patterning of the eye and bristles, several studies sought to analyze modes of action of E(spl) proteins –29. Thesse locus , 30–32, se locus , 34. As se locus  has beense locus , 29. These locus , 37, a v~100 MYR –41, a tim8 allele E(spl)D is combined with the gain of function Notch allele, Nspl [E(spl)C. The E(spl)D mutation encodes M8*, a truncated protein lacking the 56 residue C-terminal domain, the CtD [E(spl)D, indicating that CtD removal elicits hyperactivity. Studies from our laboratory, identifying CtD phosphorylation by protein kinase CK2 revealed key insights into the mechanisms underlying hyperactivity of M8*.Of relevance to studies reported here, three E(spl) members, M8, Mγ, Mδ, are expressed in the MF during R8 selection, but ectopic expression of only Mδ (at double dose) impairs R8 birth ; a singlle, Nspl . It was, the CtD , 44. ImpE(spl)D [CK2 phosphorylates M8 at Ser159 in the CtD, at a site conserved in M7 and M5 . ReplaceE(spl)D , 46, 47.E(spl)D . PhosphoE(spl)D , hallmarFollow-up studies revealed that unlike its effects at stage-2/3 of the MF, expression of M8-S159D at stage-1 has no effect on R8 birth or eye development . This reegfr-/- clones [egfr or its downstream effectors such as ras and raf [pnt-/- clones [We suspected that the putative/predicted MAPK site may be of importance because a body of work implicates EGFR/MAPK signaling in birth of patterned R8s. First, it has been well documented that cells at stage-2/3 of the MF harbor active  MAPK –54. This- clones , 56 demo and raf . However- clones . Finally- clones , a locat- clones . The tarThe studies we describe here sought to evaluate the importance of the predicted MAPK motif in M8 activity. Site-specific and deletion mutants of CK2 and MAPK sites of M8 have been expressed at different stages of the MF to reveal the roles of these two kinase motifs during R8 specification. Our studies indicate that modifications at both CK2+MAPK sites control ‘cis’-inhibition of M8. These studies are consistent with the possibility that EGFR signaling potentiates Notch pathway activity during R8 patterning, that these two signaling pathways do not always act in an antagonistic manner, and suggest a likely molecular target for activated MAPK at the first step in retinal histogenesis.S159DCD) or MAPK (P149LSP) consensus motifs were generated by inverse-PCR. M8 variants were flanked by EcoRI and BamHI sites 5’ and 3’ to the coding region, respectively. In addition, all M8 variants incorporated a KOZAK consensus (CAAC) immediately 5’ to the ATG codon for efficient expression. All constructs were fully sequenced to confirm the presence of only the intended mutations.M8 variants with Ala/Asp substitutions at the known phosphorylation site for CK2 (Ser159) and/or that predicted for MAPK (Ser151) were generated by PCR based site-directed mutagenesis. M8 variants harboring deletions of the CK2 , hH10Gal4 , egfrf2 (B2768) and Ellipse aka egfrElp or Elp1 (B5144), were obtained from Bloomington Stock Center. wdbEP3559 flies were a gift from Amita Sehgal (University of Pennsylvania).The Gal4 drivers were obtained from Bloomington Stock Center . These drivers are xas, ref ), and sc[scaGal4 . The EGFt-test and ANOVA.All crosses were performed on standard Yeast-Glucose medium at 24°C, unless indicated otherwise. Fly heads were dehydrated by sequential passage through a graded alcohol series for 24 hours each (25%-50%-75%-100%), and finally passed through Hexamethyldisalinaze. Heads were mounted on EM stubs, dried for 24 hours, sputter coated with gold and examined with a JEOL-6400 scanning electron microscope at an accelerating voltage of 10–20kV. Images were processed with Adobe Photoshop and collated in Adobe Illustrator. The reduced eye was quantified from ≥20–25 adult flies that were photographed using a Leica MZ16 stereomicroscope equipped with a Leica DFC-480 digital camera. Facet numbers were counted, as described . A similEye-antennal imaginal discs were isolated from late third instar larvae and processed as described  with minato transcriptional control [E(spl)D mutation that elicits severe loss of R8s and the eye, phenotypes fully mimicked by stage-1 expression of a UAS-M8* transgene  whose expressivity correlates to endogenous E(spl) genes  genes . The difl) genes  reflects9-68Gal4 . As repo9-68Gal4 , 46, 48,hH10Gal4) is without effect  an Asp at the CK2 site does not suffice to activate M8. First, expression of M8-S159D at stage-1 , but not at stage-1 , effects that qualitatively mimic random insertions.We adopted site-specific (ϕC31) integration to enable controlled comparisons of Ala/Asp variants at multiple kinase sites. We chose the ϕC31 site at 68E, one of ‘moderate’ expressivity , 72, as The P-domain of M8 contains four Ser residues, which are invariant in 12 Drosophila species and the stalk-eyed fly Teleopsis  that divWe previously reported that the CK2-refractory variant (M8-S159A) does not mimic unmodified M8, but elicits a rough eye due to dominant-negative (DN) activity . We thusscaGal4 did not elicit any reduced eye  and M8-S151A+S159D (refractory to MAPK but mimics CK2). Unlike M8-S159D, expression of M8-S151A+S159D with le types , 29. ThuscaGal4 at 24C or at 18C where Gal4 activity is attenuated  and the dual-kinase mimic (M8-S151D+S159D). However, both elicited embryonic lethality when expressed with tenuated . Lethaliost PNCs .109-68Gal4, a weaker stage-2/3 driver. Flies expressing all four M8 variants were viable and elicited a range of eye defects  flies or the parental M8 transgenic lines  elicits a strong loss of the eye at stage-1, whereas the CK2 mimic M8-S159D is without effect . As  of the MAPK-mimic (M8-S151D) or the dual-kinase mimic (M8-S151D+S159D) elicits a reduced eye , but is lethal when homozygous. The expectation was that if EGFR/MAPK signaling were to activate M8, then halved EGFR levels might attenuate the amount of active MAPK, and diminish phosphorylation of M8. If so, M8-S159D (CK2 mimic with an intact MAPK site) should become hypo-phosphorylated in egfrf24/+ flies, dampening the severity of its reduced eye. In contrast, the MAPK-mimic (M8-S151D) and the dual-kinase mimic (M8-S151D+S159D) should be refractory to halved EGFR levels by virtue of the Asp at the MAPK site.We next tested for a role for EGFR signaling by employing scaGal4, given the significant effect on the eye with this stage-2/3 driver. Compared to its effects in egfr+ flies , diminishing the possibility that a mutation other than that in egfr is responsible for the observed modulation.Both predictions bear out. The CK2 mimic M8-S159D was expressed with r+ flies , the redtatively  and quantatively . Staininr+ flies  elicits ckground , althougckground . Similar109-68Gal4 due to lethality upon expression with scaGal4 . Staining of eye discs revealed that these two variants elicit R8 defects indistinguishable from those described in egfr+ flies  and the dual kinase mimic (M8-S151D+S159D), but utilized the driver Gal4 see . HoweverEllipse (Elp1), an egfr gain of function allele, because Elp/+ flies on their own exhibit a reduced eye [We considered, but did not conduct studies on M8 variants in the presence of uced eye , 77, 78,uced eye , 47.M8-ΔCK2/+ nor M8-ΔMAPK/+ flies exhibit a perturbed eye (yellow inset in As M8* and MAPK variants (M8-S151D or M8-S151D+S159D) elicit R8/eye defects at stage-1 Figs , we wishscaGal4 also elicited embryonic lethality, but no such effect was observed with M8-ΔCK2 , M8-ΔCK2 is largely inactive, whereas M8-ΔMAPK elicits a ~50% reduction of the eye field , a PP2A regulatory subunit, suppresses (rescues) the R8/eye defects of the CK2 mimic M8-S159D . ModulaUAS-Wdb modulates the reduced eye of the MAPK-mimic (M8-S151D) or the dual-kinase mimic (M8-S151D+S159D). The reduced eye of either variant upon co-expression of UAS-Wdb  responsible for modification of the PXS151P motif. Nevertheless, direct demonstration that MAPK targets the similar (PXSP) motif in murine HES6 (m8 is expressed in the MF and E(spl)D potently impairs R8 birth and eye development [The studies described here reveal that the highly conserved MAPK site in E(spl)-M8 is important for repressor activity in the developing eye, a regulation that appears to be dispensable during bristle development. While this overall conclusion is supported by our genetic studies, we note that direct biochemical evidence for modification of M8 by MAPK remains to be established, and neither is it known which of the five Drosophila genes encodes ine HES6 , a proteelopment , and b) elopment , 79, 80.egfr mutant backgrounds  sites in wild type and nds Figs  and 3–7.nds Figs , these fnds Figs  raises tThe strong conservation of sites for these two kinases in M7, M5 and Mγ reinforce our view that this regulation has a broader impact on E(spl) proteins than has been recognized. The E(spl) members have been viewed as partially redundant dosage-dependent effectors of Notch signaling. This view, reflects the observations that the manifestation of neurogenic phenotypes needs simultaneous loss of multiple members , 34, 83,‘signal amplitude’ (amount of active M8), whereas the latter would control ‘signal duration' (how long M8 remains active). Our studies, which do not discriminate between these possibilities, nevertheless reveal that multi-site phosphorylation controls repression by M8, extending the proposal that ‘timing’ is of essence to Notch signaling [The studies we describe here expand on our understanding of the role of two kinase sites within the P-domain of M8. Our data suggest a revised model wherein conversion of ‘cis’-inhibited M8 into an active repressor of Ato depends upon phosphorylation of the CK2 and MAPK sites, and that the precise activation of this Ato repressor at stage-2/3 of the MF occurs in response to EGFR signaling . If so, ignaling , 88.We note that our analyses do not reveal the order of M8 phosphorylation, which can only be revealed by antibodies that recognize not only specific isoforms but also specific  phosphorylated states. Our efforts to raise such antibodies have not been successful , as they display cross-reactivity with unmodified M8 and other members with conserved CtDs . We discuss the implications of our findings in the context of two pathways, Notch and EGFR, which are vital not only for R8 ontogeny, but also for later stages of retinal histogenesis.the driver for lateral inhibition and cell fate determination is indisputable (reviewed in [Su(H) or the E(spl)C impairs lateral inhibition and elicits the specification of ‘twinned’ R8s and bristle SOPs. The developmental outcome of loss of Notch is more complicated, in particular, at the onset of retinal histogenesis, given dual roles of this receptor for R8 birth. As stated above D largely enhances transcript levels through stabilization [E(spl)D are unlikely to reflect earlier than normal expression. Constitutively active M8* would disrupt the Ato-Da cooperativity [E(spl)D or stage-1 expression of M8* seems consistent with our model, and underscores the prescient suggestion of Giebel and Campos-Ortega [The key role of Notch as iewed in –95). Detbove see , Notch ilization , suggestrativity . The loss-Ortega  that thiachaete scute Complex (ASC) occurs in response to pre-pattern factors [This, in turn, raises the question on why MAPK is dispensable for M8 regulation in the bristle? While the answer(s) to this question remain unclear, we speculate that this may reflect intrinsic differences in Notch functions in retinal versus bristle development. In the latter sensory organ, expression of the proneural activators encoded by the  factors , i.e., i factors , the MCs factors , and the factors . MoreoveElp allele of egfr by Baker and coworkers who have proposed that ‘some level’ of EGFR pathway activity is necessary for lateral inhibition to occur in a timely manner [egfr, ras or raf indeed elicit supernumerary R8s, no such effects are seen upon loss of pnt or yan. Analysis of R8 birth in clones of MAPKs has been precluded by cell lethality [Our work appears consistent with analysis of the y manner ,. While ethality . Neverthethality  precludiethality , and whoethality , and nucHow might MAPK precisely regulate M8 activity? Our studies on deletion variants of M8 and their MF stage-specificity provide clues. We have previously proposed that inactive M8 is ‘cis’-inhibited , 100. ThThese studies for the first time demonstrate a developmental context in which the biological role of the MAPK site in E(spl)-M8 has been revealed. As shown in


Protein phosphatase 2A (PP2A) is a tumor suppressor complex that has recently been reported as a novel and highly relevant molecular target in prostate cancer (PCa). However, its potential therapeutic value remains to be fully clarified. We treated PC-3 and LNCaP cell lines with the PP2A activators forskolin and FTY720 alone or combined with the PP2A inhibitor okadaic acid. We examined PP2A activity, cell growth, prostasphere formation, levels of PP2A phosphorylation, CIP2A and SET expression, and AKT and ERK activation. Interestingly, both forskolin and FTY720 dephosphorylated and activated PP2A, impairing proliferation and prostasphere formation and inducing changes in AKT and ERK phosphorylation. Moreover, FTY720 led to reduced CIP2A levels. Treatment with okadaic acid impaired PP2A activation thus demonstrating the antitumoral PP2A-dependent mechanism of action of both forskolin and FTY720. Levels of PP2A phosphorylation together with SET and CIP2A protein expression were studied in 24 PCa patients and both were associated with high Gleason scores and presence of metastatic disease. Altogether, our results suggest that PP2A inhibition could be involved in PCa progression, and the use of PP2A-activating drugs might represent a novel alternative therapeutic strategy for treating PCa patients. In fact, several mechanisms of PP2A inhibition in cancer cells have been described, including alterations affecting all of the PP2A subunits and deregulation of endogenous PP2A inhibitors ,2,3,4. Io models . Overexpo models . These otastasis . These rtastasis . Furthertastasis ,8. Moreotastasis ,10, and tastasis . Howevertastasis ,14,15,16tastasis ,13,14, oDespite the existence of data suggesting the relevance of PP2A activation status and its tumor-suppressor role in PCa, its potential therapeutic value as a molecular target in this disease requires clarification. Thus, it would be worthwhile to evaluate the antitumor effects of PP2A-activating drugs, which have shown their efficacy in other cancers with similar PP2A alterations, and their potential clinical use in PCa patients. In this work, we show that the PP2A activators forskolin and FTY720 (its unphosphorylated form) induced antitumor effects dependent on PP2A activation in PCa cells. The use of these drugs decreased cell growth; led to changes in PP2A, AKT, and ERK phosphorylation status and expression levels of the PP2A inhibitor CIP2A; and reduced prostasphere formation capability. Thus, these observations support the potential benefits that could be derived from the use of PP2A activators as an alternative therapeutic strategy in PCa.To study the potential therapeutic value of PP2A activation in PCa, PC-3 and LNCaP cells were treated with the PP2A activators forskolin and FTY720 or vehicle (DMSO). Phosphatase assays to quantify PP2A activity levels confirmed that forskolin and FTY720 treatment led to PP2A activation . As a coWe next used western blot to analyze whether forskolin or FTY720 treatments could affect the phosphorylation status of previously described PP2A targets. We observed that both drugs induced decreased phosphorylation (activity) of AKT and ERK without affecting their expression levels. Moreover, OA treatment rescued AKT and ERK1/2 phosphorylation in forskolin- and FTY720-treated PC-3 cells C. Of intWe next analyzed PP2A, SET and CIP2A after treatment with forskolin or FTY720 alone or in combination with OA in PC-3 and LNCaP cell lines. Whereas the PP2A phosphorylation at Y307 was negatively affected by both forskolin and FTY720 treatments, only FTY720 was able to decrease CIP2A levels. No changes were observed in SET and PP2A expression . In addiTo further investigate the potential therapeutic effects of PP2A activation in PCa, we assessed prostasphere formation in PC-3 and LNCaP cells treated with forskolin or FTY720. We observed that both drugs led to decreased prostasphere-formation capability in PC-3 and LNCaP cells A. IntereIn order to assess whether the use of PP2A-activating drugs could be proposed as an alternative therapeutic strategy for treating PCa, we next evaluated CIP2A and p-PP2A in a set of 24 PCa patients. Patient characteristics are included in Although restoration of PP2A activity has emerged as an alternative therapeutic strategy for human leukemias in recent years ,20, its As p-PP2A and CIP2A were modulated by the PP2A-activating drugs, we next analyzed their status in a set of PCa specimens. CIP2A is an endogenous PP2A inhibitor, and previous reports indicate that CIP2A overexpression is associated with high-risk Gleason PCa . InteresIn conclusion, our results suggest that PP2A is a relevant molecular target for the development of novel therapeutic strategies based on the use of PP2A activators in PCa. Moreover, PP2A hyperphosphorylation and CIP2A overexpression are molecular mechanisms that contribute to inhibit PP2A in PCa and determine a more aggressive disease. However, the small number of cases included is a limitation and impairs assessment of the potential prognostic value that these alterations could have in PCa; therefore, their usefulness for prognosis and their ability to predict response to therapy in PCa patients should be further investigated in future studies with a larger cohort of cases.2 atmosphere. Media were supplemented with penicillin G (100 U/mL), and streptomycin (0.1 mg/mL). Cells were treated with the following reagents: FTY720 (10 µM), forskolin (40 µM), and OA (2.5 nM) .The human PCa cell lines PC-3 (ATCC CRL-1435) and LNCaP (ATCC CRL-1740) were kindly provided by David Olmos . Cell lines were maintained in RPMI-1640  with 10% fetal bovine serum (FBS) and were grown at 37 °C in a 5% COThe study comprised consecutive formalin-fixed, paraffin-embedded samples of prostate tumor samples obtained from 24 patients who underwent radical prostatectomy. The samples were obtained from Fundacion Jimenez Diaz Biobank . Clinical data were collected by oncologists using patients’ clinical records. All investigations were carried out in accordance with the Declaration of Helsinki of 1975. Informed consent was provided in all cases. The ethical committee and institutional review board approved the project.g, 15 min, 4 °C), denatured, and subjected to SDS-PAGE and western blotting. The antibodies used were mouse monoclonal anti-PP2A , rabbit monoclonal anti-p-PP2A-C Y307 , rabbit polyclonal anti-AKT, rabbit polyclonal anti-ERK , rabbit polyclonal anti-SET , rabbit polyclonal anti-pAKTThr308, rabbit polyclonal anti-pERK1/2Thr202/Tyr204 , rabbit polyclonal anti-CIP2A, and mouse monoclonal anti-β actin . Proteins were detected with the appropriate secondary antibodies conjugated to alkaline phospatase  by chemiluminescence using Tropix CSPD and Tropix Nitro Block II .Protein extracts were isolated using TRIzol Reagent  following the manufacturer’s indications; these were then clarified  following the manufacturer’s indications.PP2A assays were performed with cell lysates (50 μg) using a PP2A immunoprecipitation phosphatase assay kit , following the manufacturer’s instructions.For the generation of prostaspheres, 10,000 cells were plated in 6-well ultra-low attachment plates . PC-3 and LNCaP cells were grown in serum-free medium DMEM/F12 + GlutMAX™-I  containing 1% N2 , 2% B27 , 20 ng/mL human FGF  and 50 ng/mL EGF . After 7 days, the number of colonies formed was quantified to evaluate prostasphere formation.Total RNA was isolated using RecoverAll Total Nucleic Acid Isolation kit  according to manufacturer’s instructions. Samples were reverse transcribed using the High Capacity Reverse Transcription Kit  and gene expression levels were quantified by quantitative real-time PCR using TaqMan Gene Expression Assays  specific for CIP2A and GAPDH as internal control. Relative gene expression was calculated according to the comparative cycle threshold (Ct) method.Tissue sections (3 µm) were placed on plus-charged glass slides. After deparaffinization in xylene and graded alcohols, heat antigen retrieval was performed in pH 9 EDTA-based buffer . Endogenous peroxidase was blocked by 0.03% hydrogen peroxide for 5 min. Slides were incubated with the same primary antibody against CIP2A or p-PP2A for 60 min at room temperature, followed by the appropriate anti-Ig horseradish peroxidase-conjugated polymer . Sections were visualized with 3,3′-diaminobenzidine as a chromogen. All stainings were performed in a Dako Autostainer. Sections incubated with normal non-immunized rabbit immunoglobulins were used as negative controls. CIP2A (1:5000), SET (1:5000) and p-PP2A (1:2000) antibody sensitivities had been calculated in a range of crescent dilutions of primary antibody. A semiquantitative histoscore was calculated by estimating the percentage of tumor cells positively stained with low, medium, or high staining intensity. The final score was determined after applying a weighting factor to each estimate. The following formula was used: histoscore = (low %) × 1 + (medium %) × 2 + (high %) × 3 and the results ranged from 0 to 300.t-test analyses. Data represented are mean of three independent experiments ± s.d. A P value less than 0.05 was considered statistically significant.Statistical analyses were performed using SPSS 20 for Windows . Comparisons were carried out by chi-square and 2-sided


Diastolic function (DF) is not routinely assessed by cardiac MR (CMR), but is critical for assessment of common disease states such as heart failure or hypertrophy. At present, special pulse sequences  are necessary for CMR for diastolic function assessment. The purpose of this study was to assess cine CMR volumetric assessment of DF using a 3D model-based analysis, in reference to 2D phase contrast and echocardiography (echo).In this IRB-approved retrospective study, consecutive subjects with HCM who underwent both echo and cardiac MRI from December 2011 to June 2013 were identified from the radiology database. Cine steady state free precession (SSFP) images  were analyzed using a semiautomated 3D model-based analysis of left ventricular filling curve . Transmitral flow curves were measured from 2D phase contrast (PC) using dedicated software . DF on echo was obtained from the medical records. Correlation of E/A ratio and E/E' ratio among CIM, 2D PC, and echo was calculated using Pearson's correlation coefficient. Differences in CIM E/A and E/E' ratios among DF groups were analyzed using ANOVA.64 subjects were included; 49 males (76.6%) and 15 females (23.4%), with mean age of 53.3 years. CIM E/A and E/E' ratios were analyzable in 62 patients and 38 patients, respectively. DF grades on echo were: grade 0 (n = 16), grade 1 (n = 9), grade 2 (n = 29), grade 3 (n = 6), and not available (n = 4). Due to abnormal cardiac morphology of HCM patients, CIM software had minor difficulties with semiautomated contour tracking, which was easily adjustable by the user. CIM performed well in tracking of valve motion. There was strong correlation between E/A ratio of CIM and 2D PC  and between E/A ratio of CIM and echo . There was no significant correlation between E/E' ratio of CIM and 2D PC  and between E/E' ratio of CIM and echo , likely due to smaller sample size. There was significant difference in CIM E/A ratio among DF groups on one-way ANOVA (p < 0.001). Difference in CIM E/E' ratio among DF groups approached significance (p = 0.106).Semiautomated 3D model-based analysis of left ventricular filling curve is a promising technique for evaluation of DF in patients with hypertrophic cardiomyopathy, demonstrating good correlation with 2D phase contrast and echo in assessment of E/A ratio. DF can be measured accurately using routine cine SSFP images and semiautomated analysis of left ventricular filling curve, without the need for specialized pulse sequences.Linda Chu received research support from RSNA R&E Foundation Fellow Grant.


A 73-year-old man was confirmed to have an influenza A (H7N9) virus infection, and the causative agent A/Beijing/02/2014(H7N9) virus was isolated. Genetic and phylogenetic analyses revealed that the virus belonged to a novel genotype, which probably emerged and further reassorted with other H9 or H7 viruses in poultry before transmitting to humans. This virus caused a severe infection with high levels of cytokines and neutralizing antibodies. Eventually, the patient was cured after serially combined treatments. Taken together, our findings indicated that this novel genotype of the human H7N9 virus did not evolve directly from the first Beijing isolate A/Beijing/01/2013(H7N9), suggesting that the H7N9 virus has not obtained the ability for human-to-human transmissibility and the virus only evolves in poultry and then infects human by direct contact. Hence, the major measures to prevent human H7N9 virus infection are still to control and standardize the live poultry trade. Early antiviral treatment with combination therapies, including mechanical ventilation, nutrition support and symptomatic treatment, are effective for H7N9 infection. Recent studies showed that the internal genes of the H7N9 virus have continued to undergo dynamic reassortments with the poultry H9N2 viruses345338Since March 2013, novel influenza A H7N9) viruses have emerged in China and spread quickly, causing severe respiratory disease in humans1N9 viruseA 73-year-old man who worked as a live poultry seller and butcher in Beijing, China was diagnosed with an influenza A (H7N9) virus infection confirmed by detection of the H7N9 virus in the laboratory. The patient was an alcoholic with a past medical history of chronic bronchitis and coronary heart disease. The illness began with flu-like symptoms, including a high fever (38.3 °C), cough with yellow-white phlegm and feeling fatigued on 30 January 2014. Because the detection of the influenza A virus universal antigen was negative on the throat-swab by means of the immune colloidal gold technique and the radiologic findings revealed bronchitis, the patient was treated with anti-infective therapy by an intravenous injection of moxifloxacin. However, that treatment did not take effect, and the symptoms gradually worsened.On 5 February 2014, the patient appeared with hyperpyrexia (maximum temperature 40.0 °C), coughing with bloody sputum and dyspnoea with a low oxygen saturation (88.8%). The H7N9 viral RNA was positive in the oropharynx swab confirmed by the real-time RT-PCR method according to the protocol of the Chinese CDCA H7N9 virus, A/Beijing/02/2014(H7N9) (abbreviated as BJ02 thereafter), was isolated and identified after one passage propagation in eggs. To further study the gene evolution of the H7N9 virus, the whole genome was amplified and sequenced. Genetic alignments showed that the HA, NA, PB1, PA, NP and NS genes possessed the highest genetic similarities (99.33–99.74%) with other H7N9 virus genes. However, the PB2 and M genes possessed the highest nucleotide similarities  with H9N2 isolates . FurtherThe phylogenetic analyses showed that the HA and NA gene sequences of BJ02 clustered with previously identified human-infecting H7N9 viruses . The phyThe level of serum neutralizing antibody (NAb) and cytokines in flu patients play pivotal roles in disease progression and recovery101112131415161718The evaluation of serum cytokines in the H7N9 patient revealed that the levels of IL-6, IL-12p40, MCP-1 and IP-10 were significantly increased on 7, 15 and 18 d.a.o. compared with the normal ranges, whereas the IFN-γ, IFN-α and IL-17A levels did not notably increase . The levThe present data documented that the novel genotype G2.9 of the H7N9 virus (BJ02) could infect and cause severe disease in humans. In addition to the G1.5 virus (BJ01), there are at least two genotypes of H7N9 virus existing in Beijing. To date, only one G1.5 and one G2.9 virus from humans have been isolated; and the other G2.9 strain (SC537) was first isolated from chickens. Although high homology is shared between BJ02 and SC537 , the PB2To date, a total of 29 genotypes of H7N9 viruses, including BJ01 (G1.5) and BJ02 (G2.9), have been identified according to our classification and nomenclature systemIn addition, the oseltamivir resistance mutation (R292K) in the NA protein of BJ02 could not be found after 3 days of oseltamivir usage; however, the mutation emerged in the A/Shanghai/1/2013(H7N9) strain just after one day treatment with oseltamivir10In general, viral and host factors contribute to disease severity and outcomes. A previous multivariate analysis revealed that the presence of coexisting medical conditions (such as chronic heart disease and chronic obstructive pulmonary disease) were the only independent risk factors for severe illness with H7N9 infection212324252728113110112333435Although this was a severe H7N9-infected case with cytokine storm-like appearances and multiple organ failure, the patient was eventually cured after combination therapy with antivirals, mechanical ventilation, supportive nutrition and symptomatic treatment. In our study, the 73-year-old patient was not prescribed oseltamivir until day 6 d.a.o. because the H7N9 virus was not identified at the onset of his illness. According to the clinical finding reports of the H7N9-infected patients in 2013, the median time of the initiated antiviral therapy was 7 days after the onset of illnessA previous study showed that the early and rapid induction of NAb was correlated significantly with better clinical outcomes14The H7N9 influenza virus is spreading, evolving and becoming widespread among chickens in ChinaA suspected case of H7N9 influenza virus infection was confirmed by a real-time RT-PCR assay in the Beijing Centers for Disease Control and Prevention (CDC). The epidemiologic and clinical data were collected. A confirmed case was defined as evidence of pneumonia with H7N9 viral RNA or isolation of H7N9 virus from respiratory specimens. Informed consent was obtained from all participating individuals. This experimental protocol was approved by the local Ethics Committee of Beijing Ditan Hospital, Capital Medical University. The methods were carried out in accordance with the approved guidelines.http://www.megasoftware.net). The genotype analysis of the H7N9 virus was classified by the criteria previously describedAn oropharynx swab, collected on day 8 after the onset of flu-like symptoms, was used for virus isolation. The virus was propagated in 10-day-old specific pathogen free (SPF) embryonated chicken eggs and MDCK cells for 48 to 72 hours at 35 °C, respectively. For genetic analysis, the complete gene segments were amplified using improved primers  based on38Sera from the H7N9 patient were consecutively collected on 7, 15 and 18 d.a.o. for kinetic evaluations of the neutralizing antibodies and cytokine levels. A pandemic H1N1 severe case, onset on 2 February 2014, was used as a control, and the serum was collected on 15 d.a.o. The neutralizing antibodies (NAbs) were detected by Microneutralization Assays with A/Anhui/1/2013 (H7N9) and A/California/04/2009 (H1N1) viruses in MDCK cells, according to the previously described methodHow to cite this article: Bi, Y. et al. A new reassortment of influenza A (H7N9) virus causing human infection in Beijing, 2014. Sci. Rep. 6, 26624; doi: 10.1038/srep26624 (2016).


For this purpose, the Factor V Leiden (FVL) (rs6025), Prothrombin G20210A (rs1799963), MTHFR C677T (rs1801133), PAI-1 4G/5G (rs1799889), ACE I/D (rs1799752), eNOS E298D (rs1799983), and Apo E E2/E3/E4 (rs429358) polymorphisms were genotyped and correlated in spontaneously aborted fetal materials, their mothers and fertile women. Twenty three abortion materials, 22 women with ≥1 unexplained fetal loss, and 22 control subjects with at least two healthy term infants as a control group were studied. Target SNPs for each gene were analyzed by real time-PCR technique after genomic DNA isolation from maternal blood-EDTA, control group blood-EDTA and spontaneously aborted fetal tissues. Some cases had a single thrombophilic polymorphism, but the rest of the patients and fetal materials had combined thrombophilic polymorphisms. The PAI-1 4G/5G+4G/4G (P= 0.0017), 4G/4G (P= 0.0253), eNOS 894GT+894TT (P=0.0011) genotypes and T allele (P=0.0185), Apo E E3/E4+E3/E2+E2/E4 (P<0.0001) genotypes, E2 (P<0.0001) and E4 (P<0.0001) alleles were higher in spontaneously aborted fetal materials when compared to their mothers and control group. The Factor V Leiden rs6025, Prothrombin G20210A, MTHFR C677T, ACE I/D genotypes were different for each group but not statistically significant due to relatively small size of the samples (P>0.05). Our results indicated that combined thrombophilic gene variations may be associated with increased risk for spontaneous abortions and results need to be confirmed by larger sample size.The aim of this study was to investigate the possible role of multiple inherited thrombophilic gene variations in women with unexplained spontaneous abortions Spontaneous abortion is defined as a clinically recognized pregnancy loss before the gestation week of twenty. The World Health Organization (WHO) defines it as expulsion or extraction of an embryo or fetus weighting 500 g or less . A normaRecurrent pregnancy loss (RPL) is an important obstetric complication which is defined as two or more miscarriages of under 20 weeks of gestation . ThromboFVL) rs6025, Prothrombin G20210A (rs1799963), MTHFR C677T (rs1801133), PAI-1 4G/5G (rs1799889), ACE I/D (rs1799752), eNOS E298D (rs1799983), and Apo E E2/E3/E4 (rs429358) in spontaneously aborted fetal materials with their mothers and fertile cases. This is the first study about thrombophilia polymorphisms in spontaneously aborted fetal materials.In this study, we aimed to compare the thrombophilic polymorphisms in Factor V Leiden  and twenty-two fertile women as control group were included in this study. Abortion materials were obtained from only spontaneous abortions. Maternally contaminated abortion materials  were excluded from the study . Maternal contamination was detected with correlation of fetus and mother STR markers. Control group women had no spontaneous abortions and they had two or more children. All participants were included from medical genetics clinic referred from Gynecology and Obstetry clinic in Canakkale On Sekiz Mart University Hospital.Written informed consent forms were obtained from all participants and the study was approved by the ethics committee of Canakkale On Sekiz Mart University.GenotypingFVL) (rs6025), Prothrombin G20210A (rs1799963), MTHFR C677T (rs1801133), PAI-1 4G/5G (rs1799889), ACE I/D (rs1799752), eNOS E298D (rs1799983), and Apo E E2/E3/E4 (rs429358) polymorphisms.All samples were genotyped for Factor V Leiden  and DNA template were used for real-time ampliﬁcation. The multiple PCR consisted of a denaturation step of 10 min at 95 oC, followed by 45 cycles of 5 s at 95 oC,10 s at 60 oC, and 15 s at 72 oC, and a melting step of 20 s at 95 oC, 20 s at 40 oC, a continuous mode at 85 oC, a cooling step of 30 s at 40 oC for all four regions. A software program  was used for the detection of the mutated and normal genotype proﬁles of the target gene in the groups included in the study. The wild, heterozygous and homozygous profiles were analysed in channel 640 with melting curve analysis  technique in Light Cycler 2.0  using Light Mix kits . Brieﬂy, Light Cycler, Roche Fast Start Master mix, master mix  and P-values were used to estimate the risk for the polymorphisms in the groups.Peripheral blood-EDTA samples from mothers and control cases (fertile women) and tissue samples from aborted materials were examined for genotyping in the current study. Mean age of the cases and the weeks of abortions are provided. The mean age of the mothers were 29.6 (21-40) years and the mean age of fertile women were 37.2 (30-46) years. The mean abortion week was 9.4 (5-18) weeks, twenty abortions were in first trimester, three abortions were in second trimester. There was no other diseases in the groups.FVL), protrombin G20210A,  methylenetetra-hydrofolate reductase (MTHFR) C677T, plasminogen activator inhibitor-1 (PAI-1) 4G/5G, Angiotensin-converting enzyme I/D (ACE I/D), endothelial nitric oxide synthase (eNOS) E298D, apolipoprotein E (Apo E) E2/E3/E4 and spontaneous abortions. By real time PCR technique, we evaluated these polymorphisms in the study groups , and the results were compared between the groups. The estimate risk was examined by odds ratio.The fetus, mothers and control groups in this study were designed to determine the association between point mutations in the factor V Leiden , E2 allele frequency  and E4 allele frequency (P<0.0001 for both) were higher in aborted materials comparing with their mothers and with control group, respectively   and 4G/4G genotype (p=0.0253) were higher in aborted materials comparing with their mothers . 4G/4G gectively -4. FVL, > 0.005) .MTHFR C677T and eNOS E298D mutations, five abortion materials had both MTHFR C677T and ACE I/D mutations, eight abortion materials had both PAI-1 4G/5G and ACE I/D mutations, three abortion materials had both eNOS E298D and Apo E mutations, eight abortion materials had both ACE I/D and eNOS E298D mutations.Three abortion materials had both MTHFR C677T, PAI-1 4G/5G,  ACE I/D,  eNOS E298D, Apo E E2/E3/E4. These thrombophilic factors may have an important role in influencing the pregnancy outcome. This is the first study to report the relationship between thrombophilic factors polymorphisms and pregnancy loss in spon-taneously aborted fetal materials and their mothers.Thrombophilia is one of the coagulation disorders which predisposes individuals to develop thrombosis . It inclFVL mutation in fetuses has been reported to be associated with histologically proven placental infarction  4G/4G homozygous genotype was associated with increased risk of RPL independently from the antiphospholipid antibodies (Apo E gene polymorphisms and RPL (With an overview to this study, tibodies . In a diIn conclusion, spontaneous abortion risk is related with PAI-1 4G/5G, eNOS E298D and Apo E E2/E3/E4 polymorphisms for fetal genotypes. If fetal genotypes are effective comparing with mother genotypes, this means father genotypes are also effective for pregnancy losses. Our results indicated that combined thrombophilic gene mutations may be associated with increased risk for spontaneous abortions. There is a need for larger studies to explore the effect of thrombophilic factors in family members for pregnancy losses.


Host FcγR polymorphisms may influence HIV infection and disease progression. Here, we examine FCGR2A and FCGR3A genotypes in HIV mother-to-child transmission. Infant genotypes did not impact infection or progression, but the maternal FCGR3A genotype may influence early breastfeeding transmission risk. Background. Fc-mediated effector functions have been suggested to influence human immunodeficiency virus (HIV) acquisition and disease progression. Analyzing the role of host Fc gamma receptor (FcγR) polymorphisms on HIV outcome in mother-to-child transmission (MTCT) will increase our understanding of how host genetics may alter immune responses in prevention, therapy, and disease. This study analyzed the impact of FCGR2A and FCGR3A genotypes on MTCT in a cohort in which Fc-mediated antibody functions are predictive of infant HIV outcome.Methods. Human immunodeficiency virus-positive mothers and their infants from a historical MTCT cohort were genotyped for FCGR2A and FCGR3A. We assessed the impact of these genotypes on transmission and acquisition of HIV and disease progression using χ2 tests, survival analyses, and logistic regression.Results. Among 379 mother-infant pairs, infant FCGR2A and FCGR3A genotypes were not associated with infant HIV infection or disease progression. Maternal FCGR2A was not associated with transmission, but there was a trend between maternal FCGR3A genotype and transmission (P = .07). When dichotomizing mothers into FCGR3A homozygotes and heterozygotes, heterozygotes had a 64.5% higher risk of transmission compared with homozygotes (P = .02). This risk was most evident in the early breastfeeding window, but a trend was only observed when restricting analyses to breastfeeding mothers .Conclusions. Infant FCGR2A and FCGR3A genotypes were not associated with HIV infection or disease progression, and, thus, host FcγR genotype may not significantly impact vaccination or therapeutic regimens that depend on Fc-mediated antibody functions. Maternal FCGR3A genotype may influence early breastfeeding transmission risk, but more studies should be conducted to clarify this association and its mechanism. Recent data from human and macaque studies suggest that Fc-mediated antibody functions, such as antibody-dependent cellular cytotoxicity (ADCC), may protect from human immunodeficiency virus (HIV) infection and/or disease progression (reviewed in ). These Two polymorphic FcγRs of interest are FcγRIIa and FcγRIIIa. FcγRIIa is an activating receptor found on many cell types: monocytes, macrophages, neutrophils, and platelets, among others. The receptor has a polymorphism SNP rs1801274) in its gene (FCGR2A) that encodes either a histidine (H) or arginine (R) at amino acid position 131 01274 in . The H aThese FcγR genotypes may be particularly relevant to mother-to-child transmission (MTCT) because ADCC has been implicated in transmission risk and in infected infant disease course , 22. ThiAntiretroviral naive mother-infant pairs from the Nairobi Breastfeeding Trial  were selDNA extracted from blood , breast milk, or cervical and vaginal samples was available for most individuals –29. If nFCGR2A and FCGR3A genotypes were determined using TaqMan SNP Genotyping Assays C_9077561_20 and C_25815666_10, respectively (Life Technologies). TaqMan assays were previously verified in the laboratory by sequencing a subset of samples . Control2 tests for categorical variables and t tests with Welch's correction for comparisons of means were used to determine which cohort characteristics were associated with HIV infection and transmission. The associations between FCGR2A and FCGR3A genotypes and infection and transmission were first analyzed using χ2 tests for independence. Logistic regressions controlling for appropriate covariates  were used to determine associations between FcγR genotype and infection risk. The associations between viral load and genotype were analyzed by linear regression. Cox proportional hazards models and Kaplan-Meier estimates with log-ranks tests were used to determine the association between genotype and time to infection/time to infant mortality. A Pearson's χ2 test was used to determine whether SNPs were in Hardy-Weinberg equilibrium and to determine linkage disequilibrium between the two SNPs. Viral loads were log10 transformed for all analyses. Analyses were not adjusted for multiple comparisons because our main findings (on the impact of genotypes on infection/transmission and infant progression), set a priori, were not statistically significant [χnificant . We then10 copies/mL, P < .0001), lower CD4 counts , and were more likely to be in the breastfeeding arm of the original study  (Table P = .029), and there were more deaths during follow-up in infected infants than uninfected infants . Infected infants had an average set point viral load of 5.85 log10 copies/mL. These characteristics are similar to those found in the larger trial cohort [In this study, 379 mothers and their corresponding infants from the Nairobi Breastfeeding Trial  were genl cohort , 24.Tab2 = 3.35, P = .07) and FCGR3A , and there was some evidence of linkage disequilibrium for the 2 SNPs , as has been reported by others [Of the 379 infants genotyped for FCGR2A, 88 (23.2%) were homozygous for the high-affinity allele (H/H), 178 (47.0%) were heterozygous (H/R), and 113 (29.8%) were homozygous for the low-affinity allele (R/R). Mothers had similar distributions of FCGR2A alleles: 88 (23.2%) H/H, 174 (45.9%) H/R, and 117 (30.9%) R/R. For the FCGR3A genotype, 41 (10.8%) infants were homozygous for the high-affinity allele (V/V), 173 (45.6%) were heterozygous (V/F), and 165 (43.5%) were homozygous for the low-affinity allele (F/F). Mothers also had similar distributions of FCGR3A alleles: 44 (11.6%) V/V, 152 (40.1%) V/F, and 183 (48.3%) F/F. The sample population was in Hardy-Weinberg equilibrium for both FCGR2A . Maternal-infant FCGR2A genotype concordance was associated with reduced odds of infant infection ; however, this relationship did not remain significant after adjusting for factors associated with infant infection   . Furthermore, in a logistic regression controlling for factors associated with maternal transmission , the heterozygote genotype (V/F) was significantly associated with increased odds of infant infection compared with FCGR3A low-affinity homozygotes (F/F) . However, when comparing mothers with at least 1 high-affinity allele (V/F or V/V) to those mothers with only the low-affinity allele (F/F), there was not a statistically significant association between genotype and transmission (P = .14), suggesting that the presence of the high-affinity allele alone was not associated with increased transmission. Infant FCGR3A heterozygotes were not at an increased infection risk compared with homozygotes in a Cox proportional hazards model .With regard to FCGR3A, infant genotype was not associated with HIV infection P = .7; Table 2P = .053). The Kaplan-Meier curves suggest that the majority of excess transmission risk in heterozygotes occurs during the peripartum period . However, in a Cox proportional hazards model, a trend in heterozygotes having increased risk of transmission compared with homozygotes was observed .We next examined time to infant infection using Kaplan–Meier analyses to determine whether maternal FCGR3A genotype was associated with a particular transmission mechanism. In a log-rank test, there was a trend in association between maternal FCGR3A genotype and time to infant infection  did have slightly higher median breast milk viral loads than V/V (2.61 log10 copies/mL) and F/F (2.54 log10 copies/mL) homozygotes; however, in a linear regression model, maternal FCGR3A genotype was not associated with breast milk viral load (P = .37)  that may modulate viral load P = .35) or FCGR3A (P = .70) genotypes in a linear regression model  and FCGR3A (P = .95) infant genotypes were also not associated with mortality after estimated infection . These results support Brouwer et al  who simiThe observation that infant FCGR2A and FCGR3A genotypes do not influence infant infection or disease progression has important implications for treatment and therapy. Given that Fc-mediated ADCC activity has been suggested to provide protective and/or therapeutic benefits (reviewed in ), our reIn mothers, FCGR2A genotype was not associated with transmission, but FCGR3A was associated with transmission risk. We unexpectedly found that FCGR3A heterozygotes (V/F) had increased risk of transmission compared with homozygotes (V/V or F/F). In particular, there was increased peripartum transmission in breastfeeding mothers, indicating that transmissions during the early breastfeeding period may be impacted by FCGR3A genotype. The biological mechanism for this heterozygote disadvantage is unclear. In vitro data vary, but they do not suggest that FCGR3A heterozygotes have lower antibody binding or functional  activity compared with the 2 homozygote groups , 6, 35. This study was the first to examine the impact of both FCGR2A and FCGR3A genotypes in HIV-infected mothers and their infants. Importantly, these receptor genotypes were studied in a population in which Fc-mediated ADCC activity has been shown to impact infant outcome , 22. Ove


Hypercytokinemia is the main immunopathological mechanism contributing to a more severe clinical course in influenza A (H1N1) virus infections. Most patients infected with the influenza A (H1N1) pdm09 virus had increased systemic levels of pro-inflammatory cytokines; including interleukin IL-6, IL-8, and tumor necrosis factor-alpha (TNF-α). We propose that single-nucleotide polymorphisms (SNPs) in the promoter regions of pro-inflammatory genes are associated with the severity of influenza A (H1N1) pdm09 virus infection.TNF, LT, IL1B, IL6, CCL1, and IL8) using real-time PCR; the ancestral genotype was used for comparison. Genotypes were correlated with 27 clinical severity variables. Ten cytokines  were measured on a Luminex 100.145 patients with influenza A (H1N1) (pA/H1N1), 133 patients with influenza-like illness (ILI), and 360 asymptomatic healthy contacts (AHCs) were included. Eleven SNPs were genotyped in six genes (IL6 rs1818879 (GA) heterozygous genotype was associated with severe influenza A (H1N1) virus infection , and two IL1B SNPs, rs16944 AG and rs3136558 TC, were associated with a decreased risk of infection . Genetic susceptibility was determined (pA/H1N1 vs. AHC): the LTA rs909253 TC heterozygous genotype conferred greater risk (OR = 1.9), and a similar association was observed with the IL1B rs3136558 CC genotype (OR = 1.89). Additionally, severely ill patients were compared with moderately ill patients. The TNF-238 GA genotype was associated with an increased risk of disease severity . Compared with ILIs, patients with severe pA/H1N1 infections exhibited increased serum IL-5 (p <0.001) and IL-6 (p  =  0.007) levels.The TNF gene was associated with disease severity, whereas IL1B and IL6 SNPs were associated with influenza A (H1N1) virus infection.The  In mid-April 2009, the influenza A H1N1) pdm09 virus emerged in Mexico and rapidly expanded across the globe. Worldwide, the mortality rate was approximately 0.2–1.23%; in Mexico, mortality was close to 0.4% [N1 pdm09 Hypercytokinemia ("cytokine storm") is the main immunopathological mechanism underlying a more severe clinical course in cases of seasonal influenza and is the ultimate cause of death . Most paCytokine production varies among individuals due in part to genetic factors and, particularly, the presence of polymorphisms in important regulatory regions such as promoters , 13. In CCL1 rs2282692, LTA rs909253, and TNF 1800750, were identified in a population in Mexico City. [In a previous study performed by our team, several single-nucleotide polymorphisms (SNPs) associated with the risk of influenza infection, including co City. –16.Borja et al. 2012, the fourth-wave pattern of influenza A (H1N1) observed in Mexico during the 2011–2012 season was not reported in other countries. These differences may be explained by vaccination coverage or genetic susceptibility in the Mexican population [TNF, IL6, IL8, IL1B, LTA, CCL1) and disease development, severity and death from influenza A (H1N1) virus infection in patients from the central region of Mexico.According to pulation . In thisThis case-control, ambispective study was performed from April 2009 to March 2012 using nasopharyngeal swab samples from 278 patients hospitalized with clinical symptoms of influenza-like illness.This study was reviewed and approved by both the Bioethics Committee and Science Committee in Research, with protocol number B05-10 at INER  and Institutional Review Board No. 13CI 09015 213 at IMSS  authorized by COFEPRIS/SSA.A trained and responsible physician informed the patient about a possible diagnosis of influenza-like illness (ILI). All participants were invited to participate voluntarily by a trained researcher and gave signed informed consent prior their enrollment. All patients received medical care even if they chose not to participate in the research protocol. The signed consent forms and ethics committee approval letters are documented at HLA Laboratory INER. Data were obtained using clinical records according to the Mexican Official Standard NOM-168-SSA1-1998. A clinical investigation was conducted according to the principles expressed in the Declaration of Helsinki. The ethics committees described above approved our data recording procedures.Quidel, San Diego, CA, USA). Additionally, peripheral blood from patients and asymptomatic healthy contacts (AHC) was collected; these interventions adhered to the guidelines and recommendations of the Centers for Disease Control and Prevention of the United States (CDC) and the World Health Organization (WHO). Subsequently, the RespiFinder assay was administered, and the presence of influenza A (H1N1) virus was confirmed.All patients underwent the QuickVue Influenza A + B test  infection; the influenza-negative patients were deemed to have ILI. On admission to the hospital, all patients with suspected influenza were treated with oseltamivir.In this study, most patients sought medical attention 3–4 days after the onset of symptoms. All clinical data were collected retrospectively; nonetheless, blood samples and nasopharyngeal swabs were collected at the hospital during the first 24 hours after admission. In the group of AHCs, 360 biologically unrelated, intra-household contacts were enrolled who were asymptomatic and in close, continuous contact with ILI or H1N1 patients before and during admission to the hospital . None ofThe study protocol was explained to all participants, and authorization was obtained with an informed consent form. The information collected included demographic data as well as laboratory data, medical histories, and drug treatment records.TNF rs361525, rs1800629, and rs1800750; LTA rs909253; IL1B rs16944, rs3136558, and rs2227307; IL6 rs1818879, rs2069840, and rs2066992; and CCL1 rs2282691 polymorphisms using commercial TaqMan assays . The genetic data for the SNPs are described in DNA samples were genotyped for IL6, IL1B and TNF genes associated with the risk of both H1N1 viral infection and greater clinical severity.Haploview version 4.2 was used to determine the presence of haplotypes in Serum samples were taken from patients with ILI or a suspected ILI diagnosis upon hospital admission. The enrolled patients and the healthy volunteers gave whole blood, which was allowed to clot for 30 minutes at 37°C and was stored thereafter at -70°C until use. The resulting serum was used for cytokine measurement. Ten cytokines  were measured on a Luminex 100  using a multiplex cytokine kit as well as the Cytokine Human 10-Plex Panel  in accordance with the manufacturer's instructions.Differences between the group proportions were analyzed using the chi-square test. Differences between clinical parameters, continuous variables, and each polymorphism were analyzed using the Mann-Whitney U test. The ancestral genotype was used as a reference for each polymorphism. Odds ratios (ORs) were calculated with 95% confidence intervals (CIs). The ORs were adjusted for age and disease severity in the logistic regression model. SPSS 21  and Epi-Info 7.0  were used for these analyses.For cytokine analysis, the subjects were stratified into three groups: 18 patients with A (H1N1) influenza, 15 patients with ILI, and 21 AHCs. Descriptive statistics included means and standard deviations or medians and interquartile ranges for continuous variables with normal and non-normal distributions, respectively. The Fisher exact test and the chi-square test were used for categorical variables. The Mann-Whitney U test was used for nonparametric variables. The nonparametric Wilcoxon test was used to compare two paired groups. The association between nonparametric variables was determined using the Spearman correlation coefficient (r). Any value of p<0.05 was considered statistically significant. GraphPad Prism version 5.03 for Windows  was used for these analyses.vs. 5.21%, respectively; p = 0.03). There were no statistically significant differences for the other comorbidities studied (This study compared three groups: patients with confirmed influenza A (H1N1) (pA/H1N1), subjects with influenza-like illness (ILI), and AHCs. Gender distributions were similar in the three groups of patients, and differences between the groups were not significant. Patients in the pA/H1N1 group were slightly younger (42±14 years) than those in the ILI group . In addition, the presence of comorbidities was assessed, including diabetes, hypertension, asthma, and chronic obstructive pulmonary disease (COPD); interestingly, in the pA/H1N1 group, there was a higher rate of diabetes than in the ILI group (12.5%  studied .Patients with confirmed influenza A (H1N1) consistently showed a greater disruption of clinical parameters influencing evolution toward greater disease severity .To determine the risk of severe influenza A (H1N1) virus infection, the genotypes of individual SNPs were compared in both groups of patients (pA/H1N1 and ILI).IL6 rs1818879 (GA) heterozygous genotype showed a risk association with viral infection . Two IL1B SNPs were associated with a decreased risk of infection: rs16944 AG  and rs3136558 TC  (Genotypes associated with influenza A (H1N1) virus infection were identified. The 29–0.88) .LTA rs909253 TC heterozygous genotype showed a risk association with disease susceptibility ; a similar association occurred with the IL1B rs3136558 CC genotype . In contrast, the IL6 rs2066992 GT genotype was protective  for individuals carrying the homozygous genotype for the ancestral allele  . Of the 11 SNPs studied, the = 16.06) .LTA rs909253, allele A is a risk factor for disease development , whereas the IL6 rs2066992 T allele is a protective factor . In addition, the IL1B rs16944 G allele is protective against the development of severe influenza A (H1N1) virus infection  (With respect to 50–0.98) .IL6 rs2069869 GG genotype was associated with mortality risk in patients with diabetes in the pA/H1N1 group . Only statistically significant data are shown.Thirty-one patients positive for influenza A (H1N1) virus died, whereas four patients with ILI died. None of the polymorphisms/genotypes were directly associated with mortality. We also aimed to demonstrate whether there was any additional risk besides mortality associated with the presence of comorbidities; only the IL6, TNF, and IL1B genes with their respective SNPs and considered both the pA/H1N1 group vs. the ILI group and each group individually. When comparing the two groups of patients, haplotypes were not formed, only independent alleles, in groups of patients (ILI or pA/H1N1). TNF SNPs located on chromosome 6 in the patients with ILI; these haplotypes are not in high linkage disequilibrium (r2 = 0.75), although the value was close to the minimum required (0.80). Polymorphisms evaluated in the TNF and IL6 genes met Hardy-Weinberg equilibrium  and IL-6 (p = 0.007) and similar levels of GM-CSF (p =  0.967). We found no statistically significant differences for the remaining seven analytes evaluated. These results show that severe pA/H1N1 patients secrete higher levels of serum IL-6 and IL-5 than do ILI patients, suggesting a particular mechanism of pathophysiology in pA/H1N1 infection.Although the levels of serum IL-6 in pA/H1N1 patients were higher than those of healthy controls, no statistically significant differences were observed .vs. median of 29.93 (p<0.01), respectively. Concentrations of IL-6 in obese ILI patients and in obese pA/H1N1 patients were 10.58 and 32.30, respectively. Compared with non-obese ILI patients, non-obese pA/H1N1 patients showed an increase: 19.41 vs. 49.17; however there was no significant difference (p = 0.28).Finally, we examined the correlation between cytokine levels and severe clinical parameters, including obesity and leukocyte counts. The median IL-5 concentrations for obese ILI patients and for obese pA/H1N1 patients were 3.08 and 3.07, respectively. In contrast, compared with non-obese ILI patients, non-obese pA/H1N1 patients showed a 9-fold increase: median of 3.03 Pearson’s analysis showed no significant difference between BMI and IL-5 and IL-6 concentrations or leukocyte counts and IL-5 and IL-6 in the pA/H1N1 group.TNF and LTA are associated with susceptibility to infection with the influenza A (H1N1) pdm09 virus. Interestingly, two SNPs, one in IL1B (rs16944) and one in IL6 (rs1818879), were associated with clinical severity when comparing influenza A (H1N1) patients with ILI patients.In addition to the findings previously described, in this second study of polymorphisms in pro-inflammatory cytokine genes, our group identified genetic factors associated with susceptibility to influenza A (H1N1). In late 2012, we reported that some genotypes in LTA rs909253 GA genotype is a risk factor for susceptibility to developing secondary disease after contact with the influenza A (H1N1) pdm09 virus; interestingly, the minor allele (A) was associated with risk in this same comparison group (pA/H1N1 vs. AHC).This study confirms our previous finding that the IL1B and IL6 SNPs showed significant associations, particularly rs16944, which is located in the promoter region of the gene. In agreement with what has been reported previously, the GA genotype in the TNF-238 (rs361525) promoter presented a risk for greater disease severity in patients who were admitted to the ICU compared with those who only required hospitalization [TNF-238, we may observe that the presence of allele A in the heterozygous genotype in severe influenza A (H1N1) patients increased the risk of severity.To complement these observations, an association analysis was conducted regarding the risk of severe influenza A (H1N1) infection; both -238 rs3625 promotlization . DespiteIL1A, IL1B [IL10 [TNF-238A (rs361525) and TNF-308G (rs1800629), IL10-592C, and IL10-1082A have been associated with increased disease severity [Associations have recently been reported for the 1A, IL1B  and IL101B [IL10  genes, wseverity –16.LTA and TNF are considered biologically relevant inflammatory cytokines with various functions in the immune response. Several authors have shown that SNPs present in these genes are associated with increased susceptibility for developing infectious diseases [LTA and the TNF transcription start sites are in close proximity in the TNF cluster of the HLA class III region; therefore, it is difficult to obtain associations of SNPs in this region [LTA and TNF was only identified in the latter group, and high linkage disequilibrium was not observed (r2 = 0.75). We believe that expanding the n or considering other candidate genes could improve this approach. Because cytokine production is influenced by SNPs in the promoter regions of these genes [Both diseases –24. The s region . Currentse genes , we propChiaretti et al., influenza A (H1N1) virus infection was shown to induce the early and significant up-regulation of IL-1β and IL-6 in plasma, suggesting that these cytokines are responsible for different molecular reactions that lead to inflammation of the airway and increased disease severity [vs. 10.5 pg/mL), with no significant differences. In contrast, non-obese patients in the pA/H1N1 group showed a 9-fold increase in IL-5 levels. Despite the increase observed in both obese patient groups, it is important to note that compared with ILI patients and AHCs, non-obese patients with severe clinical pictures of H1N1 influenza have increased IL-5 levels. There were no significant differences for leukocyte counts and cytokine levels in pA/H1N1 and ILI patients.Previous studies have reported a correlation between IL-1β and IL-6 overexpression and some clinical, radiological and immunological findings, such as pneumonia and ARDS, in both children with seasonal influenza and murine models –29. TNF,severity . In turnseverity . Similarseverity –7. Strikseverity ,35. We fAccording to published reports, cytokine levels are affected over time , 36. NevConcerning age at presentation, our results indicate that many patients who are not in the age groups at greatest risk of death or who have no comorbidities associated with an increased risk of death from influenza may have genetic characteristics that make them more susceptible to developing influenza and presenting with greater disease severity. With respect to the population of 2010 and 2012 and in accordance with what has been reported by other authors –40, apprTNF rs361525 GA genotype is associated with greater disease severity. SNPs in IL6 are associated with susceptibility to developing influenza A (H1N1) infection. In addition, the IL6 rs2069869 GG is associated with mortality from influenza A (H1N1) infection in the presence of diabetes. IL-6 levels are increased in both obese pA/H1N1 and ILI patients compared with AHCs, but greater than an obese patient alone. Strikingly, non-obese pA/H1N1 patients presented much higher IL-6 levels than did obese patients. Influenza A (H1N1) virus infection and its severity are determined by the genetic characteristics of the individual, especially among genes that regulate inflammation.Our observations highlight the need to take into account that any patient may progress to severe disease because of the influence of their genetic background. Therefore, influenza mortality prevention strategies should consider universal vaccination. The S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.


Aspergillus fumigatus. Here, implementation of a shotgun proteomic strategy led to the identification of non-redundant mycelial proteins (n = 414) from A. fumigatus including proteins typically under-represented in 2-D proteome maps: proteins with multiple transmembrane regions, hydrophobic proteins and proteins with extremes of molecular mass and pI. Indirect identification of secondary metabolite cluster expression was also achieved, with proteins (n = 18) from LaeA-regulated clusters detected, including GliT encoded within the gliotoxin biosynthetic cluster. Biochemical analysis then revealed that gliotoxin significantly attenuates H2O2-induced oxidative stress in A. fumigatus (p>0.0001), confirming observations from proteomics data. A complementary 2-D/LC-MS/MS approach further elucidated significantly increased abundance (p<0.05) of proliferating cell nuclear antigen (PCNA), NADH-quinone oxidoreductase and the gliotoxin oxidoreductase GliT, along with significantly attenuated abundance (p<0.05) of a heat shock protein, an oxidative stress protein and an autolysis-associated chitinase, when gliotoxin and H2O2 were present, compared to H2O2 alone. Moreover, gliotoxin exposure significantly reduced the abundance of selected proteins (p<0.05) involved in de novo purine biosynthesis. Significantly elevated abundance (p<0.05) of a key enzyme, xanthine-guanine phosphoribosyl transferase Xpt1, utilised in purine salvage, was observed in the presence of H2O2 and gliotoxin. This work provides new insights into the A. fumigatus proteome and experimental strategies, plus mechanistic data pertaining to gliotoxin functionality in the organism.A combined proteomics and metabolomics approach was utilised to advance the identification and characterisation of secondary metabolites in  A. fumigatus Af293 A. fumigatus strain, A1163 IFollowing the publication of A. fumigatus has been undertaken whereby 530 plasma membrane associated proteins were identified by utilising a combination of SDS-PAGE fractionation of total protein followed by peptide separation and identification by 2-D-LC-MS/MS A. fumigatus germination A. fumigatus following incubation with human sera A. fumigatus has the potential to provide a global overview of the pathways and biological processes active under a set of conditions. In addition, (i) bioinformatic analysis can expand the characterisation of large datasets generated by MS-based proteomics, and (ii) shotgun proteomics offers the possibility of identifying the presence of either hypothetical proteins or proteins of unknown function, whose existence may either be unclear, or only previously demonstrated at the transcript level. Furthermore, (iii) shotgun MS-based proteomics has the potential to be used for the non-targeted identification of secondary metabolite (SM) cluster expression, which, coupled with subsequent metabolomics, could result in the identification of novel cluster products MS-based or shotgun proteomics can adopt multiple approaches including, (i) direct LC-MS/MS, (ii) indirect LC-MS/MS and (iii) 2-D-LC-MS/MS  A. fumigatusA. fumigatus under various conditions 2O2, separately, have been shown to result in numerous, growth inhibitory-associated, alterations to the proteome of A. fumigatus2O2 induces oxidative stress 2O2–induced growth inhibition of A. fumigatus in a concentration-dependent manner, although the basis for this phenomenon was not investigated.Proteomic approaches may also have an application in characterizing the effect of exogenous SMs on 2O2-induced oxidative stress in A. fumigatus was explored by 2-D and LC-MS/MS analysis. Overall, these combinatorial approaches reveal new insights into the expression, functionality and dynamic nature of the A. fumigatus proteome during normal growth and consequent to attenuated oxidative stress conditions.The aim of the work presented here was to investigate the potential for shotgun MS to dissect the mycelial proteome, particularly with respect to identifying SM cluster expression, allied to cognate metabolite biosynthesis. Moreover, dissection of the molecular basis of SM (gliotoxin)-mediated relief of HA. fumigatus ATCC26933 shaking cultures were harvested after 48 h, 200 rpm, 37°C in Aspergillus minimal media (AMM) and snap-frozen in liquid nitrogen. A. fumigatus ATCC26933 mycelia were also harvested from shaking cultures grown for 72 h in Czapek-Dox media, 37°C, 200 rpm. Protein was extracted and subjected to trypsin digestion as described Aspergillus fumigatus strains Af293 (reference strain) and A1163, acquired from www.gravy-calculator.de). Using Phobius (http://phobius.cbr.su.se), the number of putative transmembrane regions present in each identified protein was determined. Identified proteins were grouped into functional categories based on the FunCat , GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) annotations, using the FungiFun application (https://www.omnifung.hki-jena.de/FungiFun/) For shotgun proteomics, mycelia from A. fumigatus ATCC26933 grown for 48 h in AMM or 72 h in Czapek-Dox, as described above. Organic extractions were carried out using a 1∶1 mixture of chloroform to culture supernatant Culture supernatants were harvested from 2O2-induced stress in A. fumigatus ATCC26933 were investigated by comparative 2-D and LC-MS/MS. A. fumigatus ATCC26933 was grown in Sabouraud dextrose media at 200 rpm, 37°C for 24 h prior to addition of one of the following four treatments: (i) Solvent control (500 µl methanol added per 50 ml culture), (ii) Gliotoxin alone , (iii) H2O2 alone , (iv) Gliotoxin and H2O2 combined . Mycelia  were harvested after 4 h and ground in liquid nitrogen. Crushed mycelia were resuspended in 10% (w/v) TCA and sonicated three times at 10% power, cycle 6, 10 sec. Samples were incubated on ice for 20 min and centrifuged at 10000 g, 4°C for 10 min. Pellets were washed with ice-cold acetone to remove excess TCA and resuspended in IEF buffer n = 20; 5 replicates/treatment) from all four treatments were aligned and subsets of treatments were compared. Spots demonstrating significant changes in abundance  were excised from gels and trypsin digested according to the method described by Shevchenko (2007) The mechanisms involved in gliotoxin-mediated relief of HA. fumigatus ATCC26933 conidia  were added to Sabouraud liquid media (4 ml/well) and incubated for 24 h at 37°C, static. Each well contained a glass microscope slide. After removal of the top layer of mycelia, H2O2  only, or with gliotoxin  was added. H2O2 alone acts as a positive control as it is a known ROS inducing agent. Gliotoxin  and an equivalent volume of MeOH were also added individually to separate wells as negative control. Plates were re-incubated 37°C, static, for 30 min, culture supernatants removed and mycelial mats washed once with 4 ml PBS for 5 min. After PBS removal, Sabouraud medium (3 ml) containing 2′,7′-dichlorodihydrofluorescein diacetate  was added to each well followed by incubation 37°C for 40 min, static. After washing twice with PBS (2×20 min each), mycelia were visualised using a fluorescent microscope (GFP filter: Ex/Em: 492–495/517–527 nm). Fluorescence was quantified by measuring Integrated Density Value (IDV) of selected areas from each image (n = 5/treatment) representing subsequent data as means ± standard error. Differences were evaluated using ANOVA, and statistical significance was accepted at p<0.05.A. fumigatus mycelial lysates, corresponding to 414 distinct A. fumigatus proteins from 405 protein groups (n = 414) spanned a theoretical pI range of 3.9 to 11.8 and a Mr range of 7.8 to 444 kDa , based on positive GRAVY scores. Additionally, GRAVY scores were computed for the entire predicted proteome of A. fumigatus and it was observed that 15.6% of the total proteome possess positive GRAVY scores  were detected. Several proteins were detected with 10 or more putative TM regions, including a plasma membrane H+-ATPase (AFUA_3G07640), an amino acid permease (Gap1) (AFUA_7G04290) and two ABC transporters (AFUA_1G14330 and AFUA_5G06070). One protein, a small oligopeptide transporter (OPT family) (AFUA_2G15240) was detected with 14 putative transmembrane regions and a GRAVY score of 0.276. Annotations were available for 89.37%, 86.47% and 49.27% of identified proteins using the FunCat, GO and KEGG schemes, respectively , energy , protein with binding function or cofactor requirement  and transcription . Proteins  were identified which have no functional classifications using the aforementioned methods.The GRAVY index for identified proteins ranged from −1.632 to 0.483, with positive scores indicating hydrophobicity . A numbeY scores . The majectively . Based oA. fumigatus chromosomes, using their gene locus identifiers (n = 15) that comprise a secondary metabolite supercluster, involved in the production of pseurotin A, fumitremorgins and fumagillin Afpes1 NRPS cluster on chromosome 1 Proteins identified by shotgun mass spectrometry were mapped based on their relative loci on each of the eight ntifiers . A numben = 13) were found to be significantly differentially abundant (p<0.05) when A. fumigatus was co-exposed to gliotoxin/H2O2 compared to H2O2 alone . Redundancy was noted, with some proteins included in multiple comparison sets, resulting in the net differential expression of 27 unique proteins  were significantly altered in abundance following co-addition relative to H2O2 alone (p<0.05)  but was unaltered by H2O2 addition alone  upon co-addition relative to H2O2 alone. A decrease in abundance of proteins associated with response to stress was observed following gliotoxin/H2O2 co-exposure, relative to H2O2 alone. Hsp90 and the oxidative stress protein Svf1 were of lower abundance in the co-addition , reflective of the relief of H2O2-induced stress  . Proteinon alone . An incrd stress . AdditioA. fumigatus against exogenous gliotoxin 2O2-mediated growth inhibition of A. fumigatus ATCC26933 and ΔgliK269332O2 induced-oxidative stress. Data in 2O2 exposure in A. fumigatus. However, co-addition of gliotoxin results in a significant reduction (p>0.0001) in the production of reactive metabolites, as judged by decreased 2′,7′-dichlorofluorescein fluorescence. This suggests that gliotoxin acts as an anti-oxidant and functions to impede H2O2-mediated growth inhibition.Previous work has demonstrated that GliT is essential to protect et al.A. fumigatus mycelia, corresponding to 10.62% of the proteins identified using shotgun mass spectrometry alone  from the gliotoxin biosynthetic cluster were identified (Verification of proteins encoded by genes within secondary metabolite clusters was achieved in this study using shotgun mass spectrometry. Proteins identified from AMM cultures are putatively encoded by six clusters involved in the production of up to nine secondary metabolites (SM) entified , in lineentified . Enlisti2O2-induced stress. Proteins (n = 13) were differentially abundant following exposure to a combination of H2O2 (2 mM) and gliotoxin (10 µg/ml), relative to H2O2 alone (2 mM), which facilitates dissection of the mechanisms involved in gliotoxin-mediated relief of H2O2-induced stress , with 3.5 and 1.9 fold increase in abundance, respectively. In addition to a key role in the gliotoxin biosynthetic process, GliT also mediates self-protection against the harmful effects of gliotoxin 2O2 (4.8 fold) relative to the solvent control  and this demonstrates that GliT abundance is not regulated by H2O2 and increased levels in the co-addition condition is solely a result of gliotoxin presence. Choi et al.2O2 reduction, mediated by the mammalian thioredoxin redox system and proposed that gliotoxin replaces 2-cys peroxiredoxin as an electron acceptor, in the reduction of H2O2 to H2O in mammalian cells.Proteomics also revealed changes in protein abundance associated with SM (gliotoxin)-mediated relief of Hd stress . Increas control . There w2O2, relative to H2O2 alone. Moreover, abundance of this protein was also further increased in the co-addition condition, relative to gliotoxin alone (7.4 fold) and the solvent control  and not by exposure to gliotoxin alone. Clearly, a combination of H2O2 with gliotoxin leads to further induction of this protein. PCNA is involved in the process of DNA-repair following H2O2-mediated damage 2O2, alone or coupled with gliotoxin, may therefore be indicative of H2O2-induced DNA damage in these conditions. Furthermore, additional induction of PCNA abundance in the co-addition condition relative to H2O2, alone, may account for the recovery of growth, due to enhanced DNA repair capacity.The proliferating cell nuclear antigen (PCNA) exhibited a 2.4 fold increase in abundance following incubation with a combination of gliotoxin and H2O2 alone. A decrease in abundance of Hsp90 (2.7 fold) and the oxidative stress protein Svf1 (1.6 fold) was noted. Hsp90 displayed increased abundance in the presence of H2O2 alone, relative to the solvent control (2.0 fold), with this response reversing upon co-incubation with gliotoxin. In accordance with these observations at the proteomic level, the transcript of Hsp90 was also reported to be up-regulated in A. fumigatus in response to exogenous H2O22O2 alone. Indeed, a significant reduction (p<0.0001) in ROS levels was detected following co-application of gliotoxin and H2O2, relative to H2O2 alone , an intermediate of the purine biosynthetic process, thus linking these pathways 2O2 combined, relative to the solvent control (1.8 fold) and gliotoxin alone (1.6 fold), indicating that levels of this protein are influenced by H2O2. Indeed, Lessing et al.2O2 for 45 min. Xpt1 is involved in the purine nucleotide salvage pathway, whereby XMP and GMP are formed from precursors, xanthine and guanine, respectively de novo purine biosynthesis in the presence of gliotoxin and that the alternative salvage pathway is utilised in its place  based on gene locus (blue lines). Identification of proteins (n = 15) from a supercluster on chromosome 8, involved in the production of fumitremorgin B, pseurotin A and fumagillin (red circle).(DOC)Click here for additional data file.Table S1Proteins identified by shotgun MS analysis.(XLS)Click here for additional data file.Table S2Peptides detected from gliotoxin cluster proteins following growth in Czapek-Dox media for 72 h at 37°C.(XLS)Click here for additional data file.Table S31 in Proteins undergoing significant differential abundanceA. fumigatus2O2, separately or combined, relative to the solvent control. ATCC26933 following exposure to gliotoxin and H Protein identification was achieved by 2D-PAGE and LC-MS/MS.(DOC)Click here for additional data file.Table S41 in Proteins undergoing significant differential abundanceA. fumigatus2O2 (co-addition), relative to the control, gliotoxin alone or H2O2 alone. ATCC26933 following exposure to a combination of gliotoxin and H Protein identification was achieved by 2D-PAGE and LC-MS/MS.(DOC)Click here for additional data file.Text S1Supplementary Discussion.(DOC)Click here for additional data file.


Drosophila polyploid endocycling cells (G/S cycle) repress the apoptotic response to DNA damage through at least two mechanisms. First, the expression of all the Drosophila p53 protein isoforms is strongly repressed at a post-transcriptional step. Second, p53-regulated pro-apoptotic genes are epigenetically silenced in endocycling cells, preventing activation of a paused RNA Pol II by p53-dependent or p53-independent pathways. Over-expression of the p53A isoform did not activate this paused RNA Pol II complex in endocycling cells, but over-expression of the p53B isoform with a longer transactivation domain did, suggesting that dampened p53B protein levels are crucial for apoptotic repression. We also find that the p53A protein isoform is ubiquitinated and degraded by the proteasome in endocycling cells. In mitotic cycling cells, p53A was the only isoform expressed to detectable levels, and its mRNA and protein levels increased after irradiation, but there was no evidence for an increase in protein stability. However, our data suggest that p53A protein stability is regulated in unirradiated cells, which likely ensures that apoptosis does not occur in the absence of stress. Without irradiation, both p53A protein and a paused RNA pol II were pre-bound to the promoters of pro-apoptotic genes, preparing mitotic cycling cells for a rapid apoptotic response to genotoxic stress. Together, our results define molecular mechanisms by which different cells in development modulate their apoptotic response, with broader significance for the survival of normal and cancer polyploid cells in mammals.Apoptotic cell death is an important response to genotoxic stress that prevents oncogenesis. It is known that tissues can differ in their apoptotic response, but molecular mechanisms are little understood. Here, we show that  Drosophila, that endocycling cells do not initiate cell death in response to DNA damage. The endocycle is a cell cycle variation that is widely found in nature and conserved from plant to animals. During the endocycle, cells duplicate their genomic DNA but do not enter mitosis to segregate chromosomes, resulting in a polyploid genome content. In this study, we investigate how the apoptotic response to DNA damage is repressed in endocycling cells. We find that the Drosophila ortholog of the human p53 tumor suppressor protein is expressed at very low levels in endocycling cells. Moreover, the downstream pro-apoptotic genes that are regulated by p53 are epigenetically silenced in endocycling cells. Our results provide important insights into tissue-specific apoptotic responses in development, with possible broader impact on understanding radiation therapy response and cancer of different tissues.In order to maintain genome integrity, eukaryotic cells have evolved multiple ways to respond to DNA damage stress. One of the major cellular responses is apoptosis, during which the cell undergoes programmed cell death in order to prevent the propagation of the damaged genome to daughter cells. Although clinical observations and other studies have shown that tissues can differ in their apoptotic response, the molecular mechanisms underlying these differences are largely unknown. We have shown in our model system,  Drosophila as a model to define the mechanisms by which cells in development differ in their apoptotic response to genotoxic stress.Eukaryotic cells respond to DNA damage via multiple pathways. Checkpoints arrest the cell cycle and mobilize the DNA repair machinery to fix the damage in vivoMany investigations have focused on how cells in culture respond to genotoxic stress, but much less is known about the different stress responses of tissues Drosophila tissues that are composed of cells in the endocycle repress the apoptotic response to DNA damage We had previously shown that Drosophila endocycling cells do not apoptose in response to DNA damage because both p53-dependent and independent pathways are repressed reaper (rpr), Head involution defective (Hid), and sickle (skl) are dependent on p53 after DNA damage, and evidence suggests that at least rpr is a direct target of p53 Hid expression Our previous evidence suggested that Drosophila p53 protein levels are regulated. In mitotic cycling cells, p53 protein and a paused RNA polymerase complex at the H99 gene promoters may prepare these cells to rapidly respond to genotoxic stress. We discuss the significance of these results in the context of tissue-specific apoptotic responses in development and cancer.In this study, we investigate the molecular mechanisms underlying the different apoptotic responses of mitotic cycling and endocycling cells. Our evidence suggests that p53-dependent and p53-independent apoptotic pathways are repressed in endocycling cells through epigenetic silencing of pro-apoptotic genes at the H99 locus. In addition, we find that although p53 mRNA levels are comparable between mitotic cycling and endocycling cycling cells, p53 protein is undetectable in endocycling cells. Thus, similar to humans, rpr, hid, sickle, and grim are much lower in endocycling larval salivary gland (SG) and fat body (FB) tissues than in mitotic cycling larval brain and imaginal discs (B–D) rpr and hid. The hid 5′F-WT (hereafter hid-GFP) and rpr-11-lacZ reporters contain only the promoter and part of the enhancer regions of the rpr and hid gene, but lack the transcription units, and therefore are insensitive to post-transcriptional regulation hid-GFP reporter was expressed at very low levels in both the mitotic cycling B–D and endocycling SG cells  using antibodies against chromatin silencing and activating marks in mitotic cycling B–D and endocycling SG tissues. Both the rpr and hid enhancer-promoter regions had higher levels of the silencing mark H3K27Me3 in SG than in B–D cells (SG/B–D ∼10 fold)   . Within  p53REs) . ChIP foSG cells . ChIP fong marks , S2A. Anng marks  [45]. ToUAS:6xMyc:p53 and UAS:GFP transgenes under control of the salivary gland driver, Fkh:GAL4 (Su(var)3-9, Eggless and G9a, all resulted in salivary gland cell death Su(var)3-9 was the most severe, with three different RNAi constructs resulting in extremely small salivary glands and high levels of apoptotic cell death as evidenced by pycnotic nuclei, Caspase-3 labeling, and TUNEL (Enhancer of Polycomb (E(Pc)) (E(Pc) is known to modify the silencing of genes by Polycomb complexes, which are writers and readers of the H3K27me3 mark, and is also a suppressor of heterochromatic variegation of genes with the H3K9me3 mark E(Pc) or the H3K9 HMTs also induced some salivary gland apoptosis in the absence of p53 over-expression, which may be triggered by the constitutive heterochromatic DNA damage that is known to occur in endocycling cells (E(Pc) phenotype, however, was clearly more severe when p53 was over-expressed ) . E(Pc) ing cells  [19], . Flfunction –D.rpr and hid genes in the mitotic cycling B–D cells, even in the absence of genotoxic stress ) mutants, in which the H99 locus is almost fully replicated, endocycling SG cells still did not apoptose in response to DNA damage It was previously shown that the chromatin organization at the H99 locus impedes its DNA replication in endocycling cells rpr and hid genes , suggesting a conservation of cell cycle regulation hid-GFP fly strain was kindly provided by W. Du. A y w strain was used as wild type. For the proteasome dominant negative experiments, crosses were performed at 25°C with 24 hr egg lay. The vials were then transferred to 29°C until 3rd instar larvae.Fly strains were raised at 25°C prior to and during experimental procedures. Fly strains were obtained from the Bloomington Drosophila Stock Center  unless otherwise noted. rd larval instar brains and imaginal discs (B–D) and salivary glands (SG).The ChIP protocol was modified from previous methods  and entailed at least two biological replicates from separate isolations of 3Total RNA was isolated from hand-dissected tissues using TRIzol . 1 µg of RNA from each sample was reverse-transcribed using the QuantiTect Reverse Transcription Kit (Qiagen) according to manufacturer's instructions. qPCR analysis was done on a Stratagene  Mx3005P machine with SYBR Green Master Mix . For mRNA quantification, Act 5C was used as a reference gene to calculate the relative expression (fold difference). For ChIP-qPCR experiments, the amount of DNA in the pellet was expressed as percentage of input DNA estimated by a standard curve generated from a serial dilution of the input. The values were then normalized to a control. Antibodies used for ChIP are described in +-attB vector and then transformed into the attP docking site at 65B2 (strain 24871) UAS:6xMyc:p53A (P #44) and UAS:6xMyc:p53B (P#20).BAC CH322-178C12 from the P[acman] library was tagged with mCherry at the end of the common last exon of the p53 gene Protein extracts were prepared from hand-dissected tissues of mid-late 3rd instar larvae by standard methods using RIPA buffer For RFP nanobody IP rd instar larvae were dissected in either 1× PBS or Grace's solution, and fixed in 6% formaldehyde as previously described In Situ cell death detection kit, TMR red, Roche) was performed according to manufacture's instructions. Wide-field micrographs were taken on a Leica DMRA2 and analyzed using OpenLab (Improvision) software. Confocal micrographs were captured on a Leica SP5 confocal.Mid-late 3Larvae were irradiated with a total of 4,000 rad (40 Gy) from a Cesium source, and 4, 6 and 24 hours later labeled with anti-activated-Caspase-3.Figure S1rpr-11-lacZ reporter in 3rd instar wing imaginal discs  and salivary glands  without  or with  IR. X-gal staining was performed 4 hours after 4000 rads of gamma ray treatment. rpr-11-lacZ is expressed in no IR controls due to developmental inputs, but expression increases after IR in discs only. (E–H) Expression of the hid-GFP promoter-reporter in adult female ovaries without  or with  IR. GFP expression is shown in E and G, and corresponding DAPI staining is shown in F and H. hid-GFP is induced by IR in mitotic cycling follicle cells before stage 7 (S7), but not in endocycling follicle cells in stage 7 and later egg chambers. (I–J) Over-expression of UAS:6xMyc:p53A induces apoptosis in the mitotic cycling cells independent of Chk2 function. p53 over-expression driven by hsp70:GAL4 in control wing discs from sibling larvae heterozygous  or homozygous  for a recessive Chk2 null mutation. Shown is Caspase-3 staining , and corresponding DAPI . Scale bars are 100 microns.H99 gene promoter activity reporters are repressed in the endocycling cells. (A–D) Expression of the (TIF)Click here for additional data file.Figure S2rd instar larval brain and imaginal disc  and salivary gland  indicates that the activating mark poly AcH3 at the promoter-enhancer region of the rpr gene is lower in SG than in B–D, whereas acetylation at the Act 5C control locus was similar. X-axis: primer position relative to TSS. (B) Analysis of genome-wide ChIP-array data for H3K27Me3 enrichment in salivary gland cells from Sher et al. paper rpr and hid genes analyzed in H99 locus has a deficit of activating marks and is enriched for repressive chromatin marks in endocycling cells. (A) ChIP-qPCR of 3(TIF)Click here for additional data file.Figure S3p53, UAS:6xMyc:p53/+ ; Fkh:GAL4, UAS:GFP/+. (B-B″) Salivary gland from a larva over-expressing p53 with Su(var)3-9 knockdown, RNAiUAS:6xMyc:p53/+ ; Fkh:GAL4, UAS:GFP/UAS:Su(var)3-9.  GFP fluorescence,  TUNEL,  DAPI. Images in A–B″ were captured at 40× and scale bars are 100 microns. (C-C″) A 10× image of a salivary gland from the screening strain that over-expresses p53, UAS:6xMyc:p53/+ ; Fkh:GAL4, UAS:GFP/+. (D-D″) Salivary gland from a larva over-expressing p53 with E(Pc) knockdown, RNAi.UAS:6xMyc:p53/+ ; Fkh:GAL4, UAS:GFP/UAS:E(Pc) (E-E″) E(Pc) knockdown without p53 over-expression, RNAi.Fkh:GAL4, UAS:GFP/UAS:E(Pc)  GFP fluorescence,  anti-cleaved Caspase 3,  DAPI. Images in C–E″ were all captured at 10× and scale bars are 100 microns.RNAi against epigenetic regulators results in apoptosis in endocycling SG cells. (A-A″) Salivary gland from the screening strain that over-expresses (TIF)Click here for additional data file.Figure S4rd instar larval salivary glands after acute expression of UAS:6XMyc:p53A  or UAS:6XMyc:p53B  by SGS3:GAL4 as indicated on the left. Scale bars are 100 microns.Acute expression of p53B, but not p53A, isoform induces apoptosis in endocycling cells. (A–B′) Activated Caspase-3  and DAPI  labeling in late 3(TIF)Click here for additional data file.Figure S5rd instar larval wing discs  or salivary glands  after over-expression of UAS:6XMyc:p53A  or UAS:6XMyc:p53B  as indicated on the left. Strains were transformed by either P element transformation into random sites (“P” A–H) or targeted insertion into the same genomic docking site using Phi C31 (“PhiC” I–L). Different numbers #44, #43, #20, #28 indicate independent P element transformants. Tissues were fixed six hours after a 30 min heat pulse of expression using hsp70:GAL4. (A–D) are from Analysis of multiple strains indicates that the p53B, but not p53A, isoform induces apoptosis in endocycling cells when over-expressed. (A–L) Activated Caspase-3 labeling in 3(TIF)Click here for additional data file.Figure S6rpr gene, but p53B is better at activating elongation of a paused RNA Pol II.  Over-expressed p53A or p53B binds to p53REs in the rpr promoter-enhancer in both B–D (A) and SG (B) tissues. ChIP-qPCR analysis with anti-Myc antibody on 3rd instar B–D and SG cells over-expressing UAS:6xMyc:p53A (▪), or UAS:6xMyc:p53B (•) six hours after a 30 min heat induction with hsp70:GAL4, or in controls (▴). X axis: position of the primers relative to the TSS with p53RE in red. Y axis: qPCR value with the −6,000 in rpr defined as 1. Error bars represent the range of data from two independent biological repeats.  ChIP-qPCR analysis using anti-poly AcH4 antibody on 3rd instar B–D (C) or SG (D) cells over-expressing either UAS:6xMyc:p53A (▪) or UAS:6xMyc: p53B (•), six hours after a 30 min heat pulse with hsp70:GAL4, or control (▴). X-axis: primer position relative to TSS with p53RE in red. Y axis: qPCR value with the −212 in hid defined as 1  and SG (F) cells. ChIP-qPCR analysis using anti-phosphorylated Pol II Ser5 in 3rd instar B–D and SG cells. X-axis: primer position relative to TSS. Y axis: qPCR values with −5921 in rpr defined as 1. (G) p53B is better than p53A for promoting RNA Pol II elongation. ChIP qPCR for elongating RNA Pol II phoshorylated on Serine 2 (Ser 2) at the hid gene in SG cells over-expressing UAS:6xMyc:p53A (▪), or UAS:6xMyc:p53B (•) six hours after a 30 min heat induction with hsp70:GAL4. X-axis: primer position relative to TSS, Y axis: qPCR values with −6810 in hid defined as 1. See hid gene.Both over-expressed p53A and p53B bind and recruit acetylation to the as 1 see . Error b(TIF)Click here for additional data file.Figure S7rd instar larval wing imaginal discs treated with IR. (A) Wild type. (B) 5A-1-4p53 null mutant. (C) 5A-1-4p53 null mutant with p53 wild type BAC. (D) 5A-1-4p53 null mutant with p53-Ch BAC. Scale bars are 100 microns.BAC recombineered p53-Ch rescues p53 null mutant apoptotic response to radiation. (A–D) Anti-Cleaved-caspase-3 staining of 3(TIF)Click here for additional data file.Table S1DNA primers used in this study.(XLSX)Click here for additional data file.Text S1Supplemental materials and methods.(DOC)Click here for additional data file.